Lab 3 - I. INTRODUCTION Mesenchymal stem cells, or MSCs,...

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I. INTRODUCTION Mesenchymal stem cells, or MSCs, are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, and, as described lately, beta- pancreatic islets cell. However, in vivo results are now thought to be a misinterpretation of spontaneous cell fusion between a damaged neuron and a mesenchymal stem cell placed in the brain (Wikepedia). Bone, despite its hard and mineralized structure is alive. Osteoblasts are the cells that produce the organic and inorganic matrix of bone attract a blood supply to them and then become embedded within the bone to help maintain the organ (now, osteoblasts form osteocytes). The organic matrix of bone is composed primarily of collagen and other binding proteins and the inorganic matrix is the mineralized part consisting primarily of calcium and alkaline phosphate/hydroxy apatite (Manaster 92). When MSCs are cultured, they could potentially develop into any of the connective tissues by introducing various reagents and growth factors in the media. For our experiment, we will try to produce bone. To do so, we will use ascorbic acid (helps collagen formation) and beta glycerol phosphate (an inorganic phosphate that can signal for mineralization). During this portion of the lab, we used centrifugation in order to plate our cells into differentiation media. Cells aggregate out the media during centrifugation due to buoyant, drag, and centrifugal forces (Manaster 89). This will allow a pellet of cells to form at the bottom of the tubes, allowing us to remove all the excess media, before resuspending cells in differentiation media. This will also help increase the concentration of cells. Next to visualize our cells, we will use Von Kossa method for staining to quantitate the presence of calcium phosphate. It is a histochemical stain for an adherent cell population to determine the covered area of cells that exhibit alkaline phosphatase enzyme and those areas that have mineralized (Manaster 111). The alkaline phosphatase positive areas appear red, while the mineralized areas are brown/black in color. Next performed two assays using our lysates to visualize the differentiation of the MSC to osteoblasts and production mineralized matrix. To quantitatively assess the amount of alkaline phosphatase produced by the bone. Alkaline phosphatase catalyzes the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation of an organic radical and inorganic phosphate. For this we will specifically use p-Nitrophenyl Phosphate, a reagent to indicate concentration of alkaline phosphatase in the sample. We expect the alkaline phosphataseto peak at 2 weeks and diminish as the mineral is laid down. Then we will use Calcium Assay Kit to quantitate the amount of calcium present in our
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Lab 3 - I. INTRODUCTION Mesenchymal stem cells, or MSCs,...

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