Generation of human induced pluripotent stem cells from dermal fibroblasts

Lowry et al. 10.1073/pnas.0711983105.

Supporting Information

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SI Figure 6
SI Figure 7
SI Table 2
SI Figure 8

Fig. 6. An examination of relative amount of exogenous versus endogenous expression of the "defined" transcription factors. (A) Semiquantitative real-time RT-PCR demonstrates the relative expression of exogenous and endogenous defined factors in both iPS clones and OCT4/C-MYC clones and control NHDF1 and HSF1. In most cases, microarray expression data were employed to elaborate on the relative amounts of total (exogenous plus endogenous, i.e. coding region) gene expression. Array analysis was not performed on clone 49, thus it was not included in the histograms. For NANOG, the array did not contain a suitable probe set to describe total product; instead, RT-PCR was employed with primers recognizing strictly the coding region of the gene. All values for RT-PCR were analyzed relative to GAPDH expression. Note that appropriate primers for KLF4 real time PCR could not be generated. (B) The table summarizes all microarray expression data for the defined factors, demonstrating the relative amounts of these factors in various cell types. Listed are all the probe sets on the array that generated positive signal. Note that some of these probe sets were specific for the endogenous product, whereas others recognize both endogenous and exogenous expression (indicated as Total). Expression values are calculated relative to HSF1, and those figures in italics were found to have an absent call on the array.

Fig. 7. iPS clones have a normal karyotype and are derived from NHDF1. (A) Full DNA fingerprint analysis examining polymorphic short tandem repeat (STR) DNA regions to uniquely identify unrelated cell lines. Fifteen loci plus amelogenin for sex chromosome assignment were analyzed in the indicated cell lines. HSF1 represents a human embryonic stem cell line also cultured in the same lab (at passage 49, p49). NHDF1 and NHDF2 are human fibroblasts derived from single donors, with NHDF1 serving as the line reprogrammed. The iPS lines were analyzed at nine passages after infection with retrovirus. (B) Cytogenic analysis of iPS clones 2 and 5, HSF1, and NHDF1 at the indicated passage demonstrated normal karyotypes for each of these lines.

Fig. 8. Markers of HESC differentiation are upregulated in differentiating iPS cells. Immunostaining for SSEA1 and NESTIN was performed on the undifferentiated and differentiated HESC line HSF1 and on the differentiated iPS1. Differentiation was induced by embryoid body formation and subsequent plating in FBS or retinoic acid (RA)-containing media as indicated. Staining for the surface antigen SSEA1, a marker of HESC differentiation, demonstrates general differentiation found after the EB differentiation protocol. NESTIN, a marker of neural specification, is found in only a small percentage of HESCs before induction of differentiation but is highly up-regulated upon differentiation in the presence of RA in both HESC and IPS cells.

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