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PPAR γ 2
aP2
Coll. Type II
Coll. Type IX
Osteopontin
AP
B2M
FIGURE 2.2
In vitro differentiation of MSCs does not result in differentiation to multiple lineages. Messenger
RNA isolated from control and in vitro differentiated human MSCs was probed with cDNAs
(complementary DNAs) for adipogenic markers (PPARγ2 and aP2), chondrogenic markers (col-
lagens II and IX), and an osteogenic marker (osteopontin). Under the conditions, human MSCs
differentiated with fidelity to the desired lineage, and RNA analysis did not detect differen-
tiation to the other lineages. (Reprinted from Pittenger, M.F., A.M. Mackay, S.C. Beck, R.K.
Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, and D.R. Marshak.
1999. Science 284, no. 5411: 143-47.)
of the MSCs responding and without evidence of other lineages by mRNA
(messenger RNA) analysis (Figure 2.2) or histology ( Figure 2.1 ). These assays
indicate that MSCs require specific signaling to progress along particular
lineage pathways, and conditions for one lineage inhibit the other lineages.
That is, MSC differentiation is influenced by growth factors, cytokines, basal
nutrients, cell density, spatial organization, and mechanical forces to perpet-
uate or inhibit lineage progression. Table 2.2 provides the in vitro differentia-
tion methods for the adipogenic, chondrogenic, and osteogenic lineages.
When analyzed by low cytometry, these carefully grown multipotential
cells exhibit a regular and reproducible phenotype with the reliable pres-
ence of many surface molecules and the absence of others ( Table 2.3 ). Clonal
expansion of individual prospective MSCs demonstrated that the cells could
be expanded a millionfold, approximately 22 population doublings, and
still retained their multilineage differentiation ability (Pittenger et al. 1999).
While no single marker, or even a collection of markers, can be claimed to
denote a stem cell without evidence of differentiation, the addition of evi-
dence from the in vitro multilineage assays combines to create a strong case
for the stem cell nature of these MSCs (Dominici et al. 2006; Jaiswal et al.
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