Role of pH on Lineage Fate during Pluripotent Stem Cell Differentiation

摘要 人多能干细胞(HPSCs)，包括胚胎干细胞(HESCs)和诱导多能干细胞 细胞(HiPSCs)，在分化过程中完成一系列严格受控的事件，以概括功能 我们体内的细胞。这种体外成熟过程是通过诱导中胚层、外胚层和 内胚层祖细胞，为疾病建模、药物筛选、细胞- 以治疗为基础，并揭示细胞发育的机制。然而，主要障碍依然存在，包括(1) 低效的PSC分化，(2)昂贵的、劳动密集型的促进遗传异常的方案，以及(3) 终末细胞发育不成熟和功能降低。从机制上讲，表观遗传的擦除失败 记忆留下影响HiPSC体细胞染色质结构和基因表达的残留痕迹 来源类型。因此，了解和实施优化的hPSC体外分化方案势在必行。 重建与体内细胞相匹配的基因调控和表观基因组配置程序 为采用这一非凡系统的所有应用程序实现卓越的细胞类型。 细胞因子、生长因子和趋化因子在推动hPSC分化中的作用在细胞中得到了证实。 命运决定论。此外，代谢流量和代谢物水平在重新配置 表观基因组促进hPSC分化的研究最近被发现。然而，新陈代谢产生的一个因素 活性-酸化-尚未被视为控制hPSC分化的微环境刺激因素 尽管已知它在致病和生理发育过程中驱动去分化和再分化的作用。 我们最近在外胚层中发现了不同的代谢程序和胞外酸化速率 和中胚层谱系分化。这证实了新陈代谢和酸化之间的直接联系， 并进一步表明，较低的pH值在早期血统命运获得中起到了作用。这里的总体研究目标是 揭示特定血统细胞命运中依赖pH的机制，并最终利用这些过程来 优化hPSC分化。为了验证pH敏感机制控制早期细胞命运的假设，我们 将着手于3个具体目标：(1)确定在自发的情况下，pH在谱系划分中的重要性 拟胚体模型中的PSC分化。(2)检查表观基因组构型的重塑和 由此产生的谱系特异性基因表达可识别控制早期分化的pH敏感调节因子 在低pH条件下，使用转录和表观遗传筛选。以及(3)研究pH调节的可行性 与当前的hPSC分化方法相比，丰富了功能中胚层的衍生物。在以下方面取得成功 这些研究将通过pH调控开辟新的途径，以产生更好的早期体外模型 人类发展在健康和疾病方面的许多有前途的应用。

ABSTRACT Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), complete a tightly controlled sequence of events during differentiation to recapitulate functional cells from our bodies. This in vitro maturation process initiates by induction of mesoderm, ectoderm, and endoderm lineage progenitors, and provides unique opportunities for disease modeling, drug screening, cell- based therapies, and uncovering mechanisms in cell development. Yet, key obstacles remain and include (1) inefficient PSC differentiation, (2) costly, labor-intensive protocols that promote genetic aberrations, and (3) developmental immaturity and reduced functionality of terminal cells. Mechanistically, failed erasure of epigenetic memory leaves residual marks that affects chromatin structure and gene expression from a hiPSC's somatic cell type of origin. Thus, it is imperative to understand and implement optimized in vitro hPSC differentiation protocols that reconstitute gene regulation and epigenome configuration programs matching in vivo cell counterparts to achieve superior cell types for all applications that employ this remarkable system. The role of cytokines, growth factors, and chemokines in driving hPSC differentiation is firmly established in cell fate determination. Additionally, the facilitating role of metabolic flux and metabolite levels in reconfiguring the epigenome to improve hPSC differentiation has recently been uncovered. Yet, a factor generated by metabolic activity—acidification—has not been examined as a microenvironment stimulus controlling hPSC differentiation despite its known roles in driving de- and re-differentiation during pathogenic and physiological development. We recently discovered both differential metabolic programs and extracellular acidification rates during ectoderm and mesoderm lineage differentiation. This confirms a direct connection between metabolism and acidification, and further suggests a role for lowered pH in early lineage fate acquisition. The overall study goal here is to uncover pH-dependent mechanisms in lineage-specific cell fate, and to ultimately exploit these processes for optimized hPSC differentiation. To test the hypothesis that pH-sensitive mechanisms control early cell fate, we will embark on 3 specific aims: (1) To determine the importance of pH in lineage partitioning under spontaneous PSC differentiation in an embryoid body model. (2) To examine remodeling of epigenome configuration and resulting lineage-specific gene expression to identify pH-sensitive regulators governing early differentiation under low pH using transcriptomic and epigenetic screens. And (3) to study the feasibility of pH modulation to enrich for functional mesoderm derivatives compared to current hPSC differentiation methodologies. Success in these studies will open new pathways through pH manipulations for generating superior in vitro models of early human development for numerous promising applications in health and disease.