(261d) Hypoxia Markedly Influences Pluripotent Stem Cell Differentiation to Mesoderm, Ectoderm, and Endoderm and Reduces Subsequent Teratoma Formation

Efficient
differentiation of pluripotent stem cells (PSC) to desired cell types for cell
replacement therapy remains a challenge, and potential tumor formation by residual
PSC in differentiated populations is problematic. Most PSC research is
performed in high, non-physiological O₂, but cells during embryonic
development are exposed to much lower O₂. Here we report a
wide-ranging study showing that physiological O₂ markedly influences
differentiation to all three germ layers, with enhanced differentiation to
cardiomyocytes and insulin-producing cells in particular, and reduces residual
PSC and tumor formation.

We
differentiated mouse and human embryonic stem cells (mESC and hESC) and mouse induced
pluripotent stem cells under controlled cellular O₂ environments through
adhesion culture on highly O₂-permeable silicone rubber membranes.
Low O₂ drastically increased cardiomyocyte differentiation from
these PSC. Best results were acquired by differentiation of mESC for 6 d at 5%
O₂ followed by 20% O₂ for 15 d, resulting in up to 57%
cardiomyocytes and 304 cardiomyocytes generated per initial mESC without
purification, factors of 5 and 9 higher, respectively, than differentiation
entirely at 20% O₂. Low O₂ culture for the first 6 d
increased by 3x expression of Mesp1/2, which helps restrict mesodermal cells to
the cardiovascular lineage, but did not increase Brachyury
T expression, an early mesodermal marker, compared to 20% O₂, thereby
suggesting that low O₂ acted by restricting the fate of early mesoderm
towards cardiomyocytes.

Low
O₂ decreased the fraction and number of Nestin+ cells (ectoderm) by
3x for mESC, but enhanced expression of endodermal genes Sox17, Foxa2, Hnf4a, and Pdx1, stimulating
us to examine differentiation of hESC to Insulin+ cells using a protocol
developed by ViaCyte, Inc (San Diego, CA). Differentiation
to definitive endoderm (DE) was modestly enhanced by culture at 3-8% O₂,
but performing differentiation entirely at low O₂ was detrimental
for producing Insulin+ cells. Differentiation at 5% O₂ to produce DE
with all subsequent steps at 20% O₂ achieved a population with 14% Insulin+
cells, 2x higher than differentiation entirely at 20% O₂. These
cells passively secreted C-peptide but were not glucose responsive.

Low
O₂ for extended periods after differentiation was complete
drastically reduced the amount of residual PSC within, and the tumorigenic
potential of, differentiated cell populations. Pluripotency marker (Oct4 and
Nanog) expression and the fraction and number of Oct4+ cells was reduced by up
to four orders of magnitude at low compared to high O₂. Upon
implantation into immunocompromised mice, low O₂-differentiated
cells did not form tumors or did so slower than high O₂-differentiated
cells, consistent with reduced residual PSC. Cell sorting of SSEA-4+ cells
after extended low O₂ culture of hESC further reduced residual PSC
and tumor formation rate.

These
findings establish that culture O₂ is important for almost every
aspect of PSC differentiation. We used highly-O₂ permeable silicone
rubber culture dishes for accurate control of cellular O₂ exposure.
By modulating O₂ during different stages of differentiation, we
achieved substantial increases in cardiomyocyte and Insulin+ cell yields.
Extended culture at low O₂ further decreased residual PSC by several
orders of magnitude. O₂
control, alone or combined with other methods could be applied to future cell
therapy protocols to generate and increase the safety of differentiated cells.