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Transgene Free iPSCs Yield Cells with Greater Functionality

Review of “Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells” from Stem Cells TM by Stuart P. Atkinson

In a report in Stem Cells TM the group of Ken Igawa and Junji Takeda (Osaka University, Japan) have assessed the functionality of cells derived from induced pluripotent stem cells (iPSCs) generated using the piggyBac transposon system to deliver reprogramming factors to target cells. Specifically, they have compared the morphological, functional and molecular characteristics of iPSC-derived keratinocytes derived from transgene-residual (TR), or transgene free (TF) iPSCs, and have found that TF iPSCs are the more therapeutically relevant cell type [1].

The piggyBac transposon is advantageous as it does not leave “footprint” mutations upon excision as it repairs integration sites to the original sequence [2]. The authors generated iPSCs using this system with a vector carrying the OCT4, SOX2, KLF4, cMYC, and LIN28 transgenes and normal human diploid fibroblasts (NHDFs). Screening colonies allowed the isolation of a clone with a single transgene insertion, and after excision of the transgene vector with piggyBac transposase expression plasmid in one batch of cells; the group confirmed the lack of any “footprint” mutations surrounding the integration site. Both TR and TF iPSCs had phenotypes like those of pluripotent cells in terms of gene expression, epigenetic modifications and the potential for differentiation into cells of the three germ layers. The group then differentiated iPSCs into induced keratinocytes (iKCs) with no differences in the efficiency of differentiation. However, upon extended culture, the TR iKCs took on a more spindle like morphology (See figure), and had a less methylated OCT4 promoter region (38%) compared to TF iKCs (62%) and control keratinocytes (92%). Additionally, some undifferentiated colonies of cells arose from the cultures of TR iKCs. Single cell gene expression analysis also found higher expression levels of stem cell marker genes in TR iKCs, suggesting a reactivation of transgene expression following differentiation. The researchers also found higher keratinocyte-specific gene expression in TF iKCs, while genes associated with the early stages of epidermal cell lineages were higher in TR iKCs, indicative of a less mature cellular state. These differences also translated into deficits in functionality, as TF iKCs displayed a higher reconstitution potential than TR iKCs in a 3D culturing system for keratinocytes. TF iKCs formed pluristratified epidermal structures with basal layers positive for K14, and upper layers positive for involucrin, together indicative of normal keratinocyte differentiation. Tellingly, TR iKCs did not form such structures in this assay demonstrating them to be non-functional or functionally immature.

The authors contend that the presence of transgenes can have a significant impact on the functionality of iPSC-derived cells, with immediate implications for the therapeutic use of such cells in humans. While this study concentrated one differentiated cell type – keratinocytes – it is likely that these findings represent a more generalized phenomenon. Importantly, the initial differentiation of TF and TR iPSCs into keratinocytes was not significantly altered, but the authors deem the reactivation of transgene expression at a later time point the likely culprit for the reduced functionality of the TR keratinocytes after time in culture.

References

  1. Igawa K, Kokubu C, Yusa K, et al. Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells. Stem Cells Transl Med 2014;
  2. Fraser MJ, Ciszczon T, Elick T, et al. Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol Biol 1996;5:141-151.