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The hematopoietic and vascular lineages are intimately entwined as they arise together from bipotent hemangioblasts and hemogenic endothelial precursors during human embryonic development. In vitro differentiation of human pluripotent stem cells toward these lineages provides opportunities for elucidating the mechanisms of hematopoietic genesis. We previously demonstrated the stepwise in vitro differentiation of human embryonic stem cells (hESC) to definitive erythromyelopoiesis through clonogenic bipotent primitive hemangioblasts. This system recapitulates an orderly hematopoiesis similar to human yolk sac development via the generation of mesodermal-hematoendothelial progenitor cells that give rise to endothelium followed by embryonic primitive and definitive hematopoietic cells. Here, we report that under modified feeder-free endothelial culture conditions, multipotent CD34+CD45+ hematopoietic progenitors arise in mass quantities from differentiated hESC and human induced pluripotent stem cells (hiPSC). These hematopoietic progenitors arose directly from adherent endothelial/stromal cell layers in a manner resembling in vivo hematopoiesis from embryonic hemogenic endothelium. Although fibroblast-derived hiPSC lines were previously found inefficient in hemato-endothelial differentiation capacity, our culture system also supported robust hiPSC hemato-vascular differentiation at levels comparable to hESC. We present comparative differentiation results for simultaneously generating hematopoietic and vascular progenitors from both hESC and fibroblast-hiPSC. This defined, optimized, and low-density differentiation system will be ideal for direct single-cell time course studies of the earliest hematopoietic events using time-lapse videography, or bulk kinetics using flow cytometry analyses on emerging hematopoietic progenitors. © 2012 International Society for Advancement of Cytometry
The recent advent of cell reprogramming and induced pluripotency (1, 2) has opened new avenues of investigation for disease modeling, drug discovery (3–5), and patient-specific cell therapies (6). In vitro differentiation models for dissecting the developmental processes that give rise to complex cell lineages such as the hematopoietic system have now become possible (7). The hematopoietic system develops by means of various transitory cell lineages throughout fetal life. Two blood-forming sites have been identified during human embryonic development: the extraembryonic yolk sac (YS) and the aorta-gonad-mesonephros (AGM), both of which exhibit a close juxtaposition of hematopoietic and vascular lineages (8). The existence of a common ephemeral precursor for embryonic hematopoiesis and vasculogenesis termed the hemangioblast has been supported by work on human embryonic stem cells (hESC) (9, 10). However, in the adult, intraembryonic hematopoiesis originates in the dorsal aorta (DA) from a pool of specialized endothelial cells termed hemogenic endothelium (11–14). In an effort to understand the delicate equilibrium between adult versus embryonic hematopoietic and endothelial fate through ontogeny, we and others have developed methodologies based on the differentiation of pluripotent stem cells that generate YS-like clonogenic bipotent precursors (9) or DA-like intermediate mesodermal progenitors (7, 15–17).
The derivation of engraftable vascular and hematopoietic stem cells (HSC) from patient-specific human induced pluripotent stem cell (hiPSC)-derived hemangioblasts or hemogenic endothelium may have great clinical utility for the effective, long-term treatment of hemato-vascular disorders. However, recent studies have suggested that hiPSC do not produce hemato-endothelial progeny in a manner that is quantitatively and qualitatively comparable to hESC (18). There may be several etiologies for this limitation, including the quality of reprogramming achieved in fibroblast-iPSC (due to retention of somatic donor epigenetic memory), the method of hiPSC culture used for maintaining pluripotency (e.g. on murine embryonic fibroblasts (MEF) vs. feeder-free monolayer), and the inherent efficiency of the differentiation protocol (e.g., embryoid body vs. stromal co-culture-based).
In these studies, we focused on optimizing our previously described human embryoid body (hEB)-based hemato-endothelial differentiation method for efficient hiPSC differentiation (10). We report that with rigorous culture technique for pluripotency maintenance, and optimized endothelial-supporting culture conditions, the relatively diminished hemato-endothelial differentiation capacities of fibroblast-hiPSC (18–20) can be considerably improved to levels comparable to hESC. We describe a defined differentiation system which is adapted from our previously described hEB differentiation method, and utilizes a minimal combination of recombinant growth factors [bone morphogenetic protein-4 (BMP4), vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF2)], followed by adherent low-density culture in well-defined endothelial growth medium (EGM-2). In this accessible two-dimensional (2D) culture system, multipotent hematopoietic CD34+CD45+ progenitors arose directly in mass quantities and bud off from adherent hemogenic endothelial cells. This efficient differentiation system can be used for direct time-lapse videography studies, time-course studies of hematopoietic genesis events (e.g., from various hiPSC disease models), or kinetic flow cytometry analyses of newly emerging, floating CD34+CD45+ hematopoietic progenitors.
- Top of page
- Materials and Methods
- Literature Cited
- Supporting Information
The human blood-forming system develops by a succession of dynamic events during embryonic life, and emerges from at least two independent sites: the extraembryonic YS and the AGM (8, 27). In both locations, de novo hematopoiesis involves the intimate liaison of blood-forming and angiogenic cells. However, although hematopoietic and endothelial lineages develop in YS blood islands in synchrony from a common bipotent short-lived progenitor (aka the hemangioblast) (9, 10), intraembryonic blood cells bud off of a pool of already established endothelial cells, the hemogenic endothelium (11–14).
We previously described a pluripotent stem cell model that recapitulates the human YS-like origins of embryonic hemato-endothelial development (9). Hematopoiesis arose from CD45-negative MHE progenitors that give rise to CD143/ACE+ hemangioblast progenitors, followed by sequential YS-like primitive and definitive hematopoietic waves. We delineated the kinetics for emergence of these hemangioblastic progenitors responsible for these two waves (10). Thus, our hEB-based differentiation system captures the earliest developmental steps in human hematopoietic genesis (28, 29).
Recently, this pluripotent stem cell model was validated with human embryo studies. Primitive pre-AGM mesodermal ancestors of the human intraembryonic hematopoietic system were identified in the splanchnopleura (Sp) as CD143/ACE, expressing cells (30). ACE, a key regulator of blood pressure and major component of the renin–angiotensin system (31), and also marks human blood-forming cells in the YS (hemangioblasts), hemogenic endothelium (AGM), fetal liver, and both fetal and adult bone marrow HSC (32). In the human Sp, CD143+ hematopoietic-competent cells (identified with the BB9 mAb) coexpressed OCT-4 and SSEA-1 (30), two markers of human primordial germ cells (PGC), which may indicate a possible affiliation, or at least convergence of developmental pathways. OCT-4 and SSEA-1 co-expression defines mouse ESC (but not human, which do not express SSEA-1). It is also noteworthy that only mouse ESC have thus far demonstrated the capacity to generate definitive adult hematopoietic lineages in vitro. To investigate the complex developmental relationship of blood-forming and endothelial precursors during human embryonic life, we and others have developed surrogate hESC and hiPSC in vitro differentiation systems, which tentatively mimic either YS (9, 10) or AGM hematopoiesis (7, 16, 17).
In the present work, we further improved this hEB-based hemato-endothelial differentiation protocol, and modified our developmental approach to obtain AGM-like mesodermal endothelial intermediates. We rigorously defined the minimal culture conditions to promote the emergence of mesodermal precursors from hEB, which were then guided toward endothelial commitment by adherent low-density culture in endothelial medium. The initial loss of pluripotency marker expression (TRA-1-60, TRA-1-81, SSEA4) was accompanied by the acquisition of a mesodermal phenotype (KDR, CD133, CD34, CD146, and CD31), and was highlighted by a peak of CD143/ACE expression at the 8th day of differentiation. The day 8 time point may be analogous to the in vivo emergence of pre-AGM Sp OCT4+ACE+ cells. The subsequent culture step in EGM-2 on fibronectin gave rise to multiple adherent stromal-vascular cells, distinctive both by morphology and marker expression of the acquisition of stromal, perivascular, or endothelial phenotypes. Of particular interest, adherent round, cobblestone RUNX1+ hematopoietic cell clusters emerged systematically from endothelial-shaped cell colonies, which directly released mass quantities of multipotent CD34+ CD41+CD45+CD143+ progenitors. This AGM-like hemogenic endothelium expressed RUNX1 and CD41, two markers indicative of definitive hematopoiesis potential in the mouse. However, the lack of acquisition of adult-type hemoglobins during erythropoiesis highlights the challenges yet to be overcome for generating adult-type human hematopoiesis. Considering the PGC-like phenotype of pre-AGM Sp hematopoietic ancestors (30) and the recently presumed post-implantation epiblast-like phenotype of hESC, one possibility is that human pluripotent stem cells may simply not be able per se to efficiently generate definitive adult type blood cells using contemporaneous hESC culture conditions. In contrast, murine pluripotent stem cells harbor an inner cell mass phenotype and naïve state, which may explain their propensity to generate mature blood lineages. Alternatively, blood-forming cells follow a journey through successive niches (colonization of fetal liver and bone marrow) to mature, receiving niche specific signals that must be taken into account for generating in vitro human adult-type blood.
A major factor impacting pluripotent stem cell differentiation models can be attributed to the hESC/hiPSC undifferentiated expansion stringency, as well as the robustness of the used differentiation system. We demonstrated that under rigorous culture conditions, fibroblast-hiPSC lines can preserve their pluripotency state similarly to hESC, without spontaneous differentiation, which is an essential prerequisite for efficient subsequent differentiation. Although several recent studies pointed out that hiPSC do not produce hemato-endothelial progeny in a manner that is quantitatively and qualitatively comparable to hESC (18, 33), we demonstrated here that using our robust and optimized system, fibroblast-iPSC can support simultaneous hematopoiesis and vasculogenesis, at levels comparable to an hESC line (H9; WA09) with robust hemato-endothelial capacity. However, technical variability among laboratories and the inherent efficiency of the multiple differentiation protocols (e.g., embryoid body vs. stromal co-culture) may still constrain the pluripotent state (e.g., MEF vs. feeder-free monolayer) of hESC and hiPSC.
Additionally, while some fibroblast-hiPSC differentiation limitations may be overcome using the optimized culture conditions we described herein, their reported lower differentiation efficiency may also be partially attributed to retention of somatic donor epigenetic memory (34). Indeed, while recent studies pointed out aberrant or incomplete epigenomic reprogramming in hiPSC (35–40), several reprogrammed cell types have been shown to maintain an enhanced differentiation potential toward the lineage they were derived from (41–45). Extended passages did not completely extinguish this limitation for hiPSC clones or allow them to recover an hESC-like epigenomic phenotype (42). Although residual epigenetic marks may alter the differentiation potential of iPSC lines, we show here that such limitations may be at least partially overcome by refining the expansion and differentiation method.
Recent innovative studies have similarly identified multipotent mesoderm vasculogenic precursors, (46, 47) using similar hESC/hiPSC–based methodologies. Although both studies showed the simultaneous emergence of mesenchymal/pericyte and endothelial lineages from human pluripotent cells, Vodyanik et al. demonstrated that both mesenchymal/pericyte (CD146+CD31−CD45−) and endothelial (CD31) progenies were derive from a common precursor, the “mesenchymoangioblast.” This bipotent progenitor exhibits a mesenchymal (CD105+CD73+CD90+) pericytic (CD140a+CD146+) non-endothelial nor hematopoietic (CD31−CD43−CD45−) phenotype. The mesenchymoangioblast may represent a common progenitor to mesenchymal and endothelial lineages, which might give rise to hemogenic endothelium from which in turn emerge hematopoietic progenitors (48, 49). Mesenchymogenic cells have been believed to arise from both the neural crest and the mesoderm (50). Noticeably, mesoangioblasts, a primitive CD34+ population of angiogenic and mesenchymal progenitors, emerges also from the mouse DA (51). Mesoangioblasts are believed to be the ancestors of postnatal multipotent pericytes, which have been shown to be themselves precursors of fetal and adult mesenchymal stem cells (MSC) (52). Primitive MSC have been proposed to similarly express CD34 based on in vitro models (53, 54), a phenotype shared in vivo by some rare adult bone marrow mesenchymal progenitors (55, 56) but also larger subsets of adipose pericytes (57). Further studies are required to investigate in vitro and in vivo the hierarchy of mesenchymal/pericytic, angiogenic and hematopoietic lineages, and kinetics of divergence, although our system also supports the simultaneous growth of pericyte/mesenchymal-like cells (data not shown).
Finally, the isolation of hemangioblasts from hiPSC may provide new opportunities for generating therapeutically relevant autologous transplantable HSC. However, the complex developmental signaling pathways and environments that exist in vivo will need to be recapitulated to further mature these hESC-derived primitive progenitors into adult blood cells before they can be practically used for treatment of hematological disorders.