The increasing prevalence of refractory thin endometrium (TE) is a serious issue affecting women's reproductive health. Human umbilical cord-derived mesenchymal stem cells (huMSCs) have shown therapeutic potential for TE. However, their clinical application is limited by poor cell retention, low survival rates, and unclear regenerative mechanisms. Here, we introduced an injectable 3D delivery system using clinically applicable collagen hydrogels to encapsulate huMSCs (Gel-huMSCs), offering a promising strategy for endometrial regeneration. We systematically optimized the physicochemical properties of the collagen hydrogels to enhance their efficacy in 3D stem cell culture. Our TE mouse model demonstrated that Gel-huMSCs effectively restore uterine function by increasing endometrial thickness, reconstructing immune homeostasis, promoting angiogenesis, and reversing fibrosis. Specifically, we observed significant upregulation of tissue regeneration-related genes (Vegf, Cdh4, and Foxa2) and downregulation of inflammatory response-related genes (Mstn, Tnf, Trem1, and Col4a3) in the endometrium after Gel-huMSCs therapy compared to the control group. These findings provide insights into the regenerative mechanism of Gel-huMSCs in improving endometrial repair in TE. Notably, our study confirmed the safety of Gel-huMSCs and their efficacy in enhancing fertility rates in TE treatment. In conclusion, these results suggest that Gel-huMSCs represent a promising and viable therapeutic strategy for stem cell-based treatment of TE in clinical applications. STATEMENT OF SIGNIFICANCE: Thin endometrium (TE) is a major contributor to infertility and adverse pregnancy outcomes. Human umbilical cord-derived mesenchymal stem cells (huMSCs) have shown therapeutic potential for TE, yet their clinical translation is limited by poor retention and an incomplete understanding of their regenerative mechanisms. Here, we have developed a thermosensitive injectable type I collagen hydrogel engineered to carry huMSCs (Gel-huMSCs). The hydrogel recapitulates key physicochemical and architectural features of the native endometrial extracellular matrix, preserving huMSC stemness and augmenting proliferation and angiogenesis in vitro. An AIEgen (DPTA-BT) was employed to label and track huMSCs in vivo. After intrauterine injection, Gel-huMSCs undergo controlled enzymatic degradation, gradually releasing viable cells that integrate into the endometrial epithelium and stroma. RNA sequencing of treated endometrial tissue has demonstrated significant up-regulation of genes associated with extracellular-matrix remodeling and angiogenesis, together with down-regulation of pro-inflammatory cytokine pathways. In a validated rat model of TE, Gel-huMSCs have been shown to increase endometrial thickness and restore fertility.
