Proof of iPSC induction from hematopoietic cells in a single-HSC transplantation model. (A) Schematic representation of the experimental procedure. Single CD150⁺CD34^−/low KSL cells obtained from B6 Ly5.1 mice were transplanted into lethally irradiated B6 Ly5.2 mice together with BM cells from B6 Ly5.2 mice. BM HSPCs were obtained from a recipient mouse that showed long-term (∼ 10 months) stable Ly5.1 chimerism (∼ 80%), enriched for Ly5.1⁺ cells, and subjected to iPSC generation. (B) A schematic diagram of iPSC generation from BM HSPCs. (i) Lineage markers (Lin) versus c-Kit plots are shown for cells either before (whole BM) or after (Lin⁻c-Kit⁺) purification. Note that purified HSPCs are 98% CD45-positive. (ii) A schematic diagram of iPSC generation from BM HSPCs. (C) Typical ES cell–like appearance of sHSC-iPS cell colonies (left) with high ALP activities (right). Bars represent 100 μm. (D) Determination of the cellular origin of sHSC-iPSC clones. (Top panel) Scheme of the polymerase chain reaction (PCR)–based method used, using a single-base polymorphism at Cd45 exon (EX) 25. Black triangles represent primer positions. Ly5.1 and Ly5.2 strains differ by a single base in EX 25, as shown in the presented 12-bp sequences from within the 301-bp amplicons. Treatment with the restriction enzyme KpnI leaves the Ly5.1⁺ cell–derived amplicon undigested, whereas it generates 2 smaller fragments (113 bp + 188 bp) from the Ly5.2⁺ counterpart. The gel images (bottom panel) indicate that, of 4 sHSC-iPSC clones, 1 (no. 9-1) is of Ly5.2⁺ cell origin, whereas 3 (nos. 9-5, -6, and -8) are derived from Ly5.1⁺ cells that originated from a single Ly5.1⁺ HSC. A vertical line has been inserted to indicate a repositioned gel lane. (E) Chimeric mice obtained by implantation of sHSC-iPSC clone 9-5 into ICR host blastocysts.