(489c) Variation of Oxygen in a Controlled Manner Markedly Enhances Multi-Stage Differentiation of Embryonic Stem Cells to Insulin Producing Cells

Pluripotent stem
cells (PSC) hold promise for cell replacement therapy, but efficient
differentiation to desired cell types remains a major obstacle.  Although most
PSC research is performed in high, non-physiological O₂ environments,
cells during embryonic development are actually exposed to much lower O₂.
Here we report that variation of O₂ in a controlled manner markedly
enhances multi-stage differentiation of embryonic stem cells to insulin
producing cells. We differentiated CyT49 human embryonic stem cells (hESC), using
a modification of the 5-stage protocol published by ViaCyte, Inc (D'Amour et al.,
Nat Biotech 2006, Kroon et al., Nat Biotech 2008) under different sequences of well-characterized
pO₂ environments. We controlled cellular oxygen exposure through
adhesion culture on highly O₂-permeable silicone rubber membranes so
that the cells in contact with the membrane were exposed to the same pO₂
as in the gas phase.  

Our strategy in
selecting O₂ patterns to investigate was based on two hypotheses:
(1) cells in the developing embryo are initially exposed to low O₂
levels that increase as the tissues become vascularized. (2) The effects of low
O₂ on the early stages of differentiation can be manifested in later
stages of differentiation.  At each stage there were two steps.  We started
with stage 1at six different O₂ levels (1%, 3%, 5%, 8%, 10%, &
20%). Samples at the end of stage 1 were analyzed with qPCR and flow cytometry
for markers of definitive endoderm (DE).  In the second step, we carried the
differentiation to completion. The one to three best oxygen levels that produced
the highest fraction of DE cells were then used in stage 1, and the remaining
stages were carried out at 20% O₂. Each set of complete
differentiation experiments employed a control condition of constant 20% O₂
for comparison. The best stage 1 O₂ level was
selected on the basis of the fraction c-peptide+ and/or insulin+ cells in the
stage 5 preparation as well as the extent of gene expression of relevant
pancreatic endoderm (PE) and beta cell markers, and this O₂ level
was used for all subsequent experiments.  This two-step process was repeated
sequentially with stage 2, 3, and 4; the condition for stage 5 was fixed at 20%
O₂.   Beginning with stage 3 an additional O₂ condition
of 40% was added to the conditions tested.  All together, 12 different
sequences of O₂ levels in successive stages were evaluated.

The fraction of cells
containing insulin or c-peptide was similar. Results for 9 of the 12 sequences
are summarized in Table 1 in terms of the pooled mean of the fractions of
c-peptide+ and insulin+ cells.

With normoxic conditions in
all of the stages (designated 20-20-20-20), the fraction was about 4%. 
Reduction of O₂ to 5% in stage 1caused the fraction to double.  A
similar additional reduction of O₂ in stage 2 had no further
effect.  The effect of another sequential reduction to 5% in stage 3 depended
on the oxygen level in stage 4.  If O₂ was further reduced below 5%
in stage 4, there was no change.  However, in the range of 5 to 40% O₂
for stage 4, the fraction of c-peptide+/insulin+ cells jumped by a factor of
four to a mean of 34%.  At two conditions, the fraction was about 50%, but there
was not a monotonic trend with O₂ level.

These results demonstrated
that modulation of the O₂ level to which cells are exposed can have
a pronounced effect on the outcome of directed differentiation protocols.  In
the present example, culture at reduced oxygen for the first three stages,
followed by an increase in O₂ levels for the last two stages,
produced an eight-fold increase in the fraction of c-peptide+/insulin+ cells.