Figure 1
Figure 1. Efficient generation of transgene-free iPSCs from BM mononuclear cells. (A) Schematic diagram of reprogramming protocol. (B) Kinetics of morphologic changes after blood reprogramming. (C-D) Comparison of reprogramming efficiency between blood cells and fibroblasts. (C) Left graph shows the numbers of ALP+ colonies per 1 million BM cells and fibroblasts (FB) transfected side by side with combination 19 episomal vectors. Two independent experiments, E2 and E3, are shown. Right panel shows ALP staining of colonies generated after transfection and the first passage on MEFs 2 × 105 BM cells (top) and 3.3 × 105 fibroblasts (bottom). (D) Expandable iPSC colonies obtained from 1 million FB and BM cells transfected with the same set of reprogramming factors. Black bar shows the number of iPSC colonies generated from foreskin fibroblasts in our previous studies.14 Bars in E1 and E4 show results of reprogramming of BM mononuclear cells from 2 independent experiments (E1 and E4). (E) Flow cytometric analysis of hESC-specific marker expression in 7 BM iPSC lines and 6 subclones generated from BM iPSC1. (F) Representative immunofluorescent staining of BM iPSCs with REX1 antibody. Bar indicates 50 μm. (G) H&E staining of teratoma from representative BM iPSC line (BM iPSC1M). Neuronal rosette and gastro-intestine-like structure can be seen in the left panel. Cartilage and gut epithelium can be seen in the right panel. Bars indicate 50 μm. (H) Normal karyogram representative of BM iPSC (BM iPSC1M). (I) PCR analysis of episomal and genomic DNA in subclones I-N obtained from the BM iPSC1 line. Human BM genomic DNA serves as negative control (BM), whereas DNA samples from human BM mononuclear cells transfected with the same constructs are used as a positive control (P1). T indicates that transgene specific primers were used. (J) RT-PCR analysis of expression of transgenes and endogenous pluripotency genes in subclones I-N obtained from the BM iPSC1 line. The T series of primers are transgene-specific. Negative controls (BM) are results of untransfected BM RNA. Positive controls (P1) are BM cells transfected with the same reprogramming plasmids. (K) Progressive loss of episomal plasmid from BM iPSC lines. Ten randomly selected BM iPSC lines (1-3, 7-9, 16-18, and 21) were analyzed. Vector-specific primer pairs (EBNA, middle panel) were used to examine the episomal DNA from different passages (passage 3, 4, 5, and 7) of the BM iPSC lines. Samples of passage 7 were further examined by other transgene-specific primers (right panel). Left panel shows existence of genomic DNA (human actin genomic primers) in the episomal DNA extracted using the previously published method.16

Efficient generation of transgene-free iPSCs from BM mononuclear cells. (A) Schematic diagram of reprogramming protocol. (B) Kinetics of morphologic changes after blood reprogramming. (C-D) Comparison of reprogramming efficiency between blood cells and fibroblasts. (C) Left graph shows the numbers of ALP+ colonies per 1 million BM cells and fibroblasts (FB) transfected side by side with combination 19 episomal vectors. Two independent experiments, E2 and E3, are shown. Right panel shows ALP staining of colonies generated after transfection and the first passage on MEFs 2 × 105 BM cells (top) and 3.3 × 105 fibroblasts (bottom). (D) Expandable iPSC colonies obtained from 1 million FB and BM cells transfected with the same set of reprogramming factors. Black bar shows the number of iPSC colonies generated from foreskin fibroblasts in our previous studies.14  Bars in E1 and E4 show results of reprogramming of BM mononuclear cells from 2 independent experiments (E1 and E4). (E) Flow cytometric analysis of hESC-specific marker expression in 7 BM iPSC lines and 6 subclones generated from BM iPSC1. (F) Representative immunofluorescent staining of BM iPSCs with REX1 antibody. Bar indicates 50 μm. (G) H&E staining of teratoma from representative BM iPSC line (BM iPSC1M). Neuronal rosette and gastro-intestine-like structure can be seen in the left panel. Cartilage and gut epithelium can be seen in the right panel. Bars indicate 50 μm. (H) Normal karyogram representative of BM iPSC (BM iPSC1M). (I) PCR analysis of episomal and genomic DNA in subclones I-N obtained from the BM iPSC1 line. Human BM genomic DNA serves as negative control (BM), whereas DNA samples from human BM mononuclear cells transfected with the same constructs are used as a positive control (P1). T indicates that transgene specific primers were used. (J) RT-PCR analysis of expression of transgenes and endogenous pluripotency genes in subclones I-N obtained from the BM iPSC1 line. The T series of primers are transgene-specific. Negative controls (BM) are results of untransfected BM RNA. Positive controls (P1) are BM cells transfected with the same reprogramming plasmids. (K) Progressive loss of episomal plasmid from BM iPSC lines. Ten randomly selected BM iPSC lines (1-3, 7-9, 16-18, and 21) were analyzed. Vector-specific primer pairs (EBNA, middle panel) were used to examine the episomal DNA from different passages (passage 3, 4, 5, and 7) of the BM iPSC lines. Samples of passage 7 were further examined by other transgene-specific primers (right panel). Left panel shows existence of genomic DNA (human actin genomic primers) in the episomal DNA extracted using the previously published method.16 

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