Fig 2: BNC1 function in developing epicardial cells. (A) hPSC-epi developed from TET-inducible knockdown hPSC showed more than 90% reduction in BNC1 RNA under the TET condition (Aa) and 98% reduction at the protein level by western blot (Ab) as also visualised by immunofluorescence (Ac) (n=5). (B,C) These cells showed more than 75% reduction of WT1 RNA (B) and a 5-fold increase in TCF21 RNA (C). (D,E) When BNC1 is silenced during its development, the hPSC-epi is enriched in the TCF21high population as revealed by double immunofluorescence WT1/TCF21 (D) and THY1 flow cytometry analysis [E; histograms of a representative experiment (top) and recapitulative graph (bottom) of n=5; brackets indicate the percentage of positive cells]. (F) BNC1 silencing can be achieved in human foetal primary epicardium using siRNA as shown by RT-PCR (Fa) and immunofluorescence (Fb). The knockdown of BNC1 in human foetal primary epicardium leads to a greater than 5-fold increase in TCF21 RNA (Fc) (n=3). The RT-PCR data shown in Aa, B, C, Fa and Fc were obtained by the quantitative relative standard curve protocol as described in Materials and Methods. RNA measurements were normalised to housekeeper genes porphobilinogen deaminase (PBGD) or GAPDH. Statistics were performed with Prism 7 from GraphPad with a ratio paired t-test. Error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001. Scale bars: 40 µm (Ac,D); 20 µm (Fb).

Fig 3: The THY1+ population retains CF potential. THY1-positive (THY1+) and THY1-negative (THY1-) cells were magnetically separated from GFP-positive (GFP+) hPSC-epi and mixed with regular GFP-negative hPSC-epi in known proportions measured by flow cytometry. (A) After 12 days of differentiation in SMC medium, the cultures were analysed by flow cytometry to establish the percentage of GFP-positive cells still present (Aa, n=5) and stained for CNN and TAGLN to confirm the differentiation (Ab). (B) The percentage of CNN+ or TAGLN+ cells present in the GFP+ fraction were quantified in four experiments (an average of 31 and 40 GFP+ cells from THY1+ and THY1- origin, respectively, were counted in each CNN experiment; an average of 33 and 45 GFP+ cells from THY1+ and THY1- origin, respectively, were counted in each TAGLN experiment). (C) After 12 days of differentiation in CF medium, the cultures were analysed by flow cytometry to establish the percentage of GFP-positive cells still present (Ca, n=5; ratio paired t-test performed in Prism 7 from GraphPad) and immunostained for SYT4 and POSTN (Cb). (D) The percentage of SYT4+ or POSTN+ cells amongst the GFP+ population was quantified (n=4 and ratio paired t-test performed in Prism 7 from GraphPad; an average of 215 and 67 GFP+ cells from THY1+ and THY1- origin, respectively, were counted in each SYT4 experiment; an average of 2125 and 442 GFP+ cells from THY1+ and THY1- origin, respectively, were counted in each POSTN experiment). All error bars represent s.e.m. Scale bars: 130 µm (Ab); 80 µm (Cb).

Fig 4: MSC characterization. (a) Cytometry graphs in solid black curves refer to the negative controls (cells incubated with secondary antibody) and nonfilled curves indicate the marker tested. The cell population expressed CD90, 73, and 105 and did not express CD19 and 45. (b) Brightfield images are showing that MSCs are able to differentiate in adipocytes, osteocytes, and chondrocytes. Undifferentiated cells were grown only in DMEM (A, C, and E) and show only background staining for oil red O on (A), Von Kossa on (C), and alcian blue on (E). Once incubated in differentiation-inducing culture mediums, adipose-derived MSCs showed positive staining for oil red O on (B), Von Kossa on (D), and for alcian blue on (F). Scale Bar = 200 µm. Representative images of three biological replicates that were conducted independently.

Fig 5: Trafficking for the terminal fate of transplanted ASC after local intraneural injection. (A, B) The signal intensity of ICG-bioluminescent labeling ASC was trackable and revealed a significant increase as compared to baseline (PBS injection) on day 3 but returned to baseline signal on day 7. (C) Immunofluorescent staining of the injured nerve demonstrated the colocalization of CD90+/CD105+ cells (arrow), indicating the maintenance of ASC phenotype at least on day 3 (scale bar: 50 µm; ****indicated p < 0.0001, ns indicated no significant difference; n = 3–5 for each group).