1. Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs
- Author
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Motohiko Oshima, Masamitsu Sone, Sou Nakamura, Maria Alejandra Kanashiro, Atsunori Saraya, Yasuo Harada, Kanae Hashimoto, Naoya Takayama, Koji Eto, Tomoyuki Yamaguchi, Atsushi Iwama, Sudip Kumar Paul, Akira Sawaguchi, Naoshi Sugimoto, Sachiko Umeda, Emiri Nakamura, Kyoko Tsujimura, and Harumi Ginya
- Subjects
Blood Platelets ,Cyclin-Dependent Kinase Inhibitor p21 ,p53 ,Megakaryocyte Progenitor Cells ,Induced Pluripotent Stem Cells ,bcl-X Protein ,Clone (cell biology) ,regenerative medicine ,Biology ,immortalization ,Biochemistry ,Regenerative medicine ,Cell Line ,Proto-Oncogene Proteins c-myc ,megakaryocyte ,Megakaryocyte ,Report ,Genetics ,medicine ,Humans ,Gene silencing ,Platelet ,Gene Silencing ,Induced pluripotent stem cell ,Cell Proliferation ,Polycomb Repressive Complex 1 ,platelet ,iPSC ,cyclin-dependent kinase inhibitor ,Cell Biology ,Clone Cells ,Up-Regulation ,Cell biology ,CDKN1A ,HEK293 Cells ,medicine.anatomical_structure ,Cell culture ,Gene Knockdown Techniques ,Tumor Suppressor Protein p53 ,Developmental Biology - Abstract
Summary Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology., Graphical abstract, Highlights • Expression of CDKIs is negatively correlated with MKCL proliferation potential • Silencing CDKN1A and p53 improved the induction efficiency of imMKCLs, imMKCLs derived from human iPSCs potentially overcome the many issues depending on blood donations for platelet transfusions. However, the rate of stable proliferation depends on the starting PSC clone. In this article, Takayama and colleagues show that silencing CDKN1A and p53 simultaneously with the overexpression of MYC/BMI1/BCL-XL dramatically increased the success rate of imMKCL establishment irrespective of the clone.
- Published
- 2021