It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behavior, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination, and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study, we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15) and sought to determine their gene expression signatures. To identify cell-type populations, we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 17 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene coexpression analysis. We combined this analysis with literature mining, the honey bee protein atlas, and gene ontology analysis to determine cell cluster identity. Of the cell clusters identified, 17 were related to the nervous system and sensory organs, 7 to the fat body, 19 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes, and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single-cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology at the cellular level.

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

AIMS:Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. METHODS AND RESULTS:We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. CONCLUSION:This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

Dupuytren's disease (DD) is a common, progressive fibroproliferative disease affecting the palmar fascia of the hands, causing fingers to irreversibly flex toward the palm with significant loss of function. Surgical treatments are limited; therefore, effective new therapies for DD are urgently required. To identify the key cellular and molecular pathways driving DD, we employed single-cell RNA sequencing, profiling the transcriptomes of 35,250 human single cells from DD, nonpathogenic fascia, and healthy dermis. We identify a DD-specific population of pathogenic PDPN+/FAP+ mesenchymal cells displaying an elevated expression of fibrillar collagens and profibrogenic genes. In silico trajectory analysis reveals resident fibroblasts to be the source of this pathogenic population. To resolve the processes governing DD progression, genes differentially expressed during fibroblast differentiation were identified, including upregulated TNFRSF12A and transcription factor SCX. Knockdown of SCX and blockade of TNFRSF12A inhibited the proliferation and altered the profibrotic gene expression of cultured human FAP+ mesenchymal cells, demonstrating a functional role for these genes in DD. The power of single-cell RNA sequencing is utilized to identify the major pathogenic mesenchymal subpopulations driving DD and the key molecular pathways regulating the DD-specific myofibroblast phenotype. Using this precision medicine approach, inhibition of TNFRSF12A has shown potential clinical utility in the treatment of DD.

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.

Hematopoietic stem and progenitor cells (HSPCs) develop in distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates some of these processes; however, it has proven difficult to generate functional hematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we explored single-cell RNA sequencing (scRNAseq) in combination with single-cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naive progenitors and erythroid-, megakaryocyte-, and leukocyte-committed progenitors, and we identified CD44, CD326, ICAM2/CD9, and CD18, respectively, as markers of these progenitors. Using an artificial neural network that we trained on scRNAseq derived from human fetal liver, we identified a wide range of hPSC-derived HSPCs phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded, and was completely missing in the data set that had been generated using cells selected on the basis of CD43 expression. By comparing the single-cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver, we identified transcription factors and molecular pathways that can be explored in the future to improve the in vitro production of HSCs.

Aims Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. Methods and results A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. Conclusion A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.

Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.

Aims A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. Methods and results An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreER(T2)-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. Conclusion We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs), which collects peritoneal contaminants and provides a first layer of immunological defence within the abdomen. Using single-cell RNA sequencing and spatial analysis of omental stromal cells, we reveal that the surface of FALCs are covered with specialised mesothelial cells, which we name FALC cover cells. We demonstrate that CXCL1 is expressed by FALC cover cells and that CXCL1 is critical for the retention and accumulation of neutrophils within FALCs during peritonitis. We show that protein arginine deiminase 4 mediates the formation of dense neutrophil aggregates, which are required for the neutralisation of particles present in the peritoneal cavity. Finally, we provide confirmatory evidence in humans with acute appendicitis, that the omentum is also a site of neutrophil recruitment and bacterial capture, and is thus an important component of the immunological defence against the propagation of peritoneal contaminants.

Aims: A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets. Methods: MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed in vitro following siRNA gene silencing. Results: EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts ( P <0.0001). Ten transcriptionally discrete EC clusters were defined following scRNAseq with 3 clusters predominantly composed of cells from the MI group, indicating their gene expression profiles may be relevant to neovasculogenic pathways. We selected plasmalemma vesicle associated protein (Plvap) for further study and confirmed EC-specific increased Plvap expression in ischemic border regions of human ( P =0.002) and mouse ( P =0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation ( P = 0.0038), strong evidence that Plvap can directly modulate EC function. Conclusions: Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

BACKGROUND:The delivery of alternative myogenic cell sources to enhance the efficacy of minced muscle grafts (MG) for the treatment of volumetric muscle loss (VML) injuries is a promising strategy to overcome the demand on muscle-derived donor tissue that currently limits the translation of this therapy.METHODS:Using a rat model of VML, bone marrow mononuclear cells (BMNCs) were evaluated for their ability to directly contribute to de novo muscle fiber regeneration by transplanting MG in a collagen carrier at a dose of 50% of the VML injury both with and without concomitant delivery of 5 million BMNCs derived via density gradient centrifugation from the bone marrow of a syngeneic green fluorescent protein (GFP)+ donor.RESULTS:Histological, molecular, and functional analyses revealed that BMNCs can engraft with co-delivered MG and contribute to nascent myofiber, but do so at a low magnitude without resulting in significant changes to transcription of key myogenic genes or gains in whole muscle force generation relative to MG alone.CONCLUSION:As such, co-delivery of BMNCs with MG is a promising treatment paradigm to VML that will require further investigation to identify the phenotype and therapeutic dosing of the bone marrow-derived cell populations which engraft most efficiently.

Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration.

Although skeletal muscle has a remarkable ability to repair/regenerate after most types of injuries, there is limited regeneration after volumetric muscle loss (VML). A number of scaffold materials have been used in the development of grafts to treat VML, however, there is still a need to better understand the most appropriate material with regards to its ability to maintain mechanical integrity while also supporting myogenesis. Five commonly used natural polymeric materials (Collagen I, Agarose, Alginate, Fibrin, and Collagen Chitosan) used in skeletal muscle tissue engineering grafts were evaluated for their mechanical properties and myogenic capacity. Rheological properties, water absorption rates, degradation stability, tensile characteristics, and the ability to support in vitro myogenesis were compared in all five materials. Collagen, Collagen Chitosan, and Fibrin demonstrated high elasticity and 100% stretch without failure, Agarose was the most brittle (20% max stretch), and Alginate demonstrated poor handleabilty. While Collagen was supportive of myogenesis, overall, Fibrin demonstrated the highest myogenic potential as indicated by the earliest and highest increases in myogenin and myosin heavy chain mRNA in satellite cells along with the most extensive myotube development as evaluated with immunohistochemistry. The findings herein support the notion that under the conditions used in this study, Fibrin is the most suitable scaffold for the development of scaffolds for skeletal muscle tissue engineering. Future studies are required to determine whether the differences in mechanical properties and myogenic potential observed in vitro in the current study translate to better skeletal muscle development in a VML injury model. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 672-679, 2018.

The immune response plays a key role in healing of bone and muscle injuries. An unrestrained proinflammatory response generally results in impaired healing. Extremity fracture is often complicated by traumatic volumetric muscle loss (VML), which can lead to delayed or non-union of the fracture (i.e., impaired healing). In a rat model, we recently showed that the VML injury of the tibialis anterior muscle prevented healing of the adjacent tibia fracture and induced a robust proinflammatory response in the musculature early post-injury that was not observed with the fracture alone. In the current studies, we show that a fracture treated with rhBMP-2, a highly osteoinductive protein, was impaired by the VML injury and corresponded with a heightened proinflammatory response. We utilized an autologous minced muscle graft (MMG) to repair the VML which, interestingly, reduced the proinflammatory response. Gene transcript analysis of the muscle injury indicates that MMG repair results in decreased gene expression of proinflammatory mediators including ITGAM, IL18, and CCL12, compared to non-repaired muscle. In the bone defect, MMG repair also induced a modest proinflammatory response with lower expression of IFNG and TNF than the non-repaired group. The restrained proinflammatory response corresponded with restored healing of the bone fracture and, remarkably, partial restoration of muscle function which does not normally occur clinically. Altogether, data suggest that MMG repair controls the proinflammatory response in a manner conducive to improved healing responses in both muscle and bone. These data reveal that immunomodulation to dampen proinflammatory responses could provide an effective therapy for musculoskeletal trauma.

The deleterious impact of concomitant muscle injury on fracture healing and limb function is commonly considered part of the natural sequela of orthopedic trauma. Recent reports suggest that heightened inflammation in the surrounding traumatized musculature is a primary determinant of fracture healing. Relatedly, there are emerging potential therapeutic approaches for severe muscle trauma (e.g., volumetric muscle loss [VML] injury), such as autologous minced muscle grafts (1 mm3 pieces of muscle; GRAFT), that can partially prevent chronic functional deficits and appear to have an immunomodulatory effect within VML injured muscle. The primary goal of this study was to determine if repair of VML injury with GRAFT rescues impaired fracture healing and improves the strength of the traumatized muscle in a male Lewis rat model of tibia open fracture. The most salient findings of the study were: (1) tibialis anterior (TA) muscle repair with GRAFT improved endogenous healing of fractured tibia and improved the functional outcome of muscle regeneration; (2) GRAFT repair attenuated the monocyte/macrophage (CD45+CDllb+) and T lymphocyte (CD3+) response to VML injury; (3) TA muscle protein concentrations of MCP1, IL-10, and IGF-1 were augmented in a proregenerative manner by GRAFT repair; (4) VML injury concomitant with osteotomy induced a heightened systemic presence of alarmins (e.g., soluble RAGE) and leukocytes (e.g., monocytes), and depressed IGF-1 concentration, which GRAFT repair ameliorated. Collectively, these data indicate that repair of VML injury with a regenerative therapy can modulate the inflammatory and regenerative phenotype of the treated muscle and in association improve musculoskeletal healing.