ICRS 2019 - Conference Calendar
19.1.1 - Exploring Stem Cells & Biomaterials for Regenerative Medicine of Intervertebral Disc: From Clinical Data to Innovative Concepts
Abstract
Introduction
Intervertebral disc (IVD) plays a pivotal role in spine kinematics ensuring the function of "shock absorber" against the constraints that the spine undergoes throughout life. IVD is a fibrocartilaginous tissue that grossly consists of a peripheral network of type 1 collagen fibers (namely Annulus fibrosus, AF), which surrounds a highly hydrated gel (namely Nucleus pulposus, NP) rich in type 2 collagen and proteoglycans.
IVD degeneration is one of the major causes of low back pain (LBP). Currently, LBP is primarily managed by pharmacological treatments and if unsuccessful by surgical procedures (spine fusion or arthroplasty) that are reserved for severe debilitating LBP. To clinically address LBP earlier in the degenerative cascade of IVD, biology-inspired regenerative strategies could offer less invasive and etiological alternatives to spinal reconstructive surgery1. Whilst AF mostly contains fibroblastic cells, NP contains two different cell types: the notochordal cells (NTC) and the nucleopulpocytes (NPCytes). NTC are embryonic notochord derived cells that are considered the resident progenitor/stem cells of the NP tissue. NPCytes are considered the mature cells of NP niche responsible for the production and maintenance of a mechanically competent extracellular matrix (ECM). To maintain tissue homeostasis, NTC secrete several trophic factors that ensure the proliferation, survival and ECM secretory activities of NPCytes. During the growth and maturation of IVD some still unknown cues drive the disappearance of NTC (starting around age 10 in human). This progressive loss of NTC compromises the molecular dialog between NTC and NPCytes and thereby triggers NPCytes apoptosis and inability to produce and maintain NP niche. The subsequent loss of ECM in turn results in NP dehydration, loss of IVD height and formation of tears and cracks. Viewing these data, NP regeneration by re-establishing the NTC/NPCytes molecular dialog has recently been considered as a promising strategy to clinically address LBP2.
Content
We will first share our view of the mesenchymal stromal cells (MSC)-based therapeutic approaches that have been preclinically developed and, for some of them, clinically transferred in patients with discogenic LBP. Then, we will comment on the recent biomaterial-assisted MSC therapies that recently enter the preclinical and clinical scene of IVD regeneration. Finally, we will share with you our REMEDIV project that aims at developing an injectable NP substitute containing bioactive stem cells-derived NPCytes and NTC-like cells within a hydrated biomaterial that could be percutaneously transplanted into degenerated IVDs. We will present our data regarding the generation of NPCytes from adipose-derived MSC3. We will also share our recent unpublished evidences that human induced pluripotent stem cells (iPS) can be differentiated into notochord-like cells. Finally, we will consider our ability to transplant stem cells-derived derived NPCytes and NTC using a self-setting hydrogel in various animal models from mice and rabbit to sheep4. Whether this concept could open new therapeutic windows in the management of discogenic low back pain will finally be discussed.
References
REFERENCES
N. Henry, J. Clouet,…, C. Le Visage, J. . (2018) Innovative strategies for intervertebral disc regenerative medicine: From cell therapies to multiscale delivery systems. 36(1):281-294.
J. Clouet J, M. Fusellier,…, C. Le Visage, J. . (2018) Intervertebral disc regeneration: From cell therapy to the development of novel bioinspired endogenous repair strategies. Adv Drug Deliv Rev. Apr 26. In press.
P. Colombier, J. Clouet, …, A. Camus, J. Guicheux. (2016) TGF-β1 and GDF5 act synergistically to drive the differentiation of human adipose stromal cells toward nucleus pulposus-like cells. 34(3):653-67.
L. Le Fournier, M. Fusellier, …, J. , J. Clouet. (2017) The transpedicular surgical approach for the development of intervertebral disc targeting regenerative strategies in an ovine model. Eur Spine J 26(8):2072-2083.
Acknowledgments
ACKNOWLEDGEMENTS: This work was supported by grants from INSERM, ANR REMEDIV (ANR-14-CE16-0017), FRM, AO fondation, Agence de la biomédecine, Région pays de la Loire, Fondation de l’Avenir pour la Recherche Médicale Appliquée and H2020 european projects RESPINE (Grant agreement 732163) and IPSPINE (Grant agreement ID: 825925).
19.1.2 - Progenitor Cells of the Intervertebral Disc
Abstract
Introduction
Tissue homeostasis requires local progenitor cells, and tissue repair can recruit cells to the damage sites. Like cartilage, damage to the intervertebral disc does not repair well and reason is not known (1,2). Hypotheses include a lack, or insufficiency of appropriate progenitor cells. However, there are numerous in vitrostudies showing the isolation of mesenchymal stem/stromal cells (MSCs) from the nucleus pulposus (NP) and annulus fibrosus (AF) from human and animal IVDs (3). Of interest is that the MSCs isolated from NP and AF shared very similar characteristics, even though their developmental origins are different, suggesting they may not be embryonic cells persisting in the adult IVD. Further, MSCs can be isolated from both healthy and degenerated IVDs, but MSCs from degenerated IVDs seem to be less “potent” suggesting the hostile degenerative environment could have altered the functionality of the MSCs. More recently, Tie2 was proposed as a novel marker for NP progenitor cells in human and mice with self-renewal characteristics consistent with stem cells (4). These in vitrostudies are very interesting and supportive of the need to address the presence, location and functionalities of these cells in vivo.
Content
Mouse as a model organism to study stem cell biology is well established as numerous genetic tools are available to tag and tracing fate of progenitor cells in development and tissue repair. While there are clear differences between the mouse and human IVDs, much IVD biology can be deciphered from the mouse that can be translated to the human IVD (5). Understanding the developmental process of the IVD (6), allow us to use unique Cre driver mice to tag cells in the cartilage endplate and showed that chondrocytes from the hypertrophic region of the cartilage endplate be transit to become cells of the inner AF, involved in tissue homeostasis, and are responsive to mechanical loading. Using a notochord-Cre, and a novel Cre driver mice tagging a unique population of cells along the peripheral of the NP, we showed these cells have progenitor cell characteristics, and are proliferative that contributes to all cells of the NP in a healthy IVD of young mice. These cells appear to be depleted in older mice or IVD that are degenerated, consistent with a role of progenitor cells in IVD homeostasis and the process of degeneration. Relevance to IVD biology is address through analyses of single cell transcriptomic data from cells isolated from degenerate and non-degenerate human IVDs to gain an in vivoinsight into the progenitor cells in the IVD and their function.
References
Melrose J, Smith SM, Fuller ES, et al. Biglycan and fibromodulin frag- mentation correlates with temporal and spatial annular remodelling in experimentally injured ovine intervertebral discs. Eur Spine J. 2007;16(12):2193-2205
Humzah MD, Soames RW. Human intervertebral disc: structure and function. Anat Rec. 1988;220(4):337-356
Risbud MV, Guttapalli A, Tsai TT, et al. Evidence for skeletal progeni- tor cells in the degenerate human intervertebral disc. Spine. 2007; 32(23):2537-2544
Sakai D, Nakamura Y, Nakai T, et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun. 2012;3:1264
Alini M, Eisenstein SM, Ito K, et al. Are animal models useful for studying human disc disorders/degeneration? Eur Spine J. 2008; 17(1):2-19
Chan WC et al. Coming together is a beginning: the making of an intervertebral disc. Birth Defects Res C Embryo Today. 2014;102(1): 83-100
Acknowledgments
This work was supported by funding from AOSPINE (106540), China's National Strategic Basic Research Program (“973”) Grant 2014CB942901, and the RGC Theme-based Research Scheme (T12–708/12-N)
19.1.3 - Reprogramming the Future of Cell Based Therapy for Intervertebral Disc Disease
Abstract
Introduction
Low back pain is the primary cause of disability worldwide and is associated with intervertebral disc (IVD) degeneration. No curative treatment exists, however, significant momentum is being made in cell transplantation, with initial clinical trials indicating general safety and efficacy.1,2 As such, a multitude of studies aimed at inducing nucleus pulposus cell (NPC) differentiation3 by subjecting cells to growth factors, matrix components, mechanical stimulation, etc.Content
A more direct approach would be to manipulate transcription factor(s) (TF) expression to directly alter cell transcriptome to enable NPC differentiation. Thus far SOX9 4,5 and FOXA2 6 have been reported as tools for inducing NPC phenotypes. Yang et al 4 showed that SOX9 transduction in rat adipose-derived (ad)MSCs was able to enhance COL2 and proteoglycans production similar to NPC level, but only with TGFb3 addition. Sun et al. 7 similarly reported COL2 and ACAN expression increase by SOX9 transduction in rabbit bone marrow-derived MSC, but also reported beneficial effects of SOX9 transduction compared to GFP transduced MSCs as a cell transplantation product in an induced IVD degeneration rabbit model. These results are unsurprising, as SOX9 has been reported as a master regulator for chondrogenesis 8, and thus these reprogrammed cells are likely not specifically an NPC. Later work by Zhou et al 6 applied FOXA2 (an important TF for IVD development) transduction in rat adMSCs and similarly found an enhanced COL2 and proteoglycan expression, specifically when cultured in COL2-based hydrogels. Moreover, transplantation of FOXA2 transduced adMSC showed enhancements in limiting induced IVD degeneration in a rat model.Although the reported work shows the potential of inducing NPC-like features and the potential as a strategy for enhancing cell therapies. However, it remains ambiguous which specific TF are required to induce or maintain NPC phenotypes. Therefore we applied a holistic screening approach, aimed at identifying NPC master regulator TF. Applying multiple screening tools, we were able to narrow down the total of 20.000 potential master regulator TF candidates to a specific set of TF able induce a NPC-like phenotype in both human fibroblasts and MSCs, as indicated by enhanced gene- and protein expression (e.g. ACAN, COL2A1, CD24, KRT18, KRT8, etc.), as well as chondrogenic matrix production in 3D culture settings. Moreover, notochordal cell-like vacuoles could be detected when applying a more limited set of TF.
In short, we have established a specific combination of specific TF able reprogram pluripotent and matured cells to an NPC phenotype, potentially enabling a new approach for creating an infinite NPC source, to be used as cell therapy products or as research samples. In this work, we would like to share our experience in the search for specific NPC master regulator TF and the establishment of NPC induction methodology applying our identified TFs.
References
1. Schol J, Sakai D. Cell therapy for intervertebral disc herniation and degenerative disc disease: clinical trials. International orthopaedics. 2019;43(4):1011-1025.2. Sakai D, Andersson GB. Stem cell therapy for intervertebral disc regeneration: obstacles and solutions. Nat Rev Rheumatol. 2015;11(4):243-56.
3. Sakai D, Nakamura Y, Nakai T, et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Comm.; 2012;3:1264.
4. Yang Z, Huang CY, Candiotti KA, et al. Sox-9 facilitates differentiation of adipose tissue-derived stem cells into a chondrocyte-like phenotype in vitro. Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 2011;29(8): 1291-7. eng.
5. Sun Y, Lv M, Zhou L, et al. Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation. Osteoarthritis and cartilage. 2015;23(7): 1194-203.
6. Zhou X, Ma C, Hu B, et al. FoxA2 regulates the type II collagen-induced nucleus pulposus-like differentiation of adipose-derived stem cells by activation of the Shh signaling pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2018: fj201800373R.
7. Sun W, Zhang K, Liu G, et al. Sox9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model. PloS one. 2014;9(4): e93570.
8. Wright E, Hargrave MR, Christiansen J, et al. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nature genetics. 1995;9(1): 15-20. eng.