Changes in the Transcriptome Profiles of Human Amnion-Derived Mesenchymal Stromal/Stem Cells Induced by Three-Dimensional Culture: A Potential Priming Strategy to Improve Their Properties

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.

Pre-clinical Research of Human Amnion-derived Mesenchymal Stem Cells and its First Clinical Treatment for a Severe Uremic Calciphylaxis Patient

Calciphylaxis is a rare disease characterized histologically by microvessel calcification and microthrombosis, with high mortality and no proven therapy. We reported a severe uremic calciphylaxis patient with progressive skin ischemia, large areas of painful malodorous ulcers and mummified legs. Because of her rapid progression and refractory to conventional therapy, human amnion-derived mesenchymal stem cells (hAMSCs) treatment was approved. Establishment and release inspection of hAMSCs, efficacy and safety assessment including cytokines secretory ability, immunocompetence, tumorigenicity and genetics analysis in vitro were introduced. We further performed acute and long-term hAMSC toxity evaluations in C57BL/6 mice/rats, abnormal immune response tests in C57BL/6 mice and tumorigenic tests in the neonatal NU nude mice. After pre-clinical research, she was treated by hAMSCs with intravenous and local intramuscular injection and external supernatants application to her ulcers. When followed up to 15 months, her blood-based markers of bone and mineral metabolism were improved, with regeneration of skin soft tissue and a more favorable profile of peripheral blood mononuclear cells. Skin biopsy after 1 month treatment showed vascular regeneration with mature non-calcified vessels within dermis and 20 months later re-epithelialization restored the integrity of damaged site. No infusion or local treatment related adverse events occurred. To the best of our knowledge, this is the first evidence for the clinical use of hAMSCs. These findings suggest hAMSCs warrant further investigation as a potential regenerative treatment for uremic calciphylaxis with effects of inhibiting vascular calcification, stimulating angiogenesis and myogenesis, anti-inflammatory and immune modulation, multi-differentiation, re-epithelialization and restorage of integrity.