人類胎盤絨毛膜蛻膜間葉細胞的分離、鑑別與應用

Abstract

間葉細胞（Mesenchymal stromal cells, MSCs）已知存在於許多不同的組織中，目前被認為有希望以細胞治療的形式作為再生醫學藥物。在機制上，MSCs具有抗發炎作用以及可能透過旁分泌（paracrine effect）各類生長因子來幫助改善血管新生和修復受損組織。雖然目前有許多基於MSCs治療的臨床試驗已經完成，但是對於改善MSCs的應用性，有許多議題仍值得進一步探討，包括有效製備合格的MSCs、MSCs在各種動物模型中用於臨床前試驗的生物分佈和相關的藥物動力學研究評估、鑑定不同來源MSCs中特別的生物標誌和訊息傳遞路徑等。在本研究中，我們開發了一種新型的無血清、無異種材料且具選擇性的培養方法，並成功於人類胎盤的母體部分分離出MSCs，我們將此一具有免疫調節能力的MSCs命名為placenta choriodecidual membrane-derived mesenchymal stromal cells（pcMSCs）。pcMSCs是一種新型的MSCs，在無血清培養條件下可長期於體外擴大培養（> 20次繼代培養）同時保持MSCs特點的穩定性，因此更容易取得大量細胞來供應予臨床使用，我們也因為此分離培養方法的進步性而獲得了專利申請通過。獲得了穩定供應的細胞來源後，接下來我們使用白蛋白結合的螢光納米鑽石（Fluorescent nanodiamond, FNDs），利用其生物相容性和光穩定性，將標記FND後的pcMSCs靜脈注射入迷你豬體內，而後精確定量pcMSCs於器官和組織的數量並追蹤其位置。此外，為了找出pcMSCs的特異性生物標誌和特別的訊息傳遞路徑，我們分析了pcMSCs和骨髓來源的MSCs（Bone marrow derived MSCs, BMMSCs）的全基因組基因表達譜。結果顯示，pcMSCs具有雌激素受體及黃體激素受體表現，但不表現人類絨毛膜促性腺激素（hCG）受體，然而，以上三種受體BMMSCs均不表現。另一方面，基因表達分析亦顯示，pcMSCs表現Decoy receptor 3 （DcR3），一種屬於腫瘤壞死因子受體超家族（tumor necrosis factor receptor superfamily）的可溶性蛋白，已知可抑制被Fas ligand （FasL）誘導的細胞凋亡。目前臨床研究觀察到DcR3表現量會於月經週期中變化，並在子宮內膜細胞株的體外試驗中，發現DcR3可受性荷爾蒙調節。分析人類妊娠組織也發現蛻膜的DcR3蛋白在正常妊娠比無胚胎妊娠（anembryonic pregnancy）更高。雖然pcMSCs不表現hCG受體，但我們很驚訝的發現，添加hCG會導致pcMSCs的DcR3表現量升高並積累在細胞內，此現象可能是通過TGF-β訊息傳遞路徑所引發。同時，當pcMSCs受IFN-γ和TNF-α誘導刺激後，DcR3也會升高。據我們所知，本研究是首次描述DcR3在間葉細胞上的表現、調控以及其生理功能的報告。整體而言，我們認為pcMSCs相較於其他已知的MSCs，具有特別的訊息傳遞調控，並且具有應用於治療嚴重發炎性疾病的潛力。

Mesenchymal stromal cells (MSCs) have been identified from different tissues and regarded as promising sources for cell therapy and regeneration medicine. The potential of anti-inflammatory properties and paracrine effects of MSCs may help to improve angiogenesis and to repair damaged tissue. Although different clinical trials of MSCs-based therapy have been executing, some basic information are still necessary to clarify to improve the applicability of MSCs, including the effective preparation of qualified MSCs, the biodistribution and associated pharmacokinetics studies of MSCs in various animal models for preclinical evaluation, and identification of novel biomarkers and significant signaling pathway of MSCs in different MSCs. In this study, we have successfully developed a novel serum free and xeno-free selective culture protocol to isolate MSCs from the maternal part of human term placenta and named placenta choriodecidual membrane derived MSCs (pcMSCs) which also have immunomodulatory ability. Compared with different types of MSCs, pcMSCs was a new type of MSCs and exhibited advantages for easy availability of abundance of cells number by long-term in vitro expansion process (>20 subculture) in the serum-free culture conditions and kept the stability of MSCs characteristics, that were able to an adequate supply for clinical use. The protocol and related technique have obtained the patents already. We also used a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of pcMSCs in miniature pigs by magnetic modulation in order to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. Furthermore, to reveal the specific biomarkers and significant signaling pathway of pcMSCs, we analyzed whole-genome gene expression profiles of pcMSCs and bone marrow derived MSCs (BMMSCs). The results indicated that pcMSCs expressed estrogen receptor, progesterone receptor and did not express human chorionic gonadotropin (hCG) receptor, however, all these receptors were not identified of BMMSCs. Additionally, genes expression analysis also revealed that pcMSCs could express Decoy receptor 3 (DcR3), a soluble protein of the tumor necrosis factor receptor superfamily which inhibits Fas ligand-induced apoptosis. Nevertheless, clinical study showed that DcR3 expression varies during the menstrual cycle and was regulated by sex steroid hormones in vitro in endometrial cells. Human gestational tissues also showed a differential production of DcR3 while decidual DcR3 protein was lower in anembryonic than normal pregnancies. Surprisingly, although hCG receptor did not express in pcMSCs, add-ins of hCG for pcMSCs would cause DcR3 overexpression accumulated intracellularly of pcMSCs, and, this signaling pathway maybe via TGF-β signaling pathway. Meanwhile, DcR3 also commendable increased when pcMSCs were primed with IFN-γ and TNF-α. To our knowledge, this is the first report about DcR3 expression, regulation and physiological function in MSCs. Taken altogether, we proposed that pcMSCs have novel signaling regulation rather than the other type of MSCs and have the potential for therapeutic application in severe inflammatory disease.