Rhbmp 4

Human CD34+ cells, isolated from peripheral blood of granulocyte colony-stimulating factor-mobilized healthy volunteers, were purchased from the Fred Hutchinson Cancer Research Center. The cells were maintained and differentiated as previously described^{28 (link),73} . Briefly the cells were expanded in StemSpan medium (Stem Cell Technologies Inc.) supplemented with StemSpan CC100 cytokine mix (Stem Cell Technologies Inc.) and 2% P/S for a total of 6 days. After six days of expansion the cells were stimulated for 2 h with rhBMP4 (R&D) at a final concentration of 25 ng/ml and harvested for performing all the experiments corresponding to D0 time point. For studying differentiated cells after day 6 of expansion, cells were reseeded in differentiation medium (StemSpan SFEM Medium with 2% P/S, 20 ng/ml SCF, 1 U/ml Epo, 5 ng/ml IL-3, 2 mM dexamethasone, and 1 mM b-estradiol), at a density of 0.5–13 10⁶ cells/ml. Prior to harvesting at H2, H6, D1-D8 the cells were treated with 25ng/ml hrBMP4 for 2hrs.
For testing the effects of BMP4 and dorsomorphin, cells at the beginning of the third day of differentiation were treated with either 25 ng/ml hrBMP4 or 20 μM dorsomorphin until the beginning of the fifth day of differentiation. At D5, cells were isolated for flow cytometry and qPCR analysis. Cells treated with DMSO were used for control experiments.

Mesoderm commitment was induced as previously described^{9 (link),79 (link),80 (link)} with certain optimizations. Briefly, hESC cells were maintained on Matrigel-coated 6-well plates in mTeSR Plus complete medium. At day (D) −18, mesoderm induction was initiated in X-VIVO 15 medium (Lonza, Cat. 04–418Q) supplemented with recombinant human (rh) Activin A (10 ng/ml) (R&D Systems, Cat. 338-AC-010), rhBMP4 (10 ng/ml) (R&D Systems, Cat. 314-BP-010), rhVEGF (10 ng/ml) (R&D Systems, Cat. 298-VS-005), rhFGF (10 ng/ml) (R&D Systems, Cat. 233-FB-025), and ROCK inhibitor Y-27632 dihydrochloride (10 μM) (Tocris, Cat. 1254). hESCs were plated on Matrigel coated 6-well plates at 3×10⁶ cells per well in 3ml. Medium was then changed daily with X-VIVO 15 supplemented with rhBMP4 (10 ng/ml), rhVEGF (10 ng/ml), and rhFGF (10 ng/ml). At D-14, cells were washed with PBS and detached with Accutase (Innovative Cell Technologies, Cat. AT-104) (1 mL per well, for 10 min at 37°C). Cells were harvested and hEMPs isolated by depletion of CD326+ cells by magnetic cell sorting (MACS) using CD326 (EpCAM) MicroBeads (Miltenyi, Cat. 130–061-101).

Mesoderm commitment was induced as previously described (Chin et al., 2016 (link);
Evseenko et al., 2010 ) with certain optimizations. Briefly, hESC colonies were
maintained on Matrigel-coated 6-well plates in mTesSR complete medium. hESC/hIPSC cells were then harvested as a single cell
suspension after TrypLE™ Express (Gibco Life Technologies Ref 12604–013) treatment for 5 minutes at 37°C,
washed, and counted. Cells were resuspended directly in X-VIVO 15 medium (Lonza Ref 04–418Q) supplemented with
rhActivin A (10 ng/ml) (R&D Systems, Cat. 338-AC-010), rhBMP4 (10 ng/ml) (R&D Systems, Cat. 314-BP-010), rhVEGF (10
ng/ml) (R&D Systems, Cat. 298-VS-005), rhFGF (10 ng/ml) (R&D Systems, Cat. 233-FB-025), and ROCK inhibitor Y-27632
dihydrochloride (10 uM) (Tocris, Cat. 1254). Cells were plated on Matrigel coated 6-well plates at 3×10⁶cells per well in 3ml. Medium was then changed daily with X-VIVO 15 supplemented with rhBMP4 (10 ng/ml), rhVEGF (10 ng/ml, and
rhFGF (10 ng/ml). At day 3.5, cells were washed 3 times with PBS and incubated with Accutase (Innovative Cell Technologies,
Cat. AT-104) (1 ml per well, for 10 min. at 37°C). Cells were harvested using a cell scraper, washed in PBS, and
stained with antibodies for flow cytometry. CD326⁻CD56⁺ hEMP were isolated by FACS on a FACSARIA
instrument (BD Biosciences, San Jose, CA).