Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies, achieving durable remission in patients who are refractory to conventional therapies.However, the limited persistence of infused CAR-T cells, due to T-cell exhaustion and the loss of CAR-T cells, remains a major obstacle to sustained therapeutic efficacy.Recent studies have demonstrated that CAR-T cell products enriched for less-differentiated memory T cell subsets exhibit superior longterm persistence, greater cytotoxicity, and improved clinical outcomes compared with conventional CAR-T cell products.To enrich for less-differentiated memory T cells, various strategies have been explored throughout the manufacturing process.These strategies include selecting cell sources enriched for less-differentiated memory T cells and delaying their differentiation into terminally differentiated states by regulating signaling pathways, remodeling epigenetics, and modulating metabolism.This review provides an overview of current and emerging manufacturing strategies for CAR-T cells, focusing on enhancing their prolonged persistence.Further investigations are warranted to determine the optimal composition of differentiated and lessdifferentiated T cells in CAR-T cell products under the respective clinical conditions.This could facilitate the personalized manufacturing of CAR-T cell products with the ideal cell composition, thereby maximizing the efficacy of CAR-T cell therapy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for hematological malignancies. However, it is often complicated by chronic graft-versus-host disease (cGVHD), a leading cause of late non-relapse morbidity and mortality. Early identification of patients at risk is therefore critical, yet reliable biomarkers are still lacking. In this study, we evaluated serum endothelial mediator profiles collected 1 year after allo-HSCT and their association with subsequent cGVHD development. Serum samples taken 1 year post-transplant from 82 transplanted patients, of whom 61% developed cGVHD, were analyzed for 19 mediators using Luminex and ELISA assays. Associations between mediator levels and cGVHD were assessed using statistical analysis, logistic regression, and hierarchical clustering. Six mediators, endocan, EMMPRIN, VCAM-1, MMP-1, MMP-8, and MMP-9; differed significantly between patients with and without cGVHD (P < 0.05). Endocan and VCAM-1 remained independently associated with cGVHD in multivariable regression adjusted for age, body mass index (BMI), and prior acute GVHD (aGVHD). Cluster analysis based on these mediators identified distinct patient subgroups. Clusters with generally lower mediator levels had fewer cases of cGVHD, whereas a central cluster with elevated levels showed a higher disease prevalence. Integration of clinical variables confirmed known risk factors, including older age, higher BMI, and previous aGVHD. The identified mediators are biologically linked to endothelial activation and extracellular matrix remodeling. This study is the first to demonstrate that a distinct endothelial- and matrix-remodeling-related serum mediator signature measured 1 year after allo-HSCT is associated with cGVHD. The identified patient clusters provide insight into disease biology and highlight potential biological markers of the disease. These findings support further prospective studies combining longitudinal mediator profiling with clinical data and advanced modeling to improve risk stratification and identify therapeutic targets for the prevention and treatment of cGVHD.

Human mesenchymal stromal cells derived from Wharton Jelly (hWJ-MSCs) release a broad range of bioactive components, collectively known as the secretome. This secretome includes growth factors, cytokines and extracellular vesicles (EVs). The secretome has significant therapeutic potential, such as modulating inflammation, promoting tissue repair and inhibiting tumor proliferation. The cellular microenvironment, particularly the oxygen level, plays a critical role in influencing the quantity and quality of the secretome. In this study, we originally explored the effects of long-term oxygen conditioning (7 days) at 2%, 10% and 19% oxygen on hWJ-MSCs and their secretome using stirred and controlled Ambr 250 bioreactors (Sartorius, Göttingen, Germany) and size exclusion chromatography (SEC). Our results demonstrate that cells cultured at 2% O2 exhibited a significantly enhanced proliferation and a greater cell density (2.5 × 105 ± 4.2 × 104 cells/mL) compared with those at 10% O2 (2.0 × 105 ± 2.6 × 104 cells/mL) and 19% O2 (1.6 × 105 ± 3.6 × 104 cells/mL). Furthermore, glucose uptake and lactate production were also elevated at 2% O2, indicating an increased metabolic activity. Surprisingly, despite a greater cell density and metabolic activity at 2% O2, the yield production of particles and protein per cell was reduced compared with the greater oxygen conditions. However, conditioned media and isolated EVs (EV-SEC) derived from hWJ-MSCs cultured under 2% O2 demonstrated a superior capacity to promote osteoblast and human umbilical vein endothelial cells proliferation and migration and angiogenic potential. In conclusion, although long-term hypoxic preconditioning (2% O2) may not maximize particle and protein yields, it significantly enhances the bioactivity of the secretome in supporting cell migration and potentially neovascularization. These findings highlight the importance of controlling accurately the level of oxygen in hWJ-MSC cultures to reveal an optimized regenerative potential of the secretome for therapeutic applications.