Microfabricated 3D Culture for Promoting Hepatic Differentiation of Stem Cell and Maintaining Hepatocyte Function for Liver Tissue Engineering

Many factors (such as alcohol and toxins) can cause acute and chronic liver diseases, which can further develop into liver failure. End-stage liver failure is associated with high rates of morbidity and mortality and liver transplantation is the only effective treatment. However, due to limited number of donors, at least 15% patients die while being on the waiting list for liver transplant. Liver regenerative medicine, including hepatocyte transplantation, bioartificial livers and bioengineered grafts, can be an alternative for liver transplantation. In vitro culture of hepatocytes can serve as a cell source for liver regenerative medicine. However, in most cases, the isolated primary hepatocytes rapidly dedifferentiate and lose their functions in vitro, which leads to a shortage of available hepatocytes for liver regenerative medicine. Consequently, developing a strategy to improve hepatocyte functions in vitro or supply hepatocyte or hepatocyte-like cell (HLC) derived from stem cells will advance our understanding of liver regenerative medicine and facilitate end-stage liver diseases therapy using hepatocyte and stem cells.Liver extracellular matrix (ECM) regulates the specification of liver progenitors and hepatocyte functions in vivo. Previous studies showed that decellularized ECM scaffold and liver derived-ECM hydrogel can maintain the functions of isolated hepatocyte in vitro. However, given that native ECM is composed of various component(s) that assemble into a complex structure, the effect of individual and combination of ECM component on hepatic function should be determined in order to optimize the scaffold properties. In addition, understanding the role of individual ECM component can facilitate liver regenerative medicine using hepatocyte and stem cells. Here, we explore the effects of different ECM components (i.e. type I collagen, type IV collagen, laminin, fibronectin) on hepatocyte cell polarity and hepatic function maintenance, as well as the basis of the ECM effects. However, no significant difference in terms of hepatic functions was observed in traditional monolayer culture when different ECM molecules were applied. Previous studies suggested hepatocytes in spheroids culture can better mimic their in vivo state. Thus, we try to aggregate hepatocytes to form hepatocyte spheroids, and our results suggest that spheroids culture can restore the polarity of hepatocytes. Alginate encapsulation can further enhance cell-cell interaction and improve hepatic functions. Moreover, differential effects of ECM molecules were observed in 3D spheroid encapsulation (when ECM was mixed with alginate) but not so much in other configurations. LM displayed the best individual effect on hepatocyte hepatic function maintenance in 3D culture. When ECM components were mixed in combinatorial screening, a strong positive effect of CI and a negative interaction effect of CI*LM on some hepatic functions were observed. In addition, we found that the effect of LM relies on the activation of integrin α1 and integrin α6 signals. These findings on ECM effect on hepatocyte culture have broad applications in optimizing ECM microenvironments for liver regenerative medicine.To expand the source of available hepatocytes, stem cell-derived HLC has been generated by researchers. HLC can be potential cell sources for liver tissue engineering. Nevertheless, traditional 2D differentiation methods produce HLC similar to fetal hepatocytes rather than mature hepatocytes. Therefore, promoting functional maturation of HLC is necessary. Some studies have reported 3D differentiation could promote differentiated HLC mature. However, there are also some challenges associated with 3D culture, such as insufficient oxygen and nutrient supply due to the large size of hydrogel, non-uniform spheroids size and non-uniform distributions of embryonic bodies (EB). Microfluidics technique allows high throughput generation of size-controllable microgels. Here, we utilized microfluidics technique to encapsulate ESC into CI microgels for 3D differentiation. Our results suggested uniform-sized EB were formed after ESC were encapsulated in collagen microgels that were later differentiated to HLC with high efficiency. Compared with 3D encapsulation in a bulk collagen hydrogel and 2D differentiation, our approach resulted in HLC with higher ALB gene expression and lower AFP gene expression, indicating HLC in CI microgel was more mature. Our studies on ESC hepatic differentiation can provide relatively mature and available HLC and facilitate the development of liver tissue engineering.In conclusion, our results provide a method to maintain liver function in vitro and supply functional HLC derived from stem cells. Our research can potentially help develop cell-based treatment for end-stage liver diseases.許多因素（例如酒精和毒素）都會導致急性和慢性肝病的發生，並可能進一步發展為肝衰竭。肝衰竭具有高發病率和高死亡率。目前，肝移植是肝衰竭唯一有效的治療方法。然而由於肝臟供體有限，每年至少有15％的患者在等待肝移植的過程中死亡。肝再生醫學，包括肝細胞移植，人工肝和組織工程肝組織移植，是潛在的肝移植的替代療法，而體外培養肝細胞是肝再生醫學的基礎。然而，從人體中分離的原代肝細胞在體外會迅速退分化並喪失增殖能力和肝功能，這導致了可用于肝再生醫學的肝細胞的短缺。因此，研究改善體外培養的肝細胞功能以及擴大可用肝細胞或類肝細胞樣細胞的方法將增進我們對肝再生醫學的理解，並促進利用肝細胞和幹細胞來源的類肝細胞樣細胞進行終末期肝病治療的發展。肝細胞外基質（ECM）可以調節體內肝祖細胞的分化和肝細胞功能。研究表明，脫細胞的ECM支架和肝臟來源的ECM水凝膠可以幫助分離的肝細胞維持它們的功能。然而天然ECM支架的成分十分複雜，研究具體ECM成分對於肝細胞功能的影響可以幫助我們優化出有助於肝細胞功能維持的生物材料支架，而瞭解不同ECM成分的作用也可以促進肝再生醫學的發展。本研究中，我們研究了不同ECM成分（ I型膠原（CI），IV型膠原(CIV)，層粘連蛋白(LM)，纖連蛋白(FN)）對肝細胞極性和肝功能維持的影響。然而在2D培養中，不同的ECM成分對肝細胞功能沒有明顯影響。而有研究表明，細胞團培養中的肝細胞可以更好地類比其在體狀態。因此，我們將肝細胞聚集為肝細胞團並研究不同ECM成分對於肝細胞團的肝功能的影響。結果表明，細胞團培養可以幫助肝細胞恢復細胞極性，而藻酸鹽封裝可以增強細胞間的相互作用並促進肝細胞的功能。我們的研究表明，不同ECM成分對於3D培養的肝細胞團的功能提升有著不同作用，LM能夠全方位地提升肝細胞團的肝功能，這與ITGA1和ITGA6信號的轉導有關。然而當不同ECM成分組合在一起時，CI有著明顯的促進肝功能的作用，而CI*LM的組合則有著明顯的抑制肝功能的作用。人胚胎幹細胞（hESC）衍生的類肝細胞樣細胞（HLC）是肝再生醫學潛在的細胞來源。然而，傳統的2D分化方法產生的HLC與胎兒肝細胞相似，與成熟的肝細胞相去甚遠。因此，產生成熟的具有肝功能的HLC是肝細胞再生醫學的第一步。有研究表明3D分化可以促進分化的HLC成熟，然而，3D培養中的一些困難可能會阻礙分化的肝細胞的成熟，例如傳統方法產生水凝膠過大，而其中的細胞團分佈不均勻從而導致的氧氣和營養供應不足不利於肝細胞的成熟。而微流控技術可以產生大量直徑相似的微凝膠（microgel）。因此，我們利用微流控技術將hESC封裝在CI微凝膠中然後再進行肝細胞方向分化，希望這能產生大量可用的成熟有功能的肝細胞。而我們的結果表明，hESC被包裹在膠原微凝膠可以成功分化為HLC，並且與CI水凝膠和2D分化的HLC相比微凝膠分化的HLC具有更高的ALB表達和更低的AFP表達，這表明微凝膠產生的HLC更加成熟。我們利用微流控技術包裹hESC並進行分化的方法可以產生大量成熟可用的類肝細胞樣細胞，這可能會促進肝組織工程的發展。總之，我們關於利用ECM成分幫助分離的肝細胞維持肝功能和利用微流控技術包裹hESC從而產生大量成熟可用的類肝細胞樣細胞可能有助於肝再生醫學的發展和晚期肝病的治療。DENG, Shuai.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...