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Stem Cell Res Ther. 2018 Jul 17;9(1):194. doi: 10.1186/s13287-018-0926-x.

Thrombopoietin knock-in augments platelet generation from human embryonic stem cells.

Zhang L1,2, Liu C1,2, Wang H1,2, Wu D1,2, Su P1,2, Wang M1,2, Guo J3, Zhao S1,2, Dong S1,2, Zhou W3, Arakaki C4, Zhang X1,2,4, Zhou J5,6.

Author information

1
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin, 300020, China.
2
Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Beijing, China.
3
School of Basic Medical Science and Cancer Research Institute, Central South University, Changsha, 410013, China.
4
Division of Regenerative Medicine MC 1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA, 92350, USA.
5
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin, 300020, China. zhoujx@ihcams.ac.cn.
6
Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Beijing, China. zhoujx@ihcams.ac.cn.

Abstract

BACKGROUND:

Refinement of therapeutic-scale platelet production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy. However, procedures for cost-effective and scalable platelet generation remain to be established.

METHODS:

In this study, we established human embryonic stem cell (hESC) lines containing knock-in of thrombopoietin (TPO) via CRISPR/Cas9-mediated genome editing. The expression and secretion of TPO was detected by western blotting and enzyme-linked immunosorbent assay. Then, we tested the potency for hematopoietic differentiation by coculturing the cells with mAGM-S3 cells and measured the generation of CD43+ and CD45+ hematopoietic progenitor cells (HPCs). The potency for megakaryocytic differentiation and platelet generation of TPO knock-in hESCs were further detected by measuring the expression of CD41a and CD42b. The morphology and function of platelets were analyzed with electronic microscopy and aggregation assay.

RESULTS:

The TPO gene was successfully inserted into the AAVS1 locus of the hESC genome and two cell lines with stable TPO expression and secretion were established. TPO knock-in exerts minimal effects on pluripotency but enhances early hematopoiesis and generation of more HPCs. More importantly, upon its knock-in, TPO augments megakaryocytic differentiation and platelet generation. In addition, the platelets derived from hESCs in vitro are functionally and morphologically comparable to those found in peripheral blood. Furthermore, TPO knock-in can partially replace the large quantities of extrinsic TPO necessary for megakaryocytic differentiation and platelet generation.

CONCLUSIONS:

Our results demonstrate that autonomous production of cytokines in hESCs may become a powerful approach for cost-effective and large-scale platelet generation in translational medicine.

KEYWORDS:

Early hematopoiesis; Human embryonic stem cells; Knock-in; Platelets; Thrombopoietin

PMID:
30016991
PMCID:
PMC6050740
DOI:
10.1186/s13287-018-0926-x
[Indexed for MEDLINE]
Free PMC Article

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