CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells
Zhong Zheng, Chenshuang Li, Pin Ha, Grace X. Chang, Pu Yang, Xinli Zhang, Jong Kil Kim, Wenlu Jiang, Xiaoxiao Pang, Emily A. Berthiaume, Zane Mills, Christos S. Haveles, Eric Chen, Kang Ting, Chia Soo
Zhong Zheng, Chenshuang Li, Pin Ha, Grace X. Chang, Pu Yang, Xinli Zhang, Jong Kil Kim, Wenlu Jiang, Xiaoxiao Pang, Emily A. Berthiaume, Zane Mills, Christos S. Haveles, Eric Chen, Kang Ting, Chia Soo
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Research Article Cell biology

CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells

Abstract

Tumorigenicity is a well-documented risk to overcome for pluripotent or multipotent cell applications in regenerative medicine. To address the emerging demand for safe cell sources in tissue regeneration, we established a novel, protein-based reprogramming method that does not require genome integration or oncogene activation to yield multipotent fibromodulin (FMOD)-reprogrammed (FReP) cells from dermal fibroblasts. When compared with induced pluripotent stem cells (iPSCs), FReP cells exhibited a superior capability for bone and skeletal muscle regeneration with markedly less tumorigenic risk. Moreover, we showed that the decreased tumorigenicity of FReP cells was directly related to an upregulation of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression during the FMOD reprogramming process. Indeed, sustained suppression of CDKN2B resulted in tumorigenic, pluripotent FReP cells that formed teratomas in vivo that were indistinguishable from iPSC-derived teratomas. These results highlight the pivotal role of CDKN2B in cell fate determination and tumorigenic regulation and reveal an alternative pluripotent/multipotent cell reprogramming strategy that solely uses FMOD protein.

Authors

Zhong Zheng, Chenshuang Li, Pin Ha, Grace X. Chang, Pu Yang, Xinli Zhang, Jong Kil Kim, Wenlu Jiang, Xiaoxiao Pang, Emily A. Berthiaume, Zane Mills, Christos S. Haveles, Eric Chen, Kang Ting, Chia Soo

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Figure 6

TP53 and CDKN gene–KD FReP cells have different gene expression profiles and myogenic differentiation potentials.

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TP53 and CDKN gene–KD FReP cells have different gene expression profile...
(A) Gene expression of TP53 and CDKN genes was assessed in the FReP cells derived from different KD BJ fibroblasts. Data are presented as mean ± SD and normalized to those of the BJ fibroblasts without any shRNA transfection. *P < 0.05, **P < 0.005 (analyzed by 1-way ANOVA and 1-tailed 2-sample t tests); n = 3 independent experiments performed in duplicate. Black dashed lines indicate the gene expression levels of BJ fibroblasts without any shRNA transfection (in brief, BJ fibroblasts); red dashed lines indicate the gene expression levels of BJ-iPSCs; blue dashed lines indicate the gene expression levels of FReP cells derived from BJ fibroblasts without any shRNA transfection (in brief, FReP cells); gray asterisks indicate significance in comparison with FReP cells generated from scrambled shRNA–transfected BJ fibroblasts (scramble FReP cells). (B and C) Myogenic differentiation of KD FReP cells was assessed by myogenic marker staining (B) and creatine kinase activity assay (C) in vitro. White arrowheads indicate the fusing myogenic differentiated cells. Scale bar: 100 μm (B). **P < 0.005 (analyzed by 1-tailed Mann-Whitney and Kruskal-Wallis ANOVA tests, C); n = 6.