Reprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells with Elevated Osteogenic Potency by Defined Transcription Factors

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here, we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9+/RUNX2+ cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium, mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro, but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether, we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues.published_or_final_versio

Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration

Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its fluctuating activity at the front edge. This differential RHOA activity determines polarized NC morphology and motility, and is regulated by the asymmetrically localized RhoGAP Deleted in liver cancer (DLC1) in the cytoplasm at the cell front. Importantly, the association of DLC1 with NEDD9 is crucial for its asymmetric localization and differential RHOA activity. Moreover, NC specifiers, SOX9 and SOX10, regulate NEDD9 and DLC1 expression, respectively. These results present a SOX9/SOX10-NEDD9/DLC1-RHOA regulatory axis to govern NCC migratory polarization.published_or_final_versio

The Inhibition Of GSK3β Promotes Oligodendroglial Progenitor Cell Differentiation In Spinal Cord Injury

Poster Session C40: Trauma - Spinal cord injury and regenerationAims: Demyelination is one of the major obstacles for axon regeneration and functional recovery after spinal cord injury (SCI). Previous studies have shown that resident neural progenitor cells in spinal cord can be recruited to produce progenies after injury. Our preliminary study shows that lithium and noggin stimulate oligodendrocyte differentiation of adult spinal cord-derived neural stem/progenitor cells (ASCNPCs). Lithium may exert its effect by inhibiting GSK3ß pathway. Our study aims to examine whether GSK3ß inhibition leads to endogenous oligodendroglial progenitor cell differentiation and remyelination, and results in functional recovery after SCI. Methods: Adult mice were subjected to spinal cord contusion at 8th-10th thoracic level. For neurosphere culture, injured spinal cords were harvested one day post injury (1 d.p.i.). The second passage neurosphere cells were treated with GSK3ß inhibitors ARA-014418 and lithium for seven days in differentiating medium. For in vivo experiments, injured mice were treated with lithium 21 d.p.i. for 14 days, before being sacrificed for myelin measurement. Basso Mouse Scale (BMS) was performed weekly after injury to monitor locomotion function. Results: ARA-014418 and lithium promote both oligodendroglial and neuronal differentiation of ASCNPCs. Injured mice treated with GSK3ß inhibitors exhibit better performance in BMS. GSK3ß inhibitor also significantly decreased the area and volume of demyelination in the dorsal column of contused spinal cord. Conclusions: Both GSK3ß inhibitors ARA-014418 and lithium are effective in promoting oligodendrocyte/neuron differentiation. Injured mice treated with GSK3ß inhibitors demonstrate better functional recovery. Our results indicate that GSK3ß may be a key player in impeding SCI repair

Effects of male accessory sex glands on fertilization, polyspermy and morphometry of early embryos in the golden hamster

Preimplantation embryos sired by hamsters without accessory sex glands (ASG) were found to have a higher mortality rate and a slower cleavage rate than those sired by sham-operated males at 72 h post coitum (p.c.). A time-course study of fertilization in vivo was conducted to determine whether this effect was due to delayed fertilization. Ultrastructural morphometry of 48 h embryos was also undertaken to establish the earliest manifestation of developmental anomalies. Compared to sham-operated controls (SH), ablation of all the ASG (TX), or just the ventral prostate (VPX) or ampullary gland (AGX) had no effect on the timing of sperm penetration, extrusion of the second polar body and pronuclear formation. Females mated with AGX males tended to have more polyspermic embryos (9.7%; p < 0.05). The volumes, volume fractions (V(V)) of the blastomere nuclei, mitochondria and yolk material of the four-cell embryos sired by these same groups of males were assessed using point counting techniques. No difference in the V(V) of yolk and mitochondria could be observed between groups. However, the SH group did have a significantly larger proportion of the cell occupied by the nucleus (p < 0.05), and the TX group had a higher proportion of the nucleus occupied by nucleoli when compared with the SH group (p < 0.01). Smaller nuclei and larger nucleoli in the TX group was interpreted as an early manifestation of a slower division rate of the blastomeres.link_to_subscribed_fulltex

Effects of sperm DNA damage on the levels of RAD51 and p53 proteins in zygotes and 2-cell embryos sired by golden hamsters without the major accessory sex glands

We previously reported that the male accessory sex gland (ASG) secretion is the main source of antioxidants to safeguard sperm genomic integrity and functional competence. Removal of all ASGs in the golden hamster can reduce male fertility by increasing embryo wastage. This study aims to investigate whether the oxidative DNA-damaged sperm from hamsters without all ASGs (TX) could successfully fertilize oocytes and to qualify the status of DNA repair by the expression of RAD51 and p53 proteins. Here we demonstrated a significantly higher DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine) in sperm from TX males than those from sham-operated males. Comet assays demonstrated that all female pronuclei in both zygotes were intact, but single- and double-strand DNA damage was found in decondensed sperm in TX males only. DNA damage could also be detected in both nuclei of the TX 2-cell embryos. RAD51, a DNA repair enzyme, was found to be evenly distributed in the cytoplasm and nuclei in oocytes/zygotes, while at the 2-cell stage, a strong expression of p53 protein and a larger clear perinuclear area without RAD51 expression were found in TX embryos. In conclusion, we demonstrated for the first time DNA damage in decondensed sperm of zygotes and blastomeres of 2-cell stage embryos sired by TX males, resulting in the activation of DNA repair. Sperm DNA damage could induce the increase in p53 expression and the reduction of RAD51 expression in the TX 2-cell stage embryos.link_to_subscribed_fulltex