Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells

Abstract

There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The effi cacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confi rmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration

“…Stem cells, particularly adult mesenchymal stem cells, have been proposed as good alternative candidates and there is a growing body of evidence confirming their suitability [25][26][27][28][29][30]. However, for progenitor cells to be differentiated into the correct phenotype and adequately replace or stimulate the native NP cell population, a complete understanding of the cell or cells populating this tissue is paramount.…”

Living Cell Factories ‐ Electrosprayed Microcapsules and Microcarriers for Minimally Invasive Delivery

“…Alginates are among the most frequently employed biomaterials for encapsulation due to their abundance, convenient gelling properties and high biocompatibility [28] .…”

Médecine régénératrice du disque intervertébral

“…Plusieurs pistes sont actuellement envisagées utilisant des facteurs de croissance, la modulation de la pression d'oxygène et la culture tridimensionnelle (3D). En effet, il a été montré qu'en présence de 2 % d'oxygène, de growth differentiation factor 5 (GDF5) et de transforming growth factor- (TGF-), des CSM de moelle osseuse cultivées en 3D (micromasses) synthétisent de l'agrécane et des glycosaminoglycanes [17]. Parallèlement à cette stratégie d'enrichissement d'un milieu de culture, l'utilisation d'un milieu conditionné à partir de cellules notochordales issues de NP jeunes pourrait s'avérer efficace pour différen-cier des CSM en nucléopulpocytes.…”