Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.
BACKGROUND
'Patient-specific' induced pluripotent stem cells (iPSCs) are attractive because they can generate abundant cells without the risk of immune rejection for cell therapy. Studies have shown that iPSC-derived mesenchymal stem cells (iMSCs) possess powerful proliferation, differentiation, and therapeutic effects. Recently, most studies indicate that stem cells exert their therapeutic effect mainly through a paracrine mechanism other than transdifferentiation, and exosomes have emerged as an important paracrine factor for stem cells to reprogram injured cells. The objective of this study was to evaluate whether exosomes derived from iMSCs (iMSCs-Exo) possess the ability to attenuate limb ischemia and promote angiogenesis after transplantation into limbs of mice with femoral artery excision.


METHODS
Human iPSCs (iPS-S-01, C1P33, and PCKDSF001C1) were used to differentiate into iMSCs in a modified one-step method. iMSCs were characterized by flow cytometry and multipotent differentiation potential analysis. Ultrafiltration combined with a purification method was used to isolate iMSCs-Exo, and transmission electron microscopy and Western blotting were used to identify iMSCs-Exo. After establishment of mouse hind-limb ischemia with excision of femoral artery and iMSCs-Exo injection, blood perfusion was monitored at days 0, 7, 14, and 21; microvessel density in ischemic muscle was also analyzed. In vitro migration, proliferation, and tube formation experiments were used to analyze the ability of pro-angiogenesis in iMSCs-Exo, and quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay were used to identify expression levels of angiogenesis-related molecules in human umbilical vein endothelial cells (HUVECs) after being cultured with iMSCs-Exo.


RESULTS
iPSCs were efficiently induced into iMSC- with MSC-positive and -negative surface antigens and osteogenesis, adipogenesis, and chondrogenesis differentiation potential. iMSCs-Exo with a diameter of 57 ± 11 nm and expressed CD63, CD81, and CD9. Intramuscular injection of iMSCs-Exo markedly enhanced microvessel density and blood perfusion in mouse ischemic limbs, consistent with an attenuation of ischemic injury. In addition, iMSCs-Exo could activate angiogenesis-related molecule expression and promote HUVEC migration, proliferation, and tube formation.


CONCLUSIONS
Implanted iMSCs-Exo was able to protect limbs from ischemic injury via the promotion of angiogenesis, which indicated that iMSCs-Exo may be a novel therapeutic approach in the treatment of ischemic diseases.
Department of Neurosurgery, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. hugw0625@163.com.|Jiangxi Medical College of Nanchang University, 461 BaYi Avenue, Nanchang, 330006, China. hugw0625@163.com.
Guo-wen
GW
Hu
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. liqing236@gmail.com.
Qing
Q
Li
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. niuxin999@163.com.
Xin
X
Niu
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. 6311089@qq.com.
Bin
B
Hu
Department of Neurosurgery, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. pajingsen@126.com.
Juan
J
Liu
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. zhoushumin168@hotmail.com.
Shu-min
SM
Zhou
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. 1321165979@qq.com.
Shang-chun
SC
Guo
Jiangxi Medical College of Nanchang University, 461 BaYi Avenue, Nanchang, 330006, China. langhaili1989@163.com.
Hai-li
HL
Lang
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. zhangcq@sjtu.edu.cn.
Chang-qing
CQ
Zhang
Institute of Microsurgery on Extremities, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. wangy63cn@126.com.
Yang
Y
Wang
Department of Neurosurgery, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, 200233, China. dengzf63@126.com.
Zhi-feng
ZF
Deng
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D055354:Exosomes / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D007511:Ischemia / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice