A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair, generating improved genetically...

Area of Science:

• Stem Cell Biology
• Genetics
• Molecular Biology

Background:

• Clinical application of induced pluripotent stem cells (iPSC) is challenged by genetic instability, posing long-term risks for cell transplant therapy.
• Developing genetically stable iPSC is crucial for accurate disease modeling and safe therapeutic applications.

Purpose of the Study:

• To investigate the role of CYCLIN D1 in DNA repair during iPSC reprogramming.
• To evaluate if CYCLIN D1 promotes genetic stability in iPSC compared to traditional methods.

Main Methods:

• Utilized synthetic mRNA transfection with CYCLIN D1 and base reprogramming factors (OCT3/4, SOX2, KLF4, LIN28).
• Compared CYCLIN D1-based reprogramming with the classical C-MYC method.
• Assessed genetic stability markers including multitelomeric signals, DNA double-strand breaks, RAD51 localization, and single-nucleotide polymorphism (SNP) changes.

Main Results:

• CYCLIN D1-generated iPSC exhibited reduced multitelomeric signals, fewer DNA double-strand breaks, correct RAD51 nuclear localization, and fewer SNP changes.
• CYCLIN D1 iPSC showed reduced teratoma growth and successful neural stem cell engraftment in a spinal cord injury model.
• CYCLIN D1 facilitated DNA double-stranded damage repair predominantly through homologous recombination (HR) during reprogramming.

Conclusions:

• Synthetic mRNA transfection of CYCLIN D1 promotes DNA repair via HR, leading to significantly more genetically stable human iPSC.
• This novel method offers a more reliable approach for generating iPSC for disease modeling and clinical applications.
• CYCLIN D1 reduces cell stress and can rescue reprogramming in Sirt1-deficient cells.