细胞生物学

Muscle stem cells (satellite cells), located on the surface of myofibers, are rapidly activated from a quiescent state following skeletal muscle injury. Although satellite cell activation is an initial step in muscle regeneration, the stimulation of satellite cell activation by muscle injury remains to be elucidated. We recently established an in vitro mechanical damage model of myofibers, to analyze quiescent and activated satellite cells associated with myofibers isolated from the extensor digitorum longus muscle in mice. Here, we described a protocol for the mechanical damage of myofibers and co-culture of intact healthy myofibers with damaged myofibers in a floating condition. This in vitro myofiber damage model allowed us to investigate the mechanism of satellite cell activation without contamination by interstitial cells, such as blood vessel cells and fibroblasts, as well as understand how damaged myofiber-derived factors (DMDFs) activate satellite cells.

Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in vitro. We further show how ELDA can be used on cells, before and after forced differentiation to amplify inherent differences in sphere-forming potential between mutant and control NSCs. Ultimately, ELDA exploits a difference in the ability of a single or a few seeded stem cells to self-renew, divide and form neurospheres. Importantly, the assay also allows a comparison between genetically distinct cells or between the same cells under different conditions, where the impact of target-specific drugs or other novel cancer stem cell therapies can be tested.

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.

Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells (or cancer stem cells) self-renewal potential in vitro. When single cells are plated at low density in stem cell culture medium, only the cells endowed with self-renewal are able to grow in tridimensional clusters usually named spheres. In the recent years, SFA has also been used also to test the effect of several drugs, chemical and natural compounds or microenviromental components on stem cells self-renewal capacity. Here we will illustrate a detailed protocol to assess self-renewal of human melanoma stem cells, growing as melanospheres.