Chicken fibroblasts proliferate stably in low-serum conditions. (A) Cellular morphology and EdU staining of chicken fibroblasts under different low-serum conditions. FBS: Fetal bovine serum. CS: Chicken serum. Scale bar, 200 µm. (B) Quantification of the proportion of EdU-positive cells in Figure A. Error bars indicate s.e.m. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The CCK-8 cell proliferation assay showed the proliferation of chicken fibroblasts in 1% chicken serum. Error bars indicate s.e.m, n = 3.

3D culture of chicken fibroblasts in GelMA hydrogels. (A) Microscopic images of GelMA hydrogels at different concentrations (3%wt, 5%wt, 7%wt and 9%wt) taken by scanning electron microscopy and their corresponding simplified maps of pore distributions. Scale bar, 10 µm. (B) Quantification of pore area in image A. Error bars indicate s.e.m, n = 3. ****P < 0.0001. (C) Brightfield and red fluorescent images of cells in 3D culture after PKH26 staining at different times (1hour, 1day, 3days, 5days and 9days). Scale bar, 100 µm. (D) Relative area of PKH26-linked cells in Figure C. Error bars indicate s.e.m, n = 3. *P < 0.05, ***P < 0.001, ****P < 0.0001. (E) Representative EdU staining shows the proliferation of cells in 3D culture on 1day, 3days and 5 days after cell implantation in hydrogel. Scale bar, 100 µm. (F) Quantification of the proportion of EdU-positive cells in Figure E. Error bars indicate s.e.m, n = 3. **P < 0.01.

Transdifferentiation of chicken fibroblasts into muscle cells in 3D. (A) Experimental design for fibroblast myogenic transdifferentiation in 3D culture. (B) Representative images of MHC staining showed the myogenic ability of chicken fibroblasts in 3D culture. Scale bar, 50 µm. (C) Comparison of the mean myogenic fusion index between 2D and 3D. Error bars indicate s.e.m, n = 3. *P < 0.05. (D) Three-dimensional images of MHC staining of cells cultured in 3D. The right panel is its depth-coded image which indicate different depths from the deepest (cyan) to the surface (yellow). (E) Orthogonal projections of three sets of MHC staining of cells in 3D culture at different depths. Scale bar, 50 µm. (F) Expression of skeletal muscle-related genes was determined by RT-qPCR in 2D and 3D cells upon myogenic transdifferentiation and control 3D cells without stimulation. Please note that the myogenic transdifferentiation driven by MyoD stimulate the expression of classical myogenic factors. Error bars indicate s.e.m, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Macroscopic morphology of the empty hydrogel matrix (left) and cultured meat (right). The cultured meat is the product obtained after 3D cell culture and induction of myogenesis/lipogenesis. Scale bar, 1 cm.

Myogenic transdifferentiation of fibroblasts do not produce myofibroblasts. (A) Immunofluorescence staining of 3D cultured cells showed that the skeletal muscle marker desmin was expressed only in the transdifferentiated cells but not in fibroblasts or myofibroblasts. Scale bar, 50 µm. (B) Immunofluorescence staining of 3D cultured cells showed that the myofibroblast marker α-SMA was expressed only in the myofibroblasts but not in fibroblasts or transdifferentiated cells. Scale bar, 50 µm. (C) Immunofluorescence staining of 3D cultured cells showed that the fibroblast marker vimentin was abundantly expressed in fibroblasts and myofibroblasts but greatly reduced in transdifferentiated cells. Scale bar, 50 µm. (D) RT-qPCR showed that the myogenic genes Desmin and Six1 were significantly increased upon myogenic transdifferentiation. (E) RT-qPCR showed the fibroblast marker gene Thy-1 was significantly reduced upon myogenic transdifferentiation. (F) The myofibroblast marker genes TGFβ-1, TGFβ-3 and Smad3 remain unchanged during myogenic transdifferentiation. Error bars indicate s.e.m, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not significant.

Stimulate the fat deposition in chicken fibroblasts in 3D. (A) Experimental design for fibroblast lipogenesis in 3D culture (‘F’ is for fatty acids and ‘I’ is for insulin). (B) Representative images showing the Oil-red O staining of lipid content accumulated in cells at different focal planes at the same position. The control group was normal medium without lipogenesis. Scale bar, 100 µm. (C) Relative area of lipid droplets in figure B. Error bars indicate s.e.m, n = 3. ****P < 0.0001. (D) Expression of lipid synthesis related genes determined by RT-qPCR in 2D and 3D cells upon lipogenic induction and control 3D cells without stimulation. Error bars indicate s.e.m, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Triglyceride content in the cultured meat upon different lipogenic inductions and control 3D cells without stimulation. Error bars indicate s.e.m, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Controlled fat deposition in the transdifferentiated muscle cells in 3D hydrogel. (A) Experimental design for fibroblast myogenic/lipogenic differentiation in 3D culture. (B) Representative images of MHC and Oil Red O staining of cells upon myogenesis/lipogenesis in 3D culture. Scale bar, 50 µm. (C) Orthogonal projections of three sets of MHC and Oil Red O staining of cells in 3D culture at different depths. Scale bar, 50 µm. (D) Expression of muscle-related genes (top) and lipid-related genes (bottom) in the cells with myogenesis/lipogenesis induction and control 3D cells without any stimulation were determined by RT-qPCR. (E) Triglyceride content of cultured meat under different conditions and real meat. “Meat_leg” and “Meat_breast” are taken from the leg and breast muscles of adult chickens. Error bars indicate s.e.m, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

The collagen content and expression of ECM components in cultured meat. (A) Total collagen content of cultured meat at different days of cultivation. Error bars indicate s.e.m, n = 3. *P < 0.05, ***P < 0.001. (B) Expression of ECM-related genes determined by RT-qPCR of cultured meat. Error bars indicate s.e.m, n = 3. *P < 0.05, ***P < 0.001, ****P < 0.0001. (C) Representative Laminin staining of cells in 3D culture on 1day, 3days, 5days and 7days after cell implantation in hydrogel. Scale bar, 100 µm.

Gene expression profiles during transdifferentiation and fat deposition in 3D. (A) Scheme of the RNA-Seq samples marked by different color. (B) Hierarchical clustering analysis of whole transcriptomes of 3D_fibroblasts, 3D_MyoD, 3D+FI, and 3D_MyoD+FI using Euclidean distance with ward.D cluster method. (C) PCA analysis of transcriptome changes during myogenic transdifferentiation and fat deposition (n= 10,247 genes). The ellipses group include three biological replicates in each cell type. The arrows represent the wiring of gene expression under different conditions. The routes were derived from original fibroblast toward two differentiation routes, namely “myogenic transdifferentiation” and “adipogenic transdifferentiation”. (D) Venn Diagram showing the overlap of differentially expressed genes (DEG) from 3D_MyoD, 3D+FI, and 3D_MyoD+FI compared to the original 3D_fibroblasts. (E) Heat map showed the representative genes differentially expressed between 3D_MyoD+FI and 3D_fibroblast cells (n=3 biologically independent samples). (F) GO analysis of up-regulated DEGs between 3D_MyoD+FI vs. 3D_fibroblast cells. (G) GOChord analysis of the up-regulated genes within representative pathway between 3D_MyoD+FI and 3D_fibroblast cells.