干细胞

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert et al., 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Differentiation assays have been performed with primary salivary cells in the past (Maimets et al., 2016), however this approach may sometimes be limiting due to tissue availability, labor intensity of processing the tissue samples, and/or inability to long-term passage the cells. Here we describe in detail a 3D differentiation assay to analyze the differentiation potential of a salivary gland cell line, SIMS, which was immortalized from an adult mouse submandibular salivary gland (Laoide et al., 1996). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions.

In the last years, planarians have emerged as a unique model animal for studying regeneration and stem cells biology. Although their remarkable regenerative abilities are known for a long time, only recently the molecular tools to understand the biology of planarian stem cells and the fundamentals of their regenerative process have been established. This boost is due to the availability of a sequenced genome and the development of new technologies, such as interference RNA and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular and genetic level. For these reasons, maintain a healthy and stable planarian population in the laboratory is essential to perform reproducible experiments. Here we detail the protocol used in our laboratory to maintain the planarian species Schmidtea mediterranea, the most widespread as a model.

Mesenchymal stem cells (MSCs) are currently intensively studied due to significant promise which they represent for successful implementations of future cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies of MSC effects in various murine disease models. The appropriate cell preparations with reproducible biological properties are important to minimize variability of results of experimental cell therapies. We describe here a simple protocol for isolation of murine MSCs from adipose tissues and their reproducible multi-log expansion under hypoxia conditions.

Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured cells.

Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.

Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016).

The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker’s protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016).

Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave “pits” after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive activity based on the number, size and depth of pits (Zhou et al., 2015). The pit assay protocol is separated into three sequential stages: Preparation of bone slices (1); preparation of osteoclast precursors (Ross et al., 2006; Teitelbaum et al., 2000) (2), and bone resorption pit assay (3).

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects (Bornes et al., 2014). BMSCs can be seeded within porous biomaterial scaffolds that support three-dimensional cell organization, chondrogenic differentiation and extracellular matrix deposition for the creation of engineered cartilage. This protocol describes our defined methods for isolation and expansion of human and ovine BMSCs, seeding of BMSCs within porous scaffolds and in vitro chondrogenic differentiation (Adesida et al., 2012; Bornes et al., 2015).

Mature skeletal myofibers are elongated and multinucleated cells. Many stem/progenitor cell types, including committed muscle stem (satellite cells) and progenitor (myoblasts) cells, muscle-derived stem cells, myogenic endothelial cells, and mesenchymal stem/stromal cells, have been shown to exhibit skeletal myogenesis under appropriate inductive conditions. Committed muscle stem/progenitor cells and multipotent stem/progenitor cells which have skeletal myogenic capacity can typically be differentiated into skeletal myofibers in vitro following extended low-serum exposure. Differentiated cells exhibit distinct fiber-like elongated morphology with multiple nuclei and express unique muscle molecular markers indicating myogenesis, including desmin (early) and fast- and/or slow-myosin heavy chain (mature).