Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.
To identify accessible and permissive human cell types for efficient derivation of induced pluripotent stem cells (iPSCs), we investigated epigenetic and gene expression signatures of multiple postnatal cell types such as fibroblasts and blood cells. Our analysis suggested that newborn cord blood (CB) and adult peripheral blood (PB) mononuclear cells (MNCs) display unique signatures that are closer to iPSCs and human embryonic stem cells (ESCs) than age-matched fibroblasts to iPSCs/ESCs, thus making blood MNCs an attractive cell choice for the generation of integration-free iPSCs. Using an improved EBNA1/OriP plasmid expressing 5 reprogramming factors, we demonstrated highly efficient reprogramming of briefly cultured blood MNCs. Within 14 days of one-time transfection by one plasmid, up to 1000 iPSC-like colonies per 2 million transfected CB MNCs were generated. The efficiency of deriving iPSCs from adult PB MNCs was approximately 50-fold lower, but could be enhanced by inclusion of a second EBNA1/OriP plasmid for transient expression of additional genes such as SV40 T antigen. The duration of obtaining bona fide iPSC colonies from adult PB MNCs was reduced to half (∼14 days) as compared to adult fibroblastic cells (28-30 days). More than 9 human iPSC lines derived from PB or CB blood cells are extensively characterized, including those from PB MNCs of an adult patient with sickle cell disease. They lack V(D)J DNA rearrangements and vector DNA after expansion for 10-12 passages. This facile method of generating integration-free human iPSCs from blood MNCs will accelerate their use in both research and future clinical applications.
Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Bin-Kuan
BK
Chou
Prashant
P
Mali
Xiaosong
X
Huang
Zhaohui
Z
Ye
Sarah N
SN
Dowey
Linda Ms
LM
Resar
Chunlin
C
Zou
Y Alex
YA
Zhang
Jay
J
Tong
Linzhao
L
Cheng
D018952:Antigens, CD34 / Q000378:metabolism; D001773:Blood Cells / Q000166:cytology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D065150:Cellular Reprogramming; D044127:Epigenesis, Genetic; D005312:Fetal Blood / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D020869:Gene Expression Profiling; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins
D018952:Antigens, CD34; D002352:Carrier Proteins; C432687:EBNA1BP2 protein, human; D016601:RNA-Binding Proteins
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