人類胚胎幹細胞培養、分化及冷凍之研究

Cultivation, Differentiation, and Cryopreservation of Human Embryonic Stem Cells

林佩宜(Pei-Yi Lin)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/博士(2013年)

摘要

人類胚胎幹細胞由於其自我更新及多能性的細胞特性，使其成為再生醫學、細胞治療及臨床移植上一個重要的細胞來源。然而，在真正進入實際應用之前，它仍有許多亟需克服的障礙。在本研究中將分別針對：體外培養、誘導分化及冷凍保存三個方向著手進行改善。
首先，我們利用經合成胜肽-丙烯聚酯修飾的表面材質(Synthemax®)取代Matrigel於成分確定的培養基(mTeSR™1)內進行人類胚胎幹細胞的增殖，我們進一步確認細胞經過十次的繼代後仍表現多能性的標誌以及保有分化成三胚層細胞的能力，更重要的是其仍維持正常的染色體。接著，我們建立一不含血清的誘導分化培養基能使人類胚胎幹細胞有效率地分化成為定型內胚細胞(86%)，我們甚至可讓人類胚胎幹細胞經三階段共21天的誘導培養基中順利分化成為胰島素分泌細胞(25%)，且在免疫螢光染色及定量的基因分析結果中，我們發現細胞於Matrigel或Synthemax®表面上進行分化誘導所得之效率並無顯著差異。因此，Synthemax®不僅可以作為人類胚胎幹細胞的長期培養平台，亦可在其上進行胰島素分泌細胞的誘導分化以成為未來糖尿病患者的細胞治療來源。
最後，我們利用一結合動態磁場的程式降溫儀Cell Alive System (CAS)進行人類胚胎幹細胞的冷凍保存，以減少降溫過程中冰晶所造成之冷凍傷害。我們將人類胚胎幹細胞團塊回溶於冷凍培養基中分成三組：(1) 細胞於傳統的冷凍保存容器Mr. Frosty並置於-80度冰箱降溫(MF)；(2) 細胞利用CAS降溫至-32度後改置於-80度冰箱(CAS)；(3) 細胞利用CAS降溫至-32度後改放於預冷之Mr. Frosty並置於-80度冰箱降溫(CAS-MF)。隔夜，所有冷凍管皆保存於液態氮內，一周後將人類胚胎幹細胞解凍並培養於餵養細胞上7天。由鹼性磷酸酶的染色結果計算細胞凍後貼附率，經CAS或CAS-MF冷凍保存之貼附率分別為29.0%及44.0%，皆優於經MF冷凍保存之7.0%。我們更進一步確認細胞經CAS-MF冷凍保存後仍能持續繼代並表現多能性標誌，以及保有分化成三胚層細胞的能力並維持正常的染色體。由以上結果可知CAS-MF將可提供人類胚胎幹細胞庫一個更有效率的冷凍保存方式。

關鍵字

人類胚胎幹細胞 ； 合成表面 ； 培養 ； 分化 ； 胰島素分泌細胞 ； 冷凍

並列摘要

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine, cell therapy, and clinical transplantation. However, there are still many immediate obstacles that need to be addressed before their practical application. In this study, we focus on three avenues for improvement: cultivation, differentiation, and cryopreservation.
First, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax®) in defined mTeSR™1 medium to expand undifferentiated hESCs. We confirmed that the cells still expressed pluripotency markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. We were also able to obtain 25% insulin-producing cells within 21 days by following a simple three-step protocol. Moreover, the results of immunocytochemical and quantitative gene expression analyses showed that the efficiency of induction was not significantly different between the Synthemax® surface and the Matrigel-coated surface. Thus, Synthemax® could be a stable substrate for the long-term culture of hESCs, and the differentiated insulin-producing cells could be a therapeutic resource for diabetic patients in the future.
Last, we used the Cell Alive System (CAS), which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty, and kept in a -80°C freezer (MF); (ii) cells frozen to -32°C by CAS, and then transferred to a -80°C freezer (CAS); and (iii) cells frozen to -32°C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a -80°C freezer (CAS-MF) overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder cells for 7 days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than that achieved using the MF method (7.0%). Furthermore, we confirmed that cells cryopreserved using CAS-MF could be subcultured while expressing pluripotency markers, could differentiate into the three germ layers, and could maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method for hESC banking.

並列關鍵字

Human embryonic stem cells ； Synthetic surface ； Cultivation ； Differentiation ； Insulin-producing cells ； Cryopreservation

參考文獻

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