Olivier Preynat-Seauve

Chronic wounds are ulcers unable to heal due to vascular insufficiency, diabetes, or obesity. Adipose-derived stromal cells (ASCs) are promising candidates for regenerative therapies owing to their pro-healing and angiogenic properties. Compared with autologous approaches, allogeneic ASC therapies offer the opportunity for off-the-shelf use, enabling immediate availability, standardized qualification, and consistent potency. Gelatin sponges have been shown to reprogram ASCs toward a highly angiogenic phenotype. However, because this activation also modulates some immune-related genes, including MHC, its impact on immunogenicity is unknown and could be critical for allogeneic applications. This study evaluated whether ASCs embedded in a gelatin sponge could be used in an allogeneic setting for ischemic wound repair. To mimic clinical allogeneic conditions, a controlled MHC mismatch was introduced in a rat ischemic wound model: donor ASCs carrying RT1^n or RT1^l haplotypes were implanted into outbred RT1^a recipients. Embedding ASCs within the gelatin sponge upregulated MHC class I but not class II expression, without inducing systemic or local alloreactivity. Serum acute-phase proteins remained unchanged, and no CD3⁺ T-cell infiltration was detected. Histology confirmed efficacy on ischemic wounds, with increased granulation tissue thickness, red blood cell infiltration, and enhanced vessel density versus controls. Allogeneic ASCs activated by a gelatin scaffold promote wound revascularization without eliciting immune rejection, supporting their development as standardized, off-the-shelf therapies for chronic ischemic wounds.

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells.

Although obesity is a major risk factor for cancer, it may also improve the response to cancer therapy. Here we investigated the impact of obesity on the efficacy of immune checkpoint inhibitors (ICI). In male mice, obesity promoted tumor growth but enhanced the response to ICI. This was associated with higher expression of immune-related genes within the tumor and enhanced infiltration of tumor-specific CD8 ⁺ T cells. Further, obesity in mice was associated with higher estrogen levels and enrichment of estrogen response genes in the tumor, and anti–programmed cell death 1 (anti–PD-1) efficacy was reduced upon administration of the aromatase inhibitor letrozole, which blocks the production of estrogens. Mechanistically, adipocyte-derived estrogens increased antigen presentation by dendritic cells and tumor-specific CD8 ⁺ T cell cytotoxicity. Last, overweight and obese men with melanoma responded better to ICI, with high estrogen levels being associated with improved response and survival. Our results suggest that estrogens may serve as a predictive factor of response to ICI in men with melanoma.

Adipose-derived stromal cells (ASCs) possess significant regenerative potential, playing a key role in tissue repair and angiogenesis. During wound healing, ASC interacts with the extracellular matrix by recognizing arginylglycylaspartic acid (RGD) motifs, which are crucial for mediating these functions. This study investigates how RGD exposure influences ASC behavior, with a focus on angiogenesis. To mimic the wound-healing environment, ASC were cultured in a porcine gelatin sponge, an RGD-exposing matrix. Transcriptomics revealed that ASC cultured in gelatin exhibited an upregulated expression of genes associated with inflammation, angiogenesis, and tissue repair compared to ASC in suspension. Pro-inflammatory and pro-angiogenic factors, including IL-1, IL-6, IL-8, and VEGF, were significantly elevated. Functional assays further demonstrated that ASC-conditioned media enhanced endothelial cell migration, tubulogenesis, and reduced endothelial permeability, all critical processes in angiogenesis. Notably, ASC-conditioned media also promoted vasculogenesis in human vascular organoids. The inhibition of ASC-RGD interactions using the cyclic peptide cilengitide reversed these effects, underscoring the essential role of RGD-integrin interactions in ASC-mediated angiogenesis. These findings suggest that gelatin sponges enhance ASC’s regenerative and angiogenic properties via RGD-dependent mechanisms, offering promising therapeutic potential for tissue repair and vascular regeneration. Understanding how RGD modulates ASC behavior provides valuable insights into advancing cell-based regenerative therapies.

Spheroids and organoids are important novel players in medical and life science research. They are gradually replacing two-dimensional (2D) cell cultures. Indeed, three-dimensional (3D) cultures are closer to the in vivo reality and open promising perspectives for academic research, drug screening, and personalized medicine. A large variety of cells and tissues, including tumor cells, can be the starting material for the generation of 3D cultures, including primary tissues, stem cells, or cell lines. A panoply of methods has been developed to generate 3D structures, including spontaneous or forced cell aggregation, air–liquid interface conditions, low cell attachment supports, magnetic levitation, and scaffold-based technologies. The choice of the most appropriate method depends on (i) the origin of the tissue, (ii) the presence or absence of a disease, and (iii) the intended application. This review summarizes methods and approaches for the generation of cancer spheroids and organoids, including their advantages and limitations. We also highlight some of the challenges and unresolved issues in the field of cancer spheroids and organoids, and discuss possible therapeutic applications.

This study evaluates the influence of a gelatin sponge on adipose-derived stromal cells (ASC). Transcriptomic data revealed that, compared to ASC in a monolayer, a cross-linked porcine gelatin sponge strongly influences the transcriptome of ASC. Wound healing genes were massively regulated, notably with the inflammatory and angiogenic factors. Proteomics on conditioned media showed that gelatin also acted as a concentrator and reservoir of the regenerative ASC secretome. This secretome promoted fibroblast survival and epithelialization, and significantly increased the migration and tubular assembly of endothelial cells within fibronectin. ASC in gelatin on a chick chorioallantoic membrane were more connected to vessels than an empty sponge, confirming an increased angiogenesis in vivo. No tumor formation was observed in immunodeficient nude mice to which an ASC gelatin sponge was transplanted subcutaneously. Finally, ASC in a gelatin sponge prepared from outbred rats accelerated closure and re-vascularization of ischemic wounds in the footpads of rats. In conclusion, we provide here preclinical evidence that a cross-linked porcine gelatin sponge is an optimal carrier to concentrate and increase the regenerative activity of ASC, notably angiogenic. This formulation of ASC represents an optimal, convenient and clinically compliant option for the delivery of ASC on ischemic wounds.

Chronic wounds, ie, non-healing ulcers, have a prevalence of ~1% in the general population. Chronic wounds strongly affect the quality of life and generate considerable medical costs. A fraction of chronic wounds will heal within months of appropriate treatment; however, a significant fraction of patients will develop therapy-refractory chronic wounds, leading to chronic pain, infection, and amputation. Given the paucity of therapeutic options for refractory wounds, cell therapy and in particular the use of adipose-derived stromal cells (ASC) has emerged as a promising concept. ASC can be used as autologous or allogeneic cells. They can be delivered in suspension or in 3D cultures within scaffolds. ASC can be used without further processing (stromal vascular fraction of the adipose tissue) or can be expanded in vitro. ASC-derived non-cellular components, such as conditioned media or exosomes, have also been investigated. Many in vitro and preclinical studies in animals have demonstrated the ASC efficacy on wounds. ASC efficiency appears to occurs mainly through their regenerative secretome. Hitherto, the majority of clinical trials focused mainly on safety issues. However more recently, a small number of randomized, well-controlled trials provided first convincing evidences for a clinical efficacy of ASC-based chronic wound therapies in humans. This brief review summarizes the current knowledge on the mechanism of action, delivery and efficacy of ASC in chronic wound therapy. It also discusses the scientific and pharmaceutical challenges to be solved before ASC-based wound therapy enters clinical reality.

Not all antibodies against SARS-CoV-2 inhibit viral entry, and hence, infection. Neutralizing antibodies are more likely to reflect real immunity; however, certain tests investigate protein/protein interaction rather than the fusion event. Viral and pseudoviral entry assays detect functionally active antibodies but are limited by biosafety and standardization issues. We have developed a Spike/ACE2-dependent fusion assay, based on a split luciferase. Hela cells stably transduced with Spike and a large fragment of luciferase were co-cultured with Hela cells transduced with ACE2 and the complementary small fragment of luciferase. Cell fusion occurred rapidly allowing the measurement of luminescence. Light emission was abolished in the absence of Spike and reduced in the presence of proteases. Sera from COVID-19-negative, non-vaccinated individuals or from patients at the moment of first symptoms did not lead to a significant reduction of fusion. Sera from COVID-19-positive patients as well as from vaccinated individuals reduced the fusion. This assay was more correlated to pseudotyped-based entry assay rather than serology or competitive ELISA. In conclusion, we report a new method measuring fusion-inhibitory antibodies in serum, combining the advantage of a complete Spike/ACE2 interaction active on entry with a high degree of standardization, easily allowing automation in a standard bio-safety environment.

Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the underlying mechanisms of the disease are still poorly understood. In this study, we investigated fusion inhibitory antibodies (fiAbs) in elderly and younger COVID-19 patients and analyzed predictive factors for their occurrence. Methods: Data and samples were collected in two cohorts of hospitalized patients. A fusion assay of SARS-CoV-2 spike-expressing cells with ACE2-expressing cells was used to quantify fiAbs in the serum of patients. Results: A total of 108 patients (52 elderly (mean age 85 ± 7 years); 56 young (mean age 52 ± 10 years)) were studied. The concentrations of fiAbs were lower in geriatric patients, as evidenced at high serum dilutions (1/512). The association between fiAbs and anti-Spike Ig levels was weak (correlation coefficient < 0.3), but statistically significant. Variables associated with fusion were the delay between the onset of symptoms and testing (HR = −2.69; p < 0.001), clinical frailty scale (HR = 4.71; p = 0.035), and WHO severity score (HR = −6.01, p = 0.048). Conclusions: Elderly patients had lower fiAbs levels after COVID-19 infection. The decreased fiAbs levels were associated with frailty.

Angiogenesis and bone regeneration are closely interconnected processes. Whereas type-H blood vessels are abundantly found in the osteogenic zones during endochondral long bone development, their presence in flat bones’ development involving intramembranous mechanisms remains unclear. Here, we hypothesized that type-H-like capillaries that highly express CD31 and Endomucin (EMCN), may be present at sites of intramembranous bone development and participate in the control of osteogenesis. A rabbit model of calvarial bone augmentation was used in which bone growth was controlled over time (2–4 weeks) using a particulate bone scaffold. The model allowed the visualization of the entire spectrum of stages throughout bone growth in the same sample, i.e., active ossification, osteogenic activity, and controlled inflammation. Using systematic mRNA hybridization, the formation of capillaries subpopulations (CD31–EMCN staining) over time was studied and correlated with the presence of osteogenic precursors (Osterix staining). Type-H-like capillaries strongly expressing CD31 and EMCN were identified and described. Their presence increased gradually from the regenerative zone up to the osteogenic zone, at 2 and 4 weeks. Type-H-like capillaries may thus represent the initial vascular support encountered in flat bones’ development and which organize osteogenic niches.