Mesenchymal Stem Cells - Interest Group
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.
However, the degree to which the culture will differentiate varies among individuals and how
differentiation is induced, e.g. chemical vs. mechanical, and it is not clear whether this variation is
due to a different amount of "true" progenitor cells in the culture or variable differentiation capacities
of individuals' progenitors.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, β-glycerophosphate, and
dexamethasone for 2–3 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-β. 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].

Intra- versus Extra-Mesenchymal Lineages:

Mesenchymal Stem Cells (MSCs) can be directed toward either Intra-Mesenchymal Lineages or
Extra-Mesenchymal Lineages.


References:

  • Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal
    stem cells. Science 1999;284:143–147.[Abstract/Free Full Text]

  • Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow
    mesenchymal progenitor cells. J Cell Physiol 1999;181:67–73.[CrossRef][Medline]

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