Cytotherapy, 2015; 17: 344e358

REVIEW

Progress and challenges in generating functional hematopoietic stem/progenitor cells from human pluripotent stem cells

SENQUAN LIU1,2, YULIN XU1, ZIJING ZHOU2, BO FENG2,3,* & HE HUANG1,* 1

Bone Marrow Transplantation Centre, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China, 2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China, and 3SBS Core Laboratory, Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China Abstract The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) in vitro holds great potential for providing alternative sources of donor cells for clinical HSC transplantation. However, the low efficiency of current protocols for generating blood lineages and the dysfunction identified in hPSC-derived hematopoietic cells limit their use for full hematopoietic reconstitution in clinics. This review outlines the current understanding of in vitro hematopoietic differentiation from hPSCs, emphasizes the intrinsic and extrinsic molecular mechanisms that are attributed to the aberrant phenotype and function in hPSC-derived hematopoietic cells, pinpoints the current challenges to develop the truly functional HSCs from hPSCs for clinical applications and explores their potential solutions. Key Words: hematopoietic differentiation, hematopoietic reconstitution, hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells

Introduction Hematopoietic stem cell (HSC) transplantation has been broadly used for the treatment of life-threatening hematopoietic disorders, including deadly genetic diseases and malignant leukemia, with matchless therapeutic benefits. Since the early 1960s, autologous HSCs obtained from bone marrow (BM) or mobilized peripheral blood (M-PB) have been used for transplantations in clinics to treat a wide variety of hematopoietic diseases, such as myeloma and autoimmune diseases [1,2]. As an alternative and extension, allogeneic HSCs isolated from tissue-matched BM, MPB or umbilical cord blood (UCB) have been used and proven to be beneficial in treating a wider range of pathological conditions such as leukemia, myeloproliferative disorders, lymphoma, myeloma and other solid tumors [3,4]. However, although much effort has been put into banking BM and cord blood cells, the limited

availability of suitable donors still severely restricts the application of this powerful approach in clinical therapies because of the heavy demand of large numbers of HSCs for transplantation. Hence, establishing new sources and generating a large number of transplantable HSCs has been a long sought-after goal for treating hematologic conditions. A recent advance in stem cell research has opened a new avenue for HSC transplantation. A novel source, human pluripotent stem cells (hPSCs), can be potentially used in hematopoietic therapies to develop HSCs. hPSCs include human embryonic stem cells (hESCs) derived from early embryos at the blastocyst stage and induced pluripotent stem cells (hiPSCs) generated from somatic cells through epigenetic reprogramming. hESCs and hiPSCs exhibit highly similar, if not identical, features, having the ability to differentiate into various somatic cell types while

*These authors contributed equally to this work. Correspondence: Bo Feng, MD, The Chinese University of Hong Kong, School of Biomedical Sciences, Room 105A, LIBSB, Area 39, Shatin, N.T., Hong Kong SAR, China. E-mail: [email protected]; He Huang, MD, Bone Marrow Transplantation Centre, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China. E-mail: [email protected] (Received 23 October 2014; accepted 6 January 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.01.003

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the development of technology for the de novo generation of clinically-applicable HSCs.

undergoing robust self-renewal in culture [5,6]. Hence, they provide a renewable source for generating blood lineage cells. iPSCs reprogrammed from somatic cells in particular broaden the horizons in generating patient-specific stem cells, thus having enormous potential for the treatment of hematologic diseases without restriction from immune incompatibility and ethical concerns (reviewed in van Bekkum et al. [7]). Currently, hematopoietic differentiation strategies have enabled the generation of multiple desired blood cell types [8]. However, in vitro derivation of human HSCs with robust capacity to sustain multilineage engraftment has not been achieved. Therefore, understanding the differences between hPSC-derived hematopoietic cells and their in vivo counterparts and uncovering the reasons why hPSC-derived HSC-like cells lack the multilineage engraftment potential have become major issues for the research in this field. In this Review, we provide an overview of the recent progress in improving hematopoietic differentiation from hPSCs, pinpoint the current challenges to the further application of these cells and discuss potential solutions that may facilitate

HSC development at a glance Mammalian hematopoietic development comprises a sequential series of cell fate decisions, which results in the progressive loss of cellular plasticity as well as gradual specification into single or multiple lineages (Figure 1). In embryos at the blastocyst stage, pluripotent inner cell mass first segregates into primitive endoderm and epiblast. The posterior epiblast then gives rise to a transient structure called the primitive streak (PS), which extends and differentiates into three primary germ layers (ectoderm, mesoderm and endoderm) through gastrulation [9]. Hematopoietic cells originate from the mesoderm, emerging as blood islands in the yolk sac (YS) that primarily support the primitive hematopoiesis at early embryonic stages (E 7.5 in mouse) [10,11]. Around day 10 of mouse gestation, hematopoiesis shifts to an intra-embryonic site—the aorta-gonad-mesonephros (AGM) region—where definitive hematopoietic T lymphocytes

Thymus

B lymphocytes CLP

NK cells

Quiescent LT-HSC

Dendritic cells Activated LT-HSC

ST-HSC

MPP

Macrophages GMP

Bone marrow

Granulocytes Platelets

CMP

RBC

MEP Self-renewal

Fetal liver

LT-HSC

AGM Primitive streak

Epiblasts

Progenitors HSC

Lateral plate mesoderm

Mesoderm

Hemogenic endothelium

AGM/ Placenta

Self-renewal

Blood island Extraembryonic mesoderm

Definitive blood cells (small fraction)

Primitive myeloid

Primitive hemangioblasts

Primitive progenitors

Nucleated erythrocytes

Yolk Sac

Figure 1. Overview of normal hematopoietic development in vivo. This model depicts normal hematopoietic development in the mammalian system. Primitive hematopoietic progenitors in yolk sac initiate transient hematopoiesis for the developing embryo [11,28]. Definitive HSCs originate in AGM and move to bone marrow [12,29]. Long-term HSCs in bone marrow sustain life-long hematopoiesis and provide a continuous supply of various blood cell types [15,16]. ST-HSC, short-term HSCs; MPP, multipotent progenitors; CLP, common lymphoid progenitors; CMP, common myeloid progenitors; MEP, megakaryocyte/erythrocyte progenitors; GMP, granulocyte/macrophage progenitors; RBC, red blood cells.

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precursors are generated from hemogenic endothelium (HE) through the endothelial-to-hematopoietic transition [12,13]. The first long-term HSCs (LTHSCs) are generated in the AGM region, from which they enter the circulation system and migrate to the fetal liver (FL) to further develop, expand and differentiate into all blood lineages [14,15]. During the late developmental stages, LT-HSCs further migrate and seed the BM, where they support hematopoiesis throughout adult life (reviewed in Mikkola et al. [16] and Medvinsky et al. [17]). The molecular mechanisms regulating hematopoietic development during embryogenesis have been gradually unraveled by extensive studies through the use of animal models [18]. Key transcription factors and morphogenic signals have been identified to regulate a cascade of events leading to the formation of HSCs. First, a T-box transcription factor Brachyury (T) expressed in the entire PS was found to be essential to the development of both mesoderm and endoderm [19]. Subsequently, the hematopoietic mesoderm is specified from the posterior PS by the expression of kinase insert domain receptor (Kdr) (also named fetal liver kinase 1, Flk1) [10]; and stem cell leukemia factor (Scl), a basic helix-loop-helix transcription factor, further specifies Kdrþ cells to HE [13]. Studies have indicated that Kdr and Scl are both essential to primitive as well as definitive hematopoiesis [20,21]. Other than these early determinants, critical hematopoietic regulators such as c-Kit, Fli1, and Runx1 have also been identified from the AGM-HE [22e24], and factors from the GATA family (Gata2 and Gata3) were found to play important roles in regulating HSC development [25,26] (also reviewed in Wilkinson et al. [27]). The studies of mouse embryogenesis and hematopoiesis have laid the foundation for understanding human hematopoietic development. Scarce but consistent evidence suggests that the sequence of key events described in mouse embryos also applies to the human hematopoietic development [28,29]. The aforementioned determinant transcription factors in mouse HSC development have also been found to be expressed in the potential hematopoietic territory during human embryonic development [30], which suggests that hematopoietic developments in human and mouse are largely conserved. One the other hand, extensive studies have also revealed profound differences between hematopoietic developments in humans and mice (reviewed in Mestas et al. [31]). As it is widely noticed, endogenous hematopoietic cells from humans and mice at an equivalent stage often showed distinct phenotypes. For example, CD34 antigen is expressed highly in murine short-term

HSCs, but it decreases in LT-HSCs, whereas in human BM, CD34 expression is associated with the self-renewable LT-HSCs that are capable of reconstituting the hematopoietic system in recipients [32]. To date, the widely accepted identification scheme for human BM-derived HSCs is LinCD34þCD38 CD45RA, whereas for mouse BM-HSCs, Lin Sca1þcKitþCD150þ is used (reviewed in Mestas et al. [31] and Doulatov et al. [33]). In vitro hematopoietic differentiation from hPSCs In vitro hematopoietic differentiation was first noticed in mouse ESC aggregates (embryoid bodies, EB) cultured in suspension, which differentiated spontaneously and produced a small fraction of blood lineage cells [34]. Thereafter, various methods have been attempted to induce hematopoietic differentiation from human ESCs and iPSCs, with a particular focus on enriching the engraftable hematopoietic stem cells and progenitors. The EBmediated stepwise induction approach provides a basic setting [35], whereas combining the usages of hematopoiesis-promoting factors and co-culture with stromal cell feeders such as OP9 cells or FL cells largely promoted the efficiency [36e39]. In numerous studies, hematopoietic differentiation from hESCs and hiPSCs has been found to closely mimic the stepwise hematopoietic development during embryogenesis. The sequential emergence of several types of early hematopoietic precursors has been identified; these include Brachyury (T)þ cells that indicate PS formation, Kdrþ cells representing emergence of hematopoietic mesoderm, as well as CD34þCD43þCD45 hematopoietic precursors and CD43þCD45þ progenitors [36,40,41]. Hence, on the basis of both the developmental studies and EB studies, a general stepwise induction scheme has been established by dividing hematopoietic differentiation of hPSCs into three steps (Figure 2) [36,42,43]. First, cooperative action of WNT/b-catenin signaling together with activin/nodal and bone morphogenetic protein 4 (BMP4) signaling pathways induces the formation of PS-mesoderm progenitors, which are characterized by the expression of T cells [44,45]. Extensive studies have investigated the roles of individual signaling pathways as well as their combinatory effects at this transient and highly dynamic stage. In various proposed regulatory models, the enhanced WNT/b-catenin signaling and appropriate exposure to activin A or BMP4 were all shown to increase the expression of T or production of Tþ cells and were required for the formation of Tþ cells [45e47]. Second, vascular endothelial growth factor (VEGF) synergizes with fibroblast growth factor (FGF) to specify Kdrþ

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Engraftable HSC?

Embryoblast

ESCs

BMP4 Wnt3a Activin

SCF FLT3L TPO IL3 IL6

VEGF bFGF

NK cells B cells T cells Erythrocytes

Brachyury

Somatic cells

Virus-encoding factors

Primitive streak/ Early mesoderm

Hemangioblast

HE-precusor cells

BRACHYURY+

BRACHYURY+

KDR+

CD34+

KDR+

CD34+

CD45+

iPSCs SSEA4+ NANOG+ TRA-1-60+

Hematopoietic progenitor cells

Megakaryocytes Granulocytes Monocytes Macrophages

CD43−

OCT4+

Figure 2. Stepwise hematopoietic differentiation from hPSCs. Schematic representation shows the generation of hematopoietic cells from hESCs and hiPSCs in vitro. Primitive streak-like cells and early mesoderm cells emerged at the first stage on induction by BMP4 and activin [44,47]. VEGF and FGF induce hemangioblasts and then hemogenic endothelium precursors into hematopoietic cells at the second stage [48,49]. Finally, hematopoietic cytokines, such as SCF, FLT3L, TPO, IL-3 and IL-6, further promote the maturation of and induce CD45þ hematopoietic progenitor cells [36,50].

hemangioblasts and subsequently, the HE-like precursors and progenitors, which could generate hematopoietic colonies in culture [48,49]. Third, the cultivation of CD34þ cells in a hematopoietic culture medium, supplemented with HSC expansion factors Scf, Flt-3L, thrombopoietin (TPO), interleukin (IL)3 and IL-6 [50], promotes their maturation into CD45þ hematopoietic progenitors. In addition to this basic scheme, other morphogenic signals such as noncanonical WNT, Hedgehog and Notch were also shown to be involved in the hematopoietic differentiation, in a stage-specific and time-controlled manner [51e53]. Collectively, with the current induction and culture systems, the hematopoietic differentiation from hPSCs represents a highly dynamic non-stop process. On the other hand, because the high variability of EB formation and poorly defined factors associated with feeder cells have largely hindered further applications of these differentiation methods, researchers have been keen to develop a precisely defined serum-free system to generate hematopoietic cells that could be potentially used in clinics. Hence, the previous inducing scheme has been adapted to monolayer-cultured hESCs in a chemically defined condition for hematopoietic induction [49,54]. Although the current efficiency of hematopoietic differentiation in this method, as measured by the production of CD34þ cells, was lower than the induction via EBs [36], it is still an attractive approach because of the well-defined nature for investigating the molecular mechanisms and the underlying process of hematopoietic development. This, in turn, holds the potential to further improve the functionality in the cells obtained.

Molecular features and functionality of hPSC-derived hematopoietic cells To date, hematopoietic progenitor cells have been derived from hESCs and hiPSCs through various approaches and have shown the ability to differentiate into diverse mature blood lineages [55e57], including lymphocytes [36,58e60], by use of in vitro colonyformation unit assay. However, it has been difficult to generate functional HSCs that can engraft in adult host animals and thereafter support long-term hematopoietic reconstitution [61,62]. To uncover the reasons behind this, researchers have made efforts to analyze the differences between hPSC-derived hematopoietic cells and their in vivo counterparts isolated from endogenous tissues by studying their gene expression, microRNA regulation, surface antigens and epigenetic modifications.

hPSC-derived hematopoietic cells resemble embryonic progenitors but differ from adult HSCs With the common observation that hematopoietic differentiation in vitro resembles the in vivo development, accumulating data have further suggested that the process of hematopoietic differentiation from hPSCs is more similar to the hematopoiesis at early embryonic stages in YS and FL than that in BM. Through the use of high-density microarrays, gene profiling analysis by Lu et al. [63] showed that hESCderived CD34þCD38 cells maintained a higher activity of proliferation compared with endogenous HSCs derived from BM; they exhibited a very low expression of genes associated with BM-HSCs, such as TIE1, FLT3, BCL2, GATA2, ERG2 and several of

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the major histocompatibility complex genes. Consistently, the Zambidis et al. [41] study revealed striking similarity between the stepwise hematopoietic differentiations in EBs and in YS in vivo, in regard to the cellular and molecular kinetics. Increased levels of SCL/TAL1, GATA1, GATA2, CD34 and CD31, which correlate with the initiation of embryonic hematopoiesis, were detected, indicating that in vitro hematopoiesis from hPSCs resembles the earliest events of embryonic hematopoiesis in human YS development [41]. More importantly, studies showed that the hPSCderived hematopoietic cells exhibit the features of primitive hematopoiesis in the subsequent in vitro and in vivo differentiation. Through either a coculture on feeder stromal cells or an EB formation, hematopoietic progenitors derived from both hESCs and hiPSCs were found to give rise to erythroid cells that primarily express the embryonic globin z- and ε-chains and the fetal g-chain but barely the adult bchain [55,64,65]. This resembles the primitive hematopoiesis in the embryonic YS and FL (reviewed in Mazurier et al. [66]). These studies suggest that hESC-derived hematopoietic cells have not yet acquired the identity and function of definitive HSCs. For an unidentified reason, the in vitro hematopoietic differentiation halted at a stage similar to the embryonic YS and FL, and it failed to progress to further maturation. Aberrant gene regulation in hematopoietic cells derived from hESCs On the other hand, studies also revealed the aberrant characteristics of the hPSC-derived hematopoietic cells, suggesting that they are distinct from embryonic hematopoietic cells to a certain extent. In the Salvagiotto et al. [67] study, hESC-derived hematopoietic cells were found to show distinct gene expression from that in HSC-enriched FL cells, including a reduced expression of genes associated with HSC self-renewal (EZH2, MEIS1 and MLL) and survival (ARHGAP1, ETV6 and HLF) and a higher expression of certain noncoding RNAs. The cell adhesion molecule CXCR4, which is known to be important in ensuring the homing of competent HSCs, was also not detected [67]. This is consistent with a study by McKinney-Freeman et al. [68], which showed that mouse ESC-derived HSC-like cells exhibited a unique cell-surface phenotype, which was distinct from any in vivo hematopoietic cell populations. They found that ESC-derived cells expressed surface markers CD41 and CD150 but not CD34 and were heterogeneous for CD45, which were distinct from the earlier embryonic hematopoietic cells expressing CD41 and CD34 as well as

the mature HSCs that express CD45 and CD150 [68]. This suggested that the ESC-derived HSC-like cells might represent a mixture of developmentally immature cells transitioning between embryonic and adult phenotypes. More recently, Schnerch et al. [69] performed more thorough gene expression analysis to compare hESC hematopoietic cells with various in vivo HSCs purified from embryonic, fetal and adult human samples, including the in vivo explants of human HSCs 8 weeks after transplantation [69]. They found that hESC derivatives and fetal HSCs were clustered with undifferentiated hESCs as the result of the expression of embryonic genes. Moreover, a set of epigenetic transcriptional regulators, including core PcG and TrxG genes, EZH1, ASH1L and PCGF6, were found to be present in hESC derivatives but absent in endogenous HSCs that are capable of hematopoietic repopulation. This suggests that the persistent expression of these epigenetic regulators might predispose hESC derivatives to an intermediate ESC/ HSC cell fate and result in a primitive hematopoietic phenotype lacking in vivo reconstitution capacity. Impaired lymphopoietic activity in hESC-derived hematopoietic cells At the same time, another strand of evidence pinpointed that hPSC-derived hematopoietic cells exhibited impaired lymphopoietic activity. In a study by Melichar et al. [70], lymphoid differentiations from various hESC-derived and fetal tissueederived hematopoietic cells were compared. In contrast to the endogenous hematopoietic precursors, none of the hESC-derived CD34þ precursors developed into T cells. Consistently, Martin et al. [71] showed that hESC-derived hematopoietic progenitor cells favored the commitment to natural killer (NK) cell lineage but had limited potential to give rise to T and B cells. Their analysis further identified the aberrant expression of ID family genes and the constitutive activation of transcriptional targets of stem cell factor (SCF)-induced signaling, which is known to inhibit T-cell development and to promote NK cell development. Furthermore, Dravid et al. [72] revealed that the inhibitory regulators of lymphopoiesis, such as the adaptor protein LNK, CCAAT/ enhancer-binding protein-a (CEBPa) and transcription factors commonly detected during B-lymphoid development, were aberrantly expressed in hESCderived CD34þ cells. Collectively, these results suggested that there were certain differences between hESC-derived hematopoietic progenitors and analogous primary human cells and that aberrant gene expression might be a reason that blocked the hESCs from undergoing complete definitive hematopoiesis.

Generating functional HSCs/progenitor cells from hPSCs Interaction between hPSC-derived hematopoietic cells and niche after transplantation Other than the altered gene expression and differentiation potential, recent studies have also suggested aberrant responses of hiPSC-derived hematopoietic cells after transplantation. A study by Schnerch et al. [69] found that hESC- and hiPSC-derived hematopoietic cells, which showed similar expression of marker genes but a lower capacity to generate colonyforming units (CFU), completely failed in hematopoietic reconstitution after transplantation. Through the use of quantitative polymerase chain reaction (PCR) arrays, they identified that in contrast to BMHSCs, hiPSC-derived cells did not downregulate the microRNAs involved in hematopoiesis, including miR-19b, 146a, 149, 191, 223, 324-3p, 490 and 585, after being transplanted in vivo [61]. This indicated that hPSC-derived hematopoietic cells exhibited aberrant microRNA regulation after transplantation, and this might be also a reason that led to the failure of hematopoietic reconstitution by these cells. In support of this notion, Tian et al. [62] further suggested that hESC-derived hematopoietic cells exhibited different developmental potentials in vitro and in vivo. With the use of hESCs expressing firefly luciferase (luc), Tian et al. [62] performed noninvasive, real-time bioluminescent imaging with hESCderived CD34þ cells transplanted into the livers of neonatal immunodeficient mice. The results of serial imaging showed stable engraftment and the in vivo expansion of the lucþ cells over several months. However, these bi-potent CD34þ cells were found to differentiate preferentially into endothelial cells in vivo, with only a small number of hematopoietic cells generated [62]. Collectively, hPSC-derived hematopoietic precursors exhibited a transcriptional profile in an embryonic pattern and with a fraction of dysregulation; they exhibited an impaired activity for primitive hematopoiesis and failed to reconstitute the hematopoietic system after being transplanted into adult mice. This suggested an incomplete differentiation and a skewed commitment to definitive hematopoiesis, probably caused by the persistence of “transcriptional memory.”

Restrictions of the use of human iPSCs for the generation of hematopoietic cells iPSCs have been derived from various somatic cell populations by introduction of defined factors, through different delivery approaches including the virus-mediated ectopic expression [6,73], the transfection of synthetic modified messenger RNA [74] and the direct delivery of recombinant proteins

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[75,76]. Moreover, on the basis of the findings that the chemical manipulation of signaling pathways, such as stimulation of WNT [77], inhibition of transforming growth factor-b [78] and suppression of p53 [79], could promote the reprogramming process, recent studies have demonstrated complete reprogramming of mouse fibroblasts solely through stimulation by use of chemicals [80]. These studies suggest the use of human iPSCs as a convenient tool for hematopoietic study and therapy because they can be robustly obtained from human somatic cells with minimum ethical concerns, compared with hESCs (Figure 3). Efficient differentiation of hiPSCs into functional HSCs is one of the major requisites for future HSCbased cell therapies. Although extensive studies have demonstrated that iPSCs derived from various somatic cell types were all pluripotent and highly resembled ESCs in regard to their molecular signatures and differentiation potentials [81], recent findings have suggested that the reprogramming process might not completely erase the somatic cell identity that had been gradually deposited through the long course of development [82,83] (reviewed in Narsinh et al. [84]). Polo et al. [85] further analyzed iPSC lines obtained from genetically matched mouse fibroblasts and hematopoietic and myogenic cells. Their results showed that iPSCs from different origins exhibited a minor distinction in their transcriptional and epigenetic patterns as well as biased differentiation potentials into embryoid bodies and different hematopoietic cell types, even though these epigenetic memories were transient and could been erased on continuing passaging [85]. Consistently, Pfaff et al. [86] found that mouse iPSCs derived from undifferentiated BM cells could differentiate into hematopoietic lineages with higher efficiency compared with iPSCs from other origins, which suggests that cellular origins might influence the potency of iPSCs. Studies further investigated whether iPSCs, which might carry the epigenetic memory or other aberrance, could undergo efficient hematopoietic differentiation. Although a comparison analysis showed that hiPSCs generated hematopoietic cells with a decreased efficiency compared with hESCs [87], another study showed that hESCs and hiPSCs can be differentiated into red blood cells with a similar efficiency [55]. Further reports suggested the imperfection of reprogramming technology in iPSCs could lead to the decreased hematopoietic potency. Seiler et al. [88] found that the residual expression of reprogramming factors in poorly reprogrammed mouse cells negatively correlated with hematopoietic differentiation. Consistently, Ramos-Mejia et al. [89] showed that failure in silencing reprogramming

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How to prove the functionality of derived hematopoietic cells?

Expected hematopoietic cells

How to get a large number of functional blood cells?

Reprogr am

Genetically corrected iPSCs

ming

Somatic cells

What is the best approach?

Retroviral Lentiviral Adenoviral Sendi viral Protein mRNA Small RNA Chemical molecules

Repair of diseasecausing mutation

Patient-specific iPSCs How to optimize cultivation condition of iPSCs?

Figure 3. Potential applications of iPSCs in hematological therapy. Reprogramming technology and iPSCs have the potential to be used to model and treat human blood diseases [8,81]. Schematic representation shows the potential route and remaining challenges to application of iPSC technology for studying and treating human hematological conditions.

factors inhibited the differentiation of human iPSCs to both hematopoietic and neuroectodermal lineages, whereas the successful excision of reprogramming factor transgenes from these iPSCs improved their differentiation capacity [89]. These studies suggest that it is important to use high-quality iPSCs for the generation of functional cell types as well as in the subsequent applications (reviewed in van Bekkum et al. [7], Seiler et al. [90] and Peters et al. [91]). It is noteworthy that with the rapid progress in the iPSC field, the newly emerged non-integral reprogramming methods, such as Sendai virus, Episomal vectors and direct transfection of modified messenger RNAs, have made it possible to generate fully reprogrammed and transgene-free iPSCs in routine practices [74,92,93]. On the other hand, the accumulating reports of differential features and functionalities between hESCs and hiPSCs have led to the question of whether these two cell types are truly equivalent. A recent study by Bock et al. [94] performed a comprehensive analysis of a large number of human ESC and iPSC lines. The results showed that a minor but stable cell lineespecific deviation exists, that is, each of the cell lines was different from the others in a small number of genes at the DNA methylation or gene expression levels, but no unique epigenetic or transcriptional deviation was found to be shared

by all tested hPSC lines [94]. This is consistent with a previous study in which only a subtle difference was observed among the globally similar gene expression profiles between hESCs and hiPSCs [95]. Given that hiPSCs and hESCs share the important properties of self-renewal and pluripotency and exhibit similar characteristics in regard to their morphology, signal dependence, surface marker expression, global gene expression profiles and differentiation capacity, further investigation is needed to understand whether the cell lineespecific deviation and minor differences between hESCs and hiPSCs have any implication in their hematopoietic differentiation.

Progress toward generating engraftable HSCs from hPSCs Reinforcing the definitive hematopoiesis through ectopic expression of key transcription factors Intensive studies on mouse hematopoietic development have identified genes that are associated with definitive hematopoiesis and promote the development of HSCs capable of hematopoietic reconstitution. As demonstrated by Kyba et al. [96] and Chan et al. [97], the ectopic expression of HoxB4 could switch primitive hematopoietic progenitors isolated from FL or derived from mouse ESCs to the

Generating functional HSCs/progenitor cells from hPSCs definitive HSC phenotype, and it resulted in robust multilineage hematopoietic reconstitution in the subsequent transplantations. In addition, Cdx4 [98], Sox17 [99] and Runx1 [22] were also found to play a critical function in the development of definitive hematopoiesis in vivo; other genes, such as Stat5a [100], Lhx2 [101] and Mixl1 [102], were found to enhance the production of definitive HSCs during mouse ESC differentiation and endow them with a repopulating ability in primary and secondary recipients. The hematopoietic differentiation of human PSCs was shown to resemble the hematopoietic development at the YS stage [41,66], which is believed to reflect a defect during the transition to definitive hematopoiesis. Hence, attempts have been made to enforce the definitive hematopoietic differentiation from hPSCs through manipulating the expression of key definitive hematopoiesis genes. In consistence with the mouse studies, human HOXB4 was found to promote the development of definitive hematopoiesis during hESC differentiation, either by stable overexpression or application of recombinant proteins during EB [103]. However, rather surprisingly, HOXB4 overexpression in hESCs did not improve the in vivo engraftment of the derived hematopoietic cells [104]; this suggests that further investigation is needed to identify determinant regulators for inducing definitive hematopoiesis during hPSC differentiation. Through a screening approach, Nakajima-Takagi et al. [105] identified SOX17 as a key transcriptional regulator involved in the endothelial-tohematopoietic transition, the critical stage that marks the emergence of definitive hematopoietic precursors, during the hematopoietic differentiation of hESCs [105]. They found that SOX17 overexpression led to the expansion of a population of CD34þCD43þ cells, which resembled definitive HSC precursors by expressing HE marker VEcadherin, and could give rise to differentiated hematopoietic progenies in multiple lineages [105]. Similarly, Ran et al. [106] showed that the ectopic expression of RUNX1a, an isoform of RUNX1, also positively regulated the expression of mesoderm and hematopoietic development-related genes in hESCs and hiPSCs and promoted definitive hematopoiesis during their differentiation [106]. Hematopoietic progenitors derived from RUNX1a-overexpressing hPSCs showed enhanced expansion ability, gave rise to erythrocytes with b-globin production and demonstrated improved repopulation with multilineage hematopoiesis after transplantation [106]. In line with this effort, a recent study by Elcheva et al. [107] examined 27 candidate factors and further identified two groups of transcriptional

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regulators that are capable of inducing distinct hematopoietic programs from hESCs and hiPSCs. GATA2/TAL1 induced HE-like cells with restricted erythro-megakaryocytic and macrophage potential, and ETV2/GATA2 induced HE-like cells with panmyeloid potential. This result provides further insight into the transcription network leading to the formation of HE with different functional capacities; however, the obtained cells failed to establish a meaningful engraftment in the adult mouse recipients, which indicates that definitive HSCs have not yet been induced [107]. Generation of transplantable hematopoietic cells from hiPSCs through teratoma formation Although it has been widely noticed that human ESCs and iPSCs differentiate into cells and tissues from all three germ layers and form teratoma after being injected into immune deficient mice, hematopoietic differentiation was not examined in detail until recently. Amabile et al. [108] performed conventional histological analysis of the BM-like tissues observed in teratomas derived from hiPSCs, and they detected the presence of CD34þCD45þ hematopoietic progenitors as well as more differentiated myeloid and lymphoid cells, including neutrophils, lymphocytes and megakaryocytes. This study, for the first time, showed that hematopoietic progenitors derived from hPSCs were able to reconstitute a human immune system and give rise to functional B and T cells when transplanted into NOD.CgPrkdcscid Il2rgtm1Wjl/SzJ immunocompromised (NSG) mice subcutaneously or intramuscularly [108]. These data suggest that the teratomas provided a permissive niche for high-quality definitive hematopoietic differentiation; thus, it established a novel route to derive functional hematopoietic progenitors close to endogenous HSCs. The finding by Amabile et al. has been supported by another study published by Suzuki et al. [109]; they performed a similar analysis on the teratomamediated differentiation. Through injection into NOD/SCID mice (5e7 weeks old) subcutaneously, the result showed that the hematopoietic cells yielded in teratomas derived from either mouse or human iPSCs could successfully repopulate the hematopoietic system after transplantation into irradiated immune-deficient mice. Moreover, either supplementation with cytokines through a micro-osmotic pump or co-injection with OP9 cells could improve the hematopoietic differentiation in teratomas significantly, whereas the combination of both cytokines and OP9 showed an additive effect [109]. Collectively, these two new reports demonstrated a possible path for generating truly functional HSCs

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Table I. Engraftment of in vitroe and in vivoederived hematopoietic cells in animal models.

Cell sources

Differentiation methods

hESC-derived hematopoietic cells

Co-culture with S17 stromal cells

hESC-derived hematopoietic cells

Co-culture with AGM and FL stromal cell lines EB formation, HOXB4 overexpression Co-culture with S17 stromal cells

hESC-derived hematopoietic cells hESC-derived hematopoietic cells hESC/hiPSC-derived hematopoietic cells

Transplantation methods Intravenous and intrafemoral injection Intrafemoral injection

Intravenous and intrafemoral injection Intraperitoneal injection Intrafemoral injection

Long-term engraftment potential

Reference

NOD/SCID mice

Inefficient

[110]

NOD.CgPrkdcscidIl2rgtm1Wjl (NSG) mice NOD/SCID or NOD/ SCID b2m/ mice

Inefficient

[111]

Inefficient

[104]

Inefficient

[119]

Inefficient

[112]

Inefficient

[121]

0.1e1.7% reconstitution in primary recipient mice; 0.04%0.01% in secondary recipients 0.2e5% CD45þ cells after 12 weeks

[108]

Animal models

Fetal sheep (11 months; failed in adults Reconstitution in primary recipient newborn mice for >11 months; and in secondary recipients for >6 months

[124]

[126]

[127]

(continued)

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Table I. Continued

Cell sources

Differentiation methods

Transplantation methods

Long-term engraftment potential

Animal models

Human FL cells

e

Intrahepatic injection

Human FL cells

e

Human UCB cells

e

Intraperitoneal injection in utero Intrahepatic injection

Human BM cells

e

Intravenous injection

Hematopoietic cells from human FL, UCB, BM

e

Intravenous injection

Newborn Rag2 gc mice and NOD.CgPrkdcscidIl2rgtm1Wjl (NSG) mice Fetal sheep (48e54 days of gestation) Newborn Rag2e/egce/e mice NOD/SCID and NOD.CgPrkdcscidIl2rgtm1Wjl (NSG) mice NOD.CgPrkdcscidIl2rgtm1Wjl (NSG) mice e/e

e/e

Reference

Overall long-term reconstitution

[128,129]

Reconstitution for >2 years Successful reconstitution Robust immune reconstitution

[117,118]

Successful reconstitution for >4 months

[130,131] [116]

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Summary of engraftment results of various hPSC-derived HSC-like cells as well as hematopoietic stem/progenitor cells obtained from mouse YS cells, human FL, human UCB and BM.

with authentic engraftment potential. The successful generation of reconstitution-permissive hematopoietic cells with the use of teratomas also suggested that current in vitro differentiation conditions did not fully mimic the complex in vivo hematopoietic development, thereby leading to the failure of definitive HSC specification from hPSCs.

Selection of animal models for hematopoietic transplantations Hematopoietic cells derived from human ESCs and iPSCs have been transplanted into immune-deficient mice to assess their functionality. However, although transplantation through different routes into various immune-deficient mice has been investigated, these hPSC-derived hematopoietic cells largely failed to reconstitute the hematopoietic systems in the recepients [61,62,110e112]. BM engraftment was observed at low efficiency (approximately 0.1e2%) and was lacking lymphoid reconstruction, which is apparently different from that repopulated by endogenous HSCs obtained from human FL [113], UCB [114], M-PB [115] and BM [116] (Table I). Given the fact that a complex and dynamic molecular cross-talk between HSCs and their endogenous microenvironment (niche) plays a critical role in directing their cell states between self-renewal, differentiation and quiescence, lack of a match between the transplanted cells and the BM microenvironment is likely to be a direct cause of the engraftment failure. In line with this idea, Risueno et al. [61] noticed that hiPSC-derived hematopoietic cells could actually survive in the BM of the recipient NSG mice (8e10 weeks old) after intrafemoral injection, but they failed

to demonstrate hematopoietic differentiation. Once removed from the recipient BM, the grafted cells were capable of in vitro multilineage hematopoietic differentiation, thus suggesting that mouse BM imparts a restriction and blocks the engrafted cells from undergoing hematopoietic differentiation [61]. Consistently, compared with endogenous HSCs obtained from various developmental stages and tissue origins, hPSC-derived hematopoietic cells were found to exhibit embryonic features similar to cells in the YS or FL rather than those in adult BM [41,63]. This might explain why hPSC derivatives failed in the hematopoietic reconstitution after being engrafted [61], because mature adult mouse BM might not be able to support the maintenance and differentiation of primitive hematopoietic cells. On the other hand, the similarity between hPSC derivatives and primitive hematopoietic cells also suggested that transplantation into embryonic recipients might be a promising approach to accommodate hPSC derivatives by providing age-matched microenvironments. Indeed, a human/fetal sheep xenograft model, which has been established to assess the in vivo hematopoietic activity of various endogenous human HSCs including the primitive hematopoietic progenitors from FL [117,118], was found to be supportive for the engraftment and differentiation of hPSC-derived hematopoietic cells [119]. In this study, CD34þLin or CD34þCD38 cells derived from hESCs through coculture with S17 cell feeders were transplanted in utero into fetal sheep at around 2 months of gestation, and they were found to successfully establish the chimerism at 5 to 17 months later, as shown by PCR and flow cytometric analysis on BM and peripheral blood samples [119]. This further suggested that the previous failures of hPSC-derived hematopoietic cells in

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reconstituting mouse hematopoietic systems might be caused, at least in part, by the lack of a match between donor cells and the host environment. In support of this notion, a recent study by Arora et al. [120] has systematically demonstrated the effect of developmental stages of donor HSCs and recipients on the transplantation outcome, through the use of the mouse system [120]. Their results showed that primitive hematopoietic progenitors from E9.5 and E10.5 preferentially engrafted neonates, whereas mature definitive HSCs from E14.5 FL or adult BM more robustly engrafted adults [120]. Collectively, these studies suggest that fetal sheep or neonatal mice might be the promising recipient models for the transplantations of in vitroederived HSC-like cells to assess their functionalities. The choice of animal models and transplantation techniques directly affects the engraftment outcome and must therefore be carefully considered for addressing a specific question. Conclusion and perspectives The discovery of hPSCs, especially the iPSCs that could be derived from adult somatic tissues, has opened up an unprecedented possibility for generating large numbers of HSCs for the clinical treatment of hematopoietic disorders. However, obstacles to harnessing this potential still remain. An immediate hurdle is the difficulty in robustly generating engraftable HSCs from hPSCs; an emerging one is the lack of appropriate transplantation models/systems for validating the obtained cells by assessing their efficacy and safety in transplantation. On the basis of the extensive studies, the failure in obtaining/proving functional HSCs from hPSC differentiation are largely due to the imperfection of induction methods and age-related incompatibility between engrafted cells and recipient animals. Although numerous studies showed that hematopoietic cells derived from hPSCs in culture highly resembled embryonic hematopoietic progenitors and exhibited aberrant and immature phenotypes, teratoma-mediated in vivo differentiation has demonstrated the intrinsic competence of hiPSCs to undergo normal hematopoietic development as well as to repopulate the hematopoietic system in recipient mice [108,109]. This indicates that the aberrant features in the in vitroederived HSC-like cells probably are introduced by the imperfection of current induction methodology. To obtain insightful understanding for improving the hematopoietic induction, further investigation is needed to address the undiscovered difference between the hematopoietic differentiations in vitro and in vivo, especially those related to the inducing signals and determinant

factors. To date, the profiling analysis of a variety of HSCs has revealed new genes enriched in definitive HSCs or implicated in HSC self-renewal and hematopoietic reconstitution [68,69], and genetic manipulation has been shown as promising to enforce further development of hPSC-derived hematopoietic cells for definitive hematopoiesis [96,121]. It is foreseeable that extensive research in this area will uncover critical regulators and promote the generation of hematopoietic cells with better reconstitution potential. On the other hand, accumulating evidence indicates that matching on age between donor HSCs and recipients is critical to the transplantation outcome [119,120]. Hence, transplantation of hPSC-derived HSC-like cells that resemble fetal hematopoietic progenitors into adult immunocompromised mice, which were commonly used in most of the studies, probably will result in the failure of hematopoietic reconstitution. In addition to the adult recipients, transplantations of hPSC-derived HSC-like cells into fetal sheep or neonatal mice might be promising and should be considered for assessing the functionalities and differentiation potentials of these cells. Besides the functionalities, it is also critical to prove the efficacy and the safety of in vitroederived HSC-like cells in transplantation before application for clinical therapies. Although “humanized mice” and human/fetal sheep xenograft models have been well-established and are valuable for verifying the reconstitution potential of various human HSCs, the in vivo microenvironment or niche provided by these models may exhibit intrinsic differences from those in human adult patients. Hence, additional models are needed to provide microenvironments that more closely resemble that in human BM. Recently, Chen et al. [122] have developed extramedullary bone and BM models with the use of human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice [122]. Through the use of this system, they have demonstrated the successful engraftment of human HSCs and leukemic cells, thus highlighting the potential of a novel in vivo model of the human BM microenvironment [122]. Further study is still needed to improve the quality of the extramedullary BM. Research in this direction is important for further optimizing the procedures for in vitro derivation of functional HSCs in the future. In conclusion, collaborative efforts from developmental, stem cell and molecular biologists are required to obtain further insight into the fundamental questions of HSC biology, and this will facilitate the development of technologies for generation of functional HSCs from hPSCs.

Generating functional HSCs/progenitor cells from hPSCs Acknowledgments We thank Yaofeng Wang for critical comments on the manuscript. This work was supported by grants from the Research Grants Council of Hong Kong (CUHK 478812 to BF) and in part by funds from the National Natural Science Foundation of China (NSFC 31171433 to BF; NSFC 81230014 to HH; NSFC 81170501 to HH; NSFC 81200338 to YLX).

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Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

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