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.

An analysis of the immunomodulatory properties of human spheroids from adipose-derived stem cells.

AIMS: Current methods to induce tolerance following allotransplantation or in autoimmunity carry significant morbidity, and research is very active in investigating alternative methods which could minimize toxicity. Spheroids from adipose stem cells (SASCs) are increasingly gaining interest, they hold a great proliferative and differentiating potential. An immunomodulatory effect has not been investigated on SASCs yet. In this study, we analysed the immunomodulatory properties of SASCs and compared them to ADSCs. MAIN METHODS: Adipose stem cells (SASCs and ADSCs) and peripheral blood mononuclear cells (PBMCs) were collected from healthy individuals. We analysed the cytokine production and proliferation of T cells co-cultured with adipose samples or conditioned medium. KEY FINDINGS: SASCs modulated cytokines production and proliferation of heterologous and autologous T cells. In the heterologous assays, we observed a reduction of IFNγ and IL-17 production and an increase of IL-9 in γδ T cells. The soluble factors present in SASCs sovranatants were also able to induce a slight reduction of IFNγ and an increase of IL-9, IL-10 and IL-17 while they could not modulate the proliferative ability of γδ T cells. In the autologous assays, we observed a reduction of the proliferative ability of T cells in co-culture at different ratios with SASCs. Analysis of the SASCs secretome showed an increased IL-5, IL-10, IL-4 and IL-13 production compared to the ADSCs one, demonstrating greater anti-inflammatory properties. SIGNIFICANCE: Our preliminary results support the idea that SASCs exert more pronounced biological immune modulation compared to the classical adherent ADSCs, especially in heterologous experimental settings.

κ-Carrageenan and PVA blends as bioinks to 3D print scaffolds for cartilage reconstruction.

3D printing of polymeric scaffolds and autologous stem cells is a promising tool for damaged facial cartilage reconstruction surgeries. To this end, suitable bioinks are needed to generate scaffolds with the required morphological and functional features. We formulated hydrogel bioinks using k-Carrageen (kC) and poly(vinyl alcohol) (PVA) in three different weight ratios. The kC gives the systems the ability to undergo rapid sol-to-gel transitions upon cooling from 60 °C and above to body temperature, while the PVA is used as rheology modifier and porogen. The latter is crosslinked after molding or printing by freeze-thaw cycling for 1 day (FT1) or 5 days (FT5). To select the most suitable formulation for 3D printing, the sol-to-gel transition and the physico-chemical, mechanical and morphological properties of obtained hydrogels were studied. Moreover, the absence of cytotoxic effects of the material on SASCs was assessed in both stemness-preserving or chondro-inductive media. Printing trials were performed to identify optimal process parameters and co-printing and post-printing seeding approaches of SASCs were evaluated. Cells were found to be viable after co-printing and also after the FT1 treatment. Viable adherent cells were also found in the FT5 system, where cells were plated after freezing and thawing treatment.

Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Injectable xyloglucan hydrogels incorporating spheroids of adipose stem cells for bone and cartilage regeneration.

Cartilage or bone regeneration approaches based on the direct injection of mesenchymal stem cells (MSCs) at the lesion site encounter several challenges, related to uncontrolled cell spreading and differentiation, reduced cell viability and poor engrafting. This work presents a simple and versatile strategy based on the synergic combination of in-situ forming hydrogels and spheroids of adipose stem cells (SASCs) with great potential for minimally invasive regenerative interventions aimed to threat bone and cartilage defects. Aqueous dispersions of partially degalactosylated xyloglucan (dXG) are mixed with SASCs derived from liposuction and either a chondroinductive or an osteoinductive medium. The dispersions rapidly set into hydrogels when temperature is brought to 37 °C. The physico-chemical and mechanical properties of the hydrogels are controlled by polymer concentration. The hydrogels, during 21 day incubation at 37 °C, undergo significant structural rearrangements that support cell proliferation and spreading. In formulations containing 1%w dXG cell viability increases up to 300% for SASCs-derived osteoblasts and up to 1000% for SASCs-derived chondrocytes if compared with control 2D cultures. The successful differentiation into the target cells is supported by the expression of lineage-specific genes. Cell-cell and cell-matrix interactions are also investigated. All formulations resulted injectable, and the incorporated cells are fully viable after injection.