Induction Of Hematopoietic Differentiation Research Articles

SETD7 promotes lateral plate mesoderm formation by modulating the Wnt/β-catenin signaling pathway

The role of SET domain containing 7 (SETD7) during human hematopoietic development remains elusive. Here, we found that deletion of SETD7 attenuated the generation of hematopoietic progenitor cells (HPCs) during the induction of hematopoietic differentiation from human embryonic stem cells (hESCs). Further analysis specified that SETD7 was required for lateral plate mesoderm (LPM) specification but dispensable for the generation of endothelial progenitor cells (EPCs) and HPCs. Mechanistically, rather than depending on its histone methyltransferase activity, SETD7 interacted with β-catenin at lysine residue 180 facilitated its degradation. Diminished SETD7 expression led to the accumulation of β-catenin and the consequent activation of the Wnt signaling pathway, which altered LPM patterning and facilitated the production of paraxial mesoderm (PM). Taken together, the findings indicate that SETD7 is related to LPM and PM patterning via posttranslational regulation of the Wnt/β-catenin signaling pathway, providing novel insights into mesoderm specification during hematopoietic differentiation from hESCs.

Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system.

IntroductionSickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD.ObjectiveGeneration of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium.MethodThis study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed.ResultsThe patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium.ConclusionIn conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross-presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8-/- iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8-/- DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898-908.

AF10 Plays a Key Role in the Survival of Uncommitted Hematopoietic Cells

Hematopoiesis is a complex process regulated by both cell intrinsic and cell extrinsic factors. Alterations in the expression of critical genes during hematopoiesis can modify the balance between stem cell differentiation and proliferation, and may ultimately give rise to leukemia and other diseases. AF10 is a transcription factor that has been implicated in the development of leukemia following chromosomal rearrangements between the AF10 gene and one of at least two other genes, MLL and CALM. The link between AF10 and leukemia, together with the known interactions between AF10 and hematopoietic regulators, suggests that AF10 may be important in hematopoiesis and in leukemic transformation. Here we show that AF10 is important for proper hematopoietic differentiation. The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors. Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors. We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.

Induced Pluripotent Stem Cells (iPSC) From Chronic Myeloid Leukemia: Study of BCR-ABL Addiction and Effect of Tyrosine Kinase Inhibitors,

Abstract 3754The tyrosine kinase inhibitors (TKI) such as imatinib, by suppressing BCR-ABL oncogene activity, are an effective therapy for chronic myeloid leukemia disease (CML). However, the majority of patients achieving remission with TKI continue have molecular evidence of persistent disease. In addition, we have reported that for patients who achieved a sustained complete molecular remission, 60% of them relapse after discontinuation of imatinib. Various mechanisms have been proposed to explain disease persistence and disease recurrence. One of the hypotheses is that primitive leukemic stem cells can survive in the presence of TKI. Little is known about the stem cells survival due to technical difficulties (small and poorly defined primary populations). Understanding the mechanisms by which these cells survive to TKI therapy will be critical to devising strategy aimed to their elimination. We propose to generate iPSC derived from CD34+ blood cells isolated from CML patient (CML-iPSC), as a model for study leukemic stem cells survival in the presence of TKI and study the mechanism of TKI resistance of the stem cells.Primary CD34+ CML patient cells were transduced by 2 excisable lentiviral vectors (both flanked by two LoxP sites), one expressing three reprogramming factors (OCT4-SOX2-KLF4) and another one with c-MYC and a shRNA against TP53. Twenty-one days after co-transduction, CML-iPSC colonies were picked and five iPS clones were characterized (expression of pluripotency markers by RT-PCR (DPPA4, NANOG, CRIPTO) and immunofluorescence (NANOG, SSEA-4, TRA1-60)). Efficiency of reprogrammation was low compared to cord blood CD34+ control cells (0.01% vs 0.1%, respectively), and delayed (21 days vs 14 days). Philadelphia chromosome (Ph) positive was observed in 4/5 clones after cytogenetic analysis. Expression of BCR-ABL (Western-blot and RT-PCR) was present at various levels. Interestingly, 1/5 clone was generated from non-leukemic cell (Ph negative) and was used as internal control for the following function assays. We used these 5 CML-iPSC clones to study their behavior in presence of TKI. All CML-iPSC clones survived to escalating concentration of imatinib (0 to 20μM) and ponatinib (0 to 50nM) for 6 days. To understand if the CML-iPSC survival was due to resistance or independence mechanisms, we performed western blot analysis of TKI targets. BCR-ABL activity was inhibited under TKI exposure (dephosphorylations of BCR/ABL and of Crkl). In order to check whether survival was due to the expression of reprogramming factors, we excised the gene cassettes by an Adenovirus expressing CRE recombinase. After proviral excision and subcloning, excised CML-iPSC continued to survive to TKI exposure. Taken together, these results demonstrate that CML-iPSC survival do not depend on BCR-ABL (oncogene independence). Upon induction of hematopoietic differentiation, CML-iPSC were able to efficiently generate progenitors of hematopoietic lineages (up to 40% of CD45+) and colony forming units in methylcellulose. TKI effect on iPSC-derived hematopoietic progenitors, to analyze the putative recovery of TKI sensibility compared to primitive CML blood cells from the same patient, are in progress.We conclude that reprogrammation of CD34BCR-ABL+ cells from CML patient is possible and that CML-iPSC lost the BCR-ABL dependency and became resistant to TKI. A specific differentiated epigenetic cell state is probably needed to maintain BCR-ABL dependency. CML-iPSC can be used to study mechanisms by which leukemic stem cells survive to TKI therapy and is a promising tool for testing and screening new therapeutic target reducing leukemic stem cell survival. Disclosures:Mahon:Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizzer: Honoraria.

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells.

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.

Partial Reversal of the Methylation Pattern of the X-linked Gene HUMARA during Hematopoietic Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT-PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.

X-Linked Clonality Analysis of Human Embryonic Stem Cells ( hESC ) Before and after induction of Hematopoietic Differentiation

Human ES cell lines represent an invaluable tool of research for the study of normal and pathological development. In this study we asked whether the human ES cell lines currently used for research purposes are derived from single outgrowths using X-linked clonality analysis. Amongst the cell lines tested, the female H9 cell line was informative at the Humara (human androgen receptor) locus by the presence of two CAG repeats allowing distinction of paternal (Xp) and maternal (Xm) X chromosomes. Clonal analysis was performed upon Hpa II digestion of DNA purified from total H9 cells (passages 27–61), single H9 cell clones generated from single cell cloning, single embryoid bodies (EB) (day 4, day 14) as well as from single hematopoietic progenitors. The same analysis was performed after induction of hematopoietic differentiation in liquid cultures in the presence of hematopoietic permissive conditions (co-culture in the presence of OP-9 cell line). Clonality was evaluated by calculating the Relative Corrected Index (RCI) which is the ratio of the intensity of peaks generated by Hpa II-digested and undigested DNA. In several experiments, all passages of H9 cell lines examined ( p27, p28, p31, p61) were found to exhibit a monoclonal pattern with disappearance of the same allele A upon Hpa II digestion (RCI > 10). 13 clonal cultures of H9 obtained by single cell cloning exhibited also the same clonal pattern with digestion of the same allele and RCI values between 6–14. Similarly, DNA purified from EB's at different stages was found to be clonal. In DNA obtained from 75 individual hematopoietic progenitors (CFU-GM, CFU-G), clonal analysis revealed the same clonal pattern in each, with disappearance of the same allele A. However, clonal analysis performed in 10 hematopoietic colonies, revealed unexpectedly, an equal digestion of Humara alleles with RCI < 2 (polyclonal pattern). Finally, in two experiments, hematopoietic cells recovered from liquid cultures exhibited an absolute clonal pattern (Total disappearance of the same allele A). Interestingly, quantification of X-Inactivation Specific Transcript (XIST) RNA in H9 and day14 EB's by RT-PCR did not show any evidence of XIST expression both before and after differentiation. Altogether, our data suggest that XCI has already been initiated in undifferentiated H9 cells and this inactivation is maintained, at least in the Humara locus in the absence of XIST. The monoclonal pattern of the cell line can be due to the fact that H9 ES cell line might have arisen from a limited number of stem cells having undergone XCI at a very early stage, with maintenance of the methylation status at this locus during mesodermal and hematopoietic commitment. Conversely, hematopoietic differentiation might occur from several mesodermal progenitors having inactivated the same X chromosome in H9 at the Humara locus. Despite the fact that hematopoietic progenitors and differentiated hematopoietic cells originating from H9 exhibited also a clonal pattern, a partial reactivation of the inactive X chromosome during hematopoietic differentiation could occur, and may explain the “polyclonal” pattern obtained in 10 CFU-GM analyzed. Overall, X-linked clonality testing could be an important issue before considering the potential clinical applications, in all female hES cell lines.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta‐Gonado‐Mesonephros Region

The hematopoietic system interacts with a supportive stromal environment allowing maintenance and differentiation of hematopoietic stem cells (HSCs). The aorta-gonado-mesonephros (AGM) region serves as a unique embryonic microenvironment, generating the first adult repopulating HSCs in the mouse embryo. To eludicate factors involved in hematopoietic support and induction of hematopoietic differentiation, we isolated more than 100 stromal cell clones derived from the AGM region of embryonic day (E) 10.5 mouse embryos for functional and genetic analysis. Selected isolated AGM stromal cell lines are highly efficient in supporting maintenance and expansion of mouse and human hematopoietic stem and progenitor cells. In addition, we can demonstrate for the first time that AGM stromal cell lines are also potent inducers of hematopoietic differentiation of murine embryonic stem cells. Stromal gene array analysis has identified genes that could play a role in hematopoietic support.

Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by 2-(allylthio) pyrazine

Induction of hematopoietic differentiation in human promyelocytic leukemia cells (HL-60) by new synthetic drugs or natural products has recently been recognized as a new strategy in the identification and testing of potential cancer chemopreventive and/or chemotherapeutic agents. 2-(Allythio) pyrazine (2-AP) is a pyrazine derivative of allysulfide, which has been suggested to be a potential cancer chemopreventive agent in previous in vivo and in vitro experiments. In the present study, we have investigated the inducing effect of granulocytic differentiation in HL-60 cells by 2-AP. Treatment of HL-60 cells with various concentrations of 2-AP (1–100 μM) for 7 days showed the induction of granulocytic differentiation following both morphological examination and NBT (nitroblue tetrazolium) testing (up to 40 and 52%, respectively). The expressions of bcl-2 and c-myc were down-regulated during granulocytic differentiation of HL-60 cells (up to 40%). The immunoblots for G1 cyclins in the G1-S phase transition (cyclin D1 and E) showed a progressive decrease of their expressions in both concentration- and time-dependent manners (up to 30 and 50%, respectively). These results suggest that 2-AP could induce the differentiation of HL-60 cells and might have potent cancer chemoprevention and/or chemotherapy roles in human leukemias.

Hematopoietic Stem Cell Maintenance and Differentiation Are Supported by Embryonic Aorta-Gonad-Mesonephros Region–Derived Endothelium

Hematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34+ cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin−CD34+Sca-1+c-Kit+phenotype and was proficient at maintaining fetal liver–derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.© 1998 by The American Society of Hematology.

Hematopoietic Stem Cell Maintenance and Differentiation Are Supported by Embryonic Aorta-Gonad-Mesonephros Region–Derived Endothelium

AbstractHematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34+ cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin−CD34+Sca-1+c-Kit+phenotype and was proficient at maintaining fetal liver–derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.© 1998 by The American Society of Hematology.