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Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-b1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. STEM CELLS 2016;34:653-667 SIGNIFICANCE STATEMENTIn the present manuscript, we investigated whether human adipose stromal cells (hASCs) can be a clinically relevant source of stem cells for the generation of phenotypically stable and biologically active NPCyte-like cells. We successfully generated NPCyte-like cells from hASCs using a reproducible, robust and accurate growth factor-based induction protocol. We also demonstrated by experimental organogenesis in nude mice subcutis, that NPCyte-like cells seeded in an instructive hydrogel survived, maintained their specialized phenotype and presented secretory activities in vivo. In addition to highlighting the robustness and reproducibility of our protocol, we also document the temporal role of Smad pathways towards NP commitment by using specific chemical inhibitors. These data strengthen the specificity of the NP cell commitment by demonstrating new insights into the role of Smad proteins.

Articular cartilage is an avascular tissue composed of chondrocytes, a unique cell type responsible for abundant matrix synthesis and maintenance. When damaged, it never heals spontaneously under physiological circumstances. Therefore, the delivery of mesenchymal stem cells using hydrogel has been considered for cartilage repair. This study aims at investigating the influence of in vitro chondrogenic differentiation of human adipose tissue-derived stem cells (hATSCs) on in vivo cartilage formation when associated with a cellulose-based self-setting hydrogel (Si-HPMC). hATSCs were characterized for their proliferation, surface marker expression, and multipotency. The in vitro chondrogenic potential of hATSCs cultured within Si-HPMC in control or chondrogenic medium was evaluated by measuring COL2A1, ACAN, SOX9, and COMP expression by real-time PCR. Alcian blue and type II collagen staining were also performed. To determine whether in vitro chondrogenically differentiated hATSCs may give rise to cartilage in vivo, cells differentiated as a monolayer or in pellets were finally associated with Si-HPMC and implanted subcutaneously into nude mice. Cartilage formation was assessed histologically by alcian blue and type II collagen staining. Our data demonstrate that hATSCs exhibited proliferation and self-renewal. hATSCs also expressed typical stem cell surface markers and were able to differentiate towards the adipogenic, osteogenic, and chondrogenic lineages. Real-time PCR and histological analysis indicated that Si-HPMC enabled chondrogenic differentiation of hATSCs in inductive medium, as demonstrated by increased expression of chondrogenic markers. In addition, histological analysis of implants showed that chondrogenically differentiated hATSCs (monolayers or pellets) have the ability to form cartilaginous tissue, as indicated by the presence of sulphated glycosaminoglycans and type II collagen. This study therefore suggests that an in vitro induction of hATSCs in 2D was sufficient to obtain cartilaginous tissue formation in vivo. Si-HPMC associated with autologous hATSCs could thus be a significant tool for regenerative medicine in the context of cartilage damage.