Desmin

Human myoblasts were isolated from biopsies and cultivated as described previously [19 (link)] in a growth medium consisting of 199 medium and DMEM (Invitrogen Carlsbad, CA) in a 1:4 ratio, supplemented with 20% FCS (Invitrogen), 2.5 ng/ml hepatocyte growth factor (Invitrogen), 0.1 μmol/l dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/ml gentamycin (Invitrogen). The myogenic purity of the populations was monitored by immunocytochemistry using desmin as marker. Enrichment of myogenic cells was performed using an immunomagnetic cell sorting system (MACS; Miltenyi Biotec, Paris, France) according to the manufacturer's instructions. Briefly, cells were labeled with anti-CD56 (a specific marker of myoblasts) microbeads, and then separated in a MACS column placed in a magnetic field. Purification was checked by immunochemistry using a desmin marker. Differentiation was induced at confluence by replacing the growth medium with DMEM supplemented with 100 μg/ml transferrin, 10 μg/ml insulin and 50 μg/ml of gentamycin (Sigma-Aldrich).

Fig. S1 shows the expression of Aldh1a1 gene among tissues, cellular characterization of ALDH-producing cells in omentum, and localization of ALDH1A2⁺ cells in the omentum. Fig. S2 shows gene expression analysis of Aldh1a2⁺ FRCs. Fig. S3 shows targeting construct of Postn-DTR allele and population analysis of omental stromal fractions. Fig. S4 includes Desmin⁺ FRC network, scRNA-seq analysis, and lymphoid organization of WT and Postn-DTR mice. Fig. S5 shows HEV populations in WT and Postn-DTR omenta and quantification of CXCL12 signal in the lumen of HEVs.

Rabbit monoclonal antibodies against ALDH1A2 (E6O6Q; Cell Signaling Technology), Desmin (Y66; Abcam), and CXCL12 (79018; R&D) were used. Antibodies against B220 (RA3-6B2), CD3 (17A2), CD4 (GK1.5), CD8a (53-6.7), CD11b (M1/70), CD16/32 (93), CD19 (6D5), CD23 (B3B4), CD45 (30-F11), CD105 (MJ7/18), F4/80 (BM8), IgM (RMM-1), MAdCAM-1 (MECA-367), PECAM-1 (390), PNAd (MECA-79), PDPN (8.1.1), TIE2 (TEK4), VCAM-1 (429), and Rat IgG2a,κ isotype control (RTK2758) were obtained from BioLegend. Antibodies against CD140a (APA5) and Msln (295D) were obtained from BD Biosciences and MDL, respectively. ATRA was obtained from Sigma-Aldrich.
For DT-mediated cell ablation, mice were i.p. injected with 100 ng of DT (Sigma-Aldrich). For CXCR4 inhibition, 200 μg of AMD3100 (Sigma-Aldrich) was i.p. injected every day for five times and omenta were harvested 24 h after the last injection. For inhibition of retinoic acid signaling, 500 nmol of pan retinoic acid receptor inverse agonist BMS493 (Tocris) was i.p. injected every day for three times and omenta were harvested 24 h after the last injection.

1Cells were seeded onto each nanofiber in triplicate and cultivated for 7 days in GM only, or for 7 days in GM followed by an additional 7 days in DM. Both GM and DM were replaced by fresh medium every 2 days. All cells from the triplicate were then harvested in 1 mL TriReagent (Sigma-Aldrich) and the total RNA was isolated according to the manufacturer’s instructions. Next, 1 μg of each total RNA sample was converted into cDNA, following the instructions in the RevertAid H minus first strand cDNA synthesis kit (Thermo Fischer Scientific). GAPDH was used as a reference gene (AGG​TCG​GTG​TGA​ACG​GAT​TTG and TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA). MyoD, Myf5, MyoG and Desmin were used as target genes with the following primers: for MyoD (GTG​GCA​GCG​AGC​ACT​ACA and GAC​ACA​GCC​GCA​CTC​TTC), for Myf5 (GCA​AAG​ACC​CGT​GAC​TTC​AC and GCA​TGT​GGA​AAA​GTG​ATA), for MyoG (TGA​GAG​AGA​AGG​GGG​AGG​AG and CGG​TAT​CAT​CAG​CAC​AGG​AG) and for Desmin (GTG​GAG​CGT​GAC​AAC​CTG​AT and ATG​TTC​TTA​GCC​GCG​ATG​GT). RT-qPCR was performed using a Corbett 3,000 device (Qiagen, Helden, Germany), using 0.4–0.8 μM of each primer, 1 μL (diluted 1:10) of each cDNA, and 5 μL of iTaq universal SYBR Green Supermix (Bio-Rad, Hercules, USA), in a final volume of 10 μL. Reactions were performed as follows: 50°C for 2 min, 95°C for 2 min, followed by 45 cycles of 94°C for 15 s, 60°C–62°C for 15 s, and 72°C for 20 s. The dissociation step was performed at the end of the amplification step. Relative gene expression was determined using REST2009 software (based on the model by Pfaffl et al., 2002 (link)).