Oxygen tension during expansion affects colony formation and the subsequent proliferative and differentiation
capacity of human mesenchymal stem cells
+1Pattappa, G; 1de Bruijn. J D; 1Lee D A
+1School of Engineering and Materials Science, Queen Mary University of London, London UK
[email protected]
Introduction
Human mesenchymal stem cells (MSCs) have the ability to differentiate
towards cartilage, bone, fat and muscle, and therefore have great
therapeutic potential [1]. They reside in vivo between 4-7% oxygen and
culture under these conditions, influences their population doubling limit
and differentiation capacity compared with cultures expanded under
normoxia [2, 3, 4, 5].
The present study investigated the effect of continuous and
uninterrupted control of oxygen tension on MSC proliferation and
differentiation. It is hypothesized that MSCs undergo greater population
doublings without senescence upon expansion at 5% or 2% oxygen,
whilst MSC chondrogenesis is enhanced and osteogenesis inhibited by
hypoxic expansion.
Materials and Methods
Human MSC isolation and oxygen culture: Human bone marrow
aspirates (Lonza, Wokingham UK) were aliquoted into cell culture
flasks within a system permitting continuous and uninterrupted control
of the oxygen environment (XvivoTM, Biospherix, Redfield, New York
USA). All cell manipulation was conducted within the system at the
relevant oxygen conditions. MSCs were fed with alpha-MEM + 8.5%
FBS pre-equilibrated at either 20%, 5% or 2% oxygen and placed into
incubators set at the relevant oxygen conditions. Medium was not
replenished until day 6 of culture and thereafter, medium was
replenished every 2-3 days until confluence. The number of colonies at
P0 and the number of cells recovered was recorded at each passage to
generate population growth curves and after five passages in culture, the
number of senescent cells was assessed via β-galatosidase histochemical
staining(Sigma-Aldrich, Poole, UK).
Oxygen, glucose and lactate measurements: 130 µl aliquots of cell
suspensions containing 1.5 x 105 MSCs from each oxygen condition at
passages 3 and 4 were resuspended in α-MEM + 8.5% FBS preequilibrated at 20% oxygen were added to individual wells of 384oxygen biosensor well plate (BD Biosciences, Oxford, UK). The plate
was sealed and the oxygen concentration within the cell suspensions was
measured using a fluorometer for two hours. Medium was removed at 1,
2, 4 and 6 hour time points: Medium glucose and lactate was measured
using standard biochemical assays.
Differentiation studies: Passage 2 MSCs were differentiated to
osteogenic and chondrogenic lineages using standard protocols under
each of their expanded oxygen tension (20%, 5% or 2%). Differentiation
towards their specific lineages for each oxygen tension was assessed
through biochemical assays (glycosaminoglycan content and Alkaline
phosphatase activity) and staining (alizarin red and toluidine blue).
Further to the described study, MSCs expanded at 20% or 2% oxygen
were differentiated at 20% and 2% oxygen.
Results
Figure 1. The number of cells per colony calculated from the number of
colonies and cell recovered at P0 for MSCs cultured at 20%. 5% and
2% oxygen (data represents mean + S.D. of donor A (n =4) and donor B
(n = 3); * p < 0.05).
There was a significant reduction in the number of colonies formed at
5% compared with 20% and 2% oxygen for both donors (p < 0.05). The
number of cells recovered was greatest at 20% oxygen suggesting a
greater number of cells per colony (Figure 1) compared with 5% and 2%
oxygen cultures (p < 0.05). The proliferation rate of MSCs cultured at
20% oxygen was initially greater than 5% and 2% oxygen, but there was
no significant difference in doubling time between the oxygen
conditions after two passages. However, there was a significant increase
in the proportion of senescent cells at 20% oxygen compared with 5%
and 2% oxygen.
(A)
(B)
(C)
Figure 2. (a) The changes in alkaline phosphatase activity for MSCs
expanded and differentiated at either 20% or 2% oxygen (data
represents mean + S.E.M. of n =6). Alizarin red staining for MSCs
differentiated at 20% oxygen after expansion at (a) 20% and (b) 2%
oxygen.
Cells cultured at 20% oxygen showed a significant increase in oxygen
consumption and decrease in lactate production compared with 5% and
2% oxygen (p < 0.05). Differentiation of MSCs towards the
chondrogenic lineage was achieved under all three oxygen conditions.
However, osteogenic differentiation was only achieved for cells
expanded and differentiated at 20% oxygen, as MSCs expanded or
differentiated at 2% oxygen did not show any indication of osteogenesis
(Figure 2).
Discussion
The results of the investigation show that the oxygen tension influences
the colony-forming efficiency of MSCs and may enable the selection of
MSC sub-populations as described in previous studies [4, 5]. There were
no significant differences in doubling time after two passages, although
there were a greater proportion of senescent cells under normoxia which
may be due to increased reactive oxygen species production, through
utilisation of oxidative phosphorylation [2]. Colony formation under
hypoxia may have influenced their differentiation capacity. The oxygen
levels during expansion did not affect chondrogenic potential but
expansion under hypoxia prevented subsequent ostoegenic
differentiation. Osteogenesis was also inhibited for MSCs expanded at
normoxia and differentiated under hypoxia. This phenomenon may be
related to the colony formation or the inherent metabolism of the cells
and the inhibition of expression of osteogenic genes under hypoxia.
References [1] Pittenger MF et. al. Science, 284(5411):143-147; [2]
Mousavvi-Harami F et .al. Iowa Orthop. J. 24:15-20 (2004); [3]
d’Ippolito G et .al. J. Cell. Sci., 117:2971-2981 (2004); [4] Grayson e.
al. Biochem.. Biophys. Res. Comm., 358(3): 948-953 (2007); [5] Fehrer
et. al. Aging Cell, 6(6): 745-757(2007)
Poster No. 724 • 56th Annual Meeting of the Orthopaedic Research Society