利用藥物高效率轉換人類皮膚纖維母細胞為誘導式間質幹細胞

Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

賴培倫(Pei-Lun Lai)

指導教授 ： 呂仁 ； 黃筱鈞

國立臺灣大學/生命科學院/基因體與系統生物學學位學程/博士(2017年)

https://doi.org/10.6342/NTU201700777

摘要

人類間質幹細胞（MSCs）具有多功能分化特性(multipotency) 、免疫調節能力、並且無致瘤性(tumorigenesis) 。因此在臨床應用上安全性佳，於再生醫學和細胞治療極有應用價值。迄今，MSCs已經批准在臨床上治療移植物抗宿主病(GVHD)，退行性關節炎(Arthritis)等疾病。目前大約有706個相關的臨床試驗對至少10種不同疾病進行測試。利用化學藥物配合生長因子轉化纖維母細胞成為特定具功能性的細胞可避免因為病毒感染或質粒轉染的插入誘變、病毒製備過程的繁瑣、以及轉染/轉導(transfection/transduction)技術上的麻煩。因此利用化學誘導方法改變細胞命運近來引發極大關注。過去並無研究報導能以化學藥物(chemicals)誘導人類皮膚纖維母細胞轉換為間質幹細胞(induced MSCs, iMSCs)。本研究首度發現利用藥物能在六天內將人類真皮之纖維母細胞(dermal fibroblasts)轉換為iMSCs，平均轉換率為38％。這些iMSCs與fibroblasts不同，與天然間質幹細胞一樣具有高度克隆性。此外，我們的微陣列分析顯示，iMSCs與其誘導來源之纖維母細胞相較，其分子特徵類似於骨髓間質幹細胞（BMMSCs）。 iMSCs滿足國際細胞治療學會（ISCT）對間質幹細胞定義的所有標準，可以進一步分化成成骨細胞，脂肪細胞和軟骨細胞，達到與BMMSCs相當的程度。且iMSC可以在常規培養基中(去除藥物狀態下)不失功能性繼代培養至少8代。在免疫調節方面，iMSCs如BMMSCs一樣能有效抑制內毒素(LPS)所誘導小鼠之急性肺損傷，可以降低肺損傷指數與致死率。另外我們發現3種藥(SB202190, SP600125, Go6983)對於產生iMSCs是不可少的，而6種藥(SP600125, SB202190, Go6983, Y-27632, PD0325901, CHIR99021)之產率最佳。總體而言，本研究揭示了一個全新的策略，誘導皮膚纖維母細胞轉換為iMSCs，於細胞生物學和再生醫學的發展與應用有極大助益。

關鍵字

間質幹細胞 ； 化學藥物 ； 細胞轉換 ； 重編程 ； 免疫調節 ； 急性肺損傷 ； 再生醫學 ； 細胞治療

並列摘要

Due to the multipotency, immune-modularity, and the safety concern without tumorigenicity, human mesenchymal stromal/stem cells (MSCs) hold great promise in regenerative medicine and cell therapies. To date, MSCs already approved to treat Graft-versus-host disease, and degenerative arthritis in human and undergoing 706 clinical trials for the treatment of at least 10 different kinds of disease. To avoid (1) insertional mutagenesis by virus infection or plasmid transfection, (2) the tedious processes of virus preparation, and (3) repeated transfection/transduction, the use of chemical/growth factors to convert fibroblasts to functional cells has drawn substantial attention recently. However, no previous study has generated induced MSC (iMSCs) from skin fibroblasts with chemicals and/or growth factors. Herein, we established the first method to generate functional iMSCs from primary human dermal fibroblasts by solely small molecules with or without growth factors. The protocol can enrich iMSCs in only 6 days with an average conversion rate of 38%. Like traditional stem cells, only iMSCs, but not fibroblasts have clonogenicity. In addition, our microarray data displayed that iMSCs generated from one neonatal and two adult fibroblasts are more similar to bone marrow MSCs (BMMSCs) while compared to their parental fibroblasts. The phenotype of iMSCs fulfills all of the criteria of traditional MSCs as determined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT). Most of all, the iMSCs can be further differentiated into osteoblasts, adipocytes, and chondrocytes to a degree comparable to BMMSCs. The chemicals can be removed, and iMSCs can expand in regular culture medium up to 8 passages. Of note, the iMSCs can suppress endotoxin (lipopolysaccharide, LPS)-mediated acute lung injury as effectively as BMMSCs by completely recuse the lethality and decrease injury score. We found 3 chemicals (SB202190, SP600125, Go6983) were essential for producing iMSCs while 6 chemicals (SP600125, SB202190, Go6983, Y-27632, PD0325901, CHIR99021) gave the best efficiency. Overall, in this study, we reveal a brand new strategy and breakthrough protocol to generate iMSCs from skin fibroblasts that mimic BMMSCs. Thus, iMSCs can be an easily accessible resource to enrich sufficient BMMSC like cells for research in cell biology and regenerative medicine.

並列關鍵字

multipotent ； mesenchymal stromal/stem cells ； fibroblasts ； chemicals ； small molecules ； conversion ； acute lung injury ； cell therapy ； regenerative medicine

參考文獻

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