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The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

Blood vessel and neurite ingrowth into the degenerating intervertebral disc (IVD) are related to pain. In reported studies, notochordal cell (NC)-conditioned medium (NCCM) induced a regenerative response of nucleus pulposus (NP) cells, but also inhibition of neurite and vessel formation. NC matrix (NCM) derived from NC-rich NP tissue, induced even stronger anabolic effects than NCCM. Thus, the aim was to investigate whether NCM has similar anti-neurogenic and -angiogenic properties as NCCM. NCM and NCCM where produced from porcine NC-rich NP tissue. Human umbilical vein endothelial cells (HUVECs) were cultured in base medium (BM, 300 mOsm), NCCM (produced at 300 and 400 mOsm), NCM, or with chondroitin sulfate (CS, positive control) in angiogenesis-inducing medium, after which vessel length was measured. Although CS alone inhibited vessel growth, NCCM (both osmolarities) stimulated vessel formation by HUVECs. NCM did not affect vessel growth relative to BM. SH-SY5Y cells were cultured in BM, NCCM, and NCM on poly-D-lysine coated and polystyrene surfaces, and analyzed for neurite length and percentage of neurite expressing cells. On coated surfaces, neither NCCM nor NCM affected neurite growth. On a polystyrene surface, NCCM and NCM induced a higher number of neurite-expressing cells. NCCM's previously reported anti-angiogenic and -neurogenic effects were not observed in this study. Although addition of CS inhibited HUVEC vessel formation, other factors may be present in NCCM and NCM that affect neurite and vessel growth. Therefore, future studies testing an NC-based regenerative strategy should carefully assess the risk of such adverse effects in an in vivo setting. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 36:3188-3195, 2018., (© 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc.)

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their native environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect on extracellular matrix production., Methods: Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 μg/mL Link N. Furthermore, BM and NCCM samples were injected with 10(6) BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, glycosaminoglycan [GAG], hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining., Results: Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC-injected groups was not higher than in the other groups after 4 weeks of culture., Discussion: This study shows that, indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of an NCCM-based treatment for intervertebral disc regeneration.

Introduction: Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs., Methods: Canine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture., Results: NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa., Conclusions: No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect., Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×10(6) cells/mL) and NPCs+BMSCs (6×10(6) cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-β1 (TGF-β1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR., Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone., Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive for designing new tissue engineering approaches for IVD regeneration. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture of NCs are not known. The purpose of this study was to investigate the effects of different culture media and osmolarity on the physiology of NCs in vitro. NC clusters isolated from canine IVDs were suspended in alginate beads and cultured at 37°C under normoxic conditions for 28 days. Three different culture conditions were investigated; (1) Dulbecco's modified Eagle's medium (DMEM)/F12 (300 mOsm/L), (2) α-MEM (300 mOsm/L), and (3) α-MEM adjusted to 400 mOsm/L to mimic a hyperosmolar environment. NC morphology, expression of genes related to NC markers, matrix production and remodeling, and DNA- and glycosaminoglycan (GAG) analyses were performed on 1, 7, 14, and 28 days in culture. Large, vesicle-containing cells organized in clusters, characterized as NCs, remained present during 28 days for all culture conditions. However, the proportion of the NC clusters decreased over time, whereas the proportion of spindle-shaped cells increased. Gene expression profiling at 7, 14, and 28 days in culture compared to day 1 indicated a initial loss of NC phenotype followed by some recovery of brachyury and aggrecan gene expression after 28 days of culture supporting a potential recovery of NC phenotype. NCs cultured in α-MEM adjusted to 400 mOsm/L showed the highest gene expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1α1 gene expression. In conclusion, NCs cultured in alginate in native cell clusters, partially retained their characteristic morphology and recovered their phenotype in long-term culture. The type of culture medium and medium osmolarity appear to be important factors for culturing NC clusters. These findings provide additional information concerning the maintenance of NCs in vitro that may aid further mechanistic inquiry into the biology of NCs.

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte-like cells (CLCs). Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non-chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non-chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/β-catenin signaling in the healthy, NC-rich NP and early degenerated, CLC-rich NP of both breed types by immunohistochemistry of β-catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c-myc. Both NCs and CLCs showed nuclear and cytoplasmic β-catenin protein expression and axin2 gene expression, but β-catenin signal intensity and Wnt target gene expression were higher in the CLC-rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/β-catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c-myc gene expression. In conclusion, Wnt/β-catenin signaling activity in the NC within the NC-rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/β-catenin signaling activity in early IVD degeneration may be a reflection of its dual role., (Copyright © 2011 Orthopaedic Research Society.)