BMP4 regulates vascular progenitor development in human embryonic stem cells through a Smad-dependent pathway

Abstract

The signals that direct pluripotent stem cell differentiation into lineage-specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum-free condition, hESCs sequentially differentiated into CD34+CD31-, CD34+CD31+, and then CD34-CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFbeta suppressed BMP4-induced CD34+CD31+ cell development, and promoted CD34+CD31- cells that failed to give rise to either endothelial or smooth muscle cells. The BMP-Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad-dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells.

Fig. 1

BMP4, VEGF, and FGF2 promote development of CD34+ cells in serum-free culture. A: HESCs were maintained on Hs27 cells. The hESC differentiation was induced by switching hESC growth medium to serum-free differentiation medium with (+) or without (−) BMP4 (50 ng/ml), VEGF (50 ng/ml), FGF2 (50 ng/ml). After 12 days of differentiation, the cells were analyzed for CD34 expression by flow cytometry. Differentiation medium containing 15% defined-FBS was used as a control. Error bars represent standard deviation (* indicates P < 0.05 vs. others). B: The differentiation of hESCs was induced in serum-free media containing different concentrations of BMP4 in the presence of VEGF and FGF2, as indicated. The differentiated cells were analyzed after 12 days for CD34 expression by flow cytometry. Data are representative of three independent experiments.

Fig. 2

Characterization of hESC-ECs and hESC-SMCs in serum-free media. The differentiation of hESCs was induced in serum-free medium containing BMP4, VEGF, and FGF2. After 12 days, the isolated CD34+cells (1 × 10⁴ cells/well) were cultured on collagen I-coated 24-well plates either in SFM medium containing EC growth supplements or in MCDB 131 medium containing SMC growth supplements for 7 days. A: The expression of EC markers, VE-cad and CD31, in hESC-ECs was analyzed by cell immunohistochemistry. DAPI was used for nuclear staining. B: The Dil-Ac-LDL incorporation assay. The hESC-ECs were incubated with 2 ng/ml of Dil-Ac-LDL for 12 h. The images were obtained with a fluorescent microscopy. C: Matrigel assay for vascular network formation. The hESC-ECs were loaded on a 24-well plate containing 200 μl matrigel. The images were taken with a phase-contrast microscopy after 16 h. D: Gene expression analysis of hESC-ECs by RT-PCR. HUVECs were used as a control in A–D. E: The expression of SMC-specific markers, including SMA, calponin, and desmin, was examined in hESC-SMCs by cell immunohistochemistry. Human primary aortic SMCs were used as a control. DAPI was used for nuclear staining.

Fig. 3

Vascular progenitor cells in CD34+CD31+ population. After 12 days of hESC differentiation, CD34−CD31− cells, CD34+CD31− cells, CD34+CD31+ cells, and CD34−CD31+ cells were sorted by FACS. Real-time PCR were used to analyze EC-specific genes: VEGFR2, Tie2, VE-cad, and vWF (A), and SMC-specific genes: SMA, SM22alpha, calponin, and caldesmon (B). Undifferentiated hESCs were used as a control (hESC). GAPDH was used to normalize the gene expression. The isolated subpopulation cells (1 × 10⁴ cells/well) were cultured on collagen I-coated 24-well plates either in EC growth medium or in SMC growth medium. The CD31 and VE-cad positive cells were measured as hESC-ECs by immunohistochemistry (C). The SMA and calponin positive cells were measured as hESC-SMCs by immunohistochemistry (D). DAPI was used for nuclear staining in C. D: Error bars represent standard deviation (* P < 0.05 vs. other groups). E: Flow cytometric analyses CD34 and CD31 expression during hESC differentiation at different time points. Data are representative of three independent experiments.

Fig. 4

Sequential effects of BMP4, VEGF, and FGF2 on CD34+CD31+ progenitor cells. The hESCs were induced to differentiation in serum-free differentiation medium containing various combinations of BMP4, VEGF, and FGF2. BMP4 (B), VEGF and FGF2 (VF), and BMP4, VEGF and FGF2 (BVF) were presented for different periods during hESC differentiation. The differentiation was set for 4 stages: day 1–3, day 4–6, day 7–9, and day 10–12. The CD34+CD31+ cells were analyzed after 12 days by flow cytometry. Error bars represent standard deviation (* P < 0.05 vs. compared group). C: Schematic diagram of sequential effects of BMP4, VEGF, and FGF2 on hESC differentiation into CD34+CD31+ cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

Distinct roles of TGFβ family members in the development of CD34+CD31+ cells. A: The hESCs were differentiated in serum-free differentiation medium containing VEGF and FGF2, and different TGFβ family members. (A) The differentiation medium contained BMP2, BMP4, BMP7, or BMP9, as indicated. After 12 days, the expression of CD34 and CD31 were analyzed by flow cytometry. Error bars represent standard deviation from 3 experiments. (B) The differentiation medium contained BMP4, TGFβ1, or activin, as indicated. After 12 days, the expression of CD34 and CD31 were analyzed by flow cytometry. Data were a representative of 3 independent experiments. (C), (D), and (E) Kinetic analyses of CD34+ cells, CD31+ cells, CD34+CD31+ cells. The differentiation medium contained BMP4, TGFβ1, or activin. The CD34+ cells and CD31+ cells were analyzed by flow cytometry at different time points.

Fig. 6

Smad-dependent pathway mediates BMP4 effect on CD34+CD31+ cell generation. A: The hESCs were stimulated by BMP4 (50 ng/ml) with or without dorsomorphin (5 μM) for 30 min. The phosphorylation of Smad1/5/8 was examined by Western blot analysis. B: The hESCs were stimulated by TGFβ1 (50 ng/ml) with or without SB431542 (5 μM) for 30 min. The phosphorylation of Smad2/3 was examined by Western blot analysis. C: The hESCs were induced to differentiation in serum-free medium containing BMP4, VEGF, and FGF2 in the presence or absence of dorsomorphin (5 μM). The expression of CD34+ and CD31+ cells was analyzed after 12 days by flow cytometry. The serum-free differentiation medium without BMP4 and inhibitors was used as a control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]