10.6084/m9.figshare.3160417.v2
Amr Alraies
Amr
Alraies
Figure 4. Effects of prolonged in vitro culture expansion on DPPC ageing characteristics.
figshare
2016
cell senescence
Cell Biology
2016-04-14 08:19:45
Figure
https://figshare.com/articles/figure/Figure_4/3160417
<p>Effects
of prolonged <i>in vitro</i> culture
expansion on DPPC ageing characteristics.
(A) Digital images representing the extent of senescence-associated
β-galactosidase
staining by DPPCs at early PDs (7-8 PDs) and towards the end of their
respective proliferative lifespans (A1 at 23
PDs, A3 at 83 PDs and B1 at 34
PDs). At 7-8 PDs, all DPPCs were morphologically similar, with
the characteristic MSC morphology and were negative for β-galactosidase
staining. At PDs towards the later
stages of their respective proliferative lifespans, higher percentages of cells stained
positive for β-galactosidase, which were larger, more stellate-like and with
prominent stress fibres. Scale bars = 100
µm. (B) Analysis of cellular surface
area throughout
the proliferative lifespan of A1,
A3 and B1, using ImageJ<sup>®</sup>
Software (average ± SE, **p<0.001). Significant
increases in each DPPC population’s surface area were identified with
proliferative lifespan. Only when high
proliferative capacity, A3, reached senescence (85 PDs) were surface areas
non-significantly different to senescent A1 populations at 22 PDs
(p>0.05). Senescent B1 surface areas
at 35 PDs did not reach the cell area sizes of senescent A1 or A3 at 22 PDs and
85 PDs, respectively. (C) Gene expression of the MSC markers, CD73, CD90 and CD105,
was gradually lost during <i>in
vitro</i> expansion. CD105 was lost
initially, based on expression levels in A1 (11 PDs) and A3 (24 PDs), whilst CD90 and CD73 expression was
subsequently lost towards the end of each population’s respective proliferative
lifespans. Whilst the premature loss of
CD105 was not as apparent with B1, all three markers were lost towards the end
of its proliferative lifespan (24 PDs). NA
indicates that no cells were available for analysis at these PDs. β-actin served as the reference housekeeping
gene.</p>