诱导人胚胎干细胞向胰腺内分泌细胞分化及其机制研究
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摘要
糖尿病是由于分泌胰岛素的β细胞缺失或功能异常而引起的糖代谢异常疾病。目前，尽管多种药物尤其是胰岛素的应用对糖尿病的治疗效果有了很大改善，但还没有一种治疗手段可以完全治愈糖尿病，阻止其并发症的发生。除了Ⅰ型糖尿病的中β细胞总数的缺乏外，新近研究发现在Ⅱ型糖尿病患者胰腺中同样存在β细胞团减少的现象，因此通过胰腺或胰岛移植是根治糖尿病的唯一手段，但是供体胰岛的严重缺乏限制了其广泛开展。干细胞因其多向分化潜能和自我更新的特性，已被越来越广泛地应用于细胞替代治疗的研究中。然而，将干细胞真正用于糖尿病的临床治疗还面临许多问题。
细胞数量和功能不足是干细胞治疗糖尿病所面临的主要挑战。尽管近年来，已经建立了使用生长因子和小分子化合物，将多能干细胞分化为胰腺内分泌细胞的方法，但是多能干细胞分化成为内分泌细胞需经过定型内胚层，胰腺祖细胞，内分泌祖细胞以及特定类型内分泌细胞五个阶段，诱导时间长，不可控制的因素多，所以，目前还没有一种方法可以获得足够数量、终末分化的功能性胰岛细胞。研究认为，胰岛的结构和胰岛细胞间的相互作用决定胰岛细胞的功能，三维(threedimentional,3D)细胞培养技术可通过恢复细胞与细胞间的相互关系弥补2D培养技术的不足，本课题拟利用3D培养技术从组织结构、细胞间相互作用的角度进行研究，尝试解决替代细胞分化效率和功能成熟的问题。
3D培养技术因其在体外构建与体内相近的细胞发育结构系统，成为研究细胞-细胞，细胞-基质间相互作用最直观的体外细胞培养模型，大量研究已证明，3D环境下的诱导分化效率高于2D环境，并且3D细胞培养可以恢复细胞的结构并提高细胞的功能。但是3D培养是否可以促进胚胎干细胞向胰岛细胞的分化，3D结构的细胞生长模式与胰岛细胞功能成熟之间的关系还不清楚。基于以上考虑，本课题分为两部分，第一部分的工作旨在探索3D细胞培养与胰岛细胞功能之间的关系；第二部分在第一部分工作的基础上，利用3D细胞培养技术，模仿体内胰岛生长微环境，探讨3D细胞培养对人胚胎干细胞（human embryonic stemcells, hESC）向胰岛细胞分化过程中的影响，并初步分析其作用机制，为体外获得有功能的种子细胞提供技术支持和理论基础。
一、三维细胞培养对胰岛细胞功能的影响及其机制研究：通过悬浮培养小鼠β细胞系MIN6细胞，形成类组织样结构的MIN6细胞团(3D-MIN6)，葡萄糖刺激胰岛素释放(GSIS)实验证实，3D-MIN6较2D-MIN6在葡萄糖刺激下可以释放更多的胰岛素，q-PCR显示3D-MIN6中胰腺内分泌细胞相关基因表达上调，提示3D细胞培养可以增强β细胞的基因表达和GSIS功能。RhoA和ROCK功能阻断实验表明，3D-MIN6细胞通过抑制RhoA/ROCK通路，使F-actin重构，易化胰岛素释的释放过程，另外，3D细胞培养和RhoA/ROCK功能阻断可使细胞缝隙连接蛋白36（Connexin36, Cx36）的表达增高，而通过siRNA技术阻断Cx36功能后，MIN6细胞的GSIS明显降低，即使抑制RhoA/ROCK活性也不能恢复MIN6的胰岛素释放功能，提示Rho/ROCK信号通路通过Cx36发挥调节胰岛素释放的作用。第一部分的研究表明3D细胞培养通过抑制RhoA/ROCK的活性，可以促进Cx36的表达，进而加强MIN6细胞的GSIS功能。
二、诱导人胚胎干细胞向胰腺内分泌细胞分化及其机制研究：本研究首先利用细胞因子和小分子化合物组合建立了诱导hESC向胰腺内分泌细胞(pancreaseendocrine cell, PEC)的分化体系，通过27天的诱导，hESC来源的PEC可以合成内分泌激素胰岛素、胰高血糖素和生长抑素，并且能在Fosklin、KCL的刺激下分泌胰岛素，但是对与葡萄糖的刺激反应微弱。在此基础上，我们分别对诱导细胞进行了简单的悬浮3D培养和基质材料Matrigel包埋的3D细胞培养，与常规2D培养相比，3D培养加强了hESC来源的PEC的内分泌特异性，使胰岛素阳性细胞的分化效率提高到23.7%，细胞内胰岛素的含量明显增多，并且具备了GSIS的能力。通过对影响细胞间相互作用的粘着斑激酶(focal adhesion kinase, FAK)研究发现，3D组诱导细胞中FAK活性明显低于2D组，抑制FAK或其下游ROCK的表达，可以促进内分泌细胞定向分化的相关基因NGN3、NKX6.1、ISL1的上调表达，说明FAK和ROCK通路参与了3D培养促进内分泌细胞定向分化的过程。另外TGFβ信号通路已被证实具有调节内分泌细胞定向分化的作用，我们Western blot结果显示，下调FAK活性亦可抑制TGFβ下游Smad2的活性。在3D组诱导细胞中还发现Cx36表达明显上调，抑制FAK和ROCK通路同样可以提高Cx36基因合成和蛋白表达，说明3D培养通过FAK、ROCK信号通路调控Cx36的合成与表达，进而增强细胞GSIS的功能。小鼠体内试验证实移植入体内的PEC可以合成胰岛素并根据体内血糖水平调节其释放。
综上所述，本课题的研究表明，3D细胞培养条件有利于小鼠β细胞系MIN6胰腺发育相关基因的表达和GSIS功能的发挥。在hESC向PEC的分化过程中，3D细胞培养通过抑制FAK及其下游信号通路ERK、ROCK、Smad2，可以提高PEC的分化效率和增强GSIS功能。该研究不仅为3D环境可以增强β细胞功能和促进hESC向PEC分化提供了新的理论和技术基础，也给糖尿病患者应用细胞替代治疗带来了新的希望。
Diabetes is a devastating chronic disease afflicting hundreds of millions ofindividuals. Although modern medicine has made considerable progress in thetreatment of diabetes, the morbidity and increased mortality associated with diabeticcomplications are still the major concerns. Beta cell mass and function are decreased tovarying degrees in both type1and type2diabetes. Pancreas transplantation and islettransplantation emerged as effective treatments for patients with diabetes, but thesignificant shortage of donor organs remains an issue. Stem cells are self-renewing,clonogenic and multipotent cells having tremendous potential for the treatment ofseveral human diseases and potential source for regenerative medicine. In the future,the promise of stem-cell-derived islet cell replacement or regeneration therapy maythus offer therapeutic benefit to people with diabetes, but there are major challenges tobe overcome before clinical application.
Several promising approaches to generate new β-cells have been developed inrecent years. These include directed differentiation of pluripotent cells such asembryonic stem (ES) cells. High-yield methods to differentiate cell populationsintodefinitive endoderm, pancreatic progenitors, and β-cells havebeen establishedusing growth factors and small molecules. However, the final step of directeddifferentiation to generate functional, mature β-cells in sufficient quantities is yet to beachieved in vitro. Beside the need of transplantation medicine, a renewable source ofβ-cells would also be important in terms of present topographical structures,architecture and mimicking the natural surroundings of islet and to seek for alternativetreatment, finally, generating functional, mature β-cells.
Three-dimensional (3D) cell cultures that mimic in vivo microenvironmentscontribute to study tissue structure related to organ differentiation and function.several studies have proved that3D culture can promote stem cell differentiation, andrestore cell function. In this study, our first aim was to verify the relationship between3D culture and function of β-cell. The second aim was to study the effects of3Dculture on ES cell differentiation.
Part1Three Dimension Culture enhance function of β cell line MIN6: In thisstudy, mouse insulinoma6(MIN6) cells were cultured in a rotated3D culture systemto form islet-like aggregates, and glucose stimulated insulin secretion (GSIS) has beentested accordingly. We investigated whether the RhoA/ROCK pathway was involvedin the effect of3D culture on MIN6cells. The results demonstrated that moreendocrine-specific genes expressed and GSIS increased in the MIN6cells under3Dculture condition than that in monolayer culture system. We found that RhoA/ROCKinactivation led to F-actin remodeling in MIN6cell aggregates and more insulinexocytosis. Meanwhile, we also found that the gap junction forming protein,connexin36(Cx36), was increased in MIN6cell aggregates and the monolayer cellswith RhoA/ROCK inactivation,and GSIS dramatically decreased when Cx36wasknockdown by siRNA and couldn’t be reversed by RhoA/ROCK inactivation. Inconclusion, the RhoA/ROCK signaling pathway was involved in the insulin releasethrough increasing Cx36gap junctions in3D culture condition.
Part2Three Dimension Culture Promotes Pancreas Endocrine Specificationsfrom hESC: Stem cell-based tissue engineering is a promising technology in the effortto create functional tissues of choice. To establish an efficient approach for generatingpancreas endocrine cells (PEC) directly from human embryonic stem cells (hESC) andto study the effects of three-dimensional (3D) culture on hESC differentiation, wecultured hESC on ultra-low attachment plates or biomimetic metrigel. Celldifferentiation was evaluated by Q-PCR, microscopy and flow cytometry analysis witha variety of pancreas specific markers. Our data indicate that hESC differentiated on3D culture developed multicell aggregates similar to those islet like structure; and theefficiency of PEC generation on3D, as indicated by the expression of variousPEC-specific surface markers (INS, GCG, SST), and INS positive cell wasreproducibly increased (23.7%) over their2D counterparts(16%). Moveover, insulinproduction stimulated by glucose in3D culture was found to be significantly higherthan that in2D differentiation system. We further demonstrate that3D culture inhibitsthe activation of focal adhesion kinase (FAK), and selective inhibition of this kinase issufficient to induce early endocrine commitment based on increased expression ofNGN3, PDX1and ISL1. Additional studies inhibit one of FAK downstream regulatorROCK suggest that ROCK pathway can also induce early endocrine specification. Wepropose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGF-β/Smad2/3pathway. Moreover, we show that inhibitionof FAK and/or ROCK signaling promtes Cx36expression, this result hints thatincreases the expression of Cx36may contribute insulin secreation of late endocrinecells. Finally, we assessed the competence of hESC–derived PEC in vivo. The serumlevels of glucose-regulated C-peptide and glucose-tolerance tests of mice indicated thatthe hESC–derived PEC are functional and sufficient to control levels of glucose inserum.
In summary, the results presented here demonstrated that3D culture contribute toGSIS and gene expression of mouse β-cell line MIN6.3D culture also provides ascalable system to achieve hESC proliferation and differentiation into PEC, wherebythe efficiency of ESC differentiation increased within3D systems, in terms of insulinproductivity, in comparison with2D differentiation systems. The mechanism is viainhibion FAK and its downstream singling way ERK, ROCK and Smad2. Thisresearch not only propose a theory and metherd for ESC differentiate to PEC, but alsoprovide a compelling evidence that hESC may serve as a renewable source of islets fordiabetes cell-replacement therapies.
细胞数量和功能不足是干细胞治疗糖尿病所面临的主要挑战。尽管近年来，已经建立了使用生长因子和小分子化合物，将多能干细胞分化为胰腺内分泌细胞的方法，但是多能干细胞分化成为内分泌细胞需经过定型内胚层，胰腺祖细胞，内分泌祖细胞以及特定类型内分泌细胞五个阶段，诱导时间长，不可控制的因素多，所以，目前还没有一种方法可以获得足够数量、终末分化的功能性胰岛细胞。研究认为，胰岛的结构和胰岛细胞间的相互作用决定胰岛细胞的功能，三维(threedimentional,3D)细胞培养技术可通过恢复细胞与细胞间的相互关系弥补2D培养技术的不足，本课题拟利用3D培养技术从组织结构、细胞间相互作用的角度进行研究，尝试解决替代细胞分化效率和功能成熟的问题。
3D培养技术因其在体外构建与体内相近的细胞发育结构系统，成为研究细胞-细胞，细胞-基质间相互作用最直观的体外细胞培养模型，大量研究已证明，3D环境下的诱导分化效率高于2D环境，并且3D细胞培养可以恢复细胞的结构并提高细胞的功能。但是3D培养是否可以促进胚胎干细胞向胰岛细胞的分化，3D结构的细胞生长模式与胰岛细胞功能成熟之间的关系还不清楚。基于以上考虑，本课题分为两部分，第一部分的工作旨在探索3D细胞培养与胰岛细胞功能之间的关系；第二部分在第一部分工作的基础上，利用3D细胞培养技术，模仿体内胰岛生长微环境，探讨3D细胞培养对人胚胎干细胞（human embryonic stemcells, hESC）向胰岛细胞分化过程中的影响，并初步分析其作用机制，为体外获得有功能的种子细胞提供技术支持和理论基础。
一、三维细胞培养对胰岛细胞功能的影响及其机制研究：通过悬浮培养小鼠β细胞系MIN6细胞，形成类组织样结构的MIN6细胞团(3D-MIN6)，葡萄糖刺激胰岛素释放(GSIS)实验证实，3D-MIN6较2D-MIN6在葡萄糖刺激下可以释放更多的胰岛素，q-PCR显示3D-MIN6中胰腺内分泌细胞相关基因表达上调，提示3D细胞培养可以增强β细胞的基因表达和GSIS功能。RhoA和ROCK功能阻断实验表明，3D-MIN6细胞通过抑制RhoA/ROCK通路，使F-actin重构，易化胰岛素释的释放过程，另外，3D细胞培养和RhoA/ROCK功能阻断可使细胞缝隙连接蛋白36（Connexin36, Cx36）的表达增高，而通过siRNA技术阻断Cx36功能后，MIN6细胞的GSIS明显降低，即使抑制RhoA/ROCK活性也不能恢复MIN6的胰岛素释放功能，提示Rho/ROCK信号通路通过Cx36发挥调节胰岛素释放的作用。第一部分的研究表明3D细胞培养通过抑制RhoA/ROCK的活性，可以促进Cx36的表达，进而加强MIN6细胞的GSIS功能。
二、诱导人胚胎干细胞向胰腺内分泌细胞分化及其机制研究：本研究首先利用细胞因子和小分子化合物组合建立了诱导hESC向胰腺内分泌细胞(pancreaseendocrine cell, PEC)的分化体系，通过27天的诱导，hESC来源的PEC可以合成内分泌激素胰岛素、胰高血糖素和生长抑素，并且能在Fosklin、KCL的刺激下分泌胰岛素，但是对与葡萄糖的刺激反应微弱。在此基础上，我们分别对诱导细胞进行了简单的悬浮3D培养和基质材料Matrigel包埋的3D细胞培养，与常规2D培养相比，3D培养加强了hESC来源的PEC的内分泌特异性，使胰岛素阳性细胞的分化效率提高到23.7%，细胞内胰岛素的含量明显增多，并且具备了GSIS的能力。通过对影响细胞间相互作用的粘着斑激酶(focal adhesion kinase, FAK)研究发现，3D组诱导细胞中FAK活性明显低于2D组，抑制FAK或其下游ROCK的表达，可以促进内分泌细胞定向分化的相关基因NGN3、NKX6.1、ISL1的上调表达，说明FAK和ROCK通路参与了3D培养促进内分泌细胞定向分化的过程。另外TGFβ信号通路已被证实具有调节内分泌细胞定向分化的作用，我们Western blot结果显示，下调FAK活性亦可抑制TGFβ下游Smad2的活性。在3D组诱导细胞中还发现Cx36表达明显上调，抑制FAK和ROCK通路同样可以提高Cx36基因合成和蛋白表达，说明3D培养通过FAK、ROCK信号通路调控Cx36的合成与表达，进而增强细胞GSIS的功能。小鼠体内试验证实移植入体内的PEC可以合成胰岛素并根据体内血糖水平调节其释放。
综上所述，本课题的研究表明，3D细胞培养条件有利于小鼠β细胞系MIN6胰腺发育相关基因的表达和GSIS功能的发挥。在hESC向PEC的分化过程中，3D细胞培养通过抑制FAK及其下游信号通路ERK、ROCK、Smad2，可以提高PEC的分化效率和增强GSIS功能。该研究不仅为3D环境可以增强β细胞功能和促进hESC向PEC分化提供了新的理论和技术基础，也给糖尿病患者应用细胞替代治疗带来了新的希望。
Diabetes is a devastating chronic disease afflicting hundreds of millions ofindividuals. Although modern medicine has made considerable progress in thetreatment of diabetes, the morbidity and increased mortality associated with diabeticcomplications are still the major concerns. Beta cell mass and function are decreased tovarying degrees in both type1and type2diabetes. Pancreas transplantation and islettransplantation emerged as effective treatments for patients with diabetes, but thesignificant shortage of donor organs remains an issue. Stem cells are self-renewing,clonogenic and multipotent cells having tremendous potential for the treatment ofseveral human diseases and potential source for regenerative medicine. In the future,the promise of stem-cell-derived islet cell replacement or regeneration therapy maythus offer therapeutic benefit to people with diabetes, but there are major challenges tobe overcome before clinical application.
Several promising approaches to generate new β-cells have been developed inrecent years. These include directed differentiation of pluripotent cells such asembryonic stem (ES) cells. High-yield methods to differentiate cell populationsintodefinitive endoderm, pancreatic progenitors, and β-cells havebeen establishedusing growth factors and small molecules. However, the final step of directeddifferentiation to generate functional, mature β-cells in sufficient quantities is yet to beachieved in vitro. Beside the need of transplantation medicine, a renewable source ofβ-cells would also be important in terms of present topographical structures,architecture and mimicking the natural surroundings of islet and to seek for alternativetreatment, finally, generating functional, mature β-cells.
Three-dimensional (3D) cell cultures that mimic in vivo microenvironmentscontribute to study tissue structure related to organ differentiation and function.several studies have proved that3D culture can promote stem cell differentiation, andrestore cell function. In this study, our first aim was to verify the relationship between3D culture and function of β-cell. The second aim was to study the effects of3Dculture on ES cell differentiation.
Part1Three Dimension Culture enhance function of β cell line MIN6: In thisstudy, mouse insulinoma6(MIN6) cells were cultured in a rotated3D culture systemto form islet-like aggregates, and glucose stimulated insulin secretion (GSIS) has beentested accordingly. We investigated whether the RhoA/ROCK pathway was involvedin the effect of3D culture on MIN6cells. The results demonstrated that moreendocrine-specific genes expressed and GSIS increased in the MIN6cells under3Dculture condition than that in monolayer culture system. We found that RhoA/ROCKinactivation led to F-actin remodeling in MIN6cell aggregates and more insulinexocytosis. Meanwhile, we also found that the gap junction forming protein,connexin36(Cx36), was increased in MIN6cell aggregates and the monolayer cellswith RhoA/ROCK inactivation,and GSIS dramatically decreased when Cx36wasknockdown by siRNA and couldn’t be reversed by RhoA/ROCK inactivation. Inconclusion, the RhoA/ROCK signaling pathway was involved in the insulin releasethrough increasing Cx36gap junctions in3D culture condition.
Part2Three Dimension Culture Promotes Pancreas Endocrine Specificationsfrom hESC: Stem cell-based tissue engineering is a promising technology in the effortto create functional tissues of choice. To establish an efficient approach for generatingpancreas endocrine cells (PEC) directly from human embryonic stem cells (hESC) andto study the effects of three-dimensional (3D) culture on hESC differentiation, wecultured hESC on ultra-low attachment plates or biomimetic metrigel. Celldifferentiation was evaluated by Q-PCR, microscopy and flow cytometry analysis witha variety of pancreas specific markers. Our data indicate that hESC differentiated on3D culture developed multicell aggregates similar to those islet like structure; and theefficiency of PEC generation on3D, as indicated by the expression of variousPEC-specific surface markers (INS, GCG, SST), and INS positive cell wasreproducibly increased (23.7%) over their2D counterparts(16%). Moveover, insulinproduction stimulated by glucose in3D culture was found to be significantly higherthan that in2D differentiation system. We further demonstrate that3D culture inhibitsthe activation of focal adhesion kinase (FAK), and selective inhibition of this kinase issufficient to induce early endocrine commitment based on increased expression ofNGN3, PDX1and ISL1. Additional studies inhibit one of FAK downstream regulatorROCK suggest that ROCK pathway can also induce early endocrine specification. Wepropose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGF-β/Smad2/3pathway. Moreover, we show that inhibitionof FAK and/or ROCK signaling promtes Cx36expression, this result hints thatincreases the expression of Cx36may contribute insulin secreation of late endocrinecells. Finally, we assessed the competence of hESC–derived PEC in vivo. The serumlevels of glucose-regulated C-peptide and glucose-tolerance tests of mice indicated thatthe hESC–derived PEC are functional and sufficient to control levels of glucose inserum.
In summary, the results presented here demonstrated that3D culture contribute toGSIS and gene expression of mouse β-cell line MIN6.3D culture also provides ascalable system to achieve hESC proliferation and differentiation into PEC, wherebythe efficiency of ESC differentiation increased within3D systems, in terms of insulinproductivity, in comparison with2D differentiation systems. The mechanism is viainhibion FAK and its downstream singling way ERK, ROCK and Smad2. Thisresearch not only propose a theory and metherd for ESC differentiate to PEC, but alsoprovide a compelling evidence that hESC may serve as a renewable source of islets fordiabetes cell-replacement therapies.
引文
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[2] Blum B, Hrvatin S S, Schuetz C, et al. Functional beta-cell maturation is marked by anincreased glucose threshold and by expression of urocortin3. Nat Biotechnol,2012,30:261-264.
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