Fig. 7: ROCK inhibition promotes the generation of definitive HSPCs from hESCs.

a, b Expression profiles of genes significantly upregulated or downregulated along the pseudotime progression. Based on changing profile, genes were clustered hierarchically into four different patterns (b). Lines show the mean values of scaled expression levels (TPM, z-normalized) in each pattern. c Featured GO terms enriched in genes of each pattern. Color displays P-value (Log-scaled) of GO terms. d KEGG enriched genes in pattern 1 indicated in b. e Expression profiles of ROCK pathway-related genes along the pseudotime. Each dot represents one cell and color represents pseudotime value. Line shows the mean expression level (TPM, Log-scaled). f Violin plots show expression levels (TPM, Log-scaled) of these ROCK signaling genes in early definitive and primitive HSPCs identified in human embryo. g qRT-PCR analysis of the ROCK pathway-related genes in CD43⁺CD44⁺ or CD43⁺CD44⁻ HPCs sorted at differentiation day 8. The significance level was determined using unpaired two-tailed Student’s t-tests, *P < 0.05, **P < 0.01, ***P < 0.001. The data represent mean ± SD from three independent replicates (n = 3). h Analysis of ROCK inhibition in hESC differentiation. The chemical inhibitor, Y-27632, was added in EHT medium at differentiation day 4 as indicated. The adherent or floating cells at differentiation day 8 were analyzed by FACS for the expression of CD31, CD43, and CD44. Right panel: quantitative data of each indicated population at differentiation day 8 of hESCs. The significance level was determined using unpaired two-tailed Student’s t-tests, ***P < 0.001. The data represent the mean ± SD from three independent replicates (n = 3). i Function Analysis of CD43⁺ HSPCs generated under ROCKi treatment. Y-27632 was added in EHT medium at differentiation day 4 as indicated. The floating CD43⁺ cells at differentiation day 8 were counted and co-cultured with MS5 with 10,000 cells per well. Human CD45⁺ cells were gated 2 weeks after differentiation and analyzed for the known lineage markers as indicated. My, myeloid; Ly, lymphoid. The significance level was determined using unpaired two-tailed Student’s t-tests, *P < 0.05. The data represent mean ± SD from three independent replicates (n = 3).