Functional Analysis of FOXO3A Involved in Erythroid Differentiation

Abstract Abstract 4731 FOX (Forkhead box) proteins are a family of transcription factors that emerged as playing an important role in the embryonic development, cell cycle, carbohydrate and fatty acid metabolism and immune response. It was found that FOXO3A (also known as FOXO3) involved in erythroid differentiation, yet the mechanism for regulating hematopoietic stem cells (HSCs) differentiation is unknown. We analyzed the dynamics of genome-wide transcriptome (mRNA-Seq) of human undifferentiated embryonic stem cells (HESC), erythroid cells derived from ES cells (ESER), human fetal erythroid liver cells (FLER) and peripheral CD34+derived erythroid cells (PBER) using high throughput sequencing technology. The transcriptome analysis showed that FOXO3 was barely expression in HESC while was observably up-regulated in ESER. However, FOXO3 was down-regulated in FLER and PBER compare with ESER, the erythroid cells at early developmental stage. We presumed that FOXO3 plays an important role in primitive erythropoiesis and built up the interactions network in which FOXO3 acts as a central node by Gene Ontology (GO), correlation analysis and Ingenuity Pathways Analysis (IPA). In addition, we analyzed the profiles of histone methylation in the four types of cells by ChIP-Seq to study the chromatin conformation in the vicinity of FOXO3. More histone 3 lysine 4 (H3K4) trimethylation was found near the promoter region of FOXO3 in ESER compared with the other cells, which is coincided with the mRNA-seq results. We performed a series of experiment to identify the roles of FOXO3 in regulating erythroid differentiation. The results showed that the expression level of ε and γ globin were up-regulated in FOXO3-over-expressed 293T and Hela cells and the expression level of FOXO1 and CAT in predicted network were increased by quantitative real-time PCR detection. In addition, when FOXO3 knocked down in K562 cells, the expression level of ε and γ globin were down-regulated. The expression level of CAT, BCL2L1 and other factors in predicted network, were also decreased. These results indicate FOXO3 plays an important role in globin expression and identify the credibility of our predicted networks in which FOXO3 acts as a central node. FOXO3 binding sites (GTAAACA or ATAAACA) were predicted on the upstream of CAT and BCL2L1. We are trying to prove CAT or BCL2L1 is a direct FOXO3 target in vitro and in vivo. In conclusion, we have demonstrated FOXO3 plays a key role in erythroid differentiation and globin expression. We will further determine the enriched profiles of FOXO3 by ChIP-seq in HESC, ESER, FLER and PBER to find more targets of FOXO3. Since the zebrafish is a powerful model system for investigating vertebrate hematopoiesis. We will identify the role of Foxo3b, the homologous gene of human FOXO3, in erythroid differentiation and study the dynamic transcriptomes of Foxo3b morphants in zebrafish. We are trying to make a whole picture to elaborate the molecular mechanism of FOXO3 involved in regulation of erythroid differentiation. Disclosures: No relevant conflicts of interest to declare.

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LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

Blood ◽

10.1182/blood.v122.21.4846.4846 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4846-4846

Author(s):

Yue Jin ◽

Yidi Guo ◽

Dongxue Liang ◽

Yue Li ◽

Zhe Li ◽

...

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Es Cells ◽

Regulatory Element ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Mouse Es Cells ◽

Murine Erythroleukemia

Abstract GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated. In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival. Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures: No relevant conflicts of interest to declare.

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Expression Cloning of Genes Enabling Erythropoietin -Independent Erythropoiesis in Vitro.

Blood ◽

10.1182/blood.v114.22.3608.3608 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3608-3608

Author(s):

Tomoko Yokoo ◽

Ryo Kurita ◽

Atsushi Takahashi ◽

Michiyo Okada ◽

Hirotaka Kawano ◽

...

Keyword(s):

Stem Cells ◽

Target Genes ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Apoptotic Cell ◽

Es Cells ◽

Embryonic Stem ◽

Ips Cells ◽

Expression Cloning ◽

Hematopoietic Stem

Abstract Abstract 3608 Poster Board III-544 Erythropoietin (EPO) is essential during both ontogeny and the course of erythropoiesis. In primitive (yolk sac) erythropoiesis, however, the role of EPO is not fully understood. Elucidation of such role in primitive erythropoiesis would be very helpful for the development of ex vivo red blood cell expansion system from embryonic stem (ES) cells. Recently, we reported the establishment of the ex vivo induction of hematopoietic stem cells from common marmoset ES cells using lentiviral-Tal1/Scl gene transfer in the absence of any stromal cells (Stem Cells 24: 2014-2022, 2006). This method should also be applicable to both of human ES cells and induced pluripotential stem (iPS) cells, but the efficiency will be very critical for its clinical application. In the present study, we proceed further to find out unknown factors which accelerate ex vivo proliferation and differentiation of erythroid cells, constructed the human fetal liver cDNA expression lentiviral library (Mol Cell Biochem 319: 181-187, 2008) and screened for cDNAs which confer EPO independency to an EPO-dependent cell line, UT-7/Epo ( Blood 82: 456-464, 1993). Among twenty-two candidate genes cloned after screening of 6×10∧5 cDNA, we particularly focused on two full-length genes, ribosomal protein L11 (RPL11) and retinol dehydrogenase 11 (RDH11). Two candidate gene-transduced-UT-7/Epo cells, respectively named LV-RPL11 and LV-RDH11, showed complete EPO-independent survival and proliferation, increased expression of fetal γ-globin, and decreased expression of adult β-globin compared with parental UT-7/Epo cells in the presence of EPO. Cell cycle and apoptosis analyses showed decreased apoptotic cell death and increased S/G2/M cells in LV-RPL11 and LV-RDH11 cells compared with UT-7/Epo cells in the absence of Epo. Moreover, STAT5 phosphorylation and upregulation of its target genes, c-Myc, cyclin D and Pim, were observed in LV-RPL11, LV-RDH11 cells in the absence of EPO. In conclusion, the findings suggest that RPL11 and RDH11 play a role in EPO-independent erythropoiesis and might be applicable to ex vivo expansion of red blood cells from ES/iPS cells. Disclosures: No relevant conflicts of interest to declare.

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Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽

10.1182/blood-2005-09-3621 ◽

2006 ◽

Vol 107 (4) ◽

pp. 1265-1275 ◽

Cited By ~ 51

Author(s):

Abby L. Olsen ◽

David L. Stachura ◽

Mitchell J. Weiss

Keyword(s):

Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Basic Research ◽

Cell Types ◽

Patient Specific ◽

Es Cell ◽

Blood Disorders ◽

Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

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Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽

10.1182/blood.v87.7.2740.bloodjournal8772740 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2740-2749 ◽

Cited By ~ 83

Author(s):

CD Helgason ◽

G Sauvageau ◽

HJ Lawrence ◽

C Largman ◽

RK Humphries

Keyword(s):

Stem Cells ◽

Es Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Homeobox Genes ◽

Embryoid Bodies ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

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Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin

Blood ◽

10.1182/blood-2005-11-011874 ◽

2006 ◽

Vol 108 (5) ◽

pp. 1515-1523 ◽

Cited By ~ 97

Author(s):

Kai-Hsin Chang ◽

Angelique M. Nelson ◽

Hua Cao ◽

Linlin Wang ◽

Betty Nakamoto ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Molecular Mechanisms ◽

Embryoid Body ◽

Fetal Liver ◽

Embryonic Stem ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Promising Tool

Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (ϵ) and fetal (γ) globins, with little or no adult globin (β). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.

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Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

Journal of Polymer Engineering ◽

10.1515/polyeng-2016-0193 ◽

2017 ◽

Vol 37 (7) ◽

pp. 647-660 ◽

Cited By ~ 5

Author(s):

Saradaprasan Muduli ◽

Li-Hua Chen ◽

Meng-Pei Li ◽

Zhao-wen Heish ◽

Cheng-Hui Liu ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Extracellular Matrix ◽

Stem Cell ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Poly Vinyl Alcohol ◽

Hematopoietic Stem ◽

Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

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BCR-ABL Activates STAT3 Via MEK Kinase 1 in Embryonic Stem Cells.

Blood ◽

10.1182/blood.v104.11.4320.4320 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4320-4320

Author(s):

Yukinori Nakamura ◽

Toshiaki Yujiri ◽

Ryouhei Nawata ◽

Kozo Tagami ◽

Yukio Tanizawa

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Es Cells ◽

Embryonic Stem ◽

Myelogenous Leukemia ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mek Kinase

Abstract BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate STAT3 and to promote self-renewal in ES cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction (Oncogene22:7774, 2003). To investigate the role of MEKK1 in Bcr-Abl-induced transformation of ES cells, p210 Bcr-Abl was stably transfected into wild type (WT+p210) and MEKK1−/− (MEKK1−/−+p210) ES cells. Bcr-Abl enhanced both MEKK1 expression and activation in ES cells, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT+p210 cells showed constitutive STAT3 activation and formed compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1−/−+p210 cells, by contrast, showed less STAT3 activity than WT+p210 cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays an essential role in Bcr-Abl-induced STAT3 activation and in the capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells.

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Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.1462.1462 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1462-1462

Author(s):

Michael Tallack ◽

Thomas Whitington ◽

Brooke Gardiner ◽

Eleanor Wainwright ◽

Janelle Keys ◽

...

Keyword(s):

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Es Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Gene Promoters ◽

Erythroid Cells ◽

Red Cell Membrane ◽

Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

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Characterization of Histone Modifications on Lineage-Affiliated Genes During Hematopoietic Stem Cell Differentiation

Blood ◽

10.1182/blood.v116.21.4784.4784 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4784-4784

Author(s):

Chen Fangping ◽

Huarong Tang

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Histone Modifications ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Low Level ◽

Lineage Specific Genes

Abstract Abstract 4784 Hematopoietic stem cells (HSCs) are multipotent stem cells capable of self-renewal and multi-lineage differentiation. Though it has been shown that multiple factors take part in the maintenance of HSCs’ multipotency and differentiation potential, the mechanisms are unclear. Recent studies showed that histone modifications play an important role in maintenance of embryonic stem cells pluripotency and differentiation. To characterize the histone modification patterns of different lineages, HSCs were collected from umbilical cord blood and induced to differentiate to granulocytic, erythroid, and megakarytic in vitro. genes during HSC differentiation. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) technology was adopted to investigate the dynamic changes of histone modifications on lineage specific transcription factors and lineage–affiliated genes. Our results showed a certain level of H4 acetylation and H3 acetylation together with high level of H3K4me2 and low level of H3K4me3, H3K9me3 and H3K27me3 were present in lineage specific genes in CD34+CD38- HSCs. As CD34+CD38- cells differentiated, the modification level of acH3, acH4, H3K4me2, H3K9me3 and H3K27me3 on lineage specific genes remained the same, while H3K4me3 level increased greatly. In non-lineage specific genes, the acH3 and acH4 levels decreased, and H3K4me3 level remain at low level, while H3K9me3 and H3K27me3 levels increased. Thus, our data suggested that histone modifications played an important role in maintenaning the multipotency and differentiation capability of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Efficient Erythropoiesis From Human Embryonic Stem Cells Through Dimerization of Intracellular MPL.

Blood ◽

10.1182/blood.v120.21.2291.2291 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2291-2291

Author(s):

William Sang Kim ◽

Gautam G. Dravid ◽

Yuhua Zhu ◽

Chintan Parekh ◽

Qiming Deng ◽

...

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Total Yield ◽

Embryonic Stem ◽

Erythroid Differentiation ◽

Akt Signaling ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Enucleated Rbc ◽

Negative Controls

Abstract Abstract 2291 Objectives: Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic rather than adult or fetal hemoglobins. Therefore, efficient clinical translation of this strategy is critically dependent on the development of novel methods to enhance the generation of functional RBCs from PSC. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods: Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results: F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. The increased yield of erythroid cells was not due to an overall increase in cell proliferation as the total yield of cells was not statistically different between treated and untreated cultures. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.0058; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. This indicates that CID has a greater effect on terminal erythroid differentiation than EPO. We then investigated the signaling mechanism activated by F36V-MPL dimerization and found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.0442; n=2) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Conclusion: F36V-MPL dimerization during hESC-derived hematopoiesis induces EPO-independent erythroid differentiation through AKT signaling, by both generating erythroid progenitors and promoting maturation of RBC. MPL dimerization also is more potent than EPO in inducing erythropoiesis from hESC and has an additive effect when combined with EPO, making this a potential strategy for the generation of therapeutically relevant levels of functional enucleated RBCs from PSC. Disclosures: No relevant conflicts of interest to declare.

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