TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells

Abstract

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.

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc

“…NP specific marker genes (compared to AC or AF) are increasingly used to evaluate stem cell differentiation towards an NP phenotype [12,30]. It is preferable to use unique NP marker genes that are not expressed in AF and AC tissue.…”

“…In addition, NP specific marker genes are increasingly used as readout to develop stem cell differentiation protocols for NP regeneration [11,12]. However, a recent report described large variation for NP and AF marker genes in human cell isolates, which prevented separation of NP and AF tissue based on phenotypic markers [13].…”

Human umbilical cord derivatives regenerate intervertebral disc

“…Mesenchymal stem cells (MSCs) derived from adult tissues such as bone marrow (BM) and adipose MSCs have also been studied for their potential to differentiate into chondrogenic or NP-like cells capable of producing extracellular matrix in vitro. MSCs were reported to be induced by various growth factors such as transforming growth factor (TGF)β, insulin-like growth factor 1 and growth differentiation factor (GDF)6 to express NP-specific markers (Bian and Sun, 2015;Clarke et al, 2014;Colombier et al, 2016;Gupta et al, 2011), while coculturing with BM-MSCs with NP cells resulted in differentiation into cells that express NP-specific markers including aggrecan (ACAN), Versican, Sox9, Col2 and Col6 (Shim et al, 2016;Strassburg et al, 2010). Similarly, differentiation of human BM-MSCs into NP phenotypes was achieved by porcine notochordal cells (Korecki et al, 2010;Purmessur et al, 2011).…”

Protective effect of estrogen against intervertebral disc degeneration is attenuated by miR-221 through targeting estrogen receptor

“…Extensive evidence shows that TGF-β drives the differentiation of human mesenchymal stem cells toward NF-like cells under simulated microgravity conditions, supports extracellular matrix formation by AF cells, and prevents abnormal calcification of CEP through increasing Ank expression [15,16]. TGF-β inhibits NF-κB and MAPK pathways, thereby serving as a strong immune suppressor in IVD [17].…”