Nucleus Pulposus Progenitor Cells Research Articles

Assessment of Tie2-Rejuvenated Nucleus Pulposus Cell Transplants from Young and Old Patient Sources Demonstrates That Age Still Matters.

Cell transplantation is being actively explored as a regenerative therapy for discogenic back pain. This study explored the regenerative potential of Tie2+ nucleus pulposus progenitor cells (NPPCs) from intervertebral disc (IVD) tissues derived from young (<25 years of age) and old (>60 years of age) patient donors. We employed an optimized culture method to maintain Tie2 expression in NP cells from both donor categories. Our study revealed similar Tie2 positivity rates regardless of donor types following cell culture. Nevertheless, clear differences were also found, such as the emergence of significantly higher (3.6-fold) GD2 positivity and reduced (2.7-fold) proliferation potential for older donors compared to young sources. Our results suggest that, despite obtaining a high fraction of Tie2+ NP cells, cells from older donors were already committed to a more mature phenotype. These disparities translated into functional differences, influencing colony formation, extracellular matrix production, and in vivo regenerative potential. This study underscores the importance of considering age-related factors in NPPC-based therapies for disc degeneration. Further investigation into the genetic and epigenetic alterations of Tie2+ NP cells from older donors is crucial for refining regenerative strategies. These findings shed light on Tie2+ NPPCs as a promising cell source for IVD regeneration while emphasizing the need for comprehensive understanding and scalability considerations in culture methods for broader clinical applicability.

Characterization of the Nucleus Pulposus Progenitor Cells via Spatial Transcriptomics.

Loss of refreshment in nucleus pulposus (NP) cellularity leads to intervertebral disc (IVD) degeneration. Nevertheless, the cellular sequence of NP cell differentiation remains unclear, although an increasing body of literature has identified markers of NP progenitor cells (NPPCs). Notably, due to their fragility, the physical enrichment of NP-derived cells has limited conventional transcriptomic approaches in multiple studies. To overcome this limitation, a spatially resolved transcriptional atlas of the mouse IVD is generated via the 10x Genomics Visium platform dividing NP spots into two clusters. Based on this, most reported NPPC-markers, including Cathepsin K (Ctsk), are rare and predominantly located within the NP-outer subset. Cell lineage tracing further evidence that a small number of Ctsk-expressing cells generate the entire adult NP tissue. In contrast, Tie2, which has long suggested labeling NPPCs, is actually neither expressed in NP subsets nor labels NPPCs and their descendants in mouse models; consistent with this, an in situ sequencing (ISS) analysis validated the absence of Tie2 in NP tissue. Similarly, no Tie2-cre-mediated labeling of NPPCs is observed in an IVD degenerative mouse model. Altogether, in this study, the first spatial transcriptomic map of the IVD is established, thereby providing a public resource for bone biology.

DEVELOPING REGENERATION THERAPIES FOR THE INTERVERTEBRAL DISC

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration.Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors.Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering.

Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro.

The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

An esterase-responsive ibuprofen nano-micelle pre-modified embryo derived nucleus pulposus progenitor cells promote the regeneration of intervertebral disc degeneration.

Stem cell-based transplantation is a promising therapeutic approach for intervertebral disc degeneration (IDD). Current limitations of stem cells include with their insufficient cell source, poor proliferation capacity, low nucleus pulposus (NP)-specific differentiation potential, and inability to avoid pyroptosis caused by the acidic IDD microenvironment after transplantation. To address these challenges, embryo-derived long-term expandable nucleus pulposus progenitor cells (NPPCs) and esterase-responsive ibuprofen nano-micelles (PEG-PIB) were prepared for synergistic transplantation. In this study, we propose a biomaterial pre-modification cell strategy; the PEG-PIB were endocytosed to pre-modify the NPPCs with adaptability in harsh IDD microenvironment through inhibiting pyroptosis. The results indicated that the PEG-PIB pre-modified NPPCs exhibited inhibition of pyroptosis in vitro; their further synergistic transplantation yielded effective functional recovery, histological regeneration, and inhibition of pyroptosis during IDD regeneration. Herein, we offer a novel biomaterial pre-modification cell strategy for synergistic transplantation with promising therapeutic effects in IDD regeneration.

New Frontiers towards Regeneration of the Intervertebral Disc: On Progenitor Cells, Growth Factors and Biomaterials

This Special Issue on intervertebral disc (IVD) regeneration focuses on novel advances in understanding the cell sources and culture conditions of various cell types, i.e., progenitor and IVD cells. The issue consists of seven articles that provide a comprehensive overview of recently applied research insights: (1) into how IVD herniation can be provoked in a controlled in vitro biomechanical testing set-up, (2) how cells can be used for IVD repair, (3) the physiological conditions of IVD cells and (4) how hyaluronic acid could be used for IVD repair, and (5) how nucleus pulposus progenitor cells (NPPCs) and mesenchymal stromal cells (MSCs) shall be cultured and expanded towards a possible cell therapy.

The nucleus pulposus microenvironment in the intervertebral disc: the fountain of youth?

The intervertebral disc (IVD) is a complex tissue, and its degeneration remains a problem for patients, without significant improvement in treatment strategies. This mostly age-related disease predominantly affects the nucleus pulposus (NP), the central region of the IVD. The NP tissue, and especially its microenvironment, exhibit changes that may be involved at the outset or affect the progression of IVD pathology. The NP tissue microenvironment is unique and can be defined by a variety of specific factors and components characteristic of its physiology and function. NP progenitor cell interactions with their surrounding microenvironment may be a key factor for the regulation of cellular metabolism, phenotype, and stemness. Recently, celltransplantation approaches have been investigated for the treatment of degenerative disc disease, highlighting the need to better understand if and how transplanted cells can give rise to healthy NP tissue. Hence, understanding all the components of the NP microenvironment seems to be critical to better gauge the success and outcomes of approaches for tissue engineering and future clinical applications. Knowledge about the components of the NP microenvironment, how NP progenitor cells interact with them, and how changes in their surroundings can alter their function is summarised. Recent discoveries in NP tissue engineering linked to the microenvironment are also reviewed, meaning how crosstalk within the microenvironment can be adjusted to promote NP regeneration. Associated clinical problems are also considered, connecting bench-to-bedside in the context of IVD degeneration.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells.

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

Towards Tissue-Specific Stem Cell Therapy for the Intervertebral Disc: PPARδ Agonist Increases the Yield of Human Nucleus Pulposus Progenitor Cells in Expansion

(1) Background: Low back pain (LBP) is often associated with intervertebral disc degeneration (IVDD). Autochthonous progenitor cells isolated from the center, i.e., the nucleus pulposus, of the IVD (so-called nucleus pulposus progenitor cells (NPPCs)) could be a future cell source for therapy. The NPPCs were also identified to be positive for the angiopoietin-1 receptor (Tie2). Similar to hematopoietic stem cells, Tie2 might be involved in peroxisome proliferator-activated receptor delta (PPARδ) agonist-induced self-renewal regulation. The purpose of this study was to investigate whether a PPARδ agonist (GW501516) increases the Tie2+ NPPCs’ yield within the heterogeneous nucleus pulposus cell (NPC) population. (2) Methods: Primary NPCs were treated with 10 µM of GW501516 for eight days. Mitochondrial mass was determined by microscopy, using mitotracker red dye, and the relative gene expression was quantified by qPCR, using extracellular matrix and mitophagy-related genes. (3) The NPC’s group treated with the PPARδ agonist showed a significant increase of the Tie2+ NPCs yield from ~7% in passage 1 to ~50% in passage two, compared to the NPCs vehicle-treated group. Furthermore, no significant differences were found among treatment and control, using qPCR and mitotracker deep red. (4) Conclusion: PPARδ agonist could help to increase the Tie2+ NPCs yield during NPC expansion.

Spheroid-Like Cultures for Expanding Angiopoietin Receptor-1 (aka. Tie2) Positive Cells from the Human Intervertebral Disc.

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs’ population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.

Fluorescence-Activated Cell Sorting Is More Potent to Fish Intervertebral Disk Progenitor Cells Than Magnetic and Beads-Based Methods.

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2+), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in downstream functional differentiation in adipogenesis, osteogenesis, and chondrogenesis. The cell yields among the isolation methods differed widely, with FACS presenting the highest yield (5.0% ± 4.0%), followed by MACS (1.6% ± 2.9%) and pluriSelect (1.1% ± 1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2- NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2- populations in differentiation assays. To conclude, the isolation of NPPCs was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells. Impact Statement Tissue-specific progenitor cells such as nucleus pulposus progenitor cells of the IVD could become an ultimate cell source for tissue engineering strategies as these cells are presumably best adapted to the tissue's microenvironment. Fluorescence-activated cell sorting seemed to outcompete magnetic-activated cell sorting and pluriSelect concerning selecting a rare cell population from IVD tissue as could be demonstrated by improved cell yield and functional differentiation assays.

Differentiation of Pluripotent Stem Cells into Nucleus Pulposus Progenitor Cells for Intervertebral Disc Regeneration.

Low back pain (LBP) is one of the world's most common musculoskeletal diseases and is frequently associated with intervertebral disc degeneration (IDD). While the main cause of IDD is commonly attributed to a reduced number of nucleus pulposus (NP) cells, current treatment strategies (both surgical and more conservative) fail to replenish NP cells or reverse the pathology. Cell replacement therapies are an attractive alternative for treating IDD. However, injecting intervertebral disc (IVD) cells, chondrocytes, or mesenchymal stem cells into various animal models of IDD indicate that transplanted cells generally fail to survive and engraft into the avascular IVD niche. Whereas pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), hold great potential for revolutionizing regenerative medicine, current protocols for differentiating these cells into NP-like cells are inadequate. Nucleus pulposus progenitor cells (NPPCs), which are derived from the embryonic notochord, can not only survive within the harsh hypoxic environment of the IVD, but they also efficiently differentiate into NP-like cells. Here we provide an overview of the latest progress in repairing degenerated IVDs using PSCs and NPPCs. We also discuss the molecular pathways by which PSCs differentiate into NPPCs in vitro and in vivo and propose a new, in vivo IDD therapy.

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species.

BackgroundRecently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.PurposeHere, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence‐activated cell sorting‐based methodology to sort and analyze Tie2+ NPCs.MethodsWe present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented.ResultsOur protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.ConclusionsCurrent identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

Sciatic nerve regeneration by transplantation of in vitro differentiated nucleus pulposus progenitor cells.

To assess the applicability of mouse intervertebral disc-derived nucleus pulposus (NP) progenitor cells as a cell source for sciatic nerve regeneration. P0-Cre/Floxed-EGFP-transgenic mouse-derived NP progenitor cells were differentiated to Schwann-like cells in conventional induction medium. Schwann-like cells were subsequently transplanted into a mouse model of sciatic nerve transection, and nerve regeneration assessed by immunohistochemistry, electron microscopy and functional walking track analysis and heat stimulus reflex. NP progenitor cells differentiated into Schwann-like cells. Transplantation of these cells promoted myelinated axon formation, morphology restoration and nerve function improvement. NP progenitor cells have the capacity to differentiate into neuronal cells and are candidates for peripheral nerve regeneration therapy.

EGR1 controls divergent cellular responses of distinctive nucleus pulposus cell types.

BackgroundImmediate early genes (IEGs) encode transcription factors which serve as first line response modules to altered conditions and mediate appropriate cell responses. The immediate early response gene EGR1 is involved in physiological adaptation of numerous different cell types. We have previously shown a role for EGR1 in controlling processes supporting chondrogenic differentiation. We recently established a unique set of phenotypically distinct cell lines from the human nucleus pulposus (NP). Extensive characterization showed that these NP cellular subtypes represented progenitor-like cell types and more functionally mature cells.MethodsTo further understanding of cellular heterogeneity in the NP, we analyzed the response of these cell subtypes to anabolic and catabolic factors. Here, we test the hypothesis that physiological responses of distinct NP cell types are mediated by EGR1 and reflect specification of cell function using an RNA interference-based experimental approach.ResultsWe show that distinct NP cell types rapidly induce EGR1 exposure to either growth factors or inflammatory cytokines. In addition, we show that mRNA profiles induced in response to anabolic or catabolic conditions are cell type specific: the more mature NP cell type produced a strong and more specialized transcriptional response to IL-1β than the NP progenitor cells and aspects of this response were controlled by EGR1.ConclusionsOur current findings provide important substantiation of differential functionality among NP cellular subtypes. Additionally, the data shows that early transcriptional programming initiated by EGR1 is essentially restrained by the cells’ epigenome as it was determined during development and differentiation. These studies begin to define functional distinctions among cells of the NP and will ultimately contribute to defining functional phenotypes within the adult intervertebral disc.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-0979-x) contains supplementary material, which is available to authorized users.

Tie2+ Cells of the Bovine Nucleus Pulposus are Progenitor Cells Capable of Differentiating into Osteocytes and Adipocytes

Introduction The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability.1 IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin receptor (Tie2) in the nucleus pulposus were identified.2 These cells, which express type II collagen and aggrecan, were shown to have progenitor-like multipotency. Here, we isolated primary cells from bovine IVD aged 1 year and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermore, we demonstrate that in contrast to Tie2− cells, Tie2+ expressing cells can differentiate into osteogenic and adipogenic lineages in vitro. Materials and Methods NP cells were obtained from 1-year-old bovine tails by sequential digestion with Pronase for 1 hour and collagenase overnight. Sorted Tie2− and Tie2+ cells were cultured in osteogenic and adipogenic medium containing 5% fetal bovine serum for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets using alizarin red and red oil staining, respectively. Colony forming units (CFU) were prepared for both cell suspensions (Tie2− and Tie2 + ) in methylcellulose-based medium. The formed colonies (&gt; 10 cells) were analyzed macroscopically and counted after 8 days. Results After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by calcium deposition and fat droplet formation. By contrast, Tie2− cells generated a weak staining for calcium and no fat droplets were obtained ( Fig. 1 ). Sorted Tie2− and Tie2+ subpopulations of cells, both the formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape, whereas those fromTie2− cells were spread and fibroblastic. In addition, the highest frequency of the formed colonies was obtained for the Tie2+ cells after 8 days of culture in the methylcellulose-based medium. [Figure: see text] Conclusion Our data showed that Tie2+ cells of the nucleus pulposus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage. Acknowledgments This project was funded by two projects of the Swiss National Science Foundation, grant numbers IZK0Z3_154384 and 310030_153411. References Freemont AJ. The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 2009;48(1):5–10 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

Changes in Nucleus Pulposus Cell Pools in “Healer” Mice for the Repair of Intervertebral Disc Degeneration

Presentation Method This abstract was a poster presentation. Introduction Intervertebral disc (IVD) degeneration is a major cause of back pain that can also lead to sciatica, affecting the quality of life. Current treatments are limited to salvage surgical operations. Biological treatments to relieve symptoms or to restore disc are not available, as we know little about the biology of IVD degeneration and its potential to regeneration. Although most people will develop disc degeneration with aging, there are individuals who are protected even at the age (older than 50 years) when over 90% of the population would succumb to the problem, suggesting the presence of protective genes. Furthermore, maintenance of progenitor cells within the nucleus pulposus (NP) is thought to play an important role in disc homeostasis. A hypothesis is that genetic factors, which can confer a protection against disc degeneration via better maintenance of resident progenitor cells. There exist strains of “healer” mice (MRL/MpJ and LG/J) that have better regenerative potentials of cartilage tissues.1,2 Thus, we propose to address the NP progenitor cell pools in these healer mice in relation to the degeneration and potential repair/regeneration potentials of the disc. Materials and Methods Good healer (MRL and LG/J) and poor healer (C57/BL6C, and SM/J) mice were used in this study. Histological comparison of tail disc sections was assessed from 8 to 24 weeks of age. Progenitor cell pools and differentiated NP cells were assessed using immunohistochemistry using specific cell markers, Tie-2 and disialoganglioside (GD2), that were recently identified.3 Tail looping at 8 weeks of age for a fixed period was used as an environmental perturbation that will induce degeneration. Unlooping the tail after the period of looping can assess healing processes with appropriate controls. Results A comparison of MRL and C57 mice showed neither observable histological differences, nor signs of degenerative processes from 8 to 24 weeks of age. Following tail looping for 4, 5, 6, and 8 weeks, there were significant distortion of the annulus fibrosus (AF) and NP at the compressed and distended sides; in terms of loss of NP cells, AF tears and ruptures, and cell death in the AF. After the tails are unlooped for 4 weeks, there are restoration of NP and AF structures such as cell number in both MRL and C57 mice. However, superior healing is seen for mice for MRL at all time-points studied; especially in TL6/TL7, TL7/TL8, and TL8/TL9 disc levels, in which the disc structure restores better via continuous expansion of NP region, cell repopulation, and lamellae orientation recovers in the compressed AF sides with a clear NP AF boundary. In C57 mice, the AF lamellae structure remained disorganized following unlooping. Interestingly, in the absence of tail looping, SM/J tail discs already showed severe degeneration even at the age of 8 weeks, while that of LG/J mice were relatively normal, suggesting an impact on developmental or maturation in SM/J IVDs. Immunohistochemistry analysis of progenitors-related marker Tie2, GD2, and Angiopoietin 1 shows different expression pattern in MRL and C57. Current data show that GD2 positive cells may be the functioning NP cell in disc maintenance, and the extracellular matrix play an importance role in regulating progenitor cell functions. Conclusion By comparing the genetically different healer MRL, LG/J, and nonhealer C57 and SM/J, we have shown that a population of novel-marked NP progenitors may play a key role in the maintaining, healing, or regenerative of IVD structure and function. Abnormal persistent mechanical loading of the spine has negative effects on the healing processes. Furthermore, there appears to be threshold for the disc to self-repair, likely to be related to the onset of fibrotic events. References Clark LD, Clark RK, Heber-Katz E. A new murine model for mammalian wound repair and regeneration. Clin Immunol Immunopathol 1998;88(1):35–45 Rai MF, Hashimoto S, Johnson EE, et al. Heritability of articular cartilage regeneration and its association with ear wound healing in mice. Arthritis Rheum 2012;64(7):2300–2310 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

Sciatic Nerve Regeneration in Mice Induced by Transplantation of in Vitro Differentiated Nucleus Pulposus Progenitor Cells

Sciatic Nerve Regeneration in Mice Induced by Transplantation of in Vitro Differentiated Nucleus Pulposus Progenitor Cells

Roles of Progenitor Cells for Intervertebral Disc Regeneration in “Healer” Mice

Introduction Intervertebral disc (IVD) degeneration is a major cause of back pain that can also lead to sciatica, affecting the quality of life. Current treatments are limited to salvage surgical operations. Biological treatments to relieve symptoms or to restore disc are not available as we know little about the biology of IVD degeneration and its potential to regeneration. While most people will develop disc degeneration with aging, there are individuals who are protected even at the age (older than 50 years) when over 90% of the population would succumb to the problem, suggesting the presence of protective genes. Furthermore, maintenance of progenitor cells within the nucleus pulposus (NP) is thought to play an important role in disc homeostasis. A hypothesis is that genetic factors can confer a protection against disc degeneration via better maintenance of resident progenitor cells. There exist strains of “healer” mice (MRL/MpJ, LG/J) that have better regenerative potentials of cartilage tissues.1,2 Thus, we propose to address the NP progenitor cell pools in these healer mice in relation to the degeneration and potential repair/regeneration potentials of the disc. Materials and Methods Good healer (MRL and LG/J) and poor healer (C57/BL6C, and SM/J) mice were used in this study. Histological comparison of tail disc sections was assessed from 8 to 24 weeks of age. Progenitor cell pools and differentiated NP cells were assessed using immunohistochemistry using specific cell markers, Tie-2 and disialoganglioside (GD2), that were recently identified.3 Tail looping at 8 weeks of age for a fixed period was used as an environmental perturbation that will induce degeneration. Unlooping the tail after the period of looping can assess healing processes with appropriate controls. Results A comparison of MRL and C57 miceshowedneither observable histological differences, nor signs of degenerative processes from 8-week to 24-week of age. Following tail looping for 4, 5, 6 and 8 weeks, there were significant distortion of the annulus fibrosus (AF) and NP at the compressed and distended sides; in terms of loss of NP cells, AF tears and ruptures, and cell death in the AF. After the tails are unlooped for 4 weeks, there are restoration of NP and AF structures such as cell number in both MRL and C57 mice. However, superior healing is seen for MRL mice at all time-points studied; especially in TL6/TL7, TL7/TL8 and TL8/TL9 disc levels, in which the disc structure restores better via continuous expansion of NP region, cell repopulation and lamellae orientation recovers in the compressed AF sides with a clear NP AF boundary. In C57 mice, the AF lamellae structure remained disorganized following unlooping. Interestingly, in the absence of tail looping, SM/J tail discs already showed severe degeneration even at 8-week-old, while that of LG/J mice were relatively normal, suggesting an impact on developmental or maturation in SM/J IVDs. Immunohistochemistry analysis of progenitors related marker GD2 and Angiopoietin 1 (Ang-1, Tie-2 ligand) shows different expression pattern in 8-week old MRL and C57, and GD2 is well co-localized with Ang-1, indicating a potential regulatory role of these progenitors in disc degeneration and repair. Conclusion By comparing the genetically different healer MRL, LG/J and non-healer C57 and SM/J, we have shown that a population of novel marked NP progentiors may play a key role in the maintaining, healing or regenerative of IVD structure and function. Abnormal persistent mechanical loading of the spine has negative effects on the healing processes. Furthermore, there appears to be threshold for the disc to self-repair, likely to be related to the onset of fibrotic events. Disclosure of Interest None declared References Clark LD, Clark RK, Heber-Katz E. A new murine model for mammalian wound repair and regeneration. Clin Immunol Immunopathol 1998;88(1):35–45 Rai MF, Hashimoto S, Johnson EE, et al. Heritability of articular cartilage regeneration and its association with ear wound healing in mice. Arthritis Rheum 2012;64(7):2300–2310 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

Annulus Fibrosus Repair

Intervertebral disc (IVD) herniation is a common origin of disabled spine and low back pain (LBP) which cause enormous health care toll. The annulus fibrosus (AF), which constitutes the outer part ...