Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations. Invited review following the FEBS Anniversary Prize received on 5 July 2009 at the 34th FEBS Congress in Prague

Authors


I. Moreno-Gimeno, Centro de Investigación Príncipe Felipe, Valencia 46012, Spain
Fax: 00 34 963289701
Tel: 00 34 963289680
E-mail: imoreno@cipf.es

Abstract

Human embryonic stem cells (hESCs) and induced pluripotent stem cells are excellent models for the study of embryonic hematopoiesis in vitro, aiding the design of new differentiation models that may be applicable to cell-replacement therapies. Adult and fetal hematopoietic stem cells are currently being used in biomedical applications; however, the latest advances in regenerative medicine and stem cell biology suggest that hESC-derived hematopoietic stem cells are an outstanding tool for enhancing immunotherapy and treatments for blood disorders and cancer, for example. In this review, we compare various methods used for inducing in vitro hematopoietic differentiation from hESCs, based on co-culture with stromal cells or formation of embryoid bodies, and analyse their ability to give rise to hematopoietic precursors, with emphasis on their engraftment potential as a measure of their functionality in vivo.

Abbreviations
AGM

aorta–gonad–mesonephros

BFU-E

burst forming unit-erythrocyte

BMP4

Bone morphogenetic protein 4

CFU-E

colony forming unit-erythrocyte

FBS

fetal bovine serum

hESC

human embryonic stem cell

mESC

mouse embryonic stem cell

HSC

hematopoietic stem cells

hiPSC

human induced pluripotent stem cells

NK

natural killer

NOD/SCID

non-obese diabetic/severe combined immuno-deficient

VEGF

vascular endothelial growth factor

Why use human embryonic stem cells or induced pluripotent stem cells for hematopoiesis studies?

The first paper describing the derivation of human embryonic stem cells (hESCs) was published in 1998 by Thomson et al. [1]. Since then, many studies have been performed using with hESCs in order to better understand embryonic development and stem cell biology, with the possibility of clinical application as well as their use as tools for pharmaceutical research and drug discovery being a major impetus for such investigations [2]. The study of human hematopoiesis using hESCs and induced pluripotent stem cells (hiPSCs) has been one of the most successful fields to date. It is known that hESCs, characterized by their pluripotency and theoretically unlimited proliferation ability, are capable of producing all blood cell types when differentiated under suitable conditions [3–8]. Various studies have shown that hESC differentiation to hematopoietic lineages closely mimics embryonic hematopoiesis [9,10], making them an incomparable tool for the study of hematopoiesis during embryonic development in states of health or disease [11–13]. There is hope that the insights gained by better understanding embryonic hematopoiesis can meet clinical needs (in the fields of AIDS, immunotherapy, blood disorders and cancer treatment, for example) by helping to improve existing therapies currently based on transfusions or allogeneic hematopoietic stem cell transplantation [14].

HIV infections are known to affect the hematopoietic system by specifically targeting white blood cells, substantially weakening the immune system and eventually progressing to AIDs, and resulting in death, usually as a result of secondary opportunistic infection [15]. Therefore, cell-replacement therapy based on reconstitution of the leukocytic hematopoietic compartment using a CD34+ hESC-derived starting population has been considered as a potential AIDS therapy, and as a way to alleviate secondary effects produced by anti-retroviral drugs [16]. Various studies have now shown that functional B cells, natural killer (NK) cells, dendritic cells and macrophages can be derived from hESCs and used for immune therapy [4,7,17–20]. Furthermore, by combining cellular and gene therapy, anti-HIV-1 genes can be transduced into CD34+ hESC-derived macrophages and T cells, rendering them insensitive to HIV infection, and therefore giving hope that these types of cells could eventually restore normal immune system function. [21]. Additionally, recent studies have highlighted the importance of chemokine receptor 5 (CCR5) in HIV-1 infection; silencing of CCR5 in HSCs (by RNA interference technology) yields HIV-1-resistant hematopoietic progenitors, potentially capable of restoring a healthy hematopoietic compartment [22]. Moreover, Hütter et al. [23] showed the viability of this type of treatment by transplanting CD34+ stem cells expressing an inactive form of CCR5 receptor (homozygous to the CCR5 Δ32 deletion) into an individual suffering from AIDS and acute myeloid leukemia. Following transplantation and discontinuation of anti-retroviral therapy, no HIV-1 virus was detected in the patient over a 20-month period [22,23]. A combination of gene and cell therapy has also been developed to generate hESC derived dendritic cells with enhanced in vitro antigen-presenting function [19]. These hESC-derived dendritic cells could potentially be used in clinical immune therapy in order to induce immune responses in an antigen-specific manner in patients suffering from cancer or viral infections [20].

Cancer therapy is a principal goal of the current clinical research on hESCs. Recently, regression of metastatic melanoma tumors was achieved by transplantation of adaptive T cells specific for tumor antigens; however, this technique performed poorly in human trials, as only 10% of the patients retained the genetically engineered cells 1 year after infusion [24,25]. A new anti-cancer therapeutic strategy involves targeting of the innate immune system through the cytolytic activity of NK cells. A recent study by Kaufman et al. showed that hESC-derived NK cells are competent effector cells that can kill human tumor cells in vivo. The authors compared hESC-derived NK cells to those generated from umbilical cord blood, and found that hESC-derived cells form a more mature and homogeneous population with an increased ability to kill tumor cells, and showed higher expression of effector molecules as well as cytolytic competence, thus representing a great advance in the use of hematopoietic stem cells in anti-tumor therapy [26].

The latest studies in stem cell biology have shown how somatic cells can be reprogrammed back to a pluripotent state similar to that in hESCs [27,28]. These hiPSCs have opened new avenues for creating in vitro disease models that can be used to help understand the pathology of many genetic diseases and to design new drugs. They are also considered a potential source of allogenic cells for cell replacement therapy, as hiPSCs could be derived specifically for each patient. The clinical capacity of hiPSCs has been recently described in mouse models. In 2007, Hanna et al. reported the first proof that combined gene and cell therapy could be used in the treatment of blood disorders by transplanting modified cells into a humanized mouse model of sickle cell anemia carrying the human mutant variant of the β-globin gene (βS) that is responsible for the disease. Fibroblasts from a diseased animal were reprogrammed into iPSCs, and the sickle cell anemia was corrected by homologous recombination. These cells differentiated into hematopoietic progenitors, and, following irradiation of the donor mouse resulting in the destruction of the hematopoietic compartment, were transplanted successfully back into the mouse, resulting in recovery and correction of the disease phenotype [29]. Using similar methodology, another study has shown phenotypic correction of the clotting factor VIII disorder in a hemophilia A mouse model [30]. Hemophilia A is a genetic blood disorder characterized by mutations in the factor VIII gene that provoke impaired clotting and spontaneous hemorrhages that may result in death in severe cases. Tail-tip fibroblasts from healthy mice were reprogrammed into pluripotent cells and subsequently differentiated into endothelial cells producing factor VIII, and were transplanted into mice suffering from hemophilia A. The transplanted cells engrafted and expressed endogenous factor VIII protein in vivo, resulting in phenotypic correction of the disease [30]. Together, these two studies suggest that iPSCs are a good cell source for correcting blood diseases (especially monogenic disorders); however, more work is required to examine engraftment efficiency and functionality in various animal models of diseases before clinical medical application can become a reality.

Ontogeny of human hematopoiesis

Hematopoietic ontogeny has been the subject of intensive investigation by several groups. As early as 1970, Moore and Metcalf [31] demonstrated that primitive hematopoiesis starts with the formation of yolk sac blood islands in mouse embryos, and later studies by Medvinsky and Dzierzak indicated that the aorta–gonad–mesonephros (AGM) region of the embryo is a primary source of definitive hematopoietic progenitors [32,33]. These subsequently enter the circulation and colonize the fetal liver (and other hematopoiesis-supporting organs), where they mature before migrating to the bone marrow [34,35]. Definitive hematopoiesis is initiated in the aortic endothelium region of the embryonic AGM, where blood cells emerge into the aortic lumen, from CD31+ endothelial cells lining the lumen, in a process that could be understood as hematopoietic transition. Both nascent cells express CD31, but only cells budding into the aortic lumen specifically express CD34, c-kit and CD41, and, at a later stage, CD45 [34] (Fig. 1). This process of budding has been described recently in mice and zebrafish [36,37].

Figure 1.

 Schematic representation of hematopoietic development in the embryo. The surface markers proving hematopoietic commitment at each stage of the differentiation process are shown. Transcription factors required for differentiation are shown above the blue arrows where appropriate. Runx1, runt-related transcription factor 1; Scl = Tal1, T-cell acute lymphocytic leukemia 1.

Because HSCs always emerge in close association with endothelial cells, a common origin for these two cell types has been hypothesized, a parent population known as hemangioblasts [38,39]. Using the differentiation of mouse ESC as an in vitro model, Keller's group isolated blast colony-forming cells that were shown to be responsible for the generation of endothelial and hematopoietic precursors, and hypothesized to represent the in vitro equivalent of hemangioblasts [40]. Similarly, Bhatia’s group identified a population of human cells derived from hESCs that were hypothesized to be the in vitro human equivalent of the hemangioblast, characterized by expression of the endothelial markers PECAM-1 (CD31), Flk-1 (KDR) and VE-cadherin (CD144), but not the hematopoietic marker CD45 (termed CD45negPFV cells), and hemogenic bi-phenotypic differentiation capacity [9].

Although the existence of the hemangioblast in vivo is widely accepted and has been described in Drosophila, zebrafish and mice [41–43], identification of a human hemangioblast in vivo has not yet been achieved, and is hindered largely by the difficulty of obtaining early human embryonic tissues. Nonetheless, insights gained from work on human adult cells do suggest the existence of a human hemangioblast. For example, the BCR/ABL fusion gene has been found not only in bone marrow, but also in the endothelial cells of patients suffering from chronic myelogenous leukemia, suggesting that translocation of these genes occurred in a progenitor common to them both [44]. Further supporting this hypothesis, both hematopoietic and endothelial cell lineages can be derived from single CD34+ KDR+ cells isolated from human bone marrow and cord blood [45]. Single-cell tracing and cell imaging of the aortic lumen in vitro have shown that hemogenic endothelial cells, that were initially defined by VE-cadherin and claudin expression, acetylated low density lipoprotein uptake and formation of tight junctions, start to co-express CD41 and c-kit (early markers of definitive hematopoiesis) and change morphology over time. These cells convert from adherent growth as endothelial colonies towards a more ‘hematopoietic’ phenotype, characterized by loss of their characteristic ‘hemogenic’ expression profile whilst concurrently acquiring expression of the pan-leukocyte marker CD45, and switching to growth in suspension. Thus these studies firmly prove the emergence of definitive blood cells from aortic endothelium [35,46,47]. These findings have been confirmed by many studies in mouse and human showing that definitive human hematopoiesis originates from the aortic embryonic endothelium [36,48].

Molecular studies have identified a number of transcriptional factors that are key regulators of HSC development. For instance, the stem cell leukemia factor Scl, has been shown to play a pivotal role in endothelial and hematopoietic differentiation. This transcription factor is usually altered in patients suffering from T-cell leukemia, and it has been shown that mESCs lacking Scl are unable to undergo hematopoiesis [49]. Other studies have shown that Scl is not required for hemangioblast generation, but is necessary for subsequent differentiation, as Scl mutants cannot generate hemogenic endothelium from the hemangioblast and consequently no hematopoietic or endothelial cells can be produced [50]. Another important transcription factor required for the generation of blood cells is Runx1. Alterations affecting Runx1 are involved in acute myeloid leukemia and pediatric acute lymphoblastic leukemia. In vitro studies using Runx1 null cells have shown that this transcription factor is required for the production of definitive hematopoietic cells, but not primitive hematopoietic or endothelial cells, from hemogenic endothelium [51]. Despite this progress, the complete developmental program that facilitates the onset and progression of human embryonic hematopoiesis remains to be further investigated.

Although the AGM region is considered the first site of definitive hematopoiesis, recent studies have indicated that the placenta acts as an additional extramedullary hematopoietic organ during embryonic and fetal development [52,53]. Hematopoietic precursors found in the human placenta can give rise to erythrocytic and myelocytic lineages, and can also reconstitute the hematopoietic system of myelo-ablated mice. These findings suggest the possibility of using placenta as a source of human hematopoietic progenitors for clinical use. Following these investigations, Serikov et al. [54] developed a protocol for cryopreservation and thawing of the placenta to optimize the recovery of hematopoietic precursors, demonstrating that placenta can yield higher amounts of HSCs than umbilical cord blood, and further suggesting that placenta could be banked and used for clinical transplant in a similar way to umbilical cord blood.

Which methods drive differentiation of hESC to hematopoietic lineages?

In vitro differentiation of hESCs into HSCs has been achieved through various methods, some based on culturing hESCs in the presence of stromal cells that mimic the embryonic hematopoietic developmental environment, others using methods involving the formation of three-dimensional cellular clumps termed embryoid bodies. Various hematopoietic differentiation methods are summarized in Table 1.

Table 1.   Comparison of hematopoietic differentiation methods reported previously.
MethodhESC linesDifferentiation mediaStagesCytokines% CD34+ in cultureReferences
S17 co-cultureH1, H1.1, H9.2FBS added1No1–2% at day 17Kaufman et al. (2001) [3]
OP9 co-cultureH1 and H9FBS added1No20% at day 7Vodyanik et al. (2005) [4]
Hematopoietic embryonic nichesH1, H9, hES-NCL1FBS added1No16% in AGM co-cultureLedran et al. (2008) [58]
Embryoid bodiesH1, H9FBS added1SCF, Flt-3, IL-3, IL-6, G-CSF, BMP420% at day 15Chadwick et al. (2003) [59]
Embryoid bodiesH1, HES2Serum-free1BMP4, bFGFKennedy et al. (2007) [61]
2VEGF
Spin embryoid bodieshES2, hES3, hES4Serum-free1BMP4, VEGF, SCF23% at day 11Ng et al. (2005, 2008) [62,63]
2SCF, VEGF, IL-3, IL-6, Tpo, Epo

Co-culture with stromal cells

In 2001, work by Kaufman et al. [3] showed that co-culture of hESCs with the cells lines S17 (derived from murine bone marrow) or C166 (from murine yolk sac endothelium) can induce differentiation towards blood cells. After 17 days of co-culture, ∼ 2% of the hESCs had formed hematopoietic progenitors as evaluated by expression of CD34, an embryonic and adult hematopoietic stem cell marker. The kinetics of hematopoiesis in co-culture with the S17 cell line showed increased CD34 expression in two distinct stages of the differentiation process. The first peak appeared in conjunction with CD31 expression, and is proposed to mark a hemogenic endothelium state, and the second started at day 14, together with expression of the definitive hematopoietic marker CD45, and was coincident with observation of the first colonies in hematopoietic colony assays [55]. The hematopoietic transcription factors SCL and GATA-2 were also expressed in these cells between days 7 and 21 of differentiation. Moreover, when hESCs co-cultured with the S17 cell line or CD34+ cells derived from these co-cultures were transferred to semi-solid medium, they produced erythroid, myeloid and megakaryocyte colonies, arising between days 14 and 17, demonstrating the functionality of hESC-derived CD34+ precursors.

Using a similar approach, hESCs have also been co-cultured with the mouse bone marrow stromal cell line OP9, which is deficient in macrophage colony-stimulating factor [56]. This method generates a higher level of hematopoietic differentiation than co-culture with the S17 cell line: almost 20% of initial hESCs differentiated into CD34+ progenitors after 7 days of co-culture [4]. The kinetics of OP9 differentiation show expression of CD34 from days 3 to 7, and the CD34+ population showed enhanced CD43 and CD41 expression at approximately day 5, followed by CD45 expression at day 8. The expression of CD43 has been found to mark the earliest hematopoietic cells arising from hemato-endothelial commitment [57]. Using this method, expression of hematopoietic transcription factors GATA-1 and GATA-2 was observed from days 2 and 3 of differentiation, coincident with the onset of CD34 and SCL expression on days 3 and 4, respectively. Additionally, colony-forming cells also arose from CD34+ cells from day 4, and, after subsequent co-culture with MS-5 cells, showed production of both lymphoid and myeloid lineages [4].

In our group, we used stromal cells derived from embryonic hematopoietic niches to direct the differentiation of hESCs towards hematopoietic progenitors with greater efficiency. With this in mind, we compared the ability of primary stromal cells from the AGM and fetal liver regions, as well as three established cell lines derived from the AGM region (AM20.1B4), urogenital ridge (UG26.1B6) and fetal liver (EL08.1D2), to induce hematopoietic differentiation of hESCs. Expression of CD34 was found in all cases from day 6 and peaked at day 18, matching the peak of activity of colony-forming cells. Co-culture of hESCs with primary AGM stromal cells produced the highest level of CD34+ and CD45+ cells at day 18. Most importantly, we found that some of these hESC-derived hematopoietic cells engrafted both primary and secondary immunocompromised mice (treated with a sub lethal dose of ionising radiation) at substantially higher levels than described previously [58].

Formation of embryoid bodies

Differentiation of hESCs in the form of embryoid bodies (EBs) is an easily modifiable alternative method that is often used to obtain differentiated cell types in serum-free and defined media, depending on the cytokines and/or growth factors added. Many methods of EB-mediated in vitro hematopoiesis have been described. Chadwick et al. described the use of hematopoietic cytokines in combination with BMP4 to induce hematopoiesis. Using this method, the proportion of CD34+ cells at day 15 of differentiation increased from 10% to 20% when BMP4 and cytokines were added to the medium, and 90% of the CD34+ cells co-expressed CD45. During this differentiation process, expression of hematopoietic transcription factors was detected, and hematopoietic colonies including macrophages, granulocytes, megakaryocytes and erythrocytes emerged [59,60].

In 2007, Keller’s group developed a new two-step approach for EB-mediated hematopoietic differentiation, the first driven by BMP4 and basic fibroblast growth factor, and the second by addition of VEGF165. The kinetics of EB-derived hematopoiesis showed expression of SCL, GATA-1 and RUNX1 from day 3, consistent with up-regulation of CD34 expression, detectable from day 4 of differentiation. The first hematopoietic colony-forming cells colonies arose at day 5, and consisted predominantly of primitive erythroid progenitors, followed by erythroid colonies and macrophages that emerged at approximately day 8 of differentiation. It is noteworthy that development of hematopoietic progenitors from EBs in these conditions was dependent on the presence of BMP4 [61].

A further improvement in EB-mediated hematopoietic differentiation was described in 2005 when spin EBs were first described [62]. Spin EBs are embryoid bodies produced from a defined number of cells that are centrifuged to produce tight cell compaction, thus improving experimental reproducibility compared to standard EB derivation methods. The number of cells used for EB formation and various augmenting factors were found to be critical; optimum hematopoietic differentiation was achieved when spin EBs were formed from at least 1000 hESCs, and different cytokines were required at each of two distinct differentiation stages for efficient differentiation. Onset of CD34 expression using this protocol was detected at day 6, concurrent with expression of the RUNX1 transcription factor. CD34 expression peaked at approximately day 10 and was maintained at this level until day 26, when approximately 30% of cells expressed CD45. More primitive blast forming units erythroid colonies (BFU-E) emerged on day 6, whereas more mature colony forming unit (CFU-E) erythropoietic colonies and myeloid cells arose later, probably from CD34+ hematopoietic progenitors [62,63].

Successes and failures of hematopoietic engraftment

The only definitive way to evaluate the full functionality of hESC-derived hematopoietic cells is transplantation into immunocompromised animals in order to test their ability to engraft and provide long-term multi-lineage hematopoietic reconstitution. Various mouse models have been used to study human hematopoiesis in vivo; the most commonly used are genetically modified strains of the non-obese diabetic/severe combined immuno-deficient (NOD/SCID) mouse.

To date, only a few studies have described the engraftment potential of hESC-derived hematopoietic cells. In 2005, Bhatia’s group tested EB-differentiated cells by transplanting these cells intravenously into the tail vein of sub-lethally irradiated NOD/SCID mice. However, more than 60% of the transplanted mice died after the transplantation due to aggregation of human ESC-derived hematopoietic cells in the presence of rodent serum, resulting in the formation of pulmonary emboli. To solve this problem, the authors performed intra-femoral injections to deliver the cells directly into the bone marrow. These animals showed hematopoietic reconstitution 8 weeks after injection; however, the level of human engraftment was low at ∼ 1% [64].

A second study used hematopoietic progenitors derived from hESC co-culture with S17 cells for intravenous and intra-femoral transplantation into sub-lethally irradiated NOD/SCID mice (treated with an antibody against NK cells in order to avoid immune rejection of the hESC-derived cells). Long-term engraftment was achieved using both methods as shown by expression of human CD45 3–6 months post-transplantation. Additionally, secondary engraftment was achieved following intravenous transplantation of bone marrow from primary recipients, but once again the level of human engraftment was very low (< 0.5% independently of intravenous or intra-femoral injection) [65].

Our group has successfully used the NOD/LtSz-SCID IL2rγnull (NOG) mouse model [58] to test the hematopoietic potential of hESCs differentiated in various embryonic hematopoietic environments by intra-femoral injection. We found that hESC-derived hematopoietic cells engrafted primary and secondary recipients with multiple hematopoietic lineages for up to 12 weeks following transplantation. Most importantly, engraftment levels were higher compared to previous studies, reaching 16% when hESCs were co-cultured with AM20.1B4, an established cell line derived from the AGM embryonic region [58].

Future therapeutic applications for hESC-derived hematopoietic cells will require a significant improvement in the engraftment levels achieved to date. Low engraftment may result from multiple factors, including possible rejection by the host immune system, the quantity administered, the developmental stage of the cells derived from hESC differentiation, and the quality and/or viability of the transplanted cells. Moreover, the variables affecting the study of human hematopoiesis in ‘humanized’ mice have been studied, and demonstrate the importance of selecting the appropriate mouse strain when designing experimental work. For instance, truncation of IL2 receptor gamma chain (IL2rγ as in NSG mice) in the genetic background of NOD/SCID mice has been shown to increase the engraftment ability of NOD/SCID re-populating cells, and consequently significantly improve human hematopoietic cell engraftment [66–68]. Host age is also important, as it has been shown that newborn mice support higher levels of engraftment than adults because HSCs can better mature and develop in an age-matched micro-environment [69]. This hypothesis is in agreement with the results of a study of the long-term engraftment of primary and secondary recipients (22 months after transplantation) by Narayan et al., who studied HSC engraftment in fetal sheep by injecting HSCs in utero into the fetal peritoneal cavity [70]. The site of transplantation (and therefore the differentiation cues provided by the niche) is also very important, as a recent study has shown than hESC-derived CD34+ cells differentiate preferentially to endothelial cells upon transplantation into the liver of immunocompromised recipients, suggesting that the environment surrounding the transplanted cells could determine their final lineages [71].

Advantages and disadvantages of using hESCs and hiPSCs for therapeutic applications

The successful use of stem cells for clinical purposes is one of the most appealing prospects in regenerative medicine today. HSCs can be isolated from peripheral blood, bone marrow and umbilical cord blood for example, but problems arising from immunogenic matching, resulting in rejection or graft-versus-host disease, as well as the overall lack of donors, impair use of these cell types for therapy. For these reasons, in vitro production of hESC-derived HSCs represents a remarkable opportunity for regenerative medicine, as they represent not only a theoretically unlimited source of more closely tissue-matched donor cells, but can also reduce immune responses following transplant of other cell types derived from the same hESCs if hematopoietic chimerism is first induced [60,72]. However, there are many advantages and disadvantages regarding the use of hESCs in medical practice, including some ethical objections regarding the use of human embryos for research, even when discarded embryos are used.

Use of hiPSCs bypasses these ethical issues [27,28], and several tissue-specific cells have been successfully obtained using hiPSC differentiation, including hematopoietic and endothelial cells [73]. In theory, hiPSC technology could allow the production of specific cell types for the treatment of various patients or diseases [74], but the suitability of these cells for therapeutic use is still open to debate [75,76]. Experiments by Choi et al. [73] suggest that HSCs derived from various hiPSC lines differentiate following the same profile than those derived from hESCs. However, recent work by Lanza’s group showed that endothelial and hematopoietic cells obtained from hiPSC-derived hemangioblasts show decreased proliferation potential, an apoptotic phenotype and early senescence, features that were not observed in hESC-derived counterparts. The same features were observed in hiPSC-derived retinal pigment epithelial cells and cardiomyocytes [77,78], evidence against a differentiated cell type-specific phenomenon. The mechanism that results in such abnormalities is still unknown, but may be related to the integration of viral vectors carrying the transcriptional factors required for hiPSC reprogramming, or the result of a perturbed epigenome.

A new method to reprogram hiPSCs based on episomal vectors, which avoid the expression of transgenes in the iPSC lines generated, was described recently [79]. However, it has been shown that the vectors are not responsible for the differences in effectiveness of hiPSC versus hESC differentiation, as neural cells obtained from lentiviral, retroviral or episomal vector iPSCs exhibit the same variability compared to their hESC counterparts [80]. In order to clarify this, comparative studies at the level of genomic DNA, coding RNA, microRNA and epigenetic events have recently been performed [81–83]. These findings demonstrated that, although very similar, hiPSCs and hESCs are not identical in either their genomic DNA, gene expresion pattern, epigenetic profile, teratoma formation efficiency or latency [84]. In agreement with this, two recently published studies using mouse models have shown that iPSCs retain a temporary transcriptional and epigenetic memory of their original somatic lineages, leading to preferential differentiation of these cells towards their original tissue type [85,86]. This phenomenon was only observed when pluripotent stem cells were obtained by exogenous expression of reprogramming transcription factors, but not by somatic cell nuclear transfer or embryonic stem cells derived from fertilized embryos [85]. These findings suggest that certain sets of somatic cells may be selected prior to reprogramming with the aim of enhancing the iPSC differentiation capacity using their tissue-specific epigenetic memory. However, in many cases, full pluripotency may be advisable or required prior to re-differentiation, and therefore erasing the epigenetic/transcriptional memory from iPSCs is an important goal. Work towards achieving this by serial reprogramming or by treating iPSCs with chromatin-modifying chemicals has been described [85]. An alternative method for avoiding problems associated with partially retained cellular epigenetic memory, by reprogramming human cord blood un-restricted somatic stem cells, has recently been suggested [87]. It was found that the iPSCs obtained from these cells are very similar to hESCs in terms of morphology, molecular signature and differentiation potential; nonetheless, further studies on epigenetics and comparative analysis using iPSCs obtained from other cells are required [87].

Finally, some recent studies have suggested that hiPSCs might potentially be oncogenic. It is known that some transcriptional factors used for hiPSC reprogramming, such as Lin28 and c-Myc, are tumorigenic, and although their expression should be abolished in hiPSCs, it is not known whether they can be activated again at any time during differentiation [77,88]. Another recent study showed that the p53 pathway acts not only as a tumor suppressor, but also suppresses iPSC generation as determined using small interfering RNA and p53 mutation [89]. These findings indicate the need to improve hiPSC technology as well as for a complete evaluation of the possible consequences of use of hiPSCs for drug screening, as disease models and for medical purposes.

Conclusions

The use of stem cells for the treatment of human diseases is one of the principal goals in regenerative medicine today. hESCs and hiPSCs, capable of directed differentiation into specific cell types with theoretically no limits on cell availability, are a promising source of cells, but detailed investigation is required to enable their application in clinical situations. In vitro differentiation of hESCs to hematopoietic progenitors has been successfully achieved by various methods and groups, but the cells obtained using these approaches are not completely functional in vivo, as demonstrated by the low proportion of cells able to reconstitute the hematopoietic system of immunodeficient animals. It is worth highlighting that co-culture of hESCs with the stromal cell line AM20.1B4, derived from the embryonic AGM region, yields the highest engraftment level in NOD/LtSz-SCID/IL2rγnull (NOG) mice, increasing this proportion from < 1% to 16% [58]; however, this is still not sufficient for any useful clinical application.

Human iPSCs are a promising alternative to the use of hESCs for hematopoietic studies and in drug development, but reasonable doubts regarding the suitability of these cells for clinical therapies are starting to emerge. The main objection to the potential clinical use of hiPSC-derived cells is the use of viral vectors required for initial reprogramming, which could confer tumorigenic potential to these cells and their derivatives. However, this is not the only problem: low differentiation efficiencies and early senescence have also been detected in hiPSC-derived cells.

For these reasons, future studies should strive to improve differentiation protocols, in order to attain higher engraftment levels. Additionally, the development of safer and more efficient reprogramming methods for obtaining pluripotent stem cells may guarantee the future safe application of hiPSC technologies for clinical therapy.

Acknowledgements

The authors are grateful to financial support received by from Leukemia and Lymphoma UK, Fanconi Hope UK and the Fanconi Anemia Research Fund USA, and funds for research in the field of regenerative medicine through the collaboration agreement between the Conselleria de Sanidad (Generalitat Valenciana) and the Instituto de Salud Carlos III (Ministry of Science and Innovation).

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