University of Heidelberg

iPSC - Background

  • In 2007, Yamanaka et al. revolutionized the stem cell field by showing that ectopic expression of 4 pluripotency-related factors - Oct4, Klf4, Sox2 and c-Myc (OKSM) - in somatic cells (MEF) yielded "induced pluripotent stem cells" (iPSC) that resemble embryonic stem cells morphologically (round shape, large nucleoli, scant cytoplasm), molecularly (marker expression) and phenotypically (infinite self-renewal and pluripotency). To date, many groups reprogrammed other somatic cells (human foreskin fibroblasts, keratinocytes, hematopoietic stem and progenitor cells, hepatocytes) using OKSM or other cocktails including NANOG and LIN28 together with Oct4 and Sox2, Oct-4 alone, miRNA clusters alone or in combination with the Yamanaka factors, or an shRNA against p53 to substitute c-Myc (Gonzalez et al., 2011).
  • Like embryonic stem cells, iPSC are a practically infinite source of stem cells, yet they overcome ethical concerns and tissue rejection issues when used in cell therapy. Thus, iPSC now truly pave the way to reach the holy grail in personalized regenerative medicine, i.e. to derive patient-specific pluripotent stem cells capable of self-renewal and differentiation into any cell type of the body. This hope is fostered by a wealth of recent first pre-clinical successes with iPSC, including differentiation of murine iPSC into neuronal cells which improved symptoms of Parkinson's disease in rats. Others reported phenotypically relevant gene correction in iPS-derived hematopoietic stem cells in a humanized sickle cell anemia mouse model, or exemplified the vast potential of iPSC for hemophilia A treatment. 
  • Still, a major technical hurdle that remains concerns the vector for delivery of the reprogramming factors. The originally used retroviral and lentiviral vectors are efficient but their integration into the host DNA may dysregulate oncogenes or tumor suppressor genes, even when Cre-deletable or non-conservative transposon remobilization systems are used. To overcome this issue, other delivery systems have been developed in the recent past, including adenoviral vectors, DNA minicircle vectors, episomal non-replicative vectors, RNA virus-based vectors, or modified mRNA or protein delivery. All of these have overcome the integration problem but they are difficult to apply under routine conditions due to low infection or transfection efficiencies, reduced cell survival, long reprogramming kinetics and poor reproducibility.
  • Notably, another critical problem arising from potential integration of the reprogramming vectors is that efficient induction of pluripotency requires that the reprogramming factors are only expressed during a certain initial window and then shut down. The latter can occur through transcriptional silencing which is indeed observed. However, this phenomenon is due to inherent cellular processes that are hard to control exogenously and thus not sufficiently useful and safe for translational applications. This further strongly argues for the use of non-integrating vector systems that can mediate potent delivery yet transient expressionof the reprogramming factors.
  • Two additional problems that have not been solved satisfactorily are (1) a sparsity of potent and safe systems to detect fully re-programmed iPSC and to unanimously distinguish them from their non-pluripotent ancestors, and (2) likewise a need for new methods to comprehensively eliminate remaining pluripotent and hence potentially tumorigenic cells after differentiation of iPSC into new somatic cells. Albeit it is principally possible to achieve these goal via FACS sorting based on cell-specific surface markers, it may be more desirable to develop and use viral vectors for this purpose. Lentiviral vectors have indeed been reported that encode fluorescence markers tagged with binding sites for miRNAs that are specifically regulated in somatic versus pluripotent cells; yet, these vectors pose an inherent safety problem.
  • Last but not least, a further challenge for future research in the field of iPSC and regenerative medicine is to devise and apply efficient, specific and safe methods for genetic engineering of the stem cells prior to their differentiation into new somatic cells for transplantation. Examples include the correction of disease-associated genetic defects (e.g. mutations within tumor suppressor genes), or the stable integration and expression of anti-viral RNAi sequences as a novel means to protect progeny cells from infection with a particular pathogen. In all these strategies, a major requirement will be a vector and targeting strategy that is utmost specific to alleviate safety concerns from potential random integration of the foreign sequences into the iPS or progeny cells.
Contact: E-Mail (Last update: 26/02/2012)