Initial or continuous coculture with umbilical cord-derived mesenchymal stromal cells facilitates in vitro expansion of human regulatoryT-cell subpopulations

Clinical trials have demonstrated the safety and potential efficacy of ex vivo expanded regulatory T cells (Tregs) for immune-mediated diseases. Nonetheless, achieving consistent and timely Treg yield and purity remains challenging. We aimed to evaluate the potential to enhance culture expansion of primary human total Treg (CD4⁺/CD25⁺/CD127^lo) and Treg subpopulations through coculture with human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). In 14- to 21-day anti-CD3/anti-CD28-, interleukin-2-, and rapamycin-containing cultures, fluorescence-activated cell sorting (FACS)-purified total Treg underwent 4-fold greater expansion following hUC-MSC coculture. Potency to suppress T effector cell (Teff) proliferation was equivalent for hUC-MSC-cocultured and control Tregs and correlated with the expression of HLA-DR, CD39, and inducible costimulator (ICOS). The impact of hUC-MSC coculture on ex vivo expansion of 3 FACS-purified Treg subpopulations [CD45RA⁺ (Subtype I), CD45RA−HLA-DR⁺ (Subtype II), and CD45RA−HLA-DR⁻ (Subtype III)] was then investigated. Both initial and continuous hUC-MSC coculture yielded significantly higher fold expansion of each Treg subpopulation compared to control. However, the magnitude of enhancement was substantially greater for non-naive (Subtypes II and III) than for naive (Subtype I) Treg. Coculture with hUC-MSC increased HLA-DR expression of all 3 expanded Treg subpopulations while maintaining comparable Teff suppressive potency. For non-naive Treg (Subtypes II and III), both initial and continuous hUC-MSC coculture also increased the final %Foxp3⁺ and %Helios⁺. Thus, coculture with clinical-grade hUC-MSC substantially enhances the ex vivo yield, preserves the suppressive potency, and modulates HLA-DR expression of FACS-purified Treg subpopulations with greatest effect on non-naive (CD45RA⁻) Treg. The findings have potential to facilitate identification, functional characterization, and manufacturing of Treg subpopulations with distinct therapeutic benefits.