TY - JOUR
AU - Jackson, Melany
AB - Abstract Pluripotent stem cells can be differentiated into hematopoietic lineages in vitro and hold promise for the future treatment of hematological disease. Differentiation strategies involving defined factors in serum‐free conditions have been successful in producing hematopoietic progenitors and some mature cell types from mouse and human embryonic stem cells and induced pluripotent cells. However, these precisely defined protocols are relatively inefficient and have not been used successfully to produce hematopoietic stem cells capable of multilineage long‐term reconstitution of the hematopoietic system. More complex differentiation induction strategies including coculture with stromal cells derived from sites of hematopoietic activity in vivo and enforced expression of reprogramming transcription factors, such as HOXB4, have been required to increase the efficiency of the differentiation procedure and to produce these most potent hematopoietic stem cells. We review the studies that have used HOXB4 to improve hematopoietic differentiation from pluripotent cells focusing on studies that have provided some insight into its mechanism of action. A better understanding of the molecular pathways involved in the action of HOXB4 might lead to more defined culture systems and safer protocols for clinical translation. Disclosure of potential conflicts of interest is found at the end of this article. Embryonic stem cells, Differentiation, Hematopoietic induction, HOXB4 Introduction Diseases of the hematopoietic system have been treated for decades with cell‐based therapies such as bone marrow transplantation and blood transfusion. These procedures are completely reliant on a limited supply of donor tissue, and hence a common goal for many researchers has been to develop strategies to address this problem. One method resides in the production of hematopoietic stem cells (HSCs) from a bankable and limitless source of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) and to expand and differentiate these HSCs in vitro [1]. Although there are numerous reports on the production of mature hematopoietic cells and hematopoietic progenitors cells (HPCs) from ESCs and iPSCs [2, 3], this field faces some significant challenges. It has proven particularly difficult to produce the most potent HSCs capable of long‐term reconstitution HSCs (LTR‐HSCs) for stem cell transplantation and to generate fully mature, functional cells such as enucleated erythrocytes for transfusion [4, 5]. Furthermore, the differentiation protocols that have been used to date are relatively inefficient and it has not been possible to produce large quantities of any desired cell type whether it be LTR‐HSCs, HPCs, or mature cells with a specific function. It is reasonable to assume that the production of fully functional blood cells in vitro should use strategies that aim to mimic the steps involved in their production in vivo. A recent review very elegantly describes the production of hematopoietic cells during embryonic development [6]. They are derived from the mesoderm germ layer and the first transient wave of primitive hematopoiesis first appear as “blood islands” in the yolk sac containing primitive monocytes and nucleated erythrocytes. Definitive HSCs capable of long‐term reconstitution first arise in the aorta‐gonad‐mesonephros (AGM) region of the developing embryo [7] followed by their appearance in the fetal liver and placenta. LTR‐HSCs eventually reside in the adult bone marrow where their proliferation and differentiation are controlled by intrinsic genetic programs together with extrinsic signals provided by the hematopoietic niche [8]. It was reported more than 25 years ago that the primitive wave of hematopoiesis could be replicated in differentiating ESCs; cystic embryoid bodies produced from mouse ESCs contained pockets of nucleated erythrocytes surrounded by endothelial cells [9]. This primitive hematopoiesis can be recapitulated in defined in vitro conditions by the stepwise addition of growth factors to mouse ESCs: bone morphogenetic protein (BMP4) promotes the efficient formation of mesodermal precursors, basic fibroblast growth factor (bFGF) and ActivinA promotes the further differentiation into hemangioblasts, and then vascular endothelial growth factor (VEGF) stimulates the production of fully committed hematopoietic progenitors [10]. A comparable combination of growth factors has been used for human ESCs and iPSCs in three‐dimensional embryoid body (EB) culture [11] and more recently in two‐dimensional culture on a matrix of collagen IV and fibronectin [3]. However, similar to the hematopoietic progenitor cells in the yolk sac, these primitive ESC‐derived cells are not capable of long‐term reconstitution [12]. Stromal cells derived from a number of hematopoietic sites in vivo including bone marrow, fetal liver, and AGM region have been widely used by our group and others to enhance hematopoietic differentiation and to produce LTR‐HSCs from ESCs and from yolk sac progenitors [2, 13] [14]. Indeed, one study led by our collaborators demonstrated the production of human ESC (hESC)‐derived LTR‐HSCs (albeit at low frequencies) using an AGM‐derived stromal cell line [15]. However, the molecular mechanisms responsible for the hematopoietic inductive activity or HSCs maintenance of stromal cells are not understood and only a limited number of studies have addressed this problem [16, 17]. Identification of the key signaling pathways responsible for the inductive and maintenance of LTR‐HSC by stromal cells will undoubtedly lead to the development of differentiation protocols using defined growth factor combinations. Another commonly used strategy to increase ESC‐derived hematopoiesis and to derive LTR‐HSCs from ESCs is enforced expression of homeobox genes including Hoxb4, Cdx4, and Hoxa4 [18–20]. These transcription factors have been considered both to reprogram cells into a definitive phenotype and to enhance the proliferation of hematopoietic progenitors without altering their differentiation potential. In one study, enforced expression of Hoxb4 was also shown to confer repopulating to yolk sac progenitors supporting the idea that these transcription factors can indeed reprogram cells to a specific phenotype [18]. We review the current literature on the use of Hoxb4 to enhance the production of hematopoietic lineages from pluripotent stem cells, focusing on those studies that have provided some insights into their mechanisms of action. Induction of Hematopoietic Differentiation by Enforced Expression of HOXB4 Homeobox genes play a crucial role in hematopoietic development with expression of Hoxa and Hoxb gene families largely restricted to stem and precursor populations and downregulated upon lineage commitment [21–25]. A number of homeobox genes have the capacity to enhance HSC self‐renewal [26], but Hoxb4 is particularly interesting because ex vivo expansion of adult HSCs can be promoted by enforced expression or by using a recombinant TAT‐HOXB4 protein [27–29]. HSCs retained their multipotency when expanded using this strategy and did not induce leukemia in mouse bone marrow chimeras leading to the hypothesis that it could be used to expand HSCs for clinical application [27, 30]. This characteristic might be specific for the Hox4 paralog group as Hoxa4 has also been shown to efficiently expand ESC‐derived hematopoietic progenitors and adult HSC [20, 31]. HOXB4 has been linked to key elements of the cell cycle machinery in hematopoietic cells with initial studies showing that upregulation of myc, AP‐1, Jun‐N, Fra‐1, and cyclin D1 were associated with the proliferative effects of HOXB4 in HSCs [32–34]. However, it is unlikely that HOXB4 acts simply as an enhancer of cell proliferation, and there is cumulating evidence to indicate that it can promote specific differentiation pathways. Indeed, high levels of HOXB4 was shown to reduce cell proliferation and therefore appears to affect cell growth in a dose‐dependent manner by sensitizing cells toward extrinsic signals [35]. Ectopic expression of HOXB4 confers long‐term repopulating activity on cells from the early embryonic yolk sac and from differentiating mouse ESCs suggesting that HOXB4 might be involved in programming definitive hematopoietic cells [18]. Furthermore, data from our own lab indicate that HOXB4 may play a role in the mesoderm patterning during differentiation of ESCs before HPC arise [36] Since the first report showing that overexpression of HOXB4 can enhance the differentiation of definitive hematopoiesis from mouse ESCs [37], the strategy has been used widely to enhance the production of hematopoietic cells from mouse ESCs and hESCs [16, 19, 28, 37‐45] (Table 1). Despite variations in the timing and level of exogenous HOXB4, the majority of mouse ESC studies have shown an increase in hematopoietic differentiation, with some demonstrating the production of definitive LTR‐HSCs [18, 19] (Table 1). The effect of HOXB4 on the hematopoietic differentiation of human ESCs is less consistent between hESC lines and experimental systems, and the production of therapeutic LTR‐HSCs from hESCs using HOXB4 has not yet been demonstrated to date [46] (Table 1). Most of the hESC studies to date have used a constitutive expression system and, given the differences in developmental stage between mouse and human ESCs, it is possible that there will be differences in the effect of HOXB4 expression on the early stages of differentiation. One study where a recombinant tPTD‐HOXB4 protein was added to the differentiation protocol after germline specification demonstrated an increase in colony forming units‐culture (CFU‐C) production but long‐term reconstitution was not reported. However, it is not clear how efficient the recombinant tPTD‐HOXB4 protein is at switching on target genes so a definitive conclusion on the potential use of HOXB4 in hESC differentiation awaits the use of an inducible genetic system. If HOXB4 does prove to be a route to the production of hESC‐derived HSCs and mature differentiated cells, its direct use in the clinical situation might raise safety concerns because it has been demonstrated to induce leukemia in large animal models [47]. An important goal of many groups, therefore, has been to assess the cellular and molecular mechanism of action of HOXB4 with a view of generating more efficient and safer protocols for clinical translation. To this end, a number of gene expression profiling and chromatin immunoprecipitation on chip studies have been carried out in mouse ESCs to identify HOXB4 target genes and to predict the downstream signals responsible for its hematopoietic enhancing effects (Table 1) [36, 39, 48‐51]. Each study has focused on different aspects of HOXB4 activity with some addressing its effects on adult HSC and/or ES‐HSC expansion [39, 49, 51] while others (including our own) have addressed aspects of its hematopoietic inductive activity [36, 48]. Different strategies to induce HOXB4 have resulted in variable levels of expression and significant differences in the timing of HOXB4 exposure. Therefore, it is not surprising that there are significant differences in the lists of target genes identified. HOXB4 activity is dependent on a number of cofactors and partner proteins such as PBX1 and NFY that can serve to enhance or limit HOXB4 activity [52]. Thus target genes identified in any particular study will depend on the presence of these factors in the chosen cell population resulting in different cellular fates [53]. With this in mind, we review the findings of the different studies and highlight those that are most likely to be relevant to ES‐HSC induction and expansion and that might be translated to the human ESC system. Table 1 Effect of HOXB4 overexpression on the production of HPCs and HSCs from mouse and human ESCs Open in new tab Table 1 Effect of HOXB4 overexpression on the production of HPCs and HSCs from mouse and human ESCs Open in new tab Identification of HOXB4 Target Genes The first large‐scale study of HOXB4 target genes was described by Schiedlmeier et al. [39] on both adult HSCs and in ESC‐derived cells. Adult HPCs were transduced with a retroviral vector that expressed a tamoxifen‐inducible form of HOXB4, and gene expression analysis was compared between lin‐Sca1+c‐kit+ HPCs that had been expanded in the presence or absence of tamoxifen. In this study, 103 differentially expressed genes were identified including genes associated with cell proliferation, cell cycle, and apoptosis, and those critical for the self‐renewal, survival, and maintenance of adult HSCs (e.g., Cnkn1b, Mad, Foxo3a, Ptgs2, and Zfx). The same group identified a much larger number (>700) of HOXB4 target genes during the differentiation of ESCs [39]. A doxycyline‐inducible system was used to control the timing of HOXB4 activity between days 4 and 6 of embryoid body (EB) differentiation at a time when the first HSCs are detected. Genes in this large list would be predicted to include genes involved in HSC expansion as well as those associated with hematopoietic induction and nonhematopoietic lineages. A list of 52 genes that overlapped between their adult HSC and ESC dataset were identified as “universal” targets of HOXB4 and included genes involved in a wide range of cellular processes, such as signal transduction, cell cycle, apoptosis, and response to stress and transcription. A number of these shared targets were associated with specific signaling pathways that are important for controlling self‐renewal, maintenance, and differentiation of stem cells such as FGF, transforming growth factor (TGF)‐β/activin/BMP, Wnt/β‐catenin, hedgehog, and Notch [39]. Interestingly, genes upregulated in EBs, but not in adult HSCs, included genes associated with Notch signaling (Hey2, Jagged2, Nrarp, Psen1, and Skp2), Hedgehog (Ptch1, Gli, and Rab23), and TGFβ (Etv1, Dach1, Smurf2, and Dcp1a) pathways. In addition, downregulation of a key downstream target of FGF signaling (Etv5) and upregulation of two members of the FGF negative feedback loop (Dusp6 and Spry1) implicated HOXB4 as a mediator of this signaling pathway [39]. Subsequent functional studies using specific inhibitors of FGF receptors and addition of bFGF supported their hypothesis that HOXB4 could act by modulating the response of HSCs to extrinsic FGF signals [39]. In this context, it is noteworthy that many of the signaling pathways identified in response to HOXB4 activity play a role in the establishment of Hox patterns during early embryonic development and thus implicate the involvement of complex feedback mechanisms [54, 55]. Oshima et al. [51] used both chIP‐on‐chip and microarray analysis to identify HOXB4 target genes in ESC‐derived Kit+CD41+ cells that had been transduced with HOXB4 and further cultured on OP9 stromal cells for 7 days. Comparison of the array and chIP‐on‐chip dataset distinguished genes that were directly or indirectly regulated by HOXB4 [51]. A significant number of HOXB4‐regulated genes were associated with cell proliferation, the cell cycle, and chromosomal organization and biogenesis. Gata2, Runx 1, and Scl/Tal were shown to be direct targets of HOXB4 indicating that HOXB4 might act by reprogramming cells into adult‐type HSCs/HPCs by simultaneously modulating the expression of a number of hematopoietic regulators. This study also identified components of signaling pathways including Tfgb1 and Notch2 that have previously been shown to be associated with ESC‐derived hematopoiesis [15, 16]. Their list of upregulated genes contained 13 that overlapped with that reported by Schiedlmeier including genes encoding proteins associated with specific signaling pathways such as Dusp6 (FGF), Bambi (BMP/activin inhibitor), Ptch1 (sonic hedgehog receptor), and Tle1. Interestingly, none of the top 20 ranked direct targets of HOXB4 had previous association with hematopoiesis and is therefore predicted to provide a rich source of novel molecular mechanisms to test in this process. It is noteworthy that Gata2, Runx 1, and Scl/Tal‐1 were not identified as HOXB4 targets in yet another study where HOXB4 was activated in ESC‐derived c‐Kit+CD41+ [48]. Although the target cell was similar in the two studies, the level of HOXB4 expression is likely to be significantly lower (dox‐inducible iHOXB4 vs. retroviral‐mediated) and the length of time the cells were exposed to HOXB4 was shorter (4 vs. 7 days). Higher levels and prolonged exposure will inevitably result in an increased number of indirect targets. This study demonstrated that, as predicted, CD34, CD150, and c‐Mpl, the receptor for thrombopoietin, are upregulated in the transition of pre‐HSCs to embryonic HSCs [48]. Most of the above studies assumed that the inductive effect of HOXB4 would be cell autonomous and therefore they aimed to identify HOXB4 target genes in either adult HSCs or in HSC purified from differentiating ESCs [39, 48, 49, 51]. However, we had noted that HOXB4 was also expressed in the primitive streak of the gastrulating embryo and this led us to hypothesize that HOXB4 might modulate gene expression in prehematopoietic mesoderm of differentiating ESCs. Therefore, we set out to identify HOXB4 target genes in differentiating mouse ESCs at a time point during the differentiation prior to the production of HPCs. Indeed, we demonstrated that a pulse of HOXB4 prior to HPC emergence resulted in an increase in hematopoietic differentiation, and we analyzed the genes that were upregulated by HOXB4 at this stage [36]. We were somewhat surprised when our gene profiling revealed an increase in the expression of genes associated with the development of paraxial mesoderm (including Tbx6, Frzb, Dll1, Dll3, Foxc1, Fst, and Noggin) that ultimately gives rise to tissues associated with the hematopoietic niche. This suggested that HOXB4 might modulate the formation of the hematopoietic niche as well as the production of hematopoietic cells per se. We directly demonstrated this paracrine effect in cell mixing experiments and implicated the involvement of Frzb in this process by demonstrating a hematopoietic inductive effect of FRZB‐expressing stromal cell lines and abrogation of the inductive effects of HOXB4 by short hairpin RNA Frzb knockdown [36]. Interestingly, we did not observe any alteration in β‐catenin activity indicating that Frzb expression did not have the predicted inhibitory effect in this context. A number of genes associated with Wnt signaling pathway, including Frzb, were also identified in other ESC studies [39] and this is consistent with numerous studies that have implicated Wnt signaling in hematopoietic differentiation of mouse ESCs [56–58]. More recently, it has been suggested that Wnt3a exerts its canonical effects by promoting the proliferation of hESC precursors already committed to the hematopoietic lineage and these authors proposed that it is the noncanonical Wnt signaling via Wnt 11 that is involved in specification to the hematopoietic lineage [59]. The fact that the Frzb‐induced hematopoietic induction in our system was not accompanied by an increase in β‐catenin activity might suggest that it is acting through this noncanonical WNT pathway potentially by increasing the diffusibility and signaling areas of WNT ligands [60]. This hypothesis is supported by the recent finding that HSC induction in zebrafish is controlled by noncanonical WNT signaling via Wnt16 in a non‐cell autonomous manner [61]. Conclusions In conclusion, a wide range of HOXB4 target genes associated with numerous cellular processes have been identified indicating multiple modes of action of this transcription factor in the differentiation of mouse ESCs (Fig. 1). Clearly, HOXB4 can act in a cell autonomous manner by modulating the expression of genes involved in cell proliferation and survival and thus contribute to the expansion of the HPC/HSC compartment [39]. The ability of HOXB4 to directly regulate key hematopoietic transcription factors [51] suggests that it could also act as a cell autonomous HSC reprogramming factor switching pre‐HSCs (embryonic/primitive HPC) into HSCs with an adult, definitive phenotype. Additionally, the expression of genes encoding components of cellular signaling pathways are regulated by HOXB4 and this could result in the altered response of a cell to specific signaling pathways involved in hematopoietic differentiation [36, 39]. These effects could be exerted in either a cell autonomous or a paracrine manner. The importance of signaling pathways such as Notch, Wnt, and Hedgehog pathways that have been highlighted by these HOXB4 target analyses is consistent with studies testing directly the role of these pathways during ESC differentiation [62, 63]. For example, a number of studies in vivo and in ESC‐hematopoietic differentiation have concluded that Notch signaling is essential for definitive but not primitive hematopoiesis with the hematopoietic transcription factors RUNX1 and GATA2 being downstream of Notch activation [64–66]. In hESC differentiation studies, activation of Notch signaling by addition of exogenous Jagged‐1 had an inductive effect on the production of hematopoietic cells [63]. Our own work also implicated the Notch pathway in hematopoietic differentiation after mesoderm formation: treatment with the gamma secretase inhibitor, a widely used inhibitor of Notch signaling, reduced the hematopoietic inductive effects associated with both HOXB4 overexpression, and AGM stromal coculture suggesting that some molecular mechanisms might be shared between the two inductive strategies [16]. 1 Open in new tabDownload slide Mechanistic insight into the action of HOXB4‐mediated induction of hematopoietic differentiation as revealed by gene profiling and functional experiments. HOXB4 can act in both a cell autonomous and non‐cell autonomous manner to enhance hematopoietic differentiation of ESCs. Gene profiling of HOXB4 target genes in differentiating ESCs by several groups revealed the upregulation of genes associated with several processes (marked 1–4) that are involved in the production and expansion of hematopoietic cells. These include genes associated with HSC expansion (1), HSC programming (2) and those associated with a number of signaling pathways involved in the interaction of HSC with their niche (3). Our group has shown that the induction of HOXB4 at an early time point during ESC differentiation can enhance the production of paraxial mesoderm that gives rise to the endogenous ESC‐derived hematopoietic niche (4). This possibly explains the paracrine effect of HOXB4 via an increase in the production of Frzb and some hematopoietic growth factors (5). Arrows in red indicate the processes that are enhanced by enforced expression of HOXB4. See text for the appropriate citations. Abbreviations: ESC, embryonic stem cell; FGF, fibroblast growth factor; HSC, hematopoietic stem cell; TGF, transforming growth factor; WNT. 1 Open in new tabDownload slide Mechanistic insight into the action of HOXB4‐mediated induction of hematopoietic differentiation as revealed by gene profiling and functional experiments. HOXB4 can act in both a cell autonomous and non‐cell autonomous manner to enhance hematopoietic differentiation of ESCs. Gene profiling of HOXB4 target genes in differentiating ESCs by several groups revealed the upregulation of genes associated with several processes (marked 1–4) that are involved in the production and expansion of hematopoietic cells. These include genes associated with HSC expansion (1), HSC programming (2) and those associated with a number of signaling pathways involved in the interaction of HSC with their niche (3). Our group has shown that the induction of HOXB4 at an early time point during ESC differentiation can enhance the production of paraxial mesoderm that gives rise to the endogenous ESC‐derived hematopoietic niche (4). This possibly explains the paracrine effect of HOXB4 via an increase in the production of Frzb and some hematopoietic growth factors (5). Arrows in red indicate the processes that are enhanced by enforced expression of HOXB4. See text for the appropriate citations. Abbreviations: ESC, embryonic stem cell; FGF, fibroblast growth factor; HSC, hematopoietic stem cell; TGF, transforming growth factor; WNT. Future Prospectives The ultimate goal in this field of research is to establish a safe “off‐the‐shelf” source of blood cells for the treatment of hematological disorders. Can the limited donor supply in the current practice of bone marrow transplantation be overcome in the future by the production of HSCs from a bank of hESCs covering the major histocompatible subtypes? Can the problems associated with blood transfusion such as transfusion‐transmitted infections be resolved by the efficient production of red blood cells from O‐Rhesus negative hESCs in the laboratory? Despite the significant advances over the last few decades the production of therapeutic quantities of hematopoietic cells from hESCs has not been achieved. This review focuses on the use of enforced expression of HOXB4 to enhance the production of ESC‐derived hematopoietic cells and describes some of the cellular and molecular mechanisms that have been associated with its mode of action. The improved understanding of the molecular circuitry involved in blood cell production will lead to the application of novel approaches to overcome the challenges facing the field. Acknowledgements Work in the laboratory was funded by Leukemia and Lymphoma Research, European Commission Framework 6 Fungenes Consortium, and The Wellcome Trust. We thank Drs. 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TI - Mechanism of Action of HOXB4 on the Hematopoietic Differentiation of Embryonic Stem Cells
JF - Stem Cells
DO - 10.1002/stem.1036
DA - 2012-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mechanism-of-action-of-hoxb4-on-the-hematopoietic-differentiation-of-SDOSF8APIg
SP - 379
EP - 385
VL - 30
IS - 3
DP - DeepDyve
ER -