Development and Degeneration of the Intervertebral Disc-Insights from Across Species.

Back pain caused by intervertebral disc (IVD) degeneration has a major socio-economic impact in humans, yet historically has received minimal attention in species other than humans, mice and dogs. However, a general growing interest in this unique organ prompted the expansion of IVD research in rats, rabbits, cats, horses, monkeys, and cows, further illuminating the complex nature of the organ in both healthy and degenerative states. Application of recent biotechnological advancements, including single cell RNA sequencing and complex data analysis methods has begun to explain the shifting inflammatory signaling, variation in cellular subpopulations, differential gene expression, mechanical loading, and metabolic stresses which contribute to age and stress related degeneration of the IVD. This increase in IVD research across species introduces a need for chronicling IVD advancements and tissue biomarkers both within and between species. Here we provide a comprehensive review of recent single cell RNA sequencing data alongside existing case reports and histo/morphological data to highlight the cellular complexity and metabolic challenges of this unique organ that is of structural importance for all vertebrates.

Metal-Organic Framework as a Fluorescent and Colorimetric Dual-Signal Readout Biosensor Platform for the Detection of a Genetic Sequence from the SARS-CoV-2 Genome.

The quest for the development of high-accuracy, point-of-care, and cost-effective testing platforms for SARS-CoV-2 infections is ongoing as current diagnostics rely on either assays based on costly yet accurate nucleic acid amplification tests (NAAT) or less selective and less sensitive but rapid and cost-effective antigen tests. As a potential solution, this work presents a fluorescence-based detection platform using a metal-organic framework (MOF) in an effective assay, demonstrating the potential of MOFs to recognize specific targets of the SARS-CoV-2 genome with high accuracy and rapid process turnaround time. As a highlight of this work, positive detection of SARS-CoV-2 is indicated by a visible color change of the MOF probe with ultrahigh detection selectivities down to single-base mismatch nucleotide sequences, thereby providing an alternative avenue for the development of innovative detection methods for diverse viral genomes.

Intervertebral disc degeneration-Current therapeutic options and challenges.

Degeneration of the intervertebral disc (IVD) is a normal part of aging. Due to the spine's declining function and the development of pain, it may affect one's physical health, mental health, and socioeconomic status. Most of the intervertebral disc degeneration (IVDD) therapies today focus on the symptoms of low back pain rather than the underlying etiology or mechanical function of the disc. The deteriorated disc is typically not restored by conservative or surgical therapies that largely focus on correcting symptoms and structural abnormalities. To enhance the clinical outcome and the quality of life of a patient, several therapeutic modalities have been created. In this review, we discuss genetic and environmental causes of IVDD and describe promising modern endogenous and exogenous therapeutic approaches including their applicability and relevance to the degeneration process.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

Glis1 and oxaloacetate in nucleus pulposus stromal cell somatic reprogramming and survival.

Regenerative medicine aims to repair degenerate tissue through cell refurbishment with minimally invasive procedures. Adipose tissue (FAT)-derived stem or stromal cells are a convenient autologous choice for many regenerative cell therapy approaches. The intervertebral disc (IVD) is a suitable target. Comprised of an inner nucleus pulposus (NP) and an outer annulus fibrosus (AF), the degeneration of the IVD through trauma or aging presents a substantial socio-economic burden worldwide. The avascular nature of the mature NP forces cells to reside in a unique environment with increased lactate levels, conditions that pose a challenge to cell-based therapies. We assessed adipose and IVD tissue-derived stromal cells through in vitro transcriptome analysis in 2D and 3D culture and suggested that the transcription factor Glis1 and metabolite oxaloacetic acid (OAA) could provide NP cells with survival tools for the harsh niche conditions in the IVD.

Stem cells in intervertebral disc regeneration-more talk than action?

Pain and lifestyle changes are common consequences of intervertebral disc degeneration (IVDD) and affect a large part of the aging population. The stemness of cells is exploited in the field of regenerative medicine as key to treat degenerative diseases. Transplanted cells however often face delivery and survival challenges, especially in tissues with a naturally harsh microniche environment such as the intervertebral disc. Recent interest in the secretome of stem cells, especially cargo protected from microniche-related decay as frequently present in degenerating tissues, provides new means of rejuvenating ailing cells and tissues. Exosomes, a type of extracellular vesicles with purposeful cargo gained particular interest in conveying stem cell related attributes of rejuvenation, which will be discussed here in the context of IVDD.

Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution.

OBJECTIVES: Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. RESULTS: We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. CONCLUSIONS: A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.

Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD.

Severe and chronic low back pain is often associated with intervertebral disc (IVD) degeneration. While imposing a considerable socio-economic burden worldwide, IVD degeneration is also severely impacting on the quality of life of affected individuals. Cell-based regenerative medicine approaches have moved into clinical trials, yet IVD cell identities in the mature disc remain to be fully elucidated and tissue heterogeneity exists, requiring a better characterization of IVD cells. The bovine coccygeal IVD is an accepted research model to study IVD mechano-biology and disc homeostasis. Recently, we identified novel IVD biomarkers in the outer annulus fibrosus (AF) and nucleus pulposus (NP) of the mature bovine coccygeal IVD through RNA in situ hybridization (AP-RISH) and z-proportion test. Here we follow up on Lam1, Thy1, Gli1, Gli3, Noto, Ptprc, Scx, Sox2 and Zscan10 with fluorescent RNA in situ hybridization (FL-RISH) and confocal microscopy. This permits sub-cellular transcript localization and the addition of quantitative single-cell derived values of mRNA expression levels to our previous analysis. Lastly, we used a Gaussian mixture modeling approach for the exploratory analysis of IVD cells. This work complements our earlier cell population proportion-based study, confirms the previously proposed biomarkers and indicates even further heterogeneity of cells in the outer AF and NP of a mature IVD.

Klhl14 Antisense RNA is a Target of Key Skeletogenic Transcription Factors in the Developing Intervertebral Disc.

STUDY DESIGN: RNA in situ hybridization (RISH) allows for validation and characterization of the long noncoding (lnc) natural antisense RNA (NAT) Klhl14as in the embryonic murine intervertebral disc (IVD) in the context of loss-of-function mutants for key transcription factors (TFs) in axial skeleton development. OBJECTIVE: Validation of Klhl14as in the developing murine IVD. SUMMARY OF BACKGROUND DATA: The IVD is a focus of regenerative medicine; however, processes and signaling cascades resulting in the different cell types in a mature IVD still require clarification in most animals including humans. Technological advances increasingly point to implications of lnc NATs in transcription/translation regulation. Transcriptome data generation and analysis identified a protein encoding transcript and related noncoding antisense transcript as downregulated in embryos devoid of key TFs during axial skeleton development. Here, primarily, the antisense transcript is analyzed in this loss-of-function context. METHODS: 4930426D05Rik and 6330403N15Rik were identified as Klhl14as and sense, respectively, two transcripts downregulated in the vertebral column of midgestation Pax1 and Pax9 mutant mouse embryos. RISH on wildtype and mutant embryos for the TF encoding genes Pax1/Pax9, Sox5/Sox6/Sox9, and Bapx1 was used to further analyze Klhl14as in the developing IVD. RESULTS: Klhl14as and Klhl14 were the top downregulated transcripts in Pax1; Pax9 E12.5 embryos. Our data demonstrate expression of Klhl14as and sense transcripts in the annulus fibrosus (AF) and notochord of the developing IVD. Klhl14as expression in the inner annulus fibrosus (iAF) seems dependent on the TFs Pax1/Pax9, Sox6, Sox9, and Bapx1. CONCLUSION: We are the first to suggest a role for the lncRNA Klhl14as in the developing IVD. Our data link Klhl14as to a previously established gene regulatory network during axial skeleton development and contribute further evidence that lnc NATs are involved in crucial gene regulatory networks in eukaryotic cells. LEVEL OF EVIDENCE: N/A.

Potential biomarkers of the mature intervertebral disc identified at the single cell level.

Intervertebral disc (IVD) degeneration and trauma is a major socio-economic burden and the focus of cell-based regenerative medicine approaches. Despite numerous ongoing clinical trials attempting to replace ailing IVD cells with mesenchymal stem cells, a solid understanding of the identity and nature of cells in a healthy mature IVD is still in need of refinement. Although anatomically simple, the IVD is comprised of heterogeneous cell populations. Therefore, methods involving cell pooling for RNA profiling could be misleading. Here, by using RNA in situ hybridization and z proportion test, we have identified potential novel biomarkers through single cell assessment. We quantified the proportion of RNA transcribing cells for 50 genetic loci in the outer annulus fibrosus (AF) and nucleus pulposus (NP) in coccygeal bovine discs isolated from tails of four skeletally mature animals. Our data reconfirm existing data and suggest 10 novel markers such as Lam1 and Thy1 in the outer AF and Gli1, Gli3, Noto, Scx, Ptprc, Sox2, Zscan10 and LOC101904175 in the NP, including pluripotency markers, that indicate stemness potential of IVD cells. These markers could be added to existing biomarker panels for cell type characterization. Furthermore, our data once more demonstrate heterogeneity in cells of the AF and NP, indicating the need for single cell assessment by methods such as RNA in situ hybridization. Our work refines the molecular identity of outer AF and NP cells, which can benefit future regenerative medicine and tissue engineering strategies in humans.

A quantitative and qualitative RNA expression profiling assay for cell culture with single cell resolution.

Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q2PISH allows for the analysis of both qualitative and quantitative (q2) gene expression on cultured cells for quality control measures with single cell resolution. q2PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1, Col2a1, Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q2PISH serves as an accurate quality control with single cell resolution for cultured cells.

Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors.

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

An Integrative Developmental Genomics and Systems Biology Approach to Identify an In Vivo Sox Trio-Mediated Gene Regulatory Network in Murine Embryos.

Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis.

Implications for a Stem Cell Regenerative Medicine Based Approach to Human Intervertebral Disk Degeneration.

The human body develops from a single cell, the zygote, the product of the maternal oocyte and the paternal spermatozoon. That 1-cell zygote embryo will divide and eventually grow into an adult human which is comprised of ~3.7 × 1013 cells. The tens of trillions of cells in the adult human can be classified into approximately 200 different highly specialized cell types that make up all of the different tissues and organs of the human body. Regenerative medicine aims to replace or restore dysfunctional cells, tissues and organs with fully functional ones. One area receiving attention is regeneration of the intervertebral discs (IVDs), which are located between the vertebrae and function to give flexibility and support load to the spine. Degenerated discs are a major cause of lower back pain. Different stem cell based regenerative medicine approaches to cure disc degeneration are now available, including using autologous mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs) and even attempts at direct transdifferentiation of somatic cells. Here we discuss some of the recent advances, successes, drawbacks, and the failures of the above-mentioned approaches.

RNA in situ hybridization characterization of non-enzymatic derived bovine intervertebral disc cell lineages suggests progenitor cell potential.

Degeneration of the intervertebral disc (IVD) is a meritorious target for therapeutic cell based regenerative medicine approaches, however, controversy over what defines the precise identity of mature IVD cells and lack of single cell based quality control measures is of concern. Bos taurus and human IVDs are histologically more similar than is Mus musculus. The mature bovine IVD is well suited as model system for technology development to be translated into therapeutic cell based regenerative medicine applications. We present a reproducible non-enzymatic protocol to isolate cell progenitor populations of three distinct areas of the mature bovine IVD. Bovine specific RNA probes were validated in situ and employed to assess fate changes, heterogeneity, stem cell characteristics and differentiation potential of the cultures. Quality control measures with single cell resolution like RNA in situ hybridization to assess culture heterogeneity (PISH) followed by optimization of culture conditions could be translated to human IVD cell culture to increase the safety of cell based regenerative medicine.

A developmental transcriptomic analysis of Pax1 and Pax9 in embryonic intervertebral disc development.

Pax1 and Pax9 play redundant, synergistic functions in the patterning and differentiation of the sclerotomal cells that give rise to the vertebral bodies and intervertebral discs (IVD) of the axial skeleton. They are conserved in mice and humans, whereby mutation/deficiency of human PAX1/PAX9 has been associated with kyphoscoliosis. By combining cell-type-specific transcriptome and ChIP-sequencing data, we identified the roles of Pax1/Pax9 in cell proliferation, cartilage development and collagen fibrillogenesis, which are vital in early IVD morphogenesis. Pax1 is up-regulated in the absence of Pax9, while Pax9 is unaffected by the loss of Pax1/Pax9 We identified the targets compensated by a single- or double-copy of Pax9 They positively regulate many of the cartilage genes known to be regulated by Sox5/Sox6/Sox9 and are connected to Sox5/Sox6 by a negative feedback loop. Pax1/Pax9 are intertwined with BMP and TGF-B pathways and we propose they initiate expression of chondrogenic genes during early IVD differentiation and subsequently become restricted to the outer annulus by the negative feedback mechanism. Our findings highlight how early IVD development is regulated spatio-temporally and have implications for understanding kyphoscoliosis.

Bovine annulus fibrosus cell lines isolated from intervertebral discs.

The adult bovine (Bos taurus) intervertebral disc is primarily comprised of two major tissue types: The outer annulus fibrosus (AF) and the central nucleus pulposus (NP). We isolated several primary cell lineages of passage (P) 0 cells from the AF tissue omitting typically used enzymatic tissue digestion protocols. The cells grow past p10 without signs of senescence in DMEM + 10% FCS on 0.1% gelatin coated/uncoated surfaces of standard cell culture plates and survive freeze-thawing. Preliminary analysis of the AF derived cells for expression of the two structural genes Col1a1 and Col2a1 was performed by PISH recapitulating the expression observed in vivo.

Genome wide binding (ChIP-Seq) of murine Bapx1 and Sox9 proteins in vivo and in vitro.

This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq) of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null) was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.

Gene expression profiles of Bapx1 expressing FACS sorted cells from wildtype and Bapx1-EGFP null mouse embryos.

The data described in this article refers to Chatterjee et al. (2015) "In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column" (GEO GSE35649) [1]. Transcriptional profiling combined with genome wide binding data is a powerful tool to elucidate the molecular mechanism behind vertebrate organogenesis. It also helps to uncover multiple roles of a single gene in different organs. In the above mentioned report we reveal the function of the homeobox gene Bapx1 during the embryogenesis of five distinct organs (vertebral column, spleen, gut, forelimb and hindlimb) at a relevant developmental stage (E12.5), microarray analysis of isolated wildtype and mutant cells in is compared in conjunction with ChIP-Seq analysis. We also analyzed the development of the vertebral column by comparing microarray and ChIP-Seq data for Bapx1 with similarly generated data sets for Sox9 to generate a gene regulatory network controlling various facets of the organogenesis.

Plate in situ hybridization (PISH) as a time and cost effective RNA expression assay to study phenotypic heterogeneity in a population of cultured murine cells at single cell resolution.

OBJECTIVES: Regenerative medicine approaches using reprogrammed or transdifferentiated cells require efficient single cell expression profiling to analyze culture homogeneity for quality control and recipients' safety. RESULTS: While antigen-antibody based systems have been developed for several proteins, probing at the mRNA level allows for more flexibility, faster adaption to the ever increasing new data from next generation sequencing and increased specificity, especially for genes of conserved gene families. CONCLUSIONS: We developed a time and cost effective expression profiling assay for monolayer cell culture in 96-well plates based on RNA in situ hybridization, termed PISH, at single cell resolution.