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. 2023 Apr;14(2):903-914.
doi: 10.1002/jcsm.13155. Epub 2023 Feb 1.

Chemical reprogramming of melanocytes to skeletal muscle cells

Wenjun Yang  1 , Yaqi Wang  1 , Yuanyuan Du  2 , Jiyong Li  3 , Minzhi Jia  3 , Sheng Li  1 , Ruimiao Ma  1 , Cheng Li  1 , Hongkui Deng  2 , Ping Hu  1   4   5   6
Affiliations

Chemical reprogramming of melanocytes to skeletal muscle cells

Wenjun Yang et al. J Cachexia Sarcopenia Muscle. 2023 Apr.

Abstract

Background: Direct cell-fate conversion by chemical reprogramming is promising for regenerative cell therapies. However, this process requires the reactivation of a set of master transcription factors (TFs) of the target cell type, which has proven challenging using only small molecules.

Methods: We developed a novel small-molecule cocktail permitting robust skin cell to muscle cell conversion. By single cell sequencing analysis, we identified a Pax3 (Paired box 3)-expressing melanocyte population holding a superior myogenic potential outperforming other seven types of skin cells. We further validated the single cell sequencing analysis results using immunofluorescence staining, in situ hybridization and FACS sorting and confirmed the myogenic potential of melanocytes during chemical reprogramming. We used single cell RNA-seq that detect the potential converted cell type, uncovering a unique role of Pax3 in facilitating chemical reprogramming from melanocytes to muscle cells.

Results: In this study, we demonstrated that the Pax3-expressing melanocytes to be a skin cell type for skeletal muscle cell fate conversion in chemical reprogramming. By developing a small-molecule cocktail, we showed an efficient melanocyte reprogramming to skeletal muscle cells (40%, P < 0.001). The endogenous expression of specific TFs may circumvent the additional requirement for TF reactivation and form a shortcut for cell fate conversion, suggesting a basic principle that could ease cell fate conversion.

Conclusions: Our study demonstrates the first report of melanocyte-to-muscle conversion by small molecules, suggesting a novel strategy for muscle regeneration. Furthermore, skin is one of the tissues closely located to skeletal muscle, and therefore, our results provide a promising foundation for therapeutic chemical reprogramming in vivo treating skeletal muscle degenerative diseases.

Keywords: Melanocytes; Reprogramming; Skeletal muscle cells; Small molecules.

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Conflict of interest statement

Wenjun Yang, Yaqi Wang, Yuanyuan Du, Jiyong Li, Minzhi Jia, Sheng Li, Ruimiao Ma, Cheng Li, Hongkui Deng, and Ping Hu declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Skin cells can be converted to skeletal muscles by a small molecule cocktail. (A) The scheme of chemical reprogramming of skin cells. (B) Phase contrast images of cells on day 0, 4, 8, and 12 after treated by the chemical combination. Scale bars, 200 μm. (C) Representative phase contrast image of myogenic cluster in left panel. Scale bar, 200 μm. Statistical analysis of the myogenic cluster formation ability of skin cells in right panel. Error bars indicated standard error. The statistical analysis was based on three independent experiments. (D) Relative expression levels of genes enriched in skeletal muscle cells. Cells were harvested on day 12 for RT‐qPCR analysis. Error bars indicated standard error. The statistical analysis was based on three independent experiments. ***P < 0.001. (E) Relative expression of myogenic genes. Cells were harvested on day 6 and day 8 for RT‐qPCR analysis, respectively. Error bars indicated standard error. The statistical analysis was based on three independent experiments. ***P < 0.001. (F) Statistical analysis of myogenic cluster formation ability in CD73− and CD73+ skin cells. Error bars indicated standard error. The statistical analysis was based on three independent experiments.
Figure 2
Figure 2
Melanocytes displayed myogenic potential after chemical induction. (A) scRNA‐seq was performed at different time points during skin‐to‐myocytes chemical reprogramming process. (B) UMAP plot of D0 CD73− skin cells coloured by cell types. (C) Dot plot of canonical marker genes in D0 CD73− skin cells. The size of dots indicated the percentage of cells that express the designated gene. The colour of dots represented gene expression level. (D) UMAP plot of D0 CD73− skin cells coloured by the possibility of becoming myocytes. (E) The expression of Gpnmb and Tyr in D0 CD73− cell. (F) Core regulatory network of D0 CD73− melanocytes. The blue dots were genes, and the pink dots were TFs.
Figure 3
Figure 3
Melanocytes were reprogrammed to myocytes by a small molecule cocktail. (A) Immunofluorescence staining of K14 and MyHC on skin cryosection in upper panel. Green indicated K14 representing dermal cells; Red indicated MyHC representing skeletal muscle; blue indicated DAPI staining of nuclei; merge indicated the merged images of green, red, and blue. Scale bars, 50 μm. Immunofluorescence staining of Mitf and MyHC on skin cryosection in lower panel. Green indicated Mitf; Red indicated MyHC; blue indicated DAPI staining of nuclei; merge indicated the merged images of green, red, and blue. Scale bars, 20 μm. (B) Immunofluorescence staining of Runx3 and Sox10 on skin cryosection. Green indicated Runx3 in upper panel and Sox10 in lower panel; blue indicated DAPI staining of nuclei; merge indicated the merged images of green and blue. Scale bars, 20 μm. (C) RNAscope in situ RNA hybridization of Pax7 on skin cryosection in upper panel. Red indicated the hybridized RNA signal; blue indicated nuclei. Brow was from the pigments of hair bulbs. Scale bars in the left panels: 50 μm; scale bars in the right panels: 10 μm. RNAscope in situ RNA hybridization of Pax3 on skin cryosection in lower panel. Red indicated the hybridized RNA signal; blue indicated nuclei. Brow was from the pigments of hair bulbs. Scale bars in the left panels: 50 μm; scale bars in the right panels: 10 μm. (D) Scheme of chemical induced fate conversion of melanocytes. (E) FACS analysis of GFP+ MuSC population isolated from Pax7‐eGFP mice in left panel. The GFP+ cell population was subjected for GPNMB and Tyr analysis. FACS analysis of GFP− cell population isolated from Pax7‐eGFP mice in right panel. The GFP− cell population was subjected for GPNMB and Tyr analysis. (F) Immunofluorescence staining of melanocytes treated by the small molecule cocktail in left panel. FACS sorted melanocytes were treated by the small molecule cocktail for 12 days and subjected for MyHC immunofluorescence staining. Green indicated MyHC; DAPI indicated nuclei; merge indicated the merged images of green and blue. Scale bars, 100 μm. Statistical analysis of the number of nuclei presented in myotubes in right panel. Error bars indicated standard error. The statistical analysis was based on three independent experiments. ***P < 0.001.
Figure 4
Figure 4
The cell fate conversion of melanocytes was Pax7 independent. (A) Immunofluorescence staining of Pax7 in Pax7 KO and WT muscle cryosections. Red indicated Pax7; green indicated Laminin; blue indicated DAPI staining of nuclei; merge indicated the merged images of red, green, and blue; amplified indicated the high magnification images of staining. The scale bars in lower magnification images, 25 μm. Scale bars in amplified panels, 10 μm. (B) FACS analysis of GPNMB+Tyr+ melanocytes in Pax7 KO skin cells. (C) Immunofluorescence staining of MyHC in cells converted from Pax7 KO melanocytes. Melanocytes were isolated from Pax7 KO skin tissues by GPNMB+ Tyr+ FACS sorting and treated by the small molecule cocktail for 12 days. The resulted cells were subjected for immunofluorescence staining with anti‐MyHC. Green indicated MyHC; blue indicated DAPI staining; merge indicated the merged images of green and blue. Scale bars, 100 μm. Statistical comparison of the percentage of nuclei in myotubes originated from WT and Pax7 KO melanocytes. Error bars indicated standard error. The statistical analysis was based on three independent experiments. ns indicated no significant changes. (D) Melanocytes were isolated from Pax3+/− mice. RT‐qPCR was performed to detect the expression level of Pax3. (E) Pax3+/− melanocytes were treated by the small molecule cocktail to induce myogenic transdifferentiaiton. Immunofluorescence staining was performed to detect the expression of MyHC which marked the myogenic lineage cells. Green indicated MyHC staining; Blue indicated nuclei staining of DAPI; merge indicated the merged images of green and blue. Scale bars, 100 μm. Statistical analysis of the percentage myonuclei after transdifferentiation. Error bars indicated standard error. The statistical analysis was based on three independent experiments. ***P < 0.001. (F) Analysis of the Pax3 expression in skin cells and the possibility of becoming skeletal muscle cells. The left panel indicated the imputated Pax3 expression of D0 cells excluding PC Muscle MuSCs. The right panel indicated the possibility of becoming myocytes of D0 cells excluding PC Muscle MuSCs.
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