Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production

Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, we employed a stepwise hiPSC-to-iMSC differentiation method via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, we were able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, we demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo. we differentiated two iMSC subtypes via two intermediate cell types of neural crest (NC-iMSCs) and cytotrophoblast (CT-iMSCs) using serum-free chemical-defined media.

过去数十年来，间充质基质细胞（mesenchymal stromal cells, MSCs）作为治疗多种疾病的潜在治疗性细胞来源，已被广泛研究。由人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）分化得到的诱导多能干细胞来源间充质基质细胞（induced pluripotent stem cell-derived MSCs, iMSCs），为间充质基质细胞及相关生物制品的规模化生物制造提供了可扩展的技术路径。尽管iMSCs与原代间充质基质细胞（primary MSCs, pMSCs）共享典型的间充质基质细胞标志物与功能，但诸多iMSCs分化方案仍存在谱系特异性缺失的问题。本研究采用基于神经嵴（neural crest, NC）与细胞滋养层（cytotrophoblast, CT）中间细胞阶段的逐步hiPSC向iMSCs分化方法，以获得具有不同分化效率与基因表达特征的谱系特异性iMSCs。通过对早期发育细胞类型（hiPSCs、神经嵴与细胞滋养层）、两种谱系特异性iMSCs以及六种来源特异性原代间充质基质细胞开展全面比较，本研究不仅可区分间充质基质细胞与早期发育细胞间的转录组学差异，还可明确iMSCs亚型与产后或围产期原代间充质基质细胞的转录组学相似性。此外，本研究证实，不同iMSCs亚型与预处理条件会影响细胞外囊泡（extracellular vesicles, EVs）的产生、外泌体蛋白表达及细胞因子载荷。本研究采用无血清化学成分明确的培养基，通过神经嵴与细胞滋养层两种中间细胞类型，成功分化得到两种iMSCs亚型：神经嵴来源iMSCs（NC-iMSCs）与细胞滋养层来源iMSCs（CT-iMSCs）。