Adipose-Derived Stem Cell Response to Microenvironment Differences in Collagen I Hydrogels

Introduction: Cell-based therapies are gaining traction as treatment of nervous system injuries. Adipose-derived stem cells (ASCs) have been shown to secrete neurotrophic growth factors like brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF).1 Changes to the microenvironment will affect the secretion of growth factors. Importantly, we have recently shown that ASCs cultured in aligned collagen fiber hydrogels showed an upregulation in pro-angiogenic and neurotrophic growth factor secretion compared to non-aligned environments.2 Tissue microenvironments are a dynamic setting for cells with remodeling cells through matrix metalloproteinases (MMPs), secretion of nascent proteins, and extracellular vesicles (EVs). MMPs will assist in degrading the microenvironment to mimic native tissues. In non-native matrices, this remodeling can be used to influence or guide cells to damaged areas. In peripheral nerve repair this guidance coupled with neurotrophic factors secretion encourages outgrowing neurites towards the damaged nerves. The secretion of growth factors can come in a variety of forms from nascent protein secretion to EV packaging and release. To better understand how microenvironment differences influence ASC response, adipose-derived stem cells were embedded into collagen I matrices. Microenvironment remodeling, cellular secretion of EVs, and growth factors sequestered in the matrix microenvironments were investigated. The goal of this project is to understand how microenvironment differences and changes influence ASC behavior, focusing on the use of these matrices for peripheral nerve repair.

Materials and

Methods: Our lab recently developed uniaxially aligned collagen I hydrogels embedded with ASCs.2 Using this platform, uniaxially aligned collagen gels, referred to as stretched, and their non-aligned, non-stretched counterparts were cultured for 6 or 7 days in complete ASC media containing 10% v/v fetal bovine serum, 1% L-glutamine, and 0.1% gentamycin. Gels cultured for 7 days were stained with phalloidin and DAPI and imaged using confocal reflectance microscopy with fluorescence on an Olympus IX83 microscope. EVs were isolated from conditioned media replacing complete media on day 6 with serum-free media and cultured for 24 hours. EVs were then isolated from conditioned media using centrifuge filters (Sigma-Aldrich) with a molecular weight cutoff of 100kDa. Media above the filter was washed with phosphate buffer solution 3 times to remove media and residual proteins. EV size was characterized using a NanoSight NS300 with readings of each sample (n≥3) for 30 seconds per read using continuous syringe flow.

Results, Conclusions, and Discussions: Gels showed extracellular matrix remodeling by cells creating degradation channels after 7 days in culture, Figure 1. Gels with aligned fibers showed more matrix remodeling and degradation tracks than in non-aligned fiber gels. Alignment is key for directing neurite and axon growth in neural scaffolds.2–5 Higher levels of growth factors were seen sequestered in aligned (A) gels than in non-aligned (NA) gels for GDNF and b-NGF but levels of NT-3 were not significantly different. However, preliminary decellularization trials show significantly reduced concentrations across all analytes, Figure 2 bottom. Unfortunately, BDNF was not able to be quantified in the gel lysates. Additionally, analytes show significantly higher (p < 0.001) levels in conditioned media of aligned gels, suggesting a more neuroregenerative environment. EVs collected from non-aligned gel-conditioned media were smaller in size (124.1 ± 6.1nm) than the EVs collected from the stretched gel-conditioned media (140.5 ± 7.1nm). Subsequently, EVs were either lysed for EV protein cargo quantification. The protein cargo of EVs showed a concentration of 196.6 µg/mL and 145.2 µg/mL for aligned and non-aligned gels, respectively, Figure 3. As seen in Figure 2 we see higher neurotrophic factor content in the aligned gels suggesting that the larger size of EVs may result in more neurotrophic factors being carried.

Microenvironment differences show ASCs adapting and changing their behavior. ASCs are showing an increase in matrix remodeling, growth factor secretion, EV size, and EV protein cargo in aligned collagen I hydrogels. While differences in microarchitecture appear to be driving ASC behavior further investigation is needed into the behavior of ASCs remodeling the matrix, growth factor, and EV excretion. We have begun to investigate preserving matrix remodeling through the decellularization of aligned and non-aligned gels after culture. Decellularization may lead to a tissue mimic allowing for improved neurite outgrowth. Analysis of growth factors deposited in gels will be run in tandem with the decellularization ensuring neurotrophic factors can signal neurite outgrowth. Finally, a deeper analysis into EV cargos, signaling of neural cells, and biogenesis by ASCs in response to different microenvironments. However, microenvironment differences appear to drive ASC behavior in collagen I hydrogels.

Acknowledgements (Optional):

References:
1. Trzyna, A. & Banaś-Ząbczyk, A. Adipose-derived stem cells secretome and its potential application in “stem cell-free therapy”. Biomolecules vol. 11 Preprint at https://doi.org/10.3390/biom11060878 (2021).
2. Lewis, M. et al. Neuro-regenerative behavior of adipose-derived stem cells in aligned collagen I hydrogels. Mater Today Bio 22, 100762 (2023).
3. Kim, S. H. et al. Anisotropically organized three-dimensional culture platform for reconstruction of a hippocampal neural network. Nat Commun 8, (2017).
4. Guan, W. et al. Multi-scale, multi-level anisotropic silk fibroin/metformin scaffolds for repair of peripheral nerve injury. Int J Biol Macromol 246, (2023).
5. Huang, Y. A. et al. Nanoimprinted Anisotropic Topography Preferentially Guides Axons and Enhances Nerve Regeneration. Macromol Biosci 18, (2018).