Reliability of human cortical organoid generation.

The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

Low- and High-Grade Glioma-Associated Vascular Cells Differentially Regulate Tumor Growth.

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of protumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express protumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wild-type (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to antiangiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and antiangiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo cotransplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wild-type HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFß1-GPM6A signaling.

Valproic acid targets IDH1 mutants through alteration of lipid metabolism.

Histone deacetylases (HDACs) have a wide range of targets and can rewire both the chromatin and lipidome of cancer cells. In this study, we show that valproic acid (VPA), a brain penetrant anti-seizure medication and histone deacetylase inhibitor, inhibits the growth of IDH1 mutant tumors in vivo and in vitro, with at least some selectivity over IDH1 wild-type tumors. Surprisingly, genes upregulated by VPA showed no enhanced chromatin accessibility at the promoter, but there was a correlation between VPA-downregulated genes and diminished promoter chromatin accessibility. VPA inhibited the transcription of lipogenic genes and these lipogenic genes showed significant decreases in promoter chromatin accessibility only in the IDH1 MT glioma cell lines tested. VPA inhibited the mTOR pathway and a key lipogenic gene, fatty acid synthase (FASN). Both VPA and a selective FASN inhibitor TVB-2640 rewired the lipidome and promoted apoptosis in an IDH1 MT but not in an IDH1 WT glioma cell line. We further find that HDACs are involved in the regulation of lipogenic genes and HDAC6 is particularly important for the regulation of FASN in IDH1 MT glioma. Finally, we show that FASN knockdown alone and VPA in combination with FASN knockdown significantly improved the survival of mice in an IDH1 MT primary orthotopic xenograft model in vivo. We conclude that targeting fatty acid metabolism through HDAC inhibition and/or FASN inhibition may be a novel therapeutic opportunity in IDH1 mutant gliomas.

Low- and high-grade glioma endothelial cells differentially regulate tumor growth.

Background: A key feature distinguishing high-grade glioma (HGG) from low-grade glioma (LGG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor endothelial cells (TEC) from HGG are molecularly and functionally distinct from normal brain EC and secrete higher levels of pro-tumorigenic factors that promote glioma growth and progression. However, it remains unclear whether TEC from LGG also express pro-tumorigenic factors, and to what extent they functionally contribute to glioma growth. Methods: Transcriptomic profiling was conducted on tumor endothelial cells (TEC) from grade II/III (LGG, IDH-mutant) and grade IV HGG (IDH-wildtype). Functional differences between LGG- and HGG-TEC were evaluated using growth assays, resistance to anti-angiogenic drugs and radiation therapy. Conditioned media and specific factors from LGG- and HGG-TEC were tested on patient-derived gliomasphere lines using growth assays in vitro and in co-transplantation studies in vivo in orthotopic xenograft models. Results: LGG-TEC showed enrichment of extracellular matrix and cell cycle-related gene sets and sensitivity to anti-angiogenic therapy whereas HGG-TEC displayed an increase in immune response-related gene sets and anti-angiogenic resistance. LGG- and HGG-TEC displayed opposing effects on growth and proliferation of IDH-wildtype and mutant tumor cells. Asporin (ASPN), a small leucine rich proteoglycan enriched in LGG-TEC was identified as a growth suppressor of IDH-wildtype GBM by modulating TGFΒ1-GPM6A signaling. Conclusions: Our findings indicate that TEC from LGG and HGG are molecularly and functionally heterogeneous and differentially regulate the growth of IDH-wildtype and mutant tumors.

HDAC1 and HDAC6 are essential for driving growth in IDH1 mutant glioma.

Low-grade and secondary high-grade gliomas frequently contain mutations in the IDH1 or IDH2 metabolic enzymes that are hypothesized to drive tumorigenesis by inhibiting many of the chromatin-regulating enzymes that regulate DNA structure. Histone deacetylase inhibitors are promising anti-cancer agents and have already been used in clinical trials. However, a clear understanding of their mechanism or gene targets is lacking. In this study, the authors genetically dissect patient-derived IDH1 mutant cultures to determine which HDAC enzymes drive growth in IDH1 mutant gliomas. A panel of patient-derived gliomasphere cell lines (2 IDH1 mutant lines, 3 IDH1 wildtype lines) were subjected to a drug-screen of epigenetic modifying drugs from different epigenetic classes. The effect of LBH (panobinostat) on gene expression and chromatin structure was tested on patient-derived IDH1 mutant lines. The role of each of the highly expressed HDAC enzymes was molecularly dissected using lentiviral RNA interference knock-down vectors and a patient-derived IDH1 mutant in vitro model of glioblastoma (HK252). These results were then confirmed in an in vivo xenotransplant model (BT-142). The IDH1 mutation leads to gene down-regulation, DNA hypermethylation, increased DNA accessibility and H3K27 hypo-acetylation in two distinct IDH1 mutant over-expression models. The drug screen identified histone deacetylase inhibitors (HDACi) and panobinostat (LBH) more specifically as the most selective compounds to inhibit growth in IDH1 mutant glioma lines. Of the eleven annotated HDAC enzymes (HDAC1-11) only six are expressed in IDH1 mutant glioma tissue samples and patient-derived gliomasphere lines (HDAC1-4, HDAC6, and HDAC9). Lentiviral knock-down experiments revealed that HDAC1 and HDAC6 are the most consistently essential for growth both in vitro and in vivo and target very different gene modules. Knock-down of HDAC1 or HDAC6 in vivo led to a more circumscribed less invasive tumor. The gene dysregulation induced by the IDH1 mutation is wide-spread and only partially reversible by direct IDH1 inhibition. This study identifies HDAC1 and HDAC6 as important and drug-targetable enzymes that are necessary for growth and invasiveness in IDH1 mutant gliomas.

TGFß superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor ß (TGFß) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFß-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Diet induced obesity alters muscle spindle afferent function in adult mice.

Populations with obesity are more likely to fall and exhibit balance instability. The reason for this is likely multifactorial, but there is some evidence that sensory function is impaired during obesity. We tested the hypothesis that muscle proprioceptor function is compromised in a mouse model of diet induced obesity. An in vitro muscle-nerve preparation was used to record muscle spindle afferent responses to physiological stretch and sinusoidal vibration. We compared the responses of C57/Bl6 male and female mice on a control diet (10% kcal fat) with those eating a high fat diet (HFD; 60% kcal fat) for 10 weeks (final age 14-15 weeks old). Following HFD feeding, adult mice of both sexes exhibited decreased muscle spindle afferent responses to muscle movement. Muscle spindle afferent firing rates during the plateau phase of stretch were significantly lower in both male and female HFD animals as were two measures of dynamic sensitivity (dynamic peak and dynamic index). Muscle spindle afferents in male mice on a HFD were also significantly less likely to entrain to vibration. Due to the importance of muscle spindle afferents to proprioception and motor control, decreased muscle spindle afferent responsiveness may contribute to balance instability during obesity.