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. 2010 Aug 24;18(12):2173–2181. doi: 10.1038/mt.2010.179

Nodal/Activin Signaling Predicts Human Pluripotent Stem Cell Lines Prone to Differentiate Toward the Hematopoietic Lineage

Veronica Ramos-Mejia 1, Gustavo J Melen 1, Laura Sanchez 1, Ivan Gutierrez-Aranda 1, Gertrudis Ligero 1, Jose L Cortes 1, Pedro J Real 1, Clara Bueno 1, Pablo Menendez 1
PMCID: PMC2997587  PMID: 20736931

Abstract

Lineage-specific differentiation potential varies among different human pluripotent stem cell (hPSC) lines, becoming therefore highly desirable to prospectively know which hPSC lines exhibit the highest differentiation potential for a certain lineage. We have compared the hematopoietic potential of 14 human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines. The emergence of hemogenic progenitors, primitive and mature blood cells, and colony-forming unit (CFU) potential was analyzed at different time points. Significant differences in the propensity to differentiate toward blood were observed among hPSCs: some hPSCs exhibited good blood differentiation potential, whereas others barely displayed blood-differentiation capacity. Correlation studies revealed that the CFU potential robustly correlates with hemogenic progenitors and primitive but not mature blood cells. Developmental progression of mesoendodermal and hematopoietic transcription factors expression revealed no correlation with either hematopoietic initiation or maturation efficiency. Microarray studies showed distinct gene expression profile between hPSCs with good versus poor hematopoietic potential. Although neuroectoderm-associated genes were downregulated in hPSCs prone to hematopoietic differentiation many members of the Nodal/Activin signaling were upregulated, suggesting that this signaling predicts those hPSC lines with good blood-differentiation potential. The association between Nodal/Activin signaling and the hematopoietic differentiation potential was confirmed using loss- and gain-of-function functional assays. Our data reinforce the value of prospective comparative studies aimed at determining the lineage-specific differentiation potential among different hPSCs and indicate that Nodal/Activin signaling seems to predict those hPSC lines prone to hematopoietic specification.

Introduction

The most common human cell-based therapy applied today is hematopoietic stem cell (HSC) transplantation. Currently, human bone marrow, mobilized peripheral blood, and umbilical cord blood represent the major sources of transplantable HSCs, but their availability for use is limited by both compatibility between donor and recipient and required quantity. Although increasing evidence suggests that somatic HSCs can be expanded to meet current needs, their in vivo potential is concomitantly compromised after ex vivo culture.1,2,3 In contrast, human pluripotent stem cells (hPSCs) [including human embryonic stem cells (hESCs) and induced PSCs (iPS)] possess indefinite proliferative capacity in vitro and have been shown to differentiate into the hematopoietic cell fate.4,5,6,7,8

Currently, many distinct hPSC lines have been internationally derived and efforts at new derivations are still ongoing. Of these, only a limited subset of lines has been characterized in detail. It is becoming increasingly evident that the spontaneous and lineage-specific differentiation potential varies among different hESC lines likely due to, at least in part, to the variety of methods used for hESC derivation, embryo quality, culture conditions used for maintenance and passage.9,10,11 The more recent development of iPS cell lines provides a new source of cells capable of self-renewal and differentiation into all types of somatic cells, including hematopoietic lineage.5,12,13,14 However, an in-depth characterization of the differentiation potential of iPS cells still remains to be undertaken. Public stem cell banks may provide an added value if they could not only work toward the adequate deposit and release of fully characterized hESC and iPS cell lines but also be capable of advising researchers on which hPSCs exhibit the best differentiation potential for a certain lineage in order to speed up their research.15,16 In this study, we aimed at characterizing the hematopoietic differentiation potential from a relatively suitable number of hPSC lines through the embryoid body (hEB) differentiation system.

Using the hEB model, human ESC-derived hematopoietic cells emerge from a subset of embryonic endothelium expressing CD31 (PECAM-1), Flk-1, and VE-Cadherin, but lacking CD45 (CD45−CD31+ hemogenic progenitors).3,7,8,17,18 These hemogenic precursors are exclusively responsible for hematopoietic potential of differentiated hESCs. Furthermore, despite hESC-derived hematopoietic cells show colony-forming unit (CFU) capacity and a phenotype similar to somatic hematopoietic cells, several independent studies have revealed that the in vivo generation of fully functional hESC-derived HSCs capable of engrafting immunodeficient recipients still remains a challenge.8,18,19,20,21,22 and will likely depend upon further understanding of intrinsic genetic regulation and extrinsic microenvironment cues.

To better elucidate the cellular kinetics of the stepwise differentiation of hESCs toward hematopoietic lineage, we compared the in vitro hematopoietic differentiation potential of multiple hESC/iPS cell lines by analyzing the different stages of hematopoietic development: hemangioblastic progenitors, primitive and mature hematopoietic cells as well as CFU potential. In this study, some hPSC lines exhibited robust hematopoietic differentiation while others barely differentiated into blood. Interestingly, correlation studies revealed that the CFU potential robustly correlated with hemogenic progenitors (CD31+CD45−) and primitive (CD45+CD34+) but not mature blood cells (CD45+CD34−).

At the molecular level, the capacity of mesoendodermal and hematopoietic-specific transcription factors to predict the blood-differentiation potential was evaluated at different time points in the 14 hPSC lines. Surprisingly, no correlation was found between the gene expression level and trend with either hematopoietic initiation or maturation efficiency. Microarray studies revealed that while neuroectoderm-associated genes were downregulated in those hPSCs prone to hematopoietic differentiation many members of the Nodal signaling pathway were upregulated, suggesting the Nodal signaling pathway as a potential indicator capable of predicting those hPSC lines with good blood-differentiation potential. Accordingly, the H9 hESC line with low blood (mesoderm-derived tissue) differentiation potential seemed more prone to differentiate toward neuroectodermal lineages.

Results

Hematopoietic differentiation potential largely varies among different hPSC lines

To determine the propensity of multiple hPSC lines to differentiate toward hematopoietic lineage, the differentiation was induced through the formation of hEBs in the presence of the ventral mesoderm inducer bone morphogenetic protein-4 and hematopoietic cytokines. For this prospective comparison, we analyzed the emergence of hemogenic progenitors (CD31+CD45−), primitive (CD34+CD45+) and mature (CD34−CD45+) blood cells as well as CFU potential at different time points during differentiating hEBs (day 10, 15, and 22) for all 13 hESC lines and one iPS cell line (Figure 1a,b). In our EB differentiation system, the blood differentiation efficiency largely varied among the distinct hPSC lines. Several hESC lines including SHEF2, H13C, and HS181 showed an extremely poor hematopoietic initiation and maturation. Others, such as SHEF3, AND3, ES2, VAL3, VAL5, H9, and the iPS cell line MSUH001, displayed average potential of initial specification into hemogenic progenitors (CD31+CD45−), but also failed to further mature into CD45+ hematopoietic cells or to give rise to CFUs (Figure 1c–f). In contrast, some hPSCs (AND1, AND2, H1, and SHEF1) displayed good hematopoietic emergence of CD31+CD45− hemogenic progenitors, able to mature and give rise to primitive (CD34+CD45+) and mature (CD45+CD34−) blood cells. As expected, these hPSC lines with high hematopoietic differentiation potential displayed the highest potential to form clonogenic colonies (CFU) in semisolid assays. These data highlight the importance of performing prospective comparative studies aimed at determining the propensity of distinct hPSC lines to differentiate toward certain lineage.

Figure 1.

Figure 1

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Comparative study revealing that hematopoietic differentiation potential varies among different human pluripotent stem cell (hPSC) lines. (a) Schematic of the hematopoietic differentiation protocol from human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines and end point analyses. (b) Representative flow cytometry dot plots displaying how hemogenic progenitors (CD45−CD31+) and hematopoietic cells (CD45+) are identified and analyzed at day 10, 15, and 22 of embryoid body (EB) development. Differences between 13 hESC lines and one iPSC line regarding their propensity to differentiate into hemogenic progenitors (c) (CD45−CD31+), (d) primitive blood cells (CD45+CD34+), (e) mature blood cells (CD45+CD34−) and (f) differences in their colony-forming unit potential and type. Analysis was consistently performed at day 10, 15, and 22 of EB development. CFUs were plated from day 15 EBs. The horizontal dotted red lines indicate the mean value for all lines studied. All differentiation experiments were independently done at least three times. CFU, colony-forming unit.

CFU potential is closely associated to early but not late hematopoietic cells

In the adult hematopoiesis, the CFU assay functionally identifies hematopoietic progenitors displaying bona fide in vitro clonogenic potential. However, during human embryonic hematopoietic development, the embryonic hematopoietic population responsible for CFU potential still remains to be fully identified. Based on the in vitro differentiation of 14 different hPSC lines, we therefore wondered which embryonic hematopoietic cell population is enriched in CFU capacity. Consistent with our phenotypic analysis, the CFU potential varied among the different hPSC lines (Figure 1f). AND1, AND2, H1, SHEF1, and SHEF3 lines displayed CFU potential with multilineage hematopoietic colony types including erythyroid, macrophage, granulocyte, and granulocyte–macrophage progenitors (data not shown). In contrast, H9, AND3, H13C, HS181, SHEF2, VAL3, VAL5, ES2, and the iPS cell line MSHU001 barely exhibited CFU capacity (Figure 1f).

Interestingly, correlation studies between phenotype and CFU potential revealed that the CFU capacity robustly (R = 0.94; P < 0.0001) correlates with the hemogenic progenitors (CD31+CD45−) (Figure 2a) and the primitive (CD45+CD34+) (Figure 2b) but not mature blood cells (CD45+CD34−) (Figure 2c), confirming that early rather than late hESC-derived hematopoietic cell populations are enriched in and responsible for CFU potential.

Figure 2.

Figure 2

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Correlation between the total number of CFU and the percentage of embryonic hematopoietic cell subsets. (a) Hemogenic progenitors (CD45−CD31+), (b) primitive blood cells (CD45+CD34+), and (c) mature blood cells (CD45+CD34−). All CFU experiments were independently done at least two times. CFU, colony-forming unit.

Expression of hematopoietic transcription factors does not predict the hematopoietic differentiation outcome from hPSCs

To determine whether the observed cellular differences in the hematopoietic differentiation potential from distinct hPSC lines are reflected and may be predicted at the molecular level, we first evaluated the gene expression kinetics of mesoendoderm and hematopoietic transcription factors in differentiating hEBs. By real-time reverse transcriptase-PCR, the expression of Brachyury, MIXL1, SCL, HOXA9, RUNX1, GATA1, and PU.1 was analyzed at days 0, 1, 4, 7, 10, and 15 in differentiating hEBs. As shown in Figure 3, the expression of early mesendodermal markers such as Brachyury and MIXL1 emerged as early as day 1, peaked around day 4, and then switched off rapidly to become almost undetectable around day 10 of hEB differentiation. In contrast, early hematopoietic transcription factors such as SCL or HOXA9 were almost undetectable at the earliest stages of hEBs differentiation and their expression was significantly upregulated from day 4 onward (Figure 3). Finally, late hematopoietic transcription factors such as GATA1 and PU.1 were barely expressed before day 7 of hEB differentiation but their expression was robustly induced from day 7 of hEBs differentiation (Figure 3). Intriguingly, all hPSC lines exhibited similar gene expression trends while displaying different hematopoietic differentiation outcome measured by immunophenotype of bona fide blood cells (Figure 1), suggesting that the expression of neither early nor late hematopoietic transcription factors seem to identify/predict those hPSC lines with greater hematopoietic potential.

Figure 3.

Figure 3

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Relative expression trends of hematopoietic transcription factors along hEB development. Gene expression kinetics of the mesendodermal (Brachyury and MIXL1) and hematopoietic transcription factors (SCL, HOXA9, GATA1 and PU.1) during a 15-day period of differentiating hEBs from human pluripotent stem cells displaying different hematopoietic potentials. Expression or early (SCL and HOXA9) and late (GATA1 and PU.1) hematopoietic transcription factors does not identify the human embryonic stem cell lines with good hematopoietic differentiation output analyzed by flow cytometry. hEB, human embryonic body.

Because the expression trends of all the genes analyzed could not account for the observed differences in the hematopoietic differentiation propensity among the distinct hPSC lines and also due to the large variations in the levels of gene expression, we correlated the mRNA expression levels with the hematopoietic differentiation potential of the 14 hPSC lines. The expression level of each independent gene at a given time point (hEBs day 7, 10, and 15) was correlated with the percentage of each of the three embryonic hematopoietic cell populations (hemogenic progenitors, primitive and mature blood cells) analyzed at days 10, 15, and 22 of differentiating hEBs. Unexpectedly, in our cohort of 14 hPSCs none of the analyzed genes showed correlation to the embryonic hematopoietic cell populations analyzed by flow cytometry (Supplementary Figures S1–S5). Taken together, all these findings support that expression of Brachyury, MIXL1, RUNX1, SCL, HOXA9, GATA1, or PU.1 is not sufficient to predict the blood developmental potential of hPSC lines induced to differentiate in vitro.

Nodal/Activin signaling pathway is upregulated in hPSCs with high propensity to hematopoietic differentiation

In order to identify patterns of gene expression that could help to prospectively elucidate the hematopoietic potential from distinct hPSC lines, we carried out gene expression profiling on six different hESC lines grouped according to their ability to differentiate toward hematopoietic lineage: SHEF1, AND1, and H1 were considered as good-blood-differentiating lines (high CD45 expression and high CFU potential), whereas H9, HS181, and VAL3 were selected as poor-blood-differentiating lines (low CD45 expression and low CFU potential) (Figure 4a). In the microarray analysis (see Material and Methods for details), we identified 91 genes whose expression was upregulated in hESCs with poor hematopoietic potential. Interestingly, many genes involved in neural development and function were enriched in this population. In contrast, many key components of the Nodal signaling pathway (NODAL, LEFTY1, LEFTY2, and CERBERUS) were at least fourfold upregulated in good-blood-differentiating hESCs (Figure 4a). Microarray analyses were validated by real-time reverse transcriptase-PCR in all six hESC lines confirming mRNA expression levels of NODAL, LEFTY1, and LEFTY2 (Figure 4b). In addition, high levels of LEFTY2 protein were confirmed by immunohistochemistry staining in H1 but not in HS181 hESC line (Figure 4c). Furthermore, many other components of the Nodal signaling pathway (CRIPTO1, CRIPTO3, ACVR24, and FOXH1) were also upregulated 1.5- to 2-fold in the hESCs with good hematopoietic potential, suggesting that Nodal/Activin signaling pathway may segregate hESC lines with good versus poor hematopoietic developmental potential (Figure 4d). All together, these data suggest that Nodal/Activin pathway might be active early during hEB differentiation to induce mesendoderm specification23,24 and might be a potential indicator to segregate hESC lines according to their hematopoietic differentiation propensity.

Figure 4.

Figure 4

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Differential gene expression profile between human embryonic stem cell (hESC) lines with good versus poor blood differentiation potential. (a) Heatmap diagram summarizing microarray gene expression changes in hESC lines displaying good versus poor blood differentiation potential (left panel). Of note, genes involved in neural differentiation and function are upregulated in hESCs with poor hematopoietic potential while many key genes belonging to the Nodal/Activin pathway are highly upregulated in hESCs with hematopoietic differentiation propensity. Nodal genes are highlighted in red and those genes associated to neural development are shown in blue. The right panel shows in more detail the gene expression changes for each of the six hESC lines studied. (b) Real-time reverse transcriptase-PCR validating the microarray data displaying high expression levels of Nodal, Lefty1, and Lefty 2 in AND1, H1, and SHEF1 (good blood differentiation potential) as compared with H9, HS181, and VAL3 (poor-blood-differentiation potential). (c) Immunocytochemistry confirming expression of the Lefty2 protein in H1 but not in HS181. (d) Scheme of the Nodal/Activin signaling pathway highlighting those members whose expression was four- to ninefold upregulated (red), 1.5- to 2-fold upregulated (blue), or that showed no regulation (white) in hESCs displaying robust hematopoietic differentiation potential. (e) Flow cytometry expression of the early neuroectodermal marker A2B5 in H9 and AND1. Please note that the ability of hESC lines to differentiate toward ectodermal (A2B5+ cells) versus mesodermal (blood) lineages seems to be opposite.

Nodal pathway activity is essential for mesoendoderm specification and its inhibition promotes specification into neuroectoderm.23,24,25,26,27 Because the microarray studies revealed that many genes associated with neural development and function are downregulated in those hESC lines showing hematopoietic differentiation propensity, we hypothesized that hESC lines prone to differentiate toward blood (mesoderm-derived tissue) may present a reduced capacity to differentiate toward neuroectodermal lineage and vice versa. To test this, two hESC lines displaying distinct hematopoietic differentiation potential, AND1(good) and H9 (poor), were induced to differentiate into early neuroectodermal progenitors through the hEB method in neural media.28 As expected, the proportion of hESC-derived cells expressing the neural embryonic antigen A2B5 was almost threefold higher in H9 (51%) as compared to AND1 (21%) (Figure 4e), suggesting that hESC lines with higher ability to differentiate toward mesodermal lineages might have lower propensity to develop into ectodermal lineages.

Inhibition of Nodal/Activin signaling abrogates the hematopoietic differentiation of hPSCs whereas its activation augments the hematopoietic differentiation potential

Next we treated AND1 and H1 hESC cultures just for 4 days with 10 µmol/l of SB-431542, a potent and selective chemical inhibitor of ALK4, ALK5, and ALK7 receptors inducing shutdown of Nodal/Activin signaling through SMAD-2/3. Then, EBs were formed and allowed to differentiate in the presence of bone morphogenetic protein-4, stem cell factor, FMS-like tyrosine kinase-3, interleukin-3 (IL-3), IL-6, and granulocyte colony–stimulating factor. As expected, inhibition of Nodal/Activin signaling during undifferentiated hESC culture consistently resulted in up to 14-fold reduction in the proportion of CD45+ hematopoietic cells (Figure 5a,b). Conversely, a 3.5-fold increase in its hematopoietic differentiation capacity was achieved upon the treatment with exogenous Activin-A of HS181, an hESC line initially reported to be unable to differentiate into blood (Figure 5c,d). These loss- and gain-of function assays support the initial direct correlation between active Nodal/Activin signaling and hematopoietic differentiation potential, confirming that Nodal/Activin signaling reliably predict those hPSC lines with good blood-differentiation potential.

Figure 5.

Figure 5

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Loss- and gain-of-function assays revealed that inhibition of Nodal/Activin signaling during undifferentiated human embryonic stem cell (hESC) culture abrogates hematopoietic specification whereas its activation augments the hematopoietic differentiation potential. (a) Inhibition of Nodal/Activin signaling during undifferentiated hESC culture (AND1 and H1) by treatment for 4 days with 10 µmol/l of SB-431542, a potent and selective chemical inhibitor of Nodal/Activin signaling, resulted in up to 14-fold reduction of mature hematopoietic cells. (b) Flow cytometry dot plots displaying how these hematopoietic cell subsets are identified and analyzed in the presence and absence of SB-451342 treatment. (c) Four-day exogenous addition of 100 ng/ml of Activin-A to HS181 undifferentiated cultures augmented 3.5-fold times the differentiation into mature hematopoietic cells. (d) Flow cytometry dot plots depicting the effect of Activin-A treatment in the hematopoietic differentiation potential of HS181 human embryonic stem cell line.

Discussion

To the best of our knowledge, very few studies based on a limited number of hPSC lines have been prospectively conducted aimed at characterizing and comparing the lineage-specific differentiation potential among multiple hPSC lines.5,29 Here, we compared the hematopoietic differentiation capacity of 14 hPSC lines and found a large variability in their capacity to develop toward blood lineage. This study revealed that the hPSC lines evaluated could be subgrouped in three groups regarding their hematopoietic specification potential. Some hPSC lines such as HS181, H13C, and SHEF2 barely differentiate toward hematopoietic lineage giving rise to neither hemogenic progenitors nor hematopoietic cells. Other hPSC lines including AND3, H9, SHEF3, VAL3, VAL5, ES2, and the iPS cell line MSUH001 were capable of generating hemogenic progenitors but failed to produce mature blood cells. In contrast, some hPSC lines such as AND1, AND2, H1, and SHEF1 displayed high propensity to give rise to hemogenic progenitors coupled to further differentiation/maturation into CD45+ and CD45+CD34+ hematopoietic cells. Consistent with our phenotypic analysis, the observed differences in hematopoietic differentiation potential among the different hPSC lines were functionally confirmed by in vitro CFU assays. Despite hPSC-derived hematopoietic cells have been proven in vitro to be functionally equivalent to adult CD34+ hematopoietic progenitors in terms of clonogenic CFU capacity in methylcellulose assays, it remains to be determined the cell population responsible for this CFU potential during embryonic hematopoietic development in humans. Correlation analyses robustly indicated that early hPSC-derived hematopoietic cell subsets including hemogenic progenitors (CD31+CD45−) and primitive blood cells (CD45+CD34+), rather than late hPSC derived CD45+ cells are enriched in CFU potential, being therefore responsible for the developmental formation of clonogenic hematopoietic progenitors. All the hPSCs challenged in this study were maintained for over a year in a feeder-free system in conditioned media supplemented with basic fibroblast growth factor, using the same batch of serum and identical EB differentiation protocol and cytokines. In addition, the type of feeders initially used for hESC derivation did not influence the differentiation potential, further supporting that the differences observed between lines seem mainly intrinsic.

We next performed a detailed time-course quantitative gene expression analysis in differentiating hEBs for mesoendodermal (Brachyury and MIXL1), early (RUNX1, HOXA9, and SCL), and late (GATA1 and PU.1) hematopoietic transcription factors. Despite the high variability in gene expression among the 14 hPSC lines, overall, all hPSC lines exhibited a similar trend in gene expression: prompt regulation of mesoendodermal genes (Brachyury and MIXL1) which switch off early in development followed by the emergence of early hematopoietic transcription factors such as SCL and HOXA9 indicating hematopoietic commitment and the eventual upregulation of late hematopoietic transcription factors including GATA1 and PU.1 indicative of the presence of mature CD45+ cells within the differentiating hEBs. These immunophenotype and gene expression data reveal the stepwise conversion of mesendoderm into hemogenic progenitors and their further maturation into CD45+ hematopoietic cells.

Surprisingly, the developmental progression of mesoendodermal (Brachyury and MIXL1) and hematopoietic transcription factors (SCL, RUNX1, HOXA9, PU.1, and GATA1) expression revealed no correlation with either hematopoietic initiation or maturation efficiency. The different expression of these genes could not account for the observed differences in the blood differentiation predisposition among multiple hPSC lines, suggesting that the expression of hematopoietic and mesendodermal transcription factors does not seem to be a reliable indicator to predict the hematopoietic differentiation outcome from hPSCs. In order to identify patterns of gene expression that could help to prospectively elucidate the hematopoietic potential from distinct hPSC lines, we undertook for the first time microarray analysis–based gene expression profiling of six different hESC lines grouped according to their ability to differentiate toward hematopoietic lineage: SHEF1, AND1, and H1 were considered as good-blood-differentiating lines whereas H9, HS181, and VAL3 were selected as poor-blood-differentiating lines (Figure 3a). A total of 198 genes were found to be differentially expressed. Interestingly, although many neuroectoderm-related genes were found to be upregulated in those hPSCs with poor hematopoietic differentiation potential, key components of the Nodal signaling pathway (NODAL, LEFTY1, LEFTY2, CERBERUS, CRIPTO1, CRIPTO2, ACVR24, and FOXH1) were upregulated in hESC lines displaying high propensity to differentiate toward blood lineage. Nodal signaling pathway plays an important role in the maintenance of pluripotency and in early developmental cell-fate decisions by inducing mesendoderm specification while inhibiting development along the neuroectoderm default pathway in hPSCs.23,24,30 Our data therefore suggest that upregulation of the Nodal signaling pathway may represent a reliable indicator to segregate hESC lines with good or poor hematopoietic developmental potential (Figure 3a–d). Loss- and gain-of-function assays confirmed the association between Nodal/Activin signaling and hematopoietic differentiation potential (Figure 5). These data were further supported by the differential ability of H9 and AND1 lines to differentiate toward mesodermal (blood) versus ectodermal (A2B5+ cells) lineages.

Human PSCs represent an unlimited source of cells capable of developing into a wide variety of tissues, providing a powerful model system to better understand in depth the cellular and molecular basis of human hematopoietic emergence and maturation. They are envisioned to become a potentially powerful tool for modeling different aspects of human disease that cannot otherwise be addressed by patient sample analysis or mouse models. Regarding the biology of hematopoietic malignancies, there are different types of childhood acute leukemias wherein clinically significant manifestations arise in utero. The fact that cellular transformation manifests as a blockage or altered cell differentiation suggests that the in vitro and in vivo hPSCs differentiation could become a promising human-based system for studying the emergence of early transformation events by characterizing the mechanisms that drive cell transformation rather than normal lineage specification.31,32 In addition, the generation of HSCs from hPSCs has been suggested to be a viable alternative and source of transplantable cells. However, even though it has been shown that hESCs and iPS cell lines exhibit a common expression profile for a specific set of pluripotency markers, the hematopoietic differentiation potential of the majority of existing hESCs and iPS cell lines remains unknown, preventing at least in part the acceleration of research in the field of human developmental hematopoiesis and its potential future translation into blood disease modeling, drug screening, and cell therapies. Based on the large variation of the hematopoietic differentiation potential among 14 hPSC lines described here, stem cell banks should offer a platform for selecting those hPSCs that exhibit the highest blood differentiation potential. Differentiation variability among distinct hPSC lines may be due to distinct genetic backgrounds, derivation processes, reprogramming strategies, culture conditions that may endow hPSC lines with different but specific cellular, genetic, and epigenetic signatures. This study stresses the importance of establishing robust intra- and interlaboratory criteria and requirements to assess the blood differentiation potential among distinct hPSC lines before any reliable use in basic stem cell research, disease modeling, and potential future translation approaches into drug screening and cell therapy.

Material and Methods

Human ESC and iPS cell culture. Thirteen stable well-characterized hESC lines (AND1, AND2, AND3, H9, H1, H13C, HS181, ES2, SHEF1, SHEF2, SHEF3, VAL3, and VAL5) and one iPS cell (MSUH001)33,34 were maintained undifferentiated in a feeder-free culture as previously described.4,17,35,36,37,38,39 Briefly, hESCs were cultured in Matrigel (BD Biosciences, Bedford, MA)-coated T25 flasks in either human foreskin fibroblast or mesenchymal stromal cells–conditioned medium supplemented with 8 ng/ml basic fibroblast growth factor (Miltenyi, Bergisch Gladbach, Germany).39 Media was changed daily, and the cells were split weekly by dissociation with 200 U/ml of collagenase IV (Invitrogen, Edinburgh, Scotland). Human ESC and iPS cell cultures were visualized daily by phase-contrast microscopy. Approval from the Spanish National Embryo Ethical Committee was obtained to work with hESCs and iPS cells.

Human ESC and iPS cell differentiation toward hematopoietic lineage. Undifferentiated hESCs/iPS cells at confluence were treated with collagenase IV and scraped off of the Matrigel attachments. They were then transferred to low-attachment plates (Corning, Corning, NY) to allow hEB formation by overnight incubation in differentiation medium consisting of KO-Dulbecco's modified Eagle's medium supplemented with 20% non-heat-inactivated fetal bovine serum, 1% nonessential amino acids, 1 mmol/l ℓ-glutamine, and 0.1 mmol/l β-mercaptoethanol. The medium was changed the next day (day 1) with the same differentiation medium supplemented with hematopoietic cytokines (300 ng/ml stem cell factor, 300 ng/ml FMS-like tyrosine kinase-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, and 50 ng/ml granulocyte colony–stimulating factor and 25 ng/ml bone morphogenetic protein-4).4,7,17,18,36,37 Human EBs were dissociated using collagenase B (Roche Diagnostic, Basel, Schweiz, Switzerland) for 2 hours at 37 °C followed by 10 minutes incubation at 37 °C with enzyme-free Cell Dissociation Buffer (Invitrogen) at day 10, 15, and 22 of development (Figure 1a). Single-cell suspension was obtained by gentle pipetting and passage through a 70-µm cell strainer (Becton Dickinson, San Jose, CA) and the dissociated cells were stained with anti-CD34-fluorescein isothiocyanate, anti-CD31-phycoerythrin, and anti-CD45-allophycocyanin (all from Miltenyi) antibodies and 7-actinomycin D. Live cells identified by 7-actinomycin D exclusion were analyzed using a FACSCanto II flow cytometer equipped with FACSDiva software (Becton Dickinson). Hemogenic progenitors with hemangioblastic properties were identified as CD31+CD45−.7,17,18,36 Immature and mature blood cells were identified as CD45+CD34+ and CD45+CD34−, respectively (Figure 1b).7,17,18,36

In order to confirm the positive correlation between the Nodal/Activin signaling and the hematopoietic differentiation potential, the AND1 and H1 hESC lines were treated for 4 days with 10 µmol/l of SB-431542 (Sigma, St Louis, MO), a potent and selective chemical inhibitor of Nodal/Activin-A signaling or ethanol as vehicle. Alternatively, the HS181 hESC line was treated for 4 days with 100 ng/ml of Activin-A (R&D, Minneapolis, MN) or phosphate-buffered saline (PBS) as a control. Then, EBs were formed and allowed to differentiate in the presence of bone morphogenetic protein-4 and hematopoietic cytokines for 22 days. On days 10, 15, and 22 the EBs were dissociated and the proportion of hemogenic progenitors and mature blood cells were assayed by flow cytometry (Figure 5).

Early neural differentiation. For neural differentiation, the protocol was slightly modified from that described by Pankratz et al.28 Briefly, hPSC lines were grown in suspension as hEBs in hESC growth medium for 4 days. The hEBs were then cultured in neural medium composed by Dulbecco's modified Eagle's medium/F12, nonessential amino acids, 2 µg/ml heparin, and the neural cell supplement N2 (Gibco, Billings, MT) for 3 additional days. Early neural differentiation was evaluated at day 8 of culture by dissociating and staining hEBs single cells with the neural embryonic antigen A2B5 using a mouse antihuman A2B5 (1:25 dilution; Miltenyi) or the corresponding isotype control. The secondary antibody was a fluorescein isothiocyanate-conjugated goat anti-mouse.

CFU assays. Human clonogenic progenitor assays were performed by plating 10,000 cells from day 15 hEBs into methylcellulose H4230 (Stem Cell Technologies, Vancouver, British Columbia, Canada) supplemented with recombinant human growth factors: 50 ng/ml stem cell factor, 3 U/ml erythropoietin, 10 ng/ml granulocyte–macrophage colony–stimulating factor, and 10 ng/ml IL-3. Cells were incubated at 37 °C in a 5% CO2-humidified atmosphere and colonies counted at day 14 of CFU assay using standard morphological criteria.17,36,37,40

Histological analysis. Human ESC colonies were cultured in chamber slides. Cells were fixed in 4% of paraformaldehyde for 20 minutes followed by 30 minutes incubation in 10% normal goat serum in PBS. Colonies were incubated with rabbit anti-hLefty 2 (1:1,000 dilution in PBS; Abcam) overnight at room temperature. A tetramethyl rhodamine isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (1:100 dilution in PBS) was used for 1 hour at room temperature (Jackson Laboratories, Bar Harbor, ME). The slides were mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). As a negative control, the primary antibodies were replaced by PBS.

Real-time reverse transcriptase-PCR. Total RNA extraction and reverse transcriptase-PCR reactions were done as previously described.39,41 Briefly, total RNA was extracted from hEBs using Total RNA Purification Kit (Norgen Biotek, Thorold, Canada). First-strand complementary DNA synthesis was performed (First-Strand cDNA Synthesis Kit; Amersham, Pittsburgh, PA), and the resulting complementary DNA was analyzed for differential gene expression by using Platinum SYBR Green qPCR Super Mix-UDG (Invitrogen) on an Mx3005P Q-PCR System (Stratagene, La Jolla, CA). Real-time PCR conditions were as follows: 10 minutes at 95 °C, 40 cycles of 30 seconds at 95 °C followed by 60 seconds at 60 °C and 30 seconds at 72 °C. GAPDH was used as housekeeping gene. Primer sequences were as follows: hBrachyury: 5′-ATGAGCCTCGAATCCACATAGT-3′ and 5′-TCCTCGTTCTGATAAGCAGTCA-3′ hMixL1: 5′-GGATCCAGGTA TGGTTCCAG-3′ and 5′-GGAGCACAGTGGTTGAGGAT-3′ hSCL: 5′-GGATGCCTTCCCTATGTTCA-3′ and 5′-GGTGTGGGGACCATCAGT AA-3′ hHoxA9: 5′-GATCCCAATAACCCAGCAG-3′ and 5′-CCCTGGT GAGGTACATGTTG-3′ hRunX1: 5′-CCGAGAACCTCGAAGACATC-3′ and 5′-GCTGACCCTCATGGCTGT-3′ hPU.1: 5′-GTGCCCTATGACAC GGATCT-3′ and 5′-CCCAGTAATGGTCGCTATGG-3′ GATA1: 5′-TCA CTCCCTGTCCCCAATAG-3′ and 5′-GGAGAGTTCCACGAAGCTT G-3′ hNodal: 5′-GAGGAGTTTCATCCGACCAA-3′ and 5′-GCACTCT GCCATTATCCACA-3′ hLefty1: 5′-GGACCTTGGGGACTATGGAG-3′ and 5′-AGTTCTCGGCCCACTTCATC-3′ hLefty2: 5′-TGGACCTCAG GGACTATGGA-3′ and 5′-CAGTTCTTGGCCCACTTCAT-3′ and hGAPDH: 5′-TGCACCACCAACTGCTTAGC-3′ and 5′-GGCATGGACTGTGGT CATGAG-3′.

Microarray experiments and data analysis. Human ESC samples were collected during the exponential cell growth phase and stabilized in RNA later (Ambion, Austin, TX) solution until RNA extraction. RNA was isolated using Agilent Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA) and its quality checked in the Agilent 2100 Bioanalyzer platform (Agilent Technologies). A volume of 500 ng of each total RNA sample was labeled with Cy3 using the Quick-Amp Labelling kit and hybridized with the Gene Expression Hybridization kit to a Whole Human Genome Oligo Microarray (Agilent Technologies) following the manufacturer's instructions. Each sample was labeled and hybridized as independent duplicates. A gene was considered differentially expressed if it was at least twofold up- or downregulated as compared to H9 hESC line that was used as the baseline and a t-test was performed to better judge the significance of the observed regulated genes. Only those genes displaying a P value <0.05 were included in subsequent analyses. Clustering was made using the Self-Organizing Map Algorithm and arbitrary grouping the distinct human ESC lines according to their ability to differentiate into hematopoiesis (good versus poor). Genes whose expression fell within two out of four clusters (those with opposite behavior) were taken to generate the combined tree by the Hierarchical Algorithm and clustered using Pearson Centered and Ward's as the linkage rule.35,42 Microarray data have been deposited and are available at Gene Expression Omnibus accession number GSE23091 (http://www.ncbi.nlm.nih.gov/geo/).

Statistical analysis. All data are expressed as mean ± SEM. Statistical comparisons were performed with a paired Student's t-test. Statistical significance was defined as a P value <0.05. To investigate the correlation between quantitative variables, we used the Pearson correlation and the multiple regression test (Statview 4.01 software program; Abacus Concepts, Berkeley, CA). In all cases, differences were considered to be statistically significant when the P values were <0.05.43,44,45,46

SUPPLEMENTARY MATERIAL Figure S1. Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 7 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 10 of EB development. Figure S2. Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 10 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 10 of EB development. Figure S3. Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 10 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 15 of EB development. Figure S4. Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 15 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 15 of EB development. Figure S5. Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 15 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 22 of EB development.

Acknowledgments

This work was funded by the CSJA (0029/2006 and 0030/2006 to P.M.) and CICE (P08-CTS-3678 to P.M.) de la Junta de Andalucía, The Fondo de Investigaciones Sanitarias to P.M. (PI070026), C.B. (CP07/00059), and P.J.R. (CP09/0063). The MICINN to P.M. (PLE-2009-0111) and The Marie Curie IIF to V.R.-M. (PIIF-GA-2009-236430). We are indebted to all members of the BACM for their support and technical assistance and Armando Blanco (University of Granada) for his assistance with the statistics. V.R.-M. designed and performed experiments, analyzed, and interpreted the data and wrote the article. L.S., G.L., G.M., I.G.-A., J.L.C., P.J.R., and C.B. performed research. P.M. analyzed the data and interpreted the results, supervised the study, and wrote the article. The authors reported no potential conflicts of interest.

Supplementary Material

Figure S1.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 7 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 10 of EB development.

Click here for additional data file. (841.7KB, tiff)
Figure S2.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 10 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 10 of EB development.

Click here for additional data file. (864KB, tiff)
Figure S3.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 10 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 15 of EB development.

Click here for additional data file. (885.9KB, tiff)
Figure S4.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 15 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 15 of EB development.

Click here for additional data file. (914.8KB, tiff)
Figure S5.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 15 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 22 of EB development.

Click here for additional data file. (886.2KB, tiff)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 7 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 10 of EB development.

Click here for additional data file. (841.7KB, tiff)
Figure S2.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 10 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 10 of EB development.

Click here for additional data file. (864KB, tiff)
Figure S3.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 10 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 15 of EB development.

Click here for additional data file. (885.9KB, tiff)
Figure S4.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 15 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 15 of EB development.

Click here for additional data file. (914.8KB, tiff)
Figure S5.

Lack of correlation between the gene expression levels of Brachyury, MixL1, SCL, HoxA9, RunX1, PU.1 and Gata1 at day 15 of EB development and the emergence of hemogenic progenitors (CD45-CD31+), primitive blood cells (CD45+CD34+) or mature blood cells (CD45+CD34-) at day 22 of EB development.

Click here for additional data file. (886.2KB, tiff)

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