654-Timely modification of microenvironment stiffness improves chondrogenesis of single cell HIMSCS

Purpose (the aim of the study): Generation of de novo cartilage from cell types such as hiPSC-derived mesenchymal stromal cells (hiMSCs) are attractive due to their benefits as a stable and sustainable cell source for therapies and disease modelling. Nonetheless, the quality of neocartilage generated by hiMSCs is inferior compared to that of primary chondrocytes (Rodriguez Ruiz, Dicks et al. 2021). To improve hiMSC-chondrogenesis, unbiased characterization of modifying factors that can direct cell fate during differentiation at the single cell level is needed. The impact of microenvironment stiffness has previously been shown for differentiation to adipose and bone and here we apply the same principles (Kamperman, Henke et al. 2021). By encapsulating hiMSCs in single cell microgels of with precisely controlled microenvironment stiffness to improve chondrogenesis, and allow for the easy isolation of single cells for advanced characterization.
Methods: hiMSCs were generated (Rodriguez Ruiz, Dicks et al. 2021, Ramos, Tertel et al. 2022) and encapsulated as single cells in 5% Dex-TA using two microfluidic chips (one for droplet generation and another for delayed gelation) (Kamperman, Henke et al. 2017, Kamperman, Henke et al. 2021). hiMSCs were encapsulated in microgels crosslinked to 20kPa (soft) or 75kPa (stiff). Stiffness of the microgels was managed through control of the concentration of H2O2 used in the crosslinking reaction. Single cell microgels were cultured in trans-well plates and allowed to recover from encapsulation for 24 hours following culture in chondrogenic differentiation medium with bi-weekly refreshments (Rodriguez Ruiz, Dicks et al. 2021). After seven days of differentiation, a portion of the soft single cell microgels were stiffened through controlled exposure of the single cell microgels to the crosslinking enzyme and hydrogen peroxide (in a process known as post-cure) (Kamperman, Henke et al. 2021). Viability of the single cell microgels was monitored using CAM and PI staining, chondrogenesis was analyzed with RT-qPCR, and immunofluorescent staining (anabolic cartilage marker COL2A1; hypertrophy and bone mineralization markers COL10 and SPP1) before and after 21 days of chondrogenesis. Stiffness of the single cell microgels was determined through nanoindentation.
Results: Single cell microgels remained stable and supported hiMSC survival (>80%) throughout the 21 day chondrogenesis. While remaining non-proliferative, soft single cell microgels showed an increase in size of 1x (25µm) to 4x larger than initial encapsulation diameter which is not seen in stiff or post-cure microgels. Importantly, chondrogenic marker COL2 was expressed in >80% of cells (Fig. 1A) at day 21 in soft and post-cure microgels (Fig. 1B), while observed in only 35% of stiff microgels (Fig.1 C)(quantified expression is compared in Fig. 1G). Bone marker SPP1 was expressed in 60% of soft microgels (Fig. 1D) but not observed in any post-cure or stiff microgels (Fig. 1E-F)(quantified expression is compared in Fig. 1H). Hypertrophy marker COL10 was found in 85% of soft microgels (Fig. 1 D), 57% of post-cure microgels (Fig. 1F), and 20% of stiff microgels (Fig. 1E)(quantified expression is compared in Fig. 1I).
Conclusions: We display that hiMSCs differentiated in single cell post-cure microgels show improved expression of cartilage extracellular matrix proteins. Post-cure microgel embedded chondrocyte-like cells display early commitment to COL2 expression as observed in soft microgels, while preventing SPP1 expression and reducing COL10 expression similar to stiff microgels. Therefore, we posit that time-dependent control of hiMSC microenvironment during chondrogenic differentiation in single cell microgels is an opportunity for the improved generation of chondrocyte-like cells and the analysis of cell fate decisions. In ongoing analysis of multi-model single cell sequencing we will identify the key factors that drive such differences and can improve neocartilage deposited by hiMSCs.