Assessments of safety and efficacy are crucial before human ESC (hESC) therapies can move into the clinic. Two important early potential hESC applications are the use of retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration and Stargardt disease, an untreatable form of macular dystrophy that leads to early-onset blindness. Here we show long-term functional rescue using hESC-derived RPE in both the RCS rat and Elov14 mouse, which are animal models of retinal degeneration and Stargardt, respectively. Good Manufacturing Practice-compliant hESC-RPE survived subretinal transplantation in RCS rats for prolonged periods (>220 days). The cells sustained visual function and photoreceptor integrity in a dose-dependent fashion without teratoma formation or untoward pathological reactions. Near-normal functional measurements were recorded at >60 days survival in RCS rats. To further address safety concerns, a Good Laboratory Practice-compliant study was carried out in the NIH III immune-deficient mouse model. Long-term data (spanning the life of the animals) showed no gross or microscopic evidence of teratoma/ tumor formation after subretinal hESC-RPE transplantation. These results suggest that hESCs could serve as a potentially safe and inexhaustible source of RPE for the efficacious treatment of a range of retinal degenerative diseases.

Large-scale musculoskeletal wounds, such as those seen in trauma injuries, present poor functional healing prognoses. In severe trauma, when the native tissue architecture is destroyed or lost, the regenerative capacity of skeletal muscle is diminished by scar formation. Here we demonstrate that a scaffold system composed of fibrin microthreads can provide an efficient delivery system for cell-based therapies and improve regeneration of a large defect in the tibialis anterior of the mouse. Cell-loaded fibrin microthread bundles implanted into a skeletal muscle resection reduced the overall fibroplasia-associated deposition of collagen in the wound bed and promoted in-growth of new muscle tissue. When fibrin microthreads were seeded with adult human cells, implanted cells contributed to the nascent host tissue architecture by forming skeletal muscle fibers, connective tissue, and PAX7-positive cells. Stable engraftment was observed at 10 weeks postimplant and was accompanied by reduced levels of collagen deposition. Taken together, these data support the design and development of a platform for microthread-based delivery of autologous cells that, when coupled to an in vitro cellular reprogramming process, has the potential to improve healing outcomes in large skeletal muscle wounds.

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.

Fertilization in all species studied to date induces an increase in the intracellular concentration of free calcium ions ([Ca 2þ ] i ) within the egg. In mammals, this [Ca 2þ ] i signal is delivered in the form of long-lasting [Ca 2þ ] i oscillations that begin shortly after fusion of the gametes and persist beyond the time of completion of meiosis. While not fully elucidated, recent evidence supports the notion that the sperm delivers into the ooplasm a trigger of oscillations, the so-called sperm factor (SF). The recent discovery that mammalian sperm harbor a specific phospholipase C (PLC), PLCz has consolidated this view. The fertilizing sperm, and presumably PLCz promote Ca 2þ release in eggs via the production of inositol 1,4,5-trisphosphate (IP 3 ), which binds and gates its receptor, the type-1 IP 3 receptor, located on the endoplasmic reticulum, the Ca 2þ store of the cell. Repetitive Ca 2þ release in this manner results in a positive cumulative effect on downstream signaling molecules that are responsible for the completion of all the events comprising egg activation. This review will discuss recent advances in our understanding of how [Ca 2þ ] i oscillations are initiated and regulated in mammals, highlight areas of discrepancies, and emphasize the need to better characterize the downstream molecular cascades that are dependent on [Ca 2þ ] i oscillations and that may impact embryo development.