Stem cell behaviors are regulated by multiple microenvironmental cues. As an external signal, mechanical stiffness of the extracellular matrix is capable of governing stem cell fate determination, but how this biophysical cue is translated into intracellular signaling remains elusive. Here, we elucidate mechanisms by which stem cells respond to microenvironmental stiffness through the dynamics of the cytoskeletal network, leading to changes in gene expression via biophysical transduction signaling pathways in two-dimensional culture. Furthermore, a putative rapid shift from original mechanosensing to de novo cell-derived matrix sensing in more physiologically relevant three-dimensional culture is pointed out. A comprehensive understanding of stem cell responses to this stimulus is essential for designing biomaterials that mimic the physiological environment and advancing stem cell-based clinical applications for tissue engineering.

BMMSCs have drawn great interest in tissue engineering and regenerative medicine attributable to their multi-lineage differentiation capacity. Increasing evidence has shown that the mechanical stiffness of extracellular matrix is a critical determinant for stem cell behaviors. However, it remains unknown how matrix stiffness influences MSCs commitment with changes in cell morphology, adhesion, proliferation, self-renewal and differentiation. We employed fibronectin coated polyacrylamide hydrogels with variable stiffnesses ranging from 13 to 68 kPa to modulate the mechanical environment of BMMSCs and found that the morphology and adhesion of BMMSCs were highly dependent on mechanical stiffness. Cells became more spread and more adhesive on substrates of higher stiffness. Similarly, the proliferation of BMMSCs increased as stiffness increased. Sox2 expression was lower during 4h to 1 week on the 13-16 kPa and 62-68 kPa, in contrast, it was higher during 4h to 1 week on the 48-53 kPa. Oct4 expression on 13-16 kPa was higher than 48-53 kPa at 4h, and it has no significant differences at other time point among three different stiffness groups. On 62-68 kPa, BMMSCs were able to be induced toward osteogenic phenotype and generated a markedly high level of RUNX2, ALP, and Osteopontin. The cells exhibited a polygonal morphology and larger spreading area. These results suggest that matrix stiffness modulates commitment of BMMSCs. Our findings may eventually aid in the development of novel, effective biomaterials for the applications in tissue engineering.

Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young’s modulus of 13–16 kPa and 62–68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis. Osteogenic differentiation was increased on 62–68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62–68 kPa ECM. Taken together, our results provide evidence that matrix stiffness (62–68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.

With the recent approvals for the application of monoclonal antibodies that target the well-characterized immune checkpoints, immune therapy shows a great potential against both solid and hematologic tumors. The use of these therapeutic monoclonal antibodies elicits inspiring clinical results with durable objective responses and improvements in overall survival. Agents targeting programmed cell death protein 1 (PD-1; also known as PDCD1) and its ligand (PD-L1) achieve a great success in immune checkpoints therapy. However, the majority of patients are failed to respond to PD-1/PD-L1 axis inhibitors. Expression of PD-L1 on the membrane of tumor and immune cells has been shown to be associated with enhanced objective response rates to PD-1/PD-L1 inhibition. Thus, understanding of how PD-L1 expression is regulated will enable us to better define its role as predictive marker. In this review, we summarize recent findings in the regulation of PD-L1 expression.

Background: Developing efficient methods to isolate and identify human adipose-derived mesenchymal stem cells (hADSCs) remains to be one of the major challenges in tissue engineering. Methods: We demonstrate here a method by isolating hADSCs from abdominal subcutaneous adipose tissue harvested during caesarian section. The hADSCs were isolated from human adipose tissue by collagenase digestion and adherence to flasks. Results: The yield reached around 1 × 10⁶ hADSCs per gram adipose tissue. The following comprehensive identification and characterization illustrated pronounced features of mesenchymal stem cells (MSCs). The fibroblast-like hADSCs exhibited typical ultrastructure details for vigorous cell activities. Karyotype mapping showed normal human chromosome. With unique immunophenotypes they were positive for CD29, CD44, CD73, CD105 and CD166, but negative for CD31, CD34, CD45 and HLA-DR. The growth curve and cell cycle analysis revealed high capability for self-renewal and proliferation. Moreover, these cells could be functionally induced into adipocytes, osteoblasts, and endothelial cells in the presence of appropriate conditioned media. Conclusion: The data presented here suggest that we have developed high efficient isolation and cultivation methods with a systematic strategy for identification and characterization of hADSCs. These techniques will be able to provide safe and stable seeding cells for research and clinical application.