Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Aug 10.
Published in final edited form as: J Cell Physiol. 2019 Feb 10;234(9):16136–16147. doi: 10.1002/jcp.28272

Defining early hematopoietic-fated primitive streak specification of human pluripotent stem cells by the orchestrated balance of Wnt, Activin and BMP signaling

Jun Shen 1,2, Cuicui Lyu 3, Yaoyao Zhu 1, Zicen Feng 1, Shuo Zhang 1,2, Dixie L Hoyle 2, Guangzhen Ji 1, Robert A Brodsky 2, Tao Cheng 1,4,5,*, Zack Z Wang 2,*
PMCID: PMC6689260  NIHMSID: NIHMS1009458  PMID: 30740687

Abstract

Distinct regions of the primitive streak (PS) have diverse potential to differentiate into several tissues, including the hematopoietic lineage originated from the posterior region of PS. Although various signaling pathways have been identified to promote the development of PS and its mesoderm derivatives, there is a large gap in our understanding of signaling pathways that regulate the hematopoietic fate of PS. Here, we defined the roles of Wnt, Activin and BMP signaling pathways in generating hematopoietic-fated PS from human pluripotent stem cells (hPSCs). We found that the synergistic balance of these signaling pathways was crucial for controlling PS fate determination towards hematopoietic lineage via mesodermal progenitors. Although the induction of PS depends largely on Wnt and Activin signaling, the PS generated without BMP4 lacks the hematopoietic potential, indicating that BMP signaling is necessary for the PS to acquire hematopoietic property. Appropriate levels of Wnt signaling is crucial for the development of PS and its specification to the hematopoietic lineage. Although the development of PS is less sensitive to Activin or BMP signaling, the fate of PS to mesoderm progenitors and subsequent hematopoietic lineage is determined by appropriate levels of Activin or BMP signaling. Collectively, our study demonstrate that Wnt, Activin, and BMP signaling pathways play cooperative and distinct roles in regulating the fate determination of PS for hematopoietic development. Our understanding on the regulatory networks of hematopoietic-fated PS would provide important insights on early hematopoietic patterning and a possible guidance for generating functional hematopoietic cells from hPSCs in vitro.

Keywords: hematopoietic differentiation, human pluripotent stem cells, signaling pathways, primitive streak, mesoderm

1 |. INTRODUCTION

Human embryonic hematopoiesis is a complex regulated process that involves multiple developmental steps ranging from induction and patterning of primitive streak (PS)-derived mesoderm to specification of the earliest hematopoietic progenitors. Fate-mapping studies have shown that different mesodermal derivatives, such as blood, vasculature and cardiac muscle arise in an orderly manner from distinct regions of the PS (Lawson et al., 1991; Tam and Beddington, 1987). The anterior PS has the potential to form anterior mesoderm and the middle region of PS forms the lateral plate mesoderm, while the most posterior region of PS develops extra-embryonic mesoderm progenitors that give rise to hematopoietic and vascular cells (Sumi et al., 2008; Tam and Loebel, 2007).

The developmental progression from PS formation through mesoderm induction to endothelial and hematopoietic specification can be mapped, to some extent, by the expression of genes indicative of each individual stage. The formation of PS is defined by the upregulation of the T-box transcription factor, Brachyury (T), that is expressed throughout this structure in diverse organisms (Kispert and Herrmann, 1994; Lolas et al., 2014; Schulte-Merker et al., 1997). Subsequently, the mesoderm is specified from the T+ PS by the expression of kinase insert domain receptor (KDR) (Ema et al., 2006). As the mesoderm is specified to the endothelial and hematopoietic lineages, KDR+ mesoderm progenitor cells (MPs) will gradually transform into KDR+CD34+ hemato-endothelial progenitors (HEPs) that have differentiation potential to give rise to endothelial cells and hematopoietic cells (Gori et al., 2015; Wang et al., 2004). Hematopoietic cells arise from a specific subtype of endothelial cells, hemogenic endothelial (HE) cells, through a process of endothelial-to-hematopoietic transition (EHT) (Bertrand et al., 2010; Chen et al., 2009). By analyzing the kinetics of expression of specific hematopoietic markers in hPSCs differentiation, CD43 has been identified as an early marker of hematopoietic progenitor cells (HPCs) (Vodyanik et al., 2006).

The formation of PS, as well as subsequent development of mesoderm and hematopoietic lineage, is not random but highly organized and spatiotemporally controlled via the interaction of many intrinsic factors and extrinsic factors (Wang and Chen, 2016). As a result of limited access to early human embryos, it has not been possible to demonstrate a role for various factors on the specification of PS in vivo. Human pluripotent stem cells (hPSCs) possess the ability to differentiate into all cell types of our body and provide an excellent model system for studying cell fate determination in early human development in vitro (Paes et al., 2017). Recently, various recipes have been formulated to induce the differentiation of hPSCs towards PS in vitro, which greatly facilitate the elucidation of the regulatory mechanisms of different factors involved in PS specification (Brown et al., 2011; Woll et al., 2008; Yu et al., 2011). Among the factors, the transforming growth factor-β (TGF-β) superfamily members Activin/Nodal and bone morphogenetic proteins (BMPs), as well as Wnt family members, are the key regulators of this process. Although different models have defined Wnt, Activin and BMP signaling as key regulators that control PS formation (Brennan et al., 2001; McReynolds et al., 2007; Nostro et al., 2008; Walmsley et al., 2002), it is not clear whether the PS generated by different signaling stimulation have the equal hematopoietic potential and little is known about the effect of signal intensity on the development of PS and its specification to the hematopoietic lineage.

To further elucidate the regulatory pathways that control hematopoietic-fated PS specification from hPSCs, we have evaluated the distinct roles of Activin, Wnt, and BMP signaling in this process. Our findings demonstrate that synergistic Wnt and Activin, but not BMP4, are required for PS formation, whereas high levels of Wnt signaling impairs the development of PS and its subsequent hematopoietic transformation. Interestingly, increasing the Activin and BMP signaling does not affect the development of PS but it displays a strong posteriorizing effect on subsequent hematopoietic specification. High levels of Activin signaling inhibit EHT without affecting mesoderm and HEPs production, while high BMP signaling impairs PS-derived mesoderm induction. Combined Wnt, Activin/Nodal, and BMP signaling have the ability to effectively promote the formation of hematopoietic-fated PS and its hematopoietic specification.

2 |. MATERIALS AND METHODS

2.1 |. hPSC Maintenance

The H1 hESC line was obtained from the WiCell Research Institute (Madison, WI, http://www.wicell.org). To assess PS development in hPSCs, a lentiviral transduction on BC1 human induced pluripotent stem cells (hiPSCs) was performed to generate a hiPSC line, BC1-Brachyury-GFP, in which GFP expression was driven by the Brachyury (T-box) promoter as we previously described (Bai et al., 2013). The hPSCs were cultured on a Matrigel-coated plate with E8 medium (Life Technologies) and growth medium was changed every day. The hPSCs were subcultured every 3 to 4 days with a treatment of 0.5 mM EDTA (Life Technologies) for passaging and used between passage 30 and passage 50.

2.2 |. hPSC Differentiation

Single-cell suspensions of hPSCs were obtained by treating the hPSC cultures at 70%−80% confluency with TrypLE (Thermo Fisher Scientific). Single cells were plated at an optimized density at 6×103 cells/well onto vitronectin (Peprotech)-coated 12-well plates in STEMdiff APEL Medium (STEMCELL Technologies) supplemented with 3 μM GSK3β inhibitor, CHIR99021 (abm Inc), 2 ng/ml ActivinA (Peprotech), 10 ng/ml BMP4 (Peprotech) and 10 μM Rho kinase inhibitor, Y-27632 (STEMCELL Technologies) on day 0. After 48 hr (day 2), the medium was changed to STEMdiff APEL Medium supplemented with 10 ng/ml VEGF (Peprotech). On day 3, additional 10 ng/ml FGF2 (abm) was added to the cultures without aspirating the old medium. From day 4, the medium was changed to STEMdiff APEL 2 Medium supplemented with 10 ng/ml VEGF and 10 ng/ml FGF2 until day 6. From day 6, the medium was changed to STEMdiff APEL 2 Medium supplemented with 10 ng/ml VEGF, 10 ng/ml FGF2, 50 ng/ml SCF (Peprotech), 50 ng/ml Flt-3L (Peprotech), 50 ng/ml TPO (Peprotech), 10 ng/ml IL-3 (Peprotech) and 10 ng/ml IL-6 (Peprotech). Differentiation was conducted in a 1%−5% hypoxic condition from day 0 to day 6. The TrypLE was used to dissociate and collect cells for analysis.

2.3 |. Flow Cytometry

Cells were dissociated to form a single-cell suspensions by TrypLE treatment, and washed with FACS buffer (1% FBS and 1 mM EDTA in PBS). The dissociated cells were resuspended in FACS buffer, and labeled with fluorochrome-conjugated anti-human CD34-APC (clone AC136, Miltenyi Biotec), KDR-PE (clone ES8–20E6, Miltenyi Biotec), CD31-FITC (clone AC128, Miltenyi Biotec), CD144-Alexa Fluor 700 (clone 16B1,eBioscience), CD43-PerCP (clone TP1/36, abcam), CD4-APC (clone RPA-T4, eBioscience), CD8-PE (clone 3B5, eBioscience), or CD45-PE (clone 2D1, BioLegend). Dead cells were excluded by DAPI (BD Biosciences) staining. Isotype-matched control antibodies were used to determine the background staining. Flow cytometry was performed on LSR II analyzer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Inc).

2.4 |. Hematopoietic CFC Assays

Single cells of the indicated numbers in 0.1 ml IMDM (Life Technologies) with 2% FBS were mixed with 1ml MethoCult H4034 Optimum (STEMCELL Technologies). The mixture was then transferred to 2 wells of ultra-low attachment 24-well plates (Corning). The cells were incubated at 37 °C in 5% CO2 with 100% humidity for 14 days, and then the colonies were counted. Each type of colony was classified according to morphology. Each assay was performed in triplicate.

2.5 |. T Cell Differentiation

The hematopoietic cells were collected at indicated days, resuspended in T cell differentiation medium consisting of α-MEM (Thermo Fisher Scientific) supplemented with 20% FBS (Hyclone), 5 ng/ml rhIL7 (Peprotech), 5 ng/ml rhFlt-3L (Peprotech), and 10 ng/ml rhSCF (Peprotech), and then cultured on OP9-DLL1 cells as we previously described (Bai et al., 2016). Every five days co-cultures were transferred onto fresh OP9-DLL1 cells by vigorous pipetting and passaging through a 40 µm cell strainer. Cells were analyzed by flow cytometry on the days indicated.

2.6 |. Real-time PCR assay

Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen) and treated with RNase-free DNase (Qiagen). cDNAs were synthesized with random hexamers and Oligo(dT) with Superscript III Reverse Transcriptase (Invitrogen) and stored at −20°C until use. Real-time PCR was performed using a FastStart Universal SYBR Green Master (Roche) on a Step One 7500 (Applied Biosystems). Amplification of β-actin was also conducted to control the quantity of loaded cDNA in each reaction. Primers sequences are listed in supplementary Table 1.

2.7 |. Statistical analysis

Data obtained from multiple experiments were reported as the mean ±SEM. The data were subjected to statistical analysis by the Student’s t-test. Results with a value of P <0.05 were considered statistically significant.

3 |. RESULTS

3.1 |. The synergistic effect of Wnt, Activin/Nodal, and BMP signaling pathways on PS and mesoderm fate determination

To investigate signals that initiate hematopoietic program in hPSCs, we would establish a differentiation system that is sensitive to extrinsic factors. We induced hPSC differentiation by culturing hPSCs on vitronectin-coated plate as a serum-free and stroma-free monolayer culture in chemically defined medium (Figure 1A). The first recognizable stage of hematopoietic differentiation from hPSCs is the development of PS, which is capable of differentiating into cell types belonging to mesoderm and endoderm (Wang and Chen, 2016). To track the development of PS that expresses the T-box transcription factor, Brachyury (T), we employed a T-GFP reporter system (T-GFP) in BC1 hiPSCs (Figure 1B). The fate of PS towards mesoderm was further monitored by KDR expression in mesoderm progenitors (Figure 1D).

Figure 1. The synergistic balance of Wnt, Activin and BMP signaling promotes the development of hPSC-derived PS and mesoderm.

Figure 1.

Open in a new tab

(A) Schematic showing the strategy for the generation of hPSC-derived PS and mesodermal progenitors. D, Day. (B) and (C) Flow cytometric analysis of the frequency of T-GFP+ PS cells on day 2 under different combinations of CHIR99021 (3μM), ActivinA (2ng/ml) or BMP4 (10ng/ml). n=3. SSC, Side scatter. (D) and (E) Flow cytometric analysis of the frequency of KDR+ mesodermal progenitors on day 3 under different combinations of CHIR99021 (3μM), ActivinA (2ng/ml) or BMP4 (10ng/ml) during the first 2 days of hPSC differentiation. n=3. SSC, Side scatter. Data are shown as means ±SEM. Statistical analysis was done by two-tailed Student’s t test. **P<.01; ***P<.001.

Consistent with the previously described roles (Nostro et al., 2008; Sumi et al., 2008), we found that Wnt, Activin, and BMP signaling pathways were essential for the development of PS (Figure 1B). Compared with BMP4 alone, addition of either Activin A or GSK-3 inhibitor CHIR99021 (an activator of Wnt signaling pathway) on day 0 of hPSC differentiation significantly enhanced T-GFP+ cell frequency on day 2 (Figure 1B and1C). We then tested a combined effect of two factors on T-GFP+ PS development, and found that CHIR99021 had the most profound effect on PS development. In the presence of CHIR99021, the presence of either Activin A or BMP4 synergistically promoted T-GFP+ PS development on day 2 (Figure 1B and1C). To follow the mesodermal lineage development, we tested a combined factor effect on the development of KDR+ mesoderm. Interestingly, a combination of three factors of CHIR99021, BMP4, and Activin A from day 0 to day 2, but not two-factor combination, was sufficient to induce KDR+ mesoderm on day 3 (Figure 1D and1E). These results indicate that Wnt, Activin/Nodal, and BMP signaling pathways have a cooperative effect on the development of PS progenitors and their fate to KDR+ mesoderm.

3.2 |. The role of Wnt signaling in regulating PS development and the fate to mesodermal derivatives

To further determine the role of Wnt signaling in regulating PS development and the fate to mesoderm and hematopoietic lineages, we assessed the effect of different concentrations of CHIR99021 on T-GFP+ PS generation in the presence of Activin A and BMP4. Although the frequency of T-GFP+ cells were enhanced from 60% to 90% by adding 3 to 9 μM CHIR99021 (Figure 2A and2B), an increased CHIR99021 concentration as 6 or 9 μM reduced the number of T-GFP+ PS on day 2 (Figure 2C). The subsequent flow cytometry analysis of PS derivatives demonstrated that addition of 3μM CHIR99021 between day 0 and day 2 enhanced the development of KDR+ mesoderm on day 3 (Figure 2D), KDR+CD34+ HEPs on day 4 (Figure 2E), CD43+ HPCs on day 6 (Figure 2F and2G) and CD31+ / VE-cad+ (CD144+) endothelial cells on day 6 (Figure 2G), while increasing the CHIR99021 to 6 μM impaired the sequential development of mesoderm, HEPs and HPCs (Figure 2D–2G). Furthermore, we found that in the absence of CHIR99021 or increasing CHIR99021 to 9 μM, the total numbers of T-GFP+ cells on day 2 were significantly decreased (Figure 2C), resulting in an impaired hematopoietic development on day 6 (data not shown). These results indicate that the development and survival of PS are controlled by Wnt activation in a concentration-dependent manner.

Figure 2. A low level of Wnt signaling promotes the development of PS and its fate to mesoderm-derived endothelial and hematopoietic lineages.

Figure 2.

Open in a new tab

The differentiation was induced in the presence of Activin A and BMP4, and with different concentrations of CHIR99021 between day 0 and day 2. After 2 days, CHIR99021, Activin A and BMP4 were replaced by VEGF and bFGF. (A) and (B) The frequency of T-GFP+ PS cells under different concentrations of CHIR99021. Flow cytometric analyses were performed on day 2. n=3. SSC, Side scatter. (C) The number of T-GFP+ PS cells treated by different concentrations of CHIR99021. Cell numbers were counted on day 2. n=3. (D) Representative flow cytometric analysis of the frequency of KDR+ mesodermal progenitors on day 3. SSC, Side scatter. (E) Representative flow cytometric analysis of the frequency of KDR+CD34+ HEPs on day 4. (F) Representative flow cytometric analysis of the frequency of CD34+CD43+ HPCs on day 6. (G) The frequency of hematopoietic cells (CD43+) and endothelial cells (CD34+, CD31+, VE-cadherin+) on day 6. n=3. Data are shown as means ±SEM. Statistical analysis was done by two-tailed Student’s t test. **P<.01; ***P<.001.

3.3 |. Hematopoietic fate of KDR+ mesoderm is primed by low concentration of Activin A

Activin/Nodal signaling is required for the induction of PS (Brennan et al., 2001; Brown et al., 2011). To investigate the concentration-dependent effect of Activin A on the development of PS and KDR+ mesodermal fate, we applied different concentrations of Activin A for 2 days. We were particularly interested in Activin A effect in a low concentration range because the monolayer differentiation system was sensitive to extrinsic factors and high concentration of Activin A led to endodermal fate (Vallier et al., 2009). Although the frequency of T-GFP+ PS on day 2 was not promoted by the addition of Activin A (Figure 3A), the number of T-GFP+ cells was increased by adding Activin A from 2 ng/ml to 8 ng/ml, indicating that Activin A enhanced PS survival (Figure 3B).

Figure 3. The hematopoietic fate of PS is pre-determined by Activin A in a concentration-dependent manner.

Figure 3.

Open in a new tab

The differentiation was induced in the presence of CHIR99021 and BMP4, and with different concentrations of Activin A between day 0 and day 2. After 2 days, CHIR99021, Activin A and BMP4 were replaced by VEGF and bFGF. (A) The frequency of T-GFP+ PS cells on day 2. n=3. (B) The number of T-GFP+ PS cells on day 2. n=3. (C) The frequency of KDR+ mesodermal progenitors on day 3. n=3. (D) and (E) The frequency of KDR+CD34+ HEPs on day 4. n=3. (F) and (G) The frequency of CD43+ HPCs on day 6. n=3. Data are shown as means ±SEM. Statistical analysis was done by two-tailed Student’s t test. *P<.05; **P<.01; ***P<.001.

To determine the hematopoietic potential of PS primed by Activin A, we tracked KDR+ mesoderm on day 3, KDR+CD34+ HEPs on day 4, and then CD43+ HPCs on day 6. Although T-GFP+ cells were generated without Activin A (Figure 3A and3B), the development of KDR+ mesoderm on day 3 required the presence of Activin A between day 0 and day 2 in a concentration-dependent manner (Figure 3C), suggesting that the mesodermal fate of PS was determined by Activin A. However, the transition of KDR+ mesoderm to KDR+CD34+ HEPs on day 4 was not affected by Activin A in the range of 2 to 8 ng/ml (Figure 3D and E). Interestingly, the efficiency of hematopoietic differentiation on day 6 was significantly higher in the group of 2 ng/ml Activin A (Figure 3F and3G), indicating that KDR+CD34+ HEPs primed by low concentration of Activin A acquired a higher potential of EHT.

3.4 |. The role of BMP signaling in hPSC-derived hematopoiesis

The role of BMP4 in PS development and fate determination is the subject of contentious debate (Mishina et al., 1995; Miura et al., 2006). To understand whether and how BMP4 impacts the development of PS and its fate towards endothelial and hematopoietic lineages, we used BMP4 with varied concentration (between 0 to 40 ng/ml) for 2 days in the presence of CHIR99021 and Activin A. As shown in Figure 4A and4B, the frequency and number of T-GFP+ cells on day 2 were not affected by BMP4, indicating that BMP signaling was not required for the induction of PS. However, the addition of 10ng/ml BMP4 between day 0 and day 2 significantly increased KDR+ cells on day 3 (Figure 4C), indicating that low concentration of BMP4 was required for the development of mesoderm-fated PS. Increasing BMP4 concentration (20ng/ml and 40ng/ml) suppressed the development of KDR+ mesodermal cells on day 3 (Figure 4C), thereby leading to a decreased generation of KDR+CD34+ HEPs on day 4 (Figure 4D and E) and CD43+ HPCs on day6 (Figure 4F). Collectively, these findings suggest that BMP signaling specifies the hematopoietic fate at every early stage of lineage development, and the BMP concentration play a pivotal role in regulating the mesoderm-fate determination.

Figure 4. The development of hematopoietic-biased PS requires a low concentration of BMP4.

Figure 4.

Open in a new tab

The differentiation was induced in the presence of CHIR99021 and Activin A, and with different concentrations of BMP4 between day 0 and day 2. After 2 days, CHIR99021, Activin A and BMP4 were replaced by VEGF and bFGF. (A) The frequency of T-GFP+ PS cells on day 2. n=3. (B) The number of T-GFP+ PS cells on day 2. n=3. (C) The frequency of KDR+ mesodermal progenitors on day 3. n=3. (D) and (E) The frequency of KDR+CD34+ HEPs on day 4. n=3. (F) and (G) The frequency of CD43+ HPCs on day 6. n=3. Data are shown as means ±SEM. Statistical analysis was done by two-tailed Student’s t test. *P<.05; **P<.01; ***P<.001.

3.5 |. The synergistic regulation of Wnt, Activin/Nodal, and BMP signaling pathways at the PS stage effectively promotes multipotent hematopoietic differentiation

Based on the evidence that Wnt, Activin/Nodal, and BMP signaling pathways played a distinct role in determining the fate of PS, we further investigated the synergistic effect of CHIR99021, Activin A, and BMP4 on hematopoietic differentiation from hPSCs. Various combinations of low concentrations of CHIR99021, Activin A and BMP4 were included for 2 days. When CHIR99021 (3μM), Activin A (2ng/ml) and BMP4 (10ng/ml) were used alone or in two-factor combination, CD43+ HPCs were undetectable or in very low frequency (Figure 5A). The frequency and number of CD43+ cells were significantly increased when all the three factors (All factors) were combined between day 0 and day 2 (Figure 5A and5B). Under the condition of All factors, the cells underwent sequential morphological changes until producing semi-adherent hematopoietic clusters on day 6 (Figure 5C). To verify the importance of these signaling events in PS development and induction of hematopoietic lineage, we took an attempt to use small molecular inhibitors of SB-431542, IWP2 or LDN-193189 against Activin, Wnt, or BMP signaling pathway, respectively (Figure S1). As shown in Figures 5D and5E, the frequency and number of CD43+ HPCs on day 6 were significantly reduced when Activin, Wnt, or BMP pathway was inhibited. The development of PS on day 2 (Figures S2A and S2B) and subsequent MPs on day 3 (Figures S3A-S3C) and HEPs on day 4 (Figures S4A-S4C) were all impaired when inhibiting either of Activin or BMP signals. Interestingly, compared with Activin or BMP inhibition, inhibition of Wnt did not affect PS development on day 2 (Figures S2A and S2B) and MPs on day 3 (Figures S3A-S3C), but inhibited HEPs on day 4 (Figures S4A-S4C) and hematopoiesis on day 6 (Figures 5D and5E). To determine the hematopoietic potential of CD43+ HPCs generated in the condition of All factors, we extended the culture to day 8 by adding both hematopoietic and endothelial growth factors on day 6. In contrast to CD43+ HPCs in which CD45 were not expressed on day 6, CD43+ HPCs acquired the ability to develop into CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs) on day 8 (Figure 5F). Colony-forming assays indicated that the CD43+ HPCs on day 8 gave rise to significantly higher numbers of erythroid and myeloid colonies than CD43+ HPCs on day 6 (Figure 5G). T cell potential has been used as an indicator for multipotent HPCs during hPSC differentiation (Kennedy et al., 2012; Uenishi et al., 2014). To determine whether All factors-primed PS have a fate to generate multipotent HPCs, the CD43+ HPCs were collected on day 6 and day 8, and transferred onto OP9-DLL1 cells for 21 days. Compared with the day 6 CD43+ HPCs, the day 8 CD43+ HPCs had a stronger T cell differentiation potential to give rise to CD4+, CD8+ and CD4+CD8+ T cells (Figure 5H). Taken together, these data demonstrate that the cooperative balance of Wnt, Activin/Nodal, and BMP signaling effectively promotes multipotent hematopoietic differentiation of hPSCs.

Figure 5. The orchestrated regulation of Wnt, Activin/Nodal, and BMP signaling pathways at the PS stage effectively promotes the definitive hematopoietic differentiation.

Figure 5.

Open in a new tab

The differentiation was induced in the presence of different combinations of CHIR99021 (3μM), ActivinA (2ng/ml) or BMP4 (10ng/ml) between day 0 and day 2. After 2 days, CHIR99021, Activin A and BMP4 were replaced by VEGF and bFGF. On day 6, hematopoietic growth factors were included in the differentiation medium. (A) and (B) The frequency and number of CD43+ HPCs on day 6. n=3. (C) The representative images and typical morphology at different stages of hematopoietic differentiation under the combination of CHIR99021 (3μM), ActivinA (2ng/ml) and BMP4 (10ng/ml) between day 0 and day 2. Semi-adherent hematopoietic clusters were emerged at day 6. Scale bars, 50 µm. n=3. (D) and (E) The frequency and number of CD43+ HPCs on day 6 after 2 days treatment with SB-431542, IWP2, LDN-193189 during primitive streak specification (day0-day2). n=3. (F) Representative flow cytometric analysis of the frequency of CD34+CD45+ subpopulations of day 6 CD43+ cells or day 8 CD43+ cells. (G) CFC assay of hematopoietic cells of day 6 and day 8. CFUs per 2000 cells plated. n=3. BFU-E, Burst-forming unit-erythroid; CFU-M, Colony-forming unit- macrophage; CFU-GM, Colony-forming unit-granulocyte, macrophage. (H) The hematopoietic cells of day 6 and day 8 were analyzed for T cell potential after 21days of OP9-DLL1 co-culture. Data are shown as means ±SEM. Statistical analysis was done by two-tailed Student’s t test. *P<.05; **P<.01; ***P<.001.

4 |. DISCUSSION

Human embryonic hematopoiesis is a complex process controlled by the concerted actions of many signaling pathways, and elucidating underlying mechanism of hematopoietic differentiation would allow us to establish a strategy to efficiently generate functional hematopoietic cells from hPSCs. By using hPSC-derived hematopoietic differentiation system in vitro, the mechanisms involved in various stages of hematopoietic development, including mesoderm induction, HEP generation and EHT, has been well studied (Choi et al., 2012; Ditadi et al., 2015; Rafii et al., 2013; Sturgeon et al., 2014). However, as the earliest stage of hematopoietic commitment, few studies have focused on the PS stage. Although studies on developmental biology and embryology have offered a great deal of knowledge about key signaling pathways involved in PS formation, there is little known about whether the PS produced under different stimulation conditions has the same hematopoietic potential. Here, we focused on the stage of PS to explore the effects of different signaling pathways on the development of hematopoietic-fated PS. We demonstrate for the first time that the fate of hematopoietic differentiation of hPSC is determined as early as the PS stage and the specification of PS with hematopoietic potential is regulated cooperatively by balanced Wnt, Activin and BMP signaling (Figure 6).

Figure 6. Model of hematopoietic-fated PS specification from hPSCs by the orchestrated balance of Wnt, Activin and BMP signaling.

Figure 6.

Open in a new tab

Combined Wnt, Activin/Nodal, and BMP signaling effectively promote the formation of hematopoietic-fated PS and its hematopoietic specification. Increasing the levels of Activin or BMP signaling will not affect the development of PS, but displays a strong posteriorizing effect on subsequent hematopoietic specification. High levels of Activin signaling inhibit EHT without affecting mesoderm and HE production, while high BMP signaling impairs PS-derived mesoderm induction. High levels of Wnt signaling impair the development of PS and its subsequent hematopoietic transformation.

Studies using different model systems have shown that Wnt signaling is indispensable in the specification and formation of PS. Mice lacking Wnt3 (Wnt ligand), Lrp6 (Wnt receptor), or β-catenin (intracellular component of Wnt signaling) fail to develop a PS (Huelsken et al., 2000; Kelly et al., 2004; Liu et al., 1999). In mouse ES cells (mESCs), activation of Wnt signaling promotes differentiation toward PS and its derivative tissues (Lengerke et al., 2008; Nostro et al., 2008). Our results of hPSC differentiation are consistent with these previous findings. In addition, we show for the first time that increasing the levels of Wnt signaling results in a decreased number of T-GFP+ PS, and the residual PS acquires a reduced hematopoietic differentiation potential (Figure 6). Although it is unclear whether the reduced PS survival is caused by increased levels of Wnt signaling itself or a side effect of the small molecule of CHIR99021, the hematopoietic differentiation ability of the PS produced under high Wnt signaling is impaired.

Activin/Nodal signaling has long been established as essential pathway in gastrulation and upon activation of Activin/Nodal signaling, Smad2/3, together with Smad4, bind to the promoters of PS signature genes and induce their transcription, such as Brachyury (T), Eomes, and Mixl1 (Conlon et al., 1994; Fei et al., 2010; Mendjan et al., 2014). Nodal mutant or Smad2-deficient mice has been reported failing to form PS (Brennan et al., 2001; Heyer et al., 1999). In mESCs or human ES cells (hESCs), Activin/Nodal signaling is required for PS differentiation and the PS can be hindered when Activin/Nodal signaling is inhibited by the small-molecule SB-431542 or when Smad2 is knocked down (Bernardo et al., 2011; Brown et al., 2011; Singh et al., 2012). Our studies in hPSCs also support that Activin/Nodal signaling is essential for PS development. Interestingly, by varying the Activin A concentration, we found that increasing concentrations of Activin A did not affect the development of PS and its specification to mesoderm or HEPs, but inhibited hematopoiesis by impairing the EHT (Figure 6).

The role of the BMP signaling on PS formation is controversial. Targeting studies in the mouse have shown that mutations either in BMP4, BMP receptor 1a, or BMP receptor 2 compromise gastrulation, but conditional inactivation of BMP receptor 1a specifically in the epiblast does not disrupt PS formation (Beppu et al., 2000; Mishina et al., 1995; Miura et al., 2006; Winnier et al., 1995). During in vitro mESC differentiation, BMP signaling is not required for PS induction (Nostro et al., 2008), which is also confirmed in our studies using hPSCs. Interestingly, compared with BMP4-primed PS, the PS generated without BMP4 lacks the hematopoietic potential, indicating that the BMP signaling displays a strong posteriorizing effect on the PS to acquire hematopoietic property. The indirect role of BMP signaling on the development of PS may be through the endogenous activation of the Nodal and Wnt pathways (Nostro et al., 2008).

PS formation during mammalian embryogenesis is regulated by integration of multiple signaling pathways. Both Activin A and Wnt3a have been reported to accelerate PS formation, whereas inhibition of either Activin or Wnt signaling blocks this process suggesting that Activin and Wnt specify PS in a cooperative manner (Lindsley et al., 2006; Naito et al., 2006; Yu et al., 2011). In our study using hPSC system, we found that Wnt or Activin alone had the ability to promote the development of PS, and the combination of Wnt and Activin further enhanced the production of PS. However, neither a single signaling nor the combined two signaling was sufficient to make PS acquire hematopoietic properties. BMP and Wnt have been identified to specify hematopoietic fate by activation of the Cdx-Hox pathway in mESC model (Lengerke et al., 2008). Another mESC study indicated that Wnt and Activin, but not BMP4, were required for PS formation, whereas all three factors appear to function in the induction of hematopoietic lineage (Nostro et al., 2008). Although in hPSC model, Wnt and BMP signaling have been identified collaboratively to promote hematopoietic development (Wang and Nakayama, 2009; Wang et al., 2010), the hematopoietic potential of PS has not been well studied. In our study, we demonstrate for the first time that BMP4 is not required for hPSC-derived PS generation, but is crucial for PS acquiring hematopoietic potential by synergizing Wnt and Activin signaling. Collectively, our findings demonstrate that human hematopoietic-fated PS is defined by the orchestrated balance of Wnt, Activin and BMP signaling pathways, and our investigation of generating hematopoietic-fated PS would provide a new strategy for efficiently producing functional hematopoietic cells from hPSCs.

Supplementary Material

Supp Figures
Supp TableS1

ACKNOWLEDGEMENTS

This work was supported by grants from National Key Research and Development Program of China Stem Cell and Translational Research (2017YFA0103102, 2016YFA0100600, and 2017YFA0103400), Ministry of Science and Technology of China (2015CB964902), National Natural Science Foundation of China (81421002), CAMS Initiative for Innovative Medicine (2016-I2M-1–017), Chun Miao Foundation of the First Central Hospital of Tianjin (TFCHCM201808), and National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Disease grant R01DK106109 (Z.W).

Footnotes

CONFLICTS OF INTEREST

No competing financial interests exist.

ETHICAL STATEMENT AND CLINICAL TRIAL NUMBER

None

REFERENCES

  1. Bai H, Liu Y, Xie Y, Hoyle DL, Brodsky RA, Cheng L, Cheng T, Wang ZZ. 2016. Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells. Journal of cellular physiology 231(5):1065–1076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bai H, Xie YL, Gao YX, Cheng T, Wang ZZ. 2013. The balance of positive and negative effects of TGF-beta signaling regulates the development of hematopoietic and endothelial progenitors in human pluripotent stem cells. Stem cells and development 22(20):2765–2776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beppu H, Kawabata M, Hamamoto T, Chytil A, Minowa O, Noda T, Miyazono K. 2000. BMP type II receptor is required for gastrulation and early development of mouse embryos. Developmental biology 221(1):249–258. [DOI] [PubMed] [Google Scholar]
  4. Bernardo AS, Faial T, Gardner L, Niakan KK, Ortmann D, Senner CE, Callery EM, Trotter MW, Hemberger M, Smith JC, Bardwell L, Moffett A, Pedersen RA. 2011. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell stem cell 9(2):144–155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DY, Traver D. 2010. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464(7285):108–111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brennan J, Lu CC, Norris DP, Rodriguez TA, Beddington RS, Robertson EJ. 2001. Nodal signalling in the epiblast patterns the early mouse embryo. Nature 411(6840):965–969. [DOI] [PubMed] [Google Scholar]
  7. Brown S, Teo A, Pauklin S, Hannan N, Cho CH, Lim B, Vardy L, Dunn NR, Trotter M, Pedersen R, Vallier L. 2011. Activin/Nodal signaling controls divergent transcriptional networks in human embryonic stem cells and in endoderm progenitors. Stem cells 29(8):1176–1185. [DOI] [PubMed] [Google Scholar]
  8. Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA. 2009. Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature 457(7231):887–891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Choi KD, Vodyanik MA, Togarrati PP, Suknuntha K, Kumar A, Samarjeet F, Probasco MD, Tian S, Stewart R, Thomson JA, Slukvin II. 2012. Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures. Cell reports 2(3):553–567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conlon FL, Lyons KM, Takaesu N, Barth KS, Kispert A, Herrmann B, Robertson EJ. 1994. A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120(7):1919–1928. [DOI] [PubMed] [Google Scholar]
  11. Ditadi A, Sturgeon CM, Tober J, Awong G, Kennedy M, Yzaguirre AD, Azzola L, Ng ES, Stanley EG, French DL, Cheng X, Gadue P, Speck NA, Elefanty AG, Keller G. 2015. Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages. Nature cell biology 17(5):580–591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ema M, Takahashi S, Rossant J. 2006. Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. Blood 107(1):111–117. [DOI] [PubMed] [Google Scholar]
  13. Fei T, Zhu S, Xia K, Zhang J, Li Z, Han JD, Chen YG. 2010. Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells. Cell research 20(12):1306–1318. [DOI] [PubMed] [Google Scholar]
  14. Gori JL, Butler JM, Chan YY, Chandrasekaran D, Poulos MG, Ginsberg M, Nolan DJ, Elemento O, Wood BL, Adair JE, Rafii S, Kiem HP. 2015. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. The Journal of clinical investigation 125(3):1243–1254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heyer J, Escalante-Alcalde D, Lia M, Boettinger E, Edelmann W, Stewart CL, Kucherlapati R. 1999. Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis. Proceedings of the National Academy of Sciences of the United States of America 96(22):12595–12600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W. 2000. Requirement for beta-catenin in anterior-posterior axis formation in mice. The Journal of cell biology 148(3):567–578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kelly OG, Pinson KI, Skarnes WC. 2004. The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice. Development 131(12):2803–2815. [DOI] [PubMed] [Google Scholar]
  18. Kennedy M, Awong G, Sturgeon CM, Ditadi A, LaMotte-Mohs R, Zuniga-Pflucker JC, Keller G. 2012. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell reports 2(6):1722–1735. [DOI] [PubMed] [Google Scholar]
  19. Kispert A, Herrmann BG. 1994. Immunohistochemical analysis of the Brachyury protein in wild-type and mutant mouse embryos. Developmental biology 161(1):179–193. [DOI] [PubMed] [Google Scholar]
  20. Lawson KA, Meneses JJ, Pedersen RA. 1991. Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development 113(3):891–911. [DOI] [PubMed] [Google Scholar]
  21. Lengerke C, Schmitt S, Bowman TV, Jang IH, Maouche-Chretien L, McKinney-Freeman S, Davidson AJ, Hammerschmidt M, Rentzsch F, Green JB, Zon LI, Daley GQ. 2008. BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell stem cell 2(1):72–82. [DOI] [PubMed] [Google Scholar]
  22. Lindsley RC, Gill JG, Kyba M, Murphy TL, Murphy KM. 2006. Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm. Development 133(19):3787–3796. [DOI] [PubMed] [Google Scholar]
  23. Liu P, Wakamiya M, Shea MJ, Albrecht U, Behringer RR, Bradley A. 1999. Requirement for Wnt3 in vertebrate axis formation. Nature genetics 22(4):361–365. [DOI] [PubMed] [Google Scholar]
  24. Lolas M, Valenzuela PD, Tjian R, Liu Z. 2014. Charting Brachyury-mediated developmental pathways during early mouse embryogenesis. Proceedings of the National Academy of Sciences of the United States of America 111(12):4478–4483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McReynolds LJ, Gupta S, Figueroa ME, Mullins MC, Evans T. 2007. Smad1 and Smad5 differentially regulate embryonic hematopoiesis. Blood 110(12):3881–3890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mendjan S, Mascetti VL, Ortmann D, Ortiz M, Karjosukarso DW, Ng Y, Moreau T, Pedersen RA. 2014. NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency. Cell stem cell 15(3):310–325. [DOI] [PubMed] [Google Scholar]
  27. Mishina Y, Suzuki A, Ueno N, Behringer RR. 1995. Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes & development 9(24):3027–3037. [DOI] [PubMed] [Google Scholar]
  28. Miura S, Davis S, Klingensmith J, Mishina Y. 2006. BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites. Development 133(19):3767–3775. [DOI] [PubMed] [Google Scholar]
  29. Naito AT, Shiojima I, Akazawa H, Hidaka K, Morisaki T, Kikuchi A, Komuro I. 2006. Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proceedings of the National Academy of Sciences of the United States of America 103(52):19812–19817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nostro MC, Cheng X, Keller GM, Gadue P. 2008. Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell stem cell 2(1):60–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Paes B, Moco PD, Pereira CG, Porto GS, de Sousa Russo EM, Reis LCJ, Covas DT, Picanco-Castro V. 2017. Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation. Cell biology and toxicology 33(3):233–250. [DOI] [PubMed] [Google Scholar]
  32. Rafii S, Kloss CC, Butler JM, Ginsberg M, Gars E, Lis R, Zhan Q, Josipovic P, Ding BS, Xiang J, Elemento O, Zaninovic N, Rosenwaks Z, Sadelain M, Rafii JA, James D. 2013. Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition. Blood 121(5):770–780. [DOI] [PubMed] [Google Scholar]
  33. Schulte-Merker S, Lee KJ, McMahon AP, Hammerschmidt M. 1997. The zebrafish organizer requires chordino. Nature 387(6636):862–863. [DOI] [PubMed] [Google Scholar]
  34. Singh AM, Reynolds D, Cliff T, Ohtsuka S, Mattheyses AL, Sun Y, Menendez L, Kulik M, Dalton S. 2012. Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation. Cell stem cell 10(3):312–326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sturgeon CM, Ditadi A, Awong G, Kennedy M, Keller G. 2014. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nature biotechnology 32(6):554–561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sumi T, Tsuneyoshi N, Nakatsuji N, Suemori H. 2008. Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling. Development 135(17):2969–2979. [DOI] [PubMed] [Google Scholar]
  37. Tam PP, Beddington RS. 1987. The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development 99(1):109–126. [DOI] [PubMed] [Google Scholar]
  38. Tam PP, Loebel DA. 2007. Gene function in mouse embryogenesis: get set for gastrulation. Nature reviews Genetics 8(5):368–381. [DOI] [PubMed] [Google Scholar]
  39. Uenishi G, Theisen D, Lee JH, Kumar A, Raymond M, Vodyanik M, Swanson S, Stewart R, Thomson J, Slukvin I. 2014. Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions. Stem cell reports 3(6):1073–1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vallier L, Touboul T, Chng Z, Brimpari M, Hannan N, Millan E, Smithers LE, Trotter M, Rugg-Gunn P, Weber A, Pedersen RA. 2009. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PloS one 4(6):e6082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vodyanik MA, Thomson JA, Slukvin II. 2006. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Blood 108(6):2095–2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walmsley M, Ciau-Uitz A, Patient R. 2002. Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus. Development 129(24):5683–5695. [DOI] [PubMed] [Google Scholar]
  43. Wang L, Chen YG. 2016. Signaling Control of Differentiation of Embryonic Stem Cells toward Mesendoderm. Journal of molecular biology 428(7):1409–1422. [DOI] [PubMed] [Google Scholar]
  44. Wang L, Li L, Shojaei F, Levac K, Cerdan C, Menendez P, Martin T, Rouleau A, Bhatia M. 2004. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 21(1):31–41. [DOI] [PubMed] [Google Scholar]
  45. Wang Y, Nakayama N. 2009. WNT and BMP signaling are both required for hematopoietic cell development from human ES cells. Stem cell research 3(2–3):113–125. [DOI] [PubMed] [Google Scholar]
  46. Wang Y, Umeda K, Nakayama N. 2010. Collaboration between WNT and BMP signaling promotes hemoangiogenic cell development from human fibroblast-derived iPS cells. Stem cell research 4(3):223–231. [DOI] [PubMed] [Google Scholar]
  47. Winnier G, Blessing M, Labosky PA, Hogan BL. 1995. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes & development 9(17):2105–2116. [DOI] [PubMed] [Google Scholar]
  48. Woll PS, Morris JK, Painschab MS, Marcus RK, Kohn AD, Biechele TL, Moon RT, Kaufman DS. 2008. Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells. Blood 111(1):122–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yu P, Pan G, Yu J, Thomson JA. 2011. FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation. Cell stem cell 8(3):326–334. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Supp Figures
NIHMS1009458-supplement-Supp_Figures.docx (291.5KB, docx)
Supp TableS1
NIHMS1009458-supplement-Supp_TableS1.docx (13.3KB, docx)

RESOURCES