Abstract
Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.
Highlights
A change in cellular phenotype of the nucleus pulposus (NP) cells residing in the inner core of the intervertebral disc (IVD), leading to increased extracellular matrix degradation and altered matrix synthesis, is considered to be one of the major causes of IVD degeneration which is strongly associated with low back pain [1]
To date, there is no evidence to support the hypothesis of bi-directional intercellular transfer of cellular components between Mesenchymal stem cell (MSC) and NP cells during co-culture with direct cellto-cell contact. As all of these events have been previously reported to affect the phenotype of target cells, we investigated whether cell fusion or transfer of cytoplasm or membranous components may be operational during direct co-culture of MSCs and NP cells and may be responsible for the previously reported MSC differentiation toward NP cells and improvement of degenerate NP cell phenotype
CFDA labeled MSCs appeared in region R6, SNARF labeled NP cells in region R3 and double labeled cells in region R4, suggesting that these cells were a result of cell fusion between MSCs and NP cells
Summary
A change in cellular phenotype of the nucleus pulposus (NP) cells residing in the inner core of the intervertebral disc (IVD), leading to increased extracellular matrix degradation and altered matrix synthesis, is considered to be one of the major causes of IVD degeneration which is strongly associated with low back pain [1]. Traditional therapies for IVD degeneration are mainly restricted to those that treat the pain and do not target the underlying aberrant cell biology. Since autologous and/or allogeneic NP cells are not an ideal cell population, mesenchymal stem cells (MSCs) have been proposed as the preferred cell source for IVD regeneration [3,4]. MSCs are able to interact with the surrounding microenvironment and as such a variety of mechanisms by which MSCs might exert their biological effects have been postulated, including replacement of lost/degenerate cells through differentiation of MSCs into functional NP cells or provision of trophic support/stimulation for the native NP cells
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