Stem Cell

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic.

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Extracellular vesicles (EVs) are a heterogeneous group of membranous vesicles that differ on their biogenesis and release pathways, such as exosomes, microvesicles and apoptotic bodies. They are involved in cell-to-cell communication delivering signal molecules (proteins, nucleic acids, lipids, etc.) that can regulate different physiological processes, as well as the development and progression of several diseases. There are different methods and commercial kits to isolate EVs and depending on the methodology one could obtain EVs with different degrees of efficiency, purity and it can be more or less time-consuming. Then, the choice has to be according to the different advantages and disadvantages, and their use for downstream applications. Here, we describe the EVs isolation method from mesenchymal stromal cells by ultracentrifugation. This EVs isolation can be performed using common media and buffers, and only with the requirement of an analytical ultracentrifuge. Moreover, this method can be used to obtain large quantity of EVs with a good reproducibility for developing in vitro and in vivo experiments and studying their biological actions.

Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.

Mesenchymal stem cells (MSCs) have attracted significant attention as potential therapeutic cells to treat various diseases ranging from tissue injuries, graft versus host disease, degenerative diseases and cancer. Since the initial discovery of MSCs in the bone marrow cells, MSCs have been successfully isolated from various adult and neo-natal tissues, albeit the procedures are often coupled with difficulties in harvesting tissue and produce low yield of cells, requiring extensive expansion in vitro. Here, we explored extra-ocular muscle tissues obtained from patients as a novel source of MSCs which express characteristic cell surface markers of MSCs and show multilineage differentiation potential with high proliferation capacity.

Mesenchymal stem cells (MSCs) are invaluable cell sources for understanding stem cell biology and potential application in tissue engineering and regenerative medicine. The current issues of MSCs that demand to be further addressed are limited donors, tissue sources and limited capacity of ex vivo expansion. Here, we describe a simple and easy protocol for generating functional mesenchymal stem cells from human pluripotent stem cells (hPSCs) via one-step low glucose medium switch strategy in feeder-free culture system. In this protocol, human induced pluripotent stem cells (hiPSCs) and H9 human embryonic stem cells (hESCs) were successfully differentiated into MSCs, named hiPSC-MSCs and hESC-MSCs, respectively. The derived hiPSC-MSCs and hESC-MSCs exhibited common MSC characteristics as MSCs derived from human bone marrow (hBM-MSCs), including expressing MSC surface markers and possessing capability of tri-lineage differentiation in vitro (adipogenesis, osteogenesis and chondrogenesis). As compared with other available protocols, our protocol can be applied to generate a large number of MSCs from hPSCs with high efficiency, low-cost manner, moreover, not involving embryoid body, mouse feeder-cell, flow sorting, and pathway inhibitors (such as SB203580 and SB431542). We believe that this protocol could provide a robust platform to reach the future demand for producing the industrial scale of MSC from hPSCs for autologous cell-based therapy.

The 3D culture of human mesenchymal stem cells (hMSCs) represents a more physiological environment than classical 2D culture and has been used to enhance the MSC secretome or extend cell survival after transplantation. Here we describe a simple and affordable method to generate 3D spheroids of hMSCs by seeding them at high density in a low-binding 96-well plate.

Spheroids of hMSCs cultured in low-binding 96-well plates can be used to study the basic biology of the cells and to generate conditioned media or spheroids to be used in transplantation therapeutic approaches. These MSCs or their secretome can be used as a regenerative therapy and for tissue repair across multiple disease areas, including neurodegeneration.

In comparison to other methods (hanging drop, use of gels or biomaterials, magnetic levitation, etc.), the method described here is simple and affordable with no need to use specialized equipment, expensive materials or complex reagents.

Mesenchymal stem cells (MSCs) have shown profound therapeutic potential in tissue repair and regeneration. However, recent studies indicate that MSCs are largely entrapped in lungs after intravenous delivery and die shortly. The underlying mechanisms have been poorly understood. We have provided evidence to show that excess expression and activation of integrins in culture-expanded MSCs is a critical cause of MSCs adhesion to endothelial cells of the lung microarteries resulting in the entrapment of the cells (Wang et al., 2015). Therefore, it may be meaningful to test the adhesive ability of MSCs to endothelial cells in vitro before intravenous administration to avoid their lung vascular obstructions. Here we report a simple method to measure MSCs attachment to endothelial cells.

Mesenchymal stem cells (MSCs) are currently considered as ‘medicinal signaling cells’ and a promising resource in regard to cell-based regenerative therapy. Umbilical cord is a human term perinatal tissue which is easily attainable, and a promising source of stem cells with no associated ethical concerns. MSCs have been isolated from different regions of the umbilical cord and Wharton’s jelly (WJ) is the gelatinous matrix that surrounds and provides protection to the umbilical cord blood vessels. Being more primitive, MSCs from human umbilical cord exhibit greater proliferative capacity and immunosuppressive ability as compared to adult stem cells which gives them a therapeutic advantage. To meet the requirements for cell therapy, it is important to generate MSCs at a clinical scale by following steps which are not time consuming or labor intensive. Here we present a simple, efficient protocol for isolation of MSCs from WJ of human umbilical cord by explant culture method which is reproducible and also, cost effective.

Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and β-lactoglobulin have material properties that mimic the extracellular microenvironment of many cell types. Cells cultured on such amyloid fibril networks show improved attachment, spreading and in the case of mesenchymal stem cells improved differentiation. Here we describe a detailed protocol for fabricating amyloid fibril networks suitable for eukaryotic cell culture applications.

Human endometrial stem cell/stromal cells (hEnSCs) are isolated from endometrium or menstrual blood and are recognized as a valuable cell type in tissue engineering and cell therapy. Furthermore, hEnSCs, which have CD90 (a mesenchymal marker), CD105 (endoglin), CD44, CD146 (endometrial stem cell markers) and lack CD31 (Endothelial marker), CD34 (hematopoietic marker) and CD133 on the cell surface, are a new source of mesenchymal stem/stromal cells. Additionally, these cells can be encapsulated into self-assembling peptide nanofibers as a 3D scaffold for applications in the treatment of neurodegenerative diseases. Here, we describe a protocol to isolate hEnSCs from endometrium or menstrual blood.