iPSC-Derived Mesenchymal Stromal Cells

iPSC → iMSC Differentiation

I generate iMSCs using a SMAD-inhibition / EB-outgrowth strategy adapted from Wang/Lian and Kimbrel: iPSCs are aggregated into embryoid bodies (EBs) under TGFβ/BMP inhibition to commit to lateral-plate mesoderm, plated for outgrowth, and then expanded as adherent fibroblastic cells in MSC growth medium. Magnetic or FACS sorting on CD73⁺/CD105⁺/CD45⁻ yields a homogeneous population that meets ISCT criteria for MSC identity.

1. iPSC dissociation and EB formation (D0–1)

   Single-cell dissociate iPSCs with Accutase; aggregate ~3×10⁵ cells in AggreWell or low-attachment plates with ROCK-i.

2. Mesoderm commitment (D1–7)

   EB medium (KSR-based or xeno-free) + SB431542 10 µM; optionally add BMP4 10 ng/mL on D2–4 for lateral-plate bias.

3. Outgrowth (D7–14)

   Transfer EBs onto gelatin-coated plates; switch to MSC growth medium (α-MEM + 10% MSC-qualified FBS or xeno-free + FGF2 5 ng/mL).

4. Enrichment (D14–21)

   FACS or MACS sort on CD73⁺ CD90⁺ CD105⁺ CD45⁻ CD34⁻; verify pluripotency-marker absence (TRA-1-60⁻, SSEA-4⁻).

5. Banking and use (P3–P5)

   Passage 1:3–1:6; bank early passages; verify tri-lineage differentiation potential before each experimental cohort.

Tri-Lineage Differentiation & Surface Phenotype

Per ISCT criteria, iMSCs are validated by (i) plastic adherence, (ii) surface phenotype CD73⁺/CD90⁺/CD105⁺/CD45⁻/CD34⁻/CD11b⁻/CD19⁻/HLA-DR⁻, and (iii) tri-lineage differentiation: osteogenic (Alizarin Red, RUNX2/OCN), chondrogenic (Alcian Blue, SOX9/COL2A1), and adipogenic (Oil Red O, PPARγ/FABP4). Bulk RNA-seq cross-validates MSC identity and tri-lineage capacity.

Immunomodulatory & Trophic Functions

iMSCs suppress T-cell proliferation, polarize macrophages toward M2, and secrete trophic factors (HGF, VEGF, IL-6, IL-10, IDO). I read out these activities with mixed-lymphocyte reactions (MLRs), CFSE proliferation assays, cytokine multiplex panels (Luminex/MSD), and qPCR/RNA-seq of inflammation modules. iMSC-conditioned medium is used to feed neural and cardiac differentiations and to evaluate paracrine-driven phenotypes.

Fibrosis & EMT Models

Treatment with TGFβ1 (5–10 ng/mL) for 24–72 h induces a myofibroblast-like state characterized by α-SMA, COL1A1, FN1, and CTGF up-regulation. iMSC fibrosis cultures are used to screen anti-fibrotic compounds (pirfenidone, nintedanib, SMAD inhibitors) and to dissect TGFβ–SMAD signaling. Cross-lineage co-cultures with iPSC-derived alveolar (lung) or proximal-tubule (kidney) epithelium model fibrotic remodeling in IPF and chronic kidney disease.