Human-adipose-derived mesenchymal stem cells (hADMSCs) are adult stem cells and are relatively easy to access compared to other sources of mesenchymal stem cells (MSCs). They have shown immunomodulation properties as well as effects in improving tissue regeneration. To better stimulate and preserve the therapeutic properties of hADMSCs, biomaterials for cell delivery have been studied extensively. To date, hyaluronic acid (HA)-based materials have been most widely adopted by researchers around the world. PGmatrix is a new peptide-based hydrogel that has shown superior functional properties in 3D cell cultures. Here, we reported the in vitro and in vivo functional effects of PGmatrix on hADMSCs in comparison with HA and HA-based Hystem hydrogels. Our results showed that PGmatrix was far superior in maintaining hADMSC viability during prolonged incubation and stimulated expression of SSEA4 (stage-specific embryonic antigen-4) in hADMSCs. hADMSCs encapsulated in PGmatrix secreted more immune-responsive proteins than those in HA or Hystem, though similar VEGF-A and TGFβ1 release levels were observed in all three hydrogels. In vivo studies revealed that hADMSCs encapsulated with PGmatrix showed improved skin wound healing in diabetic-induced mice at an early stage, suggesting possible anti-inflammatory effects, though similar re-epithelialization and collagen density were observed among PGmatrix and HA or Hystem hydrogels by day 21.

Human-induced pluripotent stem cells (hiPSCs) have shown great potential for human health, but their growth and properties have been significantly limited by the traditional monolayer (2D) cell culture method for more than 15 years. Three-dimensional (3D) culture technology has demonstrated tremendous advantages over 2D. In particular, the 3D PGmatrix hiPSC derived from a peptide hydrogel offers a breakthrough pathway for the maintenance and expansion of physiologically relevant hiPSC 3D colonies (spheroids). In this study, the impact of 3D culture conditions in PGmatrix hiPSC on cell performance, integrity, and secretome profiles was determined across two commonly used hiPSC cell lines derived from fibroblast cells (hiPSC-F) and peripheral blood mononuclear cells (hiPSC-P) in the two most popular hiPSC culture media (mTeSR1 and essential eight (E8)). The 3D culture conditions varied in hydrogel strength, 3D embedded matrix, and 3D suspension matrix. The results showed that hiPSCs cultured in 3D PGmatrix hiPSC demonstrated the ability to maintain a consistently high cell viability that was above 95% across all the 3D conditions with cell expansion rates of 10–20-fold, depending on the 3D conditions and cell lines. The RT-qPCR analysis suggested that pluripotent gene markers are stable and not significantly affected by the cell lines or 3D PGmatrix conditions tested in this study. Mass spectrometry-based analysis of secretome from hiPSCs cultured in 3D PGmatrix hiPSC revealed a significantly higher quantity of unique proteins, including extracellular vesicle (EV)-related proteins and growth factors, compared to those in the 2D culture. Moreover, this is the first evidence to identify that hiPSCs in a medium with a rich supplement (i.e., mTeSR1) released more growth-regulating factors, while in a medium with fewer supplements (i.e., E8) hiPSCs secreted more survival growth factors and extracellular proteins. These findings offer insights into how these differences may impact hiPSC behavior, and they deepen our understanding of how hiPSCs respond to 3D culture conditions, aiding the optimization of hiPSC properties in translational biomedical research toward clinical applications.

Human induced pluripotent stem cells (hiPSCs) hold great potential for regenerative medicine. However, optimizing their differentiation into specific lineages within three-dimensional (3D) scaffold-based culture systems that mimic in vivo environments remains challenging. This study examined the trilineage differentiation of hiPSCs under various 3D conditions using synthetic peptide hydrogel matrices with and without embryoid body (EB) medium induction. hiPSC 3D colonies (spheroids), naturally formed from single cells or small clusters in 3D culture, were used for differentiation into the three germ lineages. Differentiated spheroids exhibited distinct morphological characteristics and significantly increased expression of key lineage-specific markers—FOXA2 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm)—compared to undifferentiated controls. Marker expression varied depending on the 3D culture conditions. Differentiation efficiency improved significantly, increasing from 16% to 71% for endoderm, 61% to 80% for mesoderm, and 35% to 48% for ectoderm, by selecting the appropriate 3D matrix and applying EB induction. Comprehensive data analysis from RT-qPCR, immunocytochemistry staining, and flow cytometry confirmed that the Synthegel Spheroid (SGS) is a viable 3D matrix for evaluating all three germ lineages using a commercial trilineage differentiation kit. While EB induction is essential for endodermal differentiation, it is not required for mesodermal and ectodermal lineages. These findings are valuable not only for screening initial differentiation potential at the lineage level but also for optimizing 3D differentiation protocols for deriving somatic cells from hiPSCs.