Cytotherapy

BackgroundCompetitive transplantation is essential for defining intrinsic repopulating capacity of murine hematopoietic stem and progenitor cells (HSPCs), yet comparable assays for human cells have been limited by the lack of a robust in vivo platform. MethodsHere, we describe a novel competitive transplantation method in humanized NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice that enables simultaneous engraftment and longitudinal tracking of distinct human grafts within a shared microenvironment. ResultsUsing human leukocyte antigen-mismatched donor CD34+ cells, this method facilitates standard flow cytometry panels to track multiple donor cell chimerism, lineage output, and HSPC composition. The experimental framework may be adapted to different mouse models, conditioning strategies, donor sources, and treatments. ConclusionsOverall, this humanized competitive repopulation assay fills a critical translational gap and offers a flexible foundation for advancing mechanistic discovery in human hematopoietic biology and improving clinical strategies for stem cell transplantation.

Delivery of cells or vectors in advanced therapies is probably the major challenge for genetic disorders that affect a large part of the body such as Duchenne Muscular Dystrophy (DMD). Here, we describe a novel approach for systemic cell delivery based upon an implantable bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin, able to support adhesion and extensive proliferation of mesoderm cells both in vitro and when implanted subcutaneously in a DMD mouse model. The scaffold is rapidly vascularised leading to cell entering the circulation and colonising multiple distal organs, including distant skeletal muscles and heart. Cells survive in colonized muscles and differentiate into muscle fibres that produce well detectable levels of dystrophin and -sarcoglycan. These results are game changing for cell therapy, as they allow colonization of life essential but "difficult to reach" muscles such as diaphragm and heart while avoiding invasive catheterization. Once optimised, this approach will rapidly enter clinical experimentation for DMD, other muscular dystrophies, and possibly other genetic disorders of the mesoderm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/715524v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@11dfd34org.highwire.dtl.DTLVardef@1da6599org.highwire.dtl.DTLVardef@14427f0org.highwire.dtl.DTLVardef@19a242a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Study design and therapeutic outcome. Muscle biopsies were obtained from Duchenne muscular dystrophy (DMD) patients to isolate human DMD mesangioblasts (DMD-hMabs). Cells were genetically corrected using a lentivirus carrying a snRNA able to induce exon skipping (U7snRNA), generating U7-hMabs (1). U7-hMabs were seeded onto laminin-coated polycaprolactone (Lam-PCL) nanofiber scaffolds and implanted into the back muscle of DMD-NSG mice. This platform enabled systemic distribution of hMabs cells through circulation, resulting in engraftment across multiple muscle groups, including tibialis anterior, triceps, diaphragm and heart. C_FIG

PurposeLarge quantities of human pluripotent stem cells (hPSCs) are required for clinical applications. 3D suspension cultures are suitable for large scale manufacturing of hPSCs but yield, viability and quality are affected by the hydrodynamic environment. This paper characterizes the hydrodynamic environment inside vertical wheel bioreactors (VWBRs) as a function of size and agitation rates, measures its effect on cell aggregation and proliferation, and proposes the use of Lagrangian-based shear stress and energy dissipation rate (EDR) exposures to support scale-up. MethodsIn silico: Transient, 3D, turbulent flow simulations are conducted for two VWBR sizes (100, 500 mL) at five agitation rates between 20 and 80 rpm. Trajectories of cell aggregates of sizes from 200 to 1,000 microns are calculated, and shear stress and EDR exposures are collected along these trajectories. In vitro: ESI-017 hPSCs were cultured in VWBRs for 6 days. Aggregation efficiency and daily fold ratios were calculated based on cell counts and initial inoculation density. ResultsAggregate size, agitation rate and bioreactor size modulate cell aggregate exposures to EDR and shear stress, which significantly depart from maximum or volume average metrics used for scale-up. Combined in vitro/in silico results show EDR affects aggregation efficiency, cell counts and aggregate size, and has a small effect on daily fold ratios but a significant effect on total fold ratio. ConclusionHistory of trajectory-based cell aggregate exposures to EDRs provide a better scale-up basis for VWBRs than volume-averaged EDR. Shear stress does not significantly affect hPSC aggregation, proliferation and expansion in VWBRs under the tested conditions.

Cancer remains a major therapeutic challenge despite substantial advances in diagnosis and treatment, including immune checkpoint blockade. Among emerging immunotherapeutic approaches, adoptive cell transfer (ACT) has attracted growing interest. Human peripheral V{gamma}9V{delta}2 T cells are promising candidates for ACT because they combine rapid and potent antitumor functions with major histocompatibility complex (MHC)-independent tumor recognition, enabling allogeneic use with limited risk of graft-versus-host disease. This raises the possibility of generating standardized V{gamma}9V{delta}2 T-cell banks from healthy donors for off-the-shelf immunotherapy. Here, we provide preclinical evidence supporting the suitability of allogeneic human V{gamma}9V{delta}2 T cells for ACT. We characterized peripheral blood V{gamma}9V{delta}2 T cells from healthy donors after successive antigen-specific and non-specific amplification steps, assessing their phenotype, effector functions, and metabolic state. Amplified cells maintained a strong pro-inflammatory Th1-like profile, preserved cytotoxic activity, and did not produce immunoregulatory cytokines. They also displayed high purity, a predominant effector memory phenotype, reduced expression of several inhibitory immune checkpoints, and sustained antitumor reactivity. Altogether, these findings support the development of allogeneic V{gamma}9V{delta}2 T-cell products as a scalable platform for next-generation cancer immunotherapies.

Donor organ shortage remains the major barrier to transplantation resulting in deaths on the waiting list. For lungs, aspiration-related injury is a common cause of donor organ discard and increases the risk of primary graft dysfunction. Currently, no effective therapies exist to repair damaged donor lungs prior to transplantation. Here, we investigated whether mesenchymal stromal cells (MSCs) from bone marrow or full-term amniotic fluid could restore severely injured donor lungs in a porcine model integrating ex vivo lung perfusion, transplantation and post-transplant follow-up (n=48; 24 donors, 24 recipients). MSCs were administered either once during ex vivo lung perfusion or repeatedly across lung perfusion and the early post-transplant period and compared with placebo treated controls. A single dose conferred only partial benefit, whereas repeated dosing restored graft function, normalized gas exchange and haemodynamics, and prevented graft dysfunction. MSCs from both sources were similarly effective in repeated regimens. These findings identify dosing schedule, rather than cell source, as key determinant of durable organ rescue and support perfusion-guided cell therapy as potentially generalizable regenerative strategy across solid-organ transplantation.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI

Precision-cut lung slices (PCLS) retain the native cells and extracellular matrix that contribute to the structural and functional integrity of lung tissue. This technique enables the study of cell-matrix interactions and is particularly useful for pre-clinical pharmacological studies. More specifically, PCLS are widely used to model the complex pathophysiology of pulmonary fibrosis, an uncurable and progressive interstitial lung disease. Current ex vivo pulmonary fibrosis models expose PCLS to pro-fibrotic biochemical cues over a short timeframe (hours to days) and quickly collect samples for analysis due to viability concerns. This condensed timeline is a limitation to understanding chronic disease mechanisms. To extend the utility of ex vivo pulmonary fibrosis models, PCLS were embedded in engineered hydrogels and exposed to pro-fibrotic biochemical and biophysical cues. Hydrogel-embedded PCLS maintained greater than 80% total cell viability over 3 weeks in culture. Gene expression patterns in samples exposed to pro-fibrotic cues matched trends measured in human fibrotic lung tissue. Finally, treatment with Nintedanib, a Food and Drug Administration approved pulmonary fibrosis drug, moderately reduced fibroblast activation and influenced epithelial cell differentiation. Collectively, these results show that hydrogel-embedded PCLS models of pulmonary fibrosis extend our ability to study fibrotic processes ex vivo and, when applied to human tissues, present a new approach methodology for studying lung disease and treatment.

During development, the size of the neuroepithelial cell pool plays a key role in establishing brain size, determining the numbers of derived progenitors and subsequent neuronal cell types. While early histogenesis is well modelled in brain organoids, the organ-scale geometry of the telencephalon is not accurately recapitulated. Herein, we present a new approach for generating ventral and dorsal forebrain organoids which develop a large ventricular neuroepithelium, characteristic of the closed telencephalic vesicle. Using a growth medium that supports aerobic glycolysis and is typically used for endothelial cells, we modulate neuroepithelial expansion to induce a more anatomically accurate neuroepithelial layer which, upon maturation, thickens physiologically to generate the typical neurogenic layered architecture. In addition, we present a new method for embedding organoids in miniature collagen spheres which mimics native extracellular matrix, stabilizes the ventricular geometry for dynamic culture conditions, and provides a means for incorporating vascular cells for neurovascular development. Finally, we demonstrate that human organoids grown under these conditions exhibit dramatically enlarged ventricles and delayed maturation compared to mouse. Together, this approach provides a model of the forebrain neuroepithelium with morphogenetic macroscale geometry and tissue architecture, suitable for investigating neurodevelopment and disease.

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation. Key pointsO_LIA CROPseq-based CRISPR-Cas9 screen enables functional genomics in enucleated primary human red blood cells. C_LIO_LIChloride Intracellular Channel 3 (CLIC3) and Vesicle Associated Membrane Protein 8 (VAMP8) were identified as critical for terminal erythroid differentiation and enucleation, likely acting through two distinct mechanisms. C_LI

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f059d9org.highwire.dtl.DTLVardef@9aeffforg.highwire.dtl.DTLVardef@27cd1org.highwire.dtl.DTLVardef@150b7d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

BackgroundCompanion canines need advances in therapeutic options for solid tumor malignancies. Prior studies established feasibility of autologous natural killer (NK) cell infusions in canines with solid tumors; however, autologous products are limited by dysfunctional immunity and a manufacturing process that delays care. Allogeneic NK cells offer the possibility of "off-the-shelf" therapy to be administered from healthy donors. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15. Additional experiments included IL-12 in the expansions. In vitro potency was assessed via co-culture with the D17-mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion. ResultsFlow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days of expansion. Residual T cell numbers varied based on donor. The addition of IL-12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic/refractory solid tumors were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well. ConclusionsCanine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro, and safety in vivo. Further studies will optimize the NK cell product and escalate dosing to reach the maximal tolerable dose.

Non-front-fanged snakes are abundant, diverse and represent approximately 70% of extant snakes. However, there is limited knowledge about most species and their venoms, in part due to the technical and welfare challenges associated with venom extraction, low venom yields, and the lack of cellular models available. Organoids represent an excellent opportunity to overcome these challenges. Here, we establish, for the first time, venom gland organoids from snakes of the Colubridae family and demonstrate the in vitro production of toxins.

BackgroundIn the absence of structured donor registries, social media platforms have become a dominant mechanism for blood donor recruitment in many low-resource settings. However, the implications of this shift for transfusion timeliness and system reliability remain unclear. ObjectiveTo evaluate the impact of social media-sourced donors on transfusion delay, donor reliability, and hemovigilance-related outcomes compared with conventional donor pathways. MethodsThis prospective analytical study included 400 transfusion episodes across tertiary hospitals in Bangladesh. Donor sources were categorized as social media (SM) or conventional (CON). The primary outcome was delay-to-transfusion. Secondary outcomes included donor-related irregularities, documentation completeness, near-miss events, and acute transfusion reactions. Multivariable logistic regression identified predictors of delay [&ge;]4 hours. ResultsSocial media-sourced donors were associated with significantly longer transfusion delays (5.98 vs 2.97 hours; p<0.001). Delay [&ge;]4 hours occurred in 83.6% of SM cases versus 17.6% of CON cases (OR 23.78). Donor-related irregularities were observed in 85% of SM episodes and absent in CON donors. Safety outcomes did not differ significantly between groups. Social media donor sourcing remained the strongest independent predictor of delay (adjusted OR 18.09). ConclusionUnregulated social media-based donor recruitment introduces substantial delays and undermines system reliability without improving access. Integration of digital tools into regulated donor systems is essential to strengthen transfusion timeliness and hemovigilance in resource-limited settings.

iii.Background and ObjectivesThe "Pan-European Transfusion Research InfrAstructure" (PETRA) project was established to advance the use of donor, blood product, and patient datasets in Europe, aiming to benefit both patient and donor health. Here, the initial PETRA objective was to describe the landscape of existing donor and blood establishment (BE) databases. Materials and MethodsAn online survey was circulated to the European Blood Alliances BE members. The survey collected information on the feasibility of accessing donor data, and challenges and possibilities for linking these datasets with information on the associated blood products and transfusion recipients, and donors own health records. ResultsSeventeen BEs across 16 countries completed the survey. The majority could, in principle, link their donor data to product data (13 BEs (76%)) and recipient data (10 BEs (59%)), for research purposes. However, capabilities were limited and in only 29% of the BEs was the donor to recipients linkage an automated process. BEs reported significant challenges to achieve full vein-to-vein linkage, including legal constraints and lack of consent (11 BEs) and resources (10-14 BEs). IT and data issues as well as lack of knowledge and training were cited as obstacles by a minority of BEs. ConclusionWhilst the survey results suggest considerable interest in developing linkages between blood donors, their products, and recipients, many challenges remain due to a variety of obstacles. First steps in working towards a PETRA may be assistance to navigate legal frameworks as well as investing in resources and quality and harmonisation of data collections. iv. HighlightsO_LI17 blood establishments (BEs) in 16 countries responded to a survey on obstacles and opportunities for achieving vein-to-vein datasets. C_LIO_LIIn 59% of the BEs donor-to-recipient links can be established for research improving transfusion outcomes, but only in 29% this is an automated process. C_LIO_LIIn order to work towards a "Pan-European Transfusion Research InfrAstructure" (PETRA), legal frameworks, adequate donor consent and (financial and human) resources are the most common obstacles that require addressing. C_LI

Recombinant adeno-associated virus (rAAV) vectors are a leading platform for gene delivery in basic and clinical research, yet large-scale manufacturing remains constrained by residual nucleic-acid impurities that compromise safety. In this study, we profiled the DNA species packaged within rAAV capsids and identified plasmid backbone sequences and host cell genomic DNA (hcDNA) as predominant contaminants. To mitigate this critical quality attribute, we implemented upstream strategies designed to fragment or excise backbone DNA, including TelN/TelROL excision, I-SceI meganuclease digestion, CRISPR/Cas9 cleavage, and Cre/LoxP recombination. Quantitatively, TelN/TelROL and I-SceI reduced encapsidated plasmid backbone DNA to approximately 20-30% and 20-40% of baseline levels, respectively, while CRISPR/Cas9 lowered it to about 10-20%. Notably, the Cre/LoxP system eliminated detectable plasmid backbone DNA without compromising vector-genome titers, indicating preserved genomic integrity. Additionlly, supplementating cell culture with a caspase inhibitor significantly reduced hcDNA contamination in rAAV particles to 1-5% of the baseline level. Collectively, these interventions provide practical bioprocess frameworks that markedly enhance rAAV purity via targeted DNA minimization and prevention of hcDNA fragmentation, thereby strengthening the safety profile of rAAV therapeutics in alignment with current Good Manufacturing Practice (cGMP) expectations.

BackgroundPhotobiomodulation (PBM) therapy has demonstrated therapeutic potential in promoting cellular repair, modulating inflammation, and enhancing mitochondrial function. Platelet-rich plasma (PRP) is widely used in regenerative medicine due to its concentration of growth factors and cytokines. Very small embryonic-like stem cells (VSELs), a rare population of pluripotent stem cells present in adult tissues, have emerged as a potential contributor to tissue regeneration. While PBM and PRP are used in combination, how VSELs or Multi-lineage stress enduring (MUSE) cells are at play, and the biological mechanisms underlying their synergistic effects remain incompletely characterized. ObjectiveThis exploratory pilot study aimed to evaluate whether application of the MD Biophysics laser to autologous PRP is associated with measurable changes in VSEL-related antibody marker expression, and to identify directional trends to inform future controlled studies. MethodsPRP samples were collected from participants across seven test dates (July 2024 to February 2025), yielding 18 participant-session datasets. Samples were analyzed before (Pre) and after (Post) laser application using flow cytometry conducted at a UCLA Flow Cytometry Laboratory. Four VSEL-associated antibody markers were assessed: CD45-CD34+, CXCR4+, CD133+, and SSEA-4+. Analyses were descriptive and focused on paired differences and directional trends due to the exploratory design and absence of a control group. ResultsThree of four VSEL-associated markers (CXCR4+, CD133+, and SSEA-4+) demonstrated a group-level increase in median paired differences following laser application. Directional increases were observed in 12/18 sessions for CXCR4+, 10/18 for CD133+, and 9/18 for SSEA-4+. CD45-CD34+ showed a near-equal distribution of increases and decreases. Ki-67 positivity indicated the presence of viable, proliferative cells. While no findings reached statistical significance due to limited sample size, consistent directional trends were observed across multiple markers. ConclusionApplication of PBM to autologous PRP was associated with directional increases in multiple VSEL-associated antibody markers, suggesting a potential role for stem cell activation or mobilization in the mechanism of action. Although preliminary and not statistically powered, these findings provide hypothesis-generating evidence supporting further investigation. The observed trends informed iterative protocol refinement and establish a foundation for future controlled, adequately powered studies to evaluate clinical efficacy and underlying biological mechanisms.

Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the future egg or sperm cells. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to further mature. The reason for this is unclear. Previous studies in mice revealed that several specific metabolic changes occur during the maturation of these cells, which are essential for their developmental progress. However, very little is known about the metabolic profile of human primordial germ cells. In the severe scarcity of human PGCs, hPGCLCs serve as a research model to study PGC formation. To investigate this, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLC, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the influence of metabolism on human PGC development that could help improve methods for in vitro differentiation and maturation hPGCLCs.

Prolonged cytopenias are a frequent complication of chimeric antigen receptor (CAR) T-cell therapies and are associated with increased infection risk and non-relapse mortality. Although inflammatory cytokines released during CAR-T cell activation have been implicated in immune effector cell-associated hematotoxicity (ICAHT), their direct effects on human hematopoietic stem and progenitor cells' (HSPCs) function remains incompletely understood. Here, we established a reductionist model of CAR-T-associated hematotoxicity using conditioned media (CM) derived from activated CD19 CAR-T cells. Sustained exposure of human HSPCs to CAR-T-derived inflammatory secretome impaired HSPC expansion and reduced long-term repopulating capacity in xenotransplantation assays. In contrast, short-term exposure did not abrogate HSPC function, indicating that brief inflammatory signals can initiate durable reprogramming events, with functional consequences emerging during subsequent proliferative expansion. Mechanistically, CAR-T CM induced IFN gamma- (IFNg) and TNF alpha- (TNFa) responsive transcriptional programs in HSPCs and promoted inflammatory myeloid skewing without evidence of apoptosis-dependent stem cell loss. Combined inhibition of IFNg and TNFa restored HSPC expansion, normalized lineage output, reversed inflammatory transcriptional signatures, and rescued in vivo repopulating capacity without impairing CAR-T cytotoxic activity. These findings demonstrate that CAR-T-derived inflammatory signaling can directly impair human HSC function and identify dual IFNg/TNFa blockade as a potential strategy to mitigate CAR-T-associated hematotoxicity while preserving antitumor efficacy.