Typical sensors in an android smartphone.
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Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\\n"}]',published:!0,mainMedia:{caption:"Highly Cited",originalUrl:"/media/original/117"}},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 191 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 261 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\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:"11008",leadTitle:null,fullTitle:"Sheep Farming - Herds Husbandry, Management System, Reproduction and Improvement of Animal Health",title:"Sheep Farming",subtitle:"Herds Husbandry, Management System, Reproduction and Improvement of Animal Health",reviewType:"peer-reviewed",abstract:"This book provides an overview of sheep husbandry in different parts of the world, including information on production and management systems, reproduction, and animal health. Chapters discuss different types of sheep and sheep husbandry in Poland, India, Africa, Spain, and North America, as well as zoonotic diseases such as cryptosporidiosis and their adverse impacts on the economics of sheep herding. This book is a useful resource for producers, veterinarians, animal scientists, researchers, biologists, students, and other interested readers.",isbn:"978-1-83969-711-1",printIsbn:"978-1-83969-710-4",pdfIsbn:"978-1-83969-712-8",doi:"10.5772/intechopen.95705",price:119,priceEur:129,priceUsd:155,slug:"sheep-farming-herds-husbandry-management-system-reproduction-and-improvement-of-animal-health",numberOfPages:178,isOpenForSubmission:!1,isInWos:null,isInBkci:!1,hash:"992f691327b36848b6e1137b70d921d5",bookSignature:"Manuel Gonzalez Ronquillo and Carlos Palacios Riocerezo",publishedDate:"April 28th 2022",coverURL:"https://cdn.intechopen.com/books/images_new/11008.jpg",numberOfDownloads:678,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfCrossrefCitationsByBook:0,numberOfDimensionsCitations:0,numberOfDimensionsCitationsByBook:0,hasAltmetrics:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 18th 2021",dateEndSecondStepPublish:"April 22nd 2021",dateEndThirdStepPublish:"June 21st 2021",dateEndFourthStepPublish:"September 9th 2021",dateEndFifthStepPublish:"November 8th 2021",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6",editedByType:"Edited by",kuFlag:!1,featuredMarkup:null,editors:[{id:"175967",title:"Dr.",name:"Manuel",middleName:null,surname:"Gonzalez Ronquillo",slug:"manuel-gonzalez-ronquillo",fullName:"Manuel Gonzalez Ronquillo",profilePictureURL:"https://mts.intechopen.com/storage/users/175967/images/system/175967.png",biography:"Dr. Manuel González Ronquillo obtained his doctorate degree from the University of Zaragoza, Spain, in 2001. He is a research professor at the Faculty of Veterinary Medicine and Animal Husbandry, Autonomous University of the State of Mexico. He is also a level-2 researcher. He received a Fulbright-Garcia Robles fellowship for a postdoctoral stay at the US Dairy Forage Research Center, Madison, Wisconsin, USA in 2008–2009. He received grants from Alianza del Pacifico for a stay at the University of Magallanes, Chile, in 2014, and from Consejo Nacional de Ciencia y Tecnología (CONACyT) to work in the Food and Agriculture Organization’s Animal Production and Health Division (AGA), Rome, Italy, in 2014–2015. He has collaborated with researchers from different countries and published ninety-eight journal articles. He teaches various degree courses in zootechnics, sheep production, and agricultural sciences and natural resources.\n\nDr. Ronquillo’s research focuses on the evaluation of sustainable animal diets (StAnD), using native resources of the region, decreasing carbon footprint, and applying meta-analysis and mathematical models for a better understanding of animal production.",institutionString:null,position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"Universidad Autónoma del Estado de México",institutionURL:null,country:{name:"Mexico"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:{id:"281920",title:"Prof.",name:"Carlos",middleName:null,surname:"Palacios Riocerezo",slug:"carlos-palacios-riocerezo",fullName:"Carlos Palacios Riocerezo",profilePictureURL:"https://mts.intechopen.com/storage/users/281920/images/system/281920.jpg",biography:"Dr. Carlos Palacios Riocerezo obtained a veterinary degree from Veterinarian by the University of León, Spain, in 1991. Since then, he has worked as a veterinarian and veterinary technician specializing in dairy sheep breeding, reproduction, artificial insemination, animal nutrition services, and sheep farm management. Dr. Palacios obtained his Ph.D. in Sheep Health and Management from the University of León in 2010. He is a Full Professor of Animal Production at the Faculty of Agricultural and Environmental Sciences, University of Salamanca, Spain, where he teaches degree courses in Agricultural Engineering.",institutionString:"University of Salamanca",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Salamanca",institutionURL:null,country:{name:"Spain"}}},coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"297",title:"Animal Science",slug:"animal-science"}],chapters:[{id:"79396",title:"Magallanes Sheep Farming",doi:"10.5772/intechopen.100497",slug:"magallanes-sheep-farming",totalDownloads:85,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The Magallanes region in Chilean Patagonia encompasses 13 million hectares with approximately 3.6 million used for agricultural and livestock systems. This portion is located to the east of the Andean Mountain chain in the rain shadow zone, with annual precipitation increasing along an east to west gradient from 200 to almost 1,000 mm. To fully describe sheep farming in the Magallanes region, many topics need to be addressed, including sheep production and management, existing vegetative communities, livestock-wildlife interactions, and economic diversification into agritourism and another sheep industry products. All these give shape to the story of the development of sheep farming in Magallanes, which is important at the regional and national level. Three key points are identified that together can lead to a successful future for the industry: sustainable management, human resources and the market.",signatures:"Sergio Radic-Schilling, Francisco Sales, Raúl Lira, René Muñoz-Arriagada, Paulo Corti, Nilo Covacevich, Jorge Ivelic-Sáez, Iván Ordoñez, Osvaldo Vidal, Ricardo Echeverría and Camila Sandoval",downloadPdfUrl:"/chapter/pdf-download/79396",previewPdfUrl:"/chapter/pdf-preview/79396",authors:[{id:"270986",title:"Ph.D.",name:"Sergio",surname:"Radic",slug:"sergio-radic",fullName:"Sergio Radic"},{id:"428989",title:"Dr.",name:"Francisco",surname:"Sales",slug:"francisco-sales",fullName:"Francisco Sales"},{id:"428990",title:"MSc.",name:"Raúl",surname:"Lira",slug:"raul-lira",fullName:"Raúl Lira"},{id:"428991",title:"MSc.",name:"René",surname:"Muñoz-Arriagada",slug:"rene-munoz-arriagada",fullName:"René Muñoz-Arriagada"},{id:"428992",title:"Dr.",name:"Paulo",surname:"Corti",slug:"paulo-corti",fullName:"Paulo Corti"},{id:"428993",title:"Dr.",name:"Nilo",surname:"Covacevich",slug:"nilo-covacevich",fullName:"Nilo Covacevich"},{id:"428994",title:"MSc.",name:"Jorge",surname:"Ivelic-Sáez",slug:"jorge-ivelic-saez",fullName:"Jorge Ivelic-Sáez"},{id:"428995",title:"Dr.",name:"Iván",surname:"Ordoñez",slug:"ivan-ordonez",fullName:"Iván Ordoñez"},{id:"428996",title:"Dr.",name:"Osvaldo",surname:"Vidal",slug:"osvaldo-vidal",fullName:"Osvaldo Vidal"},{id:"428997",title:"Mr.",name:"Ricardo",surname:"Echeverría",slug:"ricardo-echeverria",fullName:"Ricardo Echeverría"},{id:"428998",title:"Dr.",name:"Camila",surname:"Sandoval",slug:"camila-sandoval",fullName:"Camila Sandoval"}],corrections:null},{id:"80263",title:"Retrospective Study of Production and Commercialization of Sheep Wool from Mexico",doi:"10.5772/intechopen.101970",slug:"retrospective-study-of-production-and-commercialization-of-sheep-wool-from-mexico",totalDownloads:81,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The purpose of this chapter is to provide information on wool production from Mexico in a period from 1980 to 2019, addressing some of the problems faced by the wool market over time. An analysis of variables such as national production of wool, cost of kg of wool, import and export of wool was performed with the aim of having a complete picture of the situation in Mexico. Also, the production of Mexico was compared with that of other countries that occupy the first places in wool production and quality, to have a starting point and propose improvement scenarios for the production of Mexican wool production. Overall, wool production in Mexico cannot cover the national demand, having to resort to the import of this product. The use of native resources of the region, such as the “Chiapas sheep breed” allows the development and maintenance of traditional ancestral culture, such as the Tzotzil, and the manufacture of handicrafts typical of each of the regions of Mexico. However, the management of long-term programs through the inclusion of dual-purpose breeds, wool, and meat can be a viable alternative for the development of the wool industry in Mexico.",signatures:"Lizbeth E. Robles-Jimenez, Paola Alejandra Fernández Estrada, Jorge Osorio Avalos, Raul Perezgrovas, Oscar Chavez-Rivera, Einar Vargas-Bello-Pérez, Carlos Palacios Riocerezo and Manuel Gonzalez-Ronquillo",downloadPdfUrl:"/chapter/pdf-download/80263",previewPdfUrl:"/chapter/pdf-preview/80263",authors:[{id:"175967",title:"Dr.",name:"Manuel",surname:"Gonzalez Ronquillo",slug:"manuel-gonzalez-ronquillo",fullName:"Manuel Gonzalez Ronquillo"},{id:"281920",title:"Prof.",name:"Carlos",surname:"Palacios Riocerezo",slug:"carlos-palacios-riocerezo",fullName:"Carlos Palacios Riocerezo"},{id:"260844",title:"Dr.",name:"Einar",surname:"Vargas-Bello-Pérez",slug:"einar-vargas-bello-perez",fullName:"Einar Vargas-Bello-Pérez"},{id:"296366",title:"Dr.",name:"Jorge",surname:"Osorio Avalos",slug:"jorge-osorio-avalos",fullName:"Jorge Osorio Avalos"},{id:"420710",title:"Dr.",name:"Lizbeth E.",surname:"Robles Jimenez",slug:"lizbeth-e.-robles-jimenez",fullName:"Lizbeth E. Robles Jimenez"},{id:"420711",title:"Ms.",name:"Paola Alejandra",surname:"Fernández Estrada",slug:"paola-alejandra-fernandez-estrada",fullName:"Paola Alejandra Fernández Estrada"},{id:"420712",title:"Dr.",name:"Raul",surname:"Perezgrovas",slug:"raul-perezgrovas",fullName:"Raul Perezgrovas"},{id:"420713",title:"MSc.",name:"Oscar",surname:"Chavez Rivera",slug:"oscar-chavez-rivera",fullName:"Oscar Chavez Rivera"}],corrections:null},{id:"78336",title:"The Pastoralism in the Silesian Beskids (South Poland): In the Past and Today",doi:"10.5772/intechopen.99722",slug:"the-pastoralism-in-the-silesian-beskids-south-poland-in-the-past-and-today",totalDownloads:163,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The Silesian Beskids (Poland), the westernmost part of the Carpathian Mountains is an area with long pastoral tradition. For centuries sheep grazed in clearings located among forested ridges have been an integral part of the mountain landscape and pastoral customs have become essential elements of regional cultural heritage. In the chapter, the history and the current state of pastoralism in the Silesian Beskids are presented. The specific pastoral system developed in the region, based on annual migration of flocks between summer highland and winter lowland pastures is described. Local breeds and specific regional sheep products are depicted. Furthermore, the importance of pastoralism for the environment, landscape and plant biodiversity is analysed and efforts to recover sheep grazing in the mountains after a period of a deep recession caused by social and economic transitions connected with the collapse of the communist system are presented. The approach to restoration of pastoralism is illustrated using a case study of a pastoral centre which combines traditional sheep grazing with cheese production, education and several activities to promote pastoral tradition.",signatures:"Anna Salachna, Katarzyna Kobiela-Mendrek, Maria Kohut, Monika Rom and Jan Broda",downloadPdfUrl:"/chapter/pdf-download/78336",previewPdfUrl:"/chapter/pdf-preview/78336",authors:[{id:"104226",title:"Prof.",name:"Jan",surname:"Broda",slug:"jan-broda",fullName:"Jan Broda"},{id:"414265",title:"Dr.",name:"Anna",surname:"Salachna",slug:"anna-salachna",fullName:"Anna Salachna"},{id:"415639",title:"Dr.",name:"Katarzyna",surname:"Kobiela-Mendrek",slug:"katarzyna-kobiela-mendrek",fullName:"Katarzyna Kobiela-Mendrek"},{id:"415640",title:"Mrs.",name:"Maria",surname:"Kohut",slug:"maria-kohut",fullName:"Maria Kohut"},{id:"415641",title:"Dr.",name:"Monika",surname:"Rom",slug:"monika-rom",fullName:"Monika Rom"}],corrections:null},{id:"78586",title:"Assessment of Sustainability of Dairy Sheep Farms in Castilla y León (Spain) Based on the MESMIS Method",doi:"10.5772/intechopen.99622",slug:"assessment-of-sustainability-of-dairy-sheep-farms-in-castilla-y-le-n-spain-based-on-the-mesmis-metho",totalDownloads:86,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"A livestock system is a productive unit, but in addition, if it pursues an optimal use of natural resources, it can increase overall sustainability. In order to evaluate the fulfillment of this objective, it is necessary to carry out a diagnosis of the system to describe and evaluate its degree of sustainability. One methodological option for this diagnosis is the construction of sustainability indicators. The MESMIS method is a methodological tool that analyses the interrelations between the results of the environmental, social and economic dimensions through a battery of indicators. The aim of this work is to assess the sustainability, using the MESMIS methodology, of different management systems of a sample of dairy sheep farms in Castilla y León (Spain). In general, the semi-extensive group obtained the highest overall score (8.40), and the intensive group achieved the highest volume of productivity. In conclusion, semi-extensive systems were more sustainable than intensive or semi-intensive systems in all attributes, especially those related to stability, adaptability and equity.",signatures:"Cristina Hidalgo-González, M. Pilar Rodríguez-Fernández, Javier Plaza-Martín and Carlos Palacios-Riocerezo",downloadPdfUrl:"/chapter/pdf-download/78586",previewPdfUrl:"/chapter/pdf-preview/78586",authors:[{id:"281920",title:"Prof.",name:"Carlos",surname:"Palacios Riocerezo",slug:"carlos-palacios-riocerezo",fullName:"Carlos Palacios Riocerezo"},{id:"414424",title:"Prof.",name:"Cristina",surname:"Hidalgo-González",slug:"cristina-hidalgo-gonzalez",fullName:"Cristina Hidalgo-González"},{id:"424373",title:"Prof.",name:"Javier",surname:"Plaza Martín",slug:"javier-plaza-martin",fullName:"Javier Plaza Martín"},{id:"427859",title:"Dr.",name:"Mª Pilar",surname:"Rodríguez-Fernández",slug:"ma-pilar-rodriguez-fernandez",fullName:"Mª Pilar Rodríguez-Fernández"}],corrections:null},{id:"78303",title:"Reproductive Rates of Merino Ewes and Offspring Quality under AI Program",doi:"10.5772/intechopen.99617",slug:"reproductive-rates-of-merino-ewes-and-offspring-quality-under-ai-program",totalDownloads:103,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:1,abstract:"Reproductive wastage is a major economic burden in sheep production globally, especially within Australia as livestock production systems face increased pressure from climatic variability (e.g. prolonged droughts or flooding). Sheep are sensitive to acute changes in their environment such as heat stress, which if not adequately monitored will result in significant production losses such as reproductive failure, increased parasite and worm burden, morbidity and mortality risks. Through basic and applied research in the areas of stress and reproductive physiology our team has made significant advancements in the understanding of sheep behaviour and physiological responses to acute and chronic stressors. Using minimally invasive hormone monitoring technology in combination with field based assessment of sheep health and productivity traits, our team has delivered new knowledge on how sheep react to acute environmental stress and how it impacts on sheep reproduction. In this chapter, we evaluated the fertility rates and embryo quality of Merino ewes under AI breeding program. We discuss factors such as heat stress that can impact on ewe and offspring quality.",signatures:"Edward Narayan, Gregory Sawyer, Natalie Hoskins and Greg Curren",downloadPdfUrl:"/chapter/pdf-download/78303",previewPdfUrl:"/chapter/pdf-preview/78303",authors:[{id:"259298",title:"Dr.",name:"Edward",surname:"Narayan",slug:"edward-narayan",fullName:"Edward Narayan"},{id:"356772",title:"Mr.",name:"Gregory",surname:"Sawyer",slug:"gregory-sawyer",fullName:"Gregory Sawyer"},{id:"423935",title:"Ms.",name:"Natalie",surname:"Hoskins",slug:"natalie-hoskins",fullName:"Natalie Hoskins"},{id:"423936",title:"Mr.",name:"Greg",surname:"Curren",slug:"greg-curren",fullName:"Greg Curren"}],corrections:null},{id:"79360",title:"Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects",doi:"10.5772/intechopen.100273",slug:"ovine-artificial-insemination-in-the-maghreb-region-present-status-and-future-prospects",totalDownloads:59,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Artificial insemination (AI) plays a key role in the genetic improvement of farm animals. Although it is widely used for cattle in the Maghreb region, it is scarcely applied in sheep at farm level. This is not only due to low fertility and irregular results that range between 30 to less than 76% for both cervical AI with fresh semen and laparoscopic insemination with frozen semen in most of studied breeds and also because of low results related to conditioning of fresh, chilled and frozen rams′ semen. An appropriately literature analysis was conducted to highlight the importance of sheep breeding in the Maghreb region particularly in Morocco, Algeria and Tunisia and to assess the efficiency of AI for Magrebin ovine breeds, the results related to different semen conditioning techniques and different AI procedures. The main factors affecting AI results are also presented. Finally, this chapter presents different strategies to improve AI efficiency at farm level in the future and the challenges to extrapolate experimental AI techniques to field conditions at a large scale.",signatures:"Moufida Atigui and Mohamed Chniter",downloadPdfUrl:"/chapter/pdf-download/79360",previewPdfUrl:"/chapter/pdf-preview/79360",authors:[{id:"287082",title:"Dr.",name:"Moufida",surname:"Atigui",slug:"moufida-atigui",fullName:"Moufida Atigui"},{id:"339973",title:"Dr.",name:"Mohamed",surname:"Chniter",slug:"mohamed-chniter",fullName:"Mohamed Chniter"}],corrections:null},{id:"80820",title:"Kendrapada Sheep: An Insight Into Productivity and Genetic Potential of this Prolific Breed",doi:"10.5772/intechopen.101289",slug:"kendrapada-sheep-an-insight-into-productivity-and-genetic-potential-of-this-prolific-breed",totalDownloads:30,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Kendrapada sheep of Odisha is a prolific, medium stature meat type breed. The Kendrapada sheep is the second prolific sheep of India after Garrole of West Bengal, which carries FecB mutation, responsible for prolificacy. The reproductive traits of this sheep is the major attribute where the ewe of this sheep comes to heat at around 10–11 months and drops its first lamb at around 15–16 months of age. The average lambing interval in these sheep is 8 months with gestation period of 150 days. The reproductive performance of these sheep is the uniqueness of this sheep population with more than 70% multiple births; 62.8% twinning, 2.3% triplet and 1% quadruplets. Thus research should be undertaken to conserve the valuable germplasm of Kendrapada sheep to improve the other breeds of India which are good in context of weight gain but lack prolificacy. As sheep are well adapted to diverse climatic conditions they can easily thrive on wide variety of grasses and crop residues thus fits well in zero input free grazing system of rearing by rural poor. However the potentiality of this Kendrapada sheep in terms of meat quality and prolificacy and resistance to diseases has been the simulating force to take up base line survey along with variety of trials to conserve this breed. Keeping the above mentioned points in mind the present study was carried out to highlight the baseline details of this neglected breed as it is one of the first review articles on Kendrapada sheep.",signatures:"Lipsa Dash, Sukanta Kumar Sahoo, Feroz H. Rahman, Simphoni Mohanty, Prasanta Kumar Nanda, Debi Prasad Kund, Surya Narayan Mishra and Susanta Kumar Dash",downloadPdfUrl:"/chapter/pdf-download/80820",previewPdfUrl:"/chapter/pdf-preview/80820",authors:[{id:"356749",title:"Dr.",name:"Lipsa",surname:"Dash",slug:"lipsa-dash",fullName:"Lipsa Dash"},{id:"425134",title:"Dr.",name:"Sukanta Kumar",surname:"Sahoo",slug:"sukanta-kumar-sahoo",fullName:"Sukanta Kumar Sahoo"},{id:"425135",title:"Dr.",name:"Feroz H",surname:"Rahman",slug:"feroz-h-rahman",fullName:"Feroz H Rahman"},{id:"425137",title:"Dr.",name:"Simphoni",surname:"Mohanty",slug:"simphoni-mohanty",fullName:"Simphoni Mohanty"},{id:"425138",title:"Mr.",name:"Prasanta Kumar",surname:"Nanda",slug:"prasanta-kumar-nanda",fullName:"Prasanta Kumar Nanda"},{id:"425139",title:"Dr.",name:"Debi Prasad",surname:"Kund",slug:"debi-prasad-kund",fullName:"Debi Prasad Kund"},{id:"425140",title:"Dr.",name:"Surya Narayan",surname:"Mishra",slug:"surya-narayan-mishra",fullName:"Surya Narayan Mishra"},{id:"425148",title:"Prof.",name:"Susanta Kumar",surname:"Dash",slug:"susanta-kumar-dash",fullName:"Susanta Kumar Dash"}],corrections:null},{id:"78547",title:"The Role of Cryptosporidiosis in Sheep Welfare",doi:"10.5772/intechopen.99876",slug:"the-role-of-cryptosporidiosis-in-sheep-welfare",totalDownloads:72,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Welfare in animal production has been defined as the optimal mental and physiological state of the animals. It has been recently redefined according to animals’ freedoms. As systems, individual sheep and herds are dynamic with constant interaction with each other and the environment. In this interaction, diseases play a fundamental role in welfare. Parasitism is common in sheep, and several management practices have been established to maintain the herds healthy. Cryptosporidium represents a special case, because it is a highly resistant environmental parasite, that can easily infect lambs, producing weakening diarrheas and even death. In this chapter, the role of cryptosporidiosis in sheep welfare and economic loss will be analyzed, as means of providing information on how to minimize and deal with the infection.",signatures:"María Uxúa Alonso Fresán and Alberto Barbabosa Pliego",downloadPdfUrl:"/chapter/pdf-download/78547",previewPdfUrl:"/chapter/pdf-preview/78547",authors:[{id:"415785",title:"Dr.",name:"María",surname:"Uxúa Alonso Fresán",slug:"maria-uxua-alonso-fresan",fullName:"María Uxúa Alonso Fresán"},{id:"425184",title:"Dr.",name:"Alberto",surname:"Barbabosa Pliego",slug:"alberto-barbabosa-pliego",fullName:"Alberto Barbabosa Pliego"}],corrections:null}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},subseries:null,tags:null},relatedBooks:[{type:"book",id:"6384",title:"Animal Husbandry and Nutrition",subtitle:null,isOpenForSubmission:!1,hash:"45e3ab6f834a3efc7836eb8b3c8e3427",slug:"animal-husbandry-and-nutrition",bookSignature:"Banu Yücel and Turgay Taşkin",coverURL:"https://cdn.intechopen.com/books/images_new/6384.jpg",editedByType:"Edited by",editors:[{id:"191429",title:"Prof.",name:"Banu",surname:"Yucel",slug:"banu-yucel",fullName:"Banu Yucel"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"9356",title:"European Local Pig Breeds - Diversity and Performance",subtitle:"A study of project TREASURE",isOpenForSubmission:!1,hash:"182fe65256f9a0bbc25b0b7576412b0e",slug:"european-local-pig-breeds-diversity-and-performance-a-study-of-project-treasure",bookSignature:"Marjeta Candek-Potokar and Rosa M. 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In a typical BAN, the localization sensors measure certain characteristics related to the location of the phone to position it on a map [1]. These sensors either provide us information on the distance from reference points to help us calculate the absolute location, such as Received Signal Strength (RSS), Time-of-Arrival (TOA), Angle-of-Arrival (AOA) of RF signals or they provide us with the velocity and direction of movement from the motion sensors for calculation of displacement or the relative location of the object. One of the fundamental differences between the two methods is that the absolute localization needs an infrastructure of reference points but relative localization position the object relative to its previous location. Figure 1 shows two examples of the results of localization using absolute and relative positioning systems [2] on a square route in a typical office building. Figure 1b shows the results of absolute Wi-Fi localization. The estimated locations (dots) are randomly distributed around the path of movement (solid line) of the object. Figure 1b shows the results of localization using an Inertia Motion Unit (IMU) device measuring the speed and direction of the movement. The estimated path of movement follow a straight line but, because of errors in angle and speed of movements, the location estimate gradually drifts from the actual path. Hybrid algorithms can combine the results of the two classes of sensors to achieve a more reliable navigation. These algorithms use the absolute localization to reduce the drift of IMU and use the IMU result to smooth the location estimates of the absolute location.
Results of localization with two classes of location sensing in square corridor route of an office (a) absolute localization using RSS of Wi-Fi access points (b) relative localization using IMU devices.
The most popular absolute positioning systems, such as Global Positioning System (GPS) and Wi-Fi Positioning System (WPS), sense features of the RF signals to measure the distance between the object and the reference points. The 26 satellites used as the reference points for GPS infrastructure are deployed for the purpose of positioning, while WPS opportunistically uses the Wi-Fi and cell phone signals for localization. Since absolute RF localization is the most fundamental and the most complex element of positioning, it is crucial to have a deep understanding of the behavior of RSS- and TOA-based ranging in different environments, which is well discussed in a great number of existing literature [3].
On the other hand, a typical application for relative positioning is Inertial Navigation Systems (INS). An INS uses a computer, motion sensors (accelerometers), rotation sensors (gyroscopes), and occasionally magnetic sensors (magnetometers) to continuously calculate by dead reckoning the position, the orientation, and the velocity (direction and speed of movement) of a moving object without the need for external references. With the development of technology, other methods can also be used for relative positioning. The speed of movement can be measured by number of rotations of the wheel (speedometer), Doppler spectrum of a received RF signal, the number of steps that a pedestrian walks (step counter), or similarities between the consecutive pictures taken by a camera.
Recently, Internet-of-Things (IOT) becomes a new fashion and opens up a new door to BAN localization. Large amount of IoT sensors have been deployed inside buildings, each of which has the ability to communicate and share location information to one other. Since the smartphone is able to communicate with both WiFi- and Bluetooth-based devices, its role in BAN localization becomes more and more important.
In this chapter, we present the way smartphone system visualize the location. We will also illustrate all the localization sensors in smartphone platform and discuss the characteristics of each. Examples are provided to show how these sensors are applied in the localization system and their accuracy is investigated as well.
In order to position a location on a map we need a coordinate system to visualize the location information. For global positing of a location, we need to refer to the global 3D coordinates of the earth. The most commonly 3D coordinates of the earth (Figure 2) are the geographical polar coordinates commonly referred to as: longitude, latitude and altitude
Earth 3D polar coordinate system with elevation, longitude and latitude.
Ground navigation systems for outdoor and indoor areas track movements of the vehicles or robots on 2D maps such as layout of a floor of a building or 2D map of streets of an urban area in area for which altitude does not change substantially. Even in multi-floor 3D applications we use a 2D map with the floor number (a representative of altitude). Cartesian coordinates provide a better tool for visual tracking for most of popular applications in indoor and urban areas. Therefore, we usually convert longitude, latitude, and altitude to a Cartesian form using:
In absolute positioning, a GPS or WPS location fix in polar or in Cartesian coordinate, represent a point in the 3D world frame coordinate. In relative positioning we represent the velocity and direction as vectors at each location. Accelerometers and gyroscopes are mechanical devices used for measurements of differential vectors for acceleration and rotation. We integrate the differential acceleration to determine the instantaneous velocity vector and we integrate the rotation vectors to determine the direction of motion.
In the universal Cartesian reference coordinate system, shown in Figure 3a, an acceleration or velocity vector in X-axis defines a vector tangential to the ground at the measurement sensor’s current location pointing to the East. The vector in Y-axis is tangential to the ground at the measurement sensor’s current location and pointing towards magnetic north. A vector in Z-axis points towards the sky and perpendicular to the ground at the location of the measurement sensor. However, as shown in Figure 3b, measurement sensors are MEM devices installed in a device, such as a smart phone, and their coordinates are defined relative to the device’s screen and not the universal earth coordinate. When a device is held in its default orientation, the X-axis is horizontal and points to the right side of the device, the Y-axis is vertical and points up to the top of the device, and the Z-axis points towards the outside of the screen face. In this system, coordinates behind the screen have negative Z-values.
(a) Ground axis and (b) smartphone axis.
Opportunistic localization is based on the location sensor payload of the platform we want to localize. Typical smart phones carry RF based GPS, Wi-Fi, Cell Phone, and iBeacon (Bluetooth Low Energy) chip sets, which support a variety of opportunistic RF localization capabilities for different environments and precision requirement. In addition, they carry a number of other sensors which can be used to enhance positioning of the device using traditional RF signals. Table 1 shows typical sensors available in an Android device and their functionality if they are used for localization. In addition to the traditional sensors such as gyroscope, magnetometer, accelerometer and barometer, there are sensors for ambient light, temperature, and humidity as well as force of gravity levels, proximity and step counts. The sensors can be beneficial for localization applications to enhance precision of RF localization. We can use the data collected by these sensors for 3D localization, calculation of speed and direction of movements and specifying the environment of operation. Positioning algorithms use RF location and motion information obtained from a variety of sensors. These sensors perform differently in different environments. For example, GPS which provides very reliable information for outdoor navigation does not operate deep in indoor areas. Therefore, in addition to the algorithm to determine the location, we need to have an algorithm to detect the environment to distinguish between the indoor and the outdoor environments. The data from all sensors is available as a resource to a positioning engineer to design different algorithms to achieve the precision requirements in the environment of operation.
The detailed description of these localization sensors is presented in the following sections as well as how accurate they can achieve in locating objects.
Accelerometer sensor measuring the acceleration (differential rate of variations of velocity) applied to a device in meter per square second
(a) Basic concept inside accelerometer (b) basic concept behind a gyroscope and (c) example of acceleration and rotation calculation.
Conceptually, an accelerometer measures the forces applied to the sensor in different directions and using Newton’s second equation calculates the acceleration by dividing the force by mass of the sensor. When an IMU device rests in a place on a table in parallel to the earth surface, the accelerometer of the device should read a magnitude of
In order to measure the real acceleration of the device, also referred to as linear-acceleration, the contribution of the force of gravity must be eliminated. Applying a high-pass filter to the results of accelerometer readings eliminates the slow changes of the readings caused by gravity and detects the fast changes introduced by movements of the device. Conversely, using a low-pass filter isolates the force of gravity and eliminates the fast changes in the acceleration caused by the movements of the device.
Gyroscope sensor measure the differential rotation momentum around the local x, y, z axis of the device Cartesian coordinates in radians/second. Figure 4b shows the basic concept behind a gyroscope. A cylindrical mass is hanging around a central shaft like a wheel, as we change the direct wheel tits and we measure the angular displacement of the wheel. The coordinate system is the same as the one is used for the acceleration sensor. The right side of Figure 4c shows how we can measure the rotation in 3D around each of three axis of the device by placing three gyroscopes in orthogonal directions. The output of the gyroscope is integrated over time to calculate a rotation describing the change of angles over time.
Rotation is positive in the counter-clockwise direction. That is, an observer looking from some positive location on the x, y or z axis at a device positioned on the origin would report positive rotation if the device appeared to be rotating counter clockwise. If we refer to angular speed around the x-axis as
(a) Definition of angular rotations: roll, pitch and yaw (b) a typical IMU device with local device coordinates.
Figure 5b shows a typical IMU device with both the device and the earth-fix coordinates. To find the location from the above equation we need to take a double integral. The first integration provides the velocity in each direction and the second integral provides the distance traveled by the device in each direction. Since smartphones are equipped with all the sensors mentioned above, we can consider them as a kind of smart IMU, and movement can be determined in the same way.
In practice, the gyroscope noise and offset will introduce some errors which need to be compensated for. This is usually done using the information from other sensors. The gyroscope cannot be emulated based on magnetometers and accelerometers, as this would cause it to have reduced local consistency and responsiveness. It must be based on a usual gyroscope chip. Most recent systems are of the strap down type, where the IMU sensor outputs are taken directly and processed to compute the orientation and displacement of the vehicle.
A simple laboratory experiment moving the IMU device shown in Figure 6a forward and then turning to the right. The IMU device is a relatively expensive device purchased in mid-2000 for approximately
(a) Scenario of IMU movement, (b) readings of gyroscope angular movement and (c) readings of accelerometer.
(a) Integral of the rotated movement (b) double integral representing motions in X and Y axis (c) trajectory of movement.
Magnetometer is a magnetic field sensor measuring the ambient magnetic field along the 3 sensor device local x, y and z axis in micro-Tesla (
Rather than high pass filtering, we low pass filter the measurements from an accelerometer we measure the force of gravity in the device coordinate. The magnitude of this vector is
(a) Basic concept of a magnetometer and (b) implementation of a 3D magnetometer.
Step counter counts the number of steps taken by the pedestrian carrying a smart device such as a smartphone or a health monitoring band. The value is in integer and it reset to zero on a system reboot. The timestamp of the event is set to the time when the last step for that event was taken. The obvious application of this sensor is in fitness tracking applications but it is also useful for indoor geolocation using smart devices.
We can implement a step counter by using the measurements from an accelerometer. Figure 9 shows hundred samples of magnitude of the accelerometer measurements for an Android phone. Considering Eq. 7.2, the magnitude of the acceleration is
(a) Step count using peak detection and (b) step count using zero crossing.
Using the step counter and the time stamps associated with each step, the average speed and the average step size can be determined and with that we can estimate the linear distance traveled by a pedestrian carrying a device with an accelerometer. To position the location of a pedestrian in a 2D map indoors or outdoors we also need to measure the rotations. Rotation can be measured with the results of gyroscope or the compass.
Figure 10 shows the results of an example relative localization system using the step counter and gyroscope of a typical smartphone at the third floor of the Atwater Kent Laboratory at the Worcester Polytechnic Institute. The schematic of corridors of this floor of building and layout of the exterior of the building in shown on the right figure. A user carrying the smartphone enters the floor from right to and walks straight and then around the central corridors before turning back to the original location. User holds the smartphone in front of the torso in parallel to the ground. In this posture the z-axis of the smartphone is towards the ceiling and the only rotation of the device is around this axis. Figure 10a shows the results of gyroscope readings in the three dimensions. There are four jumps in rotations around the z-axis (Azimuth or yaw) associated with four 900 turns in the route of movement of the device. The red line in Figure 10b illustrates the estimated path of movement when the results of step counts is complemented with the gyroscope readings. Typical increasing pattern of the drift of the location estimate away from the actual path of movement is observed. This type of error pattern is caused from using speed and direction of movement for relative localization and it is similar to the error pattern [6, 7, 8].
(a) Results of a 3D gyroscope measurement and (b) localization using step count and gyroscope.
Compass is one of the oldest mechanical magnetometer historically used for navigation. If we lay a compass in parallel to the earth surface the heading of the compass magnet points to the magnetic north. As a results, by rotating the compass to point towards a specific direction we measure the angle of that direction with respect to the magnetic north. This angle provides us with direction for relative navigation. A compass takes advantage of the earth electromagnetic field to determine the rotation angle of the device. Earth acts like a large dipole magnet with magnetic field lines originating from magnetic south pole (near the geographic south pole) and terminating in magnetic north pole located near the geographic north pole (Figure 11a). A compass has a small magnet in the center aligning with the magnetic north pole in all locations on the surface of the earth. The angle between the compass heading (the magnetic north) and the geographic north is called the magnetic declination angle (Figure 11b). The value of the decantation angle changes in location as well as in time. If we know the direction of geographical north in a location we can align the compass to that and measure the declination angle as the angle between the heading of the compass magnet and direction of the geometric north. In the east coast of United States inclination angle can go as low as
(a) Magnetic field of the Earth and North/South poles and (b) a traditional compass and heading angle.
The National Oceanic and Atmospheric Administration (NOAA) has a public web calculator for calculation of magnetic declination angle around the Globe. Using this web site we read
All popular smartphones in the market carry an electronically emulated compass application. These applications typically emulate the compass using the results of magnetometer, commonly available as a part of the IMU chip of the smartphone. Figure 11a shows a typical electronic compass implementation on an iPhone when the device is facing upward (z-axis of the device and the earth fix coordinates are aligned). The scaled round shape on top of the figure allows visualization of the angle between the north axis (y-axis) of the earth and the device to emulate the appearance of a compass. The Hx and Hy measurements of the magnetometer (Figure 12b) define the direction of compass heading in degrees using:
(a) Electronic compass application in smartphone and (b) compass heading and Earth coordinates.
As we turn the device around its z-axis, while laying the device on a table, the reading of the angle changes. For
Barometer is a pressure sensor measuring the atmospheric pressure in hPa (millibar). Barometer is used to estimate elevation changes and consequently floor number of operation inside a multi-floor building. The relation between air pressure and height is given by [9]:
Using Eq. (4) and parameters in Table 2, we can calculate altitude from air pressure. Figure 13 shows the results of a smartphone barometer measurements in a typical 3-story office building. Figure 13a is produced from the results obtained in an elevator elevating three floors of the building and Figure 13b shows the results for climbing one floor of the building using stairs. Both figures demonstrate gradual change of the barometer reading during climbing up and down among the floors regardless of the speed and the pattern of motion. Range of variation of measurements in the elevator experiment for climbing three floors are approximately three times larger than that of the measurements for obtained from climbing one floor on the stairs. These observations demonstrate that barometer can be utilized to measure the change in altitude inside the buildings. Figure 14 shows results of barometer measurements from a smartphone when a user carries the device in four floors of a typical office building in two different time of the same day. Pattern of measurement in different floors are clearly different allowing us to distinguish the floor of operation. However, results of barometer are corrupted by additive Gaussian noise, bias, and it is sensitive to the time of measurement [10, 11]. Methods to mitigate these effects and have a better estimate of the floor number in an office building is discussed in [9].
Sensor name | Description | Type | Common uses |
---|---|---|---|
Barometer | Measures the atmospheric pressure in hPa (millibar) | Environment | Elevation for floor numbering |
Ambient temperature | Measures ambient room temperature in degree Celsius | Environment | Detecting environment changes |
Ambient light | Measures ambient light level in SI lux. | Environment | Detecting environment changes |
Relative humidity | Measures relative ambient air humidity in percent | Environment | Detecting environment changes |
Accelerometer | Measures the acceleration force in m/s2 that is applied to a device on all three physical axes (x, y, and z), including the force of gravity | Mechanical | Motion detection (shake, tilt, etc.) |
Gyroscope | Measures a device’s rate of rotation in rad/s around each of the three physical axes (x, y, and z) | Mechanical | Rotation detection (spin, turn, etc.) |
Step counters | Counts the number of steps taken by the person carrying the device | Mechanical | Measuring traveled distance |
Magnetometer | Measures ambient geomagnetic field strength along the x,y,z axes in micro-Tesla (μT) | Electromagnetic | Creating a compass |
Gravity | Measures the force of gravity in m/s2 that is applied to a device on all three physical axes (x, y, z) | Electromagnetic | Motion detection (shake, tilt, etc.) |
Proximity | Measures distance from the sensor to the closest visible surface measured in centimeters | Electromagnetic | Mapping the environment |
Typical sensors in an android smartphone.
Parameter | Description | Value |
---|---|---|
Standard atmospheric pressure | 101,325 Pa | |
Universal gas constant | 8.31447 J/(mol·K) | |
Sea level standard temperature | 288.15 K | |
Gravitation acceleration | 9.81 | |
Molar mass of dry air | 0.0289644 kg/mol |
Parameters used for calculation of relation between height,
Barometric measurement in a typical 3-story building (a) elevating three floors of the building and (b) climbing floors using stairs.
Barometric reading at different time.
Mesenchymal stem/stromal cells (MSCs), a multipotent stem/progenitor cell type, were initially described in bone marrow by Friedenstein et al. as rapid adherence to tissue culture vessels and the discrete “fibroblast” colonies approximately 50 years ago [1, 2]. Julius Cohnheim, a German-Jewish pathologist, firstly proposed that a fibroblast-like cell population for nonhematopoietic cells in bone marrow were involved in wound repair over 150 years ago [3]. In the late 1980s, Caplan firstly coined the name “mesenchymal stem cell (MSC)” [4]. Since then, MSCs have gained much attention over the last three decades. Many laboratories focusing on MSCs have developed diverse methods to isolate and expand MSCs from a variety of tissues. While the assessment of characteristics of MSCs is necessitated in different platforms/laboratories, most researchers come to acknowledge the lack of a universally accepted criteria to define MSCs. To address this question of cell equivalence, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposes three minimal criteria to define MSCs [5]: property of MSC plastic adherence, the expression of specific cellular surface antigen, and capacity for trilineage mesenchymal differentiation (osteogenesis, chondrogenesis and adipogenesis).
Human MSCs from different tissues have the varied phenotypic features, the morphologic inconsistency, and heterogeneous functional behavior [6, 7, 8]. Indeed, the properties of stem cell have not been well established yet. Due to the unknown
MSCs have been considered as a promising therapeutic tool in tissue engineering and regenerative medicine. MSCs are well known to be present in almost every type of adult tissues, such as bone marrow [10, 11, 12], adipose tissue [10, 13, 14], lung [11, 15], synovial tissue [16, 17], dental pulp and periodontal ligament [18]. Notably, it has become apparent that MSCs are identified in the various human embryonic tissues, such as fetal bone marrow [19], fetal liver [20], aorta gonad-mesonephros and yolk sac [21], as well as multiple neonatal birth-associated tissues, such as placenta [10, 22, 23], amniotic and chorionic membrane [23, 24], umbilical cord tissue [10, 23, 24, 25], and umbilical cord blood [26, 27]. Therefore, different platforms/laboratories may use different type of tissue sources and methodologies for isolation and expansion of MSCs. This chapter firstly outlines protocols for standardized isolation and expansion of human bone marrow-derived MSCs (BM-MSCs), a major source of human MSCs, as well as BM-MSCs’ characteristics, cryopreservation and thawing. Protocols for the preparation of MSCs derived from the other tissue types are similar to that of BM-MSCs, except tissue sample processing differentially. Human BM-MSCs are estimated at a very low frequency at approximately 0.001–0.01% of total nucleated cells [28, 29], and, therefore, human BM-MSCs are likely to be kind of difficult to isolate and harvest. This chapter will then focuses on optimal functional assays and application on the basis of our previous studies, which would be useful for researchers working with MSCs in basic research and translational and clinical applications, such as osteogenesis, chondrogenesis, adipogenesis, colony forming unit-fibroblast (CFU-F) assay, 3-D cellular co-culture, MSC homing and migration. Last but not least, the long-term culture associated alterations of MSCs’ properties will be also discussed in this chapter.
Growth medium (pH 7.1–7.5): Dulbecco’s modified Eagle’s medium (DMEM)-low glucose (Sigma-Aldrich, St Louis, MO), 10% fetal bovine serum (FBS, heat-inactivated) (Atlanta Biologicals, Lawrenceville, GA), 0.37 g% sodium bicarbonate, and 1% penicillin–streptomycin. Adjust growth medium to pH 7.1–7.5.
Ficoll-Paque (Density gradient medium) (Stem Cell Technologies, Cambridge, MA).
1 × Phosphate buffered saline (PBS), pH 7.2.
1 × PBS + 2% + 1 mM ethylenediaminetetraacetic acid (EDTA).
Trypan blue, 0.4%.
Freezing medium: 90% FBS and 10% (v/v) dimethyl sulphoxide (DMSO).
Trypsin–EDTA solution 0.25% (Sigma, St. Louis, MO).
RosetteSep™ Human Mesenchymal Stem Cell Enrichment Cocktail (Stem Cell Technologies, Cambridge, MA).
Nalgene® Cryo 1°C Freezing Container.
Cryogenic storage vials.
Suitable box for storage in liquid nitrogen.
Tissue culture ware: T25 flasks, T75 flasks, 100-mm tissue culture dishes, 6-well plates.
Hemocytometer.
Water bath at 37°C.
Biological safety cabinet class II.
Bench centrifuge with swinging bucket rotor.
Inverted phase microscope.
Sterile conical centrifuge tubes (15 and 50 mL).
Sterile cell culture plastic pipettes (2, 5, 10, 25 mL).
Automatic pipettor.
1.5 mL eppendorf tubes.
Make sure BM sample, 1 × PBS + 2% FBS + 1 mM EDTA, Ficoll-Paque and centrifuge are all at room temperature.
Spray the sample tube with 70% ethanol and conduct the isolation of BM-MSCs in the Biological Safety cabinet.
For 10 mL size of BM, divide BM sample into two 50 mL conical tubes at room temperature. Add 50 μl RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail per mL of bone marrow and mix well.
Incubate for 20 minutes at room temperature.
Dilute sample with about same volume of 1 × PBS + 2% FBS + 1 mM EDTA solution; mix gently.
Prepare two 50 mL conical tubes with 10 mL Ficoll-Paque each tube. Layer the diluted sample on top of Ficoll-Paque. Be careful to minimize mixing of Ficoll-Paque and sample. Tilt the tube to 45 degree angle and slowly add the sample drop by drop to form a layer on top.
Centrifuge at 1200 × g for 10 minutes at room temperature in a swinging bucket rotor and set the centrifuge to brake on.
Remove the enriched cells from the Ficoll-Paque. Collect the interphase containing the mononuclear cells. Be sure not to touch the red pellet at the bottom. It is advisable to remove some of the Ficoll-Paque and a bit of upper plasma layer in order to endure complete recovery of the desired cells.
Wash enriched cells with the 5× volume of 1 × PBS + 2% FBS + 1 mM EDTA solution. Spin 300 × g for 10 minutes.
Carefully discard the supernatant and resuspend cells in 1 mL of MSC growth medium per tube. Perform a viable cell count with a hemocytometer using Trypan blue.
Seed cells into one T-75 flask finally for 10 mL size marrow (a final cell concentration of 0.5–1.5 × 106 cells/cm2). 12 mL of MSC growth medium is supplemented.
Put the flask in incubator at 37°C with 5% humidified CO2 for 48 hours to allow cells to attach.
After 48 hours, observe with phase contrast microscopy and then remove growth medium and non-adherent cells.
Wash cells twice with pre-warmed medium and add 13 mL of fresh MSC growth medium. Return the flask to the incubator.
Change growth medium every 3 days and observe the cellular colony forming.
CFU-F become in the next 3–5 days. Continue to culture until the cells reach 80% confluence in the 2 weeks.
Remove the medium and wash with PBS 2–3 times.
Add 3–4 mL pre-warmed trypsin–EDTA solution to cover cells in the flask. Return the flask to the incubator for 5 minutes.
Check with phase contrast microscopy. When most cells become detached, gently tap the side of the flask.
Add 5 mL growth medium to the flask to stop Trypsin–EDTA action. Resuspend cells by pipetting and transfer the entire cell suspension into a 15 mL conical tube.
Centrifuge at 400 × g for 5 minutes.
Remove the supernatant and resuspend the cells in 2–3 mL pre-warmed PBS. Centrifuge at 400 × g for 5 minutes.
Repeat step 22.
Harvest cells. This culture is considered as passage 0.
Count cells and reseed cells at an optimum density of 5000 cells/cm2 in the appropriate tissue culture ware.
After 24 hours, remove the growth medium and wash the cells attached to the plate once with PBS.
Add the appropriate volume of fresh culture medium and incubate the cells for 2–3 days.
When the culture reaches 80–90% confluence, remove the culture medium and wash with PBS 2–3 times.
For trypsinization, add an appropriate volume of pre-warmed trypsin–EDTA solution to cover the entire cellular surface. Return the flask to the incubator for 5 minutes.
Observe under a phase contrast microscope. When most cells become detached, gently tap the side of the flask.
Add the appropriate volume of growth medium to stop Trypsin–EDTA action. Mix and collect the mixture of the entire cell suspension into a 15 mL conical tube.
Centrifuge at 400 × g for 5 minutes at room temperature.
Remove the supernatant without disturbing the cell pellet and resuspend the cells with an appropriate volume of pre-warmed PBS.
Wash and centrifuge at 400 × g for 5 minutes again.
Harvest cells. This culture is passage 1. MSCs at passage 1 can be frozen in liquid nitrogen (see the next) or continue to serially passage.
The following antibodies are used in flow cytometry analysis of MSC characterization, CD29-PE, CD34-PE, CD44-PE, CD73-PE, CD90-PE, CD45-FITC, CD147-FITC, HLA DR-FITC, IgG1-PE, and IgG1-FITC (BD Biosciences). IgG1 immunoglobulin is used as isotype negative controls and passage 3 MSCs are characterized using PE or FITC conjugated antibodies against the cellular surface markers.
Remove the growth medium and wash cells with pre-warmed PBS twice.
Add the pre-warmed trypsin–EDTA solution to the flask. Return the flask to the incubator for 5 minutes.
When most cells become detached, gently tap the side of the flask.
Add growth medium to the flask. Mix and transfer the entire cell suspension into a 15 mL conical tube.
Centrifuge at 400 × g for 5 minutes.
Remove the supernatant and resuspend the cells in the pre-warmed PBS.
Centrifuge at 400 × g for 5 minutes.
Carefully discard the supernatant and resuspend cells with pre-warmed PBS.
Centrifuge at 400 × g for 5 minutes.
Harvest cells.
Count cells and make it a single cell suspension at concentration of 1x106 cells/mL in PBS.
Add 250 μL cellular suspension to the appropriate number of FACS tubes.
Add antibodies to the FACS tubes – 10 μL per antibody per tube.
Incubate the FACS tubes for 20–25 minutes at room temperature on a shaker to prevent aggregation.
Then spin down FACS tubes at 150 rpm for 5 minutes.
Wash twice with PBS.
Add 2 mL MSC growth media.
Centrifuge the tubes at 150 rpm for 5 minutes.
Pipette off the supernatant.
Add 250 μL MSC growth media.
Run samples through the FACS machine.
Of note, perform all following steps under sterile conditions.
Place freezing medium on ice.
Harvest the cultivated MSCs, as described above (steps 1–10 in Section 2.3).
Resuspend MSCs in freezing medium at a concentration of 5–10 × 105 cells/mL.
Transfer cells into appropriate cryogenic storage vials or tubes and close the lid.
Place the vials in the pre-cooled Cryo 1°C Freezing Container quickly and store at −80°C directly.
After 24 hours, transfer the cryogenic storage vials to the suitable boxes to liquid nitrogen for long-term storage.
Perform all following steps under sterile conditions.
Place the frozen vial of MSCs rapidly into a 37°C water bath.
Gently swirl the cryovial until the ice in the vial has melted.
After thawing, transfer the entire content of the cryovial into a sterile 15 mL conical tube containing 5 mL of pre-warmed PBS.
Gently swirl and centrifuge at 400 × g for 5 minutes.
Aspirate supernatant completely.
Wash again.
Count and reseed cells at a density of 5000 cells/cm2 in the appropriate tissue culture ware.
Osteogenic medium: MSC growth medium supplemented with 50 μM ascorbic acid phosphate (AsAP) (Wako Chemicals USA, Richmond, VA), 0.1 μM dexamethasone (Sigma-Aldrich, St Louis, MO), and 10 mM β-glycerol phosphate (Sigma-Aldrich, St Louis, MO).
Several osteogenic markers, such as alkaline phosphatase (ALP), leptin receptor, and cathepsin K, are used as the indicator of early osteogenesis [28], calcium deposition as the indicator of late-stage osteogenesis [28, 29]. ALP activity and calcium deposition are exemplified to assess osteogenic differentiation of human MSCs from passage 4 (Figure 1).
Osteogenic culture of human BM-MSCs. Representative images (10×) of human BM-MSC osteogenesis on day 12 (A), day 17 (B), and day 26 during osteogenic culture.
Collect cells from passage 4.
Prepare cellular suspension in growth medium.
Plate cellular suspension in 6-well plates at 1 × 105 cells/well in triplicate.
After 24 hours, remove the growth medium and wash cells with osteogenic medium once.
Add 2 mL osteogenic medium in each well.
Culture cells in incubator at 37°C with 5% humidified CO2.
Change osteogenic medium every 3 days.
Measure ALP activity about 1–2 weeks after MSC osteogenetic culture [29, 30]. Aspirate medium and gently wash cells twice with PBS.
Add 600 μL of lysis buffer (0.5% Triton-X 100 in molecular grade ddH2O) to each well and then scrape the cells off the surface using the end of a pipette tip.
Transfer lysates to centrifuge tubes. Prepare 15 mL conical tubes equal to the number of tubes maintaining lysates.
Dissolve the contents of a 40 mg capsule of phosphatase substrate (Sigma) in 10 mL ddH2O. Scale up if necessary.
Add 500 μL of 1.5 M alkaline buffer solution (Sigma) to each tube. Add 500 μL of phosphatase substrate solution into each tube. Keep tubes in 37°C water bath.
Vortex samples and add 100 μL of each lysate to 15 mL tubes within 30 seconds. Fifteen minutes after the first sample is added, add 1 mL of 1 N NaOH to each tube in 30 second intervals removing tubes from water bath. The reaction will take place for 15 minutes at 37°C for each tube.
Prepare standard curve (Sigma) (Note: prepare 100 nmol/mL solution of p-nitrophenol by combining 100 μL of 10 mM p-nitrophenol standard solution with 9.9 mL 0.02 N NaOH).
Add 300 μL of standards and samples in triplicate to a 96-well plate.
Measure the absorbance using excitation filter of 405 nm. One enzyme unit of ALP is defined as the quantity of enzyme which produces 1 nmol p-nitrophenol per 15 minutes [29].
Aspirate or pipette out all culture medium from each well of the 6-well culture plate that contains induced or control cells to be tested.
Rinse the cells twice with PBS. Add 200 μL of PBS to the side of each well, not to dislodge the cells. Aspirate off the PBS and re-rinse.
Add 125 μL of 0.5 N HCl to each well.
Scrape the cells off of the surface using a cell scraper and transfer the cells and HCl to a 1.5 mL polypropylene microcentrifuge tube with a tight fitting cap.
Add an additional 125 μL of 0.5 N HCl to each cell to recover any cells remaining in the well, and transfer this to the appropriate tube.
Samples may be capped tightly and stored at −20°C if they are not to be tested immediately.
Extract the calcium from the cells by shaking the tubes on an orbital shaker for 4 hours at 4°C. If using frozen samples, allow extra time for samples to thaw.
Centrifuge the sample tubes at 500 g for 2 minutes.
Carefully collect the supernatant with extracted calcium, without disrupting the cell pellets, and transfer these to a new tube.
Prepare a standard curve with the calcium standard and determine the amount of calcium in each control and osteogenesis-induced samples. Follow the instructions provided in the Stanbio Total Calcium LiquiColor® Procedure No. 0150 (Stanbio Laboratory).
Three μL of sample vs. 297 μL of assay reagent (1:100 ratio for sample to reagent) for 96-well plate is used for each calcium determination. Assay reagent is mixed by equal volume of two solutions (Color Reagent and Base Reagent) provided in the kit. Distribute the assay reagent by multipipettor after adding samples. Absorbance is read at 550 nm.
Unused sample extract may be re-frozen for future re-assay. If the reading was out of range, the sample can be diluted with ddH2O in a total volume of 3 μL (e.g. 1 μL of sample+2 μL of ddH2O) and re-assayed again.
PuraMatrix™ hydrogel (BD Biosciences) is a 16-amino acid synthetic peptide hydrogel composed of a repeating sequence of arginine, alanine, aspartate, and alanine (RADA16) [31], which is widely used for 3-D culture.
3-D culture of human BM-MSCs on PuraMatrix hydrogel as follows.
The stock of 1% peptide solution can be sonicated for 30 minutes to decrease its viscosity and then diluted with sterile ddH2O to a final concentration of 0.25% (w/v).
300 μL of 0.25% gel solution is loaded into each well of the 24-well plate until it is uniformly spread.
The gelation is initiated by slowly dripping the medium along the wall of the well. 300 μL of medium is again added carefully on top and the plate is incubated at room temperature for one hour equilibration.
After the peptide has assembled into hydrogel, the medium is changed two times over one hour to equilibrate the growth environment to physiological pH.
The equilibrated samples are stored overnight at 37°C incubator and the cells are seeded the next day.
Prepare the total of 4 × 104 cells suspended in MSC growth medium and 4 × 104 cells are seeded onto the hydrogel. The following day (Day 0), the medium will be replaced by osteogenic medium.
Von Kossa staining can be conducted at day 24 after differentiation [30]. MSCs are rinsed with the Tyrode’s balanced salt solution and fixed with 10% buffered formalin (Fisher Scientific) for 30 minutes.
Incubated with 2% silver nitrate solution for 10 minutes in the dark.
Rinse with ddH2O and expose to light for 15 minutes.
Bright-field images of stained samples are captured with an inverted microscope.
To generate a 0.25% final concentration of PuraMatrix™ hydrogel for cells encapsulation, one part of 1% PuraMatrix™ hydrogel is diluted with same volume of sterile 20% sucrose, to reach 0.5% PuraMatrix™ hydrogel in 10% sucrose, and then mix with one part of 2× concentration of cells resuspended in 10% sucrose.
For 24-well plates, 300 μL of PuraMatrix mixture is loaded into each well and 300 μL of medium is layered on top of the gel. The gelation of the PuraMatrix is completed in an incubator for 60 minutes.
Change medium the next day.
Von Kossa staining can also be conducted, as described above, or collect cells as follows for other experiments.
Mechanically disrupt BD PureMatrix™ and cells in the well or cell culture insert by pipetting the media and gel up and down.
Transfer to a 15 mL conical tube.
Rinse out the well or cell culture insert using PBS.
Centrifuge at 150 × g for 5 minutes. Discard supernatant. The pellet at the bottom of the tube contains cells and BD PuraMatrix fragments.
Resuspend pellet in 2 mL of PBS. Spin and collect pellet again.
Resuspend pellet in 1 mL of trypsin–EDTA and incubate at 37°C for 5–10 minutes. This will help separate cells that are still attached to each other.
Add 5 mL PBS to spin cell pellet again.
Aspirate the supernatant (do not disturb the gel). Resuspend pellet again in 1 mL of trypsin–EDTA and incubate at 37°C for 5–10 minutes.
Add 5 mL PBS to spin cell pellet again.
Aspirate the supernatant. Carefully take out one third of the gel pellet. Do not disturb the bottom (two-third) part of the gel.
Wash with PBS twice.
Add appropriate amount of lysis buffer to perform cell lysis and collect cell sample.
Chondrogenic medium: 95% DMEM high-glucose medium (Sigma-Aldrich, St Louis, MO), 1% 1 × ITS+1 solution (BD Biosciences, San Jose, CA), 1% Pen-Strep, 100 μg/mL sodium pyruvate (Invitrogen, Carlsbad, CA), 50 μg/mL AsAP, 40 μg/mL L-proline (Sigma-Aldrich, St Louis, MO), 0.1 μM dexamethasone, and 10 ng/mL recombinant human TGF-β3 (Lonza, Walkersville, MD).
Passage 4 human BM-MSCs are used for chondrogenic differentiation. Chondrogenic differentiation is induced by chondrogenic medium (Figure 2).
In order to form a chondrogenic pellet, approximately 2.5 × 105 human BM-MSCs are centrifuged down in a 15 mL conical tube at 150 × g for 5 minutes at room temperature.
Five hundred μL of chondrogenic medium is used to resuspend the 2.5 × 105 cells to a final concentration of 0.5 × 106 cells/mL.
Cells are centrifuged down again. Place the 15 mL conical tube in the incubator at 37°C with 5% humidified CO2.
MSCs are shaped into the pellet after 24 hours incubation without disturbance.
The cell pellet is fed every 3 days for about 2–4 weeks and, after that, the chondrogenic pellet is harvested and sample processing is described as follows for immunohistochemistry analysis (e.g. examination of the expression levels of Collagen II, X and Aggrecan) [29, 32].
Chondrogenic culture of human BM-MSCs. Representative images (10×) of immunohistochemical staining of collage II (A) and aggrecan (B) in pellet culture samples at day 17 during chondrogenesis [
Day 1: Fixation and dehydration
Rinse specimen in PBS.
Fix samples for 45 minutes in acid-formalin at 4
Rinse specimen in PBS twice.
Embed specimen in 2% agarose.
Transfer to vial and treat with 50% absolute ethanol for 1 hour at room temperature.
Transfer to 70% Absolute ethanol for 1 hour at room temperature.
Transfer to 95% Absolute ethanol for 1 hour at room temperature.
Repeat the step 7.
Transfer to 100% absolute ethanol for 1 hour at room temperature.
Leave specimen at 4
Day 2: Clearing and infiltration
Transfer to 100% CitriSolv (Fisher Scientific, catlog 22-143-975) for 1 hour at room temperature.
Transfer to 100% CitriSolv for 1 hour at 55
Transfer to 1:1 mixture of CitriSolv/Micro-cut paraffin for 1 hour at 55
Repeat the step 3.
Transfer to 100% Micro-cut paraffin for 1 hour at 55
Repeat the step 5.
Day 3: Embedding
Transfer to 100% Micro-cut paraffin for 1–2 hours at 55
Position specimen in Peel-Away mold with 100% paraffin.
Allow specimen to harden overnight.
Specimen may be sectioned the following day.
Conduct immunohistochemistry.
Adipogenic medium: DMEM (1 g/L glucose), 10% FBS, 1% penicillin/streptomycin, 10 mg/mL insulin, 1 mM dexamethasone, 0.5 mM methylxanthine, and 200 mM indomethacin.
Passage 4 human BM-MSCs are used for adipogenic differentiation. Adipogenic differentiation is induced by adipogenic medium.
Harvest cells from passage 4, as described in the previous section.
Resuspend cells in adipogenic medium carefully.
Transfer the single cell suspension in triplicate to 6-well plates (1 × 105 cells/well).
Culture cells in the incubator at 37°C with 5% humidified CO2 for 3 weeks.
Change medium every 2–3 days.
After 3 weeks, aspirate adipogenic medium and wash cells twice with PBS.
Cells are fixed in ice-cold methanol for 10 minutes.
Aspirate methanol completely and wash cells twice with ddH2O.
Add Oil Red O staining reagent to the wells to stain lipid vacuoles at room temperature and mix slowly about 20 minutes on a shaker plate.
Aspirate Oil Red O staining reagent and wash cells twice with ddH2O.
Observe and check the stained cells with phase contrast microscopy.
CFU-F assay can be used
Collect cells from passage 4.
Prepare the single cell suspension in growth medium and seed cells in the 6-well plates in triplicate at three different densities, 1.5 × 105, 2.5 × 105, and 5 × 105 cells/well, in 2 mL growth medium, respectively.
Culture cells in the incubator at 37°C with 5% humidified CO2 for two weeks.
Change medium twice each week and check with phase contrast microscopy daily to prevent overgrowth. Stop cell culture as soon as colonies are forming visibly and proceed with the Giemsa staining of CFU-F colonies on a benchtop as follows.
Wash the culture dishes twice with PBS.
Fix cells by adding 2 mL methanol to each well for 5 minutes at room temperature.
Gently remove the methanol and discard into the bio-hazardous waste.
Air dry the culture vessels and add the diluted Giemsa staining solution for 5–10 minute at room temperature.
Remove Giemsa staining solution and wash twice with ddH2O.
Count visible colonies manually with a diameter greater than 5 mm.
There are various 2-D or 3-D, dyeing or not dyeing, co-culture models of human MSCs and other cell sources to study cell–cell interaction, cell proliferation, MSCs’ immunomodulatory capacity, and the cellular contribution of each cell type. These methods making co-cultures of MSCs and other cells are well-established, such as co-cultures of MSCs and human peripheral blood mononuclear cells [33, 34], MSCs and T cells [35], MSCs and human hematopoietic stem cells [36], MSCs and umbilical vein endothelial cells [37]. MSC-cancer cell (PC-9) co-culture will be described in this section (Figure 3).
Human BM-MSCs-PC-9 co-culture. Representative images ((A) 5×; (B) 20×) of co-cultures of human BM-MSCs labeled with CellTracker™ red dye and PC-9 cells transfected with copGFP, nuclei counterstained with DAPI.
Culture MSCs in an appropriate tissue culture ware (e.g. a 6-well plate)
Remove culture medium and wash with PBS twice.
Prepare CellTracker™ working solution in MSC growth medium. Make stock solution of lipophilic tracers in DMSO at 2 mM. Dilute the stock solution in MSC growth medium at 2 μM.
Add the working solution in the tissue culture vessel to cover the entire cell surface.
Incubate for 5 minutes or less in incubator at 37°C with 5% humidified CO2.
Remove the CellTracker™ working solution.
Wash with MSC growth medium once.
Add MSC growth medium and return the tissue culture vessel to the incubator.
PC-9 cells labeling with copGFP (Santa Cruz, sc-108083) is performed using the lentiviral technique. Lentivirus is produced by using Lipofectamine™ 2000 (Invitrogen), according to the manufacturer’s instructions. After transfection, PC-9 cells expressing copGFP may be isolated via puromycin (2 μg/mL) selection.
Continue to serially passage.
Collect PC-9 cells for cell co-culture.
Harvest MSCs with CellTrcker™ red dye and wash with PBS once.
Prepare the single cell suspension in growth medium at an appropriate cell concentration and transfer the cell suspension in a 6-well plate or a special chamber.
Culture cells in the incubator at 37°C with 5% humidified CO2 for about 3 hours to allow cells to attach.
Change fresh growth medium slowly.
Add PC-9 cells in the 6-well plate (the same cell number of MSCs). Gently tap the side of the flask.
Return the 6-well plate to the incubator and culture cell overnight.
Check the cell co-culture under the contrast microscope and image under microscopy.
The intercellular communication can be executed through a direct cell–cell interaction or through paracrine signaling mediated by a combination of active molecules. The major signaling molecules include cytokines, growth factors, chemokines, which can be generated and expressed in a wide variety of cell types including tumor cells and MSCs in response to multiple signals such as inflammatory or tumor microenvironment. Circulating MSCs are driven by such signaling molecules to home and subsequently migrate into the sites of tissue injury or disease. It is critical for the ability of MSCs to migrate and identify the injury sites for tissue repair and regeneration. Clinical data are still lacking for MSCs’ homing and distribution of transplanted MSCs in the body, albeit a large number of
There are different approaches for improvement of MSC homing and migration. In this section,
Harvest MSCs and prepare cell suspension in the serum-free medium.
Transfer the cell suspension to the upper layer of a transwell insert at a density of 4 × 104 cells/cm2 and allow cells to migrate to the lower compartment containing MSC growth medium overnight in the incubator at 37°C with 5% humidifies CO2.
Cells from the upper chamber of transwell are migrated. Gently scrape the MSCs using the cotton swab at the upper layer of the membrane.
The migrated MSCs at the lower layer are stained with 0.1% crystal violet.
Check the cells and image under a light microscope.
Count the number of stained MSCs manually.
It is well known that MSCs demonstrate biological alterations in the course of
During further cultivation, MSCs demonstrate the altered common immunological surface markers. Comparison of the early and late passages of BM-MSCs reveals that no differences are observed between passage 2 and 6 MSCs in expression of CD44, CD90, CD105, HLA-ABC, and HLA-DR, while CD106 is downregulated in MSCs of passage 6 [41]. Research has also reported that the expression pattern of the common surface markers maintains consistently with consecutive passaging up to passage 8 of BM-MSCs [40]. In contrast, the positive expression of the common surface markers such as CD73, CD90 and CD105 presents at the passage 30 of human adipose-derived MSCs (AD-MSCs) [42] and human umbilical cord MSCs (UC-MSCs) [43].
MSCs exhibit a high proliferation rare at lower passage and, however, the rapid growth kinetics decrease gradually with consecutive cell passaging. A linear correlation is observed between cumulative population doubling and days in culture up to passage 6–8 of human BM-MSCs and the passage-dependent decrease in the proliferation rate is also observed beyond that period [40]. A reduction in the proliferation in the course of long-term cultivation has been reported in human dental pulp tissue-derived MSCs [44] and human tonsil-derived MSCs [45].
The differentiation ability of human BM-MSCs vary in long-term culture manifested by the significant reduction in expression levels of the osteogenic markers, such as ALP and osteocalcin, and adipogenic markers, such as fatty acid binding protein-4 and lipoprotein lipase at the late passages [45]. It has been reported that 25% samples of BM-MSCs from different donors in the 8th passage and the 20% in the 10th passage lost their osteogenic differentiation potential [46]. Similarly, 10% BM-MSC samples in the 6th passage, the 50% in the 8th passage, and the 60% in the 10th passage also lost their adipogenic differentiation [46]. In contrast, an
Replicative senescence is known as the irreversible growth arrest of the mitotic cells and is induced by telomere shortening. The expression of senescence markers such as senescence-associated β-galactosidase, heterochromatin protein-1, and p16INK4a increase during aging [47, 48]. Molecular damage and epigenetic alterations occur in aging stem cells [49], which can result in the impairment of stem cell function.
There are various signaling pathways involved in the senescence of MSCs, including oxidative damage [50, 51], age-related defects [52], and senescence associated up-regulation of microRNAs [53]. MSC senescence can be observed with long-term
Senescent cells secrete a complex combination of interleukins, chemokines, growth factors, proinflammatory/inflammatory cytokines, which compose the senescence-associated secretory phenotype (SASP) [61, 62]. One previous report has shown that conditioned medium (CM) collected from senescent BM-MSC culture at passage 10 is able to trigger senescence in young cells [63]. The key factors of senescent MSC CM needed for triggering senescence in the young MSCs have been characterized as insulin-like growth factor binding proteins 4 and 7, which are linked to cellular senescence and apoptosis [63]. Similarly, monocyte chemoattractant proten-1 (MCP-1), as a dominant component of the SASP, is markedly increased in the conditioned medium of the late-phase MSCs and MCP-1 treatment significantly increase the senescence phenotypes of umbilical cord blood-derived MSCs via its cognate receptor chemokine receptor 2 signaling cascade [64]. Senescence-associated changes are observed in the metabolome of MSCs during replicative senescence, including down-regulation of nicotinamide ribonucleotide and up-regulation of orotic acid, which may be used to monitor the cellular senescent state during culture expansion of MSCs [65].
Sarcoma represents a very heterogeneous group of relatively rare tumors and a variety of different studies have investigated to support the MSC origin of sarcoma. There are a number of cellular and molecular mechanisms of MSC transformation for better understanding of MSCs’ contribution to sarcomagenesis [66]. The majority of published research articles indicate that various sarcoma types have been shown MSCs’ origin. Several group have reported spontaneous transformation in human and murine MSCs after long term culture [67, 68, 69, 70]. For example, one study has reported that murine BM-MSCs are spontaneously transformed at passage 29 under standard conditions and that these transformed MSCs are able to generate fibrosarcoma in immunocompromised mice [70]. Accumulated chromosomal abnormalities, such as chromosome instability, chromosomal imbalances and aneuploidy, are suggested to be associated with the transformation of BM MSCs [70]. Indeed, chromosomal aberrations (chromosomal level) in
There are also studies that have not detected the transformation of MSCs in long-term culture [74, 75, 76]. One previous study reports that human BM-MSCs do not undergo malignant transformation after long-term
MSCs provide huge opportunities in translational medicine for treatment of a range of diseases or medical conditions. MSCs are multipotent stem cells and such cells can be isolated from various tissues including bone marrow, a major source of human MSCs. Given that a large number of MSCs are required for the clinical application,
This work was supported in part by Henan Provincial Engineering Research Center for Immune Cell and Stem Cell Treatment, China and Henan Key Laboratory of Stem Cell Differentiation and Modification, China.
No competing interests for this work.
AD-MSCs | Adipose-derived mesenchymal stem cell/stromal cells |
ALP | Alkaline phosphatase |
AsAP | Ascorbic acid phosphate |
BM-MSCs | Bone marrow-derived MSCs |
CM | Conditioned medium |
CFU-F | Colony forming unit-fibroblast |
DMEM | Dulbecco’s modified Eagle’s medium |
DMSO | Dimethyl sulphoxide |
EDTA | Ethylenediaminetetraacetic acid |
FBS | Fetal bovine serum |
ISCT | International Society for Cellular Therapy |
MCP-1 | Monocyte chemoattractant proten-1 |
MHC | Major histocompatibility complex |
MSCs | Mesenchymal stem cell/stromal cells |
PBS | Phosphate buffered saline |
SASP | Senescence-associated secretory phenotype |
UC-MSCs | Umbilical cord mesenchymal stem cell/stromal cells |
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In addition, it reports the involvement of antioxidant enzymes in the tolerance of plants to various stresses.",book:{id:"5066",slug:"abiotic-and-biotic-stress-in-plants-recent-advances-and-future-perspectives",title:"Abiotic and Biotic Stress in Plants",fullTitle:"Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives"},signatures:"Andréia Caverzan, Alice Casassola and Sandra Patussi Brammer",authors:[{id:"176303",title:"Dr.",name:"Alice",middleName:null,surname:"Casassola",slug:"alice-casassola",fullName:"Alice Casassola"},{id:"176409",title:"Dr.",name:"Andréia",middleName:null,surname:"Caverzan",slug:"andreia-caverzan",fullName:"Andréia Caverzan"},{id:"176410",title:"Dr.",name:"Sandra",middleName:null,surname:"Patussi Brammer",slug:"sandra-patussi-brammer",fullName:"Sandra Patussi Brammer"}]},{id:"49289",doi:"10.5772/61442",title:"Abiotic and Biotic Elicitors–Role in Secondary Metabolites Production through In Vitro Culture of Medicinal Plants",slug:"abiotic-and-biotic-elicitors-role-in-secondary-metabolites-production-through-in-vitro-culture-of-me",totalDownloads:6998,totalCrossrefCites:40,totalDimensionsCites:106,abstract:"Plant secondary metabolites are having the great application in human health and nutritional aspect. Plant cell and organ culture systems are feasible option for the production of secondary metabolites that are of commercial importance in pharmaceuticals, food additives, flavors, and other industrial materials. The stress, including various elicitors or signal molecules, often induces the secondary metabolite production in the plant tissue culture system. The recent developments in elicitation of plant tissue culture have opened a new avenue for the production of secondary metabolite compounds. Secondary metabolite synthesis and accumulation in cell and organ cultures can be triggered by the application of elicitors to the culture medium. Elicitors are the chemical compounds from abiotic and biotic sources that can stimulate stress responses in plants, leading to the enhanced synthesis and accumulation of secondary metabolites or the induction of novel secondary metabolites. Elicitor type, dose, and treatment schedule are major factors determining the effects on the secondary metabolite production. The number of parameters, such as elicitor concentrations, duration of exposure, cell line, nutrient composition, and age or stage of the culture, is also important factors influencing the successful production of biomass and secondary metabolite accumulation. This chapter reviews the various abiotic and biotic elicitors applied to cultural system and their stimulating effects on the accumulation of secondary metabolites.",book:{id:"5066",slug:"abiotic-and-biotic-stress-in-plants-recent-advances-and-future-perspectives",title:"Abiotic and Biotic Stress in Plants",fullTitle:"Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives"},signatures:"Poornananda M. Naik and Jameel M. Al–Khayri",authors:[{id:"176282",title:"Prof.",name:"Jameel M.",middleName:null,surname:"Al-Khayri",slug:"jameel-m.-al-khayri",fullName:"Jameel M. Al-Khayri"},{id:"176284",title:"Dr.",name:"Poornananda M.",middleName:null,surname:"Naik",slug:"poornananda-m.-naik",fullName:"Poornananda M. Naik"}]}],mostDownloadedChaptersLast30Days:[{id:"66996",title:"Ethiopian Common Medicinal Plants: Their Parts and Uses in Traditional Medicine - Ecology and Quality Control",slug:"ethiopian-common-medicinal-plants-their-parts-and-uses-in-traditional-medicine-ecology-and-quality-c",totalDownloads:4059,totalCrossrefCites:6,totalDimensionsCites:10,abstract:"The main purpose of this review is to document medicinal plants used for traditional treatments with their parts, use, ecology, and quality control. Accordingly, 80 medicinal plant species were reviewed; leaves and roots are the main parts of the plants used for preparation of traditional medicines. The local practitioners provided various traditional medications to their patients’ diseases such as stomachaches, asthma, dysentery, malaria, evil eyes, cancer, skin diseases, and headaches. The uses of medicinal plants for human and animal treatments are practiced from time immemorial. Stream/riverbanks, cultivated lands, disturbed sites, bushlands, forested areas and their margins, woodlands, grasslands, and home gardens are major habitats of medicinal plants. Generally, medicinal plants used for traditional medicine play a significant role in the healthcare of the majority of the people in Ethiopia. The major threats to medicinal plants are habitat destruction, urbanization, agricultural expansion, investment, road construction, and deforestation. Because of these, medicinal plants are being declined and lost with their habitats. Community- and research-based conservation mechanisms could be an appropriate approach for mitigating the problems pertinent to the loss of medicinal plants and their habitats and for documenting medicinal plants. Chromatography; electrophoretic, macroscopic, and microscopic techniques; and pharmaceutical practice are mainly used for quality control of herbal medicines.",book:{id:"8502",slug:"plant-science-structure-anatomy-and-physiology-in-plants-cultured-in-vivo-and-in-vitro",title:"Plant Science",fullTitle:"Plant Science - Structure, Anatomy and Physiology in Plants Cultured in Vivo and in Vitro"},signatures:"Admasu Moges and Yohannes Moges",authors:[{id:"249746",title:"Ph.D.",name:"Admasu",middleName:null,surname:"Moges",slug:"admasu-moges",fullName:"Admasu Moges"},{id:"297761",title:"MSc.",name:"Yohannes",middleName:null,surname:"Moges",slug:"yohannes-moges",fullName:"Yohannes Moges"}]},{id:"63148",title:"Domestic Livestock and Its Alleged Role in Climate Change",slug:"domestic-livestock-and-its-alleged-role-in-climate-change",totalDownloads:15897,totalCrossrefCites:0,totalDimensionsCites:0,abstract:"It is very old wisdom that climate dictates farm management strategies. In recent years, however, we are increasingly confronted with claims that agriculture, livestock husbandry, and even food consumption habits are forcing the climate to change. We subjected this worrisome concern expressed by public institutions, the media, policy makers, and even scientists to a rigorous review, cross-checking critical coherence and (in)compatibilities within and between published scientific papers. Our key conclusion is there is no need for anthropogenic emissions of greenhouse gases (GHGs), and even less so for livestock-born emissions, to explain climate change. Climate has always been changing, and even the present warming is most likely driven by natural factors. The warming potential of anthropogenic GHG emissions has been exaggerated, and the beneficial impacts of manmade CO2 emissions for nature, agriculture, and global food security have been systematically suppressed, ignored, or at least downplayed by the IPCC (Intergovernmental Panel on Climate Change) and other UN (United Nations) agencies. Furthermore, we expose important methodological deficiencies in IPCC and FAO (Food Agriculture Organization) instructions and applications for the quantification of the manmade part of non-CO2-GHG emissions from agro-ecosystems. However, so far, these fatal errors inexorably propagated through scientific literature. Finally, we could not find a clear domestic livestock fingerprint, neither in the geographical methane distribution nor in the historical evolution of mean atmospheric methane concentration. In conclusion, everybody is free to choose a vegetarian or vegan lifestyle, but there is no scientific basis, whatsoever, for claiming this decision could contribute to save the planet’s climate.",book:{id:"7491",slug:"forage-groups",title:"Forage Groups",fullTitle:"Forage Groups"},signatures:"Albrecht Glatzle",authors:[{id:"252990",title:"Dr.",name:"Albrecht",middleName:null,surname:"Glatzle",slug:"albrecht-glatzle",fullName:"Albrecht Glatzle"}]},{id:"66714",title:"Biotic and Abiotic Stresses in Plants",slug:"biotic-and-abiotic-stresses-in-plants",totalDownloads:5808,totalCrossrefCites:54,totalDimensionsCites:96,abstract:"Plants are subjected to a wide range of environmental stresses which reduces and limits the productivity of agricultural crops. Two types of environmental stresses are encountered to plants which can be categorized as (1) Abiotic stress and (2) Biotic stress. The abiotic stress causes the loss of major crop plants worldwide and includes radiation, salinity, floods, drought, extremes in temperature, heavy metals, etc. On the other hand, attacks by various pathogens such as fungi, bacteria, oomycetes, nematodes and herbivores are included in biotic stresses. As plants are sessile in nature, they have no choice to escape from these environmental cues. Plants have developed various mechanisms in order to overcome these threats of biotic and abiotic stresses. They sense the external stress environment, get stimulated and then generate appropriate cellular responses. They do this by stimuli received from the sensors located on the cell surface or cytoplasm and transferred to the transcriptional machinery situated in the nucleus, with the help of various signal transduction pathways. This leads to differential transcriptional changes making the plant tolerant against the stress. The signaling pathways act as a connecting link and play an important role between sensing the stress environment and generating an appropriate biochemical and physiological response.",book:{id:"8015",slug:"abiotic-and-biotic-stress-in-plants",title:"Abiotic and Biotic Stress in Plants",fullTitle:"Abiotic and Biotic Stress in Plants"},signatures:"Audil Gull, Ajaz Ahmad Lone and Noor Ul Islam Wani",authors:null},{id:"62573",title:"Introductory Chapter: Terpenes and Terpenoids",slug:"introductory-chapter-terpenes-and-terpenoids",totalDownloads:7556,totalCrossrefCites:27,totalDimensionsCites:51,abstract:null,book:{id:"6530",slug:"terpenes-and-terpenoids",title:"Terpenes and Terpenoids",fullTitle:"Terpenes and Terpenoids"},signatures:"Shagufta Perveen",authors:[{id:"192992",title:"Prof.",name:"Shagufta",middleName:null,surname:"Perveen",slug:"shagufta-perveen",fullName:"Shagufta Perveen"},{id:"192994",title:"Dr.",name:"Areej",middleName:null,surname:"Al-Taweel",slug:"areej-al-taweel",fullName:"Areej Al-Taweel"}]},{id:"62876",title:"Introduction to Phytochemicals: Secondary Metabolites from Plants with Active Principles for Pharmacological Importance",slug:"introduction-to-phytochemicals-secondary-metabolites-from-plants-with-active-principles-for-pharmaco",totalDownloads:5802,totalCrossrefCites:10,totalDimensionsCites:25,abstract:"Phytochemicals are substances produced mainly by plants, and these substances have biological activity. In the pharmaceutical industry, plants represent the main source to obtain various active ingredients. They exhibit pharmacological effects applicable to the treatment of bacterial and fungal infections and also chronic-degenerative diseases such as diabetes and cancer. However, the next step in science is to find new ways to obtain it. In this chapter, we discuss about the main groups of phytochemicals, in addition to presenting two case studies. One of the most important secondary metabolites is currently Taxol, which is a natural compound of the taxoid family and is also known for its antitumor activity against cancer located in breasts, lungs, and prostate and is also effective with Kaposi’s sarcoma. Our case studies will be about Taxol, extracted from an unexplored plant species, and the production of Taxol by its endophytic fungi.",book:{id:"6794",slug:"phytochemicals-source-of-antioxidants-and-role-in-disease-prevention",title:"Phytochemicals",fullTitle:"Phytochemicals - Source of Antioxidants and Role in Disease Prevention"},signatures:"Nadia Mendoza and Eleazar M. Escamilla Silva",authors:[{id:"51406",title:"Dr.",name:"Eleazar",middleName:"Máximo",surname:"Escamilla Silva",slug:"eleazar-escamilla-silva",fullName:"Eleazar Escamilla Silva"},{id:"243304",title:"Ph.D. Student",name:"Nadia",middleName:null,surname:"Mendoza",slug:"nadia-mendoza",fullName:"Nadia Mendoza"}]}],onlineFirstChaptersFilter:{topicId:"41",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:0,limit:8,total:null},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:9,numberOfPublishedChapters:89,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:104,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:32,numberOfPublishedChapters:318,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:12,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:11,numberOfPublishedChapters:141,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:8,numberOfPublishedChapters:129,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!0},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:113,numberOfOpenTopics:3,numberOfUpcomingTopics:1,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:11,numberOfPublishedChapters:105,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:2,numberOfUpcomingTopics:1,issn:"2753-894X",doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:5,numberOfOpenTopics:1,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!0},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:0,numberOfPublishedChapters:15,numberOfOpenTopics:5,numberOfUpcomingTopics:0,issn:null,doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. The whole process of submitting an article and editing of the submitted article goes extremely smooth and fast, the number of reads and downloads of chapters is high, and the contributions are also frequently cited.",author:{id:"55578",name:"Antonio",surname:"Jurado-Navas",institutionString:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRisIQAS/Profile_Picture_1626166543950",slug:"antonio-jurado-navas",institution:{id:"720",name:"University of Malaga",country:{id:null,name:"Spain"}}}},{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}}]},series:{item:{id:"24",title:"Sustainable Development",doi:"10.5772/intechopen.100361",issn:null,scope:"