Abstract
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.
Highlights
In the last decade, many studies have highlighted the potential use of mesenchymal stromal/stem cells (MSCs) as a therapeutic tool to be applied in the field of regenerative medicine [1,2,3,4,5]
HAMSCs were grown in parallel in two-dimensional (2D) cultures (Figure 1a–c) and in a suspended state (3D cultures), where cells spontaneously aggregated and formed compact multicellular spheroids
We analyzed the protein expression of these genes, and we found that CXCL12, leukemia inhibitory factor (LIF), vascular endothelial growth factor A (VEGF-A), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), IL6, epidermal growth factor (EGF), prostaglandin E2 (PGE2), CCL20, bone morphogenetic protein 2 (BMP2), transforming growth factor beta 1 (TGFB1), CXCL1, CCL2, growth differentiation factor 15 (GDF15), IL11, and CCL7 were more secreted by 3D human amnion-derived MSCs (hAMSCs) compared to 2D hAMSCs
Summary
Many studies have highlighted the potential use of mesenchymal stromal/stem cells (MSCs) as a therapeutic tool to be applied in the field of regenerative medicine [1,2,3,4,5]. MSC abilities have raised them as a therapeutic tool in several clinical trials for the treatment of many disorders. The results obtained from these studies are controversial [15,16,17,18]. This phenomenon is probably related to the intrinsic properties of MSCs, which are derived from different sources [9,19]. There is a need for improvement of MSC culture/production to enhance their therapeutic properties [3,20,21]
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