Professor Cornell University Ithaca, New York, United States
Introduction: Mesenchymal stromal cells (MSC) have gained increasing attention for the treatment of intervertebral disc (IVD) degeneration due to their differentiative, immunomodulatory, and trophic properties [1]. Recent clinical trials show MSCs can improve disc hydration and patient-reported outcomes through direct intradiscal injections [1]. Despite the promising results, the exact mechanisms employed by MSCs to help promote disc healing remain unclear. Previously, it has been shown that MSCs can donate mitochondria (MT) to a variety of injured cells including chondrocytes and nucleus pulposus cells (NPC) [2,3]. The goal of this study was to determine (1) whether MSCs donate MT to annulus fibrosus cells (AFCs) and (2) the extent to which one of the key inflammatory mediators in IVD degeneration [4], IL-1𝛽, can stimulate MT transfer. We hypothesize that inflammatory stimulation via IL-1𝛽 will promote MT transfer from MSCs to AFCs.
Materials and
Methods: AFCs were harvested from the tails of four donor neonatal bovids. MSCs were harvested from neonatal bovine femoral trabecular bone and transduced with a mCherry-Mito lentivirus to allow for mitochondrial visualization. Passage 2 AFCs were cultured in the presence or absence of 10ng/mL of IL-1𝛽. After 24 hours, AFCs were stained with Vibrant TM CFDA SE and passage 5 MSCs were added to the culture. Cells were co-cultured for 24 hours and imaged using a Zeiss LSM inverted confocal microscope. For each sample, 8-14 imaging fields (resolution:.21-.35 μm/pixel) containing at least one MSC were randomly chosen. All imaging fields contained 10-20 Z-stack slices. MT transfer events were identified by areas of colocalization between the CFDA (AFCs) and mCherry (MSC MT) channels, both manually and by using an established ImageJ plugin [5]. All colocalized signals above one-pixel unit in area were considered a mitochondrial transfer event and manually counted for each image (Fig.1). Cell and colocalization areas were determined using ImageJ.
Results, Conclusions, and Discussions: Confocal microscopy revealed multiple instances of red fluorescent signal within AFCs for both the control and IL-1𝛽 groups (Fig. 3), suggestive of MT transfer events. Image analysis verified apparent MT transfer by confirming colocalization (Fig. 1a-c) spanned multiple image slices, as seen through cell tracing (Fig. 1c) and orthogonal viewing of whole Z-stacks (Fig. 1d). IL-1𝛽 stimulated AFCs exhibited a 17% increase in MT transfer events compared to control AFCs (p = 0.0033, Fig. 2a). In both groups, there was strong evidence of internalized MSC MT by AFCs and examples of tunneling nanotubes (TNTs), direct contact/fusion-, and extracellular vesicle (EV)-mediated MT transfer, as seen with chondrocytes [2] and NPCs [3](Fig. 3). Ratios of colocalization area to cell area show the majority of signal colocalization occupied less than 5% of the total cell area for both the IL-1𝛽 and control groups, indicating singular or few mitochondrial transfer events per cell (Fig. 2b).
This study shows that a general inflammatory mediator can promote MT transfer in a model of IVD degeneration. To the best of our knowledge, this is the first study to show that MSCs donate MT to AFCs and that MT transfer is enhanced as a response to inflammation. Given that MSC MT transfer has shown great promise in reducing cell stress and promoting cell survival [6], it may prove to be a novel strategy for stimulating the healing of degenerative IVDs.
