Bioprinting is an emerging technique for the fabrication of living tissues that allows cells to be arranged in predetermined three-dimensional (3D) architectures. However, to date, there are limited examples of bioprinted constructs containing multiple cell types patterned at high-resolution. Here we present a low-cost process that employs 3D printing of aqueous droplets containing mammalian cells to produce robust, patterned constructs in oil, which were reproducibly transferred to culture medium. Human embryonic kidney (HEK) cells and ovine mesenchymal stem cells (oMSCs) were printed at tissue-relevant densities (107 cells mL−1) and a high droplet resolution of 1 nL. High-resolution 3D geometries were printed with features of ≤200 μm; these included an arborised cell junction, a diagonal-plane junction and an osteochondral interface. The printed cells showed high viability (90% on average) and HEK cells within the printed structures were shown to proliferate under culture conditions. Significantly, a five-week tissue engineering study demonstrated that printed oMSCs could be differentiated down the chondrogenic lineage to generate cartilage-like structures containing type II collagen.

Diffuse gliomas represent a heterogeneous group of universally lethal brain tumors characterized by minimally effective genotype-targeted therapies. Recent advances have revealed that a remarkable level of genetic, epigenetic, and environmental heterogeneity exists within each individual glioma. Together, these interconnected layers of intratumoral heterogeneity result in extreme phenotypic heterogeneity at the cellular level, providing for multiple mechanisms of therapeutic resistance and forming a highly adaptable and resilient disease. In this review, we discuss how glioma intratumoral heterogeneity and malignant cellular state plasticity drive resistance to existing therapies and look to a future in which these challenges may be overcome. Significance: Glioma intratumoral heterogeneity and malignant cell state plasticity represent formidable hurdles to the development of novel targeted therapies. However, the convergence of genotypically diverse glioma cells into a limited set of epigenetically encoded transcriptional cell states may present an opportunity for a novel therapeutic strategy we call “State Selective Lethality.” In this approach, cellular states (as opposed to genetic perturbations/mutations) are the subject of therapeutic targeting, and plasticity-mediated resistance is minimized through the design of cell state “trapping agents.”

Tumor microenvironment is critical for the maintenance of cellular states found in primary glioblastomas.

Postnatal subventricular zone (SVZ) neural stem cells generate forebrain glia, namely astrocytes and oligodendrocytes. The cues necessary for this process are unclear, despite this phase of brain development being pivotal in forebrain gliogenesis. Galectin‐3 (Gal‐3) is increased in multiple brain pathologies and thereby regulates astrocyte proliferation and inflammation in injury. To study the function of Gal‐3 in inflammation and gliogenesis, we carried out functional studies in mouse. We overexpressed Gal‐3 with electroporation and using immunohistochemistry surprisingly found no inflammation in the healthy postnatal SVZ. This allowed investigation of inflammation‐independent effects of Gal‐3 on gliogenesis. Loss of Gal‐3 function via knockdown or conditional knockout reduced gliogenesis, whereas Gal‐3 overexpression increased it. Gal‐3 overexpression also increased the percentage of striatal astrocytes generated by the SVZ but decreased the percentage of oligodendrocytes. These novel findings were further elaborated with multiple analyses demonstrating that Gal‐3 binds to the bone morphogenetic protein receptor one alpha (BMPR1α) and increases bone morphogenetic protein (BMP) signaling. Conditional knockout of BMPR1α abolished the effect of Gal‐3 overexpression on gliogenesis. Gain‐of‐function of Gal‐3 is relevant in pathological conditions involving the human forebrain, which is particularly vulnerable to hypoxia/ischemia during perinatal gliogenesis. Hypoxic/ischemic injury induces astrogliosis, inflammation and cell death. We show that Gal‐3 immunoreactivity was increased in the perinatal human SVZ and striatum after hypoxia/ischemia. Our findings thus show a novel inflammation‐independent function for Gal‐3; it is necessary for gliogenesis and when increased in expression can induce astrogenesis via BMP signaling.

The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase (Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.