Single-Cell Assays Using Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem cells (HSCs) undergo division, making two daughter cells with unique fate decision choices, that is, whether to self-renew to maintain stemness or differentiate to committed progenitors. Since HSCs are heterogeneous in nature understanding this phenomenon at the single cell level is important. In vitro single-cell assays like the paired-daughter cell and myeloid multilineage differentiation are useful to understand this unique stem cell process. Both assays are performed using cytokine combination which allows four-lineage myeloid differentiation-neutrophil, erythroid, macrophage/monocyte, and megakaryocyte. Paired-daughter cell assay examines symmetric or asymmetric retention of four myeloid lineages after first cell division in the paired-daughter cells. Thus, it defines asymmetric versus symmetric division patterns in the paired daughter cells. Thus, this assay may provide HSC fate decision cues. Myeloid multilineage differentiation assay examines the ability of a single cell to form multipotent clones containing four or less myeloid lineages. Here, we discuss in detail methodology of these assays.

3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS.

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.

Differentiation of Brain Tumor Initiating Cells.

Differentiation is a central key capability of stem cells. Their ability to be multipotent and undergo self-renewal are key identifying features of stem cells. A differentiation assay allows for study of one of the essential features of stem cells, the ability to differentiate into all of the cell types of its lineage, in order to ensure that the cells cultured and utilized in key experiments indeed have stem cell properties. Neural stem cells when plated in differentiation media, differentiate into all three neural lineages: Neurons, Astrocytes, and Oligodendrocytes. Brain tumor initiating cells (BTICs) are cells present in brain tumors that possess stem cell properties and are able to self-renew and differentiate into neural lineages. In the current chapter, we discuss protocols involved in immunofluorescence staining and identification of differentiated cells from BTIC populations.

Linear array of multi-substrate tracts for simultaneous assessment of cell adhesion, migration, and differentiation.

Cell migration, which is central to a wide variety of life processes, involves integration of the extracellular matrix (ECM) with the internal cytoskeleton and motor proteins via receptors spanning the plasma membrane. Cell migration can be induced by a variety of signals, including gradients of external soluble molecules, differences in ECM composition, or electrical gradients. Current in vitro methods to study cell migration only test one substrate at a time. Here, we present a method for assessing cell adhesion, migration, and differentiation in up to 20 different test conditions simultaneously, using only minute amounts of target substrate. Our system, which we call the linear array of multi-substrate cell migration assay (LAMA), has two configurations for direct comparison of one or two cell types in response to an array of ECM constituents under the same culture conditions. This culture model utilizes only nanogram amounts of test substrates and a minimal number of cells, which maximizes the use of limited and expensive test reagents. Moreover, LAMA can also be used for high-throughput screening of potential pharmaceuticals that target ECM-dependent cell behavior and differentiation.

In-vitro and in-vivo antiadipogenic, hypolipidemic and antidiabetic activity of Diospyros melanoxylon (Roxb).

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Diospyros melanoxylon (Roxb) belongs to the family Ebenaceae that is native to India and Sri-lanka. This is a medium-sized tree, reaching a height of 15 m and is well known for its beedi making leaves throughout the world. The purpose of the present study is to assess the effect of Diospyros melanoxylon leaves petroleum ether extract on blood glucose level, lipid level, insulin level, body weight, water and food intake in Streptozotocin (STZ) induced diabetic rats. MATERIALS AND METHODS: Two different doses of extract AK001 (250 mg/kg) and AK002 (500 mg/kg) of Diospyros melanoxylon leaves were taken to evaluate different activities. The animals were divided into five groups namely normal control, diabetic control, reference group, AK001 and AK002 each containing six animals for in-vivo study. In-vitro study for antiadipogen activity was performed on 3T3-L1 cell line. RESULTS: The extract showed dose dependent fall in Fasting Glucose Level (FSG) in experimental diabetic animals with significant reduction in food and water intake and increase in body weight. The extract exhibited hypocholesterolemic and hypotriglyceridemic effects while increased level of HDL in diabetes induced rats. In-vitro activity showed more than 75% viability of cells and significant inhibition in differentiated cells as compared to non-differentiate cells in 3T3-L1 cell line. The extract exhibited the concentration-dependent inhibitory effect with an IC50 value of 689.22 µg/ml. CONCLUSIONS: The extract exhibited significant results for antiadipogenic, antidiabetic and hypolipidemic activity both in-vivo and in-vitro and it may prove to be effective for the treatment of both types of diabetes, i.e. Insulin Dependent Diabetes Mellitus (IDDM) and Noninsulin Dependent Diabetes Mellitus (NIDDM).