Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems
Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems
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DOI：
10.1002/bit.10220
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发表时间：
2002-05-20
影响因子：
3.8
通讯作者：
Zandstra, PW
中科院分区：
文献类型：
--
作者：
Dang, SM;Kyba, M;Zandstra, PW
胚胎干（ES）细胞作为细胞疗法的细胞来源具有巨大的潜力。这种潜力的实现将取决于我们理解和操纵影响细胞命运决定的因素以及开发可扩展的细胞生产方法的能力。我们通过测量胚状体 (EB) 形成效率和细胞增殖的各个方面，并通过跟踪特定分化组织类型 - 血液使用功能（集落形成细胞）和表型（Flk-1 和 CD34 表达）测定的发展，比较了四种标准 ES 细胞分化培养系统。我们报道，单个小鼠 ES 细胞形成 EB 的效率为 42 +/- 9%，但由于 EB 聚集（两个或多个单个 ES 细胞或 EB 融合形成单个更大的细胞聚集体的过程），很少获得该值。无论 EB 是由甲基纤维素或液体悬浮培养中的单个 ES 细胞产生，还是由悬滴培养中的 ES 细胞聚集体产生，它们都会生长到每个 EB 28,000 +/- 9,000 个细胞的相似最大细胞数。在悬浮培养中产生 EB 的三种方法中，造血发育的动力学或频率没有差异。因此，用单个 ES 细胞启动 EB 并防止 EB 聚集应允许 EB 系统中分化细胞的最大产量。还使用相同的输出将 EB 分化培养物与附着分化培养物进行比较。附着的集落的细胞数量也没有类似的限制；然而，贴壁培养中的造血发育受到损害。早期 FIk-1 和 CD34 表达细胞的百分比显着低于悬浮培养的 EB，而造血集落形成几乎完全受到抑制。这些结果为开发高效、可扩展的 ES 细胞分化生物过程奠定了基础，并为生产造血组织的新方法提供了信息。 (C) 2002 年 Wiley 期刊公司
Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early FIk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues. (C) 2002 Wiley Periodicals, Inc.