Notochordal Cell Matrix As a Therapeutic Agent for Intervertebral Disc Regeneration

Vries, Stefan de; Doeselaar, Marina van; Meij, Björn P.; Tryfonidou, Marianna A.; Ito, Keita

doi:10.1089/ten.tea.2018.0026

Cited by 25 publications

(50 citation statements)

References 42 publications

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“…The TF gels were shown to provide biomechanical properties similar to the one found in the NP and promote differentiation 52. Most of the previous studies looking at the NCCM were performed in alginate beads 26, 84-86, that provide the 3D environment, however afford a relatively high surface/volume ratio and allow good factor exchange. Consequently, we used this well-establish system to test the hypothesis that our iNCs have paracrine effect on MSCs.…”

Section: Discussionmentioning

confidence: 99%

“…Notochordal conditioned medium (NCCM) including NCCM derived from porcine IVDs was previously shown to have an effect on MSCs and to promote differentiation into an NP fate 57-59. To investigate the effect of the notochordal conditioned medium generated by iNCs in vitro on BM-MSCs, we encapsulated the iNCs in alginate beads, as previously reported 26, 84-86, and cultured them in NP media in hypoxic conditions (2% O 2 ) 37. The conditioned medium was collected every 3 days, frozen in -80ºC and replaced with fresh media.…”

Section: Methodsmentioning

confidence: 99%

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Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model

et al. 2019

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Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs).Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology.Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury.Conclusion: In the present study, we report a stepwise differentiation m...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model

et al. 2019

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“…There are various ways to improve the matrix component in lumbar discs, including dynamo and biology methods . At present, the transplant of the therapeutic factor gene into nucleus pulposus cells in the lumbar disc has been widely used to increase the composition of various matrices . Cytokines, such as TGF‐β, IGF‐1, CTGF, GDF5, EGF, and BMPs, play an effective role in stimulating the karyomitosis and proteoglycan composition of lumbar disc cells.…”

Section: Discussionmentioning

confidence: 99%

The research of transgenic human nucleus pulposus cell transplantation in the treatment of lumbar disc degeneration

Wang

Jin

Kong

et al. 2019

The Kaohsiung J of Med Scie

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The present study determines whether the in vivo injection of TGFβ1 and CTGF mediated by AAV2 to transfect nucleus pulposus cells in degenerative lumbar discs can reverse the biological effects of rhesus lumbar disc degeneration. A total of 42 lumbar discs obtained from six rhesus monkeys were classified into three groups: experimental group, control group, and blank group. Degenerative lumbar discs were respectively injected with double gene-transfected human nucleus pulposus cells using minimally invasive techniques. Immumohistochemical staining, RT-PCR, and western blot were performed to observe the biological effects of double genetransfected human nucleus pulposus cells in degenerative lumbar discs on rhesus lumbar disc degeneration. At 4, 8, and 12 weeks after the transplantation of nucleus pulposus cells, the expression levels of TGF-ß1, CTGF, proteoglycan mRNA, and type-II collagen were detected by RT-PCR. The values of immumohistochemical staining and RT-PCR in the experimental group increased at 8 weeks, decreased with time at 12 weeks, and remained greater than the values in the control group, and the differences were statistically significant (P < .05). The western blot revealed that the values in the experimental group decreased with time, but remained greater than those in the PBS control group and blank control group, and the differences were statistically significant (P < .05). The double gene-transfection of human nucleus pulposus cells in degenerative lumbar discs mediated by rAAV2 can be continuously expressed in vivo after transplantation in lumbar discs of rhesus monkeys, and promotes the synthesis of proteoglycan and type II collagen, achieving the treatment purpose. K E Y W O R D S

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“…By using NCM, the NCCM incubation period, in which bioactive factors may be subject to degradation and inflammatory factors may be produced as a result of free swelling conditions is omitted. NCM induced stronger proteoglycan production and proliferation of bovine NPCs compared to NCCM, and induced a regenerative and anti‐inflammatory effect in a canine in vivo model underscoring its potential in biological IVD repair. Furthermore, NCM is directly harvested from the healthy IVD, hence it is also rich in GAGs.…”

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confidence: 83%

Notochordal cell matrix: An inhibitor of neurite and blood vessel growth?

Vries

Doeselaar

Meij

et al. 2018

Journal Orthopaedic Research

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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 9999:1-8, 2018.

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Notochordal Cell Matrix As a Therapeutic Agent for Intervertebral Disc Regeneration

Cited by 25 publications

References 42 publications

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model

The research of transgenic human nucleus pulposus cell transplantation in the treatment of lumbar disc degeneration

Notochordal cell matrix: An inhibitor of neurite and blood vessel growth?

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