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.

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.

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.

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 genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.

The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells.

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.

Making sense of Dlx1 antisense RNA.

Long non-coding RNAs (lncRNAs) have been recently recognized as a major class of regulators in mammalian systems. LncRNAs function by diverse and heterogeneous mechanisms in gene regulation, and are key contributors to development, neurological disorders, and cancer. This emerging importance of lncRNAs, along with recent reports of a functional lncRNA encoded by the mouse Dlx5-Dlx6 locus, led us to interrogate the biological significance of another distal-less antisense lncRNA, the previously uncharacterized Dlx1 antisense (Dlx1as) transcript. We have functionally ablated this antisense RNA via a highly customized gene targeting approach in vivo. Mice devoid of Dlx1as RNA are viable and fertile, and display a mild skeletal and neurological phenotype reminiscent of a Dlx1 gain-of function phenotype, suggesting a role for this non-coding antisense RNA in modulating Dlx1 transcript levels and stability. The reciprocal relationship between Dlx1as and Dlx1 places this sense-antisense pair into a growing class of mammalian lncRNA-mRNA pairs characterized by inverse regulation.

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.

BACKGROUND: Vertebrate organogenesis is a highly complex process involving sequential cascades of transcription factor activation or repression. Interestingly a single developmental control gene can occasionally be essential for the morphogenesis and differentiation of tissues and organs arising from vastly disparate embryological lineages. RESULTS: Here we elucidated the role of the mammalian homeobox gene Bapx1 during the embryogenesis of five distinct organs at E12.5 - vertebral column, spleen, gut, forelimb and hindlimb - using expression profiling of sorted wildtype and mutant cells combined with genome wide binding site analysis. Furthermore we analyzed the development of the vertebral column at the molecular level by combining transcriptional profiling and genome wide binding data for Bapx1 with similarly generated data sets for Sox9 to assemble a detailed gene regulatory network revealing genes previously not reported to be controlled by either of these two transcription factors. CONCLUSIONS: The gene regulatory network appears to control cell fate decisions and morphogenesis in the vertebral column along with the prevention of premature chondrocyte differentiation thus providing a detailed molecular view of vertebral column development.

Pax1(EGFP): new wildtype and mutant EGFP mouse lines for molecular and fate mapping studies.

The Paired box gene 1 (Pax1) transcription factor plays essential roles in the development of axial skeleton, scapula, pelvic girdle, and thymus. Delineating its pleiotropic and molecular roles in the various tissues requires the ability to track and isolate the Pax1-expressing cells for downstream high-throughput experiments such as microarray and RNA-sequencing. With these applications in mind, we have generated two new mouse lines-a Pax1 wildtype (WT) mouse line that co-expresses enhanced green fluorescent protein (EGFP) with functional Pax1, and a Pax1 knockout mouse line which expresses EGFP under the control of Pax1 promoter, using the internal ribosome entry site (IRES) and 2A-peptide multi-cistron concatenating strategies. These mouse lines facilitate the isolation and enrichment of Pax1-specific cells from Pax1-positive and Pax1-null embryos using fluorescence activated cell sorting (FACS). They can be also be used in parallel to investigate the stage- and tissue-specific molecular functions of Pax1.

A conditional mouse line for lineage tracing of Sox9 loss-of-function cells using enhanced green fluorescent protein.

Traditionally, conditional knockout studies in mouse have utilized the Cre or Flpe technology to activate the expression of reporter genes such as lacZ or PLAP. Employing these reporter genes, however, requires tissue fixation. To make way for downstream in vivo or in vitro applications, we have inserted enhanced green fluorescent protein (EGFP) into the endogenous Sox9 locus and generated a novel conditional Sox9 null allele, by flanking the entire Sox9 coding region with loxP sites and inserting an EGFP reporter gene into the 3'-UTR allowing for EGFP to be expressed upon Sox9 loss of function yet under the control of the endogenous Sox9 promoter. Mating this new allele to any Cre-expressing line, the fate of Sox9 null cells can be traced in the cell type of interest in vivo or in vitro after fluorescence-activated cell sorting.

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.

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.

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.

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.

New Bapx1(Cre-EGFP) mouse lines for lineage tracing and conditional knockout studies.

To gain insight into the roles of various genes in development and to circumvent embryonic lethality that hinders genetic studies, lineage tracing and conditional knockout techniques have been widely performed on mice using the increasing numbers of gene-targeted Cre mouse lines. Employing the internal ribosome entry site (IRES) and the 2A peptide multicistronic expression strategies, we report two new Bapx1 mouse lines with functional Bapx1 whereby Cre and enhanced green fluorescence protein (EGFP) are expressed discretely under the control of the Bapx1 promoter. These mouse lines, when mated with the Rosa26R-lacZ reporter line, can be used to trace the lineage of Bapx1-expressing cells whereas stage-specific, spatial expression of Bapx1 can be visualized by the EGFP fluorescence. In addition, both of our Bapx1(Cre-EGFP) mouse lines can be used to enrich for Bapx1-specific cells and also serve as effective conditional knockout tools to investigate gene functions in the skeleton and/or visceral organs.

Endogenous tagging of the murine transcription factor Sox5 with hemaglutinin for functional studies.

Gene expression is usually studied at the transcript level rather than at the protein level due to the lack of a specific and sensitive antibody. A way to overcome this is to fuse to the protein of interest an immunoreactive tag that has well-characterized antibodies. This epitope tagging approach is often used for in vitro experiments but for in vivo studies, the success rate of protein tagging has not been extensively analyzed and our study seeks to cover the void. A small epitope, hemaglutinin derived from the influenza virus was used to tag a transcription factor, Sox5 at the N-terminal via homologous recombination in the mouse. Sox5 is part of the Sry-related high-mobility-group box gene family and plays multiple roles in essential biological processes. Understanding of its molecular function in relation to its biological roles remains incomplete. In our study, we show that the longer isoform of Sox5 can be tagged endogenously with hemaglutinin without affecting its biological function in vivo. The tagged protein is easily and specifically detected with an anti-hemaglutinin antibody using immunohistochemistry with its expression matching the endogenous expression of Sox5. Immunoprecipitation of Sox5 was also carried out successfully using an anti-hemaglutinin antibody. The transgenic line generated from this study is predicted to be useful for future experiments such as co-immunoprecipitation and chromatin immunoprecipitation, allowing the further understanding of Sox5. Lastly, this approach can be easily employed for the investigation of other transcription factors and proteins in vivo to overcome technical limitations such as antibody cross-reactivity and to perform isoform-specific studies.

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.

Generation of mice with a novel conditional null allele of the Sox9 gene.

Sox9 is expressed in multiple tissues during mouse development and adulthood. Mutations in the Sox9 gene or changes in expression levels can be attributed to many congenital diseases. Heterozygous loss-of-function mutations in the human SOX9 gene cause Campomelic dysplasia, a semi-lethal skeletal malformation syndrome. Disruption of Sox9 by conventional gene targeting leads to perinatal lethality in heterozygous mice, hence hampering the feasibility to obtain the homozygous Sox9 null mice for in vivo functional studies. In this study, we generated a conditional allele of Sox9 (Sox9 ( tm4.Tlu )) by flanking exon 1 with loxP sites. Homozygous mice for the Sox9 ( tm4.Tlu ) allele (Sox9 ( flox/flox )) are viable, fertile and indistinguishable from wildtype (WT) mice, indicating that the Sox9 ( tm4.Tlu ) allele is a fully functional Sox9 allele. Furthermore, we demonstrated that Cre-mediated recombination using a Col2a1-Cre line resulted in specific ablation of Sox9 activity in cartilage tissues.

A more cost effective and rapid high percentage germ-line transmitting chimeric mouse generation procedure via microinjection of 2-cell, 4-cell, and 8-cell embryos with ES and iPS cells.

The long-standing traditional method of delivering embryonic stem (ES) cells adjacent to the inner cell mass (ICM) of blastocysts to generate chimeras improved with the advent of laser- or Piezo assisted 8-cell embryo microinjection. Building on this technology but omitting either the laser or the Piezo to penetrate the zona pellucida and making use of earlier embryonic stages (2-cell and 4-cell), we were able to significantly speed up and economize our ES cell microinjection and chimera production throughput. We demonstrate here that embryonic (ES) and induced pluripotent stem (iPS) cells can stay fully pluripotent when delivered into 2-cell- and 4-cell-stage embryos, long before they would naturally be incorporated into the ICM of a blastocyst (E3.5) and give rise to high percentage and germline transmitting chimeras.

A symphony of inner ear developmental control genes.

The inner ear is one of the most complex and detailed organs in the vertebrate body and provides us with the priceless ability to hear and perceive linear and angular acceleration (hence maintain balance). The development and morphogenesis of the inner ear from an ectodermal thickening into distinct auditory and vestibular components depends upon precise temporally and spatially coordinated gene expression patterns and well orchestrated signaling cascades within the otic vesicle and upon cellular movements and interactions with surrounding tissues. Gene loss of function analysis in mice has identified homeobox genes along with other transcription and secreted factors as crucial regulators of inner ear morphogenesis and development. While otic induction seems dependent upon fibroblast growth factors, morphogenesis of the otic vesicle into the distinct vestibular and auditory components appears to be clearly dependent upon the activities of a number of homeobox transcription factors. The Pax2 paired-homeobox gene is crucial for the specification of the ventral otic vesicle derived auditory structures and the Dlx5 and Dlx6 homeobox genes play a major role in specification of the dorsally derived vestibular structures. Some Micro RNAs have also been recently identified which play a crucial role in the inner ear formation.