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consensus on opinions on the seeding density required for optimal expansion of
ASCs, and laboratories currently appear to be following protocols based on in-
house evaluations.
The proliferation of ASCs can be stimulated by several exogenous supplements
including fi broblast growth factor 2 (FGF-2) via the FGF-2 receptor, sphingo-
sylphosphorylcholine via activation of c-Jun N-terminal kinase (JNK), platelet-
derived growth factors via the activation of JNK and oncostatin M via the activation
of the microtubule-associated protein kinase or extracellular-regulated kinase, and
the Janis kinase 3 or signal transducers and activators of transcription factors type 1
pathway (Chiou et al. 2006 ; Jeon et al. 2006 ; Kang et al. 2005 ; Mizuno 2009 ; Song
et al. 2005 ). On the contrary, it was suggested by Zhang and co-workers ( 2010 ) that
low-intensity and intermittent negative pressure treatment, e.g., creating a negative
pressure (vacuum) environment within the processing cabinet, could inhibit MSC
proliferation, promote cellular apoptosis, and enhance osteogenic activity. Inhibition
of proliferation could be attributed to temporal hypoxia, caused by the negative
pressure, which could cause hypoxia-inducible factor 1 (HIF-1) upregulation. The
HIF-1 heterodimer is composed of hypoxia-inducible factor 1-alpha (HIF-1α),
which is acutely regulated in response to hypoxia, and hypoxia-inducible factor
1-beta (HIF-β), which is insensitive to fl uctuations in O 2 availability and allows for
cellular adaptation to hypoxia (Zhang et al. 2010 ). This again highlights the impor-
tance of environmental factors to be included in standardized protocols.
ASCs are responsive to hypoxia, which promotes the secretion of the angiogenic
growth factor VEGF (Thangarajah et al. 2009 ). Some studies however suggest that
hypoxia reduces ASC proliferation and attenuates adipogenic, chondrogenic, and
osteogenic differentiation (Lee and Kemp 2006 ), but the literature on hypoxia and
ASCs has advanced considerably since 2006. Fotia and co-workers confi rmed that
hypoxia increases ASC proliferation while decreasing cell surface expression of
CD184 (CXCR4) and CD34 and preserves NANOG and SOX2 gene expression. In
addition to promoting proliferation and stemness, hypoxia and osteogenic stimuli
(induction media) accelerates the cell differentiation and mineralization process
(Fotia et al. 2015 ).
Recent studies have demonstrated multiple hypoxia-responsive pathways involv-
ing angiogenesis in superfi cial and deep abdominal adipose tissue. Rinkinen and
colleagues have demonstrated that mRNA levels of angiogenic chemokines
(VEGF-A, VEGF-B) and transcription factor HIF-1α signifi cantly increase in deep
abdominal tissue, in response to hypoxic culturing conditions, compared to superfi -
cial abdominal adipose tissue (Chung et al. 2012 ; Rinkinen et al. 2015 ). In addition,
increased protein expression levels (VEGF-A and protein nuclear factor K B) were
found within the ASCs derived from deep subcutaneous adipose tissue (Rinkinen
et al. 2015 ). Although notable variations in ASCs from deep and superfi cial subcu-
taneous adipose tissue are ignored during tissue harvesting, an ASC population
could be identifi ed more suited for specifi ed functionality in tissue engineering
(Rinkinen et al. 2015 ).
It was observed by Amos and colleagues ( 2008 ) that harvesting techniques not
only affect the viability of ASCs but also their level of adhesiveness to key adhesion
F.A. van Vollenstee et al.