Discovery of Muscle-Tendon Progenitor Subpopulation in Human Myotendinous Junction at Single-Cell Resolution.

The myotendinous junction (MTJ) is a complex and special anatomical area that connects muscles and tendons, and it is also the key to repairing tendons. Nevertheless, the anatomical structure and connection structure of MTJ, the cluster and distribution of cells, and which cells are involved in repairing the tissue are still unclear. Here, we analyzed the cell subtype distribution and function of human MTJ at single-cell level. We identified four main subtypes, including stem cell, muscle, tendon, and muscle-tendon progenitor cells (MTP). The MTP subpopulation, which remains the characteristics of stem cells and also expresses muscle and tendon marker genes simultaneously, may have the potential for bidirectional differentiation. We also found the muscle-tendon progenitor cells were distributed in the shape of a transparent goblet; muscle cells first connect to the MTP and then to the tendon. And after being transplanted in the MTJ injury model, MTP exhibited strong regenerative capability. Finally, we also demonstrated the importance of mTOR signaling for MTP maintenance by in vitro addition of rapamycin and in vivo validation using mTOR-ko mice. Our research conducted a comprehensive analysis of the heterogeneity of myotendinous junction, discovered a special cluster called MTP, provided new insights into the biological significance of myotendinous junction, and laid the foundation for future research on myotendinous junction regeneration and restoration.

A Cd9+Cd271+ stem/progenitor population and the SHP2 pathway contribute to neonatal-to-adult switching that regulates tendon maturation.

Tendon maturation lays the foundation for postnatal tendon development, its proper mechanical function, and regeneration, but the critical cell populations and the entangled mechanisms remain poorly understood. Here, by integrating the structural, mechanical, and molecular properties, we show that post-natal days 7-14 are the crucial transitional stage for mouse tendon maturation. We decode the cellular and molecular regulatory networks at the single-cell level. We find that a nerve growth factor (NGF)-secreting Cd9+Cd271+ tendon stem/progenitor cell population mainly prompts conversion from neonate to adult tendon. Through single-cell gene regulatory network analysis, in vitro inhibitor identification, and in vivo tendon-specific Shp2 deletion, we find that SHP2 signaling is a regulator for tendon maturation. Our research comprehensively reveals the dynamic cell population transition during tendon maturation, implementing insights into the critical roles of the maturation-related stem cell population and SHP2 signaling pathway during tendon differentiation and regeneration.

Systemic and single cell level responses to 1 nm size biomaterials demonstrate distinct biological effects revealed by multi-omics atlas.

Although ultra-small nanoclusters (USNCs, < 2 nm) have immense application capabilities in biomedicine, the investigation on body-wide organ responses towards USNCs is scant. Here, applying a novel strategy of single-cell mass cytometry combined with Nano Genome Atlas of multi-tissues, we systematically evaluate the interactions between the host and calcium phosphate (CaP) USNCs at the organism level. Combining single-cell mass cytometry, and magnetic luminex assay results, we identify dynamic immune responses to CaP USNCs at the single cell resolution. The innate immune is initially activated and followed by adaptive immune activation, as evidenced by dynamic immune cells proportions. Furthermore, using Nano Genome Atlas of multi-tissues, we uncover CaP USNCs induce stronger activation of the immune responses in the cartilage and subchondral bone among the five local tissues while promote metabolic activities in the liver and kidney. Moreover, based on the immunological response profiles, histological evaluation of major organs and local tissue, and a body-wide transcriptomics, we demonstrate that CaP USNCs are not more hazardous than the Food and Drug Administration-approved CaP nanoparticles after 14 days of injection. Our findings provide valuable information on the future clinical applications of USNCs and introduce an innovative strategy to decipher the whole body response to implants.

Potential applications of deep learning in single-cell RNA sequencing analysis for cell therapy and regenerative medicine.

When used in cell therapy and regenerative medicine strategies, stem cells have potential to treat many previously incurable diseases. However, current application methods using stem cells are underdeveloped, as these cells are used directly regardless of their culture medium and subgroup. For example, when using mesenchymal stem cells (MSCs) in cell therapy, researchers do not consider their source and culture method nor their application angle and function (soft tissue regeneration, hard tissue regeneration, suppression of immune function, or promotion of immune function). By combining machine learning methods (such as deep learning) with data sets obtained through single-cell RNA sequencing (scRNA-seq) technology, we can discover the hidden structure of these cells, predict their effects more accurately, and effectively use subpopulations with differentiation potential for stem cell therapy. scRNA-seq technology has changed the study of transcription, because it can express single-cell genes with single-cell anatomical resolution. However, this powerful technology is sensitive to biological and technical noise. The subsequent data analysis can be computationally difficult for a variety of reasons, such as denoising single cell data, reducing dimensionality, imputing missing values, and accounting for the zero-inflated nature. In this review, we discussed how deep learning methods combined with scRNA-seq data for research, how to interpret scRNA-seq data in more depth, improve the follow-up analysis of stem cells, identify potential subgroups, and promote the implementation of cell therapy and regenerative medicine measures.

Single-cell RNA-seq reveals novel mitochondria-related musculoskeletal cell populations during adult axolotl limb regeneration process.

While the capacity to regenerate tissues or limbs is limited in mammals, including humans, axolotls are able to regrow entire limbs and major organs after incurring a wound. The wound blastema has been extensively studied in limb regeneration. However, due to the inadequate characterization of ECM and cell subpopulations involved in the regeneration process, the discovery of the key drivers for human limb regeneration remains unknown. In this study, we applied large-scale single-cell RNA sequencing to classify cells throughout the adult axolotl limb regeneration process, uncovering a novel regeneration-specific mitochondria-related cluster supporting regeneration through energy providing and the ECM secretion (COL2+) cluster contributing to regeneration through cell-cell interactions signals. We also discovered the dedifferentiation and re-differentiation of the COL1+/COL2+ cellular subpopulation and exposed a COL2-mitochondria subcluster supporting the musculoskeletal system regeneration. On the basis of these findings, we reconstructed the dynamic single-cell transcriptome of adult axolotl limb regenerative process, and identified the novel regenerative mitochondria-related musculoskeletal populations, which yielded deeper insights into the crucial interactions between cell clusters within the regenerative microenvironment.

Atlas of Musculoskeletal Stem Cells with the Soft and Hard Tissue Differentiation Architecture.

Although being of utmost importance for human health and mobility, stem cell identity and hierarchical organization of musculoskeletal progenitors remain largely unexplored. Here, cells from E10.5, E12.5, and E15.5 murine limbs are analyzed by high throughput single-cell RNA sequencing to illustrate the cellular architecture during limb development. Single-cell transcriptional profiling demonstrates the identity and differentiation architecture of musculoskeletal stem cells (MSSC), soft and hard tissue progenitors through expression pattern of musculoskeletal markers (scleraxis [Scx], Hoxd13, Sox9, and Col1a1). This is confirmed by genetic in vivo lineage tracing. Moreover, single-cell analyses of Scx knockout mice tissues illustrates that Scx regulates MSSC self-renewal and proliferation potential. A high-throughput and low-cost multi-tissues RNA sequencing strategy further provides evidence that musculoskeletal system tissues, including muscle, bone, meniscus, and cartilage, are all abnormally developed in Scx knockout mice. These results establish the presence of an indispensable limb Scx+Hoxd13+ MSSC population and their differentiation into soft tissue progenitors (Scx+Col1a1+) and hard tissue progenitors (Scx+Sox9+). Collectively, this study paves the way for systematically decoding the complex molecular mechanisms and cellular programs of musculoskeletal tissues morphogenesis in limb development and regeneration.

Development and characterization of 20 polymorphic microsatellite loci in the deep sea squat lobster, Munida isos Ahyong and Poore, 2004 and cross-amplification in two congeneric species.

Munida isos is a deep sea squat lobster species that is widely distributed across the New Zealand and east Australian region, and is often associated with deep sea vulnerable marine ecosystems. To investigate its population genetic structure and patterns of regional connectivity, microsatellite loci were developed for M. isos from two genomic libraries using the Illumina HiSeq 2500 sequencing platform. Twenty-six loci amplified consistently in M. isos from the Tasman Sea, among which 20 were polymorphic and selectively neutral. Evidence of null alleles was observed at eight loci. Most loci exhibited moderate to high levels of polymorphism, with an average polymorphic information content value of 0.482. The mean number of alleles per locus was 7.45, with a mean expected heterozygosity of 0.520. Thirteen loci exhibited significant deviation from Hardy-Weinberg equilibrium, while only one locus pair was in linkage disequilibrium after false discovery rate correction for multiple testing (P < 0.05). Cross-species amplification tests revealed that the transferability of 14 loci (70%) was positive for the two congeners M. endeavourae and M. gracilis. The accessibility to new polymorphic microsatellite loci will facilitate population genetic studies and aid in developing conservation and management strategies for vulnerable marine ecosystems.

Phylogeography of the threatened tetraploid fish, Schizothorax waltoni, in the Yarlung Tsangpo River on the southern Qinghai-Tibet Plateau: implications for conservation.

The phylogeography of Schizothorax waltoni, an endemic and endangered tetraploid schizothoracine fish in the Yarlung Tsangpo River (YLTR) on southern margin of Qinghai-Tibet Plateau (QTP), was investigated using two mitochondrial DNA regions and eleven microsatellite loci. Analyses of concatenated sequences of cytochrome b (1141 bp) and the control region (712 bp) revealed high haplotype diversity and moderate nucleotide diversity. High genetic diversity was observed based on microsatellite variation. Both mtDNA and microsatellite analyses revealed significant genetic differentiation between the eastern population (Mainling) and the other four populations to the west, and non-significant genetic differentiation amongst the three central populations in the west. Significant genetic differentiation was observed between the western population (Shigatse) and the three central populations based on microsatellite analyses alone. Bayesian skyline plot analyses showed that S. waltoni experienced a pronounced population expansion 0.05 to 0.10 Ma. Hierarchical structure analyses of microsatellite data indicated that S. waltoni could be split into three groups (western, central and eastern YLTR). The results indicate that three management units should be considered for S. waltoni. Our findings highlight the need for the conservation and effective management of S. waltoni, which is a key member of the endemic and highly threatened fishes of the QTP.

Phylogeography and population genetics of Schizothorax o'connori: strong subdivision in the Yarlung Tsangpo River inferred from mtDNA and microsatellite markers.

The Qinghai-Tibet Plateau (QTP) is a biodiversity hotspot, resulting from its geological history, contemporary environment and isolation. Uplift of the QTP and Quaternary climatic oscillations are hypothesised to have influenced the genetic diversity, population structure and dynamics of all QTP endemic species. In this study, we tested this hypothesis by assaying variation at two mitochondrial DNA regions (cytochrome b and control region) and at 12 microsatellite loci of seven populations of the endemic fish, Schizothorax o'connori from the Yarlung Tsangpo River (YLTR) on the QTP. Analyses revealed one group of six populations to the west, above the Yarlung Tsangpo Grand Canyon (YTGC), and a second group to the east below the YTGC. Estimates of the timing of this east-west split indicate that these groups represent evolutionarily significant units that have evolved separately and rapidly in the middle Pleistocene, at the time of the Kunlun-Huanghe Movement A Phase and the Naynayxungla glaciation. Population dynamic analyses indicate that S. o'connori experienced a pronounced late Pleistocene expansion during the last interglacial period. The results of this study support the hypotheses that the QTP uplift and Quaternary climatic oscillations have played important roles in shaping the population genetics and dynamics of this endemic fish.