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
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
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