TAX2 peptide is a cyclic peptide that acts as an orthosteric antagonist for thrombospondin-1 (TSP-1) interaction with CD47. TAX2 was first described for its anti-angiogenic activities and showed anti-cancer efficacy in numerous preclinical models. Here, we aimed at providing an extensive molecular characterization of TAX2 mode of action, while evaluating its potential in ovarian cancer therapy. Multidisciplinary approaches were used to qualify a TAX2 drug candidate in terms of stability, solubility and potency. Then, efficacy studies, together with benchmark experiments, were performed in relevant mouse models of ovarian carcinoma. TAX2 peptide appears to be stable and soluble in clinically relevant solvents, while displaying a favorable safety profile. Moreover, clinical data mining allowed for the identification of TSP-1 as a relevant pharmacological target in ovarian cancer. In mice, TAX2 therapy inhibits ovarian tumor growth and metastatic dissemination, while activating anti-cancer adaptive immunity. Interestingly, TAX2 also synergizes when administered in combination with anti-PD-1 immune checkpoint inhibitiors. Altogether, our data expose TAX2 as an optimized candidate with advanced preclinical characterization. Using relevant syngeneic ovarian carcinoma models, we highlighted TAX2's ability to convert poorly immunogenic tumors into ones displaying effective anti-tumor T-cell immunity.

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

Currently, various kinds of materials are used for the treatment of bone defects. In general, these materials have a problem of formativeness. The three -dimensional (3D) printing technique has been introduced to fabricate artificial bone with arbitrary shapes, but poor bone replacement is still problematic.Our group has created a β⁻tricalcium phosphate (β⁻TCP) scaffold by applying 3D printing technology. This scaffold has an arbitrary shape and an internal structure suitable for cell loading, growth, and colonization. The scaffold was coated with a recombinant collagen peptide (RCP) to promote bone replacement.As indicated by several studies, cells loaded to scaffolds promote bone regeneration, especially when they are induced osteoblastic differentiation before transplantation. In this study, culture duration for bone marrow cells was optimized before being loaded to this new scaffold material.
Bone marrow cells isolated from C57BL/6J mice were subjected to osteogenic culture for 4, 7, and 14 days. The differentiation status of the cells was examined by alkaline phosphatase staining, alizarin red staining, and real-time RT-PCR for differentiation markers. In addition, the flow of changes in the abundance of endothelial cells and monocytes was analyzed by flow cytometry according to the culture period of bone marrow cells.Next, cells at days 4, 7, and 14 of culture were placed on a β-TCP/RCP scaffold and implanted subcutaneously into the back of C57BL/6J mice. Grafts were harvested and evaluated histologically 8 weeks later. Finally, Cells cultured for 7 days were also transplanted subperiosteally in the skull of the mouse with scaffolds.
Alkaline phosphatase staining was most prominent at 7 days, and alizarin red staining was positive at 14 days. Real-time RT-PCR revealed that Runx2 and Alp peaked at 7 days, while expression of Col1a1 and Bglap was highest at 14 days. Flow cytometry indicated that endothelial cells increased from day 0 to day 7, while monocytes increased continuously from day 0 to day 14. When transplanted into mice, the scaffold with cells cultured for 7 days exhibited the most prominent osteogenesis. The scaffold, which was transplanted subperiosteally in the skull, retained its shape and was replaced with regenerated bone over a large area of the field of view.
Osteoblasts before full maturation are most efficient for bone regeneration, and the pre-culture period suitable for cells to be loaded onto a β-TCP/RCP hybrid scaffold is approximately 7 days.This β-TCP/RCP hybrid scaffolds will also be useful for bone augmentation.
© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.
Copyright © 2020 Finetti, Capitani, Manganaro, Tatangelo, Libonati, Panattoni, Calaresu, Ballerini, Baldari and Patrussi.

Glycosylation is critical to megakaryocyte (MK) and thrombopoiesis in the context of gene mutations that affect sialylation and galactosylation. Here, we identify the conserved B4galt1 gene as a critical regulator of thrombopoiesis in MKs. β4GalT1 deficiency increases the number of fully differentiated MKs. However, the resulting lack of glycosylation enhances β1 integrin signaling leading to dysplastic MKs with severely impaired demarcation system formation and thrombopoiesis. Platelets lacking β4GalT1 adhere avidly to β1 integrin ligands laminin, fibronectin, and collagen, while other platelet functions are normal. Impaired thrombopoiesis leads to increased plasma thrombopoietin (TPO) levels and perturbed hematopoietic stem cells (HSCs). Remarkably, β1 integrin deletion, specifically in MKs, restores thrombopoiesis. TPO and CXCL12 regulate β4GalT1 in the MK lineage. Thus, our findings establish a non-redundant role for β4GalT1 in the regulation of β1 integrin function and signaling during thrombopoiesis. Defective thrombopoiesis and lack of β4GalT1 further affect HSC homeostasis.