Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro

Abstract

Introduction Intervertebral disc degeneration is strongly associated with low back pain. Cell transplantation has been extensively studied as a treatment option for intervertebral disc degeneration. It is often necessary to perform cell culture prior to cell transplantation; however, during cell expansion, the cells tend to dedifferentiate and lose their potency. Although the ability to suppress dedifferentiation by ROCK inhibitor (ROCKi) has recently been reported for chondrocytes, its effects on nucleus pulposus cells are still largely unknown. Methods Rat nucleus pulposus cells were cultured with or without the addition of ROCKi (Y-27632), and cell proliferation; CD24 positivity; expression of SOX9, COL2A1, Aggrecan, and COL1A1; and cell redifferentiation ability in pellet culture were evaluated. Results Although the addition of ROCKi tended to slightly increase the cell proliferative capacity, no significant differences were observed between treated and untreated conditions. The addition of ROCKi showed a trend of minimally increased COL2A1, ACAN, and SOX9 expression. Increases in COL1A1 expression was slightly suppressed by ROCKi. In pellet culture, strong increase in type II collagen deposition was observed by the addition of ROCKi. The addition of ROCKi did not significantly change the levels of CD24 positivity. The supplementation of ROCKi did not significantly enhance nucleus pulposus cell marker expression during monolayer expansion. However, ROCKi addition did result in an increased type II collagen deposition in 3D pellet culture. Conclusions Taken together, the results suggest a minimal effect by ROCKi on nucleus pulposus cell phenotype maintenance.

Wnt3a and wnt5a as Potential Chondrogenic Stimulators for Nucleus Pulposus Cell Induction: A Comprehensive Review

“…10,74,75 Moreover, standard expansion of NP cells in vitro further reduces their overall potency. 75,76 From our current review, however, neither wnt3a nor wnt5a seem to have a clear beneficial effect on NP cell proliferation induction to enhance cell numbers, maintain overall potency, or induce a chondrogenic phenotype in vitro (Figs. 3A, 4A).…”

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

“…During expansion in culture, it is well known that cells isolated from the IVD de-differentiate and become fibroblast-like both in appearance and matrix synthesis, with decreased expression of key NP marker genes and matrix proteins such as collagen type II and aggrecan and induction of collagen type I. 41,[74][75][76] Here, loss of NP cell phenotype during expansion from all species investigated was also observed, with decreased NP cell phenotype over short culture periods. With a switch in matrix expression from collagen type II and aggrecan to collagen type I rapidly in culture with significant switches seen after passage 2, while other key NP markers such as KRTs and brachyury were lost even earlier in culture during the first passage.…”

Clinical Development of Regenerative Medicine Targeted for Intervertebral Disc Disease

“…Specifically, NP and other cartilage sources have low accessibility, and often tissue sources that are obtainable are compromised by disease, age, or trauma [24]. Moreover, these chondrogenic cells types present a limited proliferation capacity and tend to lose their phenotypical features rapidly in vitro [6,79]. Although culture optimization strategies are being explored to enhance the expandability of these cell types [80][81][82].…”