A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods

Figure 2

Generated iPSCs are pluripotent. (a) The iPSC lines (representative for different reprogramming methods as episomal plasmids, Sendai virus, and STEMCCA) express endogenous pluripotency marker SOX2, OCT4, LIN28, GDF3, and FOXD3 genes at the mRNA level, which was shown by RT-PCR. iFB5 cells were used as the positive control for pluripotent iPS cells [13]. 2-FB and 4-FB are fibroblast before reprogramming. Water was used as the negative control. All other mentioned cell lines are iPSCs. (b) Expression of pluripotency markers OCT4, SOX2, LIN28, and TRA1-60 as shown by immunofluorescence staining. Scale bar: 50 μm. (c) Spontaneous differentiation capacity in vitro via embryoid bodies (EBs) of three iPSC lines, representative of each reprogramming method. Immunocytochemical staining was used for analysis of differentiation capacity by protein expression of mesodermal α-SMA, endodermal α-fetoprotein (AFP), and ectodermal βIII-tubulin exemplarily in one iPSC line generated by each reprogramming method. Scale bar: 100 μm. IPSC colonies were used for teratoma formation in immunodeficient mice to test the developmental potential of iPSCs in vivo. Representative pictures of mesodermal, endodermal, and ectodermal tissues are shown from one cell line generated by the plasmid reprogramming method. Scale bar: 20 μm. GAPDH was used as the loading control (a). Nuclear localization was marked with DAPI (blue) (b, c).

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