Mesenchymal stem cells (MSCs) are being extensively investigated for their potential in tissue engineering and regenerative medicine. However, recent evidence suggests that the beneficial effects of MSCs may be manifest by their released extracellular vesicles (EVs); typically not requiring the administration of MSCs. This evidence, predominantly from pre-clinical in vitro and in vivo studies, suggests that MSC-EVs may exhibit substantial therapeutic properties in many pathophysiological conditions, potentially restoring an extensive range of damaged or diseased tissues and organs. These benefits of MSC EVs are apparently found, regardless of the anatomical or body fluid origin of the MSCs (and include e.g., bone marrow, adipose tissue, umbilical cord, urine, etc). Furthermore, early indications suggest that the favourable effects of MSC-EVs could be further enhanced by modifying the way in which the donor MSCs are cultured (for example, in hypoxic compared to normoxic conditions, in 3D compared to 2D culture formats) and/or if the EVs are subsequently bio-engineered (for example, loaded with specific cargo). So far, few human clinical trials of MSC-EVs have been conducted and questions remain unanswered on whether the heterogeneous population of EVs is beneficial or some specific sub-populations, how best we can culture and scale-up MSC-EV production and isolation for clinical utility, and in what format they should be administered. However, as reviewed here, there is now substantial evidence supporting the use of MSC-EVs in tissue engineering and regenerative medicine and further research to establish how best to exploit this approach for societal and economic benefit is warranted.

Mesenchymal Stem Cell Derived Extracellular Vesicles for Tissue Engineering and Regenerative Medicine Applications.

Mesenchymal stem cells (MSCs) are on the cusp of regenerative medicine due to their differentiation capacity, favorable culture conditions, ability to be manipulated in vitro, and strong immunomodulatory activity. Recent studies indicate that the pleiotropic effects of MSCs, especially their immunomodulatory potential, can be largely attributed to paracrine factors. Exosomes, vesicles that are 30-150 nanometers in diameter that function in cell-cell communication, are one of the key paracrine effectors. MSC-derived exosomes are enriched with therapeutic miRNAs, mRNAs, cytokines, lipids, and growth factors. Emerging evidences support the compelling possibility of using MSC-derived exosomes as a new form of therapy for treating several different kinds of disease such as heart, kidney, immune diseases, neural injuries, and neurodegenerative disease. This review provides a summary of current knowledge and discusses engineering of MSC-derived exosomes for their use in translational medicine.

Current Knowledge and Future Perspectives on Mesenchymal Stem Cell-Derived Exosomes as a New Therapeutic Agent.

Extracellular vesicles act as shuttle vectors or signal transducers that can deliver specific biological information and have progressively emerged as key regulators of organized communities of cells within multicellular organisms in health and disease. Here, we survey the evolutionary origin, general characteristics, and biological significance of extracellular vesicles as mediators of intercellular signaling, discuss the various subtypes of extracellular vesicles thus far described and the principal methodological approaches to their study, and review the role of extracellular vesicles in tumorigenesis, immunity, non-synaptic neural communication, vascular-neural communication through the blood-brain barrier, renal pathophysiology, and embryo-fetal/maternal communication through the placenta.

https://www.mdpi.com/1422-0067/21/7/2514/pdf

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers.

Mesenchymal stem cells (MSC) are multipotent stromal cells with the potential to differentiate into several cell types. MSC-based therapy has emerged as a promising strategy for various diseases. Accumulating evidence suggests that the paracrine effects of MSC are partially exerted by the secretion of soluble factors, in particular exosomes. MSC-derived exosomes are involved in intercellular communication through transfer of proteins, RNA, DNA and bioactive lipids, which might constitute a novel intercellular communication mode. This review illustrates the current knowledge on the composition and biological functions as well as the therapeutic potential of MSC-derived exosomes in cancer, with a focus on clinical translation opportunities.

Biology and therapeutic potential of mesenchymal stem cell-derived exosomes.

Recently, mesenchymal stem/stromal cells (MSCs) and their widespread biomedical applications have attracted great consideration from the scientific community around the world. However, reports have shown that the main populations of the transplanted MSCs are trapped in the liver, spleen, and lung upon administration, highlighting the importance of the development of cell-free therapies. Concerning rising evidence suggesting that the beneficial effects of MSC therapy are closely linked to MSC-released components, predominantly MSC-derived exosomes, the development of an MSC-based cell-free approach is of paramount importance. The exosomes are nano-sized (30-100â��nm) lipid bilayer membrane vesicles, which are typically released by MSCs and are found in different body fluids. They include various bioactive molecules, such as messenger RNA (mRNA), microRNAs, proteins, and bioactive lipids, thus showing pronounced therapeutic competence for tissues recovery through the maintenance of their endogenous stem cells, the enhancement of regenerative phenotypic traits, inhibition of apoptosis concomitant with immune modulation, and stimulation of the angiogenesis. Conversely, the specific roles of MSC exosomes in the treatment of various tumors remain challenging. The development and clinical application of novel MSC-based cell-free strategies can be supported by better understanding their mechanisms, classifying the subpopulation of exosomes, enhancing the conditions of cell culture and isolation, and increasing the production of exosomes along with engineering exosomes to deliver drugs and therapeutic molecules to the target sites. In the current review, we deliver a brief overview of MSC-derived exosome biogenesis, composition, and isolation methods and discuss recent investigation regarding the therapeutic potential of MSC exosomes in regenerative medicine accompanied by their double-edged sword role in cancer.

/pdf/mesenchymal-stem-stromal-cell-derived-exosomes-in-29vvpyh7p7.pdf

Mesenchymal stem/stromal cell-derived exosomes in regenerative medicine and cancer; overview of development, challenges, and opportunities.

Mesenchymal stem/stromal cells (MSCs) display a variety of therapeutically relevant effects, such as the induction of angiogenesis, particularly under hypoxic conditions. It is generally recognized that MSCs exert their effects by secretion of paracrine factors and by stimulation of host cells. Furthermore, there is increasing evidence that some therapeutically relevant effects of MSCs are mediated by MSC-derived extracellular vesicles (EVs). Since our current knowledge on MSC-derived EVs released under hypoxic conditions is very limited, we aimed to characterize MSC-derived EVs from normoxic vs. hypoxic conditions (5% O2). Adipose-derived MSCs were grown under normoxic and hypoxic conditions, and EVs were analyzed by flow cytometry using lactadherin as a marker for EVs exposing phosphatidylserine, CD63 and CD81 as EV markers, as well as CD73 and CD90 as MSC surface markers. Particle concentration and size distribution were measured by nanoparticle tracking analysis (NTA), and the EV surface antigen signature was characterized using bead-based multiplex flow cytometry. Furthermore, we evaluated the potential of MSC-derived EVs obtained under hypoxic conditions to support angiogenesis using an in vitro assay with an hTERT-immortalized human umbilical vein endothelial cell (HUVEC) line. Proliferation and viability of MSCs were increased under hypoxic conditions. EV concentration, size, and surface signature did not differ significantly between normoxic and hypoxic conditions, with the exception of CD44, which was significantly upregulated on normoxic EVs. EVs from hypoxic conditions exhibited increased tube formation as compared to normoxic EVs or to the corresponding supernatants from both groups, indicating that tube formation is facilitated by EVs rather than by soluble factors. In conclusion, hypoxia conditioned MSC-derived EVs appear to be functionally more potent than normoxic MSC-derived EVs regarding the induction of angiogenesis.

/pdf/hypoxia-conditioned-mesenchymal-stem-cell-derived-4577o00fvd.pdf

Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation in vitro.

Extracellular vesicles (EVs) are cell-derived membrane structures exerting major effects in physiological as well as pathological processes by functioning as vehicles for the delivery of biomolecules to their target cells. An increasing number of effects previously attributed to cell-based therapies have been recognized to be actually mediated by EVs derived from the respective cells, suggesting the administration of purified EVs instead of living cells for cell-based therapies. In this review, we focus on the heterogeneity of EVs derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions. Hereby, we discuss the effects of the cell source, medium composition, 3D culture, bioreactor culture and hypoxia. Furthermore, aspects of the isolation and storage strategies influences EVs are described. Conclusively, optimization of upstream process parameters should focus on controlling MSC-derived EV heterogeneity for specific therapeutic applications. 

https://cellandbioscience.biomedcentral.com/track/pdf/10.1186/s13578-022-00786-7

Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions

/pdf/heterogeneity-of-mesenchymal-stem-cell-derived-extracellular-2ee1aptb.pdf

When starting to work with 3D cell cultures it is essential to be aware of the differences to common 2D cultures in terms of expenses and equipment as well as the type and dimension of 3D you intend to work with. The various culture methods require different equipment, efforts, and awareness on handling and analytics. Therefore, it is crucial to choose the right structure and architecture, the respective carrier/scaffold, the equipment for generating and culturing the models, and the methodology to manipulate and analyze them to be able to perform the process for the application answering the hypothesis. There is a wide range of commercially available lab equipment, bioreactors, consumables, carriers/scaffolds, and assays necessary to establish, perform, and analyze 3D cell culture. Handling, manipulation, and analysis of 3D cultures, differing significantly from 2D cultures, with the respective requirements and efforts will be considered as well. An overview on scaffold free 3D cell culture will be provided in Chap. 9.

Lab Equipment for 3D Cell Culture

This chapter will provide an overview on scaffold-free 3D models focusing on aggregates, spheroids, and organoids. While often used synonymously, the differences are presented as well as the origin and advancement until the current date. Moreover, the advantages that scaffold-free systems offer and their limitations are discussed as well as fields of applications ranging from stem cell expansion, pharmacological high-throughput tumor drug screening for personalized medicine, to human-on-a-chip systems facilitating the systemic investigation of drug metabolization reducing animal experiments by considering multiple organ functions within one setup. Furthermore, approaches and requirements for the generation and cultivation strategies are outlined.

Ciarra Almeria

Papers

Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation in vitro.

Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions

Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions

Lab Equipment for 3D Cell Culture

Application of Scaffold-Free 3D Models