Generation of Therapeutically Potent Spheroids from Human Endometrial Mesenchymal Stem/Stromal Cells

Abstract

Endometrial mesenchymal stem/stromal cells (eMSCs) hold great promise in bioengineering and regenerative medicine due to their high expansion potential, unique immunosuppressive properties and multilineage differentiation capacity. Usually, eMSCs are maintained and applied as a monolayer culture. Recently, using animal models with endometrial and skin defects, we showed that formation of multicellular aggregates known as spheroids from eMSCs enhances their tissue repair capabilities. In this work, we refined a method of spheroid formation, which makes it possible to obtain well-formed aggregates with a narrow size distribution both at early eMSC passages and after prolonged cultivation. The use of serum-free media allows this method to be used for the production of spheroids for clinical purposes. Wound healing experiments on animals confirmed the high therapeutic potency of the produced eMSC spheroids in comparison to the monolayer eMSC culture.

Keywords: cell spheroids; endometrial mesenchymal stem cells; serum-free conditions; wound healing.

Figure 1

Generation of uniform-sized eMSC spheroids. (a) Aggregation of eMSCs into spheroids on different passages (scale bar, 600 μm); (b) formation of uniform-sized eMSC spheroids in serum-containing medium supplemented with different concentrations of CDLC (scale bar, 600 μm); (c) distribution of eMSC spheroid areas obtained from the analysis of the bright-field images of spheres generated in serum-containing medium supplemented with different concentrations of CDLC; the upper labels display the area normalized to the mean value in the sample, and the lower labels display the area in absolute numbers. (d) Phase-contrast microscopy showing the aggregation of eMSCs into a spheroid in a hanging drop in serum-containing medium with and without 0.05% CDLC after 48 h (scale bar, 600 μm).

Figure 2

Viability, multipotency, cell cycle distribution and gene expression of eMSC spheroids under serum-free conditions. (a) Aggregation of eMSCs into spheroids cultured in serum-containing medium (Serum+), SFM 1 or SFM 2 (scale bar, 300 μm.); (b) viability of eMSCs in spheroids cultured in serum-containing medium (Serum+), SFM 1 or SFM 2; L—live cells, D—dead cells; (c) representative cell cycle distribution of eMSCs in spheroids cultured in serum-containing medium (Serum+), SFM 1 or SFM 2. (d) Osteogenic and adipogenic differentiation of eMSCs in spheroids cultured in serum-containing medium (Serum+), SFM 1 or SFM 2 (scale bar, 200 μm). (e) q-RT-PCR assay of TSG6, EP2 and HSF genes in eMSC spheroids and monolayer cells under serum-free conditions. Data are shown as mean ± SD (n = 3). Two-tailed Student’s t-test was utilized for pairwise comparison. * p < 0.01 vs. eMSCs in monolayer.

Figure 3

Therapeutic efficacy of eMSC spheroids in serum-free medium. (a) Macroscopic view of the wound closure in rat after transplantation of eMSCs in spheroids cultured in serum-containing medium (Serum+), SFM 1 or SFM 2, eMSCs cultured in monolayer at the same conditions and PBS as a control; (b) H&E staining of paraffin-embedded tissue section of rat wounds on the 7th day after transplantation of eMSCs in spheroids (scale bar, 100 μm). GT—granulation tissue, line—regenerating epithelium. (c) H&E staining of paraffin-embedded tissue section of rat wounds on the 7th day after transplantation of eMSCs cultured in monolayer (scale bar, 100 μm). GT—granulation tissue, line—regenerating epithelium. (d) H&E staining of paraffin-embedded tissue section of rat wounds on the 7th day after PBS injection (scale bar, 100 μm). GT—granulation tissue, line—regenerating epithelium. (e) Recovery of granulation and epithelium tissues in rat wound after eMSC transplantation. Data are shown as mean ± SD (n = 15). The differences in recovery of granulation and epithelium tissues in rat wounds were tested by the Kruskal–Wallis H-test (non-parametric ANOVA) followed by post hoc pairwise comparison using Dunn’s test. * p < 0.05 vs. PBS control; ** p < 0.05 vs. monolayer culture at the same conditions.