Impact of adipose-derived stem cells on engineering hair follicle germ-like tissue grafts for hair regenerative medicine.

Hair regenerative medicine has emerged as a promising treatment strategy for severe hair loss, such as end-stage androgenetic alopecia. Various approaches to engineering three-dimensional tissue grafts have been explored since they drive the ability to regenerate hair follicles when transplanted. In the present study, we demonstrated the assembly of human adipose-derived stem cells (hASCs) into hair follicle germ (HFG)-like aggregates for de novo hair regeneration. We mixed human dermal papilla cells (hDPCs), murine embryonic epithelial cells, and hASCs in suspension, and allowed them to form aggregates. During three days of culture, cells initially formed a single aggregate with a random distribution of the three cell types, but the epithelial and dermal papilla cells subsequently separated from each other and formed a dumbbell-shaped HFG, with hASCs localized on the hDPC aggregate side. The involvement of hASCs significantly increased gene expression associated with hair morphogenesis compared to HFGs without hASCs. The self-organization of the three cell types was observed in our scalable lab-made chip device. HFGs containing hASCs efficiently generated hair shafts upon transplantation to nude mice, while only a few shafts were generated with HFGs without hASCs. This approach may be a promising strategy for fabricating tissue grafts for hair regenerative medicine.

Potential role of host effector memory CD8+ T cells in marrow rejection after mixed chimerism induction in cynomolgus monkeys.

Mixed hematopoietic chimerism provides a powerful means of achieving transplantation tolerance. We investigated the efficacy of combined blockade of the CD40/CD154 and CD28/B7 costimulation pathways to induce sustained mixed chimerism in cynomolgus monkeys following major histocompatibility complex-mismatched bone marrow (BM) transplants. A nonmyeloablative conditioning regimen of busulfan, intravenous and intraosseous ifosfamide, and anti-thymocyte globulin was used. BM transplantation was followed by a one-week course of CTLA4-Ig/anti-CD154 monoclonal antibodies. Three recipients achieved a wide range of transient chimerism (10.8-79.8%). A rapid proliferation of host effector memory (CD28(low)CD95(high)) CD8(+) T cells was observed in conditioned animals whether or not they received allogeneic BM, and this expansion occurred concurrently with the loss of chimerism in BM recipients. CD8(+) T cells from the recipients had increased reactivity to donor stimulators vs. third-party stimulators. Additional immunosuppression with tacrolimus or deoxyspergualin after transplantation delayed post-transplant proliferation of effector memory CD8(+) T cells but did not promote chimerism. A one-month course of costimulatory blockade also did not prevent marrow rejection. These studies demonstrate that combined CD40/CD154 and CD28/B7 costimulatory blockade supports transient mixed chimerism induction following nonmyeloablative conditioning in primates, but is insufficient to overcome host immune resistance likely mediated by effector memory CD8(+) T cells.

Spontaneous Basal cell carcinoma of the submandibular gland in a rat.

At necropsy, a white nodule (about 5 × 3 mm in size) was observed in the right submandibular gland of a 10-week-old female GALAS rat. Histopathologically, oval to spindle-shaped and pale basophilic tumor cells proliferated closely, and formed variably sized foci. The nodule partially spread into or invaded the surrounding normal tissue, and necrotic foci were recognized in the tumor. Immunohistochemically, the nuclei of the tumor cells showed a diffusely positive reaction for p63, and the cytoplasm showed a diffusely positive reaction for cytokeratin and negative reaction for αSMA, vimentin, desmin and S-100. Many tumor cells were positive for PCNA. Ultrastructurally, the tumor cells contained many tonofilaments in the cytoplasm and a few desmosomes at the intercellular portion. Based on these findings, the tumor was diagnosed as a basal cell carcinoma originating from the duct in the rat submandibular gland.

Effects of methylphenidate hydrochloride on the cardiovascular system in vivo and in vitro: a safety pharmacology study.

INTRODUCTION: : We examined the effects of methylphenidate hydrochloride (MPH) on the cardiovascular system using in vivo and in vitro study methods in accordance with the ICH-S7B guideline. METHODS: MPH was orally administered at doses of 3, 10 and 30 mg/kg to unrestrained conscious dogs implanted with a telemetry transmitter and attached with body surface electrodes, and electrocardiogram (ECG) leads. The QTcF interval was determined while heart rate (HR), and blood pressure (BP) were measured. Action potentials in isolated guinea-pig papillary muscle and the rapid component of the delayed rectifier potassium current (I(Kr)) in HEK-293 cells stably transfected with hERG were also investigated at concentrations of 0.1, 0.3 and 1 microg/mL (0.37, 1.1 and 3.7 micromol/L) of MPH. RESULTS: No ECG changes were observed except for a shortening of the QT interval due to a shortening of the RR interval at the maximum dose tested, 30 mg/kg. The only observed change was an elevation of BP in dogs at the dose of 30 mg/kg, which is approximately 10 times higher than the maximum therapeutic dose for use in children with attention deficit hyperactivity disorder (ADHD). Neither APD prolongation nor I(Kr) inhibition was observed by MPH in the in vitro studies up to the maximum concentration tested, 1 microg/mL (3.7 micromol/L), which is approximately 34 times higher than the clinically attainable unbound plasma MPH concentrations in children with ADHD. DISCUSSION: These results suggest that it is unlikely that MPH affects ventricular repolarization processes at the therapeutically recommended dose levels in patients with ADHD.