胚胎幹細胞是取自於囊胚的細胞，具有｢多能性(Pluripotent)｣和｢自我更新(Self-renewal)｣的能力，能夠在體外增殖並分化出各種成體細胞。近年來，胚胎幹細胞已被證實能夠在體外誘導分化成為肝臟細胞，而且具有治療小鼠肝病的能力。由於小鼠模式的成功，我們可以預見在將來，人類將需要大規模生產肝臟細胞的製程。人類將可利用這種分化的肝臟細胞來治療肝臟疾病，還能用來作為藥物研發的篩選平台，加速新藥的開發。
　　有鑑於未來在肝臟細胞上將有大量需求，本研究探討量產具有肝分化能力的小鼠胚體及誘導分化出高肝功能的肝臟細胞。首先在初步實驗中，建立小鼠胚胎幹細胞的培養方法。本研究採用的胚胎幹細胞株為D3，與不活化的小鼠初代胚胎纖維母細胞共同培養。進行肝分化時，則將小鼠胚胎幹細胞製成胚體，隨後在貼附培養中便會進行肝分化。為了鑑定肝分化，我們利用ELISA量測胚體的白蛋白產量，並利用Indocyanine green (ICG)染色，來證實胚胎幹細胞分化出成熟的肝臟細胞。
　　｢胚體形成(Embryoid body formation)｣是讓胚胎幹細胞進入肝分化過程的重要步驟。然而，製造胚體也是生產上的瓶頸，因為傳統的胚體製造方法只能用於小規模的生產。為了解決製造胚體的問題，我們嘗試利用攪拌式生物反應器Spinner flask來生產具有肝分化能力的小鼠胚體。我們的實驗結果顯示，胚胎幹細胞在Spinner flask中能夠形成胚體。與懸滴法比較，Spinner flask所製造的胚體其大小較小，而且大小的變異性較大。這些胚體能夠在貼附培養進行肝分化，而且生長激素與誘導物質具有促進肝分化的效果。在白蛋白分泌或是肝基因表現上，Spinner flask所製造的胚體都與懸滴法所製造的胚體相似。因此，Spinner flask有潛力能作為製造胚體的系統，來供後續量產肝臟細胞。
　　在生產高肝功能的肝臟細胞方面，我們採用一連串的實驗設計法來進行培養方法的改良。我們發現，肝分化培養液在培養第7天時添加最有助於胚體產生更多白蛋白，進一步證實肝分化培養液的Dexamethasone是促進肝分化的重要因子。我們的方法能夠獲得高白蛋白產量的肝臟細胞，其白蛋白產量可達到1.90 ± 0.20 pg/h•cell，而且，這種肝臟細胞同樣具有ICG的吸收及排放能力，這顯示這些細胞具有成熟的肝臟細胞特徵，RT-PCR也證實分化出來的肝臟細胞具有成熟的肝基表現。
　　本研究利用攪拌式生物反應器，開發肝分化能力小鼠胚體的生產方法，並研究出更經濟的肝分化方法，在不使用生長激素的情況下，就能生產出具有高肝功能的肝臟細胞。本研究的成果將有助於再生醫學及醫藥開發的進步。

Embryonic stem cells (ES cells), pluripotent and self-renewal, rised from blastocysts, are able to propagate their population and differentiate into various adult cells in vitro. In recent year, ES cells are demonstrated that they can be induced to differentiate into hepatocytes in vitro. Further, the ES cell-derived hepatocytes show therapeutical efficiency to remedy the mice with liver disease. Due to the success of using the mouse model, we expect that people might need a process to produce ES cell-derived hepatocytes in large-scale. The ES cell-derived hepatocytes would be used for treatment of liver disease. They would also be used for drug screening to improve the study of drug development.
Base on the requirement of large quantity of hepatocytes in the future, we study the mass production of mouse embryoid bodies (EBs) with hepatic differentiation ability, and induction to differentiate the hepatocytes with high liver function. Initially, we established the cultivation method of mouse ES cells in preliminary study. The ES cell line D3, used in this study, was co-cultured with inactivated primary mouse embryonic fibroblasts. For hepatic differentiation, ES cells were formed EBs, and the hepatic differentiation was appeared in further attached culture. To demonstrate hepatic differentiation, we measured albumin production of EBs by ELISA. Staining by indocyanine green (ICG) was also used to demonstrate that ES cells could differentiate into mature hepatocytes.
EB formation is an important step for hepatic differentiation of ES cells. However, production of EBs is a bottleneck of the production of ES cell-derived hepatocytes, because traditional EB formation methods are just used for small-scale EB production. To this end, we attempted to produce mouse EBs with hepatic differentiation ability by a stirred tank bioreactor, “spinner flask.” Our study showed that ES cells could form EBs in spinner flask. Compare to EBs formed by hanging drop method, EBs formed by spinner flask showed in smaller size and size variation. The EBs could also proceed to hepatic differentiation in an attached culture, and promoted by growth factors and inducers. We also observed that EBs formed by spinner flask were similar to that formed by hanging drops on albumin production and hepatic gene expression. Hence, spinner flask has the potential to be an EB formation system for further mass production of ES cell-derived hepatocytes
In the production of ES cell-derived hepatocytes with high liver function, we refined the culture method via a series of experimental design studies. We observed that hepatocyte differentiation medium was suitable for EBs to produce more albumin at day 7 of cultivation. Further, we demonstrated that dexamethasone was the most important factor to improve hepatic differentiation. Our refined method could obtain the ES cell-derived hepatocytes with high albumin production reached to 1.90 ± 0.20 pg/h•cell. In addition, the ES cell-derived hepatocytes showed ICG uptake ability and mature hepatic gene expression.
In conclusion, the present study examined production method of EBs with hepatic differentiation ability by a stirred tank bioreactor. A more economical hepatic differentiation method was also examined. We could produce ES cell-derived hepatocytes with high liver function without growth factor induction. The present study will benefit for the progress of regenerative medicine and drug development.

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