Apheresis platelet rich-plasma for regenerative medicine: An in vitro study on osteogenic potential

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects’ treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-β and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets

481. Targeted Genome Editing in Mouse Hematopoietic Stem/Progenitor Cells (HSPC) To Model Gene Correction of SCID-X1

Targeted genome editing by artificial nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy with early generation integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of adverse events due to insertional mutagenesis and unregulated transgene expression. We recently reported a strategy that enabled targeted integration of a corrective cDNA into the IL2RG gene in 6% of human HSPC with high specificity. Gene corrected HSPC generated polyclonal lymphoid cells that express the IL2RG protein and have a selective growth advantage over those carrying disruptive IL2RG mutations (Genovese, Nature, 2014). Here, to model SCID-X1 disease correction, we developed a mouse model carrying the IL2RG human gene including a common disease-causing mutation in place of the murine Il2rg gene, allowing to use the same reagents utilized for gene correction of human cells. These mice have impairment in lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we first performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute at least in part the T and B cell compartments. We then developed a protocol to obtain gene correction in murine Lin- HSPC based on the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol was associated with high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR, but also with high levels of acute cytotoxicity (65% cell loss). The surviving cells remained capable of expansion in culture and preserved their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over un-corrected cells. These data indicate functional correction of the IL2RG gene by our strategy. Yet, measuring percentage of correction within myeloid cells at long-term we found that it was almost undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells stably persisted in the mice up to 7 months post transplantation within all the hematopoietic organs. Furthermore, upon challenging the transplanted mice with a murine pathogen (LCMV Arm.) we observed viral-specific γIFN production by CD8+ gene corrected cells at a similar extent as for WT mice, proving in vivo the functionality of corrected T cells. These results suggest that our protocol achieves biologically relevant levels of gene correction in progenitors that sustain long-term lymphopoiesis but is limited in multipotent HSC. Ongoing studies aim to improve murine HSC gene targeting and to compare safety and efficacy of gene correction vs gene replacement in our disease model

A novel role for CRIM1 in the corneal response to UV and pterygium development

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A > C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium

現地観測に基づく河川流の乱流特性に関する研究

博士（工学）神戸大

METODO E RELATIVO KIT PER LA DETERMINAZIONE DELLA CONCENTRAZIONE DI FOSFOETANOLAMMINA IN UNA SOLUZIONE ACQUOSA

Un metodo per la determinazione della concentrazione di fosfoetanolammina in una soluzione acquosa che comprende le fasi di: - aggiungere a tale soluzione acquosa una sostanza reagente con la fosfoetanolammina atta a modificare almeno una proprietà ottica della soluzione acquosa, - misurare l’intensità della proprietà ottica di detta soluzione a seguito dell’aggiunta di detta sostanza reagente, - determinare la concentrazione della fosfoetanolammina presente in detta soluzione in funzione dell’intensità della proprietàottica misurata