Structured coculture of mesenchymal stem cells and disc cells enhances differentiation and proliferation

Purpose: During in vivo stem cell differentiation, mature cells often induce the differentiation of nearby stem cells. Accordingly, prior studies indicate that a randomly mixed coculture can help transform mesenchymal stem cells (MSC) into nucleus pulposus cells (NPC). However, because in vivo signaling typically occurs heterotopically between adjacent cell layers, we hypothesized that a structurally organized coculture between MSC and NPC will result in greater cell differentiation and proliferation over single cell-type controls and cocultures with random organization. Methods: We developed a novel bilaminar cell pellet (BCP) system where a sphere of MSC is enclosed in a shell of NPC by successive centrifugation. Controls were made using single cell-type pellets and coculture pellets with random organization. The pellets were evaluated for DNA content, gene expression, and histology. Results: A bilaminar 3D organization enhanced cell proliferation and differentiation. BCP showed significantly more cell proliferation than pellets with one cell type and those with random organization. Enhanced differentiation of MSC within the BCP pellet relative to single cell-type pellets was demonstrated by quantitative RT-PCR, histology, and in situ hybridization. Conclusions: The BCP culture system increases MSC proliferation and differentiation as compared to single cell type or randomly mixed coculture controls.