Based on the results, MACS showed potential to optimize the sDNAfrag reduction rate, when applied to raw semen, before DGC and SU, especially in samples with low values of progressive motility, vitality and hypoosmotic swelling test.

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of hematological malignancies remains hampered by life-threatening chronic graft vs. host disease (cGVHD). Although multifactorial in nature, cGVHD has been associated with imbalances between effector and regulatory T cells (Treg). To further elucidate this issue, we performed a prospective analysis of patients undergoing unrelated donor allo-HSCT after a reduced intensity conditioning (RIC) regimen containing anti-thymocyte globulin (ATG) and the same GVHD prophylaxis, at a single institution. We studied T cell subset homeostasis over a 24-month follow-up after HSCT in a comparative analysis of patients with and without cGVHD. We also quantified naive and memory T cell subsets, proliferation and expression of the apoptosis-related proteins Bcl-2 and CD95. Finally, we assessed thymic function by T cell receptor excision circle (TREC) quantification and T cell receptor (TCR) diversity by TCRVβ spectratyping. While the total number of conventional CD4 (Tcon) and CD8 T cells was similar between patient groups, Treg were decreased in cGVHD patients. Interestingly, we also observed divergent patterns of Naive and Stem Cell Memory (SCM) subset recovery in Treg and Tcon compared to CD8. Patients with cGVHD showed impaired recovery of Naive and SCM Tcon and Treg, but significantly increased frequencies and absolute numbers of Naive and SCM were observed in the CD8 pool. Markedly increased EMRA CD8 T cells were also noted in cGVHD. Taken together, these results suggest that Naive, SCM and EMRA CD8 play a role in the emergence of cGHVD. Reduced Naive and recent thymic emigrant Tcon and Treg in cGVHD was likely due to impaired thymic output, as it was accompanied by decreased CD4 TREC and TCR diversity. On the other hand, CD8 TCR diversity was similar between patient groups. Furthermore, no correlation was observed between CD8 TREC content and Naive CD8 numbers, suggesting limited thymic production of Naive CD8 T cells in patients after transplant, especially in those developing cGVHD. The mechanisms behind the opposing patterns of CD4 and CD8 subset cell recovery in cGVHD remain elusive, but may be linked to thymic damage associated with the conditioning regimen and/or acute GVHD.

Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34 + cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34 + and CD34 + CD90 +) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.