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.

Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading

“…These projections, called neurites, mature into axons or dendrites and are critical for proper neuronal maturation and function. , PC-12 cells, which are a neuron-like rat pheochromocytoma cell line commonly used for to study neurobiology, were previously shown to have laminin-dependent neurite formation. − However, these studies largely rely on animal- or cell-line-derived laminin extracts, which have a less defined laminin content and batch-to-batch variability. While synthetic laminin peptides may be more defined, they often require specialized chemistry, buffers, or treatment to coat surfaces, in addition to extended preparation times. − LELP-based culture surface treatments provide the combined benefits of a defined and reproducible mixture of cell-attachment peptides as well as the convenient purification and coating protocols endowed by the rapid phase separation of ELPs. Brightfield images reveal that I30 ELPs facilitated cell attachment and some neurite formation (Figure E).…”

Getting to the Core: Exploring the Embryonic Development from Notochord to Nucleus Pulposus

“…Unlike articular cartilage, the human NP matrix can be distinguished by its unique GAG/hydroxyproline ratio, which remains significantly higher across all ages compared to juvenile cartilage [ 4 ]. Furthermore, NP cells (NPCs) express matrix-binding proteins, particularly integrins, and exhibit a high level of integrin-laminin binding, suggesting the crucial role of laminin in cell–matrix interactions [ 5 ]. It has been suggested that distinctive markers including an aggrecan/type II collagen expression ratio >20, and the stabilized expression of hypoxia-inducible factor 1-α (HIF-1α), glucose transporter 1 (GLUT-1), sonic hedgehog (SHH), Brachyury (TBXT), keratin (KRT)-18/19, carbonic anhydrase (CA)-XII, and cluster of differentiation (CD)-24 define the phenotype of young healthy NPCs [ 4 ].…”