Cardiac cell generation from encapsulated embryonic stem cells in static and scalable culture systems.
Cardiac cell generation from encapsulated embryonic stem cells in static and scalable culture systems.
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DOI：
10.3727/096368910x513955
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发表时间：
2010
影响因子：
3.3
通讯作者：
Tzanakakis ES
中科院分区：
文献类型：
--
作者：
Jing D;Parikh A;Tzanakakis ES
心脏病是与心脏细胞大量丧失有关的发病率和死亡率的主要原因。胚胎干细胞（ESC）产生心肌细胞样细胞，可用于心脏细胞替代疗法。大多数心源性分化方案涉及将ESC培养为胚状体（EBs）。ESC聚集体的搅拌悬浮生物反应器培养物可用于按比例扩大心肌细胞后代的生产，但EB大小的范围广以及流体动力学环境对分化EB的未知影响是严格控制分化结果的一些主要挑战。在这里，我们探讨了小鼠胚胎干细胞（mESCs）和人类胚胎干细胞（hESCs）的心脏潜能封装在聚-L-赖氨酸（pLL）涂层藻酸盐胶囊。胶囊核心的液化导致在每个珠内形成单个ESC聚集体，并且它们的平均大小取决于接种的ESC的浓度。在加入骨形态发生蛋白4（BMP 4）的无血清条件下，将包封的mESC在培养皿中沿心肌细胞谱系定向沿着。在pLL-分层的液芯（LC）藻酸盐珠中的人ESC也在含血清的培养基中向心脏细胞分化。除了稳健的细胞增殖，在包封在LC中的ESC中检测到比在固体珠中更高分数的表达心脏标志物的细胞。此外，我们首次证明了包封在pLL-层状LC藻酸盐珠中的ESC可以在搅拌悬浮生物反应器中被诱导向心脏细胞。与培养皿培养物相比，包封的ESC在生物反应器中产生更高分数的Nkx2.5-和GATA 4-阳性细胞。分化的细胞形成搏动灶，以器官型方式对变时剂作出反应。我们的研究结果保证了微囊化技术与生物反应器培养相结合的进一步开发和实施，以使干细胞衍生的心脏细胞的生产适合于临床治疗和应用。
Heart diseases are major causes of morbidity and mortality linked to extensive loss of cardiac cells. Embryonic stem cells (ESCs) give rise to cardiomyocyte-like cells, which may be used in heart cell replacement therapies. Most cardiogenic differentiation protocols involve the culture of ESCs as embryoid bodies (EBs). Stirred-suspension bioreactor cultures of ESC aggregates may be employed for scaling up the production of cardiomyocyte progeny but the wide range of EB sizes and the unknown effects of the hydrodynamic environment on differentiating EBs are some of the major challenges in tightly controlling the differentiation outcome. Here, we explored the cardiogenic potential of mouse ESCs (mESCs) and human ESCs (hESCs) encapsulated in poly-L-lysine (pLL)-coated alginate capsules. Liquefaction of the capsule core led to the formation of single ESC aggregates within each bead and their average size depended on the concentration of seeded ESCs. Encapsulated mESCs were directed along cardiomyogenic lineages in dishes under serum-free conditions with the addition of bone morphogenetic protein 4 (BMP4). Human ESCs in pLL-layered liquid core (LC) alginate beads were also differentiated towards heart cells in serum-containing media. Besides the robust cell proliferation, higher fractions of cells expressing cardiac markers were detected in ESCs encapsulated in LC than in solid beads. Furthermore, we demonstrated for the first time that ESCs encapsulated in pLL-layered LC alginate beads can be coaxed towards heart cells in stirred-suspension bioreactors. Encapsulated ESCs yielded higher fractions of Nkx2.5- and GATA4-positive cells in the biore-actor compared to dish cultures. Differentiated cells formed beating foci that responded to chronotropic agents in an organotypic manner. Our findings warrant further development and implementation of microencapsulation technologies in conjunction with bioreactor cultivation to enable the production of stem cell-derived cardiac cells appropriate for clinical therapies and applications.