Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM-Free Reprogramming to Pluripotency

Abstract

The generation of induced pluripotent stem cells (iPSCs) by forced expression of defined transcription factors has revolutionized regenerative medicine. These cells have similar features to embryonic stem cells (ESCs) regarding self-renewal and their ability to differentiate into any cell type in the body. In spite of many improvements, in using nonviral delivery reprogramming methods, there are still challenges to overcome regarding safety before patient-made iPSCs can be used in regular clinical practice. We have recently reported about a gene manipulation-free method of generating human pluripotent stem cells (PSCs), based on activation of the novel human GPI-linked glycoprotein ACA. The process of dedifferentiation of blood progenitor cells that leads to the generation of blood-derived pluripotent stem cells (BD-PSCs) is initiated upon cross-linking of this protein via activation of PLCγ/PI3K/Akt pathway. These cells are mortal, express pluripotent markers, and redifferentiate in vitro into cells of all three germ layers. The ultrastructural analysis of BD-PSCs, by means of electron microscopy, revealed them similar to human ESCs with large dense nucleolus and scarce cytoplasm. BD-PSCs are autologous stem cells and while nonteratogenic offer a new alternative that overcomes immunological, ethical, and safety concerns and opens up a new avenue in treating contemporarily intractable diseases and generally in human therapeutics.

1. Introduction

Human induced pluripotent stem cell (iPSC) technology has paved the way for new possibilities to investigate and potentially cure diseases. The iPSCs derived from patients can be used in at least two ways: regenerative medicine and drug discovery, for example, screening chemicals, natural compounds, and derivatives to identify drug candidates. This new technology promises to provide a powerful tool for modeling human pathology that allows for investigation and understanding of the underlying mechanisms and causes of various human diseases. Particularly, disease-specific iPSCs are of great potential for disease modeling and therapeutic benefits [1, 2].

iPSCs have characteristics of human embryonic stem cells (ESCs), including pluripotency and potentially unlimited self-renewal. During the last decade, patient-made iPSCs have been differentiated into a variety of functional cell types in vitro and are expected to reconstruct disease phenotypes, as already demonstrated in several animal disease models [3].

Originally, iPSCs were generated by retroviral transduction of four specific transcription factors, Oct3/4, Sox2, Klf4, and cMyc or Oct3/4 Sox2/Nanog/LIN, using retroviral or lentiviral vectors [4, 5]. Later, lentivirus was the preferred delivery method, since, unlike retrovirus can infect proliferating and nondividing cells. Viral vectors for iPSC generation are very effective for integrating exogenous genes into the genome of somatic cells; however, they could be permanently integrated into the cell’s genome, which generates serious concerns about changes in cell behavior and therefore, limiting their use in patients [6].

Despite the fact that iPSCs, as well as ESCs, are being proclaimed to have a great advantage as a source of stem cells that can be used in regenerative medicine, the ultimate goal to use them in clinical practice has not been achieved.

Cells convert one kind of signal into another through a process called signal transduction. This mechanism comprises the coupling of a ligand-receptor interaction to many intracellular events. These events include phosphorylation by tyrosine kinases and/or serine/threonine kinases. Differential localization of protein that participates in signaling pathways is essential for cells to respond efficiently to changes in their environment. In the plasma membrane, such compartmentalization is performed through lipid rafts [7] that are enriched in cholesterol, sphingolipids, and GPI-anchored proteins. During the signaling processes, various lipids are phosphorylated, recruited, and activated by different signaling components, which are essential for the regulation of cell survival and growth.

PI3Ks are a family of intracellular signal transducer enzymes involved in a variety of cellular functions like growth proliferation and differentiation. These enzymes are capable of phosphorylating position 3 of the inositol ring of phosphatidylinositol (PtdIns), and this lipid modification initiates a set of events that leads to cell activation and growth [8].

We have recently shown that human GPI-linked glycoprotein ACA, expressed in all adult stem cells, including hESCs, is involved in developmental process, which shapes the human embryo and controls adult stem cell compartments. Activation by this protein on the membrane of human blood progenitor cells drives membrane-to-nucleus signaling pathways, thus regulating pluripotency [9].

We investigate here the signaling machinery behind the antibody cross-linking activation of GPI-linked membrane glycoprotein ACA that drives the immature blood progenitor cells to pluripotency and the capacity of these cells to redifferentiate into cells belonging to different germ layers.

The newly generated human blood-derived (BD-PSCs) as well as their redifferentiated progeny was assessed by the methods of immunocytochemistry (ICC), flow cytometry, and electron microscopy (EM). Signaling competence of ACA receptor was analyzed by studying the phosphorylation pattern and real-time analysis of developmentally relevant genes, such as NOTCH, WNT, CTNNB, C-KIT, and others.

2. Materials and methods

2.1 Cell cultures

Human mononuclear cells (MNCs) were isolated from peripheral blood (PB) samples obtained from healthy donors. All of the human blood samples were used after obtaining written informed consent from the donors. PBMNCs were isolated by density gradient centrifugation using Ficoll and activated by specific antibody cross-linking as described elsewhere. Briefly, 6×10⁶ MNCs in a 15 ml polystyrol tube were incubated for 30 min with antibody (30 μg/mL in 1% PBS/BSA) to GPI-linked membrane protein ACA and further cultured and maintained in suspension in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS (Gibco Life Technology, Grand Island, NY), 0.1 mM nonessential amino acids (NEAA), 1 mM L-glutamine (all from Invitrogen, Carlsbad CA). The cells were taken at different time points for immunophenotyping (IP) by flow cytometry, ICC, and western blot (WB) analysis.

For growing of human ESCs line H9 (Wi Cell Inc., Medison, Wi) on feeder cells, mouse embryonic fibroblasts (MEFs), obtained from mouse strain CF-1 (American Type Culture Collection Manassas, VA, USA) mitotically inactivated by radiation were prepared according to standard protocol and approved (07-WO25) by the ethics committee established at Príncipe Felipe Centro de Investigación (CIPF) in Valencia (Spain), where these experiments were conducted. Human ES cells were placed on a freshly prepared MEF layer and further cultivated in ESC medium consisting of Knockout-DMEM (Invitrogen, Carlsbad CA), 100 μM β-mercaptoethanol (Sigma, St Lois), 1 mM L-glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen) and 8 ng/ml bFGF (Invitrogen), or in condition media (see below). The medium was changed every other day. Recovery of pluripotent phenotype of the differentiated H9 cells line was done by cross-linking the membrane of these cells with a GPI protein-specific antibody. Cells were maintained in IMDM supplemented with 10% FBS.

The culture of BD-PSCs on feeder-free culture dishes was performed to assess the expression of hESC markers by means of immunofluorescence. For that purpose, BD-PSCs were grown in MEF-conditioned media supplemented with 8 ng/ml bFGF on culture dishes coated with Matrigel 1:30 (BD Bioscience, Franklin Lakes, NJ).

MNCs isolated from PB were cultured in Iscove’s media supplemented with 10% FBS, preincubated with the inhibitors Et-18-CH₃ (ET) at 50 μM, LY 294002 (LY) at 20 μM or ET + LY purchased from Calbiochem (USA) for 1 h, and after activation in the presence of inhibitors cultured at 37°C, as specified. After labeling with CD34APC, CD45FITC (BD Pharmingen), and CD14PE (eBioscience), the generation of CD34 cells was daily assessed by multiple flow cytometry analyses. The nonviable cells were excluded after performing a propidium iodide (PI) assay. Conjugated isotype-matched irrelevant antibodies were used as controls.

Antibodies to SSEA-4 and TRA-1-81 purchased by Chemicon (Temecula, CA) labeled with phycoerythrin (PE) were used for phenotypic analysis of pluripotent markers expressed on BD-stem cells by means of flow cytometry. Gating was done with matched isotype control monoclonal antibodies. Conjugated isotype-matched irrelevant mAbs were used as controls.

2.2 BD-PSCs differentiation toward neuronal and hepatic cells

Differentiation to neuronal lineages was adapted from previously described protocol [10]. Briefly, BD-PSCs were grown on glass coverslips coated with 1:5 diluted poly-L-ornithin/laminin for 2 days in neuronal initiating medium N2, followed by neuronal differentiation medium (Neurobasal medium, L-glutamin, B27 supplement, nonessential amino acids (NEAA), recombinant human glial-derived neurotrophic factor (GDNF), recombinant human brain-derived neurotrophic factor (BDNF), and ascorbic acid solution. The cells were grown at 37°C, 5% CO₂ for 1−30 days. Cells were taken at D8, D20, and D30 for ICC analysis.

Differentiation of BD-PSCs toward endoderm/hepatocytes was performed on Biolaminin 111 (Biolamina Sundbyberg, Sweden) treated glass coverslips in KSR/DMSO media consisting of 80% of knockout DMEM media (KO-DMEM), 20% knockout serum replacement, 0.5% NEAA (all from Invitrogen), 0.1% β-mercaptoethanol, 1% DMSO (Sigma), and 1% penicillin–streptomycin for 7 days, followed by culturing cells in HepatoZYME maturation medium, 1% glutamax, 10 μM hydrocortisone 21-hemisuccinate sodium salt (HCC) (Biomol, Hamburg, Germany), supplemented with 10 ng/mL hepatocyte growth factor (HGF) (Life Technology) and 2 ng oncostatin M (OSM) (Biotechne), for additional 2 weeks. The cells were taken at D7 and D21 for ICC analysis.

2.3 Cell culture, inhibition, and western blot analysis

MNCs isolated from PB were cultured in Iscove’s media supplemented with 10% FBS, preincubated with the inhibitors ET at 50 μM, LY at 20 μM, and PD098059 (PD) at 10 μM, or (ET + LY + PD) purchased from Calbiochem (USA) for 1 h, and after activation in the presence of inhibitors cultured at 37°C, 5% CO₂. Cell-free extracts of these cells were subjected to western blot analysis. Nonactivated MNCs were used as controls.

2.4 Western immunoblotting

Cell lysis, protein extraction, and western blot analysis of ACA-activated PBMNCs vs. nontreated samples were performed as described elsewhere [9, 11]. Briefly, cells were lysed in Triplex buffer (50 mM Tris HCl pH 8, 120 mM NaCl, 0.1% SDS, 1% Nonidet P-40, and 0.54% deoxycholate), 300 μg of protein extracts were submitted to electrophoresis by using 10% SDS-PAGE. Immunodetection was performed by using appropriate primary antibodies followed by incubation with HPR-conjugated secondary antibodies (all purchased by cell Signaling technology, GAPDH antibody by Santa Cruz biotechnology). ECL Western blotting substrate (Pierce) was used for the detection of proteins on PVDF.

2.5 Quantitative PCR analysis

Total RNA was extracted from cells using TRIzol (Invitrogen) and transcribed into cDNA using oligo (dT) 16 and ReverTra Ace reverse transcriptase. PCR reactions were carried out by mixing 1 μL of cDNA template, 250 nM of each primer, 200 μM dNTP mixture, and 1 U of Taq DNA polymerase in a total volume of 20 μL. Samples were amplified in a thermocycler. For qPCR, each sample was analyzed in triplicate with GADH as the internal control. Amplification data were collected using ABI PRISM 7900 and analyzed using the sequence detection system 2.0 software. The primers and TaqMan probes used in this experiment are presented in Table 1.

Gene	Assay identification	Exon boundary	Assay location	NCBI reference sequence	Amplicon length
hGAPDH	Hs99999905_m1	3−3	157	NM_002046.3	122
hc-KIT	Hs00174029_m1	1−2	158	NM_001093772.1	64
hHOXB4	Hs00256884_m1	1−2	526	NM_024015.4	54
hCTNNB1	Hs00170025_m1	7−8	1351	NM_001098210.1	88
hWNT10B	Hs00559664_m1	3−4	684	NM_003394.2	63
hNOTCH	Hs00413187_m1	4−5	745	NM_017617.3	95
hBCL2	Hs00153350_m1	2−3	1079	NM_000633.2	96
hBMI	Hs00180411_m1	3−4	719	NM_005180.5	105
hTGFβ	Hs99999918_m1	4−5	1583	NM_000660.3	125

Table 1.

List of primers and TaqMan probes used in Figure 1d.

2.6 Immunocytochemistry (ICC)

Immunofluorescence analysis of cells growing on Matrigel (Corning CA) was first performed to evaluate the presence of pluripotent stem cell markers, such as SSEA-4, TRA-1-60, and TRA-1-81. All antibodies were purchased by chemicon. Secondary PE or Alexa Fluor 488 labeled antibody (Life Technology, Carlsbad CA) was used to reveal the expression of pluripotent markers on BD-PSCs.

Activated PBMNCs growing in suspension were taken at different time points during culture time period from D1−D14, transferred on glass coverslips, coated with Poly-L-lysine (Sigma-Aldrich St. Louis, USA), and the acquisition of pluripotent markers, TRA-1-60, SOX2, NANOG, and OCT3/4 monitored by means of ICC. Cells were fixed in 4% paraformaldehyde (PFA) for 15 min at RT followed by permeabilization with 0.1% Triton X-100 in PBS for 30 min in phosphate buffer saline (PBS) containing 3% BSA. All directly labeled primary antibodies were diluted in the same blocking buffer and incubated with samples overnight at 4°C. The nuclei were stained with DAPI (Sigma-Aldrich) for 3 min at RT. All images were acquired with an inverted Olympus IX71 Microscope. All antibodies used in this experiment are presented in Table 2.

antibody	Clone	Isotype	Fluorochrome
anti-human Nanog	N31–355	Mouse IgG1, ƙ	Alexa Fluor® 488
anti-Oct3/4	40/oct-3	Mouse IgG1, ƙ	Alexa Fluor® 488
anti-Sox2	245,610	Mouse IgG2a	Alexa Fluor® 647
anti-human TRA-1-60 antigen	TRA-1-60	Mouse (BALB/c) IgM, ƙ	Alexa Fluor® 488

Table 2.

Directly labeled antibodies from BD Pharmingen (California, US) are used in Figure 2.

ICC of human neuronal cells generated from BD-PSCs was performed using antibodies to Nestin, GFAP, MAP2, Neun, and Tuj1.

Cells were fixed with prewarmed fixative (PBS, PFA, MgCL₂, EGTA, and sucrose) for 15 min, then treated with 0.3% Triton X-100 in PBS containing 3% BSA for 5 min as previously described [10]. Appropriate dilution of antibodies was prepared in PBS containing 1% BSA and incubated for 1.5 h, at RT, washed three times with PBS, and anti-chicken, anti-rabbit, and anti-mouse fluorochrome-conjugated antibodies were used to reveal the expression of specific neuronal markers (antibodies used in this experiment are presented in Tables 3 and 4). DAPI was used for staining the nuclei of cells. All images were acquired with an inverted Olympus IX71 microscope.

Primary Antibodies	Clone	Isotype	Company
anti-Nestin	10C2	Mouse, IgG1, monoclonal	Antibodies online
anti-GFAP		Rabbit, polyclonal	Dako
anti-MAP2	Ap20	Mouse, IgG1, monoclonal	BD Pharmingen
anti-NeuN	1B7	Mouse, IgG2a, monoclonal	Antibodies online
anti-Tuj1		Chicken, polyclonal	Antibodies online

Table 3.

Primary antibodies used in Figure 3.

Secondary Antibodies	Isotype	Fluorochrome	Company
Goat anti-Mouse	IgG, polyclonal	DyLight 488	Antibodies online
Goat anti-Mouse	IgG, polyclonal	Alexa Fluor 647	Life Technologies
Goat anti-Rabbit	IgG, polyclonal	Alexa Fluor 488	Invitrogen
Rabbit anti-Chicken	IgG, polyclonal	Texas Red	Antibodies online

Table 4.

Secondary antibodies used in Figure 3.

BD-PSC Differentiation to human endoderm/hepatocytes was assessed by means of ICC using antibodies to alpha-fetoprotein (AFP), anti-transthyretin (TTR) (endoderm), and anti-Albumin (ALB), anti-Hepatocyte Nuclear Factor 4 alpha (HNF4 α) (hepatocytes) and their relevant fluorescent-labeled secondary antibodies (all antibodies used are presented in Tables 5 and 6). Cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton-X-100 for 3 min. DAPI was used for nuclear staining. Expressions of these markers were visualized with an inverted Olympus Microscope CKX53.

Primary Antibodies	Clone	Isotype	Company
anti-human alpha-1-Fetoprotein		Rabbit, polyclonal	DakoCytomation
anti-Transthyretin		Chicken, polyclonal	Sigma-Aldrich
anti-Albumin		Chicken, polyclonal	Sigma-Aldrich
anti-HNF-4alpha	4C19	ZooMab Rabbit, monoclonal	Merck

Table 5.

Primary antibodies used in Figure 4.

Secondary Antibodies	Isotype	Fluorochrome	Company
Goat-anti-Chicken	IgY(H + L),polyclonal	CF 488A	Sigma Aldrich
Rabbit anti-Chicken	IgG, polyclonal	Texas Red	Antibodies online
Goat anti-Rabbit	IgG, polyclonal	Alexa Fluor 488	Invitrogen

Table 6.

Secondary antibodies used in Figure 4.

2.7 Electron microscopy

Cells were seeded at 6.25×10⁵cells/cm² in 8-well Lab-Tek chamber slides (Nalgene Nunc International, Naperville, IL). Cells were fixed in 3.5% glutaraldehyde for 1 h at 37°C, postfixed in 2% OsO₄ for an additional 1 h at RT, and stained in 2% uranyl acetate in the dark at 4°C for 2 h. Finally, cells were rinsed with distilled water, dehydrated in ethanol, and embedded in Durcupan (Fluka) epoxy resin overnight. Following resin hardening, embedded cultures were detached from the chamber slide and glued to araldite blocks. Serial semi-thin sections (1.5 μm) were cut with an Ultracut UC-6 (Leica, Heidelberg, Germany), mounted onto glass slides, and lightly stained with 1% toluidine blue. Selected semi-thin sections were glued with SuperGlue-3 Loctite (Henkel, Düsseldorf, Germany) to resin blocks and detached from the glass-slide by repeated freezing (in liquid nitrogen) and thawing. Ultrathin sections (60−80 nm) were prepared with an ultramicrotome and contrasted with lead citrate. Finally, photomicrographs were obtained at 80 kV using an FEI Tecnai G² Spirit transmission electron microscope (FEI Europe, Eindhoven, and Netherlands) equipped with a Morada CCD digital camera (Olympus Soft Image Solutions GmbH, Münster, Germany).

3. Results

3.1 Membrane-to-nucleus signaling induced by ACA activation

To investigate a phosphorylation pattern across the plasma membrane, we activated GPI-linked protein by means of ACA-antibody cross-linking. The cell-free extracts were prepared from peripheral blood (PB) cells after Ficoll gradient centrifugation before and 6 days after activation. The evaluation of the expression and phosphorylation status of the proteins, presumably involved in this signaling network, was assessed by western blot analysis.

As shown in Figure 1(a), antibody cross-linking induces PI3K activation that phosphorylates and activates the known members of this pathway described in [12].

Figure 1.

Relative protein expression and phosphorylation status of mediators of ACA-signaling and its inhibition with pharmacological inhibitors. The PBMNCs activated cells were cultured either in presence or absence of inhibitors ET, LY, PD, or ET + LY. Relative protein expression and phosphorylation status of mediators of this signaling were determined by western blot analysis. Nonactivated mononuclear cells isolated from PB were used as controls. (a): Western blotting of cell lysate derived from activated PBMNCs expression and phosphorylation status of proteins involved in the signaling mechanisms. (b): The PB-generated progenitor/HSCs were cultured either in presence or absence of inhibitors ET, LY, PD, or ET + LY. Nonactivated MNCs isolated from PB were used as controls. ET, LY, and ET + LY inhibited membrane protein antibody cross-linking induced generation of CD34 cells. Flow cytometry shows the generation of CD34 cells in the presence or absence of ET, LY, or ET + LY respectively in a 1−5 days’ culture time period. (c): The total number of viable CD34 cells in cultures stimulated with or without ET, LY or ET + LY was estimated by flow cytometry. Data are presented as the mean ± SEM for three independent experiments. (d): Regulation of gene expression after GPI-anchored protein-induced generation of BD-PSCs. Living cells were isolated before (D1) and after activation at D6 and D12. The total RNA of these cells was reverse transcribed, and the expression of the following genes was studied by real-time analysis: CTNNB1 HOXB4, C-KIT, NOTCH1, WNT10, BMI1, TGFB1, and BCL2. Represented is the average of triplicate gene-expression changes measured by TaqMan as described in Methods.

The phosphoinositol-phospholipase γ (PLCγ) is a member of family of PLC enzymes consisting of various isoforms with different cellular functions. PLCγ is linked to tyrosine kinase signaling pathways with its primary function to catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphat (PIP2) to generate inositol (1,4,5)-triphosphate (PIP3) and 1,2-diacylglycerol (DAG). PIP3 initiates an increase in intracellular, whereas DAG activates protein kinase C, and the control over this important second messenger pathway is a key to changes in cellular activity and function [13]. Phosphorylation of PLCγ and its activation seemed to be the earliest event in ACA-initiated specific signaling network, leaving the hydrolytic products of this protein, like inositol phosphate and a diacyl-myristate, the latter known as the most powerful activator of PI3K.

To evaluate the function and role of PLC in the signaling pathway, we used the specific inhibitors of PLCγ, ET-18-0-CH₃ (ET), as well as the inhibitors of various kinases involved in this signaling like LY 294002 (LY) inhibitor of PI3K, and the MAP kinase inhibitor, PD098059 (PD). Peripheral blood mononuclear cells (PBMNCs) were preincubated with these inhibitors and cultured for 1−6 days after activation. Relative protein expression and phosphorylation status of various mediators of the initiated signaling were assessed by western blot analysis. Nonactivated PBMNCs were used as controls. The inhibitor of PLCγ caused a partial suppression of expression as well as reduced phosphorylation extent of various kinases involved in this signaling, whereas specific inhibitors of PI3K or MAP kinase alone had no effect on the initialization of the signaling (data not shown). Conversely, preincubation with all three inhibitors caused significant suppression of protein expression and phosphorylation extent of all participants (Figure 1(a)).

We further assessed the role of PLCγ and PI3K in membrane-to-nucleus-induced signaling, which first leads to generation of hematopoietic progenitor cells. This is an inevitable intermediate step on the described route to pluripotency [11]. Cells expressing CD34 protein are normally found in umbilical cord blood as well as in bone marrow cells, and antibodies to this protein are often used clinically to quantify the number of HSCs used in HSC transplantations [14]. CD34⁺ cells were generated in the presence or absence of their specific inhibitors, such as ET, LY, and ET + LY. The culture conditions were used to follow the effect of inhibitors on generation of CD34⁺ cells for the culture time period of 5 days.

PBMNCs were preincubated with ET, LY, and ET + LY and upon activation; newly generated cells were assessed by multiple flow cytometry analyses using antibodies to CD34, CD45, and CD14. The growing population of CD34⁺ during culture time, from D1−D5, was monitored. Upon inhibition with ET, we observed a slight decrease in the number of newly generated CD34⁺ cells, whereas LY alone had no effect on induced hematopoiesis. Most significantly, a dramatic decrease in fluorescence intensity occurred when both inhibitors were used together. Inhibition of PLCγ alone as well as inhibition of PLCγ and PI3K with their specific inhibitors ET + LY induced significant changes regarding de novo generation of CD34⁺ cells, whereas no changes were observed when IP3K was inhibited by using LY alone see Figure 1(b).

The same experiment was performed with PBMNCs from various donors confirming the previous findings (Figure 1(c)).

Herewith, we conclude that phosphorylation and activation of PLCγ are indispensable for initiation of the signaling cascade. The changes induced by ET during the generation of CD34⁺ cells are highly likely to be responsible for the initial events started by GPI-linked protein stimulation. This implies that phosphorylation and activation of PLCγ is a crucial event in this mediated signaling, while PI3K and AKT represent downstream activators, essential for induced route to pluripotency via generation of human hematopoietic progenitor cells.

3.2 Antibody activation upregulates the expression of developmentally relevant genes

In order to study these specific signal transduction networks, we used quantitative RT-PCR (TaqMan) analysis to determine the gene-expression pattern of the molecules potentially involved in this signaling.

We activated the GPI-linked protein at the surface of blood progenitor cells as described above and analyzed the way in which the initiated signaling machinery regulates the expression of genes known to play a role in human embryonic development via specific protein phosphorylation as an important regulatory mechanism in cellular processes.

We compared the gene expression profile of the PBMNCs before and after activation to assess a transcript level for candidate molecules, the most important among them NOTCH and WNT/CTNNB1 (Figure 1(d)). The signaling pathways linked to these genes are developmentally conserved and play a significant role in embryonic development as well as in the regulation of adult cell compartments [15].

Dysregulation of Wnt and Notch pathways due to their involvement in the key functions of human cells is a reason for their implication in many human diseases [16].

As shown in Figure 1(d), we demonstrated that GPI-linked glycoprotein upregulates both Notch and Wnt signaling pathways, thus acting in a hierarchical manner in the relationship to both signaling pathways. In consistency with the previous report about β-catenin as a downstream regulator of the canonical Wnt pathway [17], our results clearly show the upregulation of β-catenin as a consequence of this activation.

The involvement and significance of this GPI-linked protein in the signaling process regarding development and dedifferentiation of the human (ES) cell line H9 is shown in Figure 5, in which a spontaneously differentiated colony of ESCs was restored (dedifferentiated) to its primordial state, upon specific antibody cross-linking and culturing for 1 day in Iscove’s medium supplemented with 10% FBS.

Figure 2.

Inducing pluripotency in BD-progenitor/stem cells throughout cell culture time. (a-d): PBMNCs were activated by antibody-specific cross-linking and cultured in Iscove’s medium for 14 days. Aliquots were taken at D3, 8, and 14, incubated with fluorescent-labeled antibodies to pluripotent markers, and subjected to immunofluorescence microscopy. Immunofluorescence microscopy of cell cultures for 14 days showing the expression in vitro of the pluripotency markers, (a) TRA-1-60, (b) Nanog, (c) Oct3/4, and (d) Sox2, throughout the cell culture time. Nuclei were stained with DAPI. Scale bars 50 μm.

3.3 BD-PSCs are generated from unmanipulated steady-state PB

A PB sample (30 mL) of healthy donors was collected after obtaining informed consent. Mononuclear cells (MNCs) were isolated after Ficoll centrifugation and activated at the membrane by antibody cross-linking using specific antibodies. Figure 6 shows the steady growing new population of cells in time course modus from day 5 to 14, while the nonactivated PB cells, under identical culture conditions, show gradual deterioration of cell structure and function, which leads to disappearance and death of the majority of the cells from day 5 to 14 of culture time period.

Figure 3.

Ectodermal/neural differentiation of BD-PSCs in adherent monolayer from N8-N30. Immunocytochemical and immunophenotypic profiling of BD-neuronal cells throughout the culture time period from N8 to N30. Scale bars 100 μm (N8-N20) and 50 μm (N30).

3.4 Expression of ESC markers on BD-PSCs

As shown, membrane-to-nucleus signaling network initiates via PLCγ/PI3K/Akt/mTor/PTEN a process of de-differentiation of blood cells that leads via generation of HSCs to PSCs. By means of immunofluorescence, we analyzed the expression of pluripotent markers that BD-PSCs share with ESCs.

Human PSCs are characterized by specific cell surface markers, such as the glycolipid antigens SSEA-3 and SSEA-4, as well as the glycoprotein antigens TRA-1-60 and TRA-1-81. Stage-specific embryonic antigen (SSEA-4) is a glycosphingolipid expressed in early human embryonic development and PSCs and acts as a mediator of cell adhesion as well as a modulator of signal transduction. The expression of human SSEA-4 decreases following differentiation of ESCs [18]. Glycoprotein antigens TRA-1-60 and TRA-1-81 are expressed in early human embryonic development and PSCs; they also mark cells of the inner cell mass of preimplantation embryos [19].

Activated PBMNCs were grown in MEF-conditioned media in Matrigel-coated cell culture dishes for 2 weeks and immunocytochemistry (ICC) analysis was performed using antibodies to SSEA-4, TRA-1-60, and TRA-1-81. As shown in Figure 7(a−e), cell surface protein marker analysis demonstrated that BD-PSCs express the pluripotent markers SSEA-4 as well as TRA-1-60 and TRA-1-81.

Figure 4.

Endodermal/hepatocytes differentiation of BD-PSC in culture time period from L7 to L20. Shown is immunocytochemical and immunophenotypic analysis of endoderm/hepatocyte-specific markers through culture time period from L7 to L20. Scale bars 50 and 20 μm (L20 ALB/DAPI).

Unlike ESCs and PSCs, BD-PSCs can grow in suspension in Iscove’s medium supplemented with 10% FBS without any addition of cytokines or growth factors. Flow cytometry expression analysis of BD-PSCs grown in suspension revealed the expression of ESCs specific markers SSEA-4 and TRA-1-81 on BD-PSCs (Figure 7(f–m)).

3.5 Expression of the factors that maintain pluripotency

To further investigate the expression of pluripotency markers on BD-PSCs at the protein level, we extended the immunofluorescence analysis to the transcription factors Nanog, Sox2, and Oct3/4 that reside at the core of pluripotency network, where they can regulate their own expression and interact with a number of other pluripotency factors. Nanog, also called a pluripotency master molecule, is a unique homeobox transcription factor that is critical in regulating the cell fate of the pluripotent inner cell mass during embryonic development maintaining the pluripotency and blocking the differentiation of PSCs [20]. Activated preparations of PBMNCs were grown in suspension in a time-course manner from day 3 to 14. The newly generated cells were plated on Poly-L-lysine coated glass coverslips and ICC was performed using appropriate antibodies for specific pluripotency markers on BD-PSCs.

Expression of TRA-1-60, Sox2, Nanog, and Oct3/4 was induced following day 3, showing a rising trend from day 8 and had been completed at day 14.

Immunofluorescence analysis of the newly generated cells at different time points showed the gradual enhancement of the expression of the pluripotency markers on activated PBMNCs cultures. In contrast, nonactivated PBMNCs cultures showed no expression of these markers. These data are presented in Figure 2(a−d).

Figure 5.

Immunofluorescence analysis during redifferentiation process of spontaneously differentiated human ESCs initiated after membrane antibody cross-linking activation of human GPI-linked protein. A spontaneously differentiated ESCs colony was activated by antibody cross-linking incubated with TRA-1-81 and DAPI and micrographs were taken by confocal microscopy. (a): Brightfield micrograph of spontaneously differentiated ESC colony. (b): Nuclei stained with DAPI. (c): Brightfield/DAPI (merged). (d): Spontaneously differentiated ESC colony stained with DAPI. (e): Spontaneously differentiated ESC colony stained with antibody to TRA-1-81. (f): Spontaneously differentiated ESC colony stained with TRA-1-81/DAPI (merged). Scale bars 200 μm. Fluorescence images of restored (activated) pluripotency in spontaneously differentiated ESC colony. (g): Activated ESC colony stained with DAPI. (h): Activated ESC colony stained with antibody to TRA-1-81. (i): Activated ESC colony stained with TRA-1-81/DAPI (merged). Scale bars 100 μm.

Karyotype analysis was conducted on BD-PSCs by a qualified service provider (CELL Line Service Heidelberg, Germany) using standard G-banding methods as described elsewhere [21]. Analyses showed that BD-PSCs maintained a normal karyotype (data not shown).

3.6 Ultrastructural studies

Various blood donors provided blood samples for this study showed no differences among them in terms of cell morphology or ultrastructure. In vitro cell culture studies, after 1 day of dedifferentiation (D1), upon activation, resulted in the observation of a large cell population comprised of approximately 60% of agranular mononuclear cells and 40% of granular mononuclear cells (Figure 8A−C). Granular cells displaying deeply invaginated nuclei, scarce cytoplasm, and abundant primary and secondary granules disappeared from the culture after 5 days. Furthermore, red blood cells and platelets were detected. The population of interest for this study are agranular cells, morphologically characterized by small size and a nucleus with condensed chromatin (Figure 8B−C). These cells showed rounded shapes with slender filipodia-like cytoplasmic expansions. Their cytoplasm was electron-dense and contained few organelles, highlighting the presence of small dictiosomes, some mitochondria, and rough endoplasmic reticulum cisterns. The nucleus occasionally showed deep invaginations with the emphasis on large nucleoli and condensed chromatin, preferentially associated with the nuclear membrane. On day 5 (D5), most cells in the culture were classified as agranulocytes, which exhibited a clear decrease in the number of cytoplasmic organelles (Figure 8D). The nuclear/cytoplasmic ratio of these cells was high, similar to ESCs (Figure 8D−E). We could observe small dictiosomes, some mitochondria, and rough endoplasmic reticulum cisterns. On the other side, polyribosomes and filamentous structures were abundant. Moreover, some of these cells occasionally presented annulate lamellae (Figure 8E−F). Regarding the nucleus, we observed nuclear invaginations with abundant condensed chromatin (heterochromatin). On the cell surface, some short cytoplasmic expansions were appreciated (Figure 8E). At this stage of the cellular culture, we could occasionally see another subpopulation of large cells as well, containing a wide heterogeneity of cellular structures and highlighting the presence of lysosomes (Figure 8D). On day 16 (D16), reprogrammed cells underwent morphological changes as the culture medium was gradually changed to neuronal medium, showing signs of differentiation with the presence of lipid drops, bigger cytoplasm, and more organelles. Their shape changes to larger cells with elongated appearance resembling cells of neuroectodermal origin.

Figure 6.

Reprogramming of PBMNCs after activation. PBMNCs isolated by Ficoll gradient centrifugation were activated by specific antibody cross-linking and cultured in Iscove’s medium supplemented with 10% FBS. The brightfield images were taken at D5, 8, and 14. Nonactivated PBMNCs were used as controls. Scale bars 100 μm.

3.7 Redifferentiation of BD-PSCs to the cells belonging to different germ layers

The capability of BD-PSCs to redifferentiate in neuroectodermal layer was demonstrated by growing these cells on laminin-ornithin coated plates in N2 medium to initiate the differentiation toward neuronal cells and further cultivation in neuronal differentiation media containing B27 supplement BDNF and GDNF as described in Material and Methods. The conditions described above enable for redifferentiation of BD-PSCs toward various neuronal lineages. Depicted are different populations of cells expressing the specific markers (Figure 3). Specific neuronal lineages from BD-PSCs can be generated by slightly modifying time and culture conditions. In neuronal differentiation culture time period from D8 to D30, we observed a clear decrease in the expression of neuroepithelial stem cell protein Nestin, which is a major intermediate filament (IF) protein of embryonic central nervous system also known as neuronal progenitor marker, while the expression of MAP2, a member of neuron-specific microtubule-associated protein family, neuronal nuclear antigen NeuN, a common neuron marker, and class III β-tubulin element of tubulin family, Tuj 1 a specific marker for human neurons, significantly increases during neuronal differentiation.

Figure 7.

BD-PSCs express PSC markers. (a−e): Activated PBMNCs were grown in MEF-conditioned media on Matrigel-coated culture dishes. Immunofluorescence analysis was performed with characteristic pluripotent markers, including SSEA-4, TRA-1-81, and TRA-1-60. Immunofluorescence images were taken by confocal microscopy. DAPI was used to stain nuclei. Generation and culture of BD-PSCs in suspension. (f-m): Activated PBMNCs cultures were grown in suspension in Iscove’s medium supplemented with 10% FBS for 16 days, and flow cytometry analysis for pluripotency marker was performed using antibodies to SSEA-4 and TRA-1-8. Percentages were determined relative to appropriate isotype control.

Glial fibrillary acidic protein GFAP is a type of IF expressed in various cells belonging to central nervous system, such as glial cells and astrocytes. These cells are mainly expressed in the central nervous system, such as brain and spinal cord, contributing to astrocytes-neuron interactions as well as cell–cell communication. Using antibodies to GFAP, we confirmed that such structures are recognized in newly generated BD-neuronal cells confirming the feature of BD-PSCs to redifferentiate to neuroectoderm [22].

Capacity BD-PSCs to redifferentiate into endoderm/hepatocytes was assessed by growing the cells in appropriate medium as described in Material and Methods. Following initial differentiation into endoderm in KSR/DMSO medium, as confirmed by ICC using antibodies to AFP and TTR, in the second phase by using hepatocytes maturation medium cells turned to mature hepatocytes like cells expressing their specific marker ALB and HNF4α, (Figure 4), recapitulating liver development in vivo [23].

Figure 8.

Blood cells progressively showed morphological de-differentiation features upon activation. A, B: One day (D1) after activation in appropriate culture medium, typical blood cell types could be detected in the samples. Red blood cells, platelets, granulocytes, and agranulocytes could be found in the sample. C−E: After 5 days (D5), most cells could be classified as agranulocytes, showing some nondifferentiation characteristics, such as scarce cytoplasm, low number of organelles, and the presence of annulate lamellae (E, arrowhead). F-H: The homogeneity of the culture peaked 8 days upon activation (D8), showing similar ultrastructural characteristics as defined in D5. This state was maintained in D12 (not shown). I−K: After 16 days (D16), cells slowly started to show some differentiation signs, such as lipid drops (K, arrow). A small population of cells started to change their morphology and appeared as bigger elongated cells (L), with increased cytoplasm and more organelles. Photomicrographs in the first column correspond to toluidine blue-stained semithin (1.5 μm) sections. The center and right columns are transmission electron microscopy (TEM) images. Scale bars: A, C, F, I, K = 10 μm; B, D, G, H, J = 2 μm; E = 200 nm.

The membrane activation of human glycoprotein ACA initiates a dedifferentiation process, consecutively generating more primitive cells until the final stage of this process is reached. BD-PSCs capable of redifferentiation into all three germ layers are the final product of this dedifferentiation process initiated by the membrane glycoprotein ACA [9, 10, 11] depicted in Figure 9.

Figure 9.

Schematic presentation of dedifferentiation process that starts with human blood progenitor cells expressing CD34 via HSCs, following side population (SP) cells mainly not expressing CD34, (low HLA) and ending up with the generation of BD-PSCs expressing pluripotency marker (SSEA-4) [24].

4. Discussion

4.1 Signaling

Antibody cross-linking of a GPI-linked protein ACA initiates, via PLCγ/PI3K/Akt mTor/PTEN up-regulation of Wnt, Notch, c-Kit, and/or HoxB4 genes, among others. Signaling network linked to these genes induces dedifferentiation of blood progenitor cells leading to generation of BD-PSCs [9, 11]. Briefly, PI3K activation phosphorylates and activates Akt localizing it at the plasma membrane. Akt is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes like cell proliferation, transcription, and apoptosis [25]. The components of the PI3K/Akt Pathway, such as α, β, and γ p110 catalytic subunits, as well as subunits of 3-phosphoinositide dependent kinase 1 (PDK1), which is the major transducer of IP3 Kinase, likewise the downstream effector proteins like glycogen kinase-3 (GSK-3), which plays a central role in the regulation of the stability and synthesis of proteins involved in the cell cycle entry; C-Raf, a serine/threonine kinase, whose main role in cells is the phosphorylation and activation of the MAP kinases MEK1 and MEK2, and the mTOR complex that controls translation and acts as a critical regulator of protein synthesis, are regulated in ACA-dependent manner.

Most importantly, a lipid-protein phosphatase PTEN, also called an anti-tumor agent, which is the natural inhibitor of PI3K/Akt signaling pathway that regulates p53 protein level and activity as well. PTEN works by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol-4, 5-bisphosphat (PIP2), which limits Akt’s ability to bind to the membrane, decreasing its activity. Deletion of the PTEN tumor-suppressor gene in adult hematopoietic stem cells (HSCs) leads to myeloproliferative diseases, and recent studies showed the inactivation of PTEN in human T-ALL cell lines as well as primary cells [26, 27, 28].

PTEN is a downstream target of phosphorylation at this specific route to pluripotency. Upregulation of PTEN indicates that a proliferative control of the process that leads to generation of self-renewable BD-PSCs is tightly regulated in a GPI-linked protein-dependent manner. Our results indicate that this protein promotes the generation of self-renewing cells by activating PI3K/AKT pathway, but one of the best-conserved functions of AKT in promoting growth in this induced signaling cascade appears to be under the control of activated PTEN by preventing oncogenic outgrow.

Canonical Wnt/ß-catenin pathway is involved in the regulation of various functions like embryonic development, proliferation, survival, cell polarity, migration, and maintenance of somatic stem cells in many tissues, modulating a delicate balance between stemness and differentiation. Binding of Wnt proteins to their receptors inhibits the phosphorylation of β-catenin resulting in stabilization and accumulation of β-catenin in the cytosol and its nuclear translocation followed by transcriptional regulation of target genes [29]. Notch protein is a hetero-oligomer type of transmembrane receptor and the pathway linked to his protein is highly conserved and functionally involved in various processes from development to cell growth and cell death. Among them, the most important, together with other signaling pathways, such as Wnt, are the regulation of stem cell self-renewal, maintenance of homeostasis, cell–cell communication, regulation of cell-fate decision, neuronal function and development, and expansion of HSCs [30].

Tyrosine-protein kinase (c-Kit) is a receptor tyrosine kinase that belongs to the type III family of kinases. When the ligand binds to this receptor, the dimer is formed, which activates intrinsic kinase activity that initiates the phosphorylation of various signal-transducing molecules and propagates the signals within the cells.

C-Kit signaling is involved in various mechanisms, such as differentiation, cell survival, and proliferation. It is expressed in various cell types, most importantly in HSCs, and binding on its ligand SCF causes blood progenitor cells to grow [31].

Further downstream partner genes in the initiated signaling pathways, such as HOXB4 and BMI1, belong to the Homeobox and/or Polycomb group (PcG) family of genes involved in the development. HOXB4 gene encodes a nuclear protein with a homeobox DNA-binding domain. Ectopic expression of this protein expands HSCs and progenitor cells in vivo and in vitro, making it a potential candidate for therapeutic stem cell expansion [32]. BMI1, as a member of the PcG family of transcriptional repressors, is involved in the control of development by regulating cell growth and differentiation. It is also expressed in HSCs proven to be essential for generation of self-renewing HSCs [33].

Transforming growth factor beta (TGF-β) is a multifunctional cytokine that belongs to the transforming growth superfamily that includes endogenous growth-inhibiting proteins [34]. Activation by ACA down-regulates TGF-β, which is one of the most potent inhibitors of HSC growth in vitro. One of the features of HSCs is their relative quiescence and given the strong inhibitory properties of TGF-β, it has been proposed to be the main regulator of quiescence in vivo [35]. Anti-apoptotic BCL-2 family proteins represent a family of evolutionary conserved cytoplasmic proteins that are known for their regulation of programmed cell death and survival. In response to intracellular damage, signals initiate the proteolytic cascade that disintegrates the cells. In our findings, these genes are down-regulated compared to unmanipulated PBMNCs, indicating that apoptosis represents an important regulatory factor in the maintenance of stem cells and is a part of the molecular mechanisms regulated by GPI-anchored membrane protein.

ACA signaling network via PLCγ/IP3K/Akt/mTOR/PTEN up-regulates the critical genes that are involved in the signaling pathways that regulate human development, such as NOTCH and WNT. Moreover, due to hierarchy among them, upregulation of these genes remains under the control of tumor suppressor gene PTEN, which is also upregulated in an ACA-specific manner. Apoptosis through downregulation of BCL-2 is an additional mechanism that regulates growth and proliferation. Finally, tumor suppressor gene P53 remains constant during reprogramming by ACA (data not shown).

Notably, the highest extent of upregulation of target genes is reached with c-Kit receptor tyrosine kinase [36], which is the gene critical for proliferation and survival of HSCs, indicative of a direct link that exists between these two proteins. C-Kit, a receptor tyrosine kinase type II, activates signaling through second messengers, such as cyclic adenosine monophosphate (cAMP), which are membrane-associated and diffuse from the plasma membrane into intermembrane space, where they can reach and regulate other membrane proteins. This reaction is probably the key to molecular mechanisms regulated by GPI-anchored membrane glycoprotein.

4.2 Reprogramming by dedifferentiation

IPSCs appear to represent the greatest promise for regenerative medicine without the ethical and immunological concerns incurred by the use of ESCs. They are pluripotent and have high replicative capability. Furthermore, iPSCs have the potential to generate all the tissues of the human body and provide researchers with patient- and disease-specific cells, which can recapitulate the disease in vitro, allowing for specific drug discovery. The iPSC technology provides an opportunity to generate cells with characteristics of ESCs, including pluripotency and potentially unlimited self-renewal.

Although methods have been improved from viral integration to integration-free, there are still challenges down the road to achieving their clinical application in humans.

The use of iPSCs in autologous cell-based therapy represents an ideal approach for regenerative medicine since the patients do not require long-term immunosuppressive drugs. The derivation of iPSCs over a decade ago has been raising high expectations and enthusiasm that iPSC technology can deliver autologous cell-based therapeutics to treat a high number of degenerative diseases, but actually, autologous therapy is related to the high cost and long period of time which should be spent in the manufacturing process that includes generation, characterization, differentiation into relevant cell types, scale up, and careful validation of the generated cell product. In order to reduce production time and costs, iPSCs therapies are moving toward allogeneic approaches by establishing clinical-grade iPSC banking [37, 38].

Banking of iPSCs from healthy donors would throw the iPSC reprogramming strategy once claimed as advantageous when compared to hESCs, while autologous at its beginning background. Therefore, the reprogramming strategies entirely free of DNA-based vectors could lead to solving the problems regarding genetic induced pluripotency.

4.3 Blood-derived pluripotent stem cells

Stem cell therapy is the ultimate goal of personalized medicine and individual care for many degenerative diseases, such as Alzheimer’s disease, Parkinson, diabetes, and others. It has already been shown that human PB cells can be successfully reprogrammed into blood cells using the Yamanaka factors [39]. Blood is one of the most easily accessible sources of patient cells for reprogramming because there is no need to maintain cell cultures extensively prior to reprogramming experiments. Therefore, it is a potentially unlimited and safe source of cells.

Our own work showed recently that blood cells can be reprogrammed to PSCs without any genetic manipulation [9, 10, 11]. The present study shows that the signaling network activated by human GPI-linked protein ACA is sufficient to generate cells from circulating blood that is pluripotent, according to their morphology, pluripotent marker proteins, and differentiation potential. In fact, it is possible to reprogram adult progenitor cells that can be obtained from PB through protein activation, by means of antibody cross-linking, making them return to a similar state to that of ESCs.

Immunophenotyping of BD-PSCs by using antibodies to pluripotency markers by means of flow cytometry and immunofluorescence analysis, revealed the expression of SSEA-4, TRA-1-60, TRA-1-81, NANOG, SOX2, and OCT3/4, indicating that newly generated cells possess the properties of ES cells. Electron microscopy analysis showed the morphological changes during the culture time period from D1-D16. A scarce cytoplasm and decreased number of organelles indicate that undifferentiated characteristics appeared through culture time. Most importantly, the appearance of annulate lamellae, stacked sheets of membranes embedded with pore complexes which are frequently found in cells with high proliferative activity, such as oocytes, embryonic, and tumor cells [40], suggest that BD-PCSs correspond to an actively dividing cell population. In addition, when neuronal differentiation medium was added to the cell culture system the morphology of these cells changed to larger elongated cells with more organelles and increased cytoplasm, supporting the notion that they are able to redifferentiate [10].

Activation of a membrane protein ACA at the surface of blood progenitor cells by cross-linking at the membrane of blood cells with its specific antibody and analysis of the mode of how this signaling machinery regulates the expression of genes known to play a role in human development via specific protein phosphorylation as an important regulatory mechanism in the cellular processes related to its signaling competence showed its involvement in the processes that determine the cell type, its fate, and identity.

Our results confirm the previously published data that the initiation of GPI-linked protein ACA upon activation of PBMNCs described here is sufficient to induce signaling machinery that leads to generation of self-renewing PSCs. Moreover, it ensures the maintenance of pluripotency in ESCs as well, indicating the involvement of this protein in pluripotency signaling network in humans.

Dedifferentiation process initiated upon membrane activation follows exactly the opposite way that is known for differentiation of PB progenitor cells. It is generally accepted that the proliferation capacity is higher by hematopoietic progenitor cells and declined by more primitive cells like HSCs and SP cells. The process initiated by ACA is due to activation of tumor suppressor genes, under strict proliferative control, leading to generation of BD-PSCs and explaining the lack of teratogenicity of these cells resulting in advantage for their application in cell tissue replacement.

BD-PSCs generated through dedifferentiation process are capable of redifferentiate into cells belonging to all three germ layers.

Most importantly, an antibody that acts at the surface of PB progenitor cells initiating membrane-to-nucleus signaling pathways may have numerous potential advantages regarding clinical safety for application of these cell products in regenerative medicine.

5. Conclusions

So far today, no iPSCs-based therapy is implemented into routine clinical use [41]. The hurdles regarding use of iPSCs in clinical practice are related primarily to genetic instability of these cells that may cause mutations leading to tumor formation and cancer. Another safety concern, when it comes to their application in humans, is the presence of residual undifferentiated iPSCs, also linked to tumorigenicity [42].

Allogenic approaches by establishing clinical-grade iPSC banking must consider the populations with heterogeneous genetic background, which might be very challenging [43]. Therefore, it is very important to further improve the current iPSCs technology to minimize the possible side effects and genetic and epigenetic differences between reprogrammed cells and donors. Human ES cells derived from blastocyst imply its destruction that cause serious ethical concern. In addition, these cells are by their nature nonautologous and may cause graft-versus-host disease. They are mostly used for studying early human development using currently available hES cell lines, but they also have limited potential in medicine due to restrictions related to ethical and immunological issues [44].

Great effort is made to assure safe clinical applications using stem cell therapies. The international stem cell banking initiative (ISCB) published guidelines for the development of pluripotent stem cell stocks for clinical applications [45]. Due to complex nature of the cells to be used for therapies in regenerative medicine compared to drug therapies, standard regulations must include purity of the cells, sterility, viability, genomic stability, specific gene expression profile, functional evaluation of reprogrammed, and differentiated cells, absence of infectious pathogens and tumorigenicity [46]. The greatest attention is taken to ensure the safety of transgenic cells when compared to genetically unmodified cells, even more so when it comes to the use of more modern technology like CRISPR/Cas9 for removing randomly inserted foreign genes into human genome during reprogramming process because there is the possibility of off target mutagenesis [47, 48].

Our results reveal insight into the molecular events regulating cellular reprogramming and indicate that pluripotency may be controlled in vivo through the binding of soluble ligand(s) to ACA-protein and initiating the cascade of already known and partly characterized signaling pathways. The process of reprogramming is short (10−12 days), the source is easily accessible unmanipulated peripheral blood and no use of growth factors is necessary. BD-PSCs are autologous, capable of generating in vitro cell types of all three layers exhibiting neuronal, liver, or hematopoietic characteristics [9, 10, 11]. Due to their differentiation capacity, they could be potentially utilized to regenerate any type of tissue, and thus treat neurological and immune disorders, as well as injuries to critical organs, such as the heart and brain. Moreover, due to lack of teratogenicity, BD-PSCs can be used in situ without the necessity to be differentiated before their application [11] as is also shown in the wound healing experiment currently ongoing in our laboratory. Due to tight proliferation control, BD-PSCs do not form cell lines and therefore must be freshly prepared.

It can be expected that the standard regulations for the use of BD-PSCs would be similar to that of genetically unmodified cells implying fewer hurdles compared to iPSCs ensuring a fast and safe application of these cells in routine clinical practice.

The potential application of BD-PSCs in regenerative stem cell therapies is innovative and promising. Additional studies are underway in order to determine in vivo therapeutic potential and to ensure a safe platform for translation of basic research to new clinical therapies.

Our report provides a practical and efficient way to generate patient-specific PSCs. This will also be valuable for the generation of clinical-grade PSCs for future therapeutic applications so that the possibility to develop a truly personalized medicine becomes more realistic.

Acknowledgments

Dedicated to the memory of Dr. Rainer Saffrich.

The authors express unending gratitude to Professor Gennady T. Sukhikh for his encouragement and permanent support. We gratefully acknowledge the technical assistance provided by Patricia García-Tárraga in Valencia and by John and Oksana Greenacre in Heidelberg.

This work was supported by the Prometeo Grant for Excellence Research Groups [PROMETEO/2019/075] given to José Manuel García-Verdugo; and private funding from ACA CELL Biotech GmbH Heidelberg, Germany.

Conflict of interest

The corresponding author declares that she is a patent holder related to Novel Human GPI-linked Protein ACA, she also cofounded and works in ACA CELL Biotech GmbH. The other authors declare that there is no conflict of interest.

Abbreviations

AFP	alpha-feto-protein
ALB	albumin
APC	allophycocyanin
BCL-2	B-cell lymphoma 2
BDNF	brain-derived neurotrophic factor
BD-PSCs	blood-derived pluripotent stem cells
bFGF	basic fibroblast growth factor
BMI1	B lymphoma Mo-MLV insertion region 1 homolog
BSA	bovine serum albumin
cAMP	cyclic adenosine monophosphate
CTNNB	catenin (cadherin-associated protein), beta 1
DAG	1,2-diacylglycerol
DAPI	4′,6-Diamidin-2-phenylindol
DMEM	dulbecco’s Modified Eagle Medium
DMSO	dimethyl sulfoxide
ECL	enhanced chemiluminescence
EGTA	ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
EM	electron microscopy
ESCs	embryonic stem cells
ET	Et-18-CH3
FBS	fetal bovine serum
FITC	fluorescein isothiocyanate
GAPDH	glyceraldehyde 3-phosphate dehydrogenase
GDNF	glial-derived neurotrophic factor
GFAP	glial fibrillary acidic protein
GPI	glycosylphosphatidylinositol
GSK-3	glycogen kinase-3
HCC	hydrocortisone 21-hemisuccinate sodium salt
HGF	hepatocyte growth factor
HNF4 α	hepatocyte Nuclear Factor 4 alpha
HoxB4	homeobox protein B4
HPR	horseradish peroxidase
HSCs	hematopoietic stem cells
ICC	immunocytochemistry
IF	intermediate filament
IMDM	Iscove’s modified Dulbecco’s medium
IP	immunophenotyping
iPSCs	induced pluripotent stem cells
Klf4	kruppel-like factor 4
KSR	knockout serum replacement
LY	LY 294002
MAP2	microtubule-associated protein 2
MEFs	mouse embryonic fibroblasts
MEK1	mitogen-activated protein kinase kinase 1
MEK2	mitogen-activated protein kinase kinase 2
MNCs	mononuclear cells
mTor	mammalian target of rapamycin
NEAA	nonessential amino acids
Nestin	neuroepithelial stem cell protein
Oct3/4	octamer-binding transcription factor 3/4
OSM	oncostatin M
PB	peripheral blood
PBS	phosphate buffer saline
PcG	polycomb group
PD	PD098059
PDK1	3-phosphoinositide dependent kinase 1
PE	phycoerythrin
PFA	paraformaldehyde
PI	propidium iodide
PI3Ks	phosphoinositide 3-kinases
PIP2	phosphatidylinositol-4,5-bisphosphat
PIP3	phosphatidylinositol (3,4,5)-trisphosphate
PLCγ	phosphoinositol-phospholipase C γ
PtdIns	phosphatidylinositol
PTEN	phosphatase and tensin homolog
PVDF	polyvinylidene difluoride
SCF	stem cell factor
SDS-PAGE	sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SSEA-4	stage-specific embryonic antigen
TGF-β	transforming growth factor beta
TTR	transthyretin
Tuj1	class III β-tubulin
WB	western blot