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. 2023 Mar 10;12(6):870.
doi: 10.3390/cells12060870.

Knocking Down CDKN2A in 3D hiPSC-Derived Brown Adipose Progenitors Potentiates Differentiation, Oxidative Metabolism and Browning Process

Yasmina Kahoul  1 , Xi Yao  2 , Frédérik Oger  1 , Maeva Moreno  1 , Souhila Amanzougarene  1 , Mehdi Derhourhi  1 , Emmanuelle Durand  1 , Raphael Boutry  1 , Amélie Bonnefond  1   3 , Philippe Froguel  1   3 , Christian Dani  2 , Jean-Sébastien Annicotte  4 , Christophe Breton  1
Affiliations

Knocking Down CDKN2A in 3D hiPSC-Derived Brown Adipose Progenitors Potentiates Differentiation, Oxidative Metabolism and Browning Process

Yasmina Kahoul et al. Cells. .

Abstract

Human induced pluripotent stem cells (hiPSCs) have the potential to be differentiated into any cell type, making them a relevant tool for therapeutic purposes such as cell-based therapies. In particular, they show great promise for obesity treatment as they represent an unlimited source of brown/beige adipose progenitors (hiPSC-BAPs). However, the low brown/beige adipocyte differentiation potential in 2D cultures represents a strong limitation for clinical use. In adipose tissue, besides its cell cycle regulator functions, the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus modulates the commitment of stem cells to the brown-like type fate, mature adipocyte energy metabolism and the browning of adipose tissue. Here, using a new method of hiPSC-BAPs 3D culture, via the formation of an organoid-like structure, we silenced CDKN2A expression during hiPSC-BAP adipogenic differentiation and observed that knocking down CDKN2A potentiates adipogenesis, oxidative metabolism and the browning process, resulting in brown-like adipocytes by promoting UCP1 expression and beiging markers. Our results suggest that modulating CDKN2A levels could be relevant for hiPSC-BAPs cell-based therapies.

Keywords: 3D culture; CDKN2A; UCP1; adipocytes; brown adipose progenitor; browning; human induced pluripotent stem cells; thermogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Protocol of hiPSC-BAP differentiation into adipospheres. hiPSC-BAPs were plated into low adherent plates (ULA) and transfected with the control siRNA or the CDKN2A siRNA. Three days (D-3) after, spheroids formed and then were induced to undergo differentiation into adipospheres. Confoncal images are shown. Blue: DAPI for nuclei staining. Red: Oil Red O for lipid droplets staining. Scale: 50 µm. RNA-Seq and Pamgene analyses were performed at day 0 (D0) and day 10 (D10).
Figure 2
Figure 2
Transcriptomic analysis of 10 days-differentiated 3D adipospheres (D10 vs. D0). (A) A volcano-plot of differentially regulated gene expression. UCP1 is indicated in black. Enrichment of IPA biological process terms for down-regulated (B) and up-regulated (C) genes following differentiation. IPA terms are plotted against the negative log of corrected p-values. Most down-regulated (B) and up-regulated (C) enriched pathways.
Figure 3
Figure 3
Kinome analysis in 10 days-differentiated 3D adipospheres (D10 vs. D0). Volcano-plots of STK (A), PTK (B)-modulated peptides and STK (C), PTK (D)-modulated kinases. Green circle: hyperphosphorylated peptide (A). Red circle: hypophosphorylated peptide (A,B) and kinase (C,D). Dark circle: unmodulated peptide (A,B) and kinase (C,D).
Figure 3
Figure 3
Kinome analysis in 10 days-differentiated 3D adipospheres (D10 vs. D0). Volcano-plots of STK (A), PTK (B)-modulated peptides and STK (C), PTK (D)-modulated kinases. Green circle: hyperphosphorylated peptide (A). Red circle: hypophosphorylated peptide (A,B) and kinase (C,D). Dark circle: unmodulated peptide (A,B) and kinase (C,D).
Figure 4
Figure 4
Enrichment of IPA biological process terms for kinome analysis in 10 days-differentiated 3D adipospheres (D10 vs. D0). IPA terms are plotted against the negative log of corrected p-values. Most modulated enriched pathways in STK (A) and in PTK (B).
Figure 5
Figure 5
Transcriptomic analysis at spheroid stages of differentiation (D0) between CDKN2A-deficient and control hiPSC-BAPs. (A) A volcano-plot of differentially regulated gene expression. Enrichment of IPA biological process terms for down-regulated (B) and up-regulated (C) genes of CDKN2A-deficient D0 spheroids. IPA terms are plotted against the negative log of corrected p-values. Most down-regulated (B) and up-regulated (C) enriched pathways.
Figure 5
Figure 5
Transcriptomic analysis at spheroid stages of differentiation (D0) between CDKN2A-deficient and control hiPSC-BAPs. (A) A volcano-plot of differentially regulated gene expression. Enrichment of IPA biological process terms for down-regulated (B) and up-regulated (C) genes of CDKN2A-deficient D0 spheroids. IPA terms are plotted against the negative log of corrected p-values. Most down-regulated (B) and up-regulated (C) enriched pathways.
Figure 6
Figure 6
Kinome analysis at spheroid stages of differentiation (D0) between CDKN2A-deficient and control hiPSC-BAPs. Volcano-plots of STK (A), PTK (B)-modulated peptides and STK (C), PTK (D)-modulated kinases. Green circle: hyperphosphorylated peptide (B) and kinase (C,D). Red circle: hypophosphorylated peptide (A,B). Dark circle: unmodulated peptide (A,B) and kinase (C,D).
Figure 7
Figure 7
Enrichment of IPA biological process terms for kinome analysis at spheroid stages of differentiation (D0) between CDKN2A-deficient and control hiPSC-BAPs. IPA terms are plotted against the negative log of corrected p-values. Most modulated enriched pathways in STK (A) and in PTK (B).
Figure 8
Figure 8
Transcriptomic analysis in 10 days-differentiated (D10) 3D adipospheres following siCDKN2A silencing performed at the progenitor stage 3 days before differentiation. (A) A volcano-plot of differentially regulated gene expression. UCP1 is indicated in black. Enrichment of IPA biological process terms for down-regulated (B) and up-regulated (C) genes of CDKN2A-deficient D10 adipospheres. IPA terms are plotted against the negative log of corrected p-values. Most down-regulated (B) and up-regulated (C) enriched pathways.
Figure 8
Figure 8
Transcriptomic analysis in 10 days-differentiated (D10) 3D adipospheres following siCDKN2A silencing performed at the progenitor stage 3 days before differentiation. (A) A volcano-plot of differentially regulated gene expression. UCP1 is indicated in black. Enrichment of IPA biological process terms for down-regulated (B) and up-regulated (C) genes of CDKN2A-deficient D10 adipospheres. IPA terms are plotted against the negative log of corrected p-values. Most down-regulated (B) and up-regulated (C) enriched pathways.
Figure 9
Figure 9
Kinome analysis in 10 days-differentiated (D10) 3D adipospheres following siCDKN2A silencing performed at the progenitor stage 3 days before differentiation. Volcano-plots illustrating STK (A), PTK (B)-modulated peptides and STK (C), PTK (D)-modulated kinases. Green circle: hyperphosphorylated peptide (A,B) and kinase (C,D). Red circle: hypophosphorylated peptide (B). Dark circle: unmodulated peptide (A,B) and kinase (C,D).
Figure 10
Figure 10
Enrichment of IPA biological process terms for kinome analysis in 10 days-differentiated (D10) 3D adipospheres following siCDKN2A silencing performed at the progenitor stage 3 days before differentiation. IPA terms are plotted against the negative log of corrected p-values. Most modulated enriched pathways in STK (A) and in PTK (B).
Figure 11
Figure 11
Effects on silencing CDKN2A expression during hiPSC-BAP adipogenic differentiation in a 3D model: from spheroids (D0) to adipospheres (D10).
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