Mechanisms that govern hematopoietic lineage specification, as opposed to the expansion of committed hematopoietic progenitors, from human pluripotent stem cells (hPSCs) have yet to be fully defined. Here, we show that within the family of genes called inhibitors of differentiation (ID), ID1 and ID3 negatively regulate the transition from lineage-specified hemogenic cells to committed hematopoietic progenitors during hematopoiesis of both human embryonic stem cells (hESCs) and human induced pluripotent stem cell (hiPSCs). Upon hematopoietic induction of hPSCs, levels of ID1 and ID3 transcripts rapidly increase, peaking at the stage of hemogenic precursor emergence, and then exclusively decrease during subsequent hematopoietic commitment. Suppression of ID1 and ID3 expression in hemogenic precursors using specific small interfering RNAs augments differentiation into committed hematopoietic progenitors, with dual suppression of ID1 and ID3 further increasing hematopoietic induction compared with upon knockdown of each gene alone. This inhibitory role of ID1 and ID3 directly affects hemogenic precursors and is not dependent on non-hemogenic cells of other lineages within developing human embryoid bodies from hESCs or hiPSCs. Our study uniquely identifies ID1 and ID3 as negative regulators of the hPSC–hematopoietic transition from a hemogenic to a committed hematopoietic fate, and demonstrates that this is conserved between hESCs and hiPSCs.
Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the unique capacity to give rise to all cell types of the body, including hematopoietic cells (Murry and Keller, 2008). Thus, hPSCs have been suggested as an alternative source of cells for hematopoietic cell-based therapies, instead of adult sources such as umbilical cord blood (UCB), bone marrow and mobilized peripheral blood, for in vivo reconstitution of the hematopoietic system (Bhatia, 2007). The recently discovered hiPSCs, possessing pluripotent capacity, have potential advantages over hESCs, owing to their immunological competence for use in an autologous transplant setting (Takahashi et al., 2007; Yu et al., 2007). However, specifying hematopoietic differentiation from either hESCs or hiPSCs remains the major barrier in the therapeutical use of hematopoietic cells. Although several factors that influence both the emergence of hemogenic cells from hPSCs and the subsequent hematopoietic development have been identified (Chadwick et al., 2003; Vijayaragavan et al., 2009; Wang et al., 2004), specific pathways and genes responsible for inhibiting, in contrast with inducing, hematopoietic development, have yet to be identified and investigated.
Hematopoietic development is a complex and highly coordinated process that is controlled by spatial and temporal expression of a plethora of genes involved in specification and differentiation of blood cells (Orkin and Zon, 2008). Inhibitor of differentiation (ID) genes are helix-loop-helix (HLH) transcription factors and are ubiquitously expressed in many tissues (Benezra et al., 1990; Yokota, 2001). Mechanistically, ID proteins act as dominant-negative regulators of transcription factors and form complexes with ubiquitous E proteins (E2A, HEB and E2-2), preventing E protein homodimerization or heterodimerization with tissue-restricted basic HLH proteins, leading to cell cycle promotion and disparate regulation of terminal differentiation in various cell types (Jen et al., 1992; Norton et al., 1998). ID genes are constitutively expressed in murine and human hematopoietic cells (Buitenhuis et al., 2005; Cooper et al., 1997; Jankovic et al., 2007; Kreider et al., 1992; Leeanansaksiri and Dechsukhum, 2006; Lister et al., 1995; Martin et al., 2008). Previous gain-of-function studies have revealed that ectopic expression of ID1 enhanced myeloid and neutrophil differentiation in mouse hematopoietic stem cells (HSCs) and CD34+ cells from human UCB, respectively (Buitenhuis et al., 2005; Kreider et al., 1992). Moreover, downregulation of ID1 in mouse erythroleukemia cells inhibited terminal erythroid differentiation (Lister et al., 1995). These studies demonstrated that early hematopoietic commitment and differentiation towards myeloid and erythroid lineages could be regulated by ID proteins. ID genes are known to be direct targets of bone morphogenetic protein 4 (BMP4). BMP4 has been shown to regulate mesoderm patterning and, hence, blood development in mammals (Hollnagel et al., 1999; Miyazono and Miyazawa, 2002; Sadlon et al., 2004), and is known to promote hematopoietic differentiation of hESCs. Most interestingly, BMP4, independently of hematopoietic cytokines, supports the maintenance of primitive progenitors with enhanced self-renewal capacity in differentiating hESCs (Chadwick et al., 2003). Collectively these studies suggest ID genes might be necessary for hematopoietic development, underscoring their potential importance in hPSC hematopoiesis that has not been examined to date.
Here, we reveal a distinct temporal expression of ID1 and ID3 during hematopoietic specification compared with hematopoietic commitment, and demonstrate that ID1 and ID3 act as direct negative regulators of the hemogenic precursor transition to the hematopoietic lineage in both hESCs and human fibroblast-derived iPSCs.
BMP4 augments hematopoietic differentiation of hPSCs
Previous studies have demonstrated that BMP4 promotes hematopoietic differentiation from hESCs (Chadwick et al., 2003; Pick et al., 2007). To characterize further the cellular response of BMP4 on hESC-derived hematopoiesis, we first examined the spatiotemporal expression of the putative BMP4 receptor ALK3 (officially known as ACVR1B), in human embryoid bodies (hEBs) grown under hematopoietic-inducing culture conditions. As previously established (Vijayaragavan et al., 2009), hierarchical stages of hematopoiesis from hPSCs can be divided into two phases of hEB development: phase I (day 0–7) is characterized by the emergence and specification of bipotential hemogenic precursors (CD45negPFV); phase II (day 7–15) is the commitment phase, characterized as the period where committed hematopoietic progenitors (CD34+ CD45+) and mature hematopoietic (CD34−CD45+) cells are detected (Fig. 1A). Expression of ALK3 was maintained during both hemogenic specification and subsequent hematopoietic commitment (Fig. 1B). Both CD31 (a hemogenic precursor marker) and CD45 (a committed hematopoietic progenitor marker) were colocalized with ALK3-positive cells within hEBs (Fig. 1Bi–iii). This suggests that the majority of cells within the differentiating hEBs, which express ALK3, could be receptive to BMP4 treatment and subsequent mesodermal differentiation.
To demonstrate the specific role of BMP4 during hematopoietic differentiation from hESCs, we repressed the effect of BMP4 through its natural antagonist noggin. BMP4-treated hEBs exhibited a 4-fold increase in hemogenic precursors (Fig. 1C) and a 3.5-fold increase in hematopoietic progenitors (Fig. 1D), indicating that BMP4 treatment augments hematopoietic output. Treatment of hEBs continuously with noggin from day 0 to day 15 had no effect on the output of hemogenic precursors or committed hematopoietic cells in the absence of BMP4 (Fig. 1C–E). However, the enhanced hematopoietic differentiation induced by BMP4 addition was dramatically inhibited by noggin treatment (Fig. 1C–E). Collectively, the results indicate that exogenous BMP4 augments hematopoietic differentiation, but that hematopoietic output might not be dependent on endogenous BMP4 in hEBs given that adding noggin in the differentiation system without exogenous BMP4 had no effect on blood development.
Recent studies have shown that, similar to hESC-derived hematopoietic cells, hiPSC-derived cells can also gave rise to all types of hematopoietic colonies (Choi et al., 2009). Given the role of BMP4 in inducing hematopoietic differentiation from hESCs, we examined whether its functional role was conserved in human-fibroblast-reprogrammed iPSCs. Using a hiPSC line (MchiPSC1.1) established and characterized in our laboratory by transduction of human dermal fibroblasts with OCT4, SOX2, NANOG and LIN28 (supplementary material Fig. S1), we examined the role of BMP4 in hiPSC-derived hematopoiesis. We found that BMP4 had no effect on the number of hemogenic precursors at day 15 of differentiation, independent of the inclusion of hematopoietic growth factors (hGFs) (Fig. 1F). However, treatment with BMP4 or BMP4 plus hGFs substantially increased the proportion of committed hematopoietic cells compared with that of the control (Fig. 1F). Although the total number of cells within hEBs was not influenced by the addition of hGFs and BMP4 (Fig. 1G), the total yield of committed hematopoietic cell types was substantially higher in hEBs treated with BMP4 and BMP4 plus hGFs compared with that of the control (Fig. 1H). Addition of BMP4 plus hGFs further augments hematopoietic differentiation (Fig. 1F,H), indicating a specific role for BMP4 in hematopoietic commitment. Overall, these data suggested a conserved role for BMP4 in hESCs- and hiPSC-derived hematopoiesis.
Next, we treated hEBs with BMP4 at specific phases in order to examine whether BMP4 is effective at a specific phase of hematopoietic specification and commitment. Treatment with BMP4 at phase I or phase II of hEB development revealed that BMP4 increased both hemogenic precursor and committed hematopoietic cells only when present during the early commitment phase I (supplementary material Fig. S2). However, the enhanced effect on hematopoiesis observed upon BMP4 addition was completely blocked by addition of its natural antagonist noggin, suggesting a direct function of BMP4 on hEB development, especially during phase I. These results indicate that the BMP4 acts on hematopoietic differentiation by targeting cells at hemogenic specification phase I. Collectively, these data underscore the importance of BMP4 signals, and its downstream targets, during hematopoietic differentiation from hPSCs, thus prompting us to examine the expression profile of BMP4-targeted ID genes during hPSC-derived hematopoietic development.
Differential regulation of ID1 and ID3 transcripts between two hematopoietic phases of hPSCs
As ID genes are known to be direct targets of BMP4, a ventral mesoderm inducer that promotes hematopoietic differentiation of hESCs (Chadwick et al., 2003; Hollnagel et al., 1999; Miyazono and Miyazawa, 2002), we investigated the temporal expression of the ID family of genes (ID1–ID4) during this process. We initially examined the expression profile of ID genes in undifferentiated hESCs and hiPSCs to determine the baseline expression. Both hESCs and hiPSCs exhibited identical expression profiles for the ID genes; in the undifferentiated state higher levels of ID3 transcript were observed with no detectable expression of ID4 (Fig. 2A,B). Upon differentiation of hESCs and hiPSCs under hematopoietic culture conditions, OCT4 expression rapidly decreased, whereas ID1 and ID3 transcript levels increased and peaked at day 7, coincidental to the hemogenic specification phase (phase I; days 0–7) (Fig. 2C,D; supplementary material Fig. S3A). ID2 expression was also increased in the initial 7 days of differentiation; however, during the subsequent 7 days, its levels were constant, indicating that ID2 might represent a general marker of differentiation that is expressed independently of the hematopoietic lineage. By contrast, ID1 and ID3 transcripts were downregulated during the hematopoietic commitment phase (phase II; days 7–15) (Fig. 2C,D). To examine whether the ID genes are regulated by BMP4, the transcripts levels of ID genes were determined upon supplement of BMP4 without hGFs. The expression profiles of the ID genes were similar to that with BMP4 plus hGFs (Fig. 2C,D; supplementary material Fig. S3B), suggesting that the association of BMP with ID transcription during hematopoietic differentiation. Interestingly, ID4 expression was not detectable in either undifferentiated hPSCs or during hematopoietic development (Fig. 2A–D; supplementary material Fig. S3), suggesting that ID4 is not involved with hESC self-renewal or differentiation. The differential expression profile of ID1 and ID3 implies that these genes are uniquely regulated during the specification compared with the commitment phase in both hESC- and hiPSC-derived hematopoiesis.
To determine whether changes in ID transcript levels are associated with the hematopoietic commitment of hPSCs, we compared the levels of ID1 and ID3 transcripts between hematopoietic lineage populations and the remaining unspecified (non-hematopoietic) cells. Hemogenic precursors, hematopoietic progenitors and mature hematopoietic cells, along with the remaining hEB cells, were isolated from day 10, 15 and 20 hEBs. Analysis of highly purified subsets of different cell populations within hEBs revealed that the levels of ID1 and ID3 transcripts were diminished in hemogenic precursors and committed hematopoietic cells (Fig. 2E–G). In day 15 hEBs, although an ~50% decrease of ID1 and ID3 transcripts was seen in hemogenic precursors in comparison with that in the remaining hEB cells, a 80% decrease was seen in committed hematopoietic cells. These data indicate downregulation of ID1 and ID3 transcripts in hematopoietic populations and suggest that ID1 and ID3 might have unique regulatory roles during hematopoietic commitment of hPSCs.
ID1 and ID3 act as negative regulators during the hematopoietic commitment phase of hESCs and hiPSCs
On the basis of their distinct temporal expression patterns and effect on hematopoietic differentiation, we aimed to functionally evaluate the role of ID1, ID2 and ID3 in hESC-derived hematopoiesis by using small interfering RNA (siRNA) to silence expression at two periods of hEB hematopoietic development (day 0–2 and day 8–10) (Fig. 3A; supplementary material Fig. S4). Specific siRNA-mediated downregulation of ID1, ID2 and ID3 resulted in a 30–40% decrease in the level of the transcripts without changing the total cell number and viability of hEBs (Fig. 3B,C; supplementary material Fig. S4A). Knockdown using siRNAs against ID1, ID2 and ID3 at the early hEB stage (day 0–2) had no effects on the percentage of either hemogenic precursors or committed hematopoietic cells at day 15 of differentiation (supplementary material Fig. S4B), indicating that either hemogenic specification or subsequent hematopoietic commitment was restrained by the expression of ID genes during the hemogenic specification phase (phase I). However, treatment with siRNAs against ID1 and ID3 at the late hEB stage (day 8–10; phase II) resulted in a substantial increase in the generation of mature hematopoietic cells, whereas ID2 knockdown had no effects on the percentage of either hemogenic precursors or committed hematopoietic cells (Fig. 3D). These results provide functional evidence for the active role of ID1 and ID3 as negative regulators during hESC hematopoiesis and indicate that they function during the commitment phase of hEB hematopoietic maturation. Dual suppression of ID1 and ID3 at phase II further increased hematopoietic development from hPSCs (supplementary material Fig. S5A). Western blotting analysis further verified that siRNA effectively downregulated ID transcription and resultant protein expression as compared with when using a scrambled control (supplementary material Fig. S5B). These data suggest that ID1 and ID3 might regulate commitment through unique targets and pathways of hematopoietic maturation.
On the basis of the observed effects of single and dual siRNA knockdown of ID1 and ID3 during hematopoietic commitment in hESCs, we similarly treated hiPSC-derived hEBs at phase II of differentiation (day 8–10; Fig. 3E–G) with siRNA against ID1, ID2 and ID3. As observed using hESCs, siRNA treatment decreased the level of the transcripts by 30–40% in the hiPSC-derived hEBs without changing the total cell number and viability of the hEBs (Fig. 3E,F). Downregulation of ID1 and ID3 resulted in an increase in the generation of committed hematopoietic (CD34+ CD45+ and CD34− CD45+) cells, whereas ID2 knockdown had no effect (Fig. 3G). These results reveal that the negative regulatory role of ID1 and ID3 during hematopoietic commitment phase is conserved in both hESCs and hiPSCs.
Using the colony forming unit (CFU) assay for primitive hematopoietic cells as a measure of functional progenitors, we evaluated the multi-lineage potential of the hematopoietic progenitors from differentiating hESC or hiPSC-derived hEBs. siRNA knockdown of ID1 or ID3 in hESC- or hiPSC-derived hEBs, and subsequent isolation of the hematopoietic progenitors (CD34+ CD45+ cells), resulted in a substantially higher total number of CFUs produced by CD34+ CD45+ cells compared with that by control cells. ID2 siRNA did not affect the CFU output (Fig. 4A,B). Furthermore, ID gene knockdown did not have an effect on the distribution of CFU types generated from either hESCs- or hiPSCs-derived hematopoietic progenitors (Fig. 4C–E), suggesting the effects of ID genes are not at the level of hematopoietic lineage development, but at the point of cell fate commitment to the hematopoietic lineage.
The negative regulatory role of ID1 and ID3 is exclusively restricted to hemogenic populations
Our observations indicating enhanced hematopoietic differentiation upon suppression of ID1 and ID3 expression was limited to analysis of whole hEBs, and therefore it remains unclear whether the negative regulatory function of ID1 and ID3 is exclusively restricted to direct effects on hemogenic precursors or whether they act indirectly on cells comprising hEBs. Accordingly, we prospectively isolated hemogenic precursors and the remaining non-hemogenic cells from hESC- and hiPSC-derived hEBs (Fig. 5A,B). As the bipotent precursors emerge between days 8–10 of hEB hematopoietic differentiation (Wang et al., 2004), we isolated cells from day 12 hEBs. These subsets were then treated with ID1 and ID3 siRNAs, and effects on hematopoietic output were measured. Treatment with siRNAs decreased ID1 and ID3 expression by 40–50% in the sorted subsets of cells (Fig. 5C). Notably, knockdown of ID1 and ID3 in hemogenic precursors resulted in substantial increases in the generation of committed hematopoietic cells, whereas downregulation of ID1 and ID3 in the remaining control non-hemogenic cells had no effect, as expected (Fig. 5D). These results demonstrate that the negative regulatory role of ID1 and ID3 is direct and restricted to hemogenic precursors committed towards the hematopoietic lineage, and that it is not dependent on the presence of non-hemogenic cells in whole hEBs.
Identification of the cellular and molecular processes regulating hematopoietic development in vitro from hPSCs is an important basis for understanding complicated in utero lineage specification and applications of cell-based therapies. Our current study reveals that ID1 and ID3 regulate hematopoietic commitment, and that this regulation is under stringent temporal regulation by extrinsic stimuli, such as BMP4 and growth factors. hPSC-derived hematopoiesis can be divided into two developmental phases, hemogenic specification and hematopoietic commitment, which are temporally regulated by distinct signaling factors (Vijayaragavan et al., 2009). Using hierarchical stages of blood development from hPSCs, our study reveals that BMP4 affected hematopoietic differentiation by targeting cells during hemogenic specification and demonstrates a unique negative regulatory role for ID1 and ID3 during hematopoietic commitment from hPSCs.
ID proteins are know to act as a negative regulators by blocking DNA binding by transcription factors and the downstream activity of basic HLH proteins, and ID proteins are required for maturation and terminal differentiation in various cell types (Yokota, 2001). This inhibitory function of ID genes has been identified during both lymphoid and myeloid lineage commitment in adult human HSCs (Buitenhuis et al., 2005; Cooper et al., 1997; Jankovic et al., 2007; Kreider et al., 1992; Leeanansaksiri and Dechsukhum, 2006; Lister et al., 1995). More specifically, during hPSC cell fate regulation, ID genes have been shown to negatively regulate development of T cells from hESC-derived hematopoietic progenitors on the basis of constitutive expression of ID genes during differentiation (Martin et al., 2008). On the basis of our results, we propose a model to illustrate the role of ID1 and ID3 in the context of hPSC-hematopoietic development (Fig. 5E).
In both hESCs and hiPSCs, the regulatory role of ID1 and ID3 was restricted to the commitment phase of blood development (from hemogenic to hematopoietic progenitors) (Fig. 5E). The negative role for ID1 and ID3 during blood development was conserved between hESCs and hiPSCs, suggesting that transcriptional regulation of these genes is maintained during mesodermal differentiation of pluripotent cells in the human. Furthermore, studies have shown that ID genes also function during mouse ESC differentiation, where ID1 and ID3 transcripts are downregulated during the development of primitive blast-colony forming cells (considered to be the in vitro equivalent of the hemangioblast) into mature myeloid cells (Nogueira et al., 2000). ID1 and ID3 have been shown to regulate crucial steps during angiogenesis and vascularization (Lyden et al., 1999). Recently, James et al. (James et al., 2010) have shown that ID1 is required for expansion and maintenance of hESC-derived endothelial cells and preservation of endothelial cell commitment (James et al., 2010). As the hemogenic precursors isolated in our current study originate as hemogenic endothelial precursors and continue to mature along the endothelial lineage, the increased hematopoietic potential induced by downregulation of ID1 might be due to an inhibition of endothelial development that indirectly increases hematopoietic capacity. This suggests that ID3, as well as ID1, could influence endothelial commitment. Thus, our results suggest that BMP4 activates expression of ID genes at the hemogenic specification phase, and that other extrinsic stimuli, such as growth factors, determine the fate of hemogenic precursors as endothelial cells, whereas subsequent maturation to a hematopoietic fate requires downregulation of ID1 and ID3 for complete blood cell development. On this point, a recent study demonstrated that E2-2, one of the basic HLH family of transcription factors, negatively regulates endothelial cell specification by blocking activation of ID1 and its downstream effectors, VEGFR2, integrin-β4 and MMP2 (Tanaka et al., 2010). In addition, it has been reported that activated Notch and its downstream effector Herp2 antagonize BMP and ID1 expression (Itoh et al., 2004). Accordingly, it is possible that these molecules cooperate with ID genes to tightly regulate transitions from hemogenic endothelial, as well as hematopoietic differentiation. Additional studies will be required to clarify the mechanisms of action involving ID1 and ID3 during regulation of hemogenic endothelial specification to the hematopoietic lineage.
Unlike ID1 and ID3, ID2 levels were not altered during either hESC- or hiPSC-derived hematopoiesis. Knockdown of ID2 had no effect on the emergence of hemogenic cells or the production of committed hematopoietic cells. Moreover, levels of ID2 transcript between hemogenic precursors and committed hematopoietic cells were maintained (data not shown). In mouse-ESC-derived hematopoiesis, ID2 expression is maintained during blast cell colony differentiation, contrary to expression of ID1 and ID3 (Nogueira et al., 2000). Consistently, ID2 mRNA levels are not downregulated upon induction of differentiation of human myeloid blasts toward either granulocytes or macrophage; however, inhibition of ID2 expression blocked granulocyte differentiation from human UCB-derived CD34+ cells (Buitenhuis et al., 2005; Ishiguro et al., 1996). The differential expression profile and function of ID2, as distinguished from those of ID1 and ID3, suggest that ID2 might have a unique function during myeloid differentiation and have no effect during early blood development events. However, the exact role of ID2 during the hierarchical stages of hematopoiesis remains elusive.
Our current study reveals a negative regulatory role for ID1 and ID3 during hematopoietic commitment that is highly conserved in both hESC- and hiPSC-derived blood development, thereby demonstrating that ID genes are able to control early hematopoietic events in the human. Collectively, these findings suggest that intrinsic transcription factors are under stringent temporal regulation as a direct consequence of extrinsic stimuli, such as growth factors (BMP4) and co-culture conditions, during the derivation and expansion of hematopoietic cells. The ultimate goal is to apply this knowledge towards cell-based therapies by generating sufficient numbers and appropriately programmed hematopoietic cells from either hESCs or immunocompetent hiPSCs that have the potential for in vivo reconstitution of the hematopoietic system similar to that observed for human stem cells.
Materials and Methods
Undifferentiated hESC lines (H1 and H9) were maintained in feeder-free culture as previously described (Chadwick et al., 2003). Briefly, hESCs were cultured on Matrigel (BD Biosciences)-coated six-well plates with mouse embryonic fibroblast conditioned medium (MEF-CM) supplemented with 8 ng/ml human basic fibroblast growth factor (hbFGF) (Invitrogen). In order to maintain the undifferentiated state, the medium was changed daily and hESCs were passaged at a 1:2 split ratio every 6–7 days by enzymatic dissociation with 200 units/ml collagenase IV (Invitrogen). hESC culture was carried out at 37°C in a humidified atmosphere containing 5% CO2. H1 and H9 hESC lines were used for all experiments.
Establishment and maintenance of hiPSCs
Adult dermal fibroblasts were reprogrammed as described previously (Yu et al., 2007). In brief, lentiviruses expressing transgenes (OCT4, SOX2, NANOG and LIN28) were produced using the human embryonic kidney (HEK)-293FT cell line. Lentiviral transduction was performed with human adult dermal fibroblasts (ScienCell) for 48 hours, and then the cells were re-plated on 1:15 Matrigel-coated six-well plates in MEF-CM supplemented with 16 ng/ml hbFGF and 30 ng/ml IGF-II (Millipore). At between day 20 and 25 post-transduction of lentiviruses, iPSC colonies (Mc-hiPSC) emerged, and were expanded and maintained in the same conditions as hESCs and used for all experiments. To examine the presence of the transgenes and the pluripotency genes in the generated hiPSCs, specific primer sets were used to measure the coding regions of both the endogeneous gene and the transgene (Total), the 3′ untranslated region (Endogenous) or the region of the viral transgenes (Exogenous). A list of primers can be found in supplementary material Table S1. To examine in vivo differentiation potency, hiPSC cultures (1.0×106 cells per mouse) were injected into the testicle of NOD-SCID mice (approved by the Animal Research and Ethics Board of McMaster University), as described previously (Werbowetski-Ogilvie et al., 2009). After 8 weeks, mouse testicles were harvested, sectioned and stained with hematoxylin and eosin. Images were acquired using a ScanScope CS digital slide scanner (Aperio, CA, USA).
hEB formation and hematopoietic differentiation
Embryoid bodies (hEBs) were generated by suspension culture, as previously described (Cerdan et al., 2007; Hong et al., 2010). The medium was exchanged with hEB differentiation medium [80% Dulbecco's modified essential amino acids (DMEM) base medium plus 20% FBS supplemented with 1% non-essential amino acids, 1 mM L-glutamine and 0.1 mM β-mercaptoethanol] supplemented with hematopoietic growth factors (hGFs) [50 ng/ml granulocyte colony stimulating factor (G-CSF; Stem Cell Technologies), 300 ng/ml stem cell factor (SCF; Stem Cell Technologies), 10 ng/ml interleukin-3 (IL-3; R&D Systems), 10 ng/ml interleukin-6 (IL-6; R&D Systems), 25 ng/ml BMP4 (R&D Systems) and 300 ng/ml Flt-3 ligand (Flt-3L: R&D Systems)]. The hEBs were cultured for 15–20 days, with the hematopoietic differentiation medium, including the hGFs, changed at 3-day intervals. To investigate the effect of BMP4 on hESC-derived hematopoiesis, we blocked BMP4 signals using noggin (500 ng/ml; R&D systems) in hEB differentiation medium.
Colony forming unit assay
The colony forming unit (CFU) assay was performed by plating single-cell suspensions from dissociated hEBs into methylcellulose H4230 (Stem Cell Technologies), as previously described (Chadwick et al., 2003). Briefly, hEBs were dissociated with collagenase B and cell dissociation buffer, followed by filtration through a 40-μm cell strainer to obtain a single-cell suspension. Cells derived from dissociated hEBs were counted, and 10,000 cells were plated into methylcellulose H4230 supplemented with recombinant human growth factors [50 ng/ml SCF, 3 units/ml erythropoietin, 10 ng/ml granulocyte monocyte-colony stimulating growth factor and 10 ng/ml IL-3]. After 14 days, differential colony counts were performed on the basis of morphology.
Flow cytometry analysis
Single-cell suspensions from dissociated hEBs were resuspended at ~2×105–4×105 cells/ml in 3% fetal bovine serum (FBS) in PBS and were stained for 1 hour at 4°C with fluorochrome-conjugated monoclonal antibodies against: CD31 (phycoerythrin-conjugated; BD Pharmingen), CD34 (FITC-conjugated; Miltenyi Biotech, Bergisch Gladbach, Germany) and CD45 (allophycocyanin-conjugated; Miltenyi Biotech) or their corresponding isotype controls. Stained cells were washed twice in 3% FBS in PBS and stained with the viability dye 7-aminoactinomycin D (7-AAD) (Immunotech). Live cells were analyzed for surface marker expression using a FACSCalibur cell analyzer (Becton Dickinson Immunocytometry Systems, San Jose, CA) and Cell Quest software (BDIS).
Sorting of hEBs
To isolate CD45negPFV, CD34+ CD45+, CD34− CD45+, and the remaining hEB cells, hEBs were dissociated with collagenase B and stained with the fluorochrome-conjugated antibodies as above and 7AAD. Each cellular subset was sorted on a FACSAria (BD Pharmingen) as previously described (Wang et al., 2004).
Immunohistochemistry of hEB sections
For staining ALK3, CD31, and CD45, hEBs were washed twice with 3% FBS in PBS, fixed with 4% paraformaldehyde in PBS for 2 hours, embedded, then snap-frozen in liquid nitrogen and stored at −80°C. Cryostat sections were stained for ALK3, CD31 and CD45 proteins for detection within the same hEB region. The following primary antibody and dilutions were used: rabbit anti-ALK3 antibody (1:100; Abcam), mouse anti-CD31 antibody (1:100; BD Pharmingen) and mouse anti-human CD45 antibody (1:100; BD Pharmingen). Sections were incubated with primary antibodies for 2 hours at room temperature. Subsequently, the sections were stained for 30 minutes with Alexa-Fluor-594-conjugated goat anti-(rabbit IgG) (Molecular Probes) for ALK3 and Alexa-Fluor-488-conjugated goat anti-(mouse IgG) (Molecular Probes) for CD45 and CD31. Slides were mounted and counterstained using VECTASHIELD HardSet Mounting Medium with DAPI (Vector Labs). Images were captured with a Photometrix Cool Snap HQ2 camera and analyzed using Image-Pro 3DA version 6.0.
Cells were treated as indicated, washed with cold PBS, and then lysed for 45 minutes on ice, using Triton lysis buffer (20 mM Tris-HCl pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.5 mM dithiothreitol and protease inhibitors). The lysates were clarified by centrifugation at 18,000 g for 15 minutes, and the supernatants were stored at −80°C until use. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes, For western blotting, bands were visualized using ECL solutions.
Total RNA was extracted from hEBs and sorted cell populations using the Absolutely RNA microprep kit (Stratagene). cDNA was made with 1 μg of total RNA using the first-strand cDNA synthesis kit (Invitrogen) and subsequent quantitative real-time PCR (Q-PCR) were carried out in triplicate, using SYBR Green RT-PCR reagents (Applied Biosystems) on an Mx3000P Q-PCR System according to manufacturer instructions (Stratagene). Amplifications were performed using the following conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. All data were normalized to GAPDH. The primer sequences used are shown in supplementary material Table S2.
Knockdown of ID1, ID2 and ID3 transcripts using siRNA transfection
Whole hEBs and sorted subsets (hemogenic precursors and remaining hEB cells) were transfected with ON-TARGETplusSMARTpool siRNA targeting ID1 (100 nM, L-005051-00, Dharmacon), ID2 (100 nM, L-009864-00) or ID3 (100 nM, L-009905-00) for 24 hours using the Silencer siRNA Transfection II kit (Ambion) in KO-DMEM medium, according to the manufacturer's instructions. Non-targeting siRNA (Ambion) was used as a negative control. After siRNA transfection, culture medium was changed to fresh hEB differentiation medium. The hEBs were allowed to grow for 24 hours and lysed for quantitative RT-PCR.
All results are expressed as means±s.d. and were generated from at least three independent experiments. Statistical significance was determined using Student's t-tests and differences were considered significant when P<0.05.
We would like to thank Eva Szabo and Chantal Cerdan for helpful discussions on the manuscript. This work was supported by grants from the Canadian Institutes of Health Research and Canada Research Chair Program (to M.B.).
↵* These authors contributed equally to this work
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.077511/-/DC1
- Accepted December 17, 2010.
- © 2011.