While platelets are the cellular mediators of thrombosis, platelets are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered as bone marrow (BM) resident cells. However, platelet producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared to BM Mks. We therefore sought to define the immune functions of lung Mks. Using single cell RNA-Seq of BM and lung myeloid enriched cells, we found that lung Mks (MkL) had gene expression patterns that are similar to antigen presenting cells (APC). This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment as evidenced by BM Mks having a MkL like phenotype under the influence of pathogen receptor challenge and lung associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4 + T cell activation in a MHC II dependent manner both in vitro and in vivo. These data indicated that Mks in the lung had key immune regulatory roles dictated in part by the tissue environment.

The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(e-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffoldmediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(e-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.biomaterials | regenerative medicine | genetic engineering | gene therapy | chondrocyte

Mesenchymal stem/stromal cells (MSCs) provide an attractive cell source for cartilage repair and cell therapy; however, the underlying molecular pathways that drive chondrogenesis of these populations of adult stem cells remain poorly understood. We generated a rich data set of high-throughput RNA sequencing of human MSCs throughout chondrogenesis at 6 different time points. Our data consisted of 18 libraries with 3 individual donors as biologic replicates, with each library possessing a sequencing depth of 100 million reads. Computational analyses with differential gene expression, gene ontology, and weighted gene correlation network analysis identified dynamic changes in multiple biologic pathways and, most importantly, a chondrogenic gene subset, whose functional characterization promises to further harness the potential of MSCs for cartilage tissue engineering. Furthermore, we created a graphic user interface encyclopedia built with the goal of producing an open resource of transcriptomic regulation for additional data mining and pathway analysis of the process of MSC chondrogenesis.-Huynh, N. P. T., Zhang, B., Guilak, F. High-depth transcriptomic profiling reveals the temporal gene signature of human mesenchymal stem cells during chondrogenesis.

The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori, or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration.

Normal skeletal development requires tight coordination of transcriptional networks, signaling pathways, and biomechanical cues, and many of these pathways are dysregulated in pathological conditions affecting cartilage and bone. Recently, a significant role has been identified for long noncoding RNAs (IncRNAs) in developing and maintaining cellular phenotypes, and improvements in sequencing technologies have led to the identification of thousands of IncRNAs across diverse cell types, including the cells within cartilage and bone. It is clear that IncRNAs play critical roles in regulating gene expression. For example, they can function as epigenetic regulators in the nucleus via chromatin modulation to control gene transcription, or in the cytoplasm, where they can function as scaffolds for protein-binding partners or modulate the activity of other coding and noncoding RNAs. In this review, we discuss the growing list of IncRNAs involved in normal development and/or homeostasis of the skeletal system, the potential mechanisms by which these IncRNAs might function, and recent improvements in the methodologies available to study IncRNA functions in vitro and in vivo. Finally, we address the likely utility of IncRNAs as biomarkers and therapeutic targets for diseases of the skeletal system, including osteoarthritis, osteoporosis, and in cancers of the skeletal system.

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-β3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-β3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.

Astrocytic differentiation is developmentally impaired in patients with childhood-onset schizophrenia (SCZ). To determine why, we used genetic gain- and loss-of-function studies to establish the contributions of differentially-expressed transcriptional regulators to the defective differentiation of glial progenitor cells (GPCs) produced from SCZ patient-derived iPSCs. Negative regulators of the BMP pathway were upregulated in SCZ GPCs, including BAMBI, FST and GREM1, whose over-expression retained SCZ GPCs at the progenitor stage. SMAD4 knock-down suppressed the production of these BMP inhibitors by SCZ GPCs, and rescued normal astrocytic differentiation. In addition, the BMP-regulated transcriptional repressor REST was upregulated in SCZ GPCs, and its knock-down similarly restored normal glial differentiation. REST knockdown also rescued potassium-transport associated gene expression and K+ uptake, which were otherwise deficient in SCZ glia. These data suggest that the glial differentiation defect in childhood-onset SCZ, and its attendant disruption in K+ homeostasis, may be rescued by targeting BMP/SMAD4- and REST-dependent transcription.

The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori, or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration.