Stem Cell Processing (Stem Cells in Clinical Applications)

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forming mature adipocytes (Tang et al. 2004 ). Peroxisome proliferator-activated
receptor-γ (PPAR-γ) and CCAAT/enhancer-binding proteins (C/EBP) are key regu-
lators of adipogenesis (Kang et al. 2007 ).
In the laboratory, in vitro adipogenic differentiation takes 2–3 weeks after initiating
differentiation following exposure of the cells to the appropriate induction media. The
composition of the adipogenic induction medium is provided in the supplementary
material. Adipogenesis is associated with the accumulation of lipid droplets in the cyto-
plasm of the adipocytes. The number and size of lipid droplets increase as the ASCs
differentiate from preadipocytes to mature adipocytes. The lipid droplets are highly
organized organelles, consisting of an outer layer of amphipathic lipids such as phos-
pholipids and a core of neutral lipids including triacylglyceride and sterol esters (Guo
et al. 2009 ; Krahmer et al. 2009 ). Studies using immortalized preadipocytes, such as the
mouse 3 T3-L1 cell line, revealed that combinations of 3-isobutyl-methylxanthine with
dexamethasone or insulin induce intracellular lipid accumulation. Therefore, to promote
the effective differentiation of ASCs into adipocytes, the induction medium is supple-
mented with IBMX, insulin and dexamethasone. 3-Isobutyl-methylxanthine (IBMX), a
cAMP- phosphodiesterase inhibitor, has been shown to inhibit soluble cyclic nucleotide


Table 7.3 Minimum gene expression requirements to confirm in vitro adipogenic, osteogenic,
chondrogenic and myogenic differentiation


Lineage Gene Reference(s)
Adipogenic Adiponectin Bourin et al. ( 2013 )
C/EBPα
FABP4
Leptin
PPAR-γ
Osteogenic Alkaline phosphatase Bourin et al. ( 2013 )
Bone sialoprotein
Osteocalcin
Osterix
Runx2
Chondrogenic Aggrecan Bourin et al. ( 2013 )
Collagen type II
Sox 9
Myogenic Striateda Desmin Beier et al. ( 2011 )
Myogenic differentiation factor 1 Carvalho et al. (^2013 )
(MyoD1)
Myogenin
Myogenic enhancer factor 2 (MEF2)
Myosin heavy chain (MHC)
Smootha Alpha-smooth muscle actin
Cardiaca Sarcomeric alpha-actinin
Connexin 43
aMuscle type


7 Isolation and Characterization of Adipose-Derived Stromal Cells

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