Use of Limiting Dilution Method for Isolation of Nucleus Pulposus Mesenchymal Stem/Progenitor Cells and Effects of Plating Density on Biological Characteristics and Plasticity

Abstract

Objectives To evaluate the effects of the limiting dilution method and plating density in rat nucleus pulposus mesenchymal stem/progenitor cells (NPMSCs). Materials and Methods Nucleus pulposus tissues were isolated from 12-week-old male Sprague-Dawley rats and NPMSCs were isolated using limiting dilution method. Cells were then classified into 3 groups according to plating density. Cell morphologies were observed, and colony-forming units, migration abilities, proliferative capacities, cell cycle percentages, multilineage differentiation capacities, stem cell biomarker expression levels, and immunophenotyping were also examined in each group. Results Low density group (LD) had higher morphological homogeneity, stronger colony-forming ability, higher cell proliferation capacity, and enhanced cell migration ability relative to the other two groups (p < 0.05). Moreover, LD had more cells entering S phase, with fewer cells arrested in G0/G1 phase (p < 0.05). While all three density groups showed a multilineage differentiation potential, LD showed a higher degree of observed and semiquantified lineage specific staining (p < 0.05). Furthermore, LD displayed higher expression levels of stem cell biomarkers (Nanog, Oct4, and Sox2) and showed higher percentages of CD29+, CD44+, and CD90+ cells (p < 0.05) following flow cytometry analysis. Conclusions Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.

Icariin Improves the Viability and Function of Cryopreserved Human Nucleus Pulposus‐Derived Mesenchymal Stem Cells

“…In the past decade, supplementation with exogenous mesenchymal stem cells (MSCs), such as MSCs derived from bone marrow and adipose tissue, has shown positive outcomes for the repair of IVD degeneration [ 4 ]. And in recent years, evidence has been found that endogenous nucleus pulposus-derived mesenchymal stem cells (NPMSCs) exist naturally in the IVDs [ 5 – 7 ]. As a novel approach to repairing disc degeneration, application of endogenous NPMSCs has shown exciting prospect and attracted increasing attention [ 8 , 9 ].…”

Effect of Lentivirus-mediated GDF5 Transfection on Differentiation of Rabbit Nucleus Pulposus Mesenchymal Stem Cells

“…Lin et al cultured NPMSCs of rats in vitro and induced differentiation, and found that NPMSCs could express stem cell genes, such as SOX2, Oct4 and Nanog [15]. Hui Zhang et al compared rat NPMSCs and bone marrow mesenchymal stem cells by in vitro culture, and found that both can differentiate into bone, cartilage and fat, and can express stem cell genes Nanog, oct-4 and sox-2, with no obvious difference [16].…”

Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells

“…Lin et al cultured NPMSCs of rats in vitro and induced differentiation, and found that NPMSCs could express stem cell genes, such as SOX2, Oct4 and Nanog [15]. Hui Zhang et al compared rat NPMSCs and bone marrow MSCs by in vitro culture, and observed that both of them could differentiate into bone, cartilage and fat, and could express stem cell genes Nanog, Oct-4 and SOX-2, with no obvious difference [16].…”