綠色螢光小鼠與豬骨髓間葉幹細胞之建立與細胞治療研究

The cell-based therapeutics of marrow-derived mesenchymal stem cells isolated from transgenic mice and pigs harboring with EGFP gene

蕭士翔(Felix Shih-Hsiang Hsiao)

指導教授 ： 吳信志

共同指導教授 ： 鄭登貴(Winston Teng-Kuei Cheng)

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02772

摘要

間葉幹細胞 (mesenchymal stem cells, MSCs) 於體內為具有自我更新 (self-renew) 與複分化潛能 (multipotency) 的成體幹細胞，彼等細胞具有易於分離培養、體外增生快速與免疫調節等特性，逐年受研究者重視。迄今有關MSCs分化與移植走向之機制尚未完全明瞭，職是之故，本研究嘗試以綠色螢光小鼠與豬為模式，研究重點除建立一穩定之MSCs體外培養系統，並分析MSCs於分化前後基因體表現之差別，冀能以此調控與追蹤MSCs移植與分化走向之使用。一般而言，MSCs可藉骨髓單核細胞 (mononuclear cells) 之收集，經一次培養盤貼附 (single-step plastic-adhesion) 法分離之。然以此分離之小鼠骨髓MSCs (mMSCs) 經常伴隨造血細胞 (hematopoietic cells, HCs) 之干擾，繼使mMSC建立殊為困難。本研究發現參與mMSCs嵌合 (engaged) 的HCs群 (MSCs-engaged HCs) 為造成mMSC培養干擾的主要因素，彼等藉由胰蛋白酶 (trypsin) 之添加，trypsin不敏感之MSCs-engaged HCs仍伴隨MSCs脫離培養盤表面，並保留於下一世代 (passage)。有鑑於此，本研究嘗試藉由下列之一次低密度與短時程培養盤貼附法 (transient lower-density plastic adherence, tLDA)，迅速將mMSCs自骨髓貼附細胞群中純化分離：經擴增之小鼠骨髓細胞藉由低密度 (1.25×104 cells/cm2) 之接種可有效區隔整體貼附之mMSCs與HCs群；然為避免長程培養對mMSCs生長與分化能力之影響，研究策略係藉短時程 (3小時) 之貼附，將trypsin敏感之mMSCs群回收分離。試驗結果顯示，tLDA分離之mMSCs (tLDA-mMSCs) 可於體外連續擴增培養 (群體倍增數>20)，並具有Sca-1、CD29、CD44、CD80與MHC-I陽性以及CD11b、CD31、CD45、CD86、CD117與MHC-II陰性等抗原表現形式 (antigen profile)。在適當培養條件下，tLDA-mMSCs可分化為脂肪細胞 (adipocyte)、成骨細胞 (osteoblast) 與軟骨細胞 (chondrocyte) 等間葉系細胞，具有複分化潛能 (multipotency)。然將tLDA-mMSCs與T細胞共培養 (co-culture)發現，tLDA-mMSCs可顯著抑制同種異體 (allogeneic) T細胞與concanavalin-A誘發之T細胞增殖，此證明tLDA-mMSCs之免疫抑制 (immunosuppressive) 特性。另外經體內移植後，tLDA-mMSCs可自導引 (homing) 至骨髓並參與骨質疏鬆 (osteoporosis)小鼠骨質之形成，彼等可由追朔EGFP轉殖小鼠tLDA-mMSCs (tLDA-mMSCs-GFP) 於體內之走向證實之，此結果亦為mMSCs一次培養盤貼附分離之確效依據。
已知在骨髓微環境 (niche) 中，自MSCs衍生之骨 (bone) 與脂肪 (fat) 發育呈相互對等 (inverse relationship) 調節，而彼等調節生理與老化誘導之骨質疏鬆及所伴隨之骨髓脂化特性 (marrow adiposity) 息息相關。鑑此能有效定義MSCs於成骨生成 (osteogenesis) 與脂肪生成 (adipogenesis) 過程中參與之調節分子，對於MSCs於骨質疏鬆症研究與治療之策略將有實質之幫助。基於上述建立之HC-free mMSCs培養系統，本研究另比較其所分離之tLDA-mMSCs於早期 (0、2、6與24小時) 成骨分化與脂肪分化過程中具相互對等調控之轉錄體 (transcriptomics) 與微型核醣核酸 (microRNA, miRNAs) 表現之差別；藉由微陣列分析之結果顯示，試驗分別定義共97個與84個具顯著差異對等表現之基因群與miRNAs。另以miRanda、TargetScan與PicTar資料庫進行生物資訊分析與定量PCR確認顯示，6個經成骨分化過程顯著表現之候選miRNAs：miR-20b、miR-25、miR-29b、miR-106b、miR-125a-3p與miR-211可標的於脂肪分化顯著表現之基因群，其中包括PPAR-gamma等脂肪分化訊息路徑因子。此對等分化調節分子之定義為調控MSCs體內與體外分化走向之有效依據。
綜觀再生醫學領域之發展，動物模式之使用於疾病發展歷程中扮演重要樞紐。豬，係具有較大的體型，並與人類有相近的生理特徵，因此，豬在臨床前研究基礎上具極佳之應用價值。基於研究初步定義之功能性mMSCs與彼等參與分化調節分子之結果，另鑑於本研究團隊已成功培育出EGFP基因轉殖豬，本研究亦嘗試由EGFP轉殖豬骨髓分離其MSCs (Green fluorescent protein tagged pig bone marrow mesenchymal stem cells, pMSCs-GFP)，且建立一穩定之體外與體內功能性分析系統。研究結果顯示，自EGFP轉殖豬骨髓分離之pMSCs-GFP呈典型纖維母細胞型態，彼等貼附分離之pMSCs-GFP可於體外連續擴增培養 (群體倍增數>40)，擁有典型pMSCs抗原表現形式、複分化潛能與免疫抑制等功能，且彼等pMSCs-GFP螢光蛋白之表現亦不受體外分化程序所影響 (>99%為GFP陽性細胞)。進一步將pMSCs-GFP以原位 (in situ) 注射法注入心肌梗塞 (myocardial infarction) 疾病小鼠顯示，pMSCs-GFP可有效改善疾病小鼠之心肌厚度，並顯著提升其心功能 (heart functions)。彼等pMSCs-GFP之修復機制可由光子影像 (photon image) 系統進行追蹤，因此非常適合利用其螢光特性來進行MSCs移植後之走向分析。綜言，吾等定義由EGFP轉殖小鼠與豬骨髓MSCs之分離與分化之調控因子，此等研究成果將有助於未來臨床前以豬為模式進行細胞治療、基因治療與組織工程研究基礎之極佳用途。

關鍵字

豬 ； 小鼠 ； 骨髓 ； 間葉幹細胞 ； 綠色螢光蛋白質

並列摘要

Research into mesenchymal stem cells (MSCs) has been fueled because of their capacities to renew themselves and display general multipotentiality. Over the last several years, MSCs have generated a great deal of interest in many clinical settings, including that of regenerative medicine, immune modulation and tissue engineering. Nevertheless, assessment of therapeutics in MSCs remains controversy, largely owning to a lack of efficient MSCs tracker and in-depth understanding of the mechanisms that regulate functions and differentiations of the MSCs. Taking advantage of the green fluorescent protein (GFP) transgenic mice and pigs being generated in our laboratory, the goal of our study was to establish the optimal conditions for culturing MSCs isolated from these fluorescent animals, in which robust levels of GFP are expressed in stem cells and all differentiated progenies. In addition, the detailed mechanisms governing MSCs differentiation will also be evaluated. Classically, MSCs can be relatively easy to isolate from the bone marrow by their physical propensity to adhere to tissue culture plates and flasks. However, in mice, MSCs can be difficult to be isolated from contaminating HCs. Herein, we developed a non-antigenic based, single-step method to deplete HCs within 3 hours using the following transient lower-density plastic adherence (tLDA): cells are plated at a lower-density (1.25 × 104 cells/cm2) to allow all HCs attaching on the plastic surface and avoid overlapping with mMSCs. After a short-term of adherent period of no more than 3 hours followed by trypsin digestion, the HCs are eradicated due to the plastic-adhering HCs can not be lifted by trypsin, whereas mMSCs can. Here we show that these tLDA-isolated mMSCs, termed “tLDA-mMSCs”, can be extensively expanded (approximately 20 doubling populations) and exhibited antigen profiles that positive for Sca-1, CD29, CD44, CD80 and MHC-I and negative for CD11b, CD31, CD45, CD86, CD117 and MHC-II. Under an appropriate induction condition, tLDA-mMSCs can differentiate into adipocytes, chondrocytes and osteoblasts in vitro. When allogeneic tLDA-mMSCs was added to T cells stimulated by allogeneic lymphocytes or the potent T-cell mitogen concanavalin-A, a significant reduction in T-cell proliferation was observed, suggestive of their immunosuppressive effects. After intravenous transplantation, tLDA-mMSCs can migrate into the allogeneic bone marrow and rescued osteoporosis symptoms. We developed a simple and economic method that effectively isolated HCs-free, therapeutically functional mMSCs from marrow adherent cultures using plastic-adhesion. These cells will be suitable for various mechanistic and therapeutic studies in mouse model.
It is well established that an inverse relationship exists between bone and fat development within the marrow cavity. Developmentally, adipocytes and osteoblasts are originated from the same mesenchymal precursors, MSCs or stromal cells, in the bone marrow. Dissecting mechanism concerning the switch between adipocyte and osteoblast differentiation is crucial of treatment for osteoporosis, where an imbalanced bone/fat development in bone marrow is observed. Here, we compared the transcripotmics and microRNA (miRNAs) expression pattern of these HCs-free tLDA-mMSCs at an early step of commitment to adipocytes and osteoblasts (0, 2, 6 and 24 hours), in order to identify master miRNAs regulators responsible for the cell fate determination. Under this approach, we identified 97 genes and 84 miRNAs that showed significantly reciprocal expression during osteogenesis and adipogenesis. Using of several bioinformatic platforms to look for these potential interacted regulators, we identified 6 miRNAs namely: miR-20b, -25, -29b, -106b, 125a-3p and -211, that are activated during osteogenesis while the genes expressed during adipogenesis have target sites of these miRNAs. These candidate regulators possess potential to further elucidate the mechanism governing MSCs differentiation both in vitro and in vivo.
The pig is a very useful model animals for regenerative medicine studies because of their suitable body size and similar physiology compared to human. Since the mMSCs system could be used as references from the other species such as pigs, and in addition, the EGFP transgenic pigs had been successfully produced in our laboratory, all of their body tissues and organs expressed GFP and useful to be as a tracker in vitro and in vivo. Progressively, we would like to establish MSCs system from the bone marrow of those EGFP transgenic pigs. The pig MSCs were initially isolated from the marrow nucleated cultures using plastic-adherence and tagged with green fluorescent protein (pMSCs-GFP). These cells can be extensively expanded for more than 20 passages (approximately 40 doubling populations), exhibited homogeneous antigen profiles and have immunosuppressive effects. Under an appropriate induction condition, pMSCs-GFP can differentiate into adipocytes, chondrocytes, osteoblasts and neural cells, all of the cells expressed unform and high levels of GFP. Moreover, injection of pMSCs-GFP can dramatically increase thickness of the infracted myocardium and improve cardiac function in mice suffering myocardium infarction; these cells can be easily identified through ex vivo fluorescence imaging. In the present dissertation, an efficient, effective and easy way to identify pig MSCs after extended expansion, differentiation and transplantation in vivo has been developed and described in detail. These have further provided the opportunity to reveal the genetic changes of MSCs after transplanting to the preconditioned’ organ/tissue damaged, recipients. Based on strategies described above, optimal conditions for culture of MSCs have been identified and several potential molecular regulators involved in governing the differentiation of MSCs have been confirmed. These results would offer new insights into the biology of MSCs and it is anticipated that results derived from the present studies would have great benefit for the further MSCs therapeutics.

並列關鍵字

pig ； mouse ； bone marrow ； mesenchymal stem cells ； green fluorescent protein

參考文獻

Aggarwal, S., and M. F. Pittenger. 2005. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105: 1815-1822.

Anjos-Afonso, F., and D. Bonnet. 2007. Nonhematopoietic/endothelial ssea-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment. Blood 109: 1298-1306.

Ashton, B. A., T. D. Allen, C. R. Howlett, C. C. Eaglesom, A. Hattori, and M. Owen. 1980. Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin. Orthop. Relat. Res. 294-307.

Azizi, S. A., D. Stokes, B. J. Augelli, C. DiGirolamo, and D. J. Prockop. 1998. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts. Proc. Natl. Acad. Sci. U. S. A. 95: 3908-3913.

Bab, I., B. A. Ashton, D. Gazit, G. Marx, M. C. Williamson, and M. E. Owen. 1986. Kinetics and differentiation of marrow stromal cells in diffusion chambers in vivo. J. Cell Sci. 84: 139-151.