Human Spheroids from Adipose-Derived Stem Cells Induce Calvarial Bone Production in a Xenogeneic Rabbit Model.

ABSTRACT: Calvarial defects can result from several causes. Tissue engineering hold the potential to restore native form and protective function. We have recently shown that stemness and differentiation ability of spheroids from adipose-derived stem cells (S-ASCs) promotes osteoblasts growth within Integra in a small vertebral lesion. In our study, we aimed to test osteogenic potential of S-ASCs in aiding regeneration of a calvarial defect. Groups containing Integra showed increased bone regeneration at the calvarial defect-Integra interface compared with the control group. In particular, S-ASC-derived osteoblasts group showed a superior calvarial remodeling than undifferentiated S-ASCs group. Clusters of ossification were observed in these both groups with enhanced microvasculature density and fibrosis. In conclusion, seeding of S-ASCs in dermal regeneration templates enhanced bone healing in a rabbit calvarial defect model. These findings could prompt the elective use of S-ASCs with enhanced multilineage differentiation potential for tissue engineering purposes.

Immunomodulation in Vascularized Composite Allotransplantation: What Is the Role for Adipose-Derived Stem Cells?

Hand and face transplants are becoming increasingly common, recording progressively more penile, uterus, abdominal wall, and allotransplantation cases reported worldwide. Despite current protocols allow long-term survival of the allografts, the ultimate goal of donor-specific tolerance has not been achieved yet. In fact, the harmful adverse effects related to the lifelong administration of immunosuppressive agents are the main drawbacks for vascularized composite allotransplantations. Research is very active in investigating alternative methods to induce greater tolerance while minimizing toxicity. Adipose-derived stem cells (ASCs) represent promising cell therapies for immunomodulation in preclinical and clinical settings. Their clinical appeal is due to their easy harvest in large quantities through a noninvasive and well-accepted approach; they may well promote donor-specific tolerance and potentially reduce immunosuppression. Several experimental studies exist, but lacking review articles reporting current evidence. This work proposes a literature review on the immunomodulatory role of ASCs in vascularized composite allotransplantations. In vitro and in vivo evidence will be summarized. The role that cell passaging and upstream progenitors-the so-called spheroid ASCs-may play in modulating the immune response will also be discussed. Finally, this article will summarize current knowledge on biodistribution, migration, and homing of injected stem cells. This review may well provide useful information for preclinical and clinical studies, aiming at a breakthrough for donor-specific tolerance.

Spheroids from adipose-derived stem cells exhibit an miRNA profile of highly undifferentiated cells.

Two-dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three-dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as "spheroids," and we have previously demonstrated that spheroids from adipose-derived stem cells (S-ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S-ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S-ASCs. In this study, we investigate for the first time the miRNA expression pattern in S-ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long-term in vitro culture. The data reported in the current study suggest that S-ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.

Tumor-infiltrating γδ T cells as targets of immune checkpoint blockade in melanoma.

Melanoma is one of the most sensitive tumors to immune modulation, and the major challenge for melanoma patients' survival is immune checkpoint inhibitor (ICI) therapy. γδ T lymphocytes play an antitumoral role in a broad variety of tumors including melanoma and they are optimal candidates for cellular immunotherapy. Thus, a comprehensive analysis of the correlation between γδ T cells and immune checkpoint receptors in the context of melanoma was conducted, with the aim of devising an innovative combined immunotherapeutic strategy. In this study, using the GEPIA2.0 database, a significant positive correlation was observed between the expression of γδ T cell-related genes (TRGC1, TRGC2, TCRD) and immune checkpoint genes (PDCD1, HAVCR2, LAG3), highlighting the potential role of γδ T cells in the immune response within melanoma. Moreover, flow cytometry analysis unveiled a significant augmentation in the population of γδ T cells within melanoma lesions, which exhibited the expression of immune checkpoint receptors including LAG3, TIM3, and PD1. Analysis of single-cell RNA sequencing data revealed a significant enrichment and functional reprogramming of γδ T cell clusters in response to ICIs. Interestingly, the effects of ICI therapy varied between Vδ1 and Vδ2 γδ T cell subsets, with distinct changes in gene expression patterns. Last, a correlation analysis between γδ T cell abundance, immune checkpoint gene expression, and clinical outcomes in melanoma patients showed that low expression of immune checkpoint genes, including LAG3, HAVCR2, and PDCD1, was associated with improved 1-year overall survival, emphasizing the significance of these genes in predicting patient outcomes, potentially outweighing the impact of γδ T cell abundance. This study offers critical insights into the dynamic interaction between γδ T cells, immune checkpoint receptors, and melanoma, providing valuable perspectives for potential therapeutic avenues and predictive markers in this intricate interplay.

Spheroids of adipose derived stem cells show their potential in differentiating towards the angiogenic lineage.

INTRODUCTION: Adipose derived stem cells (ASCs) are a mesenchymal stem cell population of great scientific interest due to their abundance and easiness in obtaining them from adipose tissue. Recently, several techniques for three dimensional (3D) ASCs cultivation have been developed to obtain spheroids of adipose stem cells (SASCs). It was already proved that ASCs are able to differentiate towards the endothelial lineage thus, for the first time, we investigated the ability of our 3D SASCs to differentiate endothelially and the effects of not differentiated SASC secreted factors on specific cultured cells. MATERIALS AND METHODS: SASCs were differentiated with a specific medium towards endothelial lineage. Cell viability, gene and protein expression of typical endothelial markers were analysed. Moreover, tube formation, wound healing and migration assays were performed to investigate the ability in migration and angiogenic networks formation of endothelially differentiated cells. SASCs secretome were also tested. RESULTS: We showed the ability of SASCs to differentiate towards the endothelial lineage with an increase in cell viability of 15-fold and 8-fold at 14 and 21 days of differentiation respectively. Moreover, we showed the upregulation of VEGF-A and CD31 mRNAs of 9-fold and 1300-fold in SASCs endothelially differentiated cells, whilst protein expression was different. VEGF-A protein expression was upregulated whilst CD31 protein wasn't translated. In addition, ICAM1, VCAM1, ANGPT1, CD62E protein levels remain unchanged. SASCs were also able to organize themselves into angiogenic networks after 7 days of culturing themon ECMatrix. Secreted factors from undifferentiated 3D SASCs acted in a paracrine way on HUVECs and endothelially differentiated ASCs seeded on ECMatrix to promote angiogenic events. CONCLUSIONS: SASCs, thanks to their multilineage differentiation potential, also possess the ability to differentiate towards endothelial lineage and to organize themselves into angiogenic networks. Moreover, they are able to promote angiogenesis through their secreted factors.

Adipose tissue, angiogenesis and angio-MIR under physiological and pathological conditions.

Angiogenesis is a crucial process for the maintenance of normal tissue physiology and it is involved in tissue remodeling and regeneration. This process is essential for adipose tissue maintenance. The adipose tissue is composed by different cell types including stromal vascular cells as well as adipose stem cells (ASCs). In particular, ASCs are multipotent somatic stem cells that are able to differentiate and secrete several growth factors; they are recently emerging as a new cell reservoir for novel therapies and strategies in many diseases. Several studies suggest that ASCs have peculiar properties and participate in different disease-related processes such as angiogenesis. Furthermore, pathological expansion of adipose tissue brings to hypoxia, a major condition of unhealthy angiogenesis. Recent evidences have shown that microRNAs (miRNAs) play a crucial role also on ASCs as they take part in stemness maintenance, proliferation, and differentiation. It has been suggested that some miRNAs (MIR126, MIR31, MIR221 MIR222, MIR17-92 cluster, MIR30, MIR100 and MIR486) are directly involved in the angiogenic process by controlling multiple genes involved in this pathway. With the present review, we aim at providing an updated summary of the importance of adipose tissue under physiological and pathological conditions and of its relationship with neovascularization process. In particular, we report an overview of the most important miRNAs involved in angiogenesis focusing on ASCs. Hopefully the data presented will bring benefit in developing new therapeutic strategies.

Solitary Fibrous Tumor of the Lower Leg: A Rare and Difficult Diagnosis.

Solitary fibrous tumor (SFT) is a rare neoplasm that commonly originates in the pleura. Extrapleural locations are rare and for this reason sometimes difficult to diagnose. Malignant forms with local recurrence or distant metastases have been reported, also as a consequence of inappropriate treatment. In this article, we report the case of an SFT of the lower leg in a 37-year-old man. Leg SFT is a rare occurrence, and differential diagnosis may be difficult because they can mimic a variety of benign and malignant mesenchymal tumors; immunohistochemical analysis for CD34, CD99, vimentin, and Bcl-2 is necessary. Misdiagnosis carries a significant risk of inadequate removal with subsequent increased risk of recurrence and distant metastases.