Derivation of Mesenchymal Stem Cells through Sequential Presentation of Growth Factors via Gelatin Microparticles in Pluripotent Stem Cell Spheroids

Abstract

The use of mesenchymal stem cells (MSCs) in regenerative medicine has gained considerable attention in recent years with the development of clinically relevant MSCs from induced pluripotent stem cells (iPSCs) and embryonic stem cells. Through sequential presentations of appropriate growth factors (GFs), iPSCs can be differentiated into mesodermal cells and then into MSCs. Furthermore, the formation of 3-dimensional cell spheroids, known as embryoid bodies, can be used to mimic in vivo conditions. However, the compact nature of embryoid bodies restricts the efficient and uniform delivery of GFs, leading to the formation of necrotic zones and hindered differentiation. To address this, we developed 2 types of gelatin microparticles (GelMPs) with distinct degradation rates for sequential delivery of GFs to enhance differentiation while preventing necrotic zones. In 2-dimensional culture, bone morphogenetic protein-4 (BMP4) and fibroblast growth factor 2 (FGF2) were identified as key proteins inducing iPSC differentiation into mesodermal cells and MSCs. The sequential presentation of these GFs was optimized for a 3-dimensional culture system by engineering fast-degrading GelMPs conjugated with BMP4 and slow-degrading GelMPs conjugated with FGF2. Our approach facilitated efficient iPSC differentiation into induced mesenchymal stem cells (iMSCs), as demonstrated by enhanced expression of mesodermal markers during the early stages of differentiation and MSC-specific markers at later stages. The resulting iMSCs exhibited characteristic surface markers (e.g., CD73, CD90, CD105, and CD44) and trilineage differentiation capability and were genetically stable. Compared to adult-derived MSCs, iMSCs showed superior proliferative capacity and reduced senescence, making them advantageous for cell therapy and regenerative medicine. This innovative approach of generating iMSCs has vast potential for therapeutic applications.