Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, 50 South Drive, Bethesda, MD 20892, USA.
Mesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct.
Human MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-beta3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-beta1 and TIMP1(tissue inhibitor of metalloproteinases-1)).
Significantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFbeta groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-beta1 and decreased expression of sox-9 and collagen II relative to the TGFbeta group was observed.
Together, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.
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"According to previously published studies, the conventional chondrogenic medium was suitable for the proposed differentiation. , ,  Briefly, cells were pelleted in 5 ml polypropylene tubes (BD, NJ) by centrifugation of 200,000 cells at 400 g for 5 min. The differentiation medium contained high glucose Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies), 1% ITS+ Premix (BD Bioscience), 100 mM sodium pyruvate (Life Technologies), 10 ng/ml TGF-beta 3 (Peprotech, NJ), 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO), 50 uM magnesium L-ascorbic acid-phosphate (Sigma-Aldrich), and 100 U/ml penicillin and 100 ug/ml streptomycin (Life Technologies). "
[Show abstract][Hide abstract]ABSTRACT: Human induced pluripotent stem cells (hiPSCs) can differentiate into notochordal cell (NC)-like cells when cultured in the presence of natural porcine nucleus pulposus (NP) tissue matrix. The method promises massive production of high-quality, functional cells to treat degenerative intervertebral discs (IVDs). Based on our previous work, we further examined the effect of cell-NP matrix contact and culture medium on the differentiation, and further assessed the functional differentiation ability of the generated NC-like. The study showed that direct contact between hiPSCs and NP matrix can promote the differentiation yield, whilst both the contact and non-contact cultures can generate functional NC-like cells. The generated NC-like cells are highly homogenous regarding the expression of notochordal marker genes. A culture medium containing a cocktail of growth factors (FGF, EGF, VEGF and IGF-1) also supported the notochordal differentiation in the presence of NP matrix. The NC-like cells showed excellent functional differentiation ability to generate NP-like tissue which was rich in aggrecan and collagen type II; and particularly, the proteoglycan to collagen content ratio was as high as 12.5-17.5 which represents a phenotype close to NP rather than hyaline cartilage. Collectively, the present study confirmed the effectiveness and flexibility of using natural NP tissue matrix to direct notochordal differentiation of hiPSCs, and the potential of using the generated NC-like cells for treating IVD degeneration.
"NCconditioned medium (NCCM; medium in which NCs or NC-containing tissue is cultured for 4 days and comprises NC-secreted soluble factors) has the potential to increase NP GAG production and cell proliferation616263 . Furthermore, both NCCM and CLC + NC coculture stimulate the in vitro differentiation of MSCs into a NP-like phenotype with a high chondrogenic matrix production [36,39,42,59,60,64,65]. NCs may maintain the young NP phenotype by secreting factors that suppress cell death and by influencing genes that regulate IVD anabolic activity and matrix protection . "
[Show abstract][Hide abstract]ABSTRACT: Pain due to spontaneous intervertebral disc (IVD) disease is common in dogs. In chondrodystrophic (CD) dogs, IVD disease typically develops in the cervical or thoracolumbar spine at about 3-7 years of age, whereas in non-chondrodystrophic (NCD) dogs, it usually develops in the caudal cervical or lumbosacral spine at about 6-8 years of age. IVD degeneration is characterized by changes in the biochemical composition and mechanical integrity of the IVD. In the degenerated IVD, the content of glycosaminoglycan (GAG, a proteoglycan side chain) decreases and that of denatured collagen increases. Dehydration leads to tearing of the annulus fibrosus (AF) and/or disc herniation, which is clinically characterized by pain and/or neurological signs. Current treatments (physiotherapy, anti-inflammatory/analgesic medication, surgery) for IVD disease may resolve neurological deficits and reduce pain (although in many cases insufficient), but do not lead to repair of the degenerated disc. For this reason, there is interest in new regenerative therapies that can repair the degenerated disc matrix, resulting in restoration of the biomechanical function of the IVD. CD dogs are considered a suitable animal model for human IVD degeneration because of their spontaneous IVD degeneration, and therefore studies investigating cell-, growth factor-, and/or gene therapy-based regenerative therapies with this model provide information relevant to both human and canine patients. The aim of this article is to review potential regenerative treatment strategies for canine IVD degeneration, with specific emphasis on cell-based strategies.
Full-text · Article · Jan 2014 · BMC Veterinary Research
"Similarly, bone marrow MSCs may differentiate into NP-like or chondrocyte-like cells in vitro, in alginate three-dimensional culture or in contact with chitosan hydrogels
[17-19]. In addition, soluble factors released by NP cells or tissues were found to promote bone marrow MSC differentiation into NP-like cells in vitro using condition medium
[20,21] and co-culture methods
[22,23]. These aforementioned studies show the importance of interactions between bone marrow-derived MSCs cells and the microenvironment for regulating the NP-like phenotype. "
[Show abstract][Hide abstract]ABSTRACT: Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) - originating from the Wharton's jelly - remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system.
HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture.
Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study). Differentiated HUCMSCs under all conditions were found to contain glycosaminoglycan, expressed extracellular matrix proteins of collagen II and laminin α5, and laminin receptors (integrin α3 and β4 subunits). However, neither growth factor treatment generated distinct differences in NP-like phenotype for HUCMSC as compared with no-serum conditions.
HUCMSCs have the potential to differentiate into cells sharing features with immature NP cells in a laminin-rich culture environment and may be useful for IVD cellular therapy.
Full-text · Article · Oct 2013 · Stem Cell Research & Therapy