A Synthetic mRNA Cell Reprogramming Method Using CYCLIN D1 Promotes DNA rEpair, Generating Improved Genetically Stable Human Induced Pluripotent Stem Cells

Abstract

A key challenge for clinical application of induced pluripotent stem cells (iPSC) to accurately model and treat human pathologies depends on developing a method to generate genetically stable cells to reduce long-term risks of cell transplant therapy. Here, we hypothesized that CYCLIN D1 repairs DNA by highly efficient homologous recombination (HR) during reprogramming to iPSC that reduces genetic instability and threat of neoplastic growth. We adopted a synthetic mRNA transfection method using clinically compatible conditions with CYCLIN D1 plus base factors (OCT3/4, SOX2, KLF4, LIN28) and compared with methods that use C-MYC. We demonstrate that CYCLIN D1 made iPSC have (a) lower multitelomeric signal, (b) reduced double-strand DNA breaks, (c) correct nuclear localization of RAD51 protein expression, and (d) reduced single-nucleotide polymorphism (SNP) changes per chromosome, compared with the classical reprogramming method using C-MYC. CYCLIN D1 iPSC have reduced teratoma Ki67 cell growth kinetics and derived neural stem cells successfully engraft in a hostile spinal cord injury (SCI) microenvironment with efficient survival, differentiation. We demonstrate that CYCLIN D1 promotes double-stranded DNA damage repair predominantly through HR during cell reprogramming to efficiently produce iPSC. CYCLIN D1 reduces general cell stress associated with significantly lower SIRT1 gene expression and can rescue Sirt1 null mouse cell reprogramming. In conclusion, we show synthetic mRNA transfection of CYCLIN D1 repairs DNA during reprogramming resulting in significantly improved genetically stable footprint in human iPSC, enabling a new cell reprogramming method for more accurate and reliable generation of human iPSC for disease modeling and future clinical applications.

A Hyaluronic Acid Demilune Scaffold and Polypyrrole-Coated Fibers Carrying Embedded Human Neural Precursor Cells and Curcumin for Surface Capping of Spinal Cord Injuries

“…The iNPCs were generated as previously described [ 34 ]. Briefly, human iPSCs were reprogrammed using reprogramming factors (OCT3/4, SOX2, KLF4, LIN28) and a synthetic mRNA coding for CYCLIN D1, which supports genetic stability during the reprogramming process.…”

Human Induced Pluripotent Stem Cells: From Cell Origin, Genomic Stability, and Epigenetic Memory to Translational Medicine

“… 56 Moreover, replacing c-MYC by cyclin D1 but maintaining OCT3/4, SOX2, KLF4 and LIN28 for reprogramming improved genomic stability of iPSCs by reducing cell stress and promoting DNA damage response by error-free HR. 58 …”

Cancer cells as a new source of induced pluripotent stem cells

“…A method was adopted by Alvarez-Palomo et al that used synthetic mRNA of some Yamanaka factors along with Cyclin D1 transfection to repair DNA lesions during reprogramming. As a result of Cyclin D1 mRNA function, safer iPSCs are produced, which are sufficiently genetically stable [ 48 ]. Upregulation of cyclin-dependent kinase inhibitor 2B or P15 is associated with minimized malignancy during reprogramming dermal fibroblasts into multipotent cells.…”