Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells.
OBJECTIVE
Hemophilia A (HA) is a severe, congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years, traditional laboratory animals have been used to study HA and its therapies, although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction.


METHODS
In this study, we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4, SOX2, SV40LT and KLF4, and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells.


RESULTS
HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining, in vitro assays and in vivo assays. Importantly, we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII, but otherwise functioned normally, recapitulating the phenotype of HA disease in vitro.


CONCLUSIONS
HA-iPSCs, particular those generated from the urine using a non-viral approach, provide an efficient way for modeling HA in vitro. Furthermore, HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine.
The Center for Prenatal and Hereditary Disease Diagnosis, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, P. R. China.
Bei
B
Jia
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Shen
S
Chen
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Zhiju
Z
Zhao
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China; Department of Regeneration Medicine, School of Pharmaceutical Science, Jilin University, Changchun, P. R. China.
Pengfei
P
Liu
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Jinglei
J
Cai
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Dajiang
D
Qin
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Juan
J
Du
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Changwei
C
Wu
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Qianyu
Q
Chen
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Xiujuan
X
Cai
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Hui
H
Zhang
The Center for Prenatal and Hereditary Disease Diagnosis, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, P. R. China.
Yanhong
Y
Yu
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China.
Duanqing
D
Pei
The Center for Prenatal and Hereditary Disease Diagnosis, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, P. R. China. Electronic address: zhongmei@smu.edu.cn.
Mei
M
Zhong
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China. Electronic address: pan_guangjin@gibh.ac.cn.
Guangjin
G
Pan
D002454:Cell Differentiation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D006467:Hemophilia A / Q000235:genetics / Q000473:pathology*; D022781:Hepatocytes / Q000166:cytology / Q000473:pathology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D008297:Male; D008954:Models, Biological; D010641:Phenotype; D044968:Regenerative Medicine / Q000379:methods
C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D005169:Factor VIII
Clotting factor VIII; Directed differentiation in vitro; Hemophilia A; Induced pluripotent stem cells