Effective generation of iPS cells from CD34+ cord blood cells by inhibition of p53
Received 18 August 2009; received in revised form 22 October 2009; accepted 10 November 2009. published online 16 November 2009.
Objective
Cord blood banks provide fully human leukocyte antigen−typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively.
Materials and Methods
CD34+, mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo10 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell factor, 10 ng/mL thrombopoietin, and 20 ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like factor 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.1-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse embryonic fibroblast (MEF) or SNL76/7 feeder cells in human embryonic stem cell medium (Dulbecco's modified Eagle medium/F-12 containing 20% knockout serum replacement, 200mM l-glutamine, 1% non-essential amino acids (NEAA), 0.1mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor). Subsequently, the number of embryonic stem cell−like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the differentiation potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture.
Results
Repression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from 1 (on average) to around 100 per 2×104 cells when infected cells were grown on SNL feeder cells.
Conclusions
iPS cells can be generated easily from CD34+ cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.
aFoundation for Biomedical Research and Innovation, Kobe, Japan
bRiken Center for Developmental Biology, Kobe, Japan
Offprint requests to: Shin Kawamata, M.D., Ph.D., Foundation for Biomedical Research and Innovation, TRI308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0043, Japan
ABSTRACT