Effective generation of iPS cells from CD34+ cord blood cells by inhibition of p53.
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.


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, 200 mM L-glutamine, 1% non-essential amino acids (NEAA), 0.1 mM 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 x 10(4) 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.
Takenaka
Chiemi
C
Foundation for Biomedical Research and Innovation, Kobe, Japan.
Nishishita
Naoki
N
Takada
Nozomi
N
Jakt
Lars Martin
LM
Kawamata
Shin
S
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000166:cytology; D005312:Fetal Blood / Q000166:cytology*; D005347:Fibroblasts; D015870:Gene Expression; D018014:Gene Transfer Techniques; D016259:Genes, myc / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D055029:Inverted Repeat Sequences; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology*; D012313:RNA / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors*
D018952:Antigens, CD34; D003470:Culture Media; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D016159:Tumor Suppressor Protein p53; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA