Fig. 1

Limiting dilution assay experimental procedure and statistical analysis (A) Outline of experimental design. Myogenic progenitors were generated in vitro from mylz2-GFP or mylz2-mCherry embryos. For limiting dilution assay screening, cells were transplanted into pre-irradiated casper recipients (4–8 months old) followed by imaging of the recipient fish at 7 days post transplantation (dpt). The number of transplanted cells is indicated separately for each experiment. (B) Images of muscle engraftment visualized in the same fish by expression of the muscle-specific (mylz2-mCherry) reporter. Ten thousand cells were transplanted into casper fish and were stably observed for as long as 1 year post transplant. Mylz2, myosin light polypeptide chain 2 (red); dpt, days post transplantation; ypt, year post transplantation. n = 3. Scale bar, 500 μm. (C) Engrafted muscle-specific (mylz2-mCherry) cells are visible to the naked eye. Red, mylz2 (myosin light polypeptide chain 2). (D) Log-fraction plot of the limiting dilution model fitted to the data in Table S1. The plot represents the fraction of positive responses as a function of the dose of cells delivered in each transplantation. The triangle marks 100% engraftment efficiency with 700-cell transplantation. The line slope is the log-active cell fraction, and the two flanking dotted lines give the 95% confidence interval, as listed in the top table. See Table S1 for numbers. (E) myf5-GFP;mylz2-mCherry double-transgenic line cells were sorted into four subsets: myf5-GFP cells, mylz2-mCherry cells, double-positive cells, and double-negative cells. Each subset was evaluated for engraftment efficiency via limiting dilution assay. (F) Muscle progenitor cells (myf5-GFP) show superior muscle engraftment in recipient fish (n = 10 per cell number dose and 30 per cell population; ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, not significant; limiting dilution assay). (G) The estimated potency of engrafting cells (1/cell frequency) derived from extreme limiting dilution analysis (ELDA). Error bars represent the upper and lower confidence intervals. (H) Experimental design of the donor and muscle cell fusion assay. One million mylz2-GFP ZeMPCs were transplanted into Tg(mylz2-mCherry) recipients (4–8 months old). (I) A cross-section of a recipient fish at 7 dpt showing fusion of host and donor muscle fibers at the boundary of the engrafted region. The bottom row shows a zoomed image of the box in the top row (n = 5). Red and green, mylz2 (myosin light polypeptide chain 2); blue, nuclei (DAPI). Scale bar, 500 μm.