(F-205) Evaluating Mesenchymal Stromal Cell Mediated Extracellular Matrix Remodeling and Paracrine Signaling in Gelatin Maleimide Microgels

Introduction:: Hematopoietic stems cells (HSCs) produce the bodies entire complement of blood and immune cells via hematopoiesis, a differentiation process regulated by cellular, biochemical, and biophysical cues. Hydrogels are widely used to imitate native microenvironments due to our ability to tune these cues. Recently, granular hydrogels have gained popularity due to their ability to introduce porosity into the hydrogel environment to improve diffusive transport and cellular infiltration. Our lab has previously shown that cultures of HSCs cultured within gelatin hydrogels display enhanced maintenance and quiescence in vitro when co-cultured with MSCs. More recently we have encapsulated HSCs and MSCs within microgels to assemble granular hydrogel models of the bone marrow. By examining MSC remodeling of the extracellular matrix (ECM) and paracrine signaling, we will establish a profile from which to infer mechanisms of action in HSC-MSC co-cultures. Here, we encapsulate MSCs in bulk hydrogels and microgel suspensions to investigate the impact of encapsulation strategy upon MSC activity. We will use nascent protein labeling and mass spectrometry to build foundational knowledge of how MSCs respond to these culture conditions. This work will inform future HSC-MSC co-cultures that aim to expand HSCs and study the hematopoietic niche.

Materials and Methods:: Gelatin-maleimide (GelMAL) was synthesized by reacting N-succinimidyl 3-maleimidopropionate with gelatin (Bloom 300, Type A) to functionalize lysine residues. 3D bulk hydrogels and microgels are formed using a solution of 4wt% GelMAL and PBS. Macrogels are crosslinked in a 10 mg/mL DTT solution for 15 minutes, while microgels are formed by running the solution through a flow-focusing microfluidic device. An emulsion of 2.2 mg/mL DTT in an oil phase is used to form and crosslink microgels in the device. Mouse bone marrow mesenchymal stem cells (mMSCs, Cyagen) at a passage number of 9 were encapsulated in the bulk hydrogel or microgels by mixing with the hydrogel precursor solution prior to fabrication. Our bulk hydrogels and microgels were allowed to culture for a period of 7 or 28 days, for nascent protein labeling and mass spectrometry respectively. Metabolic labeling was used to quantify the deposition of ECM proteins in the context of time and number of mMSCs per droplet and mass spectrometry was used for protein identification.

Results, Conclusions, and Discussions:: Preliminary data quantified the extent to which mMSCs deposit extracellular matrix for days two and four of culture. The width of a 2D projected nascent protein layer was found at the two time points and analyzed on a basis of the number of cells per droplet (Fig 1A). Combined statistics show a median of 0.183um and mean of 0.940um width for day 2 and a 0.088um median and 0.860um mean for day 4 (Fig 1B). Representative images of the deposited protein (green), cell membrane (red), and the overlay from CellProfiler are also shown (Fig 1C). Ongoing degradation studies will assess overall changes in the matrix content, secreted proteins, and hydrogel diffusivity.



Preliminary data demonstrates that mMSCs are actively involved in remodeling their surrounding matrix when encapsulated in GelMAL microgels, and readily deposit protein within the first few days of encapsulation. Ongoing work will extend these preliminary experiments to 28 days, as well as incorporate additional metrics of success – mass spectrometry – to characterize the secretome during this timeframe. These metrics will be applied to the bulk hydrogels and microgel suspensions to elicit differences in cellular response due to culture conditions. Understanding MSC signaling and ECM interaction in multiple culture conditions will provide a foundational knowledge that will better inform future efforts to expand HSC populations and study the hematopoietic niche.




Acknowledgements (Optional): :

References (Optional): : [1] Miksch et al., Small. 2022

[2] Loebel et al., Advanced Functional Materials. 2020