These books synthesize perspectives of renowned scientists from the world’s most prestigious institutions - from Fukushima Renewable Energy Institute in Japan to Stanford University in the United States, including Columbia University (US), University of Sidney (AU), University of Miami (USA), Cardiff University (UK), and many others.
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This collaboration embodied the true essence of Open Access by simplifying the approach to OA publishing for Academic editors and authors who contributed their research and allowed the new research to be made available free and open to anyone anywhere in the world.
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To celebrate the 50 books published, we have gathered them at one location - just one click away, so that you can easily browse the subjects of your interest, download the content directly, share it or read online.
IntechOpen and Knowledge Unlatched formed a partnership to support researchers working in engineering sciences by enabling an easier approach to publishing Open Access content. Using the Knowledge Unlatched crowdfunding model to raise the publishing costs through libraries around the world, Open Access Publishing Fee (OAPF) was not required from the authors.
\n\n
Initially, the partnership supported engineering research, but it soon grew to include physical and life sciences, attracting more researchers to the advantages of Open Access publishing.
\n\n\n\n
These books synthesize perspectives of renowned scientists from the world’s most prestigious institutions - from Fukushima Renewable Energy Institute in Japan to Stanford University in the United States, including Columbia University (US), University of Sidney (AU), University of Miami (USA), Cardiff University (UK), and many others.
\n\n
This collaboration embodied the true essence of Open Access by simplifying the approach to OA publishing for Academic editors and authors who contributed their research and allowed the new research to be made available free and open to anyone anywhere in the world.
\n\n
To celebrate the 50 books published, we have gathered them at one location - just one click away, so that you can easily browse the subjects of your interest, download the content directly, share it or read online.
\n'}],latestNews:[{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"},{slug:"introducing-intechopen-book-series-a-new-publishing-format-for-oa-books-20210915",title:"Introducing IntechOpen Book Series - A New Publishing Format for OA Books"}]},book:{item:{type:"book",id:"6972",leadTitle:null,fullTitle:"Soybean for Human Consumption and Animal Feed",title:"Soybean for Human Consumption and Animal Feed",subtitle:null,reviewType:"peer-reviewed",abstract:"Soybean is the main oilseed crop worldwide, a staple crop for protein-rich food and feed as well as a significant source of nutraceutical compounds with many different medical benefits. Soybean for Human Consumption and Animal Feed highlights the state of research in soybean nutritional attributes as well as science-based approaches defining the future of soybean for human consumption and animal feed. Over seven chapters, this book presents a comprehensive picture of the potential of soybean for human and animal diets and health as well as quality stock for pharmaceutical and functional food industries with an emphasis on the importance of genetic improvement of soybean germplasm in enhancing healthy and safe properties of final soy products.",isbn:"978-1-83881-019-1",printIsbn:"978-1-83881-018-4",pdfIsbn:"978-1-83881-020-7",doi:"10.5772/intechopen.73719",price:119,priceEur:129,priceUsd:155,slug:"soybean-for-human-consumption-and-animal-feed",numberOfPages:160,isOpenForSubmission:!1,isInWos:1,isInBkci:!1,hash:"4bc6f95dc8630c9a8be84bb46286c445",bookSignature:"Aleksandra Sudarić",publishedDate:"December 2nd 2020",coverURL:"https://cdn.intechopen.com/books/images_new/6972.jpg",numberOfDownloads:5216,numberOfWosCitations:13,numberOfCrossrefCitations:9,numberOfCrossrefCitationsByBook:2,numberOfDimensionsCitations:25,numberOfDimensionsCitationsByBook:2,hasAltmetrics:1,numberOfTotalCitations:47,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"June 5th 2019",dateEndSecondStepPublish:"December 25th 2019",dateEndThirdStepPublish:"February 23rd 2020",dateEndFourthStepPublish:"May 13th 2020",dateEndFifthStepPublish:"July 12th 2020",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6",editedByType:"Edited by",kuFlag:!1,featuredMarkup:null,editors:[{id:"21485",title:"Dr.",name:"Aleksandra",middleName:null,surname:"Sudarić",slug:"aleksandra-sudaric",fullName:"Aleksandra Sudarić",profilePictureURL:"https://mts.intechopen.com/storage/users/21485/images/system/21485.jpg",biography:"Dr. Aleksandra Sudarić was born in Osijek, Croatia. She received an MSc in Plant Genetics and Breeding in 1996 from the University of Zagreb and a PhD in Plant Breeding in 1999 from the University of Osijek. Dr. Sudarić is a senior research scientist at the Agricultural Institute Osijek (Croatia), Department for Breeding and Genetics of Industrial Plants where she studies the breeding and seed production of soybean. She is presently coordinator of the soybean breeding program at the Institute. In addition, she completed the training for application of DNA-based marker technology at the University of Guelph in Guelph (Canada). Her main research interests are in the genetic improvement of quantity and quality characteristics of soybean and soybean adaptation to mega-environments. Dr. Sudarić has authored more than 100 papers (scientific, professional, and popular), several book chapters, books, and manuscripts about soybean as well as developed twenty-six soybean varieties.",institutionString:"Agricultural Institute Osijek",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"Agricultural Institute Osijek",institutionURL:null,country:{name:"Croatia"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"36",title:"Food Science",slug:"food-science"}],chapters:[{id:"73415",title:"Introductory Chapter: Soybean - Quality and Utilization",doi:"10.5772/intechopen.93942",slug:"introductory-chapter-soybean-quality-and-utilization",totalDownloads:525,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:null,signatures:"Aleksandra Sudarić",downloadPdfUrl:"/chapter/pdf-download/73415",previewPdfUrl:"/chapter/pdf-preview/73415",authors:[{id:"21485",title:"Dr.",name:"Aleksandra",surname:"Sudarić",slug:"aleksandra-sudaric",fullName:"Aleksandra Sudarić"}],corrections:null},{id:"69396",title:"Soybean Amino Acids in Health, Genetics, and Evaluation",doi:"10.5772/intechopen.89497",slug:"soybean-amino-acids-in-health-genetics-and-evaluation",totalDownloads:1339,totalCrossrefCites:0,totalDimensionsCites:6,hasAltmetrics:1,abstract:"Soybean is an important source of protein and amino acids for humans and livestock because of its well-balanced amino acid profile. This chapter outlines the strengths and weaknesses of soybean as a complete amino acid source as well as the relative importance of individual amino acids. Special attention is paid to the sulfur-containing amino acids, methionine and cysteine. Breeding and genetic engineering efforts are summarized to highlight previous accomplishments in amino acid improvement and potential avenues for future research. Agronomic properties and processing methods that affect amino acid levels in soybean food and feed are also explained. A brief introduction into current amino acid evaluation techniques is provided. By understanding the complexities of amino acids in soybean, protein quality for humans and livestock can be maximized.",signatures:"William Monte Singer, Bo Zhang, M.A. Rouf Mian and Haibo Huang",downloadPdfUrl:"/chapter/pdf-download/69396",previewPdfUrl:"/chapter/pdf-preview/69396",authors:[{id:"308970",title:"Mr.",name:"William",surname:"Singer",slug:"william-singer",fullName:"William Singer"},{id:"309005",title:"Dr.",name:"Bo",surname:"Zhang",slug:"bo-zhang",fullName:"Bo Zhang"},{id:"310776",title:"Dr.",name:"M.A. Rouf",surname:"Mian",slug:"m.a.-rouf-mian",fullName:"M.A. Rouf Mian"},{id:"310777",title:"Dr.",name:"Haibo",surname:"Huang",slug:"haibo-huang",fullName:"Haibo Huang"}],corrections:null},{id:"69807",title:"Improving Seed Quality of Soybean Suitable for Growing in Europe",doi:"10.5772/intechopen.89922",slug:"improving-seed-quality-of-soybean-suitable-for-growing-in-europe",totalDownloads:987,totalCrossrefCites:3,totalDimensionsCites:7,hasAltmetrics:0,abstract:"The potential of soybean for food, feed, and pharmaceutical industry arises from the composition of its seed. Since European countries import 95% of the annual demand for soybean grains, meal, and oil, causing an enormous trade deficit, the governments in Europe had started to introduce additional incentives to stimulate soybean cropping. To rebalance the sources of soybean supply in the future, production must be followed by continuous research to create varieties that would make European soybean more appealing to the processing industry and profitable enough to satisfy European farmers. This chapter is giving an overview of the European soybean seed quality research and an insight into soybean seed quality progress made at the Agricultural Institute Osijek, Croatia. The studies presented are mainly considering maturity groups suitable for growing in almost all European regions. The most important traits of soybean seed quality discussed are protein content and amino acid composition, oil content and fatty acid composition, soluble sugars, and isoflavones. Defining quality traits facilitates the parental selection in breeding programs aiming to improve the added value properties of final soybean products and enables the exchange of materials between different breeding and research institutions to introduce diversity, which is a prerequisite for genetic advance.",signatures:"Aleksandra Sudarić, Maja Matoša Kočar, Tomislav Duvnjak, Zvonimir Zdunić and Antonela Markulj Kulundžić",downloadPdfUrl:"/chapter/pdf-download/69807",previewPdfUrl:"/chapter/pdf-preview/69807",authors:[{id:"21485",title:"Dr.",name:"Aleksandra",surname:"Sudarić",slug:"aleksandra-sudaric",fullName:"Aleksandra Sudarić"},{id:"311324",title:"Dr.",name:"Maja",surname:"Matoša Kočar",slug:"maja-matosa-kocar",fullName:"Maja Matoša Kočar"},{id:"311331",title:"Dr.",name:"Tomislav",surname:"Duvnjak",slug:"tomislav-duvnjak",fullName:"Tomislav Duvnjak"},{id:"311332",title:"Dr.",name:"Zvonimir",surname:"Zdunić",slug:"zvonimir-zdunic",fullName:"Zvonimir Zdunić"},{id:"311333",title:"Dr.",name:"Antonela",surname:"Markulj Kulundžić",slug:"antonela-markulj-kulundzic",fullName:"Antonela Markulj Kulundžić"}],corrections:null},{id:"72688",title:"Respiratory Health Risks from Exposure to Dust from Soybean and Its Products",doi:"10.5772/intechopen.92839",slug:"respiratory-health-risks-from-exposure-to-dust-from-soybean-and-its-products",totalDownloads:754,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:1,abstract:"Soybean and its processed forms have become an increasingly important part of agriculture, where they are widely used as an animal feedstuff and in an extensive range of human food products. This entails transportation from producer countries, largely the USA and South America, to importer destinations such as the EU and China. Soya is recognised as a dietary allergen, containing a number of identified allergenic proteins. Inhaled soya dust generated by occupational activities also causes respiratory health problems. Reports of “asthma epidemics” in harbour cities identified ill health in the community but were related to occupational activities of unloading/loading bulk soya without appropriate dust control measures. Inhaled allergens in already-sensitised individuals can cause a range of allergic symptoms in the eyes, nose, and respiratory tract, including occupational asthma (OA). Soybean dust can also cause health problems related to lung irritancy. Endotoxin and fungal contamination associated with soya are also potential respiratory hazards. This chapter reviews published data on ill health and symptoms from airborne exposure to allergens in dust from soybean and derivative products and the levels of exposure to dust and major allergens from a range of occupational activities. Other potential health hazards associated with soybean, such as endotoxin and fungal contamination, are also highlighted.",signatures:"Howard J. Mason",downloadPdfUrl:"/chapter/pdf-download/72688",previewPdfUrl:"/chapter/pdf-preview/72688",authors:[{id:"317237",title:"Dr.",name:"Howard",surname:"Mason",slug:"howard-mason",fullName:"Howard Mason"}],corrections:null},{id:"72429",title:"The Potential Impacts of Soy Protein on Fish Gut Health",doi:"10.5772/intechopen.92695",slug:"the-potential-impacts-of-soy-protein-on-fish-gut-health",totalDownloads:592,totalCrossrefCites:6,totalDimensionsCites:12,hasAltmetrics:1,abstract:"Soy protein is the major source of protein as fishmeal replacement in fish feed because of its worldwide availability and low price. However, the presence of high carbohydrate content along with saponins, lectins, and phytates can have a negative impact on fish gut health. Based on the literature and our lab studies, dietary soybean meal can cause a dose-dependent type of distal intestine inflammation called enteritis in commercial fish species including salmonids. This leads to reduced absorptive capacity, increased mucus secretion, hyperpermeability, and leucocyte infiltration in the lamina propria and submucosa, also inducing the pro-inflammatory cytokine genes expression, including Il-1β, Il-8, and Tnf-α. In addition, dietary soy may alter the composition and population of the gut microbiota via providing nutrients and energy that preferentially support the growth of some gut bacteria. This chapter summarizes the current knowledge of the effects of soy protein on the enteritis and gut microbiota.",signatures:"Vikas Kumar, Md. Sakhawat Hossain, Janice A. Ragaza and Marina Rubio Benito",downloadPdfUrl:"/chapter/pdf-download/72429",previewPdfUrl:"/chapter/pdf-preview/72429",authors:[{id:"310392",title:"Prof.",name:"Vikas",surname:"Kumar",slug:"vikas-kumar",fullName:"Vikas Kumar"},{id:"310393",title:"MSc.",name:"Marina",surname:"Benito",slug:"marina-benito",fullName:"Marina Benito"},{id:"318034",title:"Dr.",name:"Md. Sakhawat",surname:"Hossain",slug:"md.-sakhawat-hossain",fullName:"Md. Sakhawat Hossain"},{id:"318035",title:"Dr.",name:"Janice",surname:"Ragaza",slug:"janice-ragaza",fullName:"Janice Ragaza"}],corrections:null},{id:"68701",title:"Nondestructive Evaluation of Inoculation Effects of AMF and Bradyrhizobium japonicum on Soybean under Drought Stress From Reflectance Spectroscopy",doi:"10.5772/intechopen.88673",slug:"nondestructive-evaluation-of-inoculation-effects-of-amf-and-em-bradyrhizobium-japonicum-em-on-soybea",totalDownloads:572,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Precise estimation of leaf chlorophyll content (LCC) and leaf water content (LWC) of soybean, using remote sensing technology, provides a new avenue for the nondestructive evaluation of inoculation effects of arbuscular mycorrhizal fungi (AMF) and Bradyrhizobium japonicum (BJ) on soybean growth condition. In this study, a series of pot experiments were conducted in the greenhouse, soybean inoculated with Glomus intraradices (G.i, one of AMF species), G.i and BJ, and non-inoculation were planted under drought stress (DS) and normal irrigation (NI) conditions. Leaf spectra and LCC and LWC were measured on the 28th and 56th days after inoculation. Two new simple ratio (SR) indices, derived from the first derivative spectral reflectance at λ1 nm (Dλ1) and the raw spectral reflectance at λ2 nm (Rλ2), were developed to estimate LCC and LWC. The results indicate that under DS, plants inoculated with G.i had higher LCC and LWC than the non-inoculated plants, followed by the counterparts co-inoculated with G.i and BJ. Linear estimation models, established by the D650/Rred edge and D1680/R680, achieved great improved accuracy for quantifying LCC and LWC of soybean under inoculation and drought stress treatments, with determination of coefficient of 0.63 and 0.76, respectively.",signatures:"Weiping Kong, Yinli Bi, Wenjiang Huang, Lingli Tang, Chuanrong Li and Lingling Ma",downloadPdfUrl:"/chapter/pdf-download/68701",previewPdfUrl:"/chapter/pdf-preview/68701",authors:[{id:"247987",title:"Prof.",name:"Wenjiang",surname:"Huang",slug:"wenjiang-huang",fullName:"Wenjiang Huang"},{id:"250651",title:"Dr.",name:"Weiping",surname:"Kong",slug:"weiping-kong",fullName:"Weiping Kong"},{id:"308543",title:"Dr.",name:"Yinli",surname:"Bi",slug:"yinli-bi",fullName:"Yinli Bi"}],corrections:null},{id:"73649",title:"Soybean (Glycine max (L.) Merr.) Production in the Cameroonian Cotton Basin between the Dynamics of Structuring an Agricultural Value Chain and Sustainability Issues",doi:"10.5772/intechopen.93981",slug:"soybean-em-glycine-max-em-em-l-em-em-merr-em-production-in-the-cameroonian-cotton-basin-between-the-",totalDownloads:447,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Over the past ten years, the Cameroonian cotton front, in the Sudano-Sahelian region, has experienced dynamics in soybean production, resulting in significant changes in agro-systems. From a simple hut culture not referenced in regional agricultural statistics, since 2010, soybean ranks 2nd in legumes cultivated after peanuts, followed by cowpea and voandzou, yet culturally and economically more adopted by the populations. The rapid development of cultivated areas from 6,705 ha in 2008 to 15,020 ha in 2018 is indicative of the enthusiasm shown by farmers for this speculation which, despite the almost lack of supervision on the part of public authorities, now structures a real value chain with challenges for improving farmers’ incomes, in a space where more than 80% of working people live from agricultural activities. The objective of this research is therefore to analyse the sustainability challenges of a productive and market dynamics built around speculation subject to strong demands of international competitiveness. The results show that the viability of this dynamics remains precarious through a set of constraints inherent in the proven inability of farmers to empower themselves to optimise production and control marketing, as well as the poor technical supervision of cultivation.",signatures:"Eric Joël Fofiri Nzossié and Christophe Bring",downloadPdfUrl:"/chapter/pdf-download/73649",previewPdfUrl:"/chapter/pdf-preview/73649",authors:[{id:"320682",title:"Dr.",name:"Eric Joël",surname:"Fofiri Nzossie",slug:"eric-joel-fofiri-nzossie",fullName:"Eric Joël Fofiri Nzossie"},{id:"320685",title:"Prof.",name:"Christophe",surname:"Bring",slug:"christophe-bring",fullName:"Christophe Bring"}],corrections:null}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},subseries:null,tags:null},relatedBooks:[{type:"book",id:"1484",title:"Soybean",subtitle:"Molecular Aspects of Breeding",isOpenForSubmission:!1,hash:"3bd8fd078e7df24f2eed6dc7bc226475",slug:"soybean-molecular-aspects-of-breeding",bookSignature:"Aleksandra Sudaric",coverURL:"https://cdn.intechopen.com/books/images_new/1484.jpg",editedByType:"Edited by",editors:[{id:"21485",title:"Dr.",name:"Aleksandra",surname:"Sudarić",slug:"aleksandra-sudaric",fullName:"Aleksandra Sudarić"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"10237",title:"Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products",subtitle:null,isOpenForSubmission:!1,hash:"c3a5a3c7f7999d68f04ae49ff0553f3d",slug:"innovation-in-the-food-sector-through-the-valorization-of-food-and-agro-food-by-products",bookSignature:"Ana Novo de Barros and Irene Gouvinhas",coverURL:"https://cdn.intechopen.com/books/images_new/10237.jpg",editedByType:"Edited by",editors:[{id:"260510",title:"Prof.",name:"Ana",surname:"Novo de Barros",slug:"ana-novo-de-barros",fullName:"Ana Novo de Barros"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophile",surname:"Theophanides",slug:"theophile-theophanides",fullName:"Theophile Theophanides"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. 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\r\n\tIn recent years, neurorehabilitation (neuroscience-based rehabilitation), which utilizes the brain's plasticity, has made remarkable progress and has attracted much attention. The term "neurorehabilitation" is defined as "concepts, evaluation methods, therapies, and devices that apply the knowledge of brain theory revealed by neuroscience and related research to rehabilitation medicine. The concept of neurorehabilitation is widely accepted in physical therapy, and evaluation and treatment based on this concept are being practiced. What is required in neurorehabilitation research is to analyze the changes and improvements in motor behavior and cognitive and learning abilities and the changes in brain functions that bring about these changes. This will allow us to get closer to the neural mechanisms of rehabilitation effects and is expected to develop effective methods that are more suitable for the subject.
\r\n
\r\n\tThe purpose of this book is to provide a broad introduction to neurorehabilitation, from basic research to advanced treatment and science and technology, which is also being developed in the field of physical therapy. This book hopes to cover three topics related to neurorehabilitation and physical therapy (basic research, applied research, and advanced technology). \r\n\t
",isbn:"978-1-80356-345-9",printIsbn:"978-1-80356-344-2",pdfIsbn:"978-1-80356-346-6",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,isSalesforceBook:!1,isNomenclature:!1,hash:"37f858c8b681abe60704245c7a1e89ee",bookSignature:"Prof. Hideki Nakano",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11832.jpg",keywords:"Basic Research, Fundamental Theory, Animal Study, Human Study, Applied Research, Clinical Application, Clinical Study, Translational Research, Advanced Technology, Robotics, Regenerative Medicine, Deep Learning",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 17th 2022",dateEndSecondStepPublish:"March 17th 2022",dateEndThirdStepPublish:"May 16th 2022",dateEndFourthStepPublish:"August 4th 2022",dateEndFifthStepPublish:"October 3rd 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"2 months",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dr. Nakano is a pioneering researcher in neurorehabilitation and physical therapy. He has research training experience as a JSPS Research Fellow at Neurorehabilitation Research Center, Kio University, Japan, and Queensland Brain Institute, University of Queensland, Australia. Dr. Nakano has received 13 awards from academic organizations, has authored more than 120 journal papers and 10 book chapters, and was the editor member of 7 academic journals.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"196461",title:"Prof.",name:"Hideki",middleName:null,surname:"Nakano",slug:"hideki-nakano",fullName:"Hideki Nakano",profilePictureURL:"https://mts.intechopen.com/storage/users/196461/images/system/196461.jpg",biography:"Dr. Hideki Nakano is a physical therapist and associate professor at the Neurorehabilitation Laboratory, Graduate School of Health Sciences, Kyoto Tachibana University, Japan. He received his Ph.D. in Health Science from Kio University, Japan, and has accepted research training experience as a JSPS Research Fellow at Neurorehabilitation Research Center, Kio University, Japan, and Queensland Brain Institute, University of Queensland, Australia. He specializes in neuroscience, neurophysiology, and rehabilitation science and conducts research using non-invasive brain function measurement and brain stimulation methods such as electroencephalography, transcranial magnetic stimulation, and transcranial electrical stimulation. 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1. Introduction
Blood cells are by far the most abundant cells of which our body is comprised. Red blood cells (RBCs, or erythrocytes) and platelets (Plts, or thrombocytes) circulate in the vascular system, whereas the white blood cells that form our immune system locate both in the vascular system and in the tissues. RBCs are best known for their function as oxygen transporters and for the clearance of CO2. Plts exert a crucial function in homeostasis upon vascular damage but they also function during angiogenesis, innate immunity, inflammation, wound healing, cancer, and hemostasis [1, 2]. This chapter focuses on erythropoiesis and megakaryopoiesis. RBCs in the periphery have an average life span of 120 days, constituting approximately 45% of the blood volume. To maintain the population of RBCs, humans generate daily ~2 × 1011 reticulocytes [3]. Plts are shed by megakaryocytes (MK) and live approximately 8–9 days in humans, which require a production of ~8.5 × 1010 Plts/day [4, 5]. The generation of RBCs and Plts occurs mainly in the bone marrow (BM) in adults, although the lung has also been found to host megakaryocytic progenitors as well as Plts-shedding MKs [6]. A small population of hematopoietic stem cells (HSCs) ensures the life-long generation of blood cells, although the HSCs themselves divide rarely. Mostly, HSCs that divide give rise to one new HSC and a daughter cell that develops to an actively dividing multipotent progenitor (MPP) (Figure 1) [7]. These MPPs undergo specification through reciprocal actions of transcription factors (TF) that enhance or repress expression of lineage-specific TFs and direct the cells to a lineage-specific gene expression program [7]. Erythropoiesis and megakaryopoiesis were long thought to arise from a common progenitor, the megakaryocytic-erythroid progenitor, but recent lineage tracing indicates that MKs can also differentiate directly from HSCs [8, 9, 10]. Not only MPPs, also erythroid progenitors (erythroblasts: EBLs) and megakaryocytic progenitors (megakaryoblasts: MKBLs) have extensive potential to undergo cell divisions before they commit to the final differentiation program to generate RBCs/MKs. The final differentiation stages of both lineages have unique features. Erythroid progenitors undergo 3–4 additional cell divisions with a short G1 cell cycle phase and without regaining the cell volume (i.e., loss of cell size control) [11, 12, 13]. MKBLs, instead, undergo 4–5 cell division cycles without cytokinesis, which results in a single cell with 64–128 genome copies (N = 64–128) [14]. Erythropoiesis and megakaryopoiesis also show spatiotemporal regulation. All blood cell progenitors including erythroid progenitors and MKBLs propagate in close contact with stromal cells that produce membrane-bound factors such as stem cell factor (SCF). Upon terminal differentiation, erythroid progenitors bind to central macrophages that express receptors such as CD163, VCAM1, ICAM4, and CD163 to associate with EBLs [15, 16, 17, 18, 19]. Each macrophage binds several progenitors that undergo synchronous differentiation, which ends with phagocytosis of the extruded erythroid nucleus by the macrophage and release of reticulocytes into the circulation. The mature MKs have to interact with the endothelial cells of the vasculature and protrude proplatelets into the capillaries, where shear stress contributes to the shedding of Plts [20].
Figure 1.
HSC commitment to the erythroid/megakaryocytic lineages with lineage-specific marker expression pattern.
Whereas steady state erythropoiesis and megakaryopoiesis of adult mammals take place in the BM and lung (MK), distinct anatomic sites of hematopoiesis are employed during development (Figure 2A). After gastrulation, in humans, mesodermal precursor cells arise in the primitive streak, migrate to the yolk sac, and develop into blood islands (hemangioblasts), which produce primitive RBCs, primitive MKs, and macrophages [21]. During this process, basic fibroblast growth factor (bFGF) influences the proliferation of the hemangioblast and thereby the production of hematopoietic cells [22]. bFGF is synergistic with vascular endothelial growth factor (VEGF) signaling in this process [23]. The primitive RBCs express embryonic type of hemoglobins (Hbs), retain the size of the early EBLs, and lose their nucleus only after prolonged circulation. Their erythropoietin (EPO)-dependence is unclear at this early stage of development [24, 25]. The primitive MKs are thrombopoietin (TPO) independent, have low ploidy compared to adult MKs, and produce fewer Plts, but contrary to primitive erythroid cells, these cells migrate to the fetal liver, where their polyploidization is TPO dependent [26, 27]. Erythroid-myeloid progenitors (EMPs) arise in the yolk sac from hemogenic endothelium (HE) through endothelial to hematopoietic transition (EHT) and give rise to the first intermediate definitive wave, producing RBCs with fetal type of Hbs, MKs, and other myeloid cells [21, 28]. The EMPs migrate and colonize the developing fetal liver where they transiently produce definitive fetal RBCs and MKs. Permanent definitive hematopoiesis in the fetal liver depends on the “birth” of HSC in the aorta main arteries, and more specifically in the aorta-gonad-mesonephros (AGM) region, where the first CD34+ HSC arises through EHT. These early HSCs are dependent on bone morphogenetic protein 4 (BMP4), VEGF, and bFGF secreted by “feeder cells,” which are located near the endothelial cells undergoing EHT, thereby promoting this transition [23, 29]. These HSCs home to the fetal liver to produce definitive fetal blood cells. From the fetal liver, the HSCs migrate to the final site of hematopoiesis; the BM, where they give rise to adult definitive blood cells. Perinatally, hematopoiesis also occurs in the spleen [30]. RBCs and Plts generated at distinct anatomic sites have distinct characteristics; for example, RBCs express different Hb molecules arising from different sites (Figure 2B). Hb consists of two α and two β subunits each bound to an iron-containing heme molecule. The α locus expresses ζ and α protein isoforms, the β locus expresses ε, γ (γ1 and 2), and β (β and δ) isoforms. Primitive RBCs express Hbe consisting of ζ and ε isoforms (Portland 1: ζ2γ2; Portland 2: ζ2β2; Gower 1: ζ2ε2; Gower 2: α2ε2); fetal RBCs are characterized by HbF consisting of α and γ isoforms; adult RBCs express HbA consisting of α and β isoforms (HbA1) plus a small amount of HbA2 consisting of α and δ isoforms. Hbs can be used to distinguish RBCs originated from different developmental stages; however, in the megakaryocytic lineages, there is a lack of such markers.
Figure 2.
Human erythropoiesis/megakaryopoiesis during development. (A) Schematic depiction of site-specific (yolk sac/fetal liver-AGM/bone marrow) blood production during ontogeny focusing on erythroid and megakaryocytic lineages. (B) Representative HPLC tracks, showing the Hb content of in vitro-cultured erythroid cells derived from primary sources originating from distinct anatomical sites. Fetal liver-erythroid cells express fetal Hbs (HbF). Cord blood is obtained at the time of birth when the fetal to adult Hb switch takes place, resulting a mix of HbF and HbA. PBMC/MPB-derived erythroid cells produced by the bone marrow mainly express adult hemoglobins (HbA1, HbA2).
Biochemical and molecular analysis of erythroid/megakaryocytic cells requires large cell numbers. The in vitro expansion and differentiation of erythroid and megakaryocytic progenitors from human fetal liver (HFL), cord blood (CB), BM, or peripheral blood enable the production of large cell numbers from distinct ontology and at defined stages of differentiation for basic research, drug testing, disease modeling, or translational purposes (Figure 3). Differentiation of pluripotent stem cell types such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) toward hematopoietic lineages allows to study early blood ontogeny, which is difficult to study in vivo as of ethical issues and availability of human material. The knowledge gained by using the abovementioned culture systems is subsequently of great value to control the expansion and differentiation of erythroid/megakaryocytic cells from ESCs/iPSCs leading to donor-independent blood cell production (Figure 3).
Figure 3.
Application of in vitro-derived blood products from different primary sources in basic research, drug discovery, and transfusion.
The aim of this chapter is twofold. First, we provide background information of the basic processes of erythropoiesis and megakaryopoiesis that underly the various cell culture models. Second, we provide details, interpret and compare results on current protocols to expand and differentiate erythroid and megakaryocytic progenitors.
2. Erythropoiesis/megakaryopoiesis
2.1. Growth factors and major regulators
2.1.1. Lineage- and stage-specific cytokines
Interleukins (ILs) activate cytokine receptors, which do not have enzymatic activity and recruit Janus Kinases (JAK1, JAK2, JAK3, TYK) to phosphorylate tyrosines in their intracellular tail that can subsequently recruit signaling molecules. Cytokine receptors are often expressed on a limited set of cell types/differentiation stages. IL-3 and IL-6 are cytokines that promote survival of hematopoietic stem/progenitor cells (HSPCs), but they do not act on EBL-specific stages. IL-9 synergizes with IL-3 to enhance both erythropoiesis and megakaryopoiesis [31, 32]. IL-1β is an inflammatory cytokine that signals via MyD88 to IRAK and NF-κB to inhibit cell growth, to induce expression of growth factors and the extracellular matrix, and to cause apoptosis in most tissues. In megakaryopoiesis, it enhances commitment and differentiation [33, 34].
Erythropoietin (EPO) is mainly produced by the kidney (80%) and partially in the liver (10–15%; 30% upon stress erythropoiesis) [35, 36]. EPO binds and activates EPO receptor (EPOR) and functions as a survival factor during erythropoiesis [37, 38]. Deficiency of Epo and/or Epor causes embryonic lethality due to the failure of definitive fetal liver erythropoiesis leading to the lack of mature RBCs [24, 39, 40, 41]. Fetal livers of EPO-deficient embryos contain normal numbers of erythroid progenitors that can form colony-forming unit-erythroid (CFU-E) in vitro in the presence of exogenous Epo [39]. EPO shows cross-reactivity in human and mouse.
Thrombopoietin (TPO) is the ligand for the MPL receptor. MPL is well conserved between species and TPO showing cross-reactivity between mouse and human. TPO has a direct effect on self-renewal and expansion of HSCs in the BM, but controls megakaryopoiesis as well [42]. MKs and Plts bind TPO and sequester it from the circulation. Upon activation, Plts release TPO into the plasma, thereby stimulating megakaryopoiesis. Thus, the number of Plts and their activation controls TPO levels [43]. TPO is mainly produced by hepatocytes in the human liver. TPO production is increased when hepatocytes bind damaged and aged desialylated Plts via the Ashwell-Morell receptor. In a JAK2/STAT3-dependent manner, this activates TPO transcription and thereby regulates Plt production, HSC renewal, and expansion [42, 44, 45].
The tyrosine kinase receptors directly cross-phosphorylate tyrosine residues in their own cytoplasmic tail, and they phosphorylate downstream effector molecules. They are more widely expressed compared to cytokine receptors.
Stem cell factor (SCF; first described as the Steel locus) signals via the mast/stem cell growth factor receptor KIT (CD117; White locus). SCF cooperates with other cytokines in order to maintain the viability of HSPCs, and their proliferation/differentiation ability [46, 47, 48]. SCF is produced by the stromal cells in the BM as a secreted soluble factor but also as a membrane-bound factor. A specific mutation in SCF (Steel-Dickie) disrupting the membrane form of SCF leads to severe anemia, indicating that the membrane-bound form is crucial at least for erythropoiesis [49]. In addition, Kit-deficient mice (W/W) suffer from neonatal lethality due to severe anemia [50, 51]. Besides its function on HSPC, SCF is particularly important upon blood loss, to enhance proliferation and delay differentiation of the erythroid and megakaryocytic progenitors [52, 53, 54]. In contrast to TPO and EPO, SCF is not interchangeably reactive between mouse and human—mouse SCF activating both, while human does not.
FLT-3-ligand (FL) binds to Fms-like tyrosine kinase 3 (FLT-3 (CD135)) as its receptor. FL is an important growth factor for CD34+ HSPC by controlling their proliferation. Underscoring its function, activating mutations in the FLT-3 receptor are prominent in acute myeloid leukemia [55]. FL, however, has a limited role in terminal MKBL/EBL differentiation because FLT-3 is not expressed on committed cells.
Insulin (Ins) and Insulin-like growth factor-I (IGF-I) bind the Ins receptor (InsR) and IGF-I receptor (IGF1R). Both receptors are very well conserved between species to the extent that Ins even cross-reacts between mammals and birds. The homology between the receptors suggested that Ins may also act through the IGF1R, which is not true. IGF-I has a general cell survival function mediated by PI3K activation [56]. Human erythroid progenitors (pro-EBLs, early basophilic EBLs) express the IGF1R, and not the InsR, which changes during differentiation when the InsR becomes the prominent receptor [57]. InsR signaling is particularly important to control trafficking of GLUT4 glucose transporters to the cell membrane [58]. Late EBLs and mature RBCs depend on glycolysis and EBLs express high levels of glucose transporters [59]. Upregulation of GLUT4, however, is also required for import of glutamine, required for nucleotide synthesis [60]. Because Ins and IGF-I act in physiological concentrations, their effect on in vitro cultures is not noticed in the presence of serum or plasma [61, 62].
2.1.3. Ligand-activated TFs
Steroid hormone receptors are the best known nuclear hormone receptors. In addition to these ligand-dependent TFs that bind DNA through a Zinc-finger domain, other ligand-activated TFs exist such as the aryl hydrocarbon receptor (AhR) that binds DNA through a helix-loop-helix domain.
Glucocorticoids bind the glucocorticoid receptor (GR), a nuclear hormone receptor, which translocates to the nucleus upon association with its ligand, where it functions as a transcriptional repressor and activator of gene expression. The GR homodimer binds to glucocorticoid response elements that consists of two inverted repeats. As a heterodimer with for instance STAT5, it only needs a “halfmer” GRE, i.e., a single repeat combined with the STAT5 binding site [63, 64]. The ligand of the GR is glucocorticoid (in vivo) produced by the adrenal gland. Several synthetic ligands have been designed to be used as immunosuppressive agents (e.g., dexamethasone—DEX). The GR exerts its immunosuppressive function at pharmaceutical levels of glucocorticoids, and as a monomer that binds and inhibits other TFs such as NF-κB or FOS/Jun dimers [65]. In contrast, stress erythropoiesis in vivo, and expansion of EBL cultures in vitro, is induced at physiological levels of glucocorticoids and depends on dimerization of the GR and on the ligand-dependent transcription activation domain [66, 67]. Polymorphisms of the GR may alter the expansion potential of EBLs [68].
3,5,3 -triiodothyronine (T3) binds the α and β thyroid hormone receptors (TRα, TRβ). T3 deficiency is associated with anemia, although it is not clear whether this is caused by a direct effect on erythropoiesis [69, 70]. The effect of T3 appears to be highly species and developmental stage specific. T3 has a potent differentiation promoting effect on avian erythropoiesis, but mouse EBLs are only sensitive to T3 during neonatal spleen erythropoiesis [71, 72]. In cultures of human erythroid cells, T3 enhances synchronous differentiation to enucleated reticulocytes [73].
StemRegenin 1 (SR-1) binds and inhibits the AhR, which mediates toxicity of environmental pollutants, by binding to specific DNA enhancer sequences. This receptor can be activated by endogenous or exogenous ligands and contributes to several physiological processes, among which cell migration, apoptosis, and cell growth [74, 75, 76, 77]. Importantly, AhR-deficient mice have increased numbers of BM HSCs [78]. The AhR functions in complex with the AhR Nuclear Translocator to induce differentiation by regulating genes directly such as c-MYC and C/EBP. During hematopoietic differentiation, its targets also include PU.1, β-CATENIN, CXCR4, and STAT5 [79]. Currently, the antagonist of AhR (SR-1) is used in clinical trials to expand CB-CD34+ cells prior to transplantation, thereby reducing the number of CB units needed per transplant from 5 to 1 [80]. Importantly, SR-1 also enhances in vitro proplatelet formation [81]. To achieve proper Plts production, MKs have to produce an extensive membrane system; the demarcation membrane system. This requirs extensive lipid biosynthesis which can be inhibited by AhR [82]. Thus, blocking the inhibition of AhR with SR-1 may increase the proplatelet formation, through increasing the lipid membrane biosynthesis.
2.1.4. Erythroid-specific regulation
EPO is sufficient for steady-state erythropoiesis, when proliferative signals are mediated through the EPOR-associated RON receptor and EPO-induced differentiation is dependent on STAT5 [83, 84]. Increased erythropoiesis during development and upon blood loss requires the cooperative action of the EPOR and KIT [85]. Activation of KIT prevents differentiation and propagates the long-term proliferation of erythroid progenitors through inhibition of FOXO3a and activation of mRNA translation via the PI3K/mTOR pathway [86, 87]. Activation of the GR is required for stress erythropoiesis and to inhibit in vitro differentiation. Interestingly, glucocorticoids activate largely the same growth inhibitory genes in EBLs and in immune cells, but growth inhibition is counteracted by EPOR/KIT activation [88]. Even in the presence of serum, glucocorticoids promote selective proliferation of erythroid progenitors by both supporting erythroid proliferation and inhibiting proliferation of other myeloid and lymphoid cells [57].
Erythropoiesis is also regulated by the availability of iron, which is imported into the cell as holotransferrin via the transferrin receptor (TfR; CD71), and by selenium through selenoproteins [89, 90]. In vivo, erythropoiesis is dependent on the formation of erythropoietic islands that form around a central macrophage [91, 92]. Whereas CD169 macrophages are essential for erythropoiesis in vivo, high level of enucleation can be achieved in vitro in the absence of macrophages (van den Akker and von Lindern, manuscript in preparation) [93]. The complete understanding of which macrophage signals control enucleation in vivo may further optimize the in vitro production of erythrocytes for transfusion purposes.
2.1.5. MK-specific regulation
TPO is the main regulator of megakaryopoiesis, and multiple other factors can work synergistic with it. IL-6 can, in conjunction with TPO, increase hepatic TPO synthesis [94]. This effect is through the shared usage of gp130 and amplification of the same downstream JAK pathways [95]. The direct admission of IL-6, besides its effect on TPO, results in increased polyploidization and subsequently leads to an enhanced Plts production in patients [96]. IL-11 is not constitutively expressed but was shown to be induced in thrombocytopenia patients undergoing BM transplantation. Exposure of IL-11 on HSPCs directly leads to an inhibition of NF-κB signaling, which suppresses miR-204-5p, that targets and represses the expression of TPO [97]. Because of this effect on TPO, it has a dual role in MK differentiation, first, in the expansion of HSCs, second, in the terminal differentiation of MKs. In a similar way, IL-9 synergizes with IL-3 as well as with IL-4 and SCF to increase the yield of MKBL [33, 98]. Furthermore, the addition of IL-1β promotes selective megakaryocytic differentiation. IL-1β can increase the production of Plts by enhancing the effects of SDF1 and FGF4 that are produced by BM niche cells. IL-1α was shown to induce MK rupture and is considered as a stress megakaryopoiesis regulator [99].
Inhibition of AhR by SR-1 slows the differentiation program in HSCs, leading to increased expansion, and has a direct effect on MK differentiation, by slowing and/or conditioning cells to have a more synchronized maturation [74, 79, 81]. Through its effect on PU.1, it can influence RUNX1 which is one of the TF-regulating megakaryopoiesis. The introduction of SR-1 into in vitro MK cultures results in increased cell size, higher polyploidization, and proplatelet production. In these cultures, a specific MK precursor population is identified that has an increased potential to produce proplatelets [81].
2.2. Cell culture models
2.2.1. Erythroid immortalized cell lines
2.2.1.1. Mouse cell lines
The most widely used immortalized mouse erythroid cell lines are MEL cells, which are EBLs transformed by Friend Leukemia virus. The viral gp55 protein activates the Epor to sustain cell growth, whereas integration of the virus upstream of PU.1 or Friend leukemia integration 1 (Fli-1) induces constitutive high expression and inhibits differentiation. Leukemogenesis and the establishment of cell lines require additional inactivation of tumor suppressor protein p53 [100]. Under conditions that enable transient expansion of primary mouse cultures (serum-free medium supplemented with Epo, SCF, and glucocorticoids), lack of p53 is sufficient to establish immortalized cell lines with full differentiation potential in the absence of SCF and glucocorticoids [13].
2.2.1.2. Human cell lines
Deletion of p53 is not sufficient in human to establish immortalized erythroid cell lines. However, expression of the human papilloma virus E6/E7 proteins in EBLs differentiated from iPSC gave rise to the HiDEP cell line [101]. The E6/E7 proteins inactivate both p53 and retinoblastoma tumor suppressor proteins [102]. They are expressed from a doxycycline (dox)-inducible vector allowing for unlimited growth in the presence of EPO, SCF, and dox, and differentiation in the presence of EPO but without dox and SCF. Lack of p53 does not affect differentiation, but retinoblastoma is required for terminal erythroid differentiation [103, 104, 105]. Dox-inducible expression was also used to establish immortalized erythroid cells lines from CB (HUDEP) and from adult EBLs cultured from CD34+ HSPC (BEL-A) [106, 107]. The HiDEP, HUDEP, and BEL-A express embryonic, fetal, and adult Hbs, respectively [101, 107].
2.2.2. MK-cell lines
Multiple cell lines have been generated to study megakaryopoiesis with among them; MEG-01 (suspension cells) and DAMI (adherent/suspension cells) both megakaryoblastic leukemia cell lines [108, 109]. These cell lines are mostly positive for MK-specific markers (see in Section 2.3.3) but are not a homogenous population. Although they proliferate in a MKBL-like state with some spontaneous differentiation and limited terminal differentiation, they can be induced to differentiate by the addition of phorbol myristate acetate. Under these conditions, the cells can become polyploid, increase their expression of MK-associated proteins, like von Willebrand factor, and are able to produce proplatelets although with low efficiency. Mechanistic insights were uncovered with these lines; for example, the formation of long, beaded cytoplasmic extensions of MKs that yield platelets upon shear stress. This process was also observed in normal healthy MKs in vivo, showing the usefulness of this artificial system to study fundamental processes [110]. Despite the usefulness of these lines, their genetic background (patients with genetic abnormalities) can influence megakaryopoiesis, resulting in incomplete differentiation and potential abnormalities that could possibly be linked to their immortalization process. As such, they are suboptimal models to study megakaryopoiesis and specifically MK polyploidization, synthesis of granules, and proplatelet formation.
2.3. Primary cell culture
2.3.1. Cell source and media
RBCs and MKs can be cultured for research and clinical applications from multiple primary tissue sources including HFL, CB, BM, mobilized peripheral blood (MPB), and peripheral blood mononuclear cells (PBMC). HFL is obtained from abortions on medical indication. HFL-derived erythroid cells express HbF and can be expanded to large numbers and differentiated to hemoglobinized enucleated RBCs. For MK culture, this source is less ideal, mainly because of the harshness of the isolation method. Ethical concerns rule out HFL as a general source for transfusion, but with proper consent allows research into fetal hematopoiesis development. A widely available and ethically accepted source is CB which is commonly used for production of both erythroid cells and MKs. CB is obtained at birth when Hb-switch occurs from HbF to HbA1 (γ to β switch) and both Hb types are expressed in CB-derived cultures. The presence of HSPCs with a fetal hematopoietic program in CB has notable effects in the MK cultures. MKBL expansion is high; MK polyploidization and proplatelet formation are decreased compared to cultures of adult cells. As adult hematopoietic source, either BM or PBMC can be applied. BM is a limited source that can be more difficult to obtain, but does yield large quantities of HSPCs that can differentiate to erythroid and MK lineages. HSPCs can also be isolated from PBMCs, which is less invasive therefore a less limited source. This makes PBMC an ideal source to scale-up RBC production. The HSPC percentage in PBMC is significantly lower compared to BM, which can be enhanced by leukophoresis and by mobilizing BM HSPCs using G-CSF (10 μg/kg) alone or in combination with CXCR4/CXCL12 inhibition [19, 111]. G-CSF alone leads to 5–30% mobilization [15]. HSPC mobilization is caused by the downregulation of adhesion and chemokine processes and by the loss of BM macrophages [15, 112, 113, 114, 115]. The mobilization can cause side effects for the donor, including headaches, fatigue, vomiting, muscle pain, bone pain, thrombocytopenia, citrate toxicity, etc., in which females experience the most adverse events [116]. These MPBs are ideal for RBC/Plt production because of their HSPC richness.
The MKs can be cultured in Cellgro (Corning), and both cell types can be cultured in StemSpan (Stem Cell Technologies) or other Iscove’s modified Dulbecco’s medium (IMDM)-based media. We generated a completely defined GMP-grade medium called Cell-Quin (Migliaccio et al. with minor modifications) that is highly efficient in expanding and differentiating EBLs/RBCs and MKs/Plts, with the ability to culture other hematopoietic progenitors and blood cell types [117].
2.3.2. Erythroid-specific culture system
Several parameters characterize the differentiation stage of erythroid cells. Expansion of EBL cultures is only possible when they maintain cell size control during their cell cycle, which is achieved by the cooperative action of SCF and glucocorticoids [12, 13, 118]. Terminal differentiation in the presence of EPO involves 3–4 cell divisions during which cells’ surface marker expression changes and gets smaller due to loss of cell size control until cell cycle arrest and extrusion of their nuclei, concurrently, accumulating Hb [119]. Thus, surface marker expression pattern, cell size and morphology (enucleation), Hb content, and cumulative cell numbers are a measure of differentiation (Figure 4A–C). Morphological features of the cells (nuclei-cytoplasm ratio, hemoglobinization, nuclei condensation, and polarization) are commonly assessed by cytospins coupled with Giemsa/benzidine stainings (Figure 4A). The purity of the erythroid population and its distribution over different maturation stages can be assessed by monitoring the progression of various cell surface markers. Commonly used markers are CD36, CD71 (transferrin receptor), CD117, and the erythroid-specific markers band 3 (SLC4A1) and CD235 (glycophorin A). The generally accepted dynamics of these markers during erythroid differentiation: pro-EBLs (immature EBL stage) are characterized by CD34−/CD36+/CD117+/CD71high/CD235low/−, while during expansion phase, EBLs gain CD235 expression and become CD117+/CD71+/CD235+. In terminal differentiation phase, EBLs remain positive for CD235 and lose their expression of CD117 followed by the gradual loss of CD71, which is associated with reticulocyte formation [120].
Figure 4.
Characteristics of erythroid and megakaryocytic cultures. (A) Erythroid-specific morphology by cytospin with Giemsa/benzidine staining. Left: pro-EBL, right reticulocytes. (B) Erythroid expansion growth curve from PBMCs (n = 4). (C) Flow cytometry of terminal erythroid differentiation, DNA staining by DRAQ5 resulting in three distinct populations: DRAQ5+ big cells: nucleated EBLs (red); DRAQ5+ small cells: nuclei (blue); DRAQ5− cells: enucleated reticulocytes (purple). (D) MK-specific morphology by cytospin with MGG-staining. Left: MKBL; right: polyploid MK (arrows). (E) Proplatelet-forming MK (arrows (beads on a string)).
The first human erythroid culture systems utilized the knowledge obtained from genetics, e.g., discoveries in the field of cytokines, growth factors, and their receptors. In these first protocols, HSPCs were expanded in the presence of IL-3, SCF plus or minus IL-6. It is followed by a step in which the resulting erythroid progenitors were further expanded and differentiated in the presence of EPO [121]. This protocol was modified, using low EPO concentrations in step 1 (0.5 U/ml) and high concentrations (>3 U/ml) in step 2 [122]. Others used high concentrations of EPO throughout step 1 and step 2 [123, 124]. These two-step protocols are based on the original protocol of Fibach and coworkers who employ IMDM supplemented with serum or plasma [121]. Serum and plasma contain factors that support erythropoiesis in these cultures. The major factor in the serum is transforming growth factor β (TGFβ), which is a potent differentiation factor for erythropoiesis [57, 125]. These cultures show a high degree of spontaneous differentiation, which is often used to study expansion and differentiation of EBLs carrying a genetic defect. The quality differences between serum batches and the use of different cytokines make these culture protocols difficult to compare. Two major changes increase the yield of these erythroid cultures and enable synchronous differentiation. First, glucocorticoids cooperate with SCF to retain pro-EBLs and early basophilic EBLs in their undifferentiated state [57, 126]. Second, serum-free medium avoids the differentiation promoting effect of TGFβ. However, the available serum-free media are suboptimal and require complementation with lipids [73]. Even better expansion is achieved with a serum-free medium optimized for expansion of EBLs [117]. The differentiation arrest in the presence of glucocorticoids and the absence of serum enables the expansion of a homogeneous early EBLs culture that can undergo up to 20 cell divisions to achieve a million-fold expansion [73, 119]. Using Cell-Quin, we can obtain 2 × 1010 EBLs within 16 days, starting from 5 × 107 PBMCs (Figure 4B). Expansion of EBLs in the presence of serum and in the absence of glucocorticoids irrevocably results in differentiation and transfer of the cells to differentiation conditions. Of note, addition of glucocorticoid agonists in a serum-based culture will still induce spontaneous differentiation due to the presence of TGFβ [57]. At any moment during expansion phase, cells can be transferred to differentiation conditions in which the medium is supplemented with EPO, Ins, and low level of plasma/serum [119, 120]. Although expansion of EBL cultures is achieved in serum-free medium, terminal differentiation to enucleated cells requires at least 2% serum or plasma [119, 120]. Using Cell-Quin medium, we currently obtain >90% enucleation, a deformability that corresponds to values between freshly isolated reticulocytes and erythrocytes, and normal oxygen association and dissociation values (van den Akker and von Lindern, manuscript in preparation). We use DRAQ5 staining coupled with flow cytometry analyses to quantify reticulocyte/nuclei/nucleated cell ratio (Figure 4C). Flexibility is measured on a ARCA, and oxygen binding by the Hemox analyzer [127, 128].
2.3.3. MK-specific culture system
Commitment of MKBL and differentiation of MKs can be monitored by the expression of cell surface markers and by the morphological features of the cells (Figure 4D and E). MKBLs are characterized by CD34+/CD41a+ expression and blast-like morphology. In terminal differentiation, MKs gradually lose their expression of CD34+, leading to a subdivision of stages: early MKs are CD34+/CD41a+/CD42+ and late MKs are CD34−/CD41a+/CD42+.
To obtain large numbers of MKBLs, SCF/FL and TPO are used during the first 4–7 days of cultures started from CD34+-HSPCs. TPO without SCF and FL allows terminal differentiation to proplatelet-forming cells. To increase the expansion potential, IL-3 can be included only in the initial phase as its prolonged exposure directs the HSPC toward the monocyte/granulocyte lineage. With the addition of IL-6, the MK specification and TPO signaling can be enhanced. With the addition of either IL-1β, IL-9, or IL-11 during the first phase of CD34+ differentiation, MK commitment is enhanced instead of progenitor proliferation. It is important to determine the main goal of an experiment before starting the culture: does the experiment require large numbers of MKBLs, or should MK enrichment be maximal, because a good expansion of MKBL tends to compromise terminal differentiation and vice versa (Hansen and van den Akker, unpublished results). Factors such as IL-1β and IL-9 increase polyploidization, formation of proplatelets, and Plts shedding. There is some concern about using IL-1β, because of its proinflammatory nature. Particularly, as it is closely related to IL-1α, and the increased Plt shedding may cause rupture of MKs [33, 99]. To introduce proplatelet formation, IL-6 can be used in high concentrations (>100 ng/ml), by itself or in combination with TPO. SR-1 influences megakaryopoiesis on an early and late stage of the culture, as described above, having a positive effect on the expansion of HSC and terminal differentiation of MK [80, 81]. During the terminal stages of MK cultures (during proplatelet formation), it becomes increasingly essential to prevent the activation of the MKs and produced proplatelets. The addition of heparin prevents the coagulation of plasma added to the media but cells are still able to clump together, thereby having a negative impact on the differentiation and proplatelet production. To prevent activation, signaling via the GPIIb/IIIa (ITGA2B) receptor can be blocked with tirofiban hydrochloride monohydrate. Whereas an MK sheds thousands of Plts in vivo, shedding large amounts in vitro from a single MK has not yet been achieved. In vivo of this process requires that proplatelets extrude between the endothelial cells of the blood vessel wall into the capillaries. This increases level of SP1 among others, combined with shear stress of the blood flow is required for the Plts to be released. To mimic this in vitro, several specialized bioreactors are being tested (see Section 2.5).
2.4. Erythropoiesis/megakaryopoiesis from iPSC
Pluripotent stem cells offer a novel approach for developmental studies, drug screening/discovery, disease modeling, and regenerative medicine. ESCs originate from the inner cell mass of a blastocyst stage embryo, while iPSCs are somatic cells that are reprogrammed back to this embryonic stage [129, 130, 131, 132]. Hematopoietic differentiation of ESC/iPSC cells follows the various stages of blood development from early embryonic stages (Figure 2A). This offers a valuable tool to study early human hematopoiesis which is difficult because of ethical issues and tissue availability. Besides, differentiation of iPSCs opens opportunities for large-scale manufacture of blood products with the expectancy of clinical application [133]. Several groups showed the potential of ESCs in blood cell production, the source which was later replaced by iPSCs with similar outcome including our group (Figure 5) [134, 135, 136, 137, 138, 139, 140]. The published protocols generally include four culture phases: (1) mesoderm induction, (2) hematopoietic/erythroid/megakaryocytic commitment, (3) expansion of the specific cell pool, and (4) terminal maturation. The hematopoietic differentiation phases in vitro are directed by stepwise addition of cytokines. This is commonly achieved by BMP4, bFGF, and VEGF that drive the cells toward mesoderm, followed by the addition of IL-3, IL-6, SCF, and TPO stimulating hematopoietic specification. Lineage-committed progenitors can be further differentiated toward mature cell types, which is achieved by the combination of medium, growth factors, and hormones. Although most of the differentiation protocols are following the abovementioned scheme, including the listed growth factors, there are multiple technical variations during iPSCs differentiation toward hematopoietic lineages. Two main technical details underlie the major differences in the applied protocols: (i) the induction of differentiation as a 2D monolayer versus 3D embryoid body formation and (ii) the use of coculture with feeder versus feeder-free systems [141, 142, 143, 144]. The choice of the differentiation system depends on the application need. 3D systems more closely resemble the in vivo process in comparison to 2D systems, offering a tool to study embryogenesis [141, 144, 145, 146, 147]. 2D systems, however, are relatively simple, more reproducible, and therefore suitable to scale up production, enabling clinical application [77, 148, 149, 150]. The choice of feeder-based or feeder-free differentiation similar to 2D/3D systems also depends on the purpose of the specific research question. Feeder-based coculture systems more resemble the niche, including secretome and cell-cell contact; however, for future clinical application, feeder-free systems are imperative [142, 143, 151, 152]. Protocols can also differ in timing, in the applied media and cytokine cocktails used, which makes comparisons between research groups and methods difficult. Stemline II (SIGMA) is a widely applied base medium during the first and, in some cases, the second phase in feeder-free settings [138, 149, 153]. From the second/third phase onward, the same basic medium are applied that are generally used for other definitive blood cell types such as IMDM with serum/holotransferrin/lipid/Ins supplementation or StemSpan (Stem Cell Technologies) [137, 138, 150, 153]. From the second-step onward, we apply Cell-Quin which in comparison to StemSpan was more efficient in their iPSC-erythroid expansion potential (Figure 6A). Some methods still include BMP4, VEGF, and bFGF (or either of them) at this second stage, typically in embryoid body-based system because the 3D structures are less homogenous. Therefore, the transition between phases is not entirely uniform and clear [140, 149, 154, 155]. The few feeder-free 2D systems that have been published mostly do not rely on these three additional factors during commitment phase [148, 149]. FL is also used in erythroid/MK commitment cytokine mixes to improve progenitor expansion [140, 154]. MK commitment/expansion is always based on TPO with or without the addition of multiple other cytokines (e.g., IL-1β, IL-9, IL-11) [99, 136, 156, 157, 158].
Figure 5.
Erythroid/MK differentiation of iPSC according to Hansen et al. showing the different phases of differentiation, with their corresponding growth factor combination and morphological changes [136].
Figure 6.
Optimization of iPSC-erythroid differentiation cultures. (A) iPSC-derived erythroid cells arose (D12+0) and expanded in Cell-Quin or StemSpan media. (B) Representative FACS-plots of iPSC-derived erythroid cell (D12 harvest), with or without EPO and SCF from day 6 onward.
As pointed out before, early erythropoiesis/megakaryopoiesis (yolk sac) in humans is not well studied, resulting in a lack of knowledge on the regulatory program at these developmental stages. Therefore, the generally applied cytokines might not ideally mimick the in vivo situation or the iPSC-driven hematopoietic program. This could underlie inefficient iPSC to RBC/MK differentiation. For example, EPO is applied in all systems to induce erythropoiesis; however, the role of EPO during primitive wave is not entirely clear. Disruption of EPO and/or EPOR causes embryonic lethality in mice due to the failure of the definitive fetal liver erythropoiesis with reduced primitive erythropoiesis, suggesting that EPO and EPOR are already functional in early yolk sac [24, 25, 39, 40, 41]. However, others showed that additional EPO did not affect heme synthesis in early mouse embryos [159]. Furthermore, Malik et al. found that EPOR-null embryos have normal number of primitive, early stage progenitors but subsequently develop anemia with loss of primitive EBL [24]. In line with these findings, the same group concluded that EPO signaling is not critical for the survival of human primitive erythroid progenitors, but have a less understood role to promote proliferation and maturation of these cells. Since the role of EPO is controversial in yolk sac erythropoiesis, we tested whether iPSC-erythroid commitment is EPO-dependent. We have tested the requirement of the two most important growth factors for definitive erythropoiesis in various combinations; EPO and SCF (Control), without EPO (-EPO), without SCF (-SCF), and without both cytokine (-EPO and -SCF) [136]. Without EPO, we noticed lower harvest rate/colony number and the loss of CD71/CD235 population. The erythroid commitment was not affected by the deprivation of SCF; however, the addition of SCF together with EPO resulted in a more pure CD71/CD235 population (Figure 6B). These data suggest that EPO is required to allow early erythroid commitment while the role of SCF is not entirely clear.
Introduction of erythroid/MK-specific TFs into iPSC-derived hematopoietic cells, often named “forward reprogramming”, is being pursued as an approach to improve differentiation outcome. HOXA9, ERG, RORA, SOX4, and MYB have been introduced into human pluripotent stem cells. Engraftment into NSG mice resulted in erythroid cells, which were more skewed to definitive erythropoiesis (lack of embryonic Hbs, mainly HbF and some HbA, some enucleation) compared to TF-free counterparts [160, 161]. These results suggest the possibility of more mature erythroid cell production from iPSCs if certain TFs are included; however, the in vitro feasibility is not provided presently. To direct the cells more lineage-specific, other lineage instructive TFs may be used. GATA1, FLI-1, and TAL1 (MK-specific TFs) can be overexpressed to direct iPSC to MKs, thereby achieving ~100% MK yield within 15 days [158]. Besides this set of genes, there are other combinations that are used to achieve a similar aim [156, 162].
The technical differences between published differentiation methods are leading to slight discrepancies in marker expression pattern, purity, yield, and stage of development. However, currently all published methods are limited by technical pitfalls, including the production of developmentally immature (nonadult) cell types which may be the cause of low yield and difficulty to terminally differentiate toward functional end stage blood cell types (e.g., low enucleation potential of iPSC-erythroid cells and low efficiency of iPSC-Plt formation).
2.4.1. Marker expression pattern of differentiating iPSC cells
The purity of the iPSC-derived erythroid population, and its distribution over different maturation stages can be assessed by the erythroid-specific markers used for definitive erythroid culture systems (Section 2.3.2); however, their progression differs in some aspects. Based on our differentiation scheme (Figure 5), we recognize three maturation stages: (i) an early erythroid population (harvest at day 10–14) is CD71high/CD235high/CD36med/high, which is not yet hemoglobinized and displays big nuclei [136]. Furthermore, the cells are negative for CD18 (myeloid lineage marker) confirming specification toward the erythroid lineage; (ii) a 100% pure erythroid population (day 7–9 expansion) is CD71/CD235/CD36med with some spontaneous differentiation, which is recognized by hemoglobinization and condensation of the nuclei; (iii) a mature erythroid population (D7-14 terminal differentiation) gives rise to CD235high/CD71high/med/CD36low cells. However, there is a slight CD71 decrease associated with reticulocyte formation, and iPSC-derived erythroid cells do not become CD71 negative. Morphologically, these cells were somewhat different from their definitive counterparts. Despite hemoglobinization, nuclear condensation, and polarization, we do not observe a decrease of cytoplasm size and the enucleation potential is poor. Technical variations in the published methods (timing, added growth factors) cause notable differences in the erythroid marker expression pattern; therefore, it is hard to compare and/or draw general conclusions. The emergence of CD71+/CD235+ population is generally reported with purity discrepancies. For example, Yang et al. [163] reported 80% CD71/CD235 purity (with CD34+/CD43+ preselection and OP9 coculture), Salvagiotto et al. [148] by a feeder-free monolayer system reached 40% pure population, while Kobari et al. [135] with EB-based induction reached 98–99% comparable to our findings. The pattern of CD36 expression is not entirely clear. Mao et al., for example, used a four-step differentiation scheme, including an AGM coculture induction step, and defined the following gene expression profile: early definitive EBLs derived from CD235+/CD34low/CD36−, and they develop to CD235+/CD34−/CD36−, CD235+/CD34−/CD36−/low, CD235+/CD34−/CD36− cells in sequence [164]. Others including us found high CD36 expression during the early erythroid stage [136].
The kinetics during differentiation/maturation of MK from iPSC follow the same steps as from definitive CD34+ cells, namely MKBL (CD34+/CD41a+), early MK (CD34+/CD41a+/CD42+), and late MK (CD34−/CD41a+/CD42+). The MKs can undergo some polyploidization albeit not in similar level as in vivo or primary CD34+ cultures. MKs derived from mouse iPSC can form proplatelets but also in low numbers [157, 165, 166, 167, 168]. These Plts can be activated and contribute to clot formation and wound healing when transfused to injured mice, showing that iPSC megakaryopoiesis, although still inefficient, leads to functional Plts. The MK-specific cell surface markers can be also used for iPSC-MKs with the exception of CD41a. This marker is also an early endothelial/hematopoietic marker. Therefore, it is essential to always use it in a combination, for example, with CD42 to confirm the specific MK commitment. Despite this, most groups report MK percentage only based on CD41a expression (~30–80% MK induction) [136, 150, 168]. With our method, we are able to achieve an average purity of 78% (CD41a) that can be easily used in scaleup production [136].
2.4.2. Developmental stage of iPSC-derived erythroid/MK cells
Human ESC/iPSC-derived erythropoiesis/megakaryopoiesis, with the current knowledge, do not reach the adult definitive stage, but give rise to a mixture of primitive and definitive fetal/adult cells. Very little is known about human erythropoiesis/megakaryopoiesis in the early stages, between days 17 and 23 of embryogenesis (yolk-sac, AGM region) due to the fact that abortions are primarily performed at later fetal stages and in addition have serious ethical concerns. Hbs are commonly used to distinguish between developmental waves; however, HbF expressing RBCs both arises from yolk sac and later from fetal liver, and momentarily, there is a lack of markers, which can clearly distinguish these two waves (Figure 2A and B). The iPSC-derived erythroid cells predominantly express HbF, in addition to embryonic types of Hb. We and others also showed the presence of a small portion of adult Hb [135, 136, 153, 154, 155]. From the embryonic type of globins, both the presence of Gower 1 and Gower 2 Hb has been reported, but the ratio greatly differs between methods [135]. The presence of Gower 2 Hb indicates that the cells are capable of the first globin switch (ζ to α) to provide more mature primitive-state RBCs. Interestingly, the presence of adult types of Hb also differs between protocols as some group were able to show HbA1 or the presence of β chain, whereas others, including us, observed mainly HbA2 [135, 155]. It is unknown whether primitive erythropoiesis in vivo goes through a stage that corresponds to iPSC-derived erythroid cells (Hbemid, HbFhigh, HbAlow/−) or whether globin synthesis is impaired in these cells. The globins, however, may not be the most accurate markers to define the waves. Altogether, these findings make it difficult to define the state of iPSC-RBCs with respect to their developmental stage. From a technical point of view, these cells are able to produce definitive RBCs; therefore, they potentially can give rise to the required therapeutic product. Better understanding of the underlying regulatory mechanisms such as the site where erythropoiesis/megakaryopoiesis takes place, and Hb switches during development might pave the way for the necessary improvement of the current methods.
Unlike in the erythroid lineages where the expression of stage-specific Hbs can be used to determine the ontogeny phase, this type of readout is not available in the MK lineage. There are, however, intrinsic differences between megakaryocytic cells derived at different sites during ontogeny. For instance, polyploidy is a measure of ontogeny in vivo as yolk sack MK exhibits low ploidy (4–8N), whereas adult MK reach >64N [14, 169, 170]. Besides the lower polyploidization, the number of Plts that are produced follows the same trend from a low number per MK in embryonic/fetal tissues too high numbers of in adult MK, linking polyploidization and MK size to Plts production [170]. However, ploidy levels and Plt production in vitro are generally lower compared to their in vivo counterparts; therefore, it is not a good marker to access iPSC-MK development stages. The current best approach would be to use data from erythroid cultures and their ontogeny stage/wave and extrapolated this to the MK development because of their close relationship. However, investigation of purified megakaryocytic cultures of defined ontogeny stages through, e.g., RNAseq or mass spectrometry could yield specific makers.
2.4.3. Expansion potential/yield of iPSC-erythroid/MK cells
The final yield of our method is relatively high; however, the comparison with other methods is difficult due to technical discrepancies. There are different ways to calculate the final yield, which also depends on the iPSC maintenance system (single cell vs. clumps) and on the induction system (2D or 3D), resulting various ways to report the final yield. Single cell-seeded iPSC cultures can be normalized both to the initial number of seeded cells or to colony number. Furthermore, the comparison of absolute cell number produced (harvested) between 2D and 3D systems is not entirely realistic because of the different nature of the two cultures. We use single cell-seeding, which allows to calculate the yield/iPSC and yield/colony number to represent differentiation efficiency. In our hands, one iPSC colony can give rise on average 5.6 × 106 erythroid cells after 9 days of expansion. While a single iPSC gives rise to ~8 × 103 erythroid cells (harvest) and subsequent 9 days expansion results in ~2 × 105 erythroid cells/iPSC on average [136]. The expansion potential (from harvest day) compared to definitive cell types remains relatively low, in line with other methods irrespective of culture condition (Figure 4B). The terminal maturation of iPSC-erythroid cells toward enucleating reticulocytes is inefficient with the existing methods and is currently one of the major hurdles to overcome. In our hands, matured iPSC-erythroid cultures had 30–40% enucleation rate based on their nuclei count; however, the resulting reticulocytes appeared to be instable. Altogether, iPSC-derived erythroid cells are able to expand but for limited time and length, with suboptimal enucleation capacity. Probably, this is also coming from the fact that iPSC-derived erythroid cells, based on their globin expression, do not entirely correspond to a fetal/adult definitive wave.
MK yield from iPSCs is higher than CD34+ differentiation (6.9 × 103 cells/iPSC). Unlike the erythroid system in which differentiation can be inhibited for several days by the addition of glucocorticoid analogues, the megakaryocytic system currently lacks such a specific expansion advantage. As a result, iPSC-MK yield and purity is currently low compared to the iPSC-erythroid yield. Even though the yield of MK is low, a small number of MK still could produce significantly large amount of Plts. The in vivo production of 2000–8000 Plts/MK is not achieved by far (1–50 Plts/MK in static condition) [4, 5, 81, 168]. Initiatives to increase this yield are needed but will require increasing our knowledge on the later stages of proplatelet formation, which is a current hurdle to overcome in culture conditions and further discussed in the next paragraph devoted to bioreactors (Section 2.5).
2.5. Bioreactors
The production of cultured red blood cells for transfusion purposes has been the holy grail for transfusion medicine. However, a main challenge is the well-described limitation in cell density during the expansion phase [171]. A single transfusion unit contains 2 × 1012 RBCs. Conventional cultivation systems using dishes or flasks can reach up to 10 × 106 cells/mL, meaning that more than 1000 L of culture would be required for the manufacture of a single transfusion unit [172]. Handling of such large volumes is impractical if static culture conditions are maintained. Thus, multiple bioreactor designs have been proposed to improve the volumetric productivity of the process (produced cells/volume of medium). A static culturing system mimicking BM tissue has been proposed, in which cells are grown in a porous scaffold and nutrients are continuously fed through hollow fibers, while used media containing waste from metabolism are removed [173]. This system allows to have continuous replenishment of spent components in the media, while it is possible to re-use some of the most expensive components such as growth factors and transferrin. Also, it separates cells from large shear stress sources. Although an optimal design of this system could lead to the production of transfusion units at competitive costs compared to the price of rare blood units, it would require significant improvements in transfer of nutrients and matured RBCs between the scaffold and the inflow/outflow streams [174]. Mass transfer limitations in diffusion-governed systems can be tackled with agitation. It is relevant to note that conflicting reports have been made on the effect of shear due to agitation in in vitro erythropoiesis [172, 175]. Nevertheless, important advances have been made toward culture of EBLs in stirred reactors. Enucleated RBCs were produced in microbioreactors (<20 mL) in which the effect of shear stress on expansion and enucleation of EBLs was evaluated. Gas sparging caused cell death, whereas stirring enhanced enucleation [172]. Expansion can also be performed in shake flasks (40 mL) and stirred tank bioreactors of larger volumes (500 mL) with similar growth kinetics [176]. Hybrid systems combining mechanical agitation and growth of cells in porous materials have also been proposed. This type of systems could improve mass transfer while protecting cells from shear, but harvesting of mature cells from these carriers is still a challenge that must be addressed.
The high sensitivity of MK to shear stress renders culturing and flow cytometry assays challenging. However, it can also be exploited to generate in vitro Plts. Multiple techniques can be used to induce the culturing and shedding of Plts like pipetting or the use of flow chambers [157, 165, 166, 167, 168]. These approaches mimic more the in vivo situation of Plts formation where they are shed in the vasculature, in contrast to static cultures. Plt shedding can be induced by repetitive pipetting of the cultured cells with a hand pipette (p1000) [81]. There is a risk that too much pressure is enforced on the cells, causing them to lyse instead of shed. A more sophisticated method of in vitro Plts production is the use of flow chambers, which come in different versions [167, 177]. The most basic one has a linear flow with a surface that is coated to which MKs can attach [165, 167]. These versions were further developed to chambers that include small gaps where MKs are trapped and can extend their cytoplasmic extensions through the gap where they start forming Plts [165, 177]. Another flow chamber setup is the use of pillars in the chamber instead of gaps where the MKs can attach. Coating with a various matrices like fibronectin, von Willebrand factor, or TPO can enhance the efficiency of the Plts generation. In addition, the flow rates that are produced in these chambers can be regulated. The produced Plts in these systems can be harvested and used for functional test. The aforementioned approaches can also be combined in a way that the culturing and differentiation of iPSC toward MK is performed on coated beads in a stirred spinner flask bioreactor, where subsequently also the Plt production is induced and enhanced, although with a relatively low Plt yield (1 Plt/MK) [168]. Recently, Ito et al. showed that in the vasculature, MKs are not only exposed to shear stress but also to turbulence during Plt sheading, where the process was implemented in their bioreactor [178]. By using their iPSC lines with inducible c-MYC-BMI-1 and BCL-XL expression, they were able to produce a thrombocyte transfusion unit in an 8 L system. This approach proves the possibility of large-scale Plt production but still with a discrepancy between in vitro (80–100 Plts) and in vivo (2000–8000 Plts) Plt production capacity.
2.6. Clinical trial
The ability to produce large numbers of enucleated, hemoglobinized RBCs opens the perspective of producing cultured red blood cells (cRBC) for transfusion purposes. The feasibility to do so has been demonstrated by the team of Luc Douay who cultured 1 mL of packed cRBC from CD34+ HSCs and transfused it to a healthy volunteer, with a cRBCs half-life of 26 days after injection [133, 179]. Donor-derived RBC transfusion is a cornerstone of modern medicine in the treatment of trauma, chronic anemia, and in surgery. The existence of 30 blood group systems, such as the ABO and Rhesus system, generates at least 300 distinct blood group antigens [180]. Recurrent transfusions carry an inherent risk on alloimmunization to nonidentical blood group antigens, which complicate further transfusions. Besides, this cellular therapy is dependent on donor availability with a potential risk of blood-borne diseases. In vitro derivation of cRBCs allows their thorough characterization, therefore providing access to better matched product. Improved cell culture protocols may eventually enable us to generate cRBCs for transfusion purposes from iPSCs that were selected and/or modified to lack most blood group antigens with the advance of donor-independency offered by iPSCs source.
In a recent publication, Ito et al. [178] were able to generate Plts in transfusion quantities, where the functionality of these Plts was shown in in vivo mouse experiments. This work gives a feasible prospect of clinical trials, which still requires the system to be converted to GMP grade [178].
Our culture protocol from human PBMC to RBC
day 0 expansion: purify PBMCs by Ficoll and seed in StemSpan or Cell-Quin supplemented with 1 ng/ml human IL-3, 2 U/ml EPO, 10 ng/ml hSCF, 10−6 M DEX at 10 × 106 cells/ml, 37°C, and 5% CO2.
Optional: to remove remaining RBCs after Ficoll purification, the use of RBC lysis buffer is suggested.
day 2 and 4: replace half of the medium; add all factors to the medium except for IL-3.
day 5: EBLs appear as large (nongranulated) blasts.
Optional: to remove remaining lymphocytes, purify the culture by density centrifugation on a 1.075 g/ml Percoll gradient.
day 6–20: put the cells daily or every second day to 0.5–0.7 × 106/ml medium supplemented with EPO, SCF, and DEX (concentration is same as day 0).
day 0 terminal differentiation: wash the cells twice with PBS and re-seed in medium supplemented with 10 U/ml EPO, 1 mg/ml holotransferrin, 2–5% plasma, 5 U/ml heparin at 2 × 106/ml.
day 9–14: let the cells differentiate with half media change every 2–3 days.
Our culture protocol from CD34+to MK
day 0: collect CD34+ cells by MACS isolation from CB, BM, PBMC, or MPB and seed in Cellgro or Cell-Quin with 100 ng/ml FL, 50 ng/ml hSCF, 50 ng/ml TPO, 20 ng/ml IL-6 at 1 × 106 cells/ml, 37°C, and 5% CO2.
Re-seed cells when concentration exceeds 2.5 × 106 cells/ml, otherwise keep cells as undisturbed as possible at stable CO2 levels.
day 4: cells start to commit to the MK lineage (~10–20%), cells are collected and spun down at 200 g to start terminal differentiation.
day 0 terminal differentiation: re-seed cells in media (Cellgro or Cell-Quin) supplemented with: 50 ng/ml TPO and 10 ng/ml IL-1β at 0.5 × 106 cells/ml.
During terminal differentiation, the addition of tirofiban hydrochloride monohydrate and heparin is recommended.
Pipetting should be kept to minimal.
1 μM of SR-1 can be used to increase polyploidization and Plt production.
day 11: cells will be committed to the MK lineage (80–100%) and consist of MKBLs and early MKs.
Cell collection from these days onward should be performed using 2 mL or larger pipettes and avoid the usage of hand pipettes to circumvent cell lysis and shear stress [157, 166].
Centrifugation steps are on 150 g, low ramp, and brake.
Flow cytometry techniques on unfixed MKs at this stage will induce granule release and proplatelet formation.
day 12–16: MK cells will mature to late MKs and start producing proplatelets.
Centrifugation steps are 100 g, low ramp, and brake.
CB starts proplatelet production earlier than adult sources.
Proplatelets can be harvested using Plts isolation protocols.
Proplatelets are easily activated, treat them as regular Plts.
3. Conclusions
As described in this chapter, there are multiple protocols to culture RBCs and MKs from a variety of hematopoietic tissues. Depending on the goal (fundamental research, drug screening, or clinical applications), one should consider beforehand which source can be used. For clinical applications, the use of a fully defined unlimited source would be preferred. For this, iPSC (generated with nonintegrating method) hold great potential but differentiation toward RBCs and Plts has to be improved.
Acknowledgments
We would like to thank Esther Heideveld, Joan Gallego Murillo, Department of Hematopoiesis and Laboratory for Cell Therapy (Sanquin, Amsterdam) for their experimental and scientific input.
We are grateful for the support by the Ministry of Health (PPOC: 11-035, 15-2089), the Landsteiner Foundation for Blood Transfusion Research (LSBR1141), the European Union (FA H2020-MSCA ITN-2015, RELEVANCE), and the Netherlands Organization for Scientific Research (NWO/ZonMw 40-41400-98-1327; 40-00812-98-12128).
Conflict of interest
None.
Abbreviations
AGM
aorta-gonad-mesonephros
AhR
aryl hydrocarbon receptor
bFGF
basic fibroblast growth factor
BM
bone marrow
BMP4
bone morphogenetic protein 4
CB
cord blood
cRBC
cultured red blood cell
DEX
dexamethasone
dox
doxycycline
EBL
erythroblast
EHT
endothelial to hematopoietic transition
EMP
erythroid-myeloid progenitor
EPO
erythropoietin
EPOR
EPO receptor
ESC
embryonic stem cell
FL
FLT-3 ligand
FLI-1
friend leukemia integration 1
FLT-3
Fms-like tyrosine kinase 3
GR
glucocorticoid receptor
Hb
hemoglobin
HE
hemogenic endothelium
HFL
human fetal liver
HSC
hematopoietic stem cell
HSPC
hematopoietic stem/progenitor cell
IL
interleukin
Ins
insulin
InsR
insulin receptor
iPSC
induced pluripotent stem cell
MK
megakaryocyte
MKBL
megakaryoblast
MPB
mobilized peripheral blood
MPP
multipotent progenitor
PBMC
peripheral blood mononuclear cell
Plt
platelet
RBC
red blood cell
SCF
stem cell factor
SR-1
StemRegenin 1
TF
transcription factor
TPO
thrombopoietin
T3
3,5,3′-triiodothyronine
VEGF
vascular endothelial growth factor
\n',keywords:"transfusion, cell culture model, GMP conditions, defined culture medium, cultured red blood cells, erythropoiesis, megakaryopoiesis, induced pluripotent stem cells, bioreactors, clinical trials",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/63327.pdf",chapterXML:"https://mts.intechopen.com/source/xml/63327.xml",downloadPdfUrl:"/chapter/pdf-download/63327",previewPdfUrl:"/chapter/pdf-preview/63327",totalDownloads:1574,totalViews:420,totalCrossrefCites:1,totalDimensionsCites:0,totalAltmetricsMentions:0,impactScore:0,impactScorePercentile:45,impactScoreQuartile:2,hasAltmetrics:0,dateSubmitted:"April 27th 2018",dateReviewed:"August 1st 2018",datePrePublished:"November 5th 2018",datePublished:"January 23rd 2019",dateFinished:"August 31st 2018",readingETA:"0",abstract:"Erythrocytes and platelets are the major cellular components of blood. Several hereditary diseases affect the production/stability of red blood cells (RBCs) and platelets (Plts) resulting in anemia or bleeding, respectively. Patients with such disorders may require recurrent transfusions, which bear a risk to develop alloantibodies and ultimately may result in transfusion product refractoriness. Cell culture models enable to unravel disease mechanisms, and to screen for alternative therapeutic products. Besides these applications, the ultimate goal is the large-scale production of blood effector cells for transfusion. Cultured RBCs that lack many of the common blood group antigens and Plts-lacking HLA expression would improve transfusion practice. Large numbers of RBCs and Plts can already be generated using hematopoietic stem cells derived from fetal liver, cord blood, peripheral blood, and bone marrow as starting material for cell culture. The recent advances to generate blood cells from induced pluripotent stem cells provide a donor-independent, immortal primary source for cell culture models. This enables us to study developmental switches during erythropoiesis/megakaryopoiesis and provides potential future therapeutic applications. In this review, we will discuss how erythropoiesis and megakaryopoiesis are mimicked in culture systems and how these models relate to the in vivo process.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/63327",risUrl:"/chapter/ris/63327",book:{id:"6964",slug:"cell-culture"},signatures:"Eszter Varga, Marten Hansen, Emile van den Akker and Marieke von\nLindern",authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Erythropoiesis/megakaryopoiesis",level:"1"},{id:"sec_2_2",title:"2.1. Growth factors and major regulators",level:"2"},{id:"sec_2_3",title:"2.1.1. Lineage- and stage-specific cytokines",level:"3"},{id:"sec_3_3",title:"2.1.2. Growth factor-binding tyrosine kinase receptors",level:"3"},{id:"sec_4_3",title:"2.1.3. Ligand-activated TFs",level:"3"},{id:"sec_5_3",title:"2.1.4. Erythroid-specific regulation",level:"3"},{id:"sec_6_3",title:"2.1.5. MK-specific regulation",level:"3"},{id:"sec_8_2",title:"2.2. Cell culture models",level:"2"},{id:"sec_8_3",title:"2.2.1. Erythroid immortalized cell lines",level:"3"},{id:"sec_8_4",title:"2.2.1.1. Mouse cell lines",level:"4"},{id:"sec_9_4",title:"2.2.1.2. Human cell lines",level:"4"},{id:"sec_11_3",title:"2.2.2. MK-cell lines",level:"3"},{id:"sec_13_2",title:"2.3. Primary cell culture",level:"2"},{id:"sec_13_3",title:"2.3.1. Cell source and media",level:"3"},{id:"sec_14_3",title:"2.3.2. Erythroid-specific culture system",level:"3"},{id:"sec_15_3",title:"2.3.3. MK-specific culture system",level:"3"},{id:"sec_17_2",title:"2.4. Erythropoiesis/megakaryopoiesis from iPSC",level:"2"},{id:"sec_17_3",title:"2.4.1. Marker expression pattern of differentiating iPSC cells",level:"3"},{id:"sec_18_3",title:"2.4.2. Developmental stage of iPSC-derived erythroid/MK cells",level:"3"},{id:"sec_19_3",title:"2.4.3. Expansion potential/yield of iPSC-erythroid/MK cells",level:"3"},{id:"sec_21_2",title:"2.5. Bioreactors",level:"2"},{id:"sec_22_2",title:"2.6. Clinical trial",level:"2"},{id:"sec_24",title:"3. Conclusions",level:"1"},{id:"sec_25",title:"Acknowledgments",level:"1"},{id:"sec_28",title:"Conflict of interest",level:"1"},{id:"sec_27",title:"Abbreviations",level:"1"}],chapterReferences:[{id:"B1",body:'Machlus KR, Italiano JE Jr. The incredible journey: From megakaryocyte development to platelet formation. The Journal of Cell Biology. 2013;201:785-796'},{id:"B2",body:'Franco AT, Corken A, Ware J. Platelets at the interface of thrombosis, inflammation, and cancer. Blood. 2015;126:582-588'},{id:"B3",body:'Frick PG. The erythrocyte as an example of biological fitness. Schweizerische Medizinische Wochenschrift. 1961;91:1245-1249'},{id:"B4",body:'Trowbridge EA, Martin JF, Slater DN, Kishk YT, Warren CW, Harley PJ, et al. The origin of platelet count and volume. Clinical Physics and Physiological Measurement: An Official Journal of the Hospital Physicists\' Association, Deutsche Gesellschaft fur Medizinische Physik and the European Federation of Organisations for Medical Physics. 1984;5:145-170'},{id:"B5",body:'Kaufman RM, Airo R, Pollack S, Crosby WH, Doberneck R. Origin of Pulmonary Megakaryocytes, Blood. 1965;25:767-775'},{id:"B6",body:'Lefrancais E, Ortiz-Munoz G, Caudrillier A, Mallavia B, Liu F, Sayah DM, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature. 2017;544:105-109'},{id:"B7",body:'Weiskopf K, Schnorr PJ, Pang WW, Chao MP, Chhabra A, Seita J, et al. Myeloid cell origins, differentiation, and clinical implications. Microbiology Spectrum. 2016;4:10.1128'},{id:"B8",body:'Roch A, Trachsel V, Lutolf MP. Brief report: Single-cell analysis reveals cell division-independent emergence of megakaryocytes from phenotypic hematopoietic stem cells. Stem Cells. 2015;33:3152-3157'},{id:"B9",body:'Nishikii H, Kurita N, Chiba S. The road map for megakaryopoietic lineage from hematopoietic stem/progenitor cells. Stem Cells Translational Medicine. 2017;6:1661-1665'},{id:"B10",body:'Debili N, Coulombel L, Croisille L, Katz A, Guichard J, Breton-Gorius J, et al. Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow. Blood. 1996;88:1284-1296'},{id:"B11",body:'Fraser ST, Isern J, Baron MH. Maturation and enucleation of primitive erythroblasts during mouse embryogenesis is accompanied by changes in cell-surface antigen expression. Blood. 2007;109:343-352'},{id:"B12",body:'Dolznig H, Bartunek P, Nasmyth K, Mullner EW, Beug H. Terminal differentiation of normal chicken erythroid progenitors: Shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control. Cell Growth & Differentiation: The Molecular Biology Journal of the American Association for Cancer Research. 1995;6:1341-1352'},{id:"B13",body:'von Lindern M, Deiner EM, Dolznig H, Parren-Van Amelsvoort M, Hayman MJ, Mullner EW, et al. Leukemic transformation of normal murine erythroid progenitors: V- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis. Oncogene. 2001;20:3651-3664'},{id:"B14",body:'Ma DC, Sun YH, Chang KZ, Zuo W. Developmental change of megakaryocyte maturation and DNA ploidy in human fetus. European Journal of Haematology. 1996;57:121-127'},{id:"B15",body:'Chow A, Lucas D, Hidalgo A, Mendez-Ferrer S, Hashimoto D, Scheiermann C, et al. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. The Journal of Experimental Medicine. 2011;208:261-271'},{id:"B16",body:'Fabriek BO, Polfliet MM, Vloet RP, van der Schors RC, Ligtenberg AJ, Weaver LK, et al. The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor. Blood. 2007;109:5223-5229'},{id:"B17",body:'Sadahira Y, Yoshino T, Monobe Y. Very late activation antigen 4-vascular cell adhesion molecule 1 interaction is involved in the formation of erythroblastic islands. The Journal of Experimental Medicine. 1995;181:411-415'},{id:"B18",body:'Ulyanova T, Scott LM, Priestley GV, Jiang Y, Nakamoto B, Koni PA, et al. VCAM-1 expression in adult hematopoietic and nonhematopoietic cells is controlled by tissue-inductive signals and reflects their developmental origin. Blood. 2005;106:86-94'},{id:"B19",body:'Heideveld E, Masiello F, Marra M, Esteghamat F, Yagci N, von Lindern M, et al. CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield. Haematologica. 2015;100:1396-1406'},{id:"B20",body:'Junt T, Schulze H, Chen Z, Massberg S, Goerge T, Krueger A, et al. Dynamic visualization of thrombopoiesis within bone marrow. Science. 2007;317:1767-1770'},{id:"B21",body:'Palis J. Hematopoietic stem cell-independent hematopoiesis: Emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS Letters. 2016;590:3965-3974'},{id:"B22",body:'Faloon P, Arentson E, Kazarov A, Deng CX, Porcher C, Orkin S, et al. Basic fibroblast growth factor positively regulates hematopoietic development. Development. 2000;127:1931-1941'},{id:"B23",body:'Wang C, Tang X, Sun X, Miao Z, Lv Y, Yang Y, et al. TGFbeta inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy. Cell Research. 2012;22:194-207'},{id:"B24",body:'Malik J, Kim AR, Tyre KA, Cherukuri AR, Palis J. Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts. Haematologica. 2013;98:1778-1787'},{id:"B25",body:'McGann JK, Silver L, Liesveld J, Palis J. Erythropoietin-receptor expression and function during the initiation of murine yolk sac erythropoiesis. Experimental Hematology. 1997;25:1149-1157'},{id:"B26",body:'Potts KS, Sargeant TJ, Markham JF, Shi W, Biben C, Josefsson EC, et al. A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo. Blood. 2014;124:2725-2729'},{id:"B27",body:'Li W, Johnson SA, Shelley WC, Ferkowicz M, Morrison P, Li Y, et al. Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro. Blood. 2003;102:4345-4353'},{id:"B28",body:'McGrath KE, Frame JM, Fegan KH, Bowen JR, Conway SJ, Catherman SC, et al. Distinct sources of hematopoietic progenitors emerge before HSCs and provide functional blood cells in the mammalian embryo. Cell Reports. 2015;11:1892-1904'},{id:"B29",body:'Crisan M, Solaimani Kartalaei P, Neagu A, Karkanpouna S, Yamada-Inagawa T, Purini C, et al. BMP and hedgehog regulate distinct AGM hematopoietic stem cells ex vivo. Stem Cell Reports. 2016;6:383-395'},{id:"B30",body:'Tavassoli M. Embryonic and fetal hemopoiesis: An overview. Blood Cells. 1991;17:269-281 discussion 282-266'},{id:"B31",body:'Fujiki H, Kimura T, Minamiguchi H, Harada S, Wang J, Nakao M, et al. Role of human interleukin-9 as a megakaryocyte potentiator in culture. Experimental Hematology. 2002;30:1373-1380'},{id:"B32",body:'Goswami R, Kaplan MH. A brief history of IL-9. Journal of Immunology. 2011;186:3283-3288'},{id:"B33",body:'Beaulieu LM, Lin E, Mick E, Koupenova M, Weinberg EO, Kramer CD, et al. Interleukin 1 receptor 1 and interleukin 1beta regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:552-564'},{id:"B34",body:'Ratajczak J, Marlicz W, Keidel A, Machalinski B, Ratajczak MZ, Gewirtz AM. Effect of Interleukin-1 alpha and Interleukin-1 beta on erythroid progenitor cell growth in serum free cultures: An in vitro study relevant to the pathogenesis of the anemia of chronic disease. Hematology. 1997;2:21-28'},{id:"B35",body:'Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature. 1957;179:633-634'},{id:"B36",body:'Jelkmann W. Physiology and pharmacology of erythropoietin, transfusion medicine and hemotherapy: Offizielles. Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie. 2013;40:302-309'},{id:"B37",body:'Haase VH. Regulation of erythropoiesis by hypoxia-inducible factors. Blood Reviews. 2013;27:41-53'},{id:"B38",body:'Kapur R, Zhang L. A novel mechanism of cooperation between c-Kit and erythropoietin receptor. Stem cell factor induces the expression of Stat5 and erythropoietin receptor, resulting in efficient proliferation and survival by erythropoietin. The Journal of Biological Chemistry. 2001;276:1099-1106'},{id:"B39",body:'Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83:59-67'},{id:"B40",body:'Kieran MW, Perkins AC, Orkin SH, Zon LI. Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor. Proceedings of the National Academy of Sciences of the United States of America. 1996;93:9126-9131'},{id:"B41",body:'Lin CS, Lim SK, D\'Agati V, Costantini F. Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes & Development. 1996;10:154-164'},{id:"B42",body:'de Graaf CA, Metcalf D. Thrombopoietin and hematopoietic stem cells. Cell Cycle. 2011;10:1582-1589'},{id:"B43",body:'Klinger MH, Jelkmann W. Subcellular localization of thrombopoietin in human blood platelets and its release upon thrombin stimulation. British Journal of Haematology. 2001;115:421-427'},{id:"B44",body:'Fielder PJ, Gurney AL, Stefanich E, Marian M, Moore MW, Carver-Moore K, et al. Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets. Blood. 1996;87:2154-2161'},{id:"B45",body:'Grozovsky R, Begonja AJ, Liu K, Visner G, Hartwig JH, Falet H, et al. The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nature Medicine. 2015;21:47-54'},{id:"B46",body:'Hoffman R, Tong J, Brandt J, Traycoff C, Bruno E, McGuire BW, et al. The in vitro and in vivo effects of stem cell factor on human hematopoiesis. Stem Cells. 1993;11(Suppl 2):76-82'},{id:"B47",body:'Li K, Yang M, Lam AC, Yau FW, Yuen PM. Effects of flt-3 ligand in combination with TPO on the expansion of megakaryocytic progenitors. Cell Transplantation. 2000;9:125-131'},{id:"B48",body:'Hassan HT, Zander A. Stem cell factor as a survival and growth factor in human normal and malignant hematopoiesis. Acta Haematologica. 1996;95:257-262'},{id:"B49",body:'Kapur R, Majumdar M, Xiao X, McAndrews-Hill M, Schindler K, Williams DA. Signaling through the interaction of membrane-restricted stem cell factor and c-kit receptor tyrosine kinase: Genetic evidence for a differential role in erythropoiesis. Blood. 1998;91:879-889'},{id:"B50",body:'Fleischman RA, Mintz B. Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta. Proceedings of the National Academy of Sciences of the United States of America. 1979;76:5736-5740'},{id:"B51",body:'Bernstein A, Chabot B, Dubreuil P, Reith A, Nocka K, Majumder S, et al. The mouse W/c-kit locus. Ciba Foundation Symposium. 1990;148:158-166 discussion 166-172'},{id:"B52",body:'Avraham H, Vannier E, Cowley S, Jiang SX, Chi S, Dinarello CA, et al. Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells. Blood. 1992;79:365-371'},{id:"B53",body:'Muta K, Krantz SB, Bondurant MC, Dai CH. Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation. Blood. 1995;86:572-580'},{id:"B54",body:'Broudy VC, Lin NL, Priestley GV, Nocka K, Wolf NS. Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood. 1996;88:75-81'},{id:"B55",body:'Tsapogas P, Mooney CJ, Brown G, Rolink A. The cytokine Flt3-ligand in normal and malignant hematopoiesis. International Journal of Molecular Sciences. 2017;18:1115'},{id:"B56",body:'Valentinis B, Baserga R. IGF-I receptor signalling in transformation and differentiation. Molecular Pathology: MP. 2001;54:133-137'},{id:"B57",body:'von Lindern M, Zauner W, Mellitzer G, Steinlein P, Fritsch G, Huber K, et al. The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro. Blood. 1999;94:550-559'},{id:"B58",body:'Tunduguru R, Thurmond DC. Promoting glucose transporter-4 vesicle trafficking along cytoskeletal tracks: PAK-Ing them out. Frontiers in Endocrinology. 2017;8:329'},{id:"B59",body:'Montel-Hagen A, Sitbon M, Taylor N. Erythroid glucose transporters. Current Opinion in Hematology. 2009;16:165-172'},{id:"B60",body:'Montel-Hagen A, Blanc L, Boyer-Clavel M, Jacquet C, Vidal M, Sitbon M, et al. The Glut1 and Glut4 glucose transporters are differentially expressed during perinatal and postnatal erythropoiesis. Blood. 2008;112:4729-4738'},{id:"B61",body:'Correa PN, Axelrad AA. Production of erythropoietic bursts by progenitor cells from adult human peripheral blood in an improved serum-free medium: Role of insulinlike growth factor 1. Blood. 1991;78:2823-2833'},{id:"B62",body:'Correa PN, Eskinazi D, Axelrad AA. Circulating erythroid progenitors in polycythemia vera are hypersensitive to insulin-like growth factor-1 in vitro: Studies in an improved serum-free medium. Blood. 1994;83:99-112'},{id:"B63",body:'Stocklin E, Wissler M, Gouilleux F, Groner B. Functional interactions between Stat5 and the glucocorticoid receptor. Nature. 1996;383:726-728'},{id:"B64",body:'Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991;352:497-505'},{id:"B65",body:'Karin M, Chang L. AP-1—Glucocorticoid receptor crosstalk taken to a higher level. The Journal of Endocrinology. 2001;169:447-451'},{id:"B66",body:'Wessely O, Deiner EM, Beug H, von Lindern M. The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors. The EMBO Journal. 1997;16:267-280'},{id:"B67",body:'Bauer A, Tronche F, Wessely O, Kellendonk C, Reichardt HM, Steinlein P, et al. The glucocorticoid receptor is required for stress erythropoiesis. Genes & Development. 1999;13:2996-3002'},{id:"B68",body:'Varricchio L, Migliaccio AR. The role of glucocorticoid receptor (GR) polymorphisms in human erythropoiesis. American Journal of Blood Research. 2014;4:53-72'},{id:"B69",body:'Das KC, Mukherjee M, Sarkar TK, Dash RJ, Rastogi GK. Erythropoiesis and erythropoietin in hypo- and hyperthyroidism. The Journal of Clinical Endocrinology and Metabolism. 1975;40:211-220'},{id:"B70",body:'M\'Rabet-Bensalah K, Aubert CE, Coslovsky M, Collet TH, Baumgartner C, den Elzen WP, et al. Thyroid dysfunction and anaemia in a large population-based study. Clinical Endocrinology. 2016;84:627-631'},{id:"B71",body:'Angelin-Duclos C, Domenget C, Kolbus A, Beug H, Jurdic P, Samarut J. Thyroid hormone T3 acting through the thyroid hormone alpha receptor is necessary for implementation of erythropoiesis in the neonatal spleen environment in the mouse. Development. 2005;132:925-934'},{id:"B72",body:'Schroeder C, Gibson L, Zenke M, Beug H. Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: A possible target for v-erbA oncogene action. Oncogene. 1992;7:217-227'},{id:"B73",body:'Leberbauer C, Boulme F, Unfried G, Huber J, Beug H, Mullner EW. Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood. 2005;105:85-94'},{id:"B74",body:'Jackson CS, Durandt C, Janse van Rensburg I, Praloran V, Brunet de la Grange P, Pepper MS. Targeting the aryl hydrocarbon receptor nuclear translocator complex with DMOG and Stemregenin 1 improves primitive hematopoietic stem cell expansion. Stem Cell Research. 2017;21:124-131'},{id:"B75",body:'Abdelrahim M, Smith R 3rd, Safe S. Aryl hydrocarbon receptor gene silencing with small inhibitory RNA differentially modulates Ah-responsiveness in MCF-7 and HepG2 cancer cells. Molecular Pharmacology. 2003;63:1373-1381'},{id:"B76",body:'Casado FL, Singh KP, Gasiewicz TA. Aryl hydrocarbon receptor activation in hematopoietic stem/progenitor cells alters cell function and pathway-specific gene modulation reflecting changes in cellular trafficking and migration. Molecular Pharmacology. 2011;80:673-682'},{id:"B77",body:'Smith BW, Rozelle SS, Leung A, Ubellacker J, Parks A, Nah SK, et al. The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation. Blood. 2013;122:376-385'},{id:"B78",body:'Singh KP, Garrett RW, Casado FL, Gasiewicz TA. Aryl hydrocarbon receptor-null allele mice have hematopoietic stem/progenitor cells with abnormal characteristics and functions. Stem Cells and Development. 2011;20:769-784'},{id:"B79",body:'Singh KP, Casado FL, Opanashuk LA, Gasiewicz TA. The aryl hydrocarbon receptor has a normal function in the regulation of hematopoietic and other stem/progenitor cell populations. Biochemical Pharmacology. 2009;77:577-587'},{id:"B80",body:'Wagner JE Jr, Brunstein CG, Boitano AE, DeFor TE, McKenna D, Sumstad D, et al. Phase I/II trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft. Cell Stem Cell. 2016;18:144-155'},{id:"B81",body:'Strassel C, Brouard N, Mallo L, Receveur N, Mangin P, Eckly A, et al. Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets. Blood. 2016;127:2231-2240'},{id:"B82",body:'Hu T, Wang D, Yu Q, Li L, Mo X, Pan Z, et al. Aryl hydrocarbon receptor negatively regulates lipid synthesis and involves in cell differentiation of SZ95 sebocytes in vitro. Chemico-Biological Interactions. 2016;258:52-58'},{id:"B83",body:'van den Akker E, van Dijk T, Parren-van Amelsvoort M, Grossmann KS, Schaeper U, Toney-Earley K, et al. Tyrosine kinase receptor RON functions downstream of the erythropoietin receptor to induce expansion of erythroid progenitors. Blood. 2004;103:4457-4465'},{id:"B84",body:'Grebien F, Kerenyi MA, Kovacic B, Kolbe T, Becker V, Dolznig H, et al. Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2. Blood. 2008;111:4511-4522'},{id:"B85",body:'Wessely O, Bauer A, Quang CT, Deiner EM, von Lindern M, Mellitzer G, et al. A novel way to induce erythroid progenitor self renewal: Cooperation of c-Kit with the erythropoietin receptor. Biological Chemistry. 1999;380:187-202'},{id:"B86",body:'Grech G, Blazquez-Domingo M, Kolbus A, Bakker WJ, Mullner EW, Beug H, et al. Igbp1 is part of a positive feedback loop in stem cell factor-dependent, selective mRNA translation initiation inhibiting erythroid differentiation. Blood. 2008;112:2750-2760'},{id:"B87",body:'Bakker WJ, Blazquez-Domingo M, Kolbus A, Besooyen J, Steinlein P, Beug H, et al. FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1. The Journal of Cell Biology. 2004;164:175-184'},{id:"B88",body:'Kolbus A, Blazquez-Domingo M, Carotta S, Bakker W, Luedemann S, von Lindern M, et al. Cooperative signaling between cytokine receptors and the glucocorticoid receptor in the expansion of erythroid progenitors: Molecular analysis by expression profiling. Blood. 2003;102:3136-3146'},{id:"B89",body:'Muckenthaler MU, Rivella S, Hentze MW, Galy B. A red carpet for iron metabolism. Cell. 2017;168:344-361'},{id:"B90",body:'Liao C, Carlson BA, Paulson RF, Prabhu KS. The intricate role of selenium and selenoproteins in erythropoiesis. Free Radical Biology & Medicine. 2018. In press'},{id:"B91",body:'Heideveld E, Hampton-O\'Neil LA, Cross SJ, van Alphen FPJ, van den Biggelaar M, Toye AM, et al. Glucocorticoids induce differentiation of monocytes towards macrophages that share functional and phenotypical aspects with erythroblastic island macrophages. Haematologica. 2018;103:395-405'},{id:"B92",body:'Lee SH, Crocker PR, Westaby S, Key N, Mason DY, Gordon S, et al. Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters. The Journal of Experimental Medicine. 1988;168:1193-1198'},{id:"B93",body:'Chow A, Huggins M, Ahmed J, Hashimoto D, Lucas D, Kunisaki Y, et al. CD169(+) macrophages provide a niche promoting erythropoiesis under homeostasis and stress. Nature Medicine. 2013;19:429-436'},{id:"B94",body:'Kaser A, Brandacher G, Steurer W, Kaser S, Offner FA, Zoller H, et al. Interleukin-6 stimulates thrombopoiesis through thrombopoietin: Role in inflammatory thrombocytosis. Blood. 2001;98:2720-2725'},{id:"B95",body:'Nakashima K, Taga T. gp130 and the IL-6 family of cytokines: Signaling mechanisms and thrombopoietic activities. Seminars in Hematology. 1998;35:210-221'},{id:"B96",body:'Stahl CP, Zucker-Franklin D, Evatt BL, Winton EF. Effects of human interleukin-6 on megakaryocyte development and thrombocytopoiesis in primates. Blood. 1991;78:1467-1475'},{id:"B97",body:'Wang Y, Niu ZY, Guo YJ, Wang LH, Lin FR, Zhang JY. IL-11 promotes the treatment efficacy of hematopoietic stem cell transplant therapy in aplastic anemia model mice through a NF-kappaB/microRNA-204/thrombopoietin regulatory axis. Experimental & Molecular Medicine. 2017;49:e410'},{id:"B98",body:'Tsuji K, Lyman SD, Sudo T, Clark SC, Ogawa M. Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture. Blood. 1992;79:2855-2860'},{id:"B99",body:'Nishimura S, Nagasaki M, Kunishima S, Sawaguchi A, Sakata A, Sakaguchi H, et al. IL-1alpha induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs. The Journal of Cell Biology. 2015;209:453-466'},{id:"B100",body:'Lee CR, Cervi D, Truong AH, Li YJ, Sarkar A, Ben-David Y. Friend virus-induced erythroleukemias: A unique and well-defined mouse model for the development of leukemia. Anticancer Research. 2003;23:2159-2166'},{id:"B101",body:'Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Miyoshi H, et al. Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells. PLoS One. 2013;8:e59890'},{id:"B102",body:'Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes. Blood. 1995;85:997-1005'},{id:"B103",body:'Spike BT, Dirlam A, Dibling BC, Marvin J, Williams BO, Jacks T, et al. The Rb tumor suppressor is required for stress erythropoiesis. The EMBO Journal. 2004;23:4319-4329'},{id:"B104",body:'Clark AJ, Doyle KM, Humbert PO. Cell-intrinsic requirement for pRb in erythropoiesis. Blood. 2004;104:1324-1326'},{id:"B105",body:'Zhang J, Lee EY, Liu Y, Berman SD, Lodish HF, Lees JA. pRB and E2F4 play distinct cell-intrinsic roles in fetal erythropoiesis. Cell Cycle. 2010;9:371-376'},{id:"B106",body:'Vinjamur DS, Bauer DE. Growing and genetically manipulating human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines. Methods in Molecular Biology. 2018;1698:275-284'},{id:"B107",body:'Trakarnsanga K, Griffiths RE, Wilson MC, Blair A, Satchwell TJ, Meinders M, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nature Communications. 2017;8:14750'},{id:"B108",body:'Greenberg SM, Rosenthal DS, Greeley TA, Tantravahi R, Handin RI. Characterization of a new megakaryocytic cell line: The Dami cell. Blood. 1988;72:1968-1977'},{id:"B109",body:'Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, et al. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood. 1985;66:1384-1392'},{id:"B110",body:'Saito H. Megakaryocytic cell lines. Bailliere\'s Clinical Haematology. 1997;10:47-63'},{id:"B111",body:'To LB, Levesque JP, Herbert KE. How I treat patients who mobilize hematopoietic stem cells poorly. Blood. 2011;118:4530-4540'},{id:"B112",body:'Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010;116:4815-4828'},{id:"B113",body:'Liles WC, Broxmeyer HE, Rodger E, Wood B, Hubel K, Cooper S, et al. Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood. 2003;102:2728-2730'},{id:"B114",body:'Levesque JP, Hendy J, Winkler IG, Takamatsu Y, Simmons PJ. Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from the surface of hematopoietic progenitor cells. Experimental Hematology. 2003;31:109-117'},{id:"B115",body:'Levesque JP, Takamatsu Y, Nilsson SK, Haylock DN, Simmons PJ. Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood. 2001;98:1289-1297'},{id:"B116",body:'Pulsipher MA, Chitphakdithai P, Miller JP, Logan BR, King RJ, Rizzo JD, et al. Adverse events among 2408 unrelated donors of peripheral blood stem cells: Results of a prospective trial from the National Marrow Donor Program. Blood. 2009;113:3604-3611'},{id:"B117",body:'Migliaccio G, Sanchez M, Masiello F, Tirelli V, Varricchio L, Whitsett C, et al. Humanized culture medium for clinical expansion of human erythroblasts. Cell Transplantation. 2010;19:453-469'},{id:"B118",body:'Tusi BK, Wolock SL, Weinreb C, Hwang Y, Hidalgo D, Zilionis R, et al. Population snapshots predict early haematopoietic and erythroid hierarchies. Nature. 2018;555:54-60'},{id:"B119",body:'Dolznig H, Kolbus A, Leberbauer C, Schmidt U, Deiner EM, Mullner EW, et al. Expansion and differentiation of immature mouse and human hematopoietic progenitors. Methods in Molecular Medicine. 2005;105:323-344'},{id:"B120",body:'van den Akker E, Satchwell TJ, Pellegrin S, Daniels G, Toye AM. The majority of the in vitro erythroid expansion potential resides in CD34(−) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples. Haematologica. 2010;95:1594-1598'},{id:"B121",body:'Fibach E, Rachmilewitz EA. The two-step liquid culture: A novel procedure for studying maturation of human normal and pathological erythroid precursors. Stem Cells. 1993;11(Suppl 1):36-41'},{id:"B122",body:'Ebert BL, Lee MM, Pretz JL, Subramanian A, Mak R, Golub TR, et al. An RNA interference model of RPS19 deficiency in Diamond-Blackfan anemia recapitulates defective hematopoiesis and rescue by dexamethasone: Identification of dexamethasone-responsive genes by microarray. Blood. 2005;105:4620-4626'},{id:"B123",body:'Neildez-Nguyen TM, Wajcman H, Marden MC, Bensidhoum M, Moncollin V, Giarratana MC, et al. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nature Biotechnology. 2002;20:467-472'},{id:"B124",body:'Giarratana MC, Kobari L, Lapillonne H, Chalmers D, Kiger L, Cynober T, et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nature Biotechnology. 2005;23:69-74'},{id:"B125",body:'Gandrillon O, Schmidt U, Beug H, Samarut J. TGF-beta cooperates with TGF-alpha to induce the self-renewal of normal erythrocytic progenitors: Evidence for an autocrine mechanism. The EMBO Journal. 1999;18:2764-2781'},{id:"B126",body:'Migliaccio G, Di Pietro R, di Giacomo V, Di Baldassarre A, Migliaccio AR, Maccioni L, et al. In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients. Blood Cells, Molecules & Diseases. 2002;28:169-180'},{id:"B127",body:'van Zwieten R, van Oirschot BA, Veldthuis M, Dobbe JG, Streekstra GJ, van Solinge WW, et al. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis. American Journal of Hematology. 2015;90:E35-E39'},{id:"B128",body:'Vanhille DL, Nussenzveig RH, Glezos C, Perkins S, Agarwal AM. Best practices for use of the HEMOX analyzer in the clinical laboratory: Quality control determination and choice of anticoagulant. Laboratory Hematology : Official Publication of the International Society for Laboratory Hematology. 2012;18:17-19'},{id:"B129",body:'Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-676'},{id:"B130",body:'Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861-872'},{id:"B131",body:'Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154-156'},{id:"B132",body:'Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of the National Academy of Sciences of the United States of America. 1981;78:7634-7638'},{id:"B133",body:'Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, et al. Proof of principle for transfusion of in vitro-generated red blood cells. Blood. 2011;118:5071-5079'},{id:"B134",body:'Ma F, Ebihara Y, Umeda K, Sakai H, Hanada S, Zhang H, et al. Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:13087-13092'},{id:"B135",body:'Kobari L, Yates F, Oudrhiri N, Francina A, Kiger L, Mazurier C, et al. Human induced pluripotent stem cells can reach complete terminal maturation: In vivo and in vitro evidence in the erythropoietic differentiation model. Haematologica. 2012;97:1795-1803'},{id:"B136",body:'Hansen M, Varga E, Aarts C, Wust T, Kuijpers T, von Lindern M, et al. Efficient production of erythroid, megakaryocytic and myeloid cells, using single cell-derived iPSC colony differentiation. Stem Cell Research. 2018;29:232-244'},{id:"B137",body:'Qiu C, Olivier EN, Velho M, Bouhassira EE. Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood. 2008;111:2400-2408'},{id:"B138",body:'Lu SJ, Feng Q, Park JS, Vida L, Lee BS, Strausbauch M, et al. Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood. 2008;112:4475-4484'},{id:"B139",body:'Dias J, Gumenyuk M, Kang H, Vodyanik M, Yu J, Thomson JA, et al. Generation of red blood cells from human induced pluripotent stem cells. Stem Cells and Development. 2011;20:1639-1647'},{id:"B140",body:'Lapillonne H, Kobari L, Mazurier C, Tropel P, Giarratana MC, Zanella-Cleon I, et al. Red blood cell generation from human induced pluripotent stem cells: Perspectives for transfusion medicine. Haematologica. 2010;95:1651-1659'},{id:"B141",body:'Kennedy M, D\'Souza SL, Lynch-Kattman M, Schwantz S, Keller G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood. 2007;109:2679-2687'},{id:"B142",body:'Ledran MH, Krassowska A, Armstrong L, Dimmick I, Renstrom J, Lang R, et al. Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell. 2008;3:85-98'},{id:"B143",body:'Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:10716-10721'},{id:"B144",body:'Zambidis ET, Peault B, Park TS, Bunz F, Civin CI. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood. 2005;106:860-870'},{id:"B145",body:'Ng ES, Davis RP, Azzola L, Stanley EG, Elefanty AG. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood. 2005;106:1601-1603'},{id:"B146",body:'Amit M, Itskovitz-Eldor J. Derivation and spontaneous differentiation of human embryonic stem cells. Journal of Anatomy. 2002;200:225-232'},{id:"B147",body:'Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Molecular Medicine. 2000;6:88-95'},{id:"B148",body:'Salvagiotto G, Burton S, Daigh CA, Rajesh D, Slukvin II, Seay NJ. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs. PLoS One. 2011;6:e17829'},{id:"B149",body:'Niwa A, Heike T, Umeda K, Oshima K, Kato I, Sakai H, et al. A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors. PLoS One. 2011;6:e22261'},{id:"B150",body:'Feng Q, Shabrani N, Thon JN, Huo H, Thiel A, Machlus KR, et al. Scalable generation of universal platelets from human induced pluripotent stem cells. Stem Cell Reports. 2014;3:817-831'},{id:"B151",body:'Weisel KC, Gao Y, Shieh JH, Moore MA. Stromal cell lines from the aorta-gonado-mesonephros region are potent supporters of murine and human hematopoiesis. Experimental Hematology. 2006;34:1505-1516'},{id:"B152",body:'Choi KD, Yu J, Smuga-Otto K, Salvagiotto G, Rehrauer W, Vodyanik M, et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells. 2009;27:559-567'},{id:"B153",body:'Olivier EN, Marenah L, McCahill A, Condie A, Cowan S, Mountford JC. High-efficiency serum-free feeder-free erythroid differentiation of human pluripotent stem cells using small molecules. Stem Cells Translational Medicine. 2016;5:1394-1405'},{id:"B154",body:'Chang KH, Nelson AM, Cao H, Wang L, Nakamoto B, Ware CB, et al. Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood. 2006;108:1515-1523'},{id:"B155",body:'Dorn I, Klich K, Arauzo-Bravo MJ, Radstaak M, Santourlidis S, Ghanjati F, et al. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin. Haematologica. 2015;100:32-41'},{id:"B156",body:'Takayama N, Nishimura S, Nakamura S, Shimizu T, Ohnishi R, Endo H, et al. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells. The Journal of Experimental Medicine. 2010;207:2817-2830'},{id:"B157",body:'Jiang J, Woulfe DS, Papoutsakis ET. Shear enhances thrombopoiesis and formation of microparticles that induce megakaryocytic differentiation of stem cells. Blood. 2014;124:2094-2103'},{id:"B158",body:'Moreau T, Evans AL, Vasquez L, Tijssen MR, Yan Y, Trotter MW, et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nature Communications. 2016;7:11208'},{id:"B159",body:'Cole RJ, Paul J. The effects of erythropoietin on haem synthesis in mouse yolk sac and cultured foetal liver cells. Journal of Embryology and Experimental Morphology. 1966;15:245-260'},{id:"B160",body:'Doulatov S, Vo LT, Chou SS, Kim PG, Arora N, Li H, et al. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell Stem Cell. 2013;13:459-470'},{id:"B161",body:'Sugimura R, Jha DK, Han A, Soria-Valles C, da Rocha EL, Lu YF, et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature. 2017;545:432-438'},{id:"B162",body:'Nakamura S, Takayama N, Hirata S, Seo H, Endo H, Ochi K, et al. Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell. 2014;14:535-548'},{id:"B163",body:'Yang CT, French A, Goh PA, Pagnamenta A, Mettananda S, Taylor J, et al. Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co-express gamma and beta globins. British Journal of Haematology. 2014;166:435-448'},{id:"B164",body:'Mao B, Huang S, Lu X, Sun W, Zhou Y, Pan X, et al. Early development of definitive erythroblasts from human pluripotent stem cells defined by expression of glycophorin A/CD235a, CD34, and CD36. Stem Cell Reports. 2016;7:869-883'},{id:"B165",body:'Sullenbarger B, Bahng JH, Gruner R, Kotov N, Lasky LC. Prolonged continuous in vitro human platelet production using three-dimensional scaffolds. Experimental Hematology. 2009;37:101-110'},{id:"B166",body:'Dunois-Larde C, Capron C, Fichelson S, Bauer T, Cramer-Borde E, Baruch D. Exposure of human megakaryocytes to high shear rates accelerates platelet production. Blood. 2009;114:1875-1883'},{id:"B167",body:'Pallotta I, Lovett M, Kaplan DL, Balduini A. Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes. Tissue Engineering. Part C, Methods. 2011;17:1223-1232'},{id:"B168",body:'Eicke D, Baigger A, Schulze K, Latham SL, Halloin C, Zweigerdt R, et al. Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors. Scientific Reports. 2018;8:10146'},{id:"B169",body:'Elagib KE, Brock AT, Goldfarb AN. Megakaryocyte ontogeny: Clinical and molecular significance. Experimental Hematology. 2018;61:1-9'},{id:"B170",body:'Bluteau O, Langlois T, Rivera-Munoz P, Favale F, Rameau P, Meurice G, et al. Developmental changes in human megakaryopoiesis. Journal of Thrombosis and Haemostasis: JTH. 2013;11:1730-1741'},{id:"B171",body:'Timmins NE, Nielsen LK. Manufactured RBC—Rivers of blood, or an oasis in the desert? Biotechnology Advances. 2011;29:661-666'},{id:"B172",body:'Bayley R, Ahmed F, Glen K, McCall M, Stacey A, Thomas R. The productivity limit of manufacturing blood cell therapy in scalable stirred bioreactors. Journal of Tissue Engineering and Regenerative Medicine. 2018;12:e368-e378'},{id:"B173",body:'Panoskaltsis N, Magalhães Macedo, HM, Blanco MTM, Mantalaris A, Livingston AG. 3-Dimensional hollow fibre bioreactor systems for the maintenance, expansion, differentiation and harvesting of human stem cells and their rogeny. 2012, patent EP2652119A1'},{id:"B174",body:'Misener R, Fuentes Garí M, Rende M, Velliou E, Panoskaltsis N, Pistikopoulos EN, et al. Global superstructure optimisation of red blood cell production in a parallelised hollow fibre bioreactor. Computers & Chemical Engineering. 2014;71:532-553'},{id:"B175",body:'Boehm D, Murphy WG, Al-Rubeai M. The effect of mild agitation on in vitro erythroid development. Journal of Immunological Methods. 2010;360:20-29'},{id:"B176",body:'Lee E, Han SY, Choi HS, Chun B, Hwang B, Baek EJ. Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts. Tissue Engineering. Part A. 2015;21:817-828'},{id:"B177",body:'Maeda T, Wakasawa T, Shima Y, Tsuboi I, Aizawa S, Tamai I. Role of polyamines derived from arginine in differentiation and proliferation of human blood cells. Biological & Pharmaceutical Bulletin. 2006;29:234-239'},{id:"B178",body:'Ito Y, Nakamura S, Sugimoto N, Shigemori T, Kato Y, Ohno M, et al. Turbulence activates platelet biogenesis to enable clinical scale ex vivo production. Cell. 2018;174:636-648 e618'},{id:"B179",body:'Rousseau GF, Giarratana MC, Douay L. Large-scale production of red blood cells from stem cells: What are the technical challenges ahead? Biotechnology Journal. 2014;9:28-38'},{id:"B180",body:'Mitra R, Mishra N, Rath GP. Blood groups systems. Indian Journal of Anaesthesia. 2014;58:524-528'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Eszter Varga",address:null,affiliation:'
Department of Hematopoiesis, Sanquin Research, and Landsteiner Laboratory Amsterdam UMC, Amsterdam, The Netherlands
Department of Hematopoiesis, Sanquin Research, and Landsteiner Laboratory Amsterdam UMC, Amsterdam, The Netherlands
'},{corresp:null,contributorFullName:"Emile van den Akker",address:null,affiliation:'
Department of Hematopoiesis, Sanquin Research, and Landsteiner Laboratory Amsterdam UMC, Amsterdam, The Netherlands
'},{corresp:"yes",contributorFullName:"Marieke von Lindern",address:"m.vonlindern@sanquin.nl",affiliation:'
Department of Hematopoiesis, Sanquin Research, and Landsteiner Laboratory Amsterdam UMC, Amsterdam, The Netherlands
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1. Introduction
Since the early 19th century, indentation technique has been extensively used for characterizing the mechanical properties of vast range of materials. In general, the indentation test is known to measure the hardness of materials. In conventional techniques, the mean contact pressure (MCP) upon indenting a specimen surface is evaluated. This is done on the basis of the residual area measured from the image of the indent impression and the known value of the applied load. The quantitative parameter, thus evaluated, represents the material’s response against deformation. In fact, MCP measured at the fully developed plastic zone is known as hardness [1]. With the progress in the technology and its incorporation in the experimental setup, instrumented indentation technique, particularly ‘nanoindentation’ has been evolved to assess various mechanical as well as metallurgical properties of a range of materials [2, 3, 4]. This includes characterizing elastic moduli, residual stress, creep properties, dislocation density, strain rate sensitivity etc. [5, 6, 7, 8, 9, 10, 11, 12]. Among all these developments, the potential of the nanoindentation technique in generating the indention stress (σind) – indentation strain (εind) curve is the most recent one and it is explained in detail in this present chapter [2, 3].
In nanoindentation, the associated high-resolution depth sensing technique aids to estimate the depth or size of the deformation zone. The process records the continuous response of indentation load (P) in the range of μN vs. indentation depth (h) in the magnitude of nm. The P-h curve obtained therein helps to assess the various properties of the studied materials. Unlike the conventional technique, in instrumented nanoindentation, hardness is estimated by using indirect measurement of projected contact area from P-h curve and the known geometry of the indenter tip. Similarly, elastic modulus of the material is estimated using the slope of the unloading segment in the P-h response of materials [13]. This method of analysis has been used for various scientific studies to characterize the localized mechanical properties of the samples in sub-micron scale. In fact, this revolutionary modification in the assessment methodology through nanoindentation has opened up a wide range of studies to extract the different relevant mechanical properties of materials on a small-scale.
One of the breakthroughs is the capability of this technique in generating the σind - εind response of a material of interest [3, 14, 15, 16]. This novel and recent development plays a significant role in understanding the localized deformation capability of materials system. This is particularly because stress – strain characteristics can provide an insight into the elastic – plastic mechanisms of the materials, as per the conventional notion. In fact, estimation of localized stress – strain characteristics of a material through nanoindentation can even be a substitute for typically used small-scale characterization techniques for instance, micro-pillar compression [17, 18]. Nevertheless, nanoindentation is further beneficial owing to its easier sample preparation, simplicity in experimental execution, and non-destructive nature. This technique therefore has enormous potential for evaluation of small-scale mechanical properties of materials with minimal effort.
Considering this, the present chapter is dedicated to provide a reasonable understanding for generating σind - εind data from the P-h curve of nanoindentation. To develop a more conceptual idea for a new reader, the importance of indenter tip geometry in activating different deformation modes within the indented volume are discussed at the first hand. Subsequently, the basic relationships for the indentation, the method of analysis and generation of protocol for obtaining the σind - εind curve will be discussed.
2. Role of indenter configuration
It is noteworthy that both the uni-axial tensile/compression test as well as the indentation technique are capable to assess the stress–strain characteristics of a material, however, with usually different size-scale of samples along with varying stress-states. The former provides an understanding for the degree of bond stretching induced elastic deformation and dislocation mediated plastic/permanent deformation in the material. To obtain such desired information, the strain-induced into the material should be controlled in such a way that, the material’s response reflects the gradual activation and transition from the elastic to the plastic deformation. This is realized in uni-axial deformation without any strain gradient in the specimen, at least macroscopically.
In contrary to that, upon indentation, presence multi-axial state of stress exists beneath the indenter tip. Moreover, the constraint nature of deformation induces strain gradient within the deformation volume. Hence, for assessing the elastic–plastic activity within the deformation zone, the indentation tests need to be specially designed to produce a smooth strain distribution (or gradient) along with its gradual increment. To maintain that, indenter tip geometry needs to be carefully chosen to reflect the σind - εind characteristics from the localized region. In this regard, the most suitable indenter configuration is spherical tip (or sphero-conical indenter).
Before getting into the details about the configuration of the spherical indenter tip and its importance for σind - εind generation, the reader needs to develop a comprehensive idea about the different type of indenter tips that are used in general. For the same, the geometrical aspect of indenter configuration is briefed here. From a geometrical point of view, indenters are classified into two: (i) geometrically similar indenters (GSI) and (ii) non-geometrically similar indenter (N-GSI) [4].
The most commonly used sharp pyramidal indenter such as four-sided Vickers (for micro-and macro-indentation) and three-sided Berkovich indenters (for nanoindentation) comes under the category of GSI. On the other hand, the spherical indenter falls under the category of N-GSI. The major difference in deformation characteristics experienced by a specimen surface, by indenting with any of these two categories of indenter tips can better be appreciated from Figure 1. Schematic representations in Figure 1(a-c) show the deformation modes activated in traditional elastic–plastic material while increasing the indentation load/depth, using GSI. The mathematical relation for geometrical similarity originates from the ratio of the contact radius (ac) to the maximum depth of indentation (hmax). For GSI, ac-i/hmax-i = ac-j/hmax-j = ac-k/hmax-k = constant. The subscript, i, j, k signify increasing level of h. Nevertheless, this constant ratio of ac/hmax ensures that the size of the deformation zone of indentation varies uniformly irrespective of the depth of penetration. This helps to estimate the property of the subjected material independent of the applied indentation load/depth. Nevertheless, owing to the sharp nature of the Vickers and Berkovich indenter, the strain-induced within the indentation volume is large enough to generate significant plastic deformation [1]. In that case, dislocation activity is always the dominant mechanism within the deformation volume beneath the indenter tip, irrespective of the change in depth of indentation, as apparent from Figure 1. This assists to precisely measure the hardness of a material independent of the indentation load, in the theoretical sense. However, it is realized that GSI is not adequate to assess the elastic deformation response of the indented material. In fact, while using conventional Vickers and Berkovich indenters, occurrence of prominent dislocation activity within the deformation volume negates any influence of elastic activity therein. This acts as the limitation of the most commonly using Vickers and Berkovich indenter for generating the stress–strain curve.
Figure 1.
Schematic illustration of the indentation behavior associated with traditional elastic-plastic metallic using (a-c) sharp geometrically similar indenter and (d-e) spherical non-geometrically similar indenter at various indentation depths.
On the other hand, a completely different deformation response is being experienced, while the specimen surface is indented using N-GSI (spherical tip) with increasing indentation load/depth. Figure 1(d-f), illustrate the deformation scenario within the indentation volume, in such case. It is evident from the figure that, nature of deformation is entirely different in comparison to that for GSI. This difference originates from the non-geometrical similarity of the indenter. In case, the specimen surface is indented with a N-GSI, ac-i/hmax-i < ac-j/hmax-j < ac-k/hmax-k. This essentially means with the progress of the indentation, increment in the contact radius becomes more pronounced with respect to the depth of penetration. Such movement of indenter within the material surface gradually increases the induced strain/stress into the material. Also, the blunt nature of the indenter assists in generating a smooth stress field within the indentation volume, specifically as compared to GSI. As a net effect, spherical indenter facilitates a gradual activation of elastic to the plastic deformation mechanism. This potential for gradual instigation of the deformation mechanism similar to that observed in case of uni-axial test, is exploited for σind - εind generation from nanoindentation.
Nevertheless, the most crucial part in this regard is the data analysis procedure that is necessary to convert the indentation P-h response into a reliable σind - εind curve. There have been numerous attempts to obtain a stress–strain curve from traditional indentation as well as instrumented one. In the process, the protocol for generation of indentation stress–strain curve has undergone various alterations, to precisely correlate the materials’ property. In the next section, we have briefed the different approaches adopted to appreciate the σind - εind behavior of a material. This will help to understand the scientific developments that has been materialized on this particular topic, so far.
3. Evolution of σind - εind generation protocols
The concept for the generation of σind - εind curve from indentation is introduced by Tabor in the 1950s. Tabor has measured the MCP on the specimen indented with a spherical tip to estimate the stress that is induced in the process [1]. The most crucial part, however, is the estimation of εind. Tabor defined εind by the relation (d/D), where d is the diameter of the residual impression and D is the diameter of the indenter tip. Here d is measured using the traditional approach, i.e., by imaging of residual impression after unloading. The general trend of σind - εind characteristics of materials, generated following Tabor’s protocol, resembles well with that evaluated through traditional uniaxial compression test [1]. However, this method of analysis accounts for only single σind - εind data from an indentation. So, it means that several indentation tests with different indentation parameters are necessary to be pursued, to obtain a continuous σind - εind curve for a material, making the process cumbersome.
Nevertheless, Tabor’s approach revealed the potential of the indentation technique and instigated more studies to develop a state-of-the-art protocol for generating σind - εind curve of a material. In this regard, automation through the instrumented indentation has opened up enormous possibilities to generate the σind - εind curve using a single indentation. In turn, the localized deformation behavior of a material can be precisely obtained. First among all is the Field and Swan approach [19]. They have proposed to incorporate multiple partial unload segments during each indentation. Here, the P-h responses obtained for each particular segments are used to measure the corresponding σind and εind values. The strain, on the other hand, is estimated using the relation a/Ri, where Ri represents the radius of the indenter tip. As per Field and Swan approach, the deformation associated in each unloading segment is assumed to be purely elastic. Correspondingly, the classical Hertzian elastic relationship (explained in the next section by Eq. (1)) is applied on those P-h responses to assess the contact radius, a. From the measured a value, contact area (Ac) is estimated instead of residual impression-based analysis in Tabor’s protocol.
The Field and Swan approach has much significance in the present scenario, owing to its implementation of the Hertzian contact mechanics theory. Nevertheless, interpretation of indentation strain as per both Tabor’s as well as Field and Swan approaches has been questioned for its integrity with the fundamental concept of strain. In general, strain is defined as the ratio of change in length to the initial length in a region of deformation considered. However, this fundamental relationship is not met in both these above-mentioned approaches.
In order to overcome this fundamental lacking, various studies have been conducted to formulate an adequate relationship for the εind. Among those attempts, the protocol developed by Kalidindi and Pathak has succeeded in defining εind as per the most basic concept of strain [16]. The present chapter is extensively covering the formulation and implementation of Kalidindi and Pathak protocol for the generation of σind - εind curve for a material subjected to nanoindentation. This protocol is essentially formulated based on classical Hertzian theory, which is explained below.
4. Contact mechanics for spherical tip-based indentation
Contact mechanics theory introduced by Hertz has provided a fundamental basis for the indentation technique [20]. Classical Hertzian theory predicts the elastic responses of frictionless contact between two different bodies of dissimilar geometries (with varying properties) in contact. This theory is formulated based on the assumption that material is homogenous and isotropic. In the present scenario of indentation using spherical indenter, the Hertzian theory for elastic contact between the sphere (indenter) and elastic half-space (specimen surface) is used for the formulation of σind - εind generation. In the indentation aspect, the material of interest is considered as an elastic half-space by following the criteria that indenter tip radius (Ri) should be at least ten times smaller than the horizontal dimensions of the sample [21].
As explained in previous Section 2 (see Figure 1(d-f)), indentation using spherical indenter tip facilitates the gradual activation of elastic to plastic mechanisms in the material. Therefore, for the sake of understanding, the overall deformation scenario can be categorized into (i) fully elastic and (ii) plastic following the initial elastic section. The schematic representation of these two modes of deformation and their corresponding P-h response is showed in Figure 2. In the first case, material recovers all the depth it penetrated upon the indentation (see Figure 2(a) and (c)). In the second case, some amount of permanent deformation is existing within the indentation volume (see Figure 2(b) and (d)). Hertz has provided the basis for the elastic deformation associated in two former cases using the relation below,
Figure 2.
Schematic representation of indentation of behavior of material in (a) fully elastic condition and in the pressure of (b) plastic deformation. Corresponding indentation load vs. indentation depth responses of materials are shown in (c) and (d).
P=43EeffReff12hr32E1
1Eeff=1−νs2Es+1−νi2Ei,1Reff=1Ri−1RsE2
Here P is the applied load, hr is the recoverable depth, Reff is the effective tip radius and Eeff is the effective elastic moduli. All the characteristic terms mentioned here can be appreciated from Figure 2(b). The terms hmax and hp in Figure 2 represent the maximum depth of indentation at Pmax and recurring plastic depth of indentation post-unloading (P is zero), respectively. In the Hertzian relation, the role of elastic deformation on the two mating parts is assessed using Eeff. The value of Eeff accommodates the elastic deformation associated with the hard indenter and soft sample. Eeff during the indentation is estimated using the relation (2). Similarly, Reff takes into account the influence of plastic activity on the overall deformation. It is related to the indenter tip radius (Ri) and the radius of curvature of the sample (Rs) upon the indentation. Reff of the sample is estimated using the relation (2).
All these relations derived by Hertz has laid the foundation for the formulation of σind - εind data from the nanoindentation P-h response. This is explained in details in the following section.
5. Defining the indentation stress and indentation strain
It is well understood from Section 3 that Tabor’s and Field and Swan’s protocols do not suffice to define the εind precisely. Nevertheless, Kalidindi and Pathak have defined the σind and εind by considering the size of the deformation zone formed beneath the indenter and correlated it with the fundamental Hertzian relationship [16]. This protocol has succeeded in producing comprehensive σind - εind data from the nanoindentation experiments (explained in Section 6).
As per this novel approach, eq. (1) is rearranged by incorporating the following relations:
σind=Pπa2;σind=Eeffεind;εind=43πhra≈hr2.4aE3
a=ReffhrE4
The indentation strain defined using the above relationship satisfies the general definition of strain. This can be better appreciated from Figure 3(a). In the figure, the dashed spherical shaped region beneath the indenter tip schematically shows the actual size of the deformation zone upon indentation. Based on the εind defined from Hertzian relation, the length of the deformation zone beneath the indenter tip at Pmax is noted to be ∼2.4a. Interestingly, a simulative study on the prediction of indentation behavior strongly agrees with this relation for tungsten and aluminium [16]. This has validated the new definition of εind, which is derived without any alteration of the fundamental Hertzian relation. This novel protocol is remarkably different yet comprehensive with respect to the other discussed approaches. This is primarily because it basically takes into account the actual size of the deformation zone during the indentation, rather than simply estimating the εind data using the concept of variation in indent impression.
Figure 3.
(a) Schematic representation of the deformation behaviour associated with indentation. Figure highlights the actual deformation zone of indentation and the idealized deformation zone of indentation. (b) Schematic representation of surface irregularities on a sample.
Furthermore, this novel protocol has provided a reasonable basis for the analogical comparison of indentation behavior using spherical indenter and uniaxial compression test. The overall nature of the material response upon nanoindentation can be considered as the replication of compressing up to a depth of hmax on a cylindrical sample of height 2.4a and radius a. To visualize it clearly, the idealized deformation zone of indentation and actual deformation of indentation is schematically shown in Figure 3(a). The shape of the actual deformation zone formed is schematically showed as spherical. The reader should be aware that, in reality, owing to the anisotropy in material’s properties, the actual shape of the deformation zone of indentation can be slightly different from this schematic representation. It is also noteworthy that with slight alternation in relation (4), hmax can be used instead of hr in the numerator to accommodate the plastic activity [15]. This whole theoretical concept has paved the way for generating σind - εind curve from the P-h signal in nanoindentation. To realize it in a practical scenario, the reader has to understand the necessary steps to follow for obtaining a reliable output.
6. Theoretical conceptualization to experimental execution
As mentioned in Section 1, nanoindentation typically generates a P-h response and its characteristics define the mechanical property of the material indented. Compared to any other characterization technique, particularly, the most commonly used uni-axial tests, the size of the active deformation region for nanoindentation is extremely small. Therefore, proper measures are necessary at every steps right from the precise sample preparation to the careful data analysis to obtain reliable data.
6.1 Sample preparation
The existence of an artefacts such as scratches or the presence of foreign particles on the surface can influence the P-h signal and thereby the generated σind - εind data. The poorly polished samples create a scratch on the surface, the depth of which can be in hundreds of nanometres. Data recorded from such a region will certainly influence the overall σind - εind characteristics and consequently alter the assessment of the true properties of the material. This can be visualized and understood from the schematic representation in Figure 3(b). In the figure, red coloured triangular shape and yellow coloured circular shape reveal the presence of sample surface roughness and foreign particles respectively. As per the indentation sequence, the indenter will first acquire the data from those artefacts and move to the bulk of the sample. So, actual material which is supposed to show the pure elastic response initially, is now influenced by the presence of sample surface artefacts. As a net effect, the P-h response from the bulk sample is influenced by the surface roughness/foreign particle. Hence, the assessed properties are certainly different from the true ones [1]. In case of conventional uniaxial tests, such misinterpretation of results can be obtained in case a specimen slips upon loading, or even when elastic properties are estimated from a tensile experiment, without attaching an extensometer to the test specimen.
To avoid such issues, well-polished, smooth, flat and plane-parallel specimen should be subjected to nanoindentation. The necessary steps to achieve such artefact free surface vary with the material of interest. However, colloidal silica polish for few hours (minimum 3 h) after the conventional polish using silicon carbide paper with decreasing mesh size and diamond polish is prescribed for metallic specimens, to attain a reasonably good surface condition for the σind - εind generation. Depending on the surface characteristics of the material, electropolishing may also appear to be a better option to minimize the artefacts on the sample surface.
6.2 Conversion of experimental P-h data to effective P-h data
It is noted that theoretical predictions and the experimental outcome may result to some disparities in case of the nanoindentation test. In this regard, it is noteworthy that proper data analysis plays a key role in the generation of σind - εind curve. It is highlighted in the previous section (Section 6.1) that nanoindentation experiments mandate extremely good quality surface finish. Nevertheless, obtaining the required surface finish is difficult in practice. A proper data correction route on the experimentally obtained P-h curve, on the other hand, can negate the role of artefacts on the σind - εind analysis. This step is crucial to compute a reliable stress–strain curve. For the same, effective initial contact point between the indenter tip and the specimen surface is estimated following the “zero-point correction” (ZPC). In fact, ZPC deals with discarding the data points which are influenced by unavoidable surface irregularities. In turn, the effective contact point is determined on the basis of Hertzian theory which reciprocates the material behavior. According to the type of nanoindentation instrument used, Kalidindi and Pathak have proposed two different approaches for the data correction using ZPC. One is for nanoindenter with (a) Contact Stiffness Mode, CSM (or Dynamic Mechanical Analysis, DMA) and another for (b) Non-Contact Stiffness Mode, N-CSM [22]. These two modes are slightly different in the method of experimentation.
6.2.1 CSM mode or DMA mode
In CSM or DMA mode, harmonic force is imposed in the loading and unloading segment during the indentation. This is highlighted at the inset (a) of Figure 4. It can be hypothetically viewed as if the specimen undergoes multiple indentations with minimal depth scale (2 to 4 nm) while conducting a single indentation. Displacement responses corresponding to these harmonic forces are recorded throughout the indentation. These assist in assessing the variation in contact stiffness, S (ordPdh) upon the indentation. Precise determination of S from each steps of CSM leads to estimate the continuous variation in the related properties of materials with increasing h, for example, hardness and elastic modulus changes [22].
Figure 4.
Indentation load vs. indentation depth response generated using nanoindentation. Insets in the figure highlight the method of indentation in CSM mode and N-CSM mode.
In the present scenario, the continuously varying S, hr, and P are obtained from the CSM mode of the nanoindenter and these signals are used for ZPC. For the same, the Hertzian relation (Eq. (1)) for elastic contact is rearranged into the following relationship,
P−23hrS=−23h∗S+P∗E5
Here P* and h* denote the effective indentation load and depth respectively. A linear regression analysis on relation (5) helps to trace the P* and h* values through the slope (−23h∗) and y-intercept (P*). Once the P* and h* are established, the experimentally generated P-h signal has to be corrected for obtaining an ‘effective P-h curve’, which is devoid of any influence from the surface artefacts [16].
6.2.2 Non-CSM mode
In N-CSM mode, indentation is performed without harmonic force. This is also highlighted in inset (b) of Figure 4. In this particular case, ZPC is performed by recasting the Hertzian equation as per the relation below (derived from Eq. (1),
hr−h∗=kP−P∗,k=341Eeff1√ReffE6
In the above relationship, the k value is constant in the elastic segment [15]. It is worth reiterating here that within the elastic segment, continuously varying h equates with hr whereas Reff to Ri (explained in the subsequent Section 6.3). Also, prior understanding of elastic moduli of the material makes the calculation much easier. Essentially, regression analysis on the initial elastic segment of experimentally obtained data helps to calculate the values of P* and h* and thereby the effective P-h data is estimated.
6.2.3 Selection of data segment
The above-mentioned data correction procedures for nanoindenter with CSM or N-CSM mode, ideally has to be performed on the initial elastic segment of the P-h segment. Such elastic segment dwells within few nanometers, in reality. The exact value of this elastic segment however varies with the sharpness (or bluntness) of the indenter tip and the associated variation in strain gradient [2]. The question here is how to precisely choose a segment in the P-h curve which can be used for the data correction using Eqs. (5) and (6). This can be realized through the iteration process on the initial segment with a different depth limit. For instance, for nanoindentation with hmax of 250 nm, regression analysis has to be performed in initial segments with indentation depth of 10 nm (or any other limit) to higher. By doing so, the accurate point of transition from elastic to plastic (data limit) can be approximately finalized based on the continuity nature observed in the effective P-h curve as well as the corresponding σind - εind curve (explained in next Section 6.3).
6.3 Conversion of effective P-h curve to σind - εind curve
As explained in Section 5, the Hertzian relation has provided a basis to obtain σind - εind curve from the P-h response. Once the effective P-h response is computed using the steps mentioned in Section 6.2, Eqs. (3) and (4) are used for obtaining the corresponding σind - εind values. In this conversion process, estimating the continuously varying a is important for calculating the continuous evolution in the σind and εind values. It is particularly evident from Eq. (4) that, a is the main characterizing parameter to obtain the σind and εind values.
Prior to going through further details, the physical significance of a and the mechanisms behind its alteration during indentation are explained through Figure 5. The figure schematically shows the indentation behavior of different materials with different extents of elastic–plastic activities. Sample-1 with green color indicates the material with full elastic recovery. Sample-2 (orange) and sample-3 (blue) exhibit the indentation behavior of two materials with different degrees of plastic activities along with elastic deformation. In a fully elastic material (sample-1), the indented surface recovers the whole depth upon the complete removal of load. Thereby Rs attains infinity in this case (see Figure 5). So, Reff = Ri for material with full depth recovery (see equation (2)). Similarly, owing to the full recovery, continuously recording h signal can equate with the depth recovery (hr). In short, Reff = Ri and h = hr within the elastic regime of material upon indentation.
Figure 5.
Schematic representation of the nature of deformation volume beneath the indenter tip for materials with three different degree of elastic-plastic property. Green line shows the sample with full depth recovery. The orange and light blue colors reveal the indentation behaviour of samples with different shares of elastic and plastic activities.
But, once the dislocation mediated plastic activity is instigated, Rs attains a finite value. The orange and light blue colors in Figure 5 reveal the formation of finite values of Rs in the materials due to the occurrence of plastic deformation. In these two cases, Reff is no longer equal to Ri. It is reported that Reff is significantly larger than Ri once plastic deformation initiates in the material. Almost a 100-fold increment in the Reff is reported with presence of plastic activity in aluminum sample [3].
All these physical changes are also related to hr after the unloading. While comparing sample-2 and sample-3, depth recovery is noted to be higher for the former. Correspondingly, Rs in the material also changes. It is apparent from the Figure 5 that Rs-1 > Rs-2 and hr-1 > hr-2. As a net effect of change in Reff and hr, contact between the indenter and sample deflects. This is reciprocated in the changes in a (a1 > a2). In conclusion, all three parameters are correlated which are primarily controlled by the share of elastic–plastic activities within the material of interest. Eq. (4) derived by Hertz relates all these physical phenomena and in the present scenario, it is utilized to estimate σind-εind curve using relation (3).
6.3.1 σind-εindfromCSM nanoindenter
Estimation of a from nanoindentation using CSM mode is straight forward. The interrelation between S and a are derived from Eqs. (1) and (4) according to the Hertzian theory, as shown below:
dPdh=2EeffReff1/2he1/2=2Eeffaa=S2EeffE7
The analytical significance of this mathematical derivation lies in the fact that unlike Tabor’s approach, this expression (eq. (7)) enables to assess the nature of deformation inside the material without a visual inspection of residual impression.
In the data analysis, once the evolving values of a are established using eq. (7), the final σind - εind curve is generated from Eq. (3). Figure 6(a) shows σind - εind curves obtained before and after ZPC on experimental P-h data. Interestingly, in this novel protocol, elastic moduli measured from the loading and unloading segments of the σind - εind curve are noted to resemble each other [16]. This observation has validated the new definition for εind as well as the novel protocol for reliably assessing the mechanical property via σind - εind curve.
Figure 6.
(a) σind−εind curve obtained before and after the zero-point correction. (b) Schematic representation of the P-h responses with multiple unloading segments for generating σind−εind curve in N-CSM measurement nanoindenter.
6.3.2 σind-εindfrom non-CSM nanoindenter
As compared to the CSM mode, experimentation and method of analysis is different in case of N-CSM mode of nanoindentation. In N-CSM mode, multiple unloading segments are introduced into the indentation test for measuring the evolution in a and thereby the continuous variation in σind and εind values. This is similar to Field and Swan approach in terms of experimentation. Figure 6(b) schematically shows the P-h curve obtained after the multiple unloading. Once the effective P-h curve is generated by employing ZPC, following Section 6.2, the evolving values of a are estimated from each segment. For the same, Reff value is estimated by fitting the unloading response using the modified Hertz relation as mentioned below,
hr=hmax−hp=kP2/3E8
here k is a function of Reff and Eeff (see relation (6)). Eeff can be traced from the prior understanding of elastic moduli of sample or from the initial elastic segment in the P-h curve [15]. So, from the understanding of k value of the respective alloys and the recorded value of hp with reduction in indentation load in the unloading segments, Reff is estimated by fitting using the relation (8). Once Reff is established, a can be determined from relation (4) and in turn σind - εind curve can be generated using Eq. (3). It is also important to note here that, number of data points in the resultant σind - εind curve depends on the number of unloading segments provided in the experiment.
7. Protocol for σind - εind generation in pseudoelastic shape memory alloys
Previous sections have elaborated the potential of the nanoindentation technique in appreciating the σind - εind characteristics of traditional elastic–plastic metallic systems. In a further extension, Sujith and Sen have revealed the capability of nanoindentation in assessing the unique pseudoelastic (or superelastic) properties of shape memory alloys (SMA) via σind - εind curve [2, 6]. This recent development has succeeded in the producing the specialized stress - strain characteristics of the pseudoelastic NiTi system using most commonly used N-CSM nanoindenter.
It is noteworthy at this point that as compared to the traditional elastic–plastic metallic alloys, pseudoelastic system is different owing to the occurrence of reversible stress-induced martensitic transformation (SIMT). In pseudoelastic alloys (some examples of metallic systems are NiTi, Cu-Al-Zn, Cu-Al-Ni, Ni-Ti-Fe, Fe-Mn-Si, Fe-Mn-Si-Co-Ni), parent austenitic phase transforms to product martensitic phase upon the application of stress and it reverts to the previous austenite with the release of stress. Owing to this reversible SIMT along with usual elastic deformation in the parent and product phase, the NiTi system in pseudoelastic state shows (8–10) % of recoverable strain. This is also reflected as a unique characteristic in the conventional uni-axial stress - strain curve. Hence, evaluating such unique property using nanoindentation requires special attention in terms of (a) optimizing indentation parameters as well as (b) tailored σind - εind generation protocol. This investigation by Sujith and Sen is the first of its kind to consider spherical indenter tips with varying Ri as well as Pmax levels with the aim to identify the optimum combination to precisely evaluate localized pseudoelasticity in SMA through nanoindentation. Following steps are briefed:
7.1 Optimizing indentation parameters
For optimizing the indentation parameters, a detailed analysis is performed on the P-h curve obtained from various indenter configuration (Ri of 10 μm, 20 μm and 50 μm) as well as Pmax (1 mN to 7 mN). Details of the experiments and analysis procedures are reported elsewhere [2]. However, the key observations in this method of analysis are mentioned here.
Optimization of indentation parameters is performed based on the close scrutiny of the experimentally generated P-h curve using Hertzian theoretical prediction and the understanding of the pseudoelastic behavior in the alloy system. Figure 7(a) shows the method of analysis performed on the P-h curve. The black solid and the red dashed curves in Figure 7(a) show the experimental results and Hertzian theoretical predication of indentation response, respectively. Using this comparison, overall deformation mode in the indentation is parted into different sections. Correspondingly, the depth of indentation, specifically influenced by pseudoelasticity is assessed. This can be even better appreciated from Figure 7(b). Physical variation associated with indentation volume of NiTi sample using Ri of 10 μm and 20 μm are schematically (in two halves) shown in Figure 7(b). The region influenced by reversible SIMT is highlighted as green color in the schematics. This novel method of analysis is performed using a range of combination of indentation parameter. The most adequate combination to assess the pseudoelasticity is identified based on the share of reversible SIMT activity and the overall depth recoverability (minimum 90% depth recovery). Based on this systematic analysis, spherical indenter with Ri of 20 μm and Pmax of 5 mN is noted to be most suitable combination for appreciating pseudoelasticity devoid of the influence of dominant plasticity, in NiTi system.
Figure 7.
(a) The P-h response of pseudoelastic NiTi system at its optimized nanoindentation parameter condition (Ri=20μm and Pmax=5mN). Red dotted curve shows the theoretical prediction of P-h response and the green dotted region infers the region that is dominantly influenced by reversible SIMT in the NiTi alloy. Ptr in the graph highlights the indentation load at which SIMT initiates in the material. (b) Schematic representation of share of different deformation mechanisms within the nanoindentation volume for pseudoelastic NiTi system indented using Ri of 10 μm and 20 μm. (c) σind−εind curve corresponding to the P-h response (Figure 7(a)). Here, σtr is the transformation stress to initiate SIMT [2].
7.2 σind - εind protocol
Considering the extremely high depth recoverability (≥ 90%) of pseudoelastic NiTi system, following assumption is used while generating the corresponding σind - εind curves,
Reff=Riandhr=hE9
Section 6.3 has already mentioned about the validity of this assumption when material shows full depth recovery. In the present scenario, same assumption is used with depth recovery limit of 90% of the hmax. This assists in converting the P-h response into σind - εind curve using Eq. (3), while employing relations (9) in it. Essentially, this new protocol defined the εind and a using following relation,
εind≈h2.4aa=RihE10
Prior to conversation of P-h results into σind - εind curve, ZPC is performed following Section 6.2.2. Figure 7(c) shows the σind - εind curve that is generated from the P-h response of NiTi system. Interestingly the curve has shown the signature trends of pseudoelastic system like sudden changes in the transformation strength, plateau strain, significant recovery etc. The transformation strength (σtr) of the NiTi system estimated from the nanoindentation resembles reasonably well with that derived from uni-axial compression test [2]. This has validated the present protocol for future analysis on smart characteristics of NiTi based shape memory alloys.
8. Closure
The present chapter elucidates the vast potential of nanoindentation technique to develop insights about the localized stress–strain characteristics of materials. Nevertheless, to achieve the σind - εind curve, experiments need to be carefully designed. Also, post indention analysis should be meticulously performed to obtain the reliable data. Blunt spherical indenter tip is primarily necessary to activate elastic and plastic mechanisms sequentially in the material and thereby to estimate the σind - εind curve. On the other hand, different post-indentation analysis has to be adopted based on the mode of nanoindenter and the material of interest to compute the indentation stress–strain data. Validation of the protocols are also discussed for pseudoelastic material systems. The detailed explanation provided in the present chapter based on the physical mechanism associated with different alloy system upon indentation and further data analysis can pave the way for future usage of this method of analysis in various studies.
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The suitability of continuous stiffness measurement (CSM) based nanoindenter systems along with the adaptability of the instrument without CSM for precisely evaluating the deformation behavior of specialized materials is discussed in details. In this regard, the roll of indenter tip geometry and size is greatly emphasized. The recent research in the field is reviewed thoroughly and the updated protocol generated is illustrated.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/77158",risUrl:"/chapter/ris/77158",signatures:"Indrani Sen and S. Sujith Kumar",book:{id:"10943",type:"book",title:"Elasticity of Materials",subtitle:null,fullTitle:"Elasticity of Materials",slug:null,publishedDate:null,bookSignature:"Associate Prof. Gülşen Akın Evingür and Dr. Önder Pekcan",coverURL:"https://cdn.intechopen.com/books/images_new/10943.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-83969-961-0",printIsbn:"978-1-83969-960-3",pdfIsbn:"978-1-83969-962-7",isAvailableForWebshopOrdering:!0,editors:[{id:"180256",title:"Associate Prof.",name:"Gülşen",middleName:null,surname:"Akın Evingür",slug:"gulsen-akin-evingur",fullName:"Gülşen Akın Evingür"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Role of indenter configuration",level:"1"},{id:"sec_3",title:"3. Evolution of σind - εind generation protocols",level:"1"},{id:"sec_4",title:"4. Contact mechanics for spherical tip-based indentation",level:"1"},{id:"sec_5",title:"5. Defining the indentation stress and indentation strain",level:"1"},{id:"sec_6",title:"6. Theoretical conceptualization to experimental execution",level:"1"},{id:"sec_6_2",title:"6.1 Sample preparation",level:"2"},{id:"sec_7_2",title:"6.2 Conversion of experimental P-h data to effective P-h data",level:"2"},{id:"sec_7_3",title:"6.2.1 CSM mode or DMA mode",level:"3"},{id:"sec_8_3",title:"6.2.2 Non-CSM mode",level:"3"},{id:"sec_9_3",title:"6.2.3 Selection of data segment",level:"3"},{id:"sec_11_2",title:"6.3 Conversion of effective P-h curve to σind - εind curve",level:"2"},{id:"sec_11_3",title:"6.3.1 σind-εind from CSM nanoindenter",level:"3"},{id:"sec_12_3",title:"6.3.2 σind-εind from non-CSM nanoindenter",level:"3"},{id:"sec_15",title:"7. Protocol for σind - εind generation in pseudoelastic shape memory alloys",level:"1"},{id:"sec_15_2",title:"7.1 Optimizing indentation parameters",level:"2"},{id:"sec_16_2",title:"7.2 σind - εind protocol",level:"2"},{id:"sec_18",title:"8. Closure",level:"1"}],chapterReferences:[{id:"B1",body:'D. Tabor, The Hardness of Metals, Oxford University Press, 1951.'},{id:"B2",body:'S. Kumar S, I.A. Kumar, L. Marandi, I. Sen, Assessment of small-scale deformation characteristics and stress-strain behavior of NiTi based shape memory alloy using nanoindentation, Acta Mater. 16375 (2020) 1–2. https://doi.org/10.1016/j.actamat.2020.09.080.'},{id:"B3",body:'S. Pathak, S.R. Kalidindi, Spherical nanoindentation stress-strain curves, Mater. Sci. Eng. R Reports. 91 (2015) 1–36. https://doi.org/10.1016/j.mser.2015.02.001.'},{id:"B4",body:'Antony C. fisher Cripps, Nanoindentation, Springer International Publishing, 2011.'},{id:"B5",body:'G.M. Pharr, An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments, J. Mater. Res. 7 (1992) 1564–1583. https://doi.org/10.1557/JMR.1992.1564.'},{id:"B6",body:'S. Kumar S, L. Marandi, V.K. Balla, S. Bysakh, D. Piorunek, G. Eggeler, M. Das, I. Sen, Microstructure – Property correlations for additively manufactured NiTi based shape memory alloys, Materialia. 8 (2019) 100456. https://doi.org/10.1016/j.mtla.2019.100456.'},{id:"B7",body:'J. Hu, W. Zhang, G. Peng, T. Zhang, Y. Zhang, Nanoindentation deformation of refine-grained AZ31 magnesium alloy: Indentation size effect, pop-in effect and creep behavior, Mater. Sci. Eng. A. 725 (2018) 522–529. https://doi.org/10.1016/j.msea.2018.03.104.'},{id:"B8",body:'I.C. Choi, B.G. Yoo, Y.J. Kim, J. Il Jang, Indentation creep revisited, J. Mater. Res. 27 (2012) 3–11. https://doi.org/10.1557/jmr.2011.213.'},{id:"B9",body:'S. Graça, R. Colaço, P.A. Carvalho, R. Vilar, Determination of dislocation density from hardness measurements in metals, Mater. Lett. 62 (2008) 3812–3814. https://doi.org/10.1016/j.matlet.2008.04.072.'},{id:"B10",body:'C.P. Frick, T.W. Lang, K. Spark, K. Gall, Stress-induced martensitic transformations and shape memory at nanometer scales, Acta Mater. 54 (2006) 2223–2234. https://doi.org/10.1016/j.actamat.2006.01.030.'},{id:"B11",body:'S. Sujith Kumar, I. Sen, A Comparative Study on Deformation Behaviour of Superelastic NiTi with Traditional Elastic–Plastic Alloys in Sub-micron Scale, Trans. Indian Inst. Met. (2021). https://doi.org/10.1007/s12666-021-02207-8.'},{id:"B12",body:'K. Jacob, D. Yadav, S. Dixit, A. Hohenwarter, B.N. Jaya, High pressure torsion processing of maraging steel 250: Microstructure and mechanical behaviour evolution, Mater. Sci. Eng. A. 802 (2021) 140665. https://doi.org/10.1016/j.msea.2020.140665.'},{id:"B13",body:'W.C. Oliver, G.M. Pharr, Measurement of hardness and elastic modulus by instrumented indentation: Advances in understanding and refinements to methodology, J. Mater. Res. 19 (2004) 3–20. https://doi.org/10.1557/jmr.2004.19.1.3.'},{id:"B14",body:'S. Pathak, D. Stojakovic, S.R. Kalidindi, Measurement of the local mechanical properties in polycrystalline samples using spherical nanoindentation and orientation imaging microscopy, Acta Mater. 57 (2009) 3020–3028. https://doi.org/10.1016/j.actamat.2009.03.008.'},{id:"B15",body:'S. Pathak, J. Shaffer, S.R. Kalidindi, Determination of an effective zero-point and extraction of indentation stress-strain curves without the continuous stiffness measurement signal, Scr. Mater. 60 (2009) 439–442. https://doi.org/10.1016/j.scriptamat.2008.11.028.'},{id:"B16",body:'S.R. Kalidindi, S. Pathak, Determination of the effective zero-point and the extraction of spherical nanoindentation stress-strain curves, Acta Mater. 56 (2008) 3523–3532. https://doi.org/10.1016/j.actamat.2008.03.036.'},{id:"B17",body:'N.G. Mathews, A.K. Saxena, C. Kirchlechner, G. Dehm, B.N. Jaya, Effect of size and domain orientation on strength of Barium Titanate, Scr. Mater. 182 (2020) 68–73. https://doi.org/10.1016/j.scriptamat.2020.02.039.'},{id:"B18",body:'G. Dehm, B.N. Jaya, R. Raghavan, C. Kirchlechner, Overview on micro- and nanomechanical testing: New insights in interface plasticity and fracture at small length scales, Acta Mater. 142 (2018) 248–282. https://doi.org/10.1016/j.actamat.2017.06.019.'},{id:"B19",body:'J.S. Field, M. V. Swain, A simple predictivity model for spherical indentation, J. Mater. Res. 8 (1993) 297–306. https://doi.org/10.1557/JMR.1993.0297.'},{id:"B20",body:'S.K. Kang, Y.C. Kim, Y.H. Lee, J.Y. Kim, D. Kwon, Hertz elastic contact in spherical nanoindentation considering infinitesimal deformation of indenter, Tech. Proc. 2012 NSTI Nanotechnol. Conf. Expo, NSTI-Nanotech 2012. 1 (2012) 132–135.'},{id:"B21",body:'B.C. Maji, M. Krishnan, The effect of microstructure on the shape recovery of a Fe-Mn-Si-Cr-Ni stainless steel shape memory alloy, Scr. Mater. 48 (2003) 71–77. https://doi.org/10.1016/S1359-6462(02)00348-2.'},{id:"B22",body:'X. Li, B. Bhushan, A review of nanoindentation continuous stiffness measurement technique and its applications, Mater. Charact. 48 (2002) 11–36. https://doi.org/10.1016/S1044-5803(02)00192-4.'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Indrani Sen",address:"indrani.sen@metal.iitkgp.ac.in",affiliation:'
Department of Metallurgical and Materials Engineering, Indian Institute of Technology, India
Department of Metallurgical and Materials Engineering, Indian Institute of Technology, India
Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology, India
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\r\n\tWater is not only a crucial substance needed for biological life on Earth, but it is also a basic requirement for the existence and development of the human society. Owing to the importance of water to life on Earth, early researchers conducted numerous studies and analyses on the liquid form of water from the perspectives of chemistry, physics, earth science, and biology, and concluded that Earth is a "water polo". Water covers approximately 71% of Earth's surface. However, 97.2% of this water is seawater, 21.5% is icebergs and glaciers, and only 0.65% is freshwater that can be used directly by humans. As a result, the amount of water reserves available for human consumption is limited. The development, utilization, and protection of freshwater resources has become the focus of water science research for the continued improvement of human livelihoods and society.
\r\n
\r\n\tWater exists as solid, liquid, and gas within Earth’s atmosphere, lithosphere, and biosphere. Liquid water is used for a variety of purposes besides drinking, including power generation, ecology, landscaping, and shipping. Because water is involved in various environmental hydrological processes as well as numerous aspects of the economy and human society, the study of various phenomena in the hydrosphere, the laws governing their occurrence and development, the relationship between the hydrosphere and other spheres of Earth, and the relationship between water and social development, are all part of water science. Knowledge systems for water science are improving continuously. Water science has become a specialized field concerned with the identification of its physical, chemical, and biological properties. In addition, it reveals the laws of water distribution, movement, and circulation, and proposes methods and tools for water development, utilization, planning, management, and protection. Currently, the field of water science covers research related to topics such as hydrology, water resources and water environment. It also includes research on water related issues such as safety, engineering, economy, law, culture, information, and education.
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