Differentiation:
MSCs have a large capacity for self-renewal
while maintaining their multipotency. Beyond
that, there is little that can be definitively said.
The standard test to confirm multipotency is
differentiation of the cells into osteoblasts,
adipocytes, and chondrocytes as well as
myocytes and possibly neuron-like cells.
MSCs have a large capacity for self-renewal
while maintaining their multipotency. Beyond
that, there is little that can be definitively said.
The standard test to confirm multipotency is
differentiation of the cells into osteoblasts,
adipocytes, and chondrocytes as well as
myocytes and possibly neuron-like cells.
The capacity of cells to proliferate and differentiate is known to decrease with the age of
the donor, as well as the time in culture. Likewise, whether this is due to a decrease in
the number of MSCs or a change to the existing MSCs is not known. In addition to the
identification of MSCs based on their morphologic or phenotypic characteristics, a further
way to identify supposed MSC populations is by their capacity to be induced to
differentiate into bone, fat, and cartilage in vitro. The classic method for differentiation of
MSCs to osteoblasts in vitro involves incubating a confluent monolayer of MSCs with
ascorbic acid, glycerophosphate, and dexamethasone for 3 weeks. The MSCs form
aggregates or nodules and increase their expression of alkaline phosphatase; calcium
accumulation can be seen over time. These bone nodules stain positively by alizarin red
and von Kossa techniques. These conditions, however, are unlikely to reflect the
physiological signals MSCs receive that induce osteogenesis in vivo. There have been
some recent reports investigating the role of bone morphogenic proteins (BMPs) on
osteogenesis, but there appears to be species-specific differences in the effect of BMPs
in vitro.
To promote adipogenic differentiation, MSC cultures are incubated with dexamethasone,
insulin, isobutyl methyl xanthine, and indomethacin. There is an accumulation of lipid-rich
vacuoles within cells, and they express peroxisome proliferation-activated receptor
{gamma}2, lipoprotein lipase, and the fatty acid-binding protein aP2. Eventually, the
lipid vacuoles combine and fill the cells. Accumulation of lipid in these vacuoles is assayed
histologically by oil red O staining. Having first been identified for their ability to
differentiate into bone and adipocytes, further studies have demonstrated that MSCs
can also differentiate, under appropriate in vitro conditions, to form chondrocytes,
tenocytes, skeletal myocytes, neurons, and cells of visceral mesoderm (endothelial cells).
To promote chondrogenic differentiation, MSCs are centrifuged to form a pelleted
micromass and cultured in the presence of transforming growth factor. The cell pellets
develop a multilayered, matrix-rich morphology, and histological analysis shows strong
staining with toluidine blue, indicating an abundance of glycosaminoglycans within the
extracellular matrix. The cells also produce type II collagen, which is typical of articular
cartilage.It has also been demonstrated that, when treated with 5-azacytidine and
amphotericin B, MSCs differentiate into myoblasts that fuse into rhythmically beating
myotubes. In addition, differentiation into neuron-like cells expressing markers typical for
mature neurons has been reported. However, Hofstetter and colleagues reported that
these neuron-like cells lack voltage-gated ion channels necessary for generation of
action potentials; therefore, these cells may not actually be classified as true neurons
[38].
References:
Pittenger MF, Mackay AM, Beck SC et al.
Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-
147.
Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow
mesenchymal progenitor cells. J Cell Physiol 1999;181:67-73].
More References...
the donor, as well as the time in culture. Likewise, whether this is due to a decrease in
the number of MSCs or a change to the existing MSCs is not known. In addition to the
identification of MSCs based on their morphologic or phenotypic characteristics, a further
way to identify supposed MSC populations is by their capacity to be induced to
differentiate into bone, fat, and cartilage in vitro. The classic method for differentiation of
MSCs to osteoblasts in vitro involves incubating a confluent monolayer of MSCs with
ascorbic acid, glycerophosphate, and dexamethasone for 3 weeks. The MSCs form
aggregates or nodules and increase their expression of alkaline phosphatase; calcium
accumulation can be seen over time. These bone nodules stain positively by alizarin red
and von Kossa techniques. These conditions, however, are unlikely to reflect the
physiological signals MSCs receive that induce osteogenesis in vivo. There have been
some recent reports investigating the role of bone morphogenic proteins (BMPs) on
osteogenesis, but there appears to be species-specific differences in the effect of BMPs
in vitro.
To promote adipogenic differentiation, MSC cultures are incubated with dexamethasone,
insulin, isobutyl methyl xanthine, and indomethacin. There is an accumulation of lipid-rich
vacuoles within cells, and they express peroxisome proliferation-activated receptor
{gamma}2, lipoprotein lipase, and the fatty acid-binding protein aP2. Eventually, the
lipid vacuoles combine and fill the cells. Accumulation of lipid in these vacuoles is assayed
histologically by oil red O staining. Having first been identified for their ability to
differentiate into bone and adipocytes, further studies have demonstrated that MSCs
can also differentiate, under appropriate in vitro conditions, to form chondrocytes,
tenocytes, skeletal myocytes, neurons, and cells of visceral mesoderm (endothelial cells).
To promote chondrogenic differentiation, MSCs are centrifuged to form a pelleted
micromass and cultured in the presence of transforming growth factor. The cell pellets
develop a multilayered, matrix-rich morphology, and histological analysis shows strong
staining with toluidine blue, indicating an abundance of glycosaminoglycans within the
extracellular matrix. The cells also produce type II collagen, which is typical of articular
cartilage.It has also been demonstrated that, when treated with 5-azacytidine and
amphotericin B, MSCs differentiate into myoblasts that fuse into rhythmically beating
myotubes. In addition, differentiation into neuron-like cells expressing markers typical for
mature neurons has been reported. However, Hofstetter and colleagues reported that
these neuron-like cells lack voltage-gated ion channels necessary for generation of
action potentials; therefore, these cells may not actually be classified as true neurons
[38].
References:
Pittenger MF, Mackay AM, Beck SC et al.
Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-
147.
Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow
mesenchymal progenitor cells. J Cell Physiol 1999;181:67-73].
More References...
Image: Renjith Krishnan / FreeDigitalPhotos.net
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