Fig 1.
A) Flow diagram of stages and media required to achieve all 3 cell types during the RAPID protocol (including LPD). B) Growth factor regimen for RAPID (including LPD) protocols at each day for the chondrogenic, osteogenic and cardiogenic protocols.
Fig 2.
hPSCs are directed through lateral plate lineage to limb-bud like progenitor cells.
Gene expression relative to GAPDH using 2-Δct +SEM, for transcriptional analysis of hPSCs differentiating into A) mid-primitive streak (Day2), B) lateral plate mesoderm (Day4), C) paraxial markers and D) limb-bud transition like cells (Day 6). D’Agostino-Pearson test was used to test for normality. All series tested using Kruskal-Wallis test to determine statistical significance. Significance relative to hPSCs (day 0) (* ≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). (N = 6 independent experiments).
Fig 3.
Differentiation of chondroprogenitors through to developing chondrocytes, from limb-bud like progenitor cells.
A) QRT-PCR analysis of chondrogenic gene expression during differentiation in 2D (until day 11) and in 3D pellet culture (day 11+14). Gene expression data displayed as relative to housekeeping gene GAPDH +SEM. Mann-Whitney test was used to determine statistical significance relative to hPSCs (day 0) (+ ≤0.05, ++≤0.01, +++≤0.001). # indicates significant difference to day 11 (N = 5 independent experiments). B) Volcano plot for differential gene expression in day 11 cells (N = 3 independent experiments with duplicate samples) compared to pluripotent hESCs (N = 4 independent experiments). Significant gene changes detected with log2 fold change (log2FC) ≥1 or log2FC ≤-1, with p-value ≤0.05. Red plots indicate adjusted p-value ≤0.05. Targets of interest are indicated in black and named. C) Heat map of normalised gene expression for target pluripotent, limb bud and chondrogenic gene expression targets in pluripotent (N = 4 independent experiments) and day 11 cells (N = 3 independent experiments with duplicate samples). Data displayed as percentage normalised to lowest and highest expressed for each gene of interest. D) Image at day 11(2D) +28 d in 3D for pellet. E) H&E histological stain of day 11+28 pellet, and F) enlarged view of pellet periphery in H&E including arrows indicating flattened surface cells (red arrow) and cells in lacune (green arrow). G) Alcian blue stain of day11+28 pellet. H) Picrosirius red collagen matrix stain. I) Polarised light imaging of Picrosirius red staining of day 11+28 pellet. J) Immunofluorescence images for day 11+28 pellets stained for Aggrecan (red), Lubricin (green) and K) immunohistochemistry for type-II collagen and type-I collagen (IHC). In J) DAPI and in K) haematoxylin were used for visualisation of cell nuclei. L) IgG controls for mouse and rabbit primary antibodies.
Fig 4.
Differentiation of preosteoblast like cells from limb-bud-like progenitor cells.
A) QRT-PCR gene expression analysis during osteogenic differentiation pathway up to day 28. Gene expression data displayed relative to housekeeping gene GAPDH +SEM. Mann-Whitney test was used to determine statistical significance. Significance relative to hPSCs (day 0) (* ≤0.05, **≤0.01, ***≤0.001) (N = 5 independent experiments day 0 to 14, N = 3 independent experiments for day 28). B) Alizarin Red staining for mineralisation assay of undifferentiated hPSCs (Day 0) and preosteoblast-like cells at the end of differentiation (Day 28) (N = 3 independent experiments). C) Quantification of stained area positive for Alizarin Red at day 0 and day 28 (percentage total area stained). Data presented as mean percentage area + SEM. Unpaired t-test was used to determine statistical significance (** p<0.01) D) BCIP-NBT active ALP enzyme assay for preosteoblast like cells cultured till day 28 (N = 3 independent experiments). E) Quantification of stained area positive for BCIP-NBT at day 0 and day 28 of differentiation. Data presented as mean percentage area + SEM. Unpaired t-test was used to determine statistical significance (** p<0.01).
Fig 5.
Differentiation of cardiomyocyte progenitor cells from lateral plate lineage through cardiac mesoderm.
Gene transcriptional analysis of cardiomyocyte genes during differentiation to beating cardiomyocytes. (For video of contracting tissue please see supplementary evidence). Gene expression data displayed relative to housekeeping gene GAPDH +SEM. Mann-Whitney test was used to determine statistical significance. Significance relative to hPSCs (day 0) (+ ≤0.05, ++≤0.01) (N = 5 independent experiments for days 0 to 14, N = 4 for day 28).
Fig 6.
Comparison in chondrogenic gene expression between paired existing DDP and lateral plate derived RAPID cells.
Man7 PSCs were differentiated to prechondrocytes through both the DDP and RAPID protocols. A) QRT-PCR gene expression assessment for the main transcription factors and ECM components in pair match DDP and RAPID samples. DDP samples were taken at day 14 compared to day 11 in the RAPID protocol. Data is expressed as normalised to existing DDP protocol, with connecting lines indicating paired experiments. B) Light microscopy image of pellets, Alcian blue matrix staining, immunohistochemistry for type-II collagen and aggrecan in 2D+28 day pellets. A Wilcoxen test was used to determine statistical significance. + indicates significance to hPSCs (day 0) (+ ≤0.05, ++≤0.01) (N = 6 independent experiments).