CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells
Zhong Zheng, … , Kang Ting, Chia Soo
Zhong Zheng, … , Kang Ting, Chia Soo
Published July 15, 2019
Citation Information: J Clin Invest. 2019;129(8):3236-3251. https://doi.org/10.1172/JCI125015.
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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 7

TP53 and CDKN gene KD diversely alters FReP cell proliferation, anchorage-independent survival ability in soft agar, and proto-oncogene expression in vitro.

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TP53 and CDKN gene KD diversely alters FReP cell proliferation, anchora...
(A) A cell proliferation assay was carried out in 96-well culture plates after 2000 cells per well were cultured in undifferentiating conditions for 3 days. (B and C) A soft agar colony formation assay was performed after 14 days of cultivation. Five thousand cells per well were seeded initially. (D) The expressions of multiple proto-oncogenes in scramble FReP cells and CDKN2B-KD FReP cells were compared. Data are presented as mean values (A and B) or mean values normalized with those of the BJ fibroblasts ± SD (D). **P < 0.005 (analyzed by 1-way ANOVA and 1-tailed 2-sample t tests); n = 6 (A–C) or 3 independent experiments performed in duplicate (D). Gray dashed lines indicate the original cell seeding densities (A and B); black dashed lines indicate the gene expression levels of BJ fibroblasts (D); red dashed lines indicate the gene expression levels of BJ-iPSCs (D); blue dashed lines indicate the cell densities (A and B) or the gene expression levels (D) of FReP cells, respectively; gray asterisks indicate significance in comparison with scramble FReP cells. Scale bar: 500 μm.