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

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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UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2019-127466 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2500-2500

Author(s):

Tellechea Maria Florencia ◽

Flavia S. Donaires ◽

Tiago C. Silva ◽

Lilian F. Moreira ◽

Yordanka Armenteros ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

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Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells

Stem Cells International ◽

10.1155/2021/8274936 ◽

2021 ◽

Vol 2021 ◽

pp. 1-19

Author(s):

Salam Salloum-Asfar ◽

Rudolf Engelke ◽

Hanaa Mousa ◽

Neha Goswami ◽

I. Richard Thompson ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Cell Line ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Specific Pattern ◽

Hyperosmotic Stress ◽

Granule Formation ◽

Induced Pluripotent

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.

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Experimental Limitations Using Reprogrammed Cells for Hematopoietic Differentiation

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/895086 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Katharina Seiler ◽

Motokazu Tsuneto ◽

Fritz Melchers

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Somatic Cells ◽

Embryonic Stem ◽

Clinical Settings ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Induced Pluripotent

We review here our experiences with thein vitroreprogramming of somatic cells to induced pluripotent stem cells (iPSC) and subsequentin vitrodevelopment of hematopoietic cells from these iPSC and from embryonic stem cells (ESC). While, in principle, thein vitroreprogramming and subsequent differentiation can generate hematopoietic cell from any somatic cells, it is evident that many of the steps in this process need to be significantly improved before it can be applied to human cells and used in clinical settings of hematopoietic stem cell (HSC) transplantations.

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What do we know about the neurogenic potential of different stem cell types?

Arquivos de Neuro-Psiquiatria ◽

10.1590/s0004-282x2012000700013 ◽

2012 ◽

Vol 70 (7) ◽

pp. 540-546 ◽

Cited By ~ 2

Author(s):

Guilherme Lepski

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Large Body ◽

Cell Therapies ◽

Induced Pluripotent

Cell therapies, based on transplantation of immature cells, are being considered as a promising tool in the treatment of neurological disorders. Many efforts are being concentrated on the development of safe and effective stem cell lines. Nevertheless, the neurogenic potential of some cell lines, i.e., the ability to generate mature neurons either in vitro or in vivo, is largely unknown. Recent evidence indicate that this potential might be distinct among different cell lines, therefore limiting their broad use as replacement cells in the central nervous system. Here, we have reviewed the latest advancements regarding the electrophysiological maturation of stem cells, focusing our attention on fetal-derived-, embryonic-, and induced pluripotent stem cells. In summary, a large body of evidence supports the biological safety, high neurogenic potential, and in some diseases probable clinical efficiency related to fetal-derived cells. By contrast, reliable data regarding embryonic and induced pluripotent stem cells are still missing.

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GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab246 ◽

2012 ◽

Vol 24 (1) ◽

pp. 286

Author(s):

A. Dinnyes ◽

M. K. Pirity ◽

E. Gocza ◽

P. Osteil ◽

N. Daniel ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Pluripotent Cells ◽

Domestic Species ◽

Isolation And Characterization ◽

Induced Pluripotent ◽

Pluripotency Genes

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.

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OncogenicPIK3CApromotes cellular stemness in an allele dose-dependent manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821093116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8380-8389 ◽

Cited By ~ 16

Author(s):

Ralitsa R. Madsen ◽

Rachel G. Knox ◽

Wayne Pearce ◽

Saioa Lopez ◽

Betania Mahler-Araujo ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Dependent Manner ◽

Developmental Context ◽

Epithelial Morphology ◽

Pi3k Signaling Pathway ◽

Induced Pluripotent

ThePIK3CAgene, which encodes the p110α catalytic subunit of PI3 kinase (PI3K), is mutationally activated in cancer and in overgrowth disorders known asPIK3CA-related overgrowth spectrum (PROS). To determine the consequences of geneticPIK3CAactivation in a developmental context of relevance to both PROS and cancer, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous knockin ofPIK3CAH1047R. While heterozygous iPSCs remained largely similar to wild-type cells, homozygosity forPIK3CAH1047Rcaused widespread, cancer-like transcriptional remodeling, partial loss of epithelial morphology, up-regulation of stemness markers, and impaired differentiation to all three germ layers in vitro and in vivo. Genetic analysis ofPIK3CA-associated cancers revealed that 64% had multiple oncogenicPIK3CAcopies (39%) or additional PI3K signaling pathway-activating “hits” (25%). This contrasts with the prevailing view thatPIK3CAmutations occur heterozygously in cancer. Our findings suggest that a PI3K activity threshold determines pathological consequences of oncogenicPIK3CAactivation and provide insight into the specific role of this pathway in human pluripotent stem cells.

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Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0365 ◽

2015 ◽

Vol 370 (1680) ◽

pp. 20140365 ◽

Cited By ~ 19

Author(s):

Maria Rostovskaya ◽

Nicholas Bredenkamp ◽

Austin Smith

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cells ◽

Type I ◽

Pancreatic Progenitors ◽

Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

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ASXL2 Is a Novel Mediator of RUNX1-ETO Transcriptional Function and Collaborates with RUNX1-ETO to Promote Leukemogenesis

Blood ◽

10.1182/blood.v126.23.302.302 ◽

2015 ◽

Vol 126 (23) ◽

pp. 302-302

Author(s):

Jean-Baptiste Micol ◽

Nicolas Duployez ◽

Alessandro Pastore ◽

Robert Williams ◽

Eunhee Kim ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Hematopoietic Stem Cell ◽

Binding Sites ◽

Target Genes ◽

Cell Function ◽

Rna Seq ◽

Hematopoietic Stem

Abstract Mutations in Addition of Sex Combs Like 1 (ASXL1) are common in patients with myeloid leukemias. More recently, mutations in ASXL2, a paralog of ASXL1 with ~40% shared amino acid homology, have been discovered to occur specifically in patients with acute myeloid leukemia (AML) patients bearing the RUNX1-ETO (AML1-ETO; RUNX1-RUNX1T1) translocation and are amongst the most common mutations in RUNX1-ETO AML (mutated in 20-25% of patients). Although ASXL1 is critical for Polycomb Repressive Complex 2 function in myeloid hematopoietic cells and loss of Asxl1 recapitulates key aspects of myelodysplastic syndrome (MDS), the function of ASXL2 in normal or malignant hematopoiesis is unknown. We therefore set out to perform a functional comparison of ASXL1and ASXL2on hematopoiesis and transcription and determine the functional basis for frequent mutations in RUNX1-ETO AML. In vitro analyses of ASXL2 insertion/deletion mutations revealed that these mutations resulted in substantial reduction of ASXL2 protein expression, stability, and half-life. We therefore generated Asxl2 conditional knockout (cKO) mice to delineate the effect of ASXL2 loss on hematopoiesis. Competitive (Fig. 1A) and noncompetitive transplantation revealed that Asxl2 or compound Asxl1/2 loss resulted in cell-autonomous, rapid defects of hematopoietic stem cell function, self-renewal, and number with peripheral blood leukopenia and thrombocytopenia but without any obvious MDS features- phenotypes distinct from Asxl1 cKO mice. Mice with heterozygous deletion of Asxl2 demonstrated an intermediate phenotype between control and homozygous cKO mice indicating a gene dosage effect of Asxl2 loss. RNA sequencing (RNA-seq) of hematopoietic stem/progenitor cells from Asxl2- and Asxl1-deficient mice revealed twenty-fold greater differentially expressed genes in Asxl2 cKO mice relative to Asxl1 cKO mice. Interestingly, genes differentially expressed with Asxl2 loss significantly overlapped with direct transcriptional targets of RUNX1-ETO, findings not seen in Asxl1 cKO mice (Fig. 1B). Asxl2 target genes appeared to also be targets of RUNX1, a key gene repressed by RUNX1-ETO to promote leukemogenesis. Consistent with this, genome-wide analysis of Asxl2 binding sites through anti-Asxl2 ChIP-seq revealed that Asxl2 binding sites substantially overlap with those of Runx1. Overall, the above data suggest that Asxl2 may be a critical mediator of RUNX1-ETO mediated leukemogenesis by affecting the expression of RUNX1 and/or RUNX1-ETO target genes. RNA-seq of primary RUNX1-ETO AML patient samples revealed that ASXL2-mutant RUNX1-ETO patients form a distinct transcriptional subset of RUNX1-ETO AML (Fig. 1C) suggesting a specific role of ASXL2 in leukemogenesis. To functionally interrogate the role of ASXL2 loss in RUNX1-ETO mediated leukemogenesis we first utilized an in vitro model with RNAi-mediated depletion of ASXL1 or ASXL2 in the SKNO1 cell line (the only ASXL-wildtype human RUNX1-ETO cell line). RNA-seq revealed distinct target genes dysregulated by ASXL1 versus ASXL2 loss in these cells without any significant overlap. Anti-ASXL2, RUNX1, and RUNX1-ETO ChIPSeq in SKNO1 cells revealed significant co-occupancy of ASXL2 with RUNX1 and RUNX1-ETO binding sites. Moreover, analysis of histone modification ChIPSeq revealed an enrichment in intergenic and enhancer H3K4me1 abundance following ASXL2 loss in SKNO1 cells. Next, to understand the in vivo effects of Asxl2 loss in the context of RUNX1-ETO, we performed retroviral bone marrow (BM) transplantation assays using RUNX1-ETO9a in Asxl2 cKO mice. In contrast to the failure of hematopoietic stem cell function with Asxl2 deletion alone, mice reconstituted with BM cells expressing RUNX1-ETO9a in Asxl2-deficient background had a shortened leukemia-free survival compared to Asxl2 -wildtype control. Overall, these data reveal that ASXL2 is required for hematopoiesis and has differing biological and transcriptional functions from ASXL1. Moreover, this work identifies ASXL2 as a novel mediator of RUNX1-ETOtranscriptional function and provides a new model of penetrant RUNX1-ETO AML based on genetic events found in a substantial proportion of t(8;21) AML patients. Further interrogation of the enhancer alterations generated by ASXL2 loss in RUNX1-ETO AML may highlight new therapeutic approaches for this subset of AML. Figure 1. Figure 1. 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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