Emerging concepts for the in vitro derivation of murine hematopoietic stem and progenitor cells

Eva Garcia-Alegria1, Sara Menegatti1, Kiran Batta2, Sara Cuvertino 1,3, Magdalena Florkowska2 and Valerie Kouskoff1

1Cancer Research UK Stem Cell Haematopoiesis Group, Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow road, Manchester, M20 4BX, UK.

2Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow road, Manchester, M20 4BX, UK.

3Present address: Institute of Human development, University of Manchester, St Mary's Hospital, Manchester M13 9WL, UK.

Key words: embryonic stem cells, hemogenic endothelium, reprogramming, bone marrow transplantation

Corresponding author: Valerie Kouskoff, CRUK Manchester Institute, University of Manchester, Wilmslow Road, M20 4BX, Manchester, UK.

valerie.kouskoff@cruk.manchester.ac.uk. Tel. (44) 0161 446 8381.

Abstract

Well into the second decade of the 21st century, the field of regenerative medicine is bursting with hopes and promises to heal young and old. The bespoken generation of cells is thought to offer unprecedented cures for a vast range of diseases. Hematological disorders have already benefited tremendously from stem cell therapy in the form of bone marrow transplantation. However, lack of compatible donors often means that patients remain on transplantation waiting lists for too long. The in vitro derivation of hematopoietic stem cells offers the possibility to generate tailor-made cells for the treatment of these patients. Promising approaches to generate in vitro derived blood progenitors include the directed differentiation of pluripotent stem cells and the reprogramming of somatic cells.

1. Introduction

Why there is a need to derive hematopoietic stem and progenitors from in vitro sources

Hematopoietic stem cell transplantation (HSCT) is a medical procedure that allows replacing the diseased or damaged hematopoietic system of a patient by a healthy one. HSCT has been used in the clinic for several decades now to treat many hematological disorders including leukemias, myelomas, lymphomas as well as immune deficiencies and autoimmune diseases (reviewed in [1]). Ultimately, the success of HSCTs relies on the presence of hematopoietic stem cells (HSCs) within the donor cell population. Upon transplantation HSCs colonize the host bone marrow microenvironment where these adult stem cells either differentiate to produce all lineages of the hematopoietic system or self-renew to maintain a lifelong pool of HSCs. Donor cells for transplantation can be obtained from the bone marrow through aspiration of the posterior iliac crest under local or general anesthesia, a rather invasive and painful procedure for the donor. Alternatively, HSCs can be mobilized to the peripheral blood circulation by stimulation with cytokines such as G-CSF (granulocyte-colony stimulating factor) by a mechanism not yet fully understood [2]. These mobilized HSCs are then harvested from the donor via leukapheresis a much less invasive and less painful procedure for the donor. Cells for transplantation can also be obtained from umbilical cord blood that can be conveniently preserved and banked. The source of cells for HSCT is ultimately dependent on the type of disease to treat, the age of the patient and the availability of donor cells whether it is from siblings, from registries or from cord blood banks [1]. Unless autologous transplantation is undertaken, one of the most important factors for the identification of donor cells is histocompatibility, controlled by major histocompatibility HLA (human leucocyte antigens) haplotype and minor histocompatibility loci. Depending on age and ethnicity, considerable variations (13% to 51%) occur in the likelihood to find HLA-match siblings who can provide cells for transplantation [3]. In the absence of related donor, international transplant registries have large lists of donors matching around 50% of the patients. However, too often patients in need of HSCT remain on transplantation waiting lists for too long due to a lack of histocompatible donor. In these cases, alternative sourcing of HSCs would be lifesaving. The directed differentiation of pluripotent stem cells or the reprogramming of somatic cells is thought to offer this, the possibility to generate on demand limitless quantities of hematopoietic stem and progenitor cells in vitro.

Why it is so difficult to derive HSCs in vitro

The first demonstration that mouse embryonic stem cells (mESCs) were able to generate hematopoietic cells in vitro upon differentiation was published around thirty years ago [4]. The routine in vitro generation of long-term multilineage engrafting hematopoietic cells is however still a distant goal. Why have progresses been so slow? One of the main reason to be invoked is probably a lack of fully understanding the molecular mechanisms leading to the generation of HSCs in vivo, in particular the cues provided by the microenvironment that drive this developmental process. However, we are slowly closing this gap in our knowledge and ultimately we will have a full understanding of the cellular and molecular events leading to HSCs formation. Whether we can apply this information to the in vitro differentiation of ESCs to generate functional HSCs is a different issue. Another possible explanation to account for this lack of success is that in vitro differentiating ESCs only recapitulates yolk sac hematopoiesis and that therefore adult-engrafting HSCs cannot be derived from this in vitro model system. If this is the case, the direct reprogramming of somatic cells to HSCs will be our only option to generate these cells in vitro. This promising line of investigation is however still very much in its infancy with many hurdles to address.

In this review, we discuss the progresses made toward the in vitro generation of HSCs and blood progenitors using murine cells as model system. We highlight the current limitations and roadblocks hindering the successful derivation of HSCs. The use of human ESCs in this area of investigation is discussed elsewhere in this special issue.

2. In vitro differentiation of ESCs to hematopoiesis

The first evidence that ESCs were capable of generating blood cells upon differentiation was established in the mid-eighties [4]. In the study by Doetschman and collaborators, differentiating ESCs were shown to form 3D embryoid bodies (EBs) in which structures akin to the blood islands of the yolk sac were observed. Characterisation of the timing of emergence of blood lineages in EBs was later determined via gene expression analysis and clonogenic replating assays [5-8]. Those studies revealed striking parallels in the emergence of blood lineages between early embryonic development and in vitro differentiating ESCs. In both systems, primitive erythropoiesis emerged first followed by the generation of myeloid progenitors and definitive erythropoiesis. Following on from these early studies, further dissection and understanding of the emergence and specification of blood progenitors from differentiating ESCs were performed using a plethora of approaches such as multi-parameter flow cytometry, genome-wide RNA-seq transcriptomic, ChIP-seq of transcription factors, single cell gene expression analysis, genetic modification including deletion, enforced expression and fluorescent reporter insertion to track and isolate cells expressing specific genes [9-16].

2a. Cellular steps of blood specification in vitro

During the in vitro differentiation process, blood progenitors are generated through sequential steps of specification, each characterized by the expression of defined genes as summarized in figure 1. All blood cells are derived from mesodermal progenitors expressing the T box transcription factor Brachyury [10], the homeobox mixl1 gene [17] and the fetal liver kinase 1 (Flk1) [18]. When tested in clonogenic assay, FLK1+ cells give rise to blastic colonies that contain blood, endothelium and smooth muscle lineage cells [19-21]. These FLK1+ cells, transiently detected within the differentiating EBs, have been termed blast colony forming cells (BL-CFC) and are thought to represent the in vitro equivalent of the in vivo hemangioblast in reference to century-old observations made by Sabin and others [22, 23]. Whilst hemangioblast are detected in vitro in differentiating EBs [19, 20] and ex vivo in culture of embryo-derived cells [24], there is still no evidence to demonstrate that this population of mesodermal hemangioblast do fulfil its full potential in vivo in gastrulating embryos [25, 26]. Further dissection of the differentiation of FLK1+ cells to blood revealed that VEGF stimulation promotes the generation of a cell population expressing markers of the endothelial lineage such as TIE2, VE-Cadherin, and CD31 [27, 28]. Through an endothelial to hematopoietic transition, these endothelial cells further differentiated into blood progenitors expressing first the IIb integrin (CD41, ITGA2B) then the pan-hematopoietic marker CD45 [27, 28]. This population of specialized endothelium has been termed hemogenic endothelium (HE) and has been detected in vitro but also in vivo at sites of blood emergence in the yolk sac [29] and embryo proper [30] and in all vertebrate species studied to date [31-35].

2b. Transcriptional control of blood specification in vitro

Whilst we still do not understand many aspects of the transcriptional machinery and the molecular mechanisms that control blood specification, great progresses have been achieved over the last two decades. The key transcription factors identified to date that are implicated in the specification of the hematopoietic program from the mesoderm germ layer are summarized in figure 2. The ETS transcription factor ETV2 is first expressed in Brachyury+FLK1+ mesoderm and is critical both in vitro and in vivo for the establishment of the cardiovascular system [36-38]. Embryos and ESCs deficient for ETV2 expression do not generate HE or blood cells and no vasculature is observed in Etv2-/- embryos. In line with this severe phenotype, the transcription factors Sox7 [39] and Scl [40] were shown to be direct transcriptional targets of ETV2. The expression of both regulators was completely abrogated in the absence of ETV2 [38]. SOX7 is an important regulator of endothelium specification along with other members of the SOXF family [41, 42] while SCL is a critical regulator of blood emergence. Both in vitro and in vivo, SCL deficiency leads to a total absence of primitive and definitive blood cells [43-46]. Further insight into the function of SCL has established that this factor is critical for the formation of HE [27] and that along with GATA2 and FLI1, it forms a recursive triad controlling an early hematopoietic program [47]. Downstream of SCL, the core binding factor RUNX1 acts as a master regulator of definitive hematopoiesis. There is some evidence to suggest that SCL is implicated in the transcriptional regulation of RUNX1 [48], however RUNX1 is still expressed in the absence of ETV2 and SCL [37, 38]. RUNX1 deficiency does not impair primitive erythropoiesis but all definitive blood lineages are absent in Runx1-/- embryos [49]; this phenotype was similarly observed in vitro in differentiating Runx1-/- ESCs [50]. Further studies have now established that RUNX1 is critically required at the HE stage for the transition of endothelium to hematopoiesis, again this was shown in developing embryos [51] and in differentiating ESCs [27]. During this transition, RUNX1 controls the downregulation of the endothelial program concurrently with the upregulation of the blood program. The transcriptional repressors GFI1 and GFI1B were identified as critical transcriptional targets of RUNX1, essential for switching off the endothelial identity of HE [52, 53]. To date, it is still not known what transcriptional targets of RUNX1 are critical for switching on the blood programme. One possibility is that blood specification is a dominant programme in endothelial progenitors during early embryonic development and that the shutdown of the endothelial identity by the RUNX1-GFI1 axis is sufficient for hematopoiesis to progress [54]. At the molecular level, RUNX1 was shown to orchestrate the global reorganization of lineage-specific transcription factor assemblies on the chromatin during the transition from HE to blood cells [55].

Overall, we are now starting to understand better the transcriptional control of blood specification from mesoderm and importantly both in vitro and in vivo processes have been shown to depend on the same transcriptional machinery. Blood specification is a step-wise process, occurring through a series of changes in identities from mesoderm, to endothelium then to blood. Key transcription factors have been implicated in switching on or switching off specific programs. However, the interplay between these critical players which regulates the timing and site of blood emergence is still poorly understood. To successfully derive HSCs in vitro, understanding this interplay is of utmost importance as in vivo timing and site of emergence directly correlates with the emergence of specific lineages as described elsewhere in this special issue.

3. Successes and failures in the derivation of engrafting blood cells from murine ESCs

Since the initial findings that ESCs could give rise to blood cells, intense research efforts have been directed toward the generation of long-term in vivo repopulating HSCs from in vitro differentiating ESCs. However, despite many progresses, the routine generation of bona fide in vitro derived HSCs is still a distant goal.

Several studies in the mid-nineties reported the in vitro derivation of hematopoietic progenitors with the capacity to engraft in vivo and to give rise to lymphoid lineages [56-59]. These studies were performed using various ESC differentiation conditions, immuno-phenotype of the engrafted cells and recipient mice and all clearly showed contribution to T and B cell lineages. In the same period, the first data claiming the in vitro generation of HSCs with multilineage and long term repopulation ability was published [60]. In this study, ESCs were differentiated on irradiated stroma layers and after 21 days of culture, PgP-1+ (Phagocytic glycoprotein-1, CD44) Lineage- cells were isolated and injected into sub-lethally irradiated SCID recipients. It was shown that in vitro-derived PgP-1+ Lineage- cells gave rise to all hematopoietic lineages in both primary and secondary recipients. The data presented in this study were remarkable, but they were unfortunately never reproduced by other laboratories and no follow-up studies were ever published by Palacios and collaborators. The next published study describing the in vitro derivation of repopulating cells was performed using a serum and cytokines supplemented EB differentiation culture system from which cKIT+CD45+ cells were isolated and transplanted into irradiated BALB/c recipients [61]. These in vitro derived progenitors provided long-term multilineage engraftment and were used in a subsequent study by the same group to prevent autoimmune diabetes mellitus in NOD mice [62]. However, again no other research team followed up or published studies validating this protocol for the in vitro derivation of HSCs.

In parallel, alternative approaches were developed to overcome the lack of reproducibility observed in the engraftment capacity of ESC-derived cells. The enforced expression of homeobox transcription factors in differentiating ESCs, including Hoxb4 [63-66], Cdx4 [67] and Lhx2 [68], led to the reproducible generation of repopulating blood cells with multilineage and long term engraftment. However, these approaches were not successful when translated to the differentiation of human ESCs [69, 70] and were therefore of no value to potential clinical applications. Furthermore, the enforced expression of potentially oncogenic factors would have limited the clinical use of these in vitro derived cells.

To overcome reproducibility issues, most likely originating from the use of specific batches of serum, we developed serum-free differentiating conditions in which the step-wise addition of growth factors to EB cultures led to the very efficient differentiation of hematopoietic progenitors [12, 71]. Using this well-defined culture conditions, we were able to establish that multilineage engrafting capacity arose very rapidly upon the specification of mesoderm to the blood lineage, in line with a previously published study [72]. Our study furthermore revealed that the repopulating activity was restricted to a subset of cKIT+ HE cells and was lost when these cells further differentiated [73]. While we observed long-term engraftment of the ESC-derived cells, no secondary repopulating ability was observed suggesting a limited self-renewing capacity of the in vitro derived cells.

The derivation of long-term in vivo engrafting blood progenitors from differentiating ESCs has been riddled with false hope and remains a distant goal. It is still not clear whether bona fide HSCs can ever be derived from in vitro differentiating ESCs. Two studies have however raised hope suggesting that this might be possible if the correct microenvironmental cues are provided to the differentiating ESCs [74, 75]. In both studies, the implantation of undifferentiated ESCs or iPSCs into mouse recipients led to formation of teratomas inside which HSCs were generated, demonstrating that the teratoma microenvironment is conducive to the formation of HSCs. In light of these results, it will be important to understand how the teratoma microenvironment differs from the EB microenvironment. One likely explanation is that ESCs differentiating culture conditions are highly optimized to push differentiation toward mesoderm giving rise to blood cells. As a consequence, the EB microenvironment is rather restricted and mostly contains cardiovascular derivatives, including endothelium, smooth muscle and blood cells. In contrast, teratomas typically comprise derivatives of the three germ layers including endodermal and neuronal lineages. It is likely that these derivatives are important in establishing a niche conductive to the emergence and maintenance of HSCs within the teratoma. Future research efforts will need to aim at determining what this specific microenvironment is and how it relates to the dorsal aorta microenvironment in which HSCs emerge during embryonic development.

4. Reprogramming somatic cells to blood cells

Given the difficulties encountered to date in trying to generate HSCs from in vitro differentiating ESCs, the direct reprogramming or conversion of somatic cells into hematopoietic stem and progenitor cells has recently become an attractive alternative approach. Following on from the seminal work of Yamanaka and collaborators demonstrating that somatic cells can be reprogrammed by transcription factors [76, 77], a plethora of studies have now established that somatic cells can be directly converted into specific lineages such as cardiomyocytes, hepatocytes or neurons by the enforced expression of tissue specific transcription factors (reviewed in [78]). Several studies have now also demonstrated that mouse and human somatic cells can be reprogrammed to hematopoietic lineages (Table 1).

Szabo and colleagues first demonstrated that the expression of OCT4 in human fibroblasts cultured in blood-permissive conditions resulted in lineage conversion to hematopoietic progenitors with in vitro clonogenic potential and in vivo short term engraftment [79]. In a similar approach, Pulecio and collaborators established that the expression of SOX2 and miR125b was also able to reprogram human fibroblasts to blood progenitors with limited macrophage potential both in vitro and in vivo [80]. However, these two reprogramming strategies employ pluripotent transcription factors (OCT4 or SOX2) that might lead to partial pluripotency and formation of teratoma in vivo, and hence would not be suitable for clinical applications. Using hematopoietic transcription factors to promote lineage conversion, we and others have established that mouse fibroblasts can be reprogrammed to blood progenitors via a hemogenic endothelium intermediate [81, 82]. The reprogrammed blood cells obtained by Pereira et al displayed a limited in vitro clonogenic potential and no in vivo engraftment [81] whilst our study led to the generation of blood progenitors with robust in vitro clonogenic potential but limited in vivo engraftment [82]. In a similar attempt at lineage conversion, Vereide et al demonstrated that a set of six transcription factors was able to reprogram mouse fibroblasts or program mouse ESCs into expandable hemangioblast populations [83]. Upon release from the ectopic expression of the reprogramming factors, these expandable populations gave rise to blood progenitors with in vitro clonogenic potential; the in vivo engraftment potential of these expandable hemangioblast populations was not tested [83]. It is interesting to note that these three studies used similar starting somatic cell types and similar strategies aiming at screening a large panel of hematopoietic transcription factors to promote lineage conversion. However, the set of transcription factors identified in each of these studies as able to reprogram fibroblast to blood was strikingly different with only GATA2 commonly found in all three studies and SCL /LMO2 common only between the Vereide and Batta studies (Table 1). One may speculate that in each strategy the specific stoichiometry and interplay between the set of transcription factors identified is critical for reprogramming. Further work will be required to tease out the mechanistic differences underlying fibroblast to blood conversion driven by these heterogeneous sets of transcription factors.

Two additional studies have provided further insights into somatic cell to blood cell conversion and have also established the critical importance of the microenvironment for efficient reprogramming to blood progenitors with long-term in vivo engraftment potential [84, 85]. Starting from endothelial cells, Sandler et al established that conversion to blood progenitors with long-term in vivo engraftment was achieved by the ectopic expression of four transcription factors (Table 1) followed by co-culture on a stroma layer mimicking the vascular niche [84]. This study is remarkable as it demonstrates that environmental cues are essential in directing reprogrammed cells toward a multipotent and self-renewing state. From a mechanistic standpoint, it will be important to establish whether these reprogramming conditions can be used to convert other somatic cell types or if these conditions work well only in converting endothelial cells because they are closely related to blood cells. In the second study, Riddell et al screened a set of 36 regulatory genes defined as HSC-specific for their capacity to convert committed lymphoid and myeloid progenitors back into HSCs [85]. The cleverness of this study was to use the in vivo endogenous bone marrow microenvironment to promote and select for the optimal combination of reprogramming factors. This strategy resulted in the identification of six factors able to convert committed blood progenitors back into HSCs (Table 1); two additional factors (Mycn and Meis1) were further shown to improve conversion efficiency. Similar to the Sandler study [84], these data demonstrate the critical importance of microenvironmental cues in driving the conversion to a multipotent self-renewing state. In the case of this study, the somatic cells used are even more closely related to HSCs than the endothelial cells from the Sandler study. From a mechanistic perspective, it will be important and interesting to assess if more distant somatic cells can also be converted into HSCs using this strategy. In term of clinical applications, this approach would obviously not be possible given the use of an in vivo step required for reprogramming; however, the identification and in vitro mimicking of the cues provided by the in vivo environment would certainly become a very attractive strategy to derive HSCs in vitro.

The conversion or reprogramming of somatic cells into hematopoietic stem and progenitor cells has come a long way in the last decade and has shown great promises and spectacular advances. However, to translate these approaches to clinical applications, many hurdles are still in the way with notably avoiding the use of integrating vectors for the expression of reprogramming factors which may lead to insertional mutagenesis, the ectopic expression of transcription factors often linked to leukemogenesis, the use of serum and stroma in culture conditions or the potential harmful genetic or epigenetic status of the starting cell population to be reprogrammed.

5. Conclusions and Perspectives

Tremendous progresses have pushed forward the field of in vitro generation of hematopoietic stem and progenitor cells toward the goal of producing cells usable in the clinic for regenerative medicine purposes. However, this is still a field in its infancy with many more challenges to come and to overcome. A recurrent theme has been emerging whether it is in the directed differentiation of ESCs or in the reprogramming of somatic cells and this theme is the critical importance of the microenvironment in which cells are grown or maintained. If one can understand which specific elements of the microenvironment are essential to push or maintain cells into a self-renewing multi-potential state, one might be able to use this knowledge in experimental approaches to generate HSCs in vitro. Recent progresses have been achieved in dissecting the microenvironment and inductive signals during HSCs emergence in the embryo [86-88] or how stroma layers maintain hematopoietic stem and progenitor cells [89]. These findings have yet to be translated into experimental strategies to generation HSCs in vitro.

Acknowledgements

Research in the authors’ laboratory is supported Cancer Research UK and the Biotechnology and Biological Sciences Research Council.

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Figure legends

Figure 1: Specification of embryonic stem cells to hematopoiesis. Schematic representation of the cellular intermediates leading to the generation of blood progenitors from embryonic stem cells (ESC); the temporal expression of specific markers is depicted with mesodermal markers in grey, endothelial markers in green and hematopoietic markers in red.

Figure 2: Transcriptional control of hematopoietic specification. Schematic representation of the relationships and interactions between some of the most critical transcriptional regulators of mesoderm specification to blood progenitors.

Table 1: Summary of studies reporting the successful reprogramming of somatic cells to blood progenitors

1