Neurovascular interactions (NVIs) are critical in establishing vascular patterning, barrier function, and in regulating cerebral blood flow, thereby maintaining neuronal homeostasis and brain health. While developmental programs and neuronal activity play a central role in cerebral cortex angiogenesis during a critical postnatal period, whether cellular heterogeneity causes region-specific angiogenic regulation remains unknown. Here, we combine spatial and endothelial single-cell RNAseq (scRNAseq) transcriptomic analysis to establish a molecular atlas of developing brain regions and a comprehensive list of spatiotemporal-specific NVIs. We find that the thalamic area has a higher vascular density than the other brain regions and our atlas identifies transforming growth factor β ( Tgfβ ) and Vegfc ligands enrichment in this area during a critical postnatal period. Neonatal endothelial Tgfβr1 deletion ( Tgfβr1iEKO ) induces vascular malformations and hemorrhages, primarily in the thalamic area. Mechanistically, we show that loss of endothelial TGFβ signaling increases VEGFC-induced AKT/mTOR signaling in vitro and in vivo , and that mTOR inhibitor Rapamycin efficiently inhibits intracerebral bleeding and vascular defects in Tgfβr1iEKO mice. Altogether, our data suggest that spatiotemporal-specific NVIs control brain vascular heterogeneity, which extends our knowledge of region-specific cerebrovascular development and will benefit our understanding of neurovascular disorders.

Fibrosis is a prominent pathological feature of skeletal muscle in Duchenne muscular dystrophy (DMD). The commonly used disease mouse model, mdx 5cv , displays progressive fibrosis in the diaphragm but not limb muscles. We use single-cell RNA sequencing to determine the cellular expression of the genes involved in extracellular matrix (ECM) production and degradation in the mdx 5cv diaphragm and quadriceps. We find that fibro/adipogenic progenitors (FAPs) are not only the primary source of ECM but also the predominant cells that express important ECM regulatory genes, including Ccn2, Ltbp4, Mmp2, Mmp14, Timp1, Timp2, and Loxs. The effector and regulatory functions are exerted by diverse FAP clusters which are different between diaphragm and quadriceps, indicating their activation by different tissue microenvironments. FAPs are more abundant in diaphragm than in quadriceps. Our findings suggest that the development of anti-fibrotic therapy for DMD should target not only the ECM production but also the pro-fibrogenic regulatory functions of FAPs.
© 2022 The Author(s).

Growth differentiation factor (GDF)‑5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF‑5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF‑5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF‑5 and the effects of GDF‑5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF‑5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF‑5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF‑5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF‑5, as determined by enhanced expression levels of phosphorylated p38 (p‑p38). The GDF‑5‑induced protein expression levels of p‑p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen‑activated protein kinase. These results suggested that GDF‑5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.

The cells present in amniotic fluid (AF) are currently used for prenatal diagnosis of fetal anomalies but are also a potential source of cells for cell therapy. To better characterize putative progenitor cell populations present in AF, we used culture conditions that support self-renewal to determine if these promoted the generation of stable cell lines from AF-derived cells (AFC). Cells isolated from E11.5 mouse were cultured on irradiated STO fibroblast feeder layers in human embryonic germ cell derivation conditions. The cultures grew multicellular epithelial colonies that could be repropagated from single cells. Reverse transcription semiquantitative polymerase chain reaction of established cell lines revealed that they belonged to the extraembryonic endoderm (ExEn) expressing high levels of Gata6, Gata4, Sox17, Foxa2 and Sox7 mRNA. Hierarchical clustering based on the whole transcriptome expression profile of the AFC lines (AFCL) shows significant correlation between transcription profiles of AFCL and blastocyst-derived XEN, an ExEn cell line. In vitro differentiation of AFCL results in the generation of cells expressing albumin and α-fetoprotein (AFP), while intramuscular injection of AFCL into immunodeficient mice produced AFP-positive tumors with primitive endodermal appearance. Hence, E11.5 mouse AF contains cells that efficiently produce XEN lines. These AF-derived XEN lines do not spontaneously differentiate into embryonic-type cells but are phenotypically stable and have the capacity for extensive expansion. The lack of requirement for reprogramming factors to turn AF-derived progenitor cells into stable cell lines capable of massive expansion together with the known ability of ExEn to contribute to embryonic tissue suggests that this cell type may be a candidate for banking for cell therapies.
© 2013 S. Karger AG, Basel