![Figure 1. Optimization of BMP4 concentration for hematopoietic specification of nonhuman primate iPSCs. (A) Schematic of EB method of hematopoietic differentiation specifying titration of BMP4 on day 0 of induction. On day −2, MniPSC line 3 colonies (passage 47) on iMEF feeders were treated with rock inhibitor (10μM Y-27632) and then passaged as clusters 1:2 onto GFR-matrigel in standard PSC media supplemented with bFGF and Y-27632. On day 0, MniPSCs clusters aggregated in media consisting of complete StemPro (cStemPro; StemPro-34 plus StemPro supplement, 2mM l-glutamine, 150 μg/mL transferrin, 50 μg/mL ascorbic acid, and 4 × 10−4M 1-thioglycerol) with different concentrations of BMP4 (10, 20, or 50 ng/mL, test conditions in bold type) and transferred to hypoxia (5% O2). On day 1, EBs were induced with cStemPro supplemented with BMP4 and bFGF. On day 4, media was changed to cStemPro containing VEGF and bFGF for induction. To induce hematopoietic cell expansion, EBs were suspended in cStemPro containing a 10-cytokine cocktail as indicated and placed in normoxia. (B) Comparative kinetics of hematopoietic specification as determined by flow cytometry analysis. On the indicated days of differentiation, EBs were dissociated, counted, stained with fluorophore-conjugated antibodies and analyzed by flow cytometry. (C) Absolute cell number of fluorophore cells. Absolute cell yields corresponding to the indicated hematoendothelial subsets were calculated (absolute positive cell yield = 100 × [% flurophore+ × viable cell yield]). Each number shown is ×106 and is representative of the total fluorpohore+ cell yield obtained from an input undifferentiated MniPS cell number of 1 million. (D) Hematopoietic colony-forming cell assays plated on the indicated days of differentiation. MniPSC-derived cells were dissociated and single cells plated in Methocult H4435. CFUs were enumerated and scored as a function of input cell number (CFUs per 105 cells plated). The results from 1 representative experiment of 3 conducted are shown. Error bars indicate SD of mean of triplicates. ANOVA significance levels: *P = .039 (day 8), *P = .016 (day 14).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/13/10.1182_blood-2012-05-433797/4/zh89991297090001.jpeg?Expires=1724221631&Signature=14X3P2hOHaTj0PZeelFlmg4rPw5L9JJsHhF8U8aD1N4j6ICwm3ztj8HYeQ3nLmBryW8fIYnGq2-3a2FAY24f0WKhrKKLO8NSPLDn2gdTMC8wF6PmzQC~jWmdIcE0x4UIVQ4l5F85RB-a8FuzzVXHnywVp8LobLBGwdyrng2Ihe19kr-hfwMoTIN5tdSo4iXHCQfe2Z61137jr48nQ35fwPSRKvg-cjGcB-JVciAGvdk4mEKPRKoZG5bx~UsyIZODoKXTKEbKbvXgzvK5AwxwVIyNeuHSdKZ-mbiGPqz-6qoLIR1DPvA~1mOyH0CAbAG2Yeqqr6snsGVHyVrxXfmJtw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Optimization of BMP4 concentration for hematopoietic specification of nonhuman primate iPSCs. (A) Schematic of EB method of hematopoietic differentiation specifying titration of BMP4 on day 0 of induction. On day −2, MniPSC line 3 colonies (passage 47) on iMEF feeders were treated with rock inhibitor (10μM Y-27632) and then passaged as clusters 1:2 onto GFR-matrigel in standard PSC media supplemented with bFGF and Y-27632. On day 0, MniPSCs clusters aggregated in media consisting of complete StemPro (cStemPro; StemPro-34 plus StemPro supplement, 2mM l-glutamine, 150 μg/mL transferrin, 50 μg/mL ascorbic acid, and 4 × 10−4M 1-thioglycerol) with different concentrations of BMP4 (10, 20, or 50 ng/mL, test conditions in bold type) and transferred to hypoxia (5% O2). On day 1, EBs were induced with cStemPro supplemented with BMP4 and bFGF. On day 4, media was changed to cStemPro containing VEGF and bFGF for induction. To induce hematopoietic cell expansion, EBs were suspended in cStemPro containing a 10-cytokine cocktail as indicated and placed in normoxia. (B) Comparative kinetics of hematopoietic specification as determined by flow cytometry analysis. On the indicated days of differentiation, EBs were dissociated, counted, stained with fluorophore-conjugated antibodies and analyzed by flow cytometry. (C) Absolute cell number of fluorophore cells. Absolute cell yields corresponding to the indicated hematoendothelial subsets were calculated (absolute positive cell yield = 100 × [% flurophore+ × viable cell yield]). Each number shown is ×106 and is representative of the total fluorpohore+ cell yield obtained from an input undifferentiated MniPS cell number of 1 million. (D) Hematopoietic colony-forming cell assays plated on the indicated days of differentiation. MniPSC-derived cells were dissociated and single cells plated in Methocult H4435. CFUs were enumerated and scored as a function of input cell number (CFUs per 105 cells plated). The results from 1 representative experiment of 3 conducted are shown. Error bars indicate SD of mean of triplicates. ANOVA significance levels: *P = .039 (day 8), *P = .016 (day 14).
