Transplantation of Human-Fetal-Spinal-Cord-Derived NPCs Primed with a Polyglutamate-Conjugated Rho/Rock Inhibitor in Acute Spinal Cord Injury.

Neural precursor cell (NPC) transplantation represents a promising therapy for treating spinal cord injuries (SCIs); however, despite successful results obtained in preclinical models, the clinical translation of this approach remains challenging due, in part, to the lack of consensus on an optimal cell source for human neuronal cells. Depending on the cell source, additional limitations to NPC-based therapies include high tumorigenic potential, alongside poor graft survival and engraftment into host spinal tissue. We previously demonstrated that NPCs derived from rat fetal spinal cords primed with a polyglutamate (PGA)-conjugated form of the Rho/Rock inhibitor fasudil (PGA-SS-FAS) displayed enhanced neuronal differentiation and graft survival when compared to non-primed NPCs. We now conducted a similar study of human-fetal-spinal-cord-derived NPCs (hfNPCs) from legal gestational interruptions at the late gestational stage, at 19-21.6 weeks. In vitro, expanded hfNPCs retained neural features, multipotency, and self-renewal, which supported the development of a cell banking strategy. Before transplantation, we established a simple procedure to prime hfNPCs by overnight incubation with PGA-SS-FAS (at 50 µM FAS equiv.), which improved neuronal differentiation and overcame neurite-like retraction after lysophosphatidic-acid-induced Rho/Rock activation. The transplantation of primed hfNPCs into immune-deficient mice (NU(NCr)-Foxn1nu) immediately after the eighth thoracic segment compression prompted enhanced migration of grafted cells from the dorsal to the ventral spinal cord, increased preservation of GABAergic inhibitory Lbx1-expressing and glutamatergic excitatory Tlx3-expressing somatosensory interneurons, and elevated the numbers of preserved, c-Fos-expressing, activated neurons surrounding the injury epicenter, all in a low percentage. Overall, the priming procedure using PGA-SS-FAS could represent an alternative methodology to improve the capabilities of the hfNPC lines for a translational approach for acute SCI treatment.

Isolation and phenotyping of potential stem cells from the umbilical cord of the bottlenose dolphin(Tursiops truncatus).

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.

Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification.

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.