Fig 2: The redifferentiation of ?dT-iPSCs into hematopoietic lineage. (A): A schematic diagram showing the redifferentation protocol for deriving hematopoietic progenitor cells from ?dT-iPSCs. (B): The flow cytometric analysis for a hematovascular precursor marker (APJ) in ?dT-iPSC-derived cells. The percentages of APJ-positive cells are shown. (C): The flow cytometric analysis for hematopoietic lineage markers (CD34 and CD43) in ?dT-iPSC-derived cells. The percentages of CD34/CD43 double-positive cells are shown. Abbreviations: iPSCs, induced pluripotent stem cells.

Fig 3: Co-culture of immune cell progenitors and Mk-biased HSPCs. a Experimental strategy. Schematic illustration of the transwell co-culture systems. Related to Fig. 6. b, c The proportion of CD41a+CD61+ cells derived from CD9+Lin-CD34+CD45RA- HSPCs co-cultured with pre-B cells of HC and ITP, respectively. Two-tailed Student’s t test; *p < 0.05; n = 5. d, e The proportion of CD41a+CD61+ cells derived from CD9+Lin-CD34+CD45RA- HSPCs co-cultured with NK/Tp cells of HC and ITP, respectively. Two-tailed Student’s t test; ns, not significant; n = 6

Fig 4: Single-Cell Analysis of DC3 Commitment(A) Flow cytometry analysis of bulk (500 cells) or single CD34+CD38+CD123-CD64- progenitor cells cocultured for 14 days with MS5_FS12 supplemented with recombinant human GM-CSF (MS5_FS12+recGM-CSF). Flow cytometry plots resulting from single CD34+CD38+CD123-CD64- progenitor cells with different potentials are shown as representative examples (n = 355 cells from 2 independent experiments).(B) HC of lineage potential from single CD34+CD38+CD123-CD64- progenitor cells (n = 355).(C) Bar graph and Venn diagram summarizing the frequency of the potential of mono-, bi-, tri-, or multipotent individual CD34+CD38+CD123-CD64- cells within the total wells analyzed (n = 355).(D) Bar graphs summarizing the frequency of mono-, bi-, tri-, or multipotent individual CD34+CD38+CD123-CD64- cells among DC3-generating progenitors only. An orange bar represents the frequency of DC3-restricted progenitors.(E) Cell surface phenotype of DC3-restricted progenitors before differentiation cultures inferred by index flow cytometry sorting. tSNE plots display an overlay of total CD45+ cells (gray) and DC3-restricted progenitor cells (orange) (top left). Shown is relative expression of the markers CD45RA, CD38, CD34, CD10, Clec12A, CD64, CD123, CD163, and SIRPa.(F) Validation experiment for identification of Clec12A as a marker for DC3-committed progenitors. Shown is flow cytometry analysis of bulk-sorted CD34+CD38+CD45RA+CD123-CD64-Clec12A- and Clec12A+ cells. 500 cells were cocultured with MS5_GM for 7 days. The bar graph summarizes the number of differentiated DC3s from each bulk population (n = 4 healthy donors, *p < 0.05, Mann-Whitney two-tailed t test).See also Figure S5.

Fig 5: JNK-IN-8 promotes expansion of HSCs in primary recipients.a Human CD45+ engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34+ cells on 5, 9, 12, 16 weeks posttransplantation (n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where *p < 0.05, **p < 0.01. b Representative flow plots of engraftment of J8-expanded and DMSO-expanded CD34+ cells in mouse recipients’ PB 16 weeks after transplantation. Progenies of 10,000 day 0 CD34+ cells after 10-day culture with DMSO or J8 were transplanted per mouse. c HSC frequencies of fresh CD34+ cells, J8, and DMSO cultured cells determined by LDA, calculated by ELDA software. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0055. The frequency of more than 1% human chimerism (human CD45 compared with all CD45) in the BM was regarded as positive response. d HSC frequencies presented as 1/fresh CD34+ cells equivalent for each condition from (c), the required confidence interval was 95%. See Supplementary Table S3A. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05, and **p < 0.01. See also Supplementary Fig. S2