Center for Stem Cell Research and Application, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC), Huacai Road 26, Chengdu, 610052, China.
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College(CAMS & PUMC), Tianjin, 300020, China.
Stem Cell Res Ther. 2024 Mar 5;15(1):68. doi: 10.1186/s13287-023-03624-w.
Mesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.
Flow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34 (UCB-CD34) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.
The stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34 HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34 HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34 HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34 HSPCs by antagonizing Wnt activation.
A novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell-cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.
间充质干细胞（MSCs）因其在维持造血干/祖细胞（HSPCs）功能方面的作用而具有重要的治疗价值。由于其来源丰富，因此源自人类多能干细胞的 MSCs 是一种理想的替代物。然而，源自人类多能干细胞的神经嵴起源的 MSCs 对 HSPCs 的维持作用尚未见报道。
采用流式细胞分析、RNA 测序和分化能力检测，从 3D 人脑类器官中分离基质细胞。在不同的共培养条件下，用人脐带血 CD34（UCB-CD34）细胞培养源自 3D 人脑类器官的基质细胞和脐带间充质干细胞（UC-MSCs），或不添加细胞因子鸡尾酒。通过 LTC-IC 测定法在体外和通过将脐血细胞和基质细胞共移植到免疫缺陷小鼠中来检测基质细胞的造血基质能力。采用 RNA 和蛋白质组学测序来检测 MSC 对 HSPCs 的作用。
源自 H1-hESCs 和人类诱导多能干细胞前脑类器官的基质细胞能够分化为经典的间充质来源细胞（成骨细胞、软骨细胞和脂肪细胞）。这些细胞表达 MSC 标志物，因此被命名为多能干细胞衍生的 MSC（pMSCs）。这些 pMSCs 具有神经嵴起源，在早期表达 CD271。当人 UCB-CD34 HSPCs 在 UC-MSCs 或 pMSCs 上共培养时，后者可在长期培养中大量扩增 UCB-CD34 HSPCs，并有效地维持其可移植性。通过 RNA 测序比较表明，人 UCB-CD34 HSPCs 与 pMSCs 共培养为 HSC 维持提供了一个改善的微环境。pMSCs 高度表达 Wnt 信号抑制剂 SFRP1 和 SFRP2，表明它们可能通过拮抗 Wnt 激活来帮助调节细胞周期，从而促进 UCB-CD34 HSPCs 的维持。
报道了一种在无血清培养条件下从 3D 人脑类器官中收获具有神经嵴起源的 MSC 的新方法。我们证明 pMSCs 可在体外长期培养中支持人 UCB-HSPC 的扩增，并维持其可移植能力。RNA 和蛋白质组学测序表明，pMSCs 通过细胞-细胞接触和分泌因子以及抑制 Wnt 信号传递来提供改善的 HSC 维持微环境。这代表了一种大规模生产神经嵴起源 MSC 的新方法，并为开发用于治疗血液系统疾病的人类造血基质细胞疗法提供了一种潜在方法。
