Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

Purpose To elucidate the subclinical anatomy of retinopathy of prematurity (ROP) using spectral domain optical coherence tomography (SD OCT). Design Prospective, observational case series. Participants Three low-birth-weight, severely premature infants. Methods Clinical examination was performed using a portable slit lamp and indirect ophthalmoscope. Imaging was performed by using a handheld SD OCT device and Retcam (Clarity Medical Systems, Pleasanton, CA) or video-indirect recording. Spectral domain optical coherence tomography imaging was conducted without sedation at the bedside in the neonatal intensive care unit on 1 patient. The other 2 patients had an examination under anesthesia with SD OCT imaging in the operating room. Main Outcome Measures In vivo determination of vitreoretinal morphology, anatomy, and pathology by clinical examination, imaging, and SD OCT. Results Linear and volumetric imaging was achieved with the handheld system in infant eyes despite tunica vasculosa lentis and vitreous bands. Imaging was not possible in eyes with notable vitreous hemorrhage. Analysis of SD OCT images revealed preretinal structures (ranging from 409 to 2700 μm in width and 212 to 440 μm in height), retinoschisis, and retinal detachment in the posterior pole of patients with advanced ROP. Both the retinoschisis and the preretinal structures were not identified on conventional examination or imaging by expert pediatric ophthalmologists. The preretinal structures varied in location and size, and may represent preretinal fibrovascular proliferation. Some were found in close proximity to blood vessels, whereas others were near the optic nerve. Conclusions Handheld SD OCT imaging can be performed on the sedated or nonsedated neonate and provides valuable subclinical anatomic information. This novel imaging modality can reveal the location and extent of posterior ROP pathology not evident on standard examination. This could affect future clinical decision-making if studies validate a management strategy based on findings from this imaging technique.

A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

Pathological ocular neovascularization, caused by diabetic retinopathy, age-related macular degeneration, or retinopathy of prematurity, is a leading cause of blindness, yet much remains to be learned about its underlying causes. Here we used oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) to assess the contribution of the metalloprotease-disintegrin ADAM9 to ocular neovascularization in mice. Pathological neovascularization in both the OIR and CNV models was significantly reduced in Adam9 ؊/؊ mice compared to wild-type controls. In addition, the level of ADAM9 expression was strongly increased in endothelial cells in pathological vascular tufts in the OIR model. Moreover, tumor growth from heterotopically injected B16F0 melanoma cells was reduced in Adam9 ؊/؊ mice compared to controls. In cell-based assays, the overexpression of ADAM9 enhanced the ectodomain shedding of EphB4, Tie-2, Flk-1, CD40, VCAM, and VE-cadherin, so the enhanced expression of ADAM9 could potentially affect pathological neovascularization by increasing the shedding of these and other membrane proteins from endothelial cells. Finally, we provide the first evidence for the upregulation of ADAM9-dependent shedding by reactive oxygen species, which in turn are known to play a critical role in OIR. Collectively, these results suggest that ADAM9 could be an attractive target for the prevention of proliferative retinopathies, CNV, and cancer.Ocular neovascularization is one of the leading causes of blindness in humans and is found in diverse eye diseases including diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity (3,4,6). In addition, pathological neovascularization also has critical roles in other diseases such as cancer and rheumatoid arthritis (12,14). Although proteins with crucial functions in pathological neovascularization are considered to be important targets for the treatment of tumor growth (5), proliferative retinopathies (19), and rheumatoid arthritis (12), much remains to be learned about the identity of these molecules and the mechanisms underlying their function. In this study, we focused on the contribution of a disintegrin and metalloprotease, ADAM9, to pathological neovascularization. ADAM9, one of the first ADAM proteins to be identified and characterized, is a membrane-anchored metalloproteinase containing an N-terminal prodomain followed by a metalloprotease domain, a disintegrin domain and cysteine-rich region, an epidermal growth factor (EGF) repeat, a transmembrane domain, and a cytoplasmic tail with potential SH3 ligand domains (25). ADAM9 is catalytically active in both biochemical and cell-based assays and can cleave several membrane proteins including EGF and FGFR2iiib when it is overexpressed together with these substrates (10, 15, 16). In addition, ADAM9 is thought to participate in cell-cell interactions by binding to integrins (13,30). Mice lacking ADAM9 have no evident major abnormalities during development or adult life (24) but show redu...