Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells

Abstract

BackgroundThe intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro.MethodsNucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2– and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2).ResultsAfter 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2– NPC), respectively. Sorted Tie2– and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2– cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2– cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001).ConclusionsOur results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

“…Other studies demonstrated that transplanted Tie2 + NPCs could be differentiated towards Schwan‐like cells resulting in improved functional recovery in murine sciatic peripheral nerves, again substantiating the progenitor‐like nature . Nevertheless, Tie2 + NPCs application remains limited due to the rapid reduction in ratio of Tie2 + NPCs with in vitro expansion (Figure B) . In particular, Tekari et al showed that Tie2 expression in bovine NPCs decreased from approximately 8% to less than 1% after 2.3 population doublings.…”

“…The expansion is ideally performed in αMEM with 10% (v/v) FBS under hypoxic conditions (5% O 2 ), to augment maintenance of the Tie2 + NPCs phenotype . Supplementation of 100 ng/mL FGF2, limited culture time and cell passaging can limit the loss of Tie2 expression (Figure C) . For murine specifically, it is highly recommended to expand primary isolated cells to induce Tie2 expression.…”

“…No clear effect on the frequency of CFU‐S formation is observed with days of culture. Finally, in vitro multilineage differentiation potential can be used to confirm the progenitor‐like characteristics of the isolated Tie2 + NPCs, following a previously published protocol from Tekari et al…”

Pathomechanism of intervertebral disc degeneration

“…Sakai et al further revealed that angiopoietin‐1, a ligand of Tie2, suppressed apoptosis and promoted the proliferation of Tie2 + cells, enabling the development of a strategy to stimulate ANG‐1 to enhance Tie2 + progenitor cells for prevention of IDD . Later, the usefulness of the surface marker Tie2 was validated in a bovine coccygeal model …”

Discogenic cell transplantation directly from a cryopreserved state in an induced intervertebral disc degeneration canine model

“…The superiority of the OTS treatment is likely a consequence of a loss of potency of the precultured cells, which could result in changes to the original discogenic cell phenotype. A variety of studies have indicated a change of NP cell characteristics with extended culture, which might explain the enhanced outcomes for the OTS cells . In particular considering no stimulating factors were supplemented to the media.…”