Isolation, culture and 48 h induction of neural stem cells
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For 48 h induction using serum-free conditions with growth factors, dissociated neurospheres were cultured with recombinant human (rh) BMP4 250 ng/mL , rh Activin A 100 ng/mL and rh bFGF 100 ng/mL individually as well as in combinations as indicated in Supporting Information Fig S1 and
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Blimp1−/− EpiSC Derivation and Culture
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EpiSCs were derived from E6.5 epiblasts by culturing on MEFs in medium'>N2B27 medium containing human activin A (20 ng/ml), bFGF (12 ng/ml), and KSR (20%) (
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Directed differentiation of hESC-derived cysts to RPE using transwell filters.
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Ac+/Ac−: with or without Activin A.
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Loss of REX1 within the pluripotent population primes cells for differentiation.
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n = 3 E) Fold enrichment of the percentage of GATA4 positive endoderm cells generated from TRA+VEN− cells relative to those from TRA+VEN+ population after 3 days of treatment with Activin A and BMP4 in low serum media, as observed by immunocytochemistry.
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K02288 selectively inhibits BMP signaling.
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Activin A-induced P-Smad2 in HEK293 cells was weakly inhibited by K02288 and LDN-193189.
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Differentiation from human iPS cells toward hepatic lineage cells.
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Human iPS cells were sequentially stimulated with various cytokines: (1) activin A, (2) basic FGF and BMP-4, and (3) HGF.
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WNT3 Is a Functional Biomarker for Predicting the DE Differentiation Potential (A) WNT3 levels in control and various knockdown HUES8 sublines correlate with their DE differentiation efficiency.
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The four protocols included the following treatments in a serum-free medium: (1) A - > A (Activin A only for 4 days); (2) AW - > A (Activin A and Wnt3a for 2 days followed by Activin A only for another 2 days); (3) A - > AX (Activin A only for 2 days followed by Activin A and XAV 939 for another 2 days); and (4) AW - > AX (Activin A and Wnt3a for 2 days followed by Activin A and XAV 939 for another 2 days).
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Human ES cell culture and differentiation
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Human H1, H9 and UCLA1-6 (UCLA stem cell core) [–1 bFGF .
- EBs were differentiated according to previous reports with minor modification [μM Y27632 (Rho kinase inhibitor IV, EMD Chemicals), 3 ng ml−1 Activin A , 5 ng ml−1 bFGF, 10 ng ml−1 BMP4 and 5 μM IWR-1 (EMD Chemicals), for the first 4 days.
- EBs were then cultured in StemPro 34 supplemented with 10 ng ml−1 bFGF, 5 ng ml–1 vascular endothelial growth factor (VEGF) and 5 μM IWR-1 for 5 additional days before plating onto elastic substrates.
- Usage of all human ES cell lines is approved by the UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee and the Institutional Review Boards (IRB, approval #2009-006-04).
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Induction of hESCs into primitive streak (PS) cells.
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(b) Comparison of expression levels of PS specific genes by combinatorial treatments with exogenous factors of Activin A, Wnt3a and BMP4, FGF2 , in hESC-derived PS cells.
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Differentiation of human ESC and iPSC into definitive endoderm
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Differentiation into definitive endoderm was performed as previously described.
- Briefly, pluripotent stem cells were harvested, gently triturated into single cell suspensions and seeded onto transwells (0.4μm pore size, Corning) pre-coated with human placental collagen Type IV, which has previously been shown to support airway epithelial cell growth.
- The cells were immediately treated with 100ng/ml Activin-A and 25ng/ml WNT3A for 4 consecutive days in Endoderm ifferentiation Media consisting of serum-free Knockout MEM with Glutamax , penicillin/streptomycin (GIBCO), 1 mM nonessential amino acids (GIBCO) and 0.5 mM mercaptoethanol.
- Subsequent differentiation steps were performed on the transwells.
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