Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, No.79 Qingchun Road, Hangzhou, Zhejiang, PR China.
Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang, PR China.
Stem Cell Res Ther. 2021 Jun 19;12(1):353. doi: 10.1186/s13287-021-02434-2.
Decades of efforts have attempted to differentiate the pluripotent stem cells (PSCs) into truly functional hematopoietic stem cells (HSCs), yet the problems of low differentiation efficiency in vitro and poor hematopoiesis reconstitution in vivo still exist, mainly attributing to the lack of solid, reproduced, or pursued differentiation system.
In this study, we established an in vitro differentiation system yielding in vivo hematopoietic reconstitution hematopoietic cells from mouse PSCs through a 3D induction system followed by coculture with OP9 stromal cells. The in vivo hematopoietic reconstitution potential of c-kit cells derived from the mouse PSCs was evaluated via m-NSG transplantation assay. Flow cytometry analysis, RNA-seq, and cell cycle analysis were used to detect the in vitro hematopoietic ability of endothelial protein C receptor (EPCR, CD201) cells generated in our induction system.
The c-kit cells from 3D self-assembling peptide induction system followed by the OP9 coculture system possessed apparently superiority in terms of in vivo repopulating activity than that of 3D induction system followed by the 0.1% gelatin culture. We interestingly found that our 3D+OP9 system enriched a higher percentage of CD201c-kitcells that showed more similar HSC-like features such as transcriptome level and CFU formation ability than CD201c-kitcells, which have not been reported in the field of mouse PSCs hematopoietic differentiation. Moreover, CD201 hematopoietic cells remained in a relatively slow cycling state, consistent with high expression levels of P57 and Ccng2. Further, we innovatively demonstrated that notch signaling pathway is responsible for in vitro CD201 hematopoietic cell induction from mouse PSCs.
Altogether, our findings lay a foundation for improving the efficiency of hematopoietic differentiation and generating in vivo functional HSC-like cells from mouse PSCs for clinical application.
几十年来，人们一直试图将多能干细胞（PSCs）分化为真正具有功能的造血干细胞（HSCs），但体外分化效率低和体内造血重建能力差的问题仍然存在，主要归因于缺乏可靠的、可重复的或可追踪的分化系统。
本研究通过 3D 诱导系统随后与 OP9 基质细胞共培养，从小鼠 PSCs 中建立了一个能够产生体内造血重建造血细胞的体外分化系统。通过 m-NSG 移植实验评估源自小鼠 PSCs 的 c-kit 细胞的体内造血重建潜能。采用流式细胞术分析、RNA-seq 和细胞周期分析来检测我们诱导系统中产生的内皮蛋白 C 受体（EPCR，CD201）细胞的体外造血能力。
与 3D 诱导系统随后在 0.1%明胶培养中的情况相比，来自 3D 自组装肽诱导系统随后经 OP9 共培养系统的 c-kit 细胞在体内重编程活性方面具有明显优势。我们有趣地发现，我们的 3D+OP9 系统富集了更高比例的 CD201c-kit 细胞，这些细胞在转录组水平和集落形成能力等方面表现出更类似 HSC 的特征，这在小鼠 PSCs 造血分化领域尚未有报道。此外，CD201 造血细胞仍处于相对缓慢的循环状态，与 P57 和 Ccng2 的高表达水平一致。此外，我们创新性地证明了 notch 信号通路负责从小鼠 PSCs 体外诱导 CD201 造血细胞。
总的来说，我们的研究结果为提高造血分化效率和从小鼠 PSCs 中产生体内功能类似 HSC 的细胞奠定了基础，可用于临床应用。
