Fig 2: The differentiation status of megakaryocytes in three cell culture batches (MK1-3) assessed by flow cytometry. Of the three megakaryocytic cell culture batches from three different healthy adult HSPC donors (MK1-3), MK3 has both the most megakaryocytes and the most mature megakaryocytes after 9 days of differentiation. The results were based on flow cytometry expression of the cell surface antigens CD34, CD41a, CD42a, a marker of true megakaryocytic commitment, and CD42b, a marker of more mature megakaryocytes

Fig 3: Pluripotency marker expression and trilineage differentiation capability confirmed in the established human induced pluripotent stem cell (hiPSC) lines. a Fluorescent images of live colonies stained with anti-TRA-1-60 antibody (red, left), anti-TRA-1-81 antibody (green, middle), or the CDy1 dye (red, right). Phase-contrast images are also shown in a side-by-side fashion for each picture. Scale bars indicate 200 µm. b Graph demonstrating the quantification results of OCT4 and NANOG transcripts in the TkPP2 lines, assessed by RT-qPCR analysis. The relative expression values for each gene, normalized to the level observed in the standard TkDA3-4 hiPSC sample, are shown. CD34+-enriched cells derived from the TkPP2 donor sample were used as the negative control (CD34). c Scatter plot showing a comprehensive representation of the relative expression values of OCT4 and NANOG in the 85 hiPSC lines examined. The results of 76 patient-derived hiPSCs are shown as light green plots. The results of 9 TkPP2 lines (same data in b) are shown in dark green for comparison. d Graphs showing the relative expression values of each marker gene calculated by the ??Ct method with CFX™ Manager software. The plots (14 hiPSC lines) are colored according to the culture condition shown at the bottom. “Undifferentiated” (green) represents hiPSC samples cultured in a pluripotent state. The other samples were cells cultured under differentiation conditions, with each being directed for the corresponding lineage (ectodermal, endodermal, or mesodermal)

Fig 4: Healthy donor-derived human-induced pluripotent stem cell (hiPSC) generation from non-mobilized peripheral blood (PB)-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) using SeVdp(KOSM)-302L. a Schematic diagram illustrating the schedule of hiPSC generation. b Percentages of cells expressing CD34, as assessed by FACS analysis of non-enriched peripheral blood mononuclear cells (PBMCs), a flow-through population (flow-through), and the CD34+-selected cells (CD34+ cells). The results demonstrate significant enrichment of the CD34+ cells (up to 60.8%) after immunomagnetic bead selection. c Sequential images of a representative colony derived from SeVdp(KOSM)-302L-transduced CD34+ cells, showing a phase of initial proliferation (day 1–day 4), followed by the formation of spherical colony-like structures (day 5–day 11). Also shown are images of typical hiPSC-like colonies that appeared during the subsequent expansion phase (day 17 and day 37). Magnified images are shown in insets for clarity. P1 and P4 indicate passage 1 and passage 4, respectively. d Colony formation efficiency of each seeded cell type. PBMCs, flow-through cells, and CD34+ cells were tested after infection with SeVdp(KOSM)-302L. Each bar represents the efficiency assessed in each individual well. The mean efficiency values for PBMCs (0.17%), flow-through (0%), and CD34+ PBMCs (5.58%) are shown. e Fluorescent images of live colonies stained with anti-TRA-1-60 antibody (red, top), anti-TRA-1-81 antibody (green, middle), or the CDy1 dye (red, bottom). Nuclei were stained with Hoechst 33342 dye (blue)

Fig 5: Relation between mature megakaryocytes and specific substrate uptake. The specific substrate uptake of serotonin (left y-axis) and MIBG (right y-axis) was plotted against the percentage of mature megakaryocytes (CD34-/CD41a+/CD42a+/CD42b+) in the culture batch. Data from megakaryocytic cell culture batches from three different healthy adult HSPC donors (MK1-3) are plotted (mean ± SD)