Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro

Abstract

The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2⁺ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

Keywords: Tie2; cell proliferation; iMatrix-511; intervertebral disc; laminin-511; nucleus pulposus; progenitor cell; regenerative potential; type 2 collagen.

Figure 1

Assessment of cell morphology and proliferation (a) Morphological assessment of cultured nucleus pulposus (NP) cells for 6 days on either standard (Non-coat) or iMatrix (Lami-coat) coated dishes (Scale bar represents 1 µm). (b) Proliferation rates of cells cultured on both plate types (c) A Western blot example aimed to evaluate phosphorylated-Akt and phosphorylated-Erk1/2 levels. (d) Quantification of Western blot phosphorylated-Akt and phosphorylated-Erk1/2 levels for NP cells cultured on Non-coat and Lami-coat. Note: samples marked with X involve samples from an unrelated study simultaneously applied to the Western blot but have no relation to the presented study. ** indicates a p < 0.01 between the indicated groups. For a complete overview of Western blots, we refer to the Supplemental Files.

Figure 2

Flow cytometry analysis of cell surface markers to determine the maintenance of the nucleus pulposus progenitor cell phenotype (a) Example of flow cytometry gating for Tie2 positivity of cultured nucleus pulposus (NP) cells for 6 days on either standard (Non-coat) or iMatrix (Lami-coat) coated dishes, where gating plots show Tie2^- (blue) and Tie2⁺ (purple) measurements. (b) Total yield of NP cells positive for Tie2 after Non-coat or Lami-coat culture. (n = 5) (c) The positivity rate of Tie2, GD2, and CD24 of NP cells following Non-coat or Lami-coat culture. (n = 5) * and ** indicates a p < 0.05 and p < 0.01 between the indicated groups respectively.

Figure 3

Assessment of extracellular matrix production potency of cultured nucleus pulposus (NP) cells for 6 days on either standard (Non-coat) or iMatrix (Lami-coat) coated dishes. Rate of cells presenting with positive intracellular staining against (a) proteoglycans (PG) and (b) type II collagen (Col2). (c) Western blot analysis analyzing levels of Col2 levels * indicated a p < 0.05 between the indicated groups (n = 5). Dotted line indicates relative intensity of control samples.

Figure 4

Quantification of the number of spherical colonies forming from nucleus pulposus (NP) cells cultured for 6 days on either standard (Non-coat) or iMatrix (Lami-coat) coated dishes, followed by a methylcellulose colony-forming assay. * Indicated a p < 0.05 between the indicated groups. (n = 5).

Figure 5

Assessment of integrin-α3 and -α6 involvement in the beneficial outcomes derived from laminin coating. (a) Example of a gating procedure in flow cytometry analysis involving sorting Tie2-positive (blue) and Tie2-negative (red) nucleus pulposus (NP) cells (b) Quantification of staining intensity against integrin-α3 and integrin-α6 in Tie2-positive and Tie2-negative NP cell populations. (c) Positivity rate of NP cells for intracellular Col2 following blocking of integrin-α3 or integrin-α6 compared to NP cells not subjected to any blocking antibody. * Indicated a p < 0.05 between the indicated groups. (n = 3).