Elucidating an Essential Role of Promoting Endothelial-to-Hematopoietic Transition By a Key EMT-Regulator ZEB2 Using a Human iPSC-Based Model

ZEB2 (Zinc finger E-box binding homeobox 2; also known as SMAD interacting protein-1 or SIP1) is a DNA-binding transcriptional regulator associated with epithelial-to-mesenchymal-transition (EMT). It has been reported that it is important in many other developmental processes, including mesoderm formation during gastrulation and neural crest formation. More recently, a study reported that inactivation of ZEB2 in mice results in differentiation defects in multiple hematopoietic cell lineages (1). In order to examine the roles of ZEB2 in human developmental process and hematopoiesis, we decided to apply human induced pluripotent stem cells (iPSCs) as a model with a pair of isogenic iPSCs with or without functional ZEB2 expression. We used a validated iPSC-based hematopoietic differentiation under a feeder- and xeno-free culture condition was used to study the role of ZEB2 in hematopoietic cell formation and to avoid other EMT-inducing factors in serum. We found that ZEB2 expression is abundant in either cord blood cells or human iPSCs-derived hematopoietic stem/progenitor cells (HSPCs), indicating that ZEB2 deficiency may hinder human hematopoietic differentiation. To knock out the ZEB2 gene and generate ZEB2 deficient human iPSCs, we used two different CRISPR guide RNAs as well as SpCas9 in a human iPSC line BC1. An engineered isogenic BC1 iPSC line that has both alleles 155-bp deletion in ZEB2 exon 1 (ZEB2 -/-) was successfully isolated and expanded, resulting in the frameshift and the consequent disappearance of ZEB2 protein expression. The complete ZEB2 knockout had little effects on human iPSCs expansion. However, we observed that the differentiation of ZEB-/- BC1 into CD34+CD45+ HSPCs was decreased. The number of ZEB-/- BC1-derived HSPCs was 5-fold lower than that from wide type BC1 control. Moreover, ZEB2-deleted HSPCs were defected in hematopoietic colony formation. To figure out at which hematopoietic differentiating stage the defects occurred, we applied inducible iPSC's endothelium-hematopoietic transformation (EHT) system to mimic hemogenic endothelial cells (ECs) giving rise to HSPCs. By using this established EHT system, ZEB2 deficient iPSCs were differentiated in order into CD34+CD31+CD144+ ECs, and then to HSPCs (2). We found that ZEB2 depletion did not adversely affect the formation of ECs from human iPSCs in this EHT experimental system but did impact on HSPCs generation from ECs. This study is unique in several ways to elucidate the roles of ZEB2 in human hematopoietic development. This prospective genetic model allowed us to pinpoint ZEB2 is critical at the stage of EHT during human hematopoietic cell formation. Our results indicate that contribution of ZEB2 deficiency in blood disorders might result from the inhibition of HSPC formation. The human iPSC-based hematopoietic differentiation system coupled with precisely edited isogenic iPSC lines also provide a more controlled genetic model for studying functions of others gene in their regulation of human hematopoiesis. References: 1. J Li, et al. Blood, 2017, 129(4): 460-472. 2. Hao B, et al. Stem Cells, 2016, 231(5): 1065-1076. Disclosures No relevant conflicts of interest to declare.

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The Roles of RUNX1 in Human Hematopoiesis and Megakaryopoiesis Revealed By Genome-Targeted Human iPSCs and an Improved Hematopoietic Differentiation Model

Blood ◽

10.1182/blood.v126.23.1167.1167 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1167-1167

Author(s):

Bin-Kuan Chou ◽

Hao Bai ◽

Yongxing Gao ◽

Ying Wang ◽

Zhaohui Ye ◽

...

Keyword(s):

Stem Cells ◽

Culture System ◽

Cell Formation ◽

Hematopoietic Cells ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Runx1 Gene ◽

Human Ipscs ◽

Cd34 Cells ◽

Definitive Hematopoiesis

Abstract The RUNX1 gene (also called AML1), one of the most mutated genes in acute myeloid leukemia (AML), was first identified decades ago that encodes a key regulatory transcriptional factor. Numerous studies using mouse and zebrafish models show that RUNX1 is essential for definitive hematopoiesis. In mice, its homozygous knock-out (KO) in hematopoietic stem/progenitor cells also causes defects in lymphoid and megakaryocytic (MK) development. However, heterozygous Runx1 gene mutations in laboratory mouse and zebrafish had little effects on development of hematopoietic stem/progenitor cells (HSPCs) or the MK cell lineage. In contrast, heterozygous germline mutations in RUNX1 were found in patients with familial platelet disorder (FPD) with predisposition to AML and MDS. The mechanisms underlying the observed differences between humans and small animal models remain unclear. In the past decade, we and others have utilized human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate human hematopoiesis including the roles of the RUNX1 gene. Using a feeder-free culture system, we generated human CD34+CD45+ HSPCs cells from human ESCs and iPSCs around 11-14 days after embryoid body (EB) formation. The CD34+CD45+ HSPCs were capable to form multiple types of hematopoietic cells such as myeloid, erythroid, and polyploid MK cells (Connelly et al., 2014; Liu et al., 2015). We also reported that human iPSCs derived from FPD patients containing a heterozygous RUNX1 mutation were defective in MK formation, and targeted correction of the mutated RUNX1 allele by genome editing restored the MK potential (Connelly et al., 2014). Since then, we have extended our studies by precise genomic targeting in human wildtype iPSCs to ablate exon 5 that is common in all 3 isoforms of the RUNX1 gene, or exon 1B that is unique to the RUNX1c isoform. Bi-allelic KO of RUNX1 at exon 5 completely abolished the formation of hematopoietic cells at days 11-14 after EB formation. Complete disruption of exon 1B showed little effect, indicating that the RUNX1c isoform is dispensable for definitive hematopoiesis in the presence of the RUNX1a and RUNX1b isoforms transcribed from the downstream P2 promoter. Detailed analysis of EBs at days 6-8 revealed that bi-allelic RUNX1 KO at exon 5 (ablating all 3 isoforms) did not affect the formation of CD34+/CD31+/CD144+ endothelial-like cells. However, the endothelial-to-hematopoietic transition (EHT) was completely blocked and no CD45+ hematopoietic cells emerged from the EHT culture supplemented with hematopoietic cytokines. To elucidate the functions of different RUNX1 isoforms in early steps of human hematopoiesis, we adapted the EHT culture system that uses CD34+ cells isolated from earlier stages of EB formation (day 6-8), before definite hematopoiesis was observable (after day 8, Bai et al., 2015). Two different iPSCs clones with homozygous or bi-allelic RUNX1 KO at exon 5 both failed to form CD45+ hematopoietic cells after EHT culture. Because constitutive transgene expression of RUNX1b or RUNX1c (but not RUNX1a) cDNA in human iPSCs inhibits hematopoietic differentiation, we transduced the day 6 EB cells at the beginning of the EHT culture with RUNX1-expressing vectors. Lentiviral vectors constitutively express RUNX1b or RUNX1c (but not RUNX1a) cDNA partially restored the EHT and hematopoietic (CD45+) cell formation after 4-5 days in EHT culture. We further used a lentiviral vector in which RUNX1c (as an ER fusion protein) can be conditionally activated by 4-HT induction. Induction starting at day 4 and lasting for 3 days rendered the maximal effect of hematopoietic cell formation in the EHT culture using CD34+ cells isolated at day 6 of EB formation. Our data corroborate with limited in vivo data using human fetal tissues on the possible roles of RUNX1 in definitive hematopoiesis. At present we are analyzing iPSC clones with mono-allelic disruption of exon 5 in a wild type iPSC line, comparing to iPSCs derived from FPD patients. The current study of using isogenic human iPSCs will help to understand the roles of RUNX1 in human hematopoiesis and megakaryopoiesis, and offer an amenable system to study the RUNX1 gene functions and downstream target genes. Disclosures No relevant conflicts of interest to declare.

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Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

10.1101/2021.01.13.426620 ◽

2021 ◽

Cited By ~ 1

Author(s):

Carol X.-Q. Chen ◽

Narges Abdian ◽

Gilles Maussion ◽

Rhalena A. Thomas ◽

Iveta Demirova ◽

...

Keyword(s):

Stem Cells ◽

Quality Control ◽

Cortical Neurons ◽

Genomic Stability ◽

Control Measures ◽

List Type ◽

Ipsc Line ◽

Differentiation Potential ◽

Germ Layers ◽

Human Ipscs

AbstractInduced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a four-step workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions (mTeSR1 and Essential 8) and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic stability in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based stability test, and cell-line identity authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by q-PCR for progenitor, neuronal, cortical and glial markers. Taken together, we present a standardized quality control workflow to evaluate variability and reproducibility across and between iPSCs.HighlightsValidation of culture conditions is critical in the expansion and maintenance of an iPSC line.Characterization of pluripotency and genomic stability ensures each line is free of defects at the DNA level, while maintaining its ability to be directed into any of the three germ layers.Forebrain cortical neurons can be generated from all iPSC line tested; however, the morphology and expression pattern of these neurons can vary from line to line.

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A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.09.206 ◽

2004 ◽

Vol 324 (4) ◽

pp. 1333-1339 ◽

Cited By ~ 13

Author(s):

Zhongling Feng ◽

Anand S. Srivastava ◽

Rangnath Mishra ◽

Ewa Carrier

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Embryonic Stem ◽

Regulatory Role ◽

Hematopoietic Differentiation ◽

Murine Embryonic Stem Cells

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Optimization Of Stepwise Hematopoietic Differentiation Of Human iPSCs

Blood ◽

10.1182/blood.v122.21.4840.4840 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4840-4840

Author(s):

Friedrich Schuening ◽

Narasimhachar Srinivasakumar ◽

Michail Zaboikin ◽

Tatiana Zaboikina

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Single Cells ◽

Retroviral Vectors ◽

Dermal Fibroblasts ◽

Hematopoietic Differentiation ◽

In Vitro Differentiation ◽

Hematopoietic Stem ◽

Human Ipscs

Abstract Since their discovery in 2006, induced pluripotent stem cells (iPSCs) with their ES cell-like self-renewal and differentiation capability, are set to revolutionize the field of regenerative medicine. There is tremendous interest in the field of hematology for derivation of hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPCs) by in vitro differentiation of IPSCs. IPSCs can be differentiated into HSC/HPCs by coculture on feeder cells, such as OP9, or by using stepwise differentiation protocols on defined media. Neither approach produces high yields of HSCs or HPCs. With an intention to improve this, we systematically investigated various parameters for in vitro differentiation of iPSCs into HPCs. iPSCs were derived from human adult dermal fibroblasts by transduction with the Yamanaka retroviral vectors (encoding human Klf4, Oct3/4, Sox2 and cMyc) or by electroporation with the Yamanaka Epstein–Barr virus-based episomal plasmid vectors (encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA). One iPSC clone of each variety was then subjected to a stepwise differentiation protocol described by Niwa and coworkers [PLoSOne. (2011); 6(7):e22261] followed by hematopoietic colony forming (CFU) assays in MethoCult (STEMCELL Technologies, Vancouver, Canada). The original protocol calls for the use of Stemline II serum-free medium (Sigma, St. Louis, MO) supplemented with various growth factors/cytokines. We investigated the use of APEL medium described by Ng and coworkers [Nature Protocols. (2008); 3(5): 768] as a possible substitute for Stemline II. We also tested the effect of varying the number of colonies seeded in 6-well plates and the efficiency of hematopoietic differentiation after seeding iPSCs as single cells. The results, based on the number of hematopoietic colonies obtained in MethoCult following differentiation, showed that the APEL medium (>100 CFU/100,000 cells) was a superior substitute to the Stemline II medium (<10 CFU/100,000). When IPSCs were seeded as single cells, at initial densities of 10, 100 or 1,000 cells/cm2 in the presence of Y-27632 Rock inhibitor, only the cells at starting density higher than 1,000 per cm2survived but did not yield hematopoietic CFUs in MethoCult. When seeded as colony fragments, lower density of seeding in 6-well plates (< 20 colonies/well) was superior to higher density (>50 colonies/well) for obtaining HPCs. Other parameters that can affect differentiation, such as bone-morphopoietic protein (BMP) and O2 concentration, are being investigated. Figure A. Cellular markers detected at different time points of stepwise hematopoietic differentiation. B. CFUs in MethoCult. Disclosures: No relevant conflicts of interest to declare.

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p53 Is a Crucial Regulator of Hemogenic Endothelial Cell Differentiation from Human Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2021-154243 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1085-1085

Author(s):

Jitendra K. Kanaujiya ◽

Elizabeth G. Lingenheld ◽

William C. Skarnes ◽

Hideyuki Oguro

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Pluripotent Stem Cells ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Scientific Advisory ◽

Induced Pluripotent

Abstract De novo generation of hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) could provide a virtually unlimited supply of autologous HSCs for clinical transplantation, and offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human hematopoietic development. However, the derivation of long-term self-renewing HSCs from hiPSCs in culture remains elusive. The tumor suppressor protein p53 plays important roles in normal and malignant hematopoiesis, and Trp53-deficient mice exhibit increased number of HSCs. Although activation of p53 is known to promote differentiation of hPSCs and hPSCs recurrently acquire TP53 dominant negative mutations, its role in hematopoietic differentiation of hiPSCs has not been explored. To differentiate hiPSCs into hematopoietic stem and progenitor cells (HSPCs), we used embryoid body (EB) formation method to first differentiate hiPSCs into hemogenic endothelial (HE) cells that express the CD34 highCD144 +CD73 -CD184 -CD43 -CD235a - cell-surface markers. HE cells were then transferred onto a Matrigel-coated plate to undergo endothelial-to-hematopoietic transition (EHT) to generate HSPCs that express the CD34 midCD45 mid cell-surface markers. Developed HSPCs were functionally evaluated by colony forming assay. We observed that the expression of CDKN1A, a p53 target gene, was upregulated in hiPSC-derived EBs and HSPCs over the course of differentiation. To investigate the role of p53 in the generation of HSPCs from hiPSCs, we genetically deleted TP53 in hiPSCs followed by hematopoietic differentiation. While TP53 deletion increased the growth of EBs, it resulted in severe impairment of differentiation into HE cells and overall production of HSPCs that can form colonies. During HE differentiation from hiPSCs, TP53-deficient EBs showed significant reduction of endothelial-lineage gene expression, such as ETV2, CDH5, and PECAM1, as well as expression of RUNX1, a master transcription factor required for HE specification. These results indicate the indispensable role of p53 in HE differentiation from hiPSCs. We then examined the effect of p53 activation on HE differentiation from hiPSCs by pharmacological activation of p53 in hiPSC-derived cells. Transient activation of p53 by Nutlin-3, a small molecule that inhibits the p53-HDM2 interaction and protects p53 from proteasomal degradation, only during HE differentiation but not during EHT significantly promoted HSPC generation as compared to the vehicle treated control. Our findings shed light on the importance of selecting hiPSC lines that retain normal p53 activity for HE differentiation, and provide an approach to promote hematopoietic differentiation of hiPSCs by transiently activating p53 during HE differentiation. Disclosures Kanaujiya: Synthego: Other: Scientific Advisory; eGenesis: Other: Scientific Advisory.

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IL-3 Specifies Early Hematopoietic Development from Human iPSCs and Synergizes with M-CSF and G-CSF on Myeloid Differentiation

Blood ◽

10.1182/blood.v124.21.4308.4308 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4308-4308

Author(s):

Nico Lachmann ◽

Mania Ackermann ◽

Eileen Frenzel ◽

Christine Happle ◽

Olga Klimenkova ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Terminal Differentiation ◽

Myeloid Differentiation ◽

Hematopoietic Differentiation ◽

In Vitro Differentiation ◽

Hematopoietic Development ◽

Cd34 Cells

Abstract Hematopoietic in-vitro-differentiation of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) holds great promise for disease modeling, drug testing, as well as cell- and gene-therapy strategies. Although hematopoietic differentiation of PSC has been shown to be feasible, the majority of current protocols apply a large number of different cytokines to direct differentiation. In this line, priming the differentiation process by a multitude of cytokines may alter the endogenous hematopoietic differentiation program of PSCs, thus hampering the usefulness of such protocols to gain insight into physiologic human hematopoietic development. To overcome this problem we have investigated the hematopoietic differentiation potential of human PSC, based on minimal cytokine application. Given the emerging role of IL3 as a critical factor in adult hematopoiesis and the pivotal role of M-CSF and G-CSF for terminal myeloid differentiation, we here employed IL3 in combination with either M-CSF or G-CSF on hematopoietic development. To prove our concept, human CD34+ cell-derived iPSC clones were subjected to an embryoid body (EB)-based myeloid differentiation protocol employing cytokines from day 5 onwards and yielding so-called “myeloid cell forming complexes” (MCFCs) within 7-10 days. Analysis of MCFC within 10 days of differentiation revealed expression of MIXL1, KDR1, GATA2, and RUNX1, as well as an early CD34+/CD45- population undergoing transition to a CD34+/CD45+ and thereafter CD34-/CD45+ phenotype. The hypothesis of a primitive hematopoietic cell arising from a population with dual (hematopoietic and vascular epithelial) potential was supported by co-staining of these populations with VE-cadherin (CD144). Here primarily the CD34+/CD45+/CD144- cells were capable of colony formation in vitro. Differentiation of PSC for more than 15-days resulted in the continuous shedding of hematopoietic cells from MCFCs and further differentiation along the IL3/M-CSF let to the generation of >99% pure monocytes/ macrophages (iPSC-MΦ), while IL3/G-CSF promoted granulopoiesis (iPSC-gra, purity >95%). Of note, hardly any CD34+ cells were detected among MCFC-shedded cells for the IL3/M-CSF as well as the IL3/G-CSF combination. In contrast, differentiation in IL3 only resulted in 10% MCFC-derived CD34+ cells, an observation further confirmed by a 10-times increased clonogenicity for cells shedded from MCFC exposed to IL3 only when compared to IL3/G-CSF or IL3/M-CSF cultures. Furthermore, cells cultured in IL3 maintained the capacity of subsequent M-CSF-driven terminal differentiation, whereas no suspension cells were observed following differentiation of PSC with G-CSF alone. Most strikingly, IL3/M-CSF or IL3/G-CSF cultures generated iPSC-MΦ or iPSC-gra from day 14-15 onwards over a period of 3-5 months at a quantity of 0.4-2.0 x 106 cells/week (cumulative 0.8-4.0 x 107 cells) per 3.5 cm well. For IL3/M-CSF cultures detailed characterization of mature myeloid cells demonstrated a typical MΦ-morphology of iPSC-MΦ by cytospins and a surface-marker profile of CD45, CD11b, CD14, CD163, and CD68. In addition, iPSC-MΦ had the ability to phagocytose latex-coated beads similar to peripheral blood (PB)-MΦ polarized to M2 and upon LPS stimulation secreted MCP1, IL6, IL8, and IL10, whereas IFNy, IL1b, IL4, IL5, and IL12 were absent. iPSC-gra showed surface expression of CD45, CD11b, CD16, CD15, CD66b and a differential count containing pro-myelocyte (3%), myelocyte (5%), meta-myelocyte (30%), bands (22%), eosinophils (2%), basophils (1%), and segmented-neutrophils (37%) . Moreover, iPSC-gra were able to migrate towards an IL8 or fMLP gradient, formed neutrophil extracellular traps, and up-regulated NADPH activity and ROS production upon PMA stimulation to a similar degree as PB granulocytes. In summary, we here present an in vitro differentiation protocols for human iPSC requiring minimal cytokine stimulation, which appears highly suited to model human hematopoietic development or generate cells for gene and cell-replacement strategies. We further provide evidence that IL3 constitutes a key cytokine driving the early hematopoietic specification of human PSC, whereas M-CSF and G-CSF function primarily as downstream “supporter” cytokines regulating the terminal differentiation towards macrophages and granulocytes, respectively. Disclosures No relevant conflicts of interest to declare.

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Micro-RNA Profiling Reveals the Key Role of miR-206 in the Hematopoietic Potential of Human Embryonic and Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-110729 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1275-1275

Author(s):

Stephane Flamant ◽

Jean-Claude Chomel ◽

Christophe Desterke ◽

Olivier Feraud ◽

Emilie Gobbo ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Line ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Target Genes ◽

Hematopoietic Differentiation ◽

Induced Pluripotent

Abstract Although human pluripotent stem cells (hPSCs) can theoretically be differentiated into any cell type, their ability to generate hematopoietic cells shows a major variability from one cell line to another. The reasons of this variable differentiation potential, which is constant and reproducible in a given hPSC line, are not clearly established. In order to study this phenomenon, we comparatively studied 4 human embryonic stem cell lines (hESC) and 11 human induced pluripotent stem cell (hiPSC) lines using transcriptome assays. These cell lines exhibited a significant variability to generate in vitro hematopoiesis as evaluated by day-16 embryoid body (EB) formation followed by clonogenic (CFC) assays. Four out of 11 iPSC lines (PB6, PB9, PB12.1, and PB14.3) were found to lack any hematopoietic differentiation ability whereas 7 cell lines showed variable hematopoietic potential. Among hESC lines, H9 and CL0 had low H1 and SA01 exhibited high hematopoietic potential using the above assays. Among hESC and hIPSC displaying hematopoietic potential, two sub-groups were further defined based on their hematopoietic CFC efficiency: a group of poor (generation of less than 100 CFC/105 cells, PB4 / PB10 /H9 /CL01), and high hematopoietic competency (more than 120 CFC/105 cells, PB3/ PB6.1 /PB7 /PB13 /PB17 /SA01/H1). Using global miRNome analysis performed at the pluripotency stage, the expression of 754 individual miRNAs was analyzed from 15 hPSC lines in order to explore a potential predictive marker between both sub-groups of pluripotent cells according to their hematopoietic potency. Using this approach, 27 miRNAs out of 754 appeared differentially expressed allowing the identification of a miRNA signature associated with hematopoietic-competency. The hematopoietic competency was associated with down-regulation of miR-206, miR-135b, miR-105, miR-492, miR-622 and upregulation of miR-520a, miR-296, miR-122, miR-515, miR-335. Amongst these, miR-206 harbored the most significant variation (0.04-Fold change). To explore the role of miRNA-206 in this phenomenon, we have generated a miR-206-eFGP-Puro lentiviral vector which was transfected in hESC line H1 followed by puromycin selection. As a control, H1 cell line was transfected with a Arabidopsis thaliana microRNA sequence (ath-miR-159a), which has no specific targets in mammalian cells. The correct expression of the transgenes were evaluated by flow cytometry (using GFP) and q-RT-PCR for miR-206 expression. The hematopoietic potential of H1 cell line and its miR-206-overexpressing counterpart was then tested using standard in vitro assays via d16-EB generation. We found that both CFC numbers and percentage of CD34+ were significantly lower in H1-mir-206-derived day-16 EB cells than in H1-ath- derived day-16 EB cells (p < 0.05). Thus, over-expression of miR-206 in this blood-competent hESC appeared to repress its hematopoietic potential at very early stage, since a similar lower CFC efficiency was observed in day-3 EB cells derived from miR-206 overexpressing H1 cell line. We then conducted an integrative bioinformatics analysis on miR-206 predicted target genes. To this end, 773 mRNA target transcripts of the broadly conserved (across vertebrates) miR-1-3p/206 family were identified in the TargetScan database and were integrated into the global transcriptomic analysis performed by microarray on day-16 EB cells. Using supervised ranking product analysis, 62 predicted gene targets of the miR-1-3p/206 family were found to be significantly up-regulated in hematopoietic-competent EB samples including the transcription factors RUNX1 and TAL1. Hierarchical unsupervised clustering, based on this subset of 62 predicted mir-206 target genes, fully discriminated hematopoietic-deficient from hematopoietic-competent cells. In conclusion, miRNA profiling performed at pluripotency stage could be useful to predict the ability to human iPSC to give rise to blood cell progenitors. This work emphasizes for the first time the critical role of the muscle-specific miR-206 in hematopoietic differentiation. Finally, these results suggest that genetic manipulation of hESC/iPSC could be used to enhance their hematopoietic potential and to design protocols for generation of hPSC-derived hematopoietic stem cells with long-term reconstitution ability. Disclosures No relevant conflicts of interest to declare.

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Differentiation of human induced pluripotent stem cells into erythroid cells

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01998-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Mohsen Ebrahimi ◽

Mehdi Forouzesh ◽

Setareh Raoufi ◽

Mohammad Ramazii ◽

Farhoodeh Ghaedrahmati ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Promising Alternative ◽

Human Ipscs ◽

Induced Pluripotent ◽

Human Ipsc

AbstractDuring the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.

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Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells

Blood ◽

10.1182/blood-2010-08-302711 ◽

2011 ◽

Vol 117 (7) ◽

pp. 2157-2165 ◽

Cited By ~ 41

Author(s):

Xinqiang Huang ◽

Eric Gschweng ◽

Ben Van Handel ◽

Donghui Cheng ◽

Hanna K. A. Mikkola ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Protein Tyrosine Phosphatase ◽

Embryoid Body ◽

Tyrosine Phosphatase ◽

Embryonic Stem ◽

Hematopoietic Differentiation ◽

Protein Tyrosine

Abstract MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34+ EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34+ cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.

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