Fig 1: Identification of the putative myogenic factors produced by undifferentiated adipocytes. (a) Myogenic factors of the conditioned media are thermo-sensitive. Control and conditioned media were incubated at different temperatures for 15 min. The myogenic potential of the media was evaluated microscopically on the in initial fraction. (b) CD34+/Sca1+ cells secrete proteins. CD34+/Sca1+ cells were pulsed with tritiated lysine for 24 h and the culture media were analyzed by SDS-PAGE (Stain) and autoradiography (Autorad). (c) Myogenic factors secreted by CD34+/Sca1+ cells are soluble. Conditioned media were centrifuged at 25,000 g and 100,000 g, respectively. The myogenic potential of the resulting media was then evaluated using non-centrifuged conditioned medium as a control (0). (d) CD34+/Sca1+ cell cultures require FCS to produce efficient conditioned media. Conditioned media were prepared from CD34+/Sca1+ cultured in DMEM/decomplemented FCS (+FCS) and DMEM (−FCS), respectively. The myogenic potential of the resulting conditioned media was then evaluated using as control DMEM/decomplemented FCS (+FCS) and DMEM (−FCS), respectively. Statistics: n = 3, t-test P < 0.001 (*). (e) Proteomic analyses of CD34+/Sca1+ primary cell conditioned media. Proteins were prepared as described in Methods, identified by mass spectrometry and sorted by their cellular location; extracellular (Secreted) and intracellular (Intracellular). (f) Transcriptomic analyses of CD34+/Sca1+ primary cells. Transcriptomes of CD34+/Sca1+ cells were analyzed in early proliferative state (lower efficiency of the secretome, lower rate of proliferation, Sca1 Early proliferation) and late proliferative state (higher efficiency of the secretome, higher rate of proliferation, Sca1 Late proliferation), respectively. The positive probes were sorted by the level of expression in the two culture states, arbitrary considering 2-fold changes as over-expression. The probes with higher expression in proliferative state were then analyzed against the transcriptome of CD34+/CD31+. The over-expression in CD34+/Sca1+ cells (Sca1 Late proliferation positive) relative to CD43+/CD31+ cells (CD31) was more than a 2-fold change. The gene products were then sorted by their cellular location; extracellular (Secreted) and intracellular (Intracellular). (g) Cross-analyses of proteomics and transcriptomics revealed putative myogenic candidates. Comparison of secreted proteins from proteomic and transcriptomic analyses revealed 11 proteins in common.
Fig 2: Adipogenic lineage cells are required for myogenic differentiation of competent cells. (a) Microscopic observations of the myogenic differentiation of competent cells. Cells harvested from leg muscles of 17 dpc foetuses and sorted into a CD34+ population (CD34+/depletion none), CD34+/CD31− cells (CD34+/depletion CD31+), and CD34+/Sca1− cells (CD34+/depletion Sca1+), respectively. The different cell groups were cultured for 4 days and myogenic differentiation was assessed by immuno-staining using an anti-myosin antibody (My32, red), counterstained with DAPI (blue). Bar scale corresponds to 100 μm. (b) Quantitative analyses of myogenic differentiation of competent cells. The percentage of myogenic differentiation was determined microscopically. Total CD34+ cell population was used as a reference and myogenic differentiation was arbitrarily estimated at 100%. Statistics: n = 3, t-test P < 0.0001 (**).
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