Insufficiency of Non-Canonical PRC1 Complex Cooperates with an Activating JAK2 Mutation in the Pathogenesis of Myelofibrosis

Abstract Introduction: PcG proteins form two main multiprotein complexes, Polycomb repressive complex 1 (PRC1) and PRC2. They repress the transcription of target genes. Polycomb group ring finger protein1 (PCGF1) is a component of PRC1.1, a non-canonical PRC1.1 that monoubiquitylates H2A at lysine 119 in a manner independent of H3K27me3. Several groups including ours showed that the loss of Ezh2, a component of PRC2, promotes the development of JAK2 V617F-induced Myelofibrosis (MF) in mice. However, the role of PRC1.1 in hematologic malignancies is still not fully understood. We found that the deletion of PCGF1 in mice promotes myeloid commitment of hematopoietic stem and progenitor cells (HSPCs), and eventually induces a lethal myeloproliferative neoplasm (MPN)-like disease in mice (Nakajima-Takagi Y, unpublished data). Based on these findings, we investigated the role of PCGF1 in a mouse model of JAK2V617F-induced myelofibrosis. Methods: We transplanted BM cells from Cre-ERT2, PCGF1flox/flox;Cre-ERT2, JAK2V617F;Cre-ERT2, and JAK2V617F;PCGF1flox/flox;Cre-ERT2 mice into lethally irradiated recipient mice. We deleted PCGF1 by tamoxifen administration 4 weeks after transplantation. Results: JAK2/PCGF1 KO mice developed lethal MF significantly earlier than the other genotypes (p<0.01). JAK2/PCGF1 KO mice showed progressive anemia and severe thrombocytopenia. Bone marrow analysis of JAK2/PCGF1 KO mice revealed a significant reduction in HSPCs and an increase in the number of granulocyte-macrophage progenitors (GMPs). Erythropoiesis was severely impaired at the later stages of erythroid differentiation. To understand the molecular basis of MF-initiating cells in JAK2/PCGF1 KO mouse, we performed a gene expression analysis of LSKs/GMPs/MEPs isolated from the primary recipients 1 month after TAM injection. Gene set enrichment analysis of RNA-seq data clearly showed de-repression of PRC1 target genes marked with H2AK119ub1 in hematopoietic stem and progenitor cells (HSPCs) from JAK2/PCGF1 KO mice. The gene set of megakaryocyte progenitors was also positively enriched in JAK2/PCGF1 KO HSPCs. ChIP sequencing of H2AK119Ub revealed that the levels of H2AK119Ub at promoter regions were mildly reduced in JAK2/PCGF1 KO LK cells compared with Pcgf1 KO LK cells. Among differentially expressed genes, we found that HoxA cluster genes were de-repressed in JAK2/PCGF1 KO progenitor cells including MEPs following significant reductions in H2AK119Ub levels at the promoter regions. Lin28b-Let-7-Hmga2 pathway genes that are activated in JAK2/Ezh2 KO progenitor cells were not altered in expression in JAK2/PCGF1 KO progenitor cells, suggesting different mechanisms operating in the pathogenesis of JAK2/Ezh2 KO and JAK2/PCGF1 KO MF. A selective AURKA inhibitor has been reported to promote differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF (Wen et al., Nat Med 21:1473, 2015). Following this report, we treated JAK2/PCGF1 KO mice with JAK inhibitors and/or AURKA inhibitors. Both inhibitors improved MF-related phenotypes including impaired erythroid differentiation of JAK2/PCGF1 KO mice. Conclusions: Our findings suggest that dysregulated PRC1.1 function promotes JAK2V617F-induced MF with mechanisms distinct from MF associated with PRC2 dysfunction. Disclosures Harada: Celgene: Research Funding.

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A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

Blood ◽

10.1182/blood-2019-126677 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3736-3736

Author(s):

Tanabe Mikoto ◽

Nguyen Hoang Maianh ◽

Kohei Hosokawa ◽

Noriharu Nakagawa ◽

Luis Espinoza ◽

...

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Leukemia Cell Line ◽

Serum Free Medium ◽

Free Medium ◽

Hematopoietic Stem ◽

Serum Free ◽

Company Limited ◽

Immune Mediated

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

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Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation

Blood ◽

10.1182/blood-2009-10-250894 ◽

2010 ◽

Vol 115 (22) ◽

pp. 4367-4376 ◽

Cited By ~ 16

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Target Genes ◽

Lentiviral Vector ◽

Erythroid Differentiation ◽

Down Regulation ◽

Hematopoietic Stem ◽

Progenitor Cell Proliferation ◽

Cord Blood Cells

Abstract Previously, we have shown that overexpression of an activated mutant of signal transducer and activator of transcription-5 (STAT5) induces erythropoiesis, impaired myelopoiesis, and an increase in long-term proliferation of human hematopoietic stem/progenitor cells. Because GATA1 is a key transcription factor involved in erythropoiesis, the involvement of GATA1 in STAT5-induced phenotypes was studied by shRNA-mediated knockdown of GATA1. CD34+ cord blood cells were double transduced with a conditionally active STAT5 mutant and a lentiviral vector expressing a short hairpin against GATA1. Erythropoiesis was completely abolished in the absence of GATA1, indicating that STAT5-induced erythropoiesis is GATA1-dependent. Furthermore, the impaired myelopoiesis in STAT5-transduced cells was restored by GATA1 knockdown. Interestingly, early cobblestone formation was only modestly affected, and long-term growth of STAT5-positive cells was increased in the absence of GATA1, whereby high progenitor numbers were maintained. Thus, GATA1 down-regulation allowed the dissection of STAT5-induced differentiation phenotypes from the effects on long-term expansion of stem/progenitor cells. Gene expression profiling allowed the identification of GATA1-dependent and GATA1-independent STAT5 target genes, and these studies revealed that several proliferation-related genes were up-regulated by STAT5 independent of GATA1, whereas several erythroid differentiation-related genes were found to be GATA1 as well as STAT5 dependent.

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Role of HMGA2 in human hematopoietic stem and progenitor cells

Experimental Hematology ◽

10.1016/j.exphem.2013.05.173 ◽

2013 ◽

Vol 41 (8) ◽

pp. S44

Author(s):

Praveen Kumar ◽

Aurélie Baudet ◽

Ineke De Jong ◽

Jonas Larsson

Keyword(s):

Progenitor Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2409.2409 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2409-2409

Author(s):

Yiwen Song ◽

Sonja Vermeren ◽

Wei Tong

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Ph Domain ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Stem And Progenitor Cells

Abstract ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined. Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood. Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues. We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants. Arap3 KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation. Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures: No relevant conflicts of interest to declare.

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Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v126.23.4107.4107 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4107-4107

Author(s):

Susan Hilgendorf ◽

Hendrik Folkerts ◽

Jan Jacob Schuringa ◽

Edo Vellenga

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Erythroid Differentiation ◽

Lentiviral Vectors ◽

Apoptotic Response ◽

Double Positive ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

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p53-dependent induction of P2X7 on hematopoietic stem and progenitor cells regulates hematopoietic response to genotoxic stress

Cell Death and Disease ◽

10.1038/s41419-021-04202-9 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Lin Tze Tung ◽

HanChen Wang ◽

Jad I. Belle ◽

Jessica C. Petrov ◽

David Langlais ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Transcriptional Response ◽

Genotoxic Stress ◽

Whole Body ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Stem And Progenitor Cells ◽

Hematopoietic Response

AbstractStem and progenitor cells are the main mediators of tissue renewal and repair, both under homeostatic conditions and in response to physiological stress and injury. Hematopoietic system is responsible for the regeneration of blood and immune cells and is maintained by bone marrow-resident hematopoietic stem and progenitor cells (HSPCs). Hematopoietic system is particularly susceptible to injury in response to genotoxic stress, resulting in the risk of bone marrow failure and secondary malignancies in cancer patients undergoing radiotherapy. Here we analyze the in vivo transcriptional response of HSPCs to genotoxic stress in a mouse whole-body irradiation model and, together with p53 ChIP-Seq and studies in p53-knockout (p53KO) mice, characterize the p53-dependent and p53-independent branches of this transcriptional response. Our work demonstrates the p53-independent induction of inflammatory transcriptional signatures in HSPCs in response to genotoxic stress and identifies multiple novel p53-target genes induced in HSPCs in response to whole-body irradiation. In particular, we establish the direct p53-mediated induction of P2X7 expression on HSCs and HSPCs in response to genotoxic stress. We further demonstrate the role of P2X7 in hematopoietic response to acute genotoxic stress, with P2X7 deficiency significantly extending mouse survival in irradiation-induced hematopoietic failure. We also demonstrate the role of P2X7 in the context of long-term HSC regenerative fitness following sublethal irradiation. Overall our studies provide important insights into the mechanisms of HSC response to genotoxic stress and further suggest P2X7 as a target for pharmacological modulation of HSC fitness and hematopoietic response to genotoxic injury.

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TNFα Rescues Dendritic Cell Development in Hematopoietic Stem and Progenitor Cells Lacking C/EBPα

Cells ◽

10.3390/cells9051223 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1223

Author(s):

Subramanian Anirudh ◽

Angelika Rosenberger ◽

Elke Schwarzenberger ◽

Carolin Schaefer ◽

Herbert Strobl ◽

...

Keyword(s):

Progenitor Cells ◽

Gene Expression Analysis ◽

Hematopoietic Stem ◽

Reporter Mouse ◽

Multistep Process ◽

Stem And Progenitor Cells ◽

Culture Supernatants ◽

Cytokine Profiling

Dendritic cells (DCs) are crucial effectors of the immune system, which are formed from hematopoietic stem and progenitor cells (HSPCs) by a multistep process regulated by cytokines and distinct transcriptional mechanisms. C/EBPα is an important myeloid transcription factor, but its role in DC formation is not well defined. Using a CebpaCre-EYFP reporter mouse model, we show that the majority of splenic conventional DCs are derived from Cebpa-expressing HSPCs. Furthermore, HSPCs isolated from Cebpa knockout (KO) mice exhibited a marked reduced ability to form mature DCs after in vitro culture with FLT3L. Differentiation analysis revealed that C/EBPα was needed for the formation of monocytic dendritic progenitors and their transition to common dendritic progenitors. Gene expression analysis and cytokine profiling of culture supernatants showed significant downregulation of inflammatory cytokines, including TNFα and IL-1β as well as distinct chemokines in KO HSPCs. In addition, TNFα-induced genes were among the most dysregulated genes in KO HSPCs. Intriguingly, supplementation of in vitro cultures with TNFα at least partially rescued DC formation of KO HSPCs, resulting in fully functional, mature DCs. In conclusion, these results reveal an important role of C/EBPα in early DC development, which in part can be substituted by the inflammatory cytokine TNFα.

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Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium

Blood ◽

10.1182/blood.v128.22.371.371 ◽

2016 ◽

Vol 128 (22) ◽

pp. 371-371

Author(s):

Ryohichi Sugimura ◽

Areum Han ◽

Deepak Jha ◽

Yi-Fen Lu ◽

Jeremy A Goettel ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Target Genes ◽

Conflicts Of Interest ◽

Functional Characterization ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. Disclosures No relevant conflicts of interest to declare.

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c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression

Blood ◽

10.1182/blood-2009-08-238311 ◽

2010 ◽

Vol 116 (22) ◽

pp. e99-e110 ◽

Cited By ~ 74

Author(s):

Elisa Bianchi ◽

Roberta Zini ◽

Simona Salati ◽

Elena Tenedini ◽

Ruggiero Norfo ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Fate ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Luciferase Reporter ◽

Myb Transcription Factor ◽

Promoter Regions ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb–silenced CD34+ cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

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The staying power of hematopoietic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.202109019 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Michael L. Dustin

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Cxcr4 Signaling ◽

Stem And Progenitor Cells ◽

Necessary And Sufficient

Hematopoietic stem and progenitor cells (HSPCs) use specialized adhesive structures referred to as magnupodium to stay in hematopoietic niches. Bessey et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202005085) define new characteristics of the magnupodium, including centriole polarization and the necessary and sufficient role of CXCR4 signaling.

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