Reprogramming of human CB and adult BM CD34⁺ cells to iPS cells. (A) Live culture was stained by the TRA-1-60 antibody, 3 to 4 weeks after transduction of CB CD34⁺ cells. TRA-1-60⁺ colony shown on right was first seen at week 3 and was picked at week 4 (after restaining). (B) An illustration of various colonies 4 weeks after transduction of BM CD34⁺ cells, after live staining with TRA-1-60 and a secondary fluorescent reagent. A smaller fraction of formed colonies are TRA-1-60⁺. TRA-1-60⁺ colonies were individually picked and gave rise to iPS clones. (C) Immunofluorescence staining images of expanded iPS cells from CB are shown here (BM-derived iPS cells are shown in supplemental Figure 1). In addition to TRA-1-60, they also express other pluripotency markers NANOG and SSEA4. (D) Gene expression of undifferentiated (OCT4 and NANOG) and differentiation markers in undifferentiated iPS cells (U) derived from CB and teratoma cells (T) after in vivo differentiation. The expression of α-fetoprotein (AFP, endoderm), CD34 (mesoderm), and PAX6 (ectoderm) and a housekeeping gene GAPDH was measured by RT-PCR analysis. (E) Differentiation potential of CB-derived iPS cells after in vitro differentiation by EB formation (10 days). After immunofluorescence staining, differentiated cells expressing AFP, CD34, and β-III-tubulin (an ectoderm marker) were seen. The image of specific staining is overlaid by 4,6-diamidino-2-phenylindole staining of nuclei. (F) In vivo differentiation potential after teratoma formation from CB-derived iPS cells. Hematoxylin and eosin staining of various slides after sectioning show various tissues from the 3 embryonic germ layers: gut epithelium (endoderm), cartilage (mesoderm), and glycogenated epithelium (ectoderm). Scale bar represents 200 μm.