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Accurate and Reliable Generation of Human iPSCs For Disease Modeling and Clinical Applications

Review of "A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair generating improved genetically stable human induced pluripotent stem cells" from STEM CELLS by Stuart P. Atkinson

Recent research from the laboratory of Michael J. Edel (Institut Universitari Barraquer, Bellaterra, Spain) discovered that neural stem cells derived from human induced pluripotent stem cells (iPSCs) generated using a virally transduced set of reprogramming factors that included C-MYC displayed tumorigenic potential after transplantation into the injured rat spinal cord [1]. Their subsequent research, which focused on exploring links between the REM2 GTPase in human pluripotency, suggested that CYCLIN D1 may represent a safe replacement for C-MYC in human iPSC generation [2] thanks to its reported role in DNA damage repair [3, 4].

In a new STEM CELLSarticle [5], the Edel team now report on the full characterization of human iPSCs generated by synthetic mRNA transfection [6] with the help of CYCLIN D1 and provide evidence that this reprogramming strategy will allow for the more accurate and reliable generation of human iPSCs for disease modeling and future clinical applications.

Alvarez‐Palomo et al. employed clinically compatible feeder-free conditions and the transfection of synthetic mRNA coding for the OCT3/4, SOX2, KLF4, and LIN28 reprogramming factors alongside CYCLIN D1 to generate human iPSCs from human foreskin fibroblasts (See image - CYCLIN D1 reprograms human cells to induced pluripotent stem cells (iPSC)). Synthetic mRNA transfection represents an efficient and robust method that does not involve transgene insertion or viruses and provides for low aneuploidy and a lack of genetic scarring, making it a preferred choice for clinical applications [7, 8]. Overall, the addition of CYCLIN D1 generated iPSCs with lower multitelomeric signal, reduced double‐strand DNA breaks, the correct nuclear localization of RAD51 protein, and reduced single‐nucleotide polymorphism changes per chromosome when compared with classical reprogramming using C‐MYC.

Importantly, iPSCs generated with CYCLIN D1 also displayed reduced teratogenic capabilities, while iPSC-derived neural stem cells successfully engrafted into the injured rodent spinal cord, where they survived and differentiated appropriately. At the mechanistic level, the authors demonstrated that CYCLIN D1 transfection promoted DNA double-strand break repair through homologous recombination during the reprogramming process and reduced cell stress, as evidenced by a reduction in SIRT1 gene expression.

Overall, the addition of CYCLIN D1 to reprogramming factors during synthetic mRNA-induced reprogramming creates human iPSCs with significantly improved genomic stability compared to traditional iPSC generation protocols. The authors hope that their new advance will provide a means for the more accurate and reliable generation of human iPSCs for disease modeling and future clinical applications.

For more on enhanced human iPSC generation approaches, stay tuned to the Stem Cells Portal!


References

  1. López-Serrano C, Torres-Espín A, Hernández J, et al., Effects of the Post-Spinal Cord Injury Microenvironment on the Differentiation Capacity of Human Neural Stem Cells Derived from Induced Pluripotent Stem Cells. Cell Transplantation 2016;25:1833-1852.
  2. Edel MJ, Menchon C, Menendez S, et al., Rem2 GTPase maintains survival of human embryonic stem cells as well as enhancing reprogramming by regulating p53 and cyclin D1. Genes & Development 2010;24:561-573.
  3. Chalermrujinanant C, Michowski W, Sittithumcharee G, et al., Cyclin D1 promotes BRCA2-Rad51 interaction by restricting cyclin A/B-dependent BRCA2 phosphorylation. Oncogene 2016;35:2815-2823.
  4. Jirawatnotai S, Hu Y, Michowski W, et al., A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers. Nature 2011;474:230-234.
  5. Alvarez-Palomo AB, Requena-Osete J, Delgado-Morales R, et al., A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair, generating improved genetically stable human induced pluripotent stem cells. STEM CELLS 2021;39:866-881.
  6. Warren L, Manos PD, Ahfeldt T, et al., Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell 2010;7:618-630.
  7. Stadtfeld M, Nagaya M, Utikal J, et al., Induced Pluripotent Stem Cells Generated Without Viral Integration. Science 2008;322:945.
  8. Yoshioka N, Gros E, Li H-R, et al., Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA. Cell Stem Cell 2013;13:246-254.