Maša Kandušer

A cytokine storm drives critical COVID-19, yet the determinants and long-term consequences of this exaggerated inflammatory response remain unclear. We investigated whether cytokine responsiveness remains altered 2 years after recovery from critical disease. In a cohort of 117 individuals with mild, severe, or critical COVID-19, acute-phase clinical and laboratory parameters were analyzed in moderate–severe and critical cases. Long-term immune function was assessed by ex vivo stimulation of whole blood with PMA/ionomycin, lipopolysaccharide (LPS), and phytohemagglutinin (PHA), followed by cytokine quantification and correlation with clinical parameters. A biobank of 76 patient-derived lymphoblastoid cell lines (LCL cells) was established to evaluate the stability of cytokine responses. During acute illness, critically ill patients exhibited marked inflammatory dysregulation, including lymphopenia (72%), neutrophilia (51%), elevated neutrophil-to-lymphocyte ratios, and increased C-reactive protein (median 91 mg/L) and ferritin (median 1,153 μg/L), consistent with a hyperinflammatory–hypercoagulable phenotype. Two years post-infection, individuals with prior critical disease showed more than threefold higher secretion of IL-1β, IL-6, TNF-α, and IL-8 following PMA/ionomycin (p < 0.0001) and LPS stimulation (p < 0.05) compared with younger mild cases, whereas PHA-induced responses remained comparable. PBMC counts were also higher in the moderate–severe and critical groups 2 years post-infection (p < 0.001), highlighting differences in cellular composition. LCL cells from critically ill donors secreted 79% more IL-8 and TNF-α than those from mild cases (p < 0.05). Collectively, these findings indicate differences in cytokine responsiveness 2 years after COVID-19 which correspond to prior disease severity and are associated particularly with age and innate immune activation.

Background: Tissue regeneration is a rigorously regulated physiological process that maintains tissue homeostasis and restores the functionality of injured tissues and organs. Mesenchymal stem/stromal cells (MSCs) play a key role in this process. One of the mechanisms that underpins their regenerative capacity is cell fusion. The fusion of various animal cells is well recognized and established. In contrast, the process of cell fusion between human MSCs and other somatic cell types is poorly understood, particularly regarding the mechanisms that induce membrane merging and the characteristics of the resulting hybrids, such as their regenerative potential. This review aims to systematically evaluate evidence from studies investigating cell fusion between human MSCs and various tissue-specific cells for the purpose of tissue regeneration. Methods: The search terms “cell fusion” and “mesenchymal stem cells” or “mesenchymal stromal cells” AND (humans[Filter]) NOT (cancer) were used to search for publications in PubMed, Web of Science, and Scopus in July 2024, with the search updated in June 2026. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for reporting systematic reviews were followed. Results: A total of 206 publications were identified across all three databases. After excluding the studies evaluating osteoclastogenesis, myogenesis, neurogenesis, tumorigenesis, and nonhuman samples, only 47 references remained that addressed the topic of this review, indicating that knowledge of human MSC fusion for regenerative purposes remains limited. Most of the research studies investigated fusion between adult bone marrow (BM)-derived MSCs using an in vitro coculture system to improve cardiac tissue regeneration or to treat Duchenne muscular dystrophy. However, a substantial number of studies provide evidence that fusion is not a mechanism of MSCs plasticity and transdifferentiation. Conclusions: The studies on MSC fusion reviewed here suggest the potential of the resulting hybrids for tissue regeneration, particularly in the cardiovascular, muscular, and nervous systems. However, there is also evidence that spontaneous MSC fusion is a rare event. Further studies evaluating the generation and regenerative potential of the hybrids that possess the best characteristics of both partner cells, such as self-renewal and tissue formation, are of utmost importance for developing advanced therapy medicinal products (ATMPs) for the treatment of genetic and degenerative disorders.

Background: Primary cells derived from connective tissues contain mesenchymal stem/stromal cell (MSC)–like progenitors with chondrogenic potential relevant for cartilage repair. However, donor- and tissue-specific variability and the lack of robust, high-content analytical methods limit their translational use. Objectives: This study aimed to develop and optimize a high-content imaging workflow for quantitative evaluation of chondrogenesis in three-dimensional (3D) pellets derived from primary cells. Methods: Primary human cells isolated from cartilage were chondrogenically differentiated in vitro. A systematic optimization of immunofluorescence staining parameters was performed, including staining platform, enzymatic matrix digestion, non-specific site blocking, membrane permeabilization, and nuclear counterstaining. Type II collagen was detected using an Alexa Fluor 488–conjugated antibody, and pellets were analyzed using high-content non-confocal imaging. Fluorescence intensities were adjusted to the pellet area to account for size-dependent effects. Results: Staining directly in imaging plates enabled streamlined high-content analysis. Controlled pepsin-mediated matrix digestion markedly enhanced antibody penetration, while excessive digestion compromised pellet integrity. Extended bovine serum albumin blocking improved type II collagen signal intensity and homogeneity. Triton X-100 permeabilization increased detection sensitivity but occasionally induced structural disruption in weakly organized control pellets. The optimized protocol enabled clear discrimination between chondrogenic pellets and controls, with approximately threefold higher type II collagen signal in chondrogenic samples. Conclusions: This study establishes a high-content imaging–based workflow for quantitative assessment of 3D chondrogenesis from primary cells. The approach provides a rapid, scalable platform with direct relevance for in vitro screening, potency testing, and quality control in cartilage-oriented advanced therapy development.

Mitochondria‐targeting moieties (MTMs) are molecular fragments designed to deliver covalently tethered functional cargo to mitochondria, providing a modular strategy for chemical biology tools, imaging agents, and mitochondria‐targeted therapies. Phosphonium‐ or nitrogen cation‐based MTMs are not inert vectors and exhibit intrinsic bioactivity on mitochondrial and cellular levels to various extents. Here, we systematically evaluated a panel of N ⁺ ‐based cations to determine how structural features influence subcellular distribution and inherent bioactivity. Live‐cell imaging of fluorescent dye conjugates revealed that 3,5‐diphenylpyridinium (DPPy ⁺ ) exhibits cellular uptake and mitochondrial targeting comparable to the benchmark triphenylphosphonium (TPP ⁺ ), whereas conjugates with unsubstituted pyridinium preferentially accumulate in lysosomes. Profiling of inert cargo derivatives showed that DPPy ⁺ has lower intrinsic activity on mitochondrial membrane potential and oxidative phosphorylation, as well as on cellular respiration and viability than TPP ⁺ . The combination of efficient mitochondrial delivery and low intrinsic bioactivity translated to bioactive cargo: a Kv1.3 inhibitor conjugate with DPPy ⁺ induced apoptosis in cancer cell lines and demonstrated improved cancer selectivity relative to the TPP ⁺ conjugate in pancreatic organoid models. These results position lipophilic pyridinium cations as effective TPP ⁺ surrogates with enhanced biocompatibility for mitochondria‐targeted therapeutic and diagnostic agents, while revealing the structure‐dependent competing lysosomal accumulation of permanent nitrogen cations.

Mitochondria‐targeting moieties (MTMs) are molecular fragments designed to deliver covalently tethered functional cargo to mitochondria, providing a modular strategy for chemical biology tools, imaging agents, and mitochondria‐targeted therapies. Phosphonium‐ or nitrogen cation‐based MTMs are not inert vectors and exhibit intrinsic bioactivity on mitochondrial and cellular levels to various extents. Here, we systematically evaluated a panel of N ⁺ ‐based cations to determine how structural features influence subcellular distribution and inherent bioactivity. Live‐cell imaging of fluorescent dye conjugates revealed that 3,5‐diphenylpyridinium (DPPy ⁺ ) exhibits cellular uptake and mitochondrial targeting comparable to the benchmark triphenylphosphonium (TPP ⁺ ), whereas conjugates with unsubstituted pyridinium preferentially accumulate in lysosomes. Profiling of inert cargo derivatives showed that DPPy ⁺ has lower intrinsic activity on mitochondrial membrane potential and oxidative phosphorylation, as well as on cellular respiration and viability than TPP ⁺ . The combination of efficient mitochondrial delivery and low intrinsic bioactivity translated to bioactive cargo: a Kv1.3 inhibitor conjugate with DPPy ⁺ induced apoptosis in cancer cell lines and demonstrated improved cancer selectivity relative to the TPP ⁺ conjugate in pancreatic organoid models. These results position lipophilic pyridinium cations as effective TPP ⁺ surrogates with enhanced biocompatibility for mitochondria‐targeted therapeutic and diagnostic agents, while revealing the structure‐dependent competing lysosomal accumulation of permanent nitrogen cations.

Background: Primary cells derived from connective tissues contain mesenchymal stem/stromal cell (MSC)–like progenitors with chondrogenic potential relevant for cartilage repair. However, donor‑ and tissue‑specific variability and the lack of robust, high‑throughput analytical methods limit their translational use. Objectives: This study aimed to develop and optimize a fast, reproducible high‑content imaging workflow for quantitative evaluation of chondrogenesis in three‑dimensional (3D) spheroids derived from primary cells. Methods: Primary human cells isolated from cartilage were chondrogenically differentiated in vitro. A systematic optimization of immunofluorescence staining parameters was performed, including staining platform, enzymatic matrix digestion, non‑specific site blocking, membrane permeabilization, and nuclear counterstaining. Type II collagen was detected using an Alexa Fluor 488–conjugated antibody, and spheroids were analyzed using high‑content non-confocal imaging. Fluorescence intensities were normalized to spheroid area to account for size‑dependent effects. Results: Staining directly in imaging plates enabled streamlined high‑content analysis. Controlled pepsin‑mediated matrix digestion markedly enhanced antibody penetration, while excessive digestion compromised spheroid integrity. Extended bovine serum albumin blocking improved type II collagen signal intensity and homogeneity. Triton X‑100 permeabilization increased detection sensitivity but occasionally induced structural disruption in weakly organized control spheroids. The optimized protocol enabled clear discrimination between chondrogenic spheroids and controls, with approximately threefold higher type II collagen signal in chondrogenic samples. Conclusions: This study establishes a standardized, high‑content imaging–based workflow for quantitative assessment of 3D chondrogenesis from primary cells. The approach provides a rapid, scalable platform with direct relevance for in vitro screening, potency testing, and quality control in cartilage‑oriented advanced therapy development.

Cell fusion is a complex phenomenon that is key in maintaining tissue homeostasis, particularly in aiding tissue regeneration processes. Studies show that mesenchymal stem/stromal cells (MSCs) are capable of restoring damaged tissue by adopting the phenotype of various cell types via cell fusion. As cell fusion of MSCs with cells of different origin remains poorly researched, we have developed a protocol that allows successful electrofusion between human synovium-derived MSCs and human chondrocytes. Building on from our protocol can help researchers study the cell fusion processes in the in vitro environment and could set basis for development of fusion cell-based advanced therapy medicinal products (ATMPs). In our protocol, we provide a detailed description on how to culture both of the fusion partner cells, how to carry out the modified adherence method (MAM) to achieve a high yield of successfully fused cells, and how to determine the yield of cell fusion using either methyl violet or fluorescent cell trackers.

Cell-based therapies hold great potential for cancer immunotherapy. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasability of combining gene electrotransfer and electrofusion into a single-step technique and evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity for single or combined method of gene delivery or cell fusdion. We determined the percentage of fused cells expressing green fluorescence protein (GFP) in a murine cell model of melanoma B16F1, cell line used in our previous studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single step. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Furthermore, our results suggest that cell membrane fluidity is not related to the efficiency of the gene electrotransfer and electrofusion. The protocol is compatible with microfluidic devices, however further optimization of electric pulse parameters and buffers is still needed. Graphical Abstract

Continuous treatment of patients with chronic lymphocytic leukemia (CLL) with venetoclax, an antagonist of the anti-apoptotic protein Bcl-2, can result in resistance, which highlights the need for novel targets to trigger cell death in CLL. Venetoclax also induces autophagy by perturbing the Bcl-2/Beclin-1 complex, so autophagy might represent a target in CLL. Diverse autophagy inhibitors were assessed for cytotoxic activities against patient-derived CLL cells. The AMPK inhibitor dorsomorphin, the ULK1/2 inhibitor MRT68921, and the autophagosome–lysosome fusion inhibitor chloroquine demonstrated concentration-dependent and time-dependent cytotoxicity against CLL cells, even in those from hard-to-treat patients who carried del(11q) and del(17p). Dorsomorphin and MRT68921 but not chloroquine triggered caspase-dependent cell death. According to the metabolic activities of CLL cells and PBMCs following treatments with 10 µM dorsomorphin (13% vs. 84%), 10 µM MRT68921 (7% vs. 78%), and 25 µM chloroquine (41% vs. 107%), these autophagy inhibitors are selective toward CLL cells. In these CLL cells, venetoclax induced autophagy, and addition of dorsomorphin, MRT68921, or chloroquine showed potent synergistic cytotoxicities. Additionally, MRT68921 alone induced G2 arrest, but when combined with venetoclax, it triggered caspase-dependent cytotoxicity. These data provide the rationale to target autophagy and for autophagy inhibitors as potential treatments for patients with CLL.

Induced cell fusion is a powerful method for production of hybridoma in biotechnology and cell vaccines in medical applications. Among different alternatives, physical methods have an advantage, as they do not require any additives. Among them electrofusion, an electroporation-based cell fusion method holds a great promise. Electric pulses cause cell membrane permeabilization and due to pore formation bring cell membrane into the fusogenic state. At the same time, however, they compromise cell viability. We used a train of 8 × 100 µs electric pulses, delivered at 1 Hz with strengths ranging from 400 to 1600 V/cm. We evaluated electrofusion efficiency by dual color microscopy. We determined cell viability, because during electroporation reactive oxygen species are generated affecting cell survival. The novelty of our study is evaluation of the effect of lipid antioxidant α-tocopherol on cell fusion yield and cell viability on mouse B16-F1 cells. Pretreatment with α-tocopherol slowed down dynamic of cell fusion shortly after electroporation. Twenty-four hours later, fusion yields between α-tocopherol treated and untreated cells were comparable. The viability of α-tocopherol pretreated cells was drastically improved. Pretreatment of cells with α-tocopherol improved whole electrofusion process by more than 60%. We believe that α-tocopherol holds great promise to become an important agent to improve cell electrofusion method.