Gene-Modified Blister Fluid-Derived Mesenchymal Stromal Cells for Treating Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis caused by variants in COL7A1-encoded type VII collagen, a major component of anchoring fibrils. In this study, we developed an ex vivo gene therapy for RDEB using autologous mesenchymal stromal cells (MSCs). On the basis of our previous studies, we first attempted to isolate MSCs from the blister fluid of patients with RDEB and succeeded in obtaining cells with a set of MSC characteristics from all 10 patients. We termed these cells blister fluid-derived MSCs. Blister fluid-derived MSCs were genetically modified and injected into skins of type VII collagen-deficient neonatal mice transplanted onto immunodeficient mice, resulting in continuous and widespread expression of type VII collagen at the dermal-epidermal junction, particularly when administered into blisters. When injected intradermally, the efforts were not successful. The gene-modified blister fluid-derived MSCs could be cultured as cell sheets and applied to the dermis with an efficacy equivalent to that of intrablister administration. In conclusion, we successfully developed a minimally invasive and highly efficient ex vivo gene therapy for RDEB. This study shows the successful application of gene therapy in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions.

The mouse forkhead gene Foxp2 modulates expression of the lung genes.

AIMS: Foxp2 is expressed in the lung during mouse development. A monoclonal anti-mouse Foxp2 antibody was created to determine the expression pattern in the developing lung. Next, transcriptional control of two lung genes, CC10 and surfactant protein C (SPC) genes, by Foxp2 was investigated in H441 and A549 cells. Thirdly, expression patterns of Foxp2 and Foxf2 were compared in the developing lung. Finally, Foxp2 expression was determined in the Foxf2-null mice. MAIN METHODS: Immunohistochemical staining and in situ hybridization were applied to the sections of lungs in the developing embryos. KEY FINDINGS: Monoclonal anti-Foxp2 antibody demonstrated that Foxp2 was expressed in the bronchial epithelium at E10.5 and its expression became restricted to the distal portion of the elongating bronchiolar epithelium and finally to type II alveolar epithelial cells around birth and in the adult. Foxp2 activated the SPC gene promoter in the presence of Nkx2.1 in A549 cells while it repressed the CC10 gene promoter in H441 cells. Next, the expression domains of the Foxp2 and Foxf2 were found to be exclusive in the lung. Finally, the expression of Foxp2 did not change in the lung of Foxf2-null mice. SIGNIFICANCE: The Foxp2 protein is expressed in the growing distal edge of airway epithelium. When the bronchiolus elongates, Foxp2 suppresses CC10 expression. When the lung alveolus is formed, Foxp2 modulates the Nkx2.1-mediated SPC expression in type II alveolar cells. Foxp2 and Foxf2 independently play distinct roles in the alveoli and the mesenchyme, respectively.

Neurogenesin-1 differentially inhibits the osteoblastic differentiation by bone morphogenetic proteins in C2C12 cells.

Bone morphogenetic protein (BMP) antagonists regulate the pleiotropic actions of BMPs by binding to BMPs. We previously isolated the Neurogenesin-1 (Ng1) gene and found that Ng1 protein induces neuronal differentiation in the brain. In this study, we found that Ng1 was expressed in the primordial cells of the skeleton and investigated whether Ng1 protein inhibited the BMP action to induce osteoblastic differentiation in C2C12 myoblasts. Interestingly, Ng1 protein inhibited the BMP7-induced alkaline phosphatase activity while it did not inhibit the BMP2-induced activity. All data suggest that Ng1 protein plays an important role in the embryonic bone formation by differentially regulating BMPs.

Roles of the Foxj1 and Inv genes in the left-right determination of internal organs in mice.

In Foxj1 knockout mice, half show situs solitus while the other half show situs inversus, which means a random determination of the left-right axis. In contrast, the inv mutant mice show a mirror-image configuration of the internal organs, which means a reversal of the left-right axis. Although these two mutant mice have primary cilia on the nodal cells, their phenotypes are different in laterality determination. We thus made Foxj1/inv double mutant mice and analyzed their phenotype. We found the phenotypes of Foxj1/inv double mutant mice to be more similar to those of the Foxj1 mutant mice than those of the inv mutant mice. We also found right pulmonary isomerism to be a major phenotype of the Foxj1 mutant mice and the Foxj1/inv double mutant mice, which is likely due to the absence of the Pitx2 expression at both lateral plate mesoderms. These results indicate that a random signal of laterality (Foxj1) is dominant over the reversal signal of laterality (Inv).

Roles of forkhead transcription factor Foxc2 (MFH-1) and endothelin receptor A in cardiovascular morphogenesis.

OBJECTIVE: Foxc2/MFH-1 is a member of the forkhead family of transcription factors and Foxc2-deficient mice exhibit aortic arch anomalies (type B interruption of the aortic arch). Endothelin receptor type-A (ETA) is one of the two known endothelin receptors that belong to the G-protein-coupled receptor family. ETA-deficient mice show defects in the great arteries, primarily type B interruption of the aortic arch. Based on similar phenotypes in the cardiovascular system of Foxc2- and ETA-deficient mice, we investigated whether Foxc2 and ETA have a close relationship in aortic arch patterning. METHODS: The Foxc2 and ETA homozygotes were obtained by crossing the Foxc2 and ETA heterozygotes, respectively. The double Foxc2/ETA homozygotes were obtained by crossing the double Foxc2/ETA heterozygotes. RESULTS: We investigated the expression of ETA in Foxc2-null mice and the expression of Foxc2 in ETA-null mice and found that the absence of either Foxc2 or ETA had no effect on the expression of the other. Next, we analyzed mice lacking both Foxc2 and ETA to examine the relationship between Foxc2 and ETA on aortic arch patterning in vivo. We found that the majority of Foxc2/ETA double-mutant embryos died around 11.5 dpc and that all surviving mice had persistent truncus arteriosus. CONCLUSIONS: The results suggest that Foxc2- and ETA-expressing cells additively form the aorticopulmonary septum.

Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis.

Lymphatic vessels are essential for the removal of interstitial fluid and prevention of tissue edema. Lymphatic capillaries lack associated mural cells, and collecting lymphatic vessels have valves, which prevent lymph backflow. In lymphedema-distichiasis (LD), lymphatic vessel function fails because of mutations affecting the forkhead transcription factor FOXC2. We report that Foxc2(-/-) mice show abnormal lymphatic vascular patterning, increased pericyte investment of lymphatic vessels, agenesis of valves and lymphatic dysfunction. In addition, an abnormally large proportion of skin lymphatic vessels was covered with smooth muscle cells in individuals with LD and in mice heterozygous for Foxc2 and for the gene encoding lymphatic endothelial receptor, Vegfr3 (also known as Flt4). Our data show that Foxc2 is essential for the morphogenesis of lymphatic valves and the establishment of a pericyte-free lymphatic capillary network and that it cooperates with Vegfr3 in the latter process. Our results indicate that an abnormal interaction between the lymphatic endothelial cells and pericytes, as well as valve defects, underlie the pathogenesis of LD.

Forkhead transcription factor Foxf2 (LUN)-deficient mice exhibit abnormal development of secondary palate.

The forkhead genes encode a transcription factor involved in embryogenesis and pattern formation in multicellular organisms. They are mammalian transcriptional regulators that bind DNA as a monomer through their forkhead domain. The Foxf2 (LUN) mRNA is expressed in the mesenchyme directly adjacent to the ectoderm-derived epithelium in the developing tongue and in the mesenchyme adjacent to the endoderm-derived epithelium in the gastrointestinal (GI) tract, lungs, and genitalia. To investigate the developmental role of the Foxf2 gene during embryogenesis, we disrupted the Foxf2 gene and showed that these mutant mice died shortly after birth. Mice lacking the Foxf2 gene were found to develop cleft palate and an abnormal tongue. In addition, we found that the GI tract and the lungs of Foxf2-deficient newborn mice were normal in both morphology and function. These results suggest that the Foxf2 gene plays key roles in palatogenesis by reshaping the growing tongue.