“9.61x6.69” b2815 Tissue Engineering and Nanotheranostics
Directed Differentiation of Human Pluripotent Stem Cells 79
Xu et al. demonstrated a successful feeder-free system for hESC
culture where cells were plated on Matrigel or laminin in MEF-
conditioned medium (MEF-CM).^17 These feeder-free cells had a
doubling time comparable to that of cells grown on a feeder layer.^17
While MEF-CM’s combination with Matrigel and laminin spurred
cell growth, using gelatin with the MEF-CM was incapable of
supporting the feeder-free culture. This suggests that necessary sig-
nals for stem cell proliferation were most likely coming from the
Matrigel/laminin layers in addition to the MEF-CM.^17
Matrigel was closely examined for the factors it could be contrib-
uting to in the maintenance of pluripotency. Matrigel is a basement
membrane made up of continuous layers of specialized extracellular
matrix, forming an interface between cells and their adjacent connec-
tive tissue. Basement membranes also act as a storage depot for
growth factors in vivo. Specifically, Matrigel contains laminin, colla-
gen IV, and heparin sulfate proteoglycans as its main component.^18
Combinations of the MEF-CM with either laminin or whole Matrigel
had much larger yields of undifferentiated cells than MEF-CM com-
bined with any other component of Matrigel.^19 Xu et al. suggested
laminin may be binding a receptor created from integrin α6 and β1 in
order to signal the pluripotency maintenance.^17
1.4. Mouse Embryonic Fibroblast Conditioned Media
The contents of MEF-CM remain necessary for maintaining feeder-
free, serum free hESC populations. The components of MEF-CM
have been examined by numerous groups. An uncultured medium
(UM) is often used in these analyses to determine if its application to
hESCs, combined with the minimum essential additional factors, can
substitute for MEF-CM.
The addition of bone morphogenetic protein 4 (BMP4) to mouse
ESCs helped maintain stemness; this led to similar experiments with
hESCs with contradictory results.^20 The BMP4 treatment did not
maintain pluripotency but instead caused differentiation with enhanced
expression of trophoblast markers.^21 Therefore, Noggin, a BMP
inhibitor, was explored to counteract BMP4 induced differentiation.
