Enhanced HSC-like cell generation from mouse pluripotent stem cells in a 3D induction system cocultured with stromal cells

Wei Shan; Qin Yu; Yan Long; Qian Luo; Honghu Li; Yingli Han; Yulin Xu; Shan Fu; Xiangjun Zeng; Cong Wei; Yang Gao; Xiaoqing Li; Xia Li; Lifei Zhang; Lizhen Liu; Ming Chen; Pengxu Qian; He Huang

doi:10.1186/s13287-021-02434-2

Enhanced HSC-like cell generation from mouse pluripotent stem cells in a 3D induction system cocultured with stromal cells

Stem Cell Res Ther. 2021 Jun 19;12(1):353. doi: 10.1186/s13287-021-02434-2.

Authors

Wei Shan^#^{1

2

3

4}, Qin Yu^#⁵, Yan Long^{1

2

3

4}, Qian Luo^{1

2

3

4}, Honghu Li^{1

2

3

4}, Yingli Han^{1

2

3

4}, Yulin Xu^{1

2

3

4}, Shan Fu^{1

2

3

4}, Xiangjun Zeng^{1

2

3

4}, Cong Wei^{1

2

3

4}, Yang Gao⁶, Xiaoqing Li^{1

2

3

4}, Xia Li^{1

2

3

4}, Lifei Zhang⁶, Lizhen Liu^{1

2

3

4}, Ming Chen^{2

7}, Pengxu Qian^{8

9

10

11}, He Huang^{12

13

14

15}

Affiliations

¹ Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, No.79 Qingchun Road, Hangzhou, Zhejiang, PR China.
² Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang, PR China.
³ Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, Zhejiang, PR China.
⁴ Zhejiang Laboratory for Systems & Precison Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, PR China.
⁵ College of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, PR China.
⁶ Department of Hematology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, No. 3 Qingchun East Rd., Hangzhou, 310016, Zhejiang, PR China.
⁷ Department of Bioinformatics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China.
⁸ Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang, PR China. axu@zju.edu.cn.
⁹ Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, Zhejiang, PR China. axu@zju.edu.cn.
¹⁰ Zhejiang Laboratory for Systems & Precison Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, PR China. axu@zju.edu.cn.
¹¹ Center of Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, 310012, PR China. axu@zju.edu.cn.
¹² Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, No.79 Qingchun Road, Hangzhou, Zhejiang, PR China. huanghe@zju.edu.cn.
¹³ Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang, PR China. huanghe@zju.edu.cn.
¹⁴ Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, Zhejiang, PR China. huanghe@zju.edu.cn.
¹⁵ Zhejiang Laboratory for Systems & Precison Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, PR China. huanghe@zju.edu.cn.

^# Contributed equally.

Abstract

Background: 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.

Methods: 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.

Results: 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 CD201⁺c-kit⁺cells that showed more similar HSC-like features such as transcriptome level and CFU formation ability than CD201^-c-kit⁺cells, 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.

Conclusions: 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.

Keywords: 3D system; CD201; Hematopoiesis; Notch; Pluripotent stem cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation
Coculture Techniques
Hematopoiesis
Hematopoietic Stem Cells*
Mice
Pluripotent Stem Cells*
Stromal Cells