Stem Cells, Development, and Cancer Laboratory, GENYO: Centro de Genómica e Investigación Oncológica Pfizer-Universidad de Granada-Junta de Andalucía, Granada, Spain.
Stem Cells Dev. 2012 Jun 10;21(9):1549-58. doi: 10.1089/scd.2011.0400. Epub 2011 Oct 27.
The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.
人类胚胎干细胞 (hESC) 作为人类发育性造血的模型，以及在潜在的细胞替代策略中，其实现依赖于对调节造血特异性 hESC 分化的外在和内在因素的更好理解。人类间充质干细胞 (hMSC) 是多能细胞，起源于中胚层，构成造血干细胞龛的一部分，通过分泌因子的产生和/或细胞间相互作用，在调节造血中发挥重要作用。我们之前已经表明，可以使用 hMSC 条件培养基 (MSC-CM) 成功无饲养层维持 hESC。在这里，我们假设在 MSC-CM 中维持的 hESC 比在标准人包皮成纤维细胞条件培养基中生长的 hESC 更倾向于向造血谱系分化。我们报告说，在 MSC-CM 中维持的 hESC 系向造血祖细胞的特化以及随后的造血分化和克隆形成祖细胞能力得到了强有力的增强。有趣的是，将 hESC 与 hMSC 共培养完全消除了 hESC 的造血特化，因此表明改善的造血分化是由 MSC 分泌的因子介导的，而不是由 MSC-hESC 物理相互作用介导的。为了研究该过程中涉及的分子机制，我们分析了全基因组 (LINE-1) 甲基化和全基因组启动子 DNA 甲基化。在 MSC-CM 中生长的 hESC 显示出全基因组 DNA 甲基化降低了 17%，并且启动子 DNA 甲基化特征由 45 个常见低甲基化基因和 102 个频繁高甲基化基因组成。我们的数据表明，在 MSC-CM 中维持 hESC 可显著增强造血特化，并且该过程似乎由 MSC 分泌的因子介导，赋予未分化的 hESC 一种 DNA 甲基化特征，这可能影响进一步向造血特化的倾向。
