Figure 2. Rejuvenation of adult myogenesis, and albumin-independent effects of TPE. One day after the NBE, muscle was injured at two sites per TA by cardiotoxin; 5 days later muscle was isolated and cryosectioned at 10 μm. (A) Representative H&E and eMyHC IF images of the injury site. Scale bar = 50 μm. (B) Regenerative index: the number of centrally nucleated myofibers per total nuclei. OO vs. ONBE p = 0.000001, YY vs ONBE non-significant p = 0.4014; Fibrotic index: white devoid of myofibers areas. OO vs ONBE p = 0.000048, YY vs YNBE non-significant p = 0.1712. Minimal Feret diameter of eMyHC+ myofibers is normalized to the mean of YY . OO vs. ONBE p= 3.04346E-05, YY vs. YNBE p=0.009. Data-points are TA injury sites of 4-5 YNBE and 5 ONBE animals. Young and Old levels (detailed in Supplementary Figure 1) are dashed lines. Representative images for YY versus YNBE cohorts are shown in Supplementary Figure 6. (C) Automated microscopy quantification of HSA dose response, as fold difference in BrdU+ cells from OPTI-MEM alone (0 HSA). There was no enhancement of myogenic proliferation at 1-16% HSA. N=6. (D) Meta-Express quantification of BrdU+ cells by automated high throughput microscopy for myoblasts cultured with 4% PreTPE versus PostTPE serum and (E) for these cells cultured with 4% of each: PreTPE serum + HSA or PostTPE serum + HSA. Significant increase in BrdU positive cells is detected in every subject 1, 2, 3, and 4 for TPE-treated serum (p=0.011, <0.0001, <0.0001, 0.0039, respectively), as well as for TPE-treated serum when 4%HSA is present (p<0.0001, <0.0001, <0.0001, =0.009 respectively). N=6. (F) Scatter plot with Means and SEM of all Pre-TPE, Post-TPE, +/- HSA cohorts shows significant improvement in proliferation in Pre TPE as compared to and Post TPE cohorts (p*=0.033), as well as Pre+HSA and Post+HSA cohorts (p*=0.0116). In contrast, no significant change was observed when comparing Pre with Pre+HSA (p=0.744) or Post with Post+HSA (p=0.9733). N=4 subjects X 6 independent assays for each, at each condition. (G) Representative BrdU IF and Hoechst staining in sub-regions of one of the 9 sites that were captured by the automated microscopy. Blood serum from old individuals diminished myogenic cell proliferation with very few BrdU+ cells being visible (illustrated by one positive cell in Pre-TPE and arrowhead pointing to the corresponding nucleus); TPE abrogated this inhibition but HSA did not have a discernable effect.