A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.
Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications.
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Bin-Kuan
BK
Chou
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Haihui
H
Gu
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Yongxing
Y
Gao
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Sarah N
SN
Dowey
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Ying
Y
Wang
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Jun
J
Shi
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Yanxin
Y
Li
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Zhaohui
Z
Ye
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Tao
T
Cheng
Division of Hematology, Department of Medicine, and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin, People's Republic of China; Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA; Key Laboratory of Pediatric Hematology/Oncology of Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China lcheng2@jhmi.edu.
Linzhao
L
Cheng
D000328:Adult; D001773:Blood Cells / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics*; D065150:Cellular Reprogramming / Q000235:genetics*; D016895:Culture Media, Serum-Free; D061252:Feeder Cells / Q000166:cytology; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000637:transplantation; D007963:Leukocytes, Mononuclear / Q000166:cytology; D019096:Vitronectin / Q000008:administration & dosage
D016895:Culture Media, Serum-Free; D019096:Vitronectin
Episomal vectors; Feeder-free; Human blood mononuclear cells; Human iPS cells; Plasmid expression; Reprogramming; Xeno-free cell culture