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

Stem Cell Research & Therapy
Lei-Sheng ZhangJiaxi Zhou

Abstract

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. 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. 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. ...Continue Reading

References

Nov 6, 1998·Science·J A ThomsonJ M Jones
Sep 6, 2001·Proceedings of the National Academy of Sciences of the United States of America·D S KaufmanJ A Thomson
Jul 23, 2003·Journal of Thrombosis and Haemostasis : JTH·K Kaushansky
May 15, 2013·Trends in Biotechnology·Thomas GajCarlos F Barbas
Dec 10, 2013·Cell Stem Cell·Yuxuan WuJinsong Li
Sep 30, 2014·Cell·Randall J PlattFeng Zhang
Jan 13, 2015·Seminars in Hematology·Kenneth Kaushansky
Jan 22, 2015·Blood·Jonathan N ThonJoseph E Italiano
Jun 11, 2016·Critical Reviews in Clinical Laboratory Sciences·Xiaohong Ruby XuHeyu Ni
Jun 25, 2016·Scientific Reports·Jian-Ping ZhangXiao-Bing Zhang
Oct 22, 2016·Thrombosis Journal·Kenneth Kaushansky
Nov 30, 2016·Molecular Therapy. Nucleic Acids·Xianglian GeFeng Gu
Jul 29, 2017·Journal of Thrombosis and Haemostasis : JTH·N Sugimoto, K Eto
Oct 7, 2017·Arteriosclerosis, Thrombosis, and Vascular Biology·Sara BorstPaul Gadue

❮ Previous
Next ❯

Citations

Aug 31, 2021·Frontiers in Cell and Developmental Biology·Huicong LiuFangfang Zhu
Dec 7, 2021·Frontiers in Public Health·Hongjun ChenDonghang Zhang

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
transfection
enzyme-linked immunosorbent assay
electron microscopy
confocal microscopy
electrophoresis
electron micrography
flow cytometry
flow

Software Mentioned

IVIS
GraphPad Prism

Related Concepts

Related Feeds

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

Blood And Marrow Transplantation

The use of hematopoietic stem cell transplantation or blood and marrow transplantation (bmt) is on the increase worldwide. BMT is used to replace damaged or destroyed bone marrow with healthy bone marrow stem cells. Here is the latest research on bone and marrow transplantation.