A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair generating improved genetically stable human induced pluripotent stem cells
Por:
A. ALVAREZ-PALOMO, J. REQUENA-OSETE, R. DELGADO-MORALES, V. MORENO-MANZANO, C. GRAU-BOVE, A. TEJERA, M. OTERO, C. BARROT, I. SANTOS-BARRIOPEDRO, A. VAQUERO, J. MEZQUITA-PLA, S. MORAN, C. NAYA, I. GARCIA-MARTINEZ, F. PEREZ, M. BLASCO, M. ESTELLER and M. EDEL
Publicada:
1 jul 2021
Ahead of Print:
1 mar 2021
Resumen:
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.
Filiaciones:
A. ALVAREZ-PALOMO:
Univ Barcelona, Hosp Clin, Mol Genet & Control Pluripotency Lab, Dept Biomed,Inst Neurosci,Fac Med, Barcelona, Catalonia, Spain
Banc Sang & Teixits BST, Cell Therapy Serv, Barcelona, Spain
J. REQUENA-OSETE:
Univ Barcelona, Hosp Clin, Mol Genet & Control Pluripotency Lab, Dept Biomed,Inst Neurosci,Fac Med, Barcelona, Catalonia, Spain
Univ Oslo, Inst Clin Med, Oslo, Norway
NORMENT, Div Mental Hlth & Addict, Ctr Mental Disorders Res, Oslo, Norway
R. DELGADO-MORALES:
Bellvitge Biomed Res Inst, Canc Epigenet & Biol Program PEBC, Lhospitalet De Llobregat, Spain
Maastricht Univ, Sch Mental Hlth & Neurosci MHeNs, Dept Psychiat & Neuropsychol, Maastricht, Netherlands
:
Principe Felipe Res Ctr, Neuronal & Tissue Regenerat Lab, Valencia, Spain
C. GRAU-BOVE:
Univ Barcelona, Hosp Clin, Mol Genet & Control Pluripotency Lab, Dept Biomed,Inst Neurosci,Fac Med, Barcelona, Catalonia, Spain
A. TEJERA:
Spanish Natl Canc Ctr CNIO 3, Telomeres & Telomerase Grp, Mol Oncol Program, Madrid, Spain
M. OTERO:
Hosp Clin, Dept Clin Immunol, Biomed Diagnost Ctr CDB, Villarroel, Catalonia, Spain
C. BARROT:
Univ Barcelona, Legal Med Dept, Forens Genet Lab, Fac Med, Barcelona, Spain
I. SANTOS-BARRIOPEDRO:
Bellvitge Biomed Res Inst IDIBELL, Canc Epigenet & Biol Program PEBC, Chromatin Biol Lab, Barcelona, Catalonia, Spain
A. VAQUERO:
Bellvitge Biomed Res Inst IDIBELL, Canc Epigenet & Biol Program PEBC, Chromatin Biol Lab, Barcelona, Catalonia, Spain
J. MEZQUITA-PLA:
Inst Invest Biomed August Pi i Sunyer IDIBAPS, Barcelona, Catalonia, Spain
S. MORAN:
Bellvitge Biomed Res Inst, Canc Epigenet & Biol Program PEBC, Lhospitalet De Llobregat, Spain
C. NAYA:
Banc Sang & Teixits BST, Congenital Coagulopathies Dept, Barcelona, Spain
Univ Autonoma Barcelona VHIR UAB, Vall dHebron Res Inst, Transfus Med, Barcelona, Spain
I. GARCIA-MARTINEZ:
Banc Sang & Teixits BST, Congenital Coagulopathies Dept, Barcelona, Spain
Univ Autonoma Barcelona VHIR UAB, Vall dHebron Res Inst, Transfus Med, Barcelona, Spain
F. PEREZ:
Banc Sang & Teixits BST, Congenital Coagulopathies Dept, Barcelona, Spain
Univ Autonoma Barcelona VHIR UAB, Vall dHebron Res Inst, Transfus Med, Barcelona, Spain
CIBER Enfermedades Cardiovasc CIBERCV, Madrid, Spain
M. BLASCO:
Spanish Natl Canc Ctr CNIO 3, Telomeres & Telomerase Grp, Mol Oncol Program, Madrid, Spain
M. ESTELLER:
Josep Carreras Leukemia Res Inst IJC, Barcelona, Catalonia, Spain
Ctr Invest Biomed Red Canc CIBEROnc, Madrid, Spain
Inst Catalana Recerca & Estudis Avancats ICREA, Barcelona, Catalonia, Spain
Univ Barcelona UB, Sch Med & Hlth Sci, Physiol Sci Dept, Barcelona, Catalonia, Spain
M. EDEL:
Univ Barcelona, Hosp Clin, Mol Genet & Control Pluripotency Lab, Dept Biomed,Inst Neurosci,Fac Med, Barcelona, Catalonia, Spain
Victor Chang Cardiac Res Inst, Sydney, NSW, Australia
Univ Western Australia, Sch Med & Pharmacol, Harry Perkins Res Inst, Ctr Cell Therapy & Regenerat Med CCTRM, Perth, WA, Australia
Univ Autonoma Barcelona, Inst Univ Barraquer, Ctr Oftalmol Barraquer, Bellaterra, Spain
hybrid
|