Angiogenesis is defined as the generation of new blood vessels or the sprouting of endothelial cells from a pre-existing vascular network. Angiogenesis occurs during the growth and development of an organism, the response of organs or tissues to injury, and during cancer development and progression. The majority of studies on stem-cell-derived extracellular vesicles (EVs) have used cell lines, and have primarily focused on well-known solitary proteins. Here, we isolated stem cells from human adipose tissue (ADSCs), and we isolated EVs from them (ADSC-EVs). The ADSC-EVs were characterised and 20 angiogenic proteins were analysed using an angiogenic antibody array. Furthermore, we analysed the ability of ADSC-EVs to induce angiogenesis in vitro and in vivo. ADSC-EVs were positive for CD81 and negative for GM130, calnexin, and cytochrome-C. ADSC-EVs showed typical EV spherical morphology and were ~200 nm in size. ADSC-EVs were found to contain angiogenic proteins as cargo, among which interleukin 8 (IL-8) was the most abundant, followed by chemokine (C-C motif) ligand 2 (CCL2), a tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and vascular endothelial growth factor-D (VEGF-D). ADSC-EVs treatment increased the proliferation, migration, total vessel length, total number of junctions, and junction density of endothelial cells in vitro. The results of an in vivo Matrigel plug assay revealed that ADSC-EVs induced more blood vessels in the Matrigel compared with the control. These results demonstrate that ADSC-EVs contain angiogenic proteins as cargo and promote angiogenesis in vitro and in vivo. Therefore, ADSC-EVs have potential for therapeutic use in ischaemia.

Stem cells can be defined as the cells that have the capacity to both self-renew and give rise to differentiated cells. Under the right conditions and signals, depending on their origin and bio-plasticity, stem cells can differentiate into multiple cell lineages and develop into various mature cells. Stem cell therapy is a fast-developing branch of medicine that includes the most innovative regenerative therapies for the restoration of cell and tissue function in individuals with severe diseases. Stem cell research has resulted in the emergence of cell-based therapies for disorders that are resistant to conventional drugs and therapies, and they are considered under the category of an Advanced Therapeutic Medicinal Product (ATMP). The FDA and the European Medicines Agency (EMA) devised a new strategy in 2017 with the aim of unifying the standards for development of ATMPs such that it is easy to exchange information at the international level. In this review, we discuss the evolution of mesenchymal stem cell-based therapy as an ATMP in the global and Indian scenarios, along with the guidelines governing their usage and clinical application of these therapeutics.

A complete redevelopment of the skin remains a challenge in the management of acute and chronic wounds. Recently, the application of extracellular vesicles (EVs) for soft tissue wound healing has received much attention. As fibroblasts are fundamental cells for soft tissues and skin, we investigate the proangiogenic factors in human normal fibroblast-derived EVs (hNF-EVs) and their effects on wound healing. Normal fibroblasts were isolated from human skin tissues and characterized by immunofluorescence (IF) and Western blotting (WB). hNF-EVs were isolated by ultracentrifugation and characterized using transmission electron microscopy and WB. The proangiogenic cargos in hNF-EVs were identified by a TaqMan assay and a protein array. Other in vitro assays, including internalization assays, cell counting kit-8 analysis, scratch wound assays, WBs, and tube formation assays were conducted to assess the effects of hNF-EVs on fibroblasts and endothelial cells. A novel scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue was applied onto full-thickness skin wounds in mice. The wound healing therapeutical effect of hNF-EVs was assessed by calculating the rate of wound closure and through histological analysis. Isolated hNF was confirmed by verifying the expression of the fibroblast markers vimentin, αSMA, Hsp70, and S100A4. Isolated hNF-EVs showed intact EVs with round morphology, enriched in CD81 and CD63, and devoid of the cell markers GM130, Calnexin, and Cytochrome C. Our TaqMan assay showed that hNF-EVs were enriched in miR130a and miR210, and protein arrays showed enriched levels of the proangiogenic proteins’ vascular endothelial growth factor (VEGF)-D and CXCL8. Next, we found that the internalization of hNF-EVs into hNF increased the proliferation and migration of hNF, in addition to increasing the expression of bFGF, MMP2, and αSMA. The internalization of hNF-EVs into the endothelial cells increased their proliferation and tube formation. A scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue accelerated the wound healing rate in full-thickness skin wounds in mice, and the treatments increased the cellular density, deposition, and maturation of collagens in the wounds. Moreover, the scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue increased the VEGF and CD31 expression in the wounds, indicating that hNF-EVs have an angiogenic ability to achieve complete skin regeneration. These findings open up for new treatment strategies to be developed for wound healing. Further, we offer a new approach to the efficient, scaffold-free noninvasive delivery of hNF-EVs to wounds.

The two-dimensional (2D) monolayer culture as a conventional method has been widely applied in molecular biology fields, but it has limited capability to recapitulate real cell environments, being prone to misinterpretation with poor prediction of in vivo behavior. Recently, the three-dimensional (3D) spheroid culture has been studied extensively. Spheroids are self-assembled cell aggregates that have biomimicry capabilities. The behavior of thyroid cancer under the 3D spheroid culture environment has been studied; however, there are no reports regarding differences in the degree of thyroid cancer cell differentiation under 2D and 3D culture conditions. This study investigated the expression of thyroid differentiation proteins related to iodide-metabolizing mechanisms in thyroid cancer cells under different culture conditions. Four thyroid cancer cell lines and one thyroid follicular epithelial cell line were grown in adherent 2D cell culture and 3D spheroid culture with agarose-coated plates. We observed changes in proliferation, hypoxia, extracellular matrix (ECM), cytoskeleton, thyroid-specific proteins, and thyroid transcription factors. All cell lines were successfully established in the spheroid following cell aggregation. Proliferation considerably decreased, while hypoxia-inducible factor 1-α(HIF1-α) was promoted in 3D spheroids; moreover, 3D spheroids with thyroid cancers showed diminished thyroid differentiation markers, but thyroid follicular epithelial cells revealed either a maintenance or weak decline of protein expression. We verified that the 3D spheroid culture environment can be similar to in vivo conditions because of its alterations in numerous cellular and functional activities, including morphology, cellular proliferation, viability, hypoxia, ECM, cytoskeleton, and thyroid differentiation, compared to the conventional 2D monolayer culture environment. An in vitro experimental study using 3D spheroid culture is ideal for the faster discovery of new drugs.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been demonstrated to deliver therapeutic drugs in preclinical studies. However, their use is limited, as they lack the ability to specifically deliver drugs to tumor tissues in vivo. In the present study, we propose the use of a targeting peptide, IL-4R-binding peptide (IL4RPep-1), to specifically deliver intravenously (i.v.) infused EVs to thyroid tumors. In vivo, a xenograft tumor model was treated with either the control peptide (NSSSVDK) or IL4RPep-1-Flamma; mice were fluorescently imaged (FLI) using an in vivo imaging system at 0–3 h post-treatment. EVs (labeled with DiD dye) were conjugated with IL4RPep-1 through a DOPE-NHS linker and administered to mice intravenously. FLI was performed 0–24 h post-injection, and the animals were sacrificed for further experiments. The morphology and size of EVs, the presence of EV markers such as CD63 and ALIX, and the absence of the markers GM130 and Cyto-C were confirmed. In vivo, FLI indicated an accumulation of i.v. injected IL4RPep-1-Flamma at the tumor site 90 min post-injection. No accumulation of NSSSVDK-Flamma was detected. In vivo, IL4RPep-1-EVs targeted the Cal-62 tumor 2 h post-injection. NSSSVDK-EVs were not even detected in the tumor 24 h post-injection. The quantification of FLI showed a significant accumulation of MSC-EVs in the tumor 2 h, 3 h, and 24 h post-injection. Furthermore, ex vivo imaging and an IF analysis confirmed the in vivo findings. Our results demonstrate the use of the IL4RPep-1 peptide as a targeting moiety of EVs for IL-4R-expressing anaplastic thyroid tumors.

Alopecia is a common medical condition affecting both sexes. Dermal papilla (DP) cells are the primary source of hair regeneration in alopecia patients. Therapeutic applications of extracellular vesicles (EVs) are restricted by low yields, high costs, and their time-consuming collection process. Thus, engineered nanovesicles (eNVs) have emerged as suitable therapeutic biomaterials in translational medicine. We isolated eNVs by the serial extrusion of fibroblasts (FBs) using polycarbonate membrane filters and serial and ultracentrifugation. We studied the internalization, proliferation, and migration of human DP cells in the presence and absence of FB-eNVs. The therapeutic potential of FB-eNVs was studied on ex vivo organ cultures of human hair follicles (HFs) from three human participants. FB-eNVs (2.5, 5, 7.5, and 10 µg/mL) significantly enhanced DP cell proliferation, with the maximum effect observed at 7.5 µg/mL. FB-eNVs (5 and 10 µg/mL) significantly enhanced the migration of DP cells at 36 h. Western blotting results suggested that FB-eNVs contain vascular endothelial growth factor (VEGF)-a. FB-eNV treatment increased the levels of PCNA, pAKT, pERK, and VEGF-receptor-2 (VEGFR2) in DP cells. Moreover, FB-eNVs increased the human HF shaft size in a short duration ex vivo. Altogether, FB-eNVs are promising therapeutic candidates for alopecia.