Soft Elasticity-Associated Signaling and Bone Morphogenic Protein 2 Are Key Regulators of Mesenchymal Stem Cell Spheroidal Aggregates

Abstract

Cell therapy with adult mesenchymal stem cells (MSCs) is a promising approach to regenerative medicine and autoimmune diseases. There are various approaches to improve the efficacy of MSC-based therapeutics, and MSC preparation as spheroidal aggregates, or MSC spheroids, is a novel preparatory and delivery method. Spheroid formation induces a dramatic change in the gene expression profile of MSCs. Self-activation of interleukin-1 (IL1) signaling was shown to be upstream of both pro-and anti-inflammatory genes in MSC spheroids, but the molecular pathways that initiate IL1 signaling remain unknown. As bone morphogenic protein (BMP)2 upregulation precedes that of IL1B expression during spheroid formation, we hypothesized that BMP2 signaling triggers IL1 signaling in MSC spheroids. Contrary to expectations, BMP2 signaling decreased expression of IL1B and downstream genes in a SMAD6-dependent manner. Conversely, IL1B signaling enhanced BMP2 expression. Another major difference between two-dimensional (2D) monolayer culture and three-dimensional (3D) spheroid culture is the Young's elasticity modulus, or stiffness, of the materials surrounding the cells, as there is a million-fold difference between a plastic surface for standard 2D culture (GPa) and 3D spheroidal aggregates (0.1 kPa). We tested another hypothesis that soft elasticity-associated mechanosignaling initiates the gene expression change during spheroid formation. Results showed that both BMP2 expression and inflammatory signaling are upregulated in an elasticity-associated signaling-dependent manner in MSCs. Lastly, BMP2 signaling enhanced cell survival and cell spreading of MSC spheroids. In summary, our study suggests that soft elasticity and BMP2 signaling are critical for MSC spheroids.

Aggregation‐induced integrated stress response rejuvenates culture‐expanded human mesenchymal stem cells

“…Interestingly, significantly increased caspase 3/7 activity was observed in aggregates which result in increased cellular apoptosis. This increase in caspase activity was also observed previously by our group (Tsai et al, 2015) and others (Bazhanov et al, 2016;Cesarz et al, 2016). The increased ISR activity has previously been shown to increase caspase 3/7 activity and regulate the apoptosis instead of necrosis (Young et al, 2016).…”

“…Identifying the mechanism that regulates the responses of MSCs to external stimuli has important implications in designing optimal expansion and delivery strategies for MSC therapeutic applications (Galipeau & Sensebe, 2018;Yin et al, 2019). MSCs are highly responsive to microenvironmental stimuli arising from biological parameters, for example, immunomodulatory cytokines (Gao et al, 2016;Zhao, Ren, & Han, 2016), biomechanical regulation, for example, culture surface tension or stiffness (Bijonowski et al, 2019;Cesarz, Funnell, Guan, & Tamama, 2016), and physiological modulation, for example, oxygen tension (Hu, Zhao, Wu, & Li, 2019;Peck et al, 2019). Our previous studies have indicated that the significant alterations of MSC aggregates are due to different levels of cortical compaction-induced metabolic reconfiguration rather than hypoxic core within aggregates (Bijonowski et al, 2019;Liu et al, 2017).…”

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

“…Nowadays, a high number of MSC lines that display specific characteristics and differentiation capabilities have been generated and are valuable tools as part of models of disease and tissue repairing strategies. Different MSC lines have been employed for testing [17,[20][21][22][23][24][25] or producing [26,27] engineered scaffolds for skeletal applications, and for both investigating the MSC differentiation process [28][29][30][31][32][33][34] and finding new ways to improve it [35][36][37][38][39]. Additionally, these cell lines have also been used for analyzing functional makers [19,40] or even for exploring their roles in different diseases, such as osteoarthritis [41,42].…”

Size-Dependent Cortical Compaction Induces Metabolic Adaptation in Mesenchymal Stem Cell Aggregates

“…By contrast, hMSC aggregates with and without polymer encapsulation have enhanced secretory profiles; suggesting that cell-cell aggregation rather than a diffusion barrier is required for hMSC functional enhancement. 11,12 Elasticity plays a significant role in regulating hMSC fate and gene expression profiles, 13 which hMSCs can sense via cellular contraction. 14 Aggregation induces a lower cellular elastic niche than planar culture, and when hMSCs were cultured on plates matching the modulus of aggregates they showed similar upregulation of select proteins, and the induction of some pro-and anti-inflammatory genes, though to a lesser extent than after aggregation, suggesting the importance of cell-cell contact in conjunction with surface elasticity.…”

“…14 Aggregation induces a lower cellular elastic niche than planar culture, and when hMSCs were cultured on plates matching the modulus of aggregates they showed similar upregulation of select proteins, and the induction of some pro-and anti-inflammatory genes, though to a lesser extent than after aggregation, suggesting the importance of cell-cell contact in conjunction with surface elasticity. 13 Compared with diffusion-induced molecular gradients, little is known about the role of biomechanical forces, and the characteristics of their associated gradients in 3D aggregates in regulating hMSC properties. Aggregated hMSCs display a heterogenous morphology with a cortical region of flattened cells and a more regular and rounded morphology in the core.…”