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Oral Communications 8
16.2 - Differentiation of embryonic stem cells under modulated oxygen conditions increase stage 4 NKX6.1+ cells and in vivo maturation to beta cells
Presenter: Amanda, DiIenno, Cambridge , United States Authors: Amanda DiIenno1,2, Anna Kokensparger1,2, Jennifer Hollister-Lock1,2, Susan Bonner-Weir1,2, Clark Colton1,2
Differentiation of embryonic stem cells under modulated oxygen conditions increase stage 4 NKX6.1+ cells and in vivo maturation to beta cells
Amanda DiIenno1,2, Anna Kokensparger1,2, Jennifer Hollister-Lock1,2, Susan Bonner-Weir1,2, Clark Colton1,2
1Chemical Engineering, Massachusetts Institute of Technology, Cambridge , MA, United States; 2Section on Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Boston, MA, United States
Pluripotent stem cells (PSC) hold promise for cell replacement therapy and investigation of embryonic development. However, efficient differentiation to desired cell types remains a major obstacle. Most PSC differentiation 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 insulin-producing cells. We differentiated CyT49 human embryonic stem cells (hESC) under different, well-characterized pO2 environments, controlling cellular oxygen exposure through adhesion culture on highly O2-permeable silicone rubber membranes,using a modification of the 5-stage protocol reported by ViaCyte, Inc (San Diego, CA) (D’Amour 2006 Nature Biotech). Each stage was examined at multiple controlled high and low oxygen levels, and O2 conditions were identified that increased the fraction of the appropriate intermediate cell type by flow cytometry or increased expression of appropriate genetic markers by real-time PCR. The best differentiation was produced by an oxygen-modulated protocol. Differentiation under 5% O2 from hESC to definitive endoderm (stage 1), primitive gut tube (stage 2), and to posterior foregut (stage 3), then under 20% O2 to pancreatic endoderm (stage 4) and insulin-producing cells (stage 5) gave rise to a cell population that was 43% positive for NKX6.1, after stage 4, and was 10% positive for both c-peptide and NKX6.1 after stage 5. In comparison, differentiation of cells at normoxic oxygen (20% O2) gave rise to a population that is 33% positive for NKX6.1 after stage 4 but 3% positive for both c-peptide and NKX6.1 after stage 5. Both normoxic and the modulated oxygen differentiations produced cells that passively secreted c-peptide into the medium but were not glucose responsive. Pancreatic endoderm markers NKX6.1 and PDX1were increased by a factor of two and four respectively for the controlled-hypoxia (5% stage 1-3, 20% stage 4-5) when compared to the normoxic condition (20% stage 1-5). After differentiation to pancreatic endoderm (stage 4) under the modulated oxygen condition or normoxia, 1 million were implanted under the kidney capsule of SCID/beige mice to allow maturation into functional beta cells. Human c-peptide was detected in serum of 2/8 animals containing oxygen-modulated grafts (one at 12 weeks, the other at 20 weeks post implantation) and 0/8 of animals with normoxic grafts 60 min after stimulation with glucose. Grafts from the same 2 mice transplanted with modulated oxygen differentiation cells had cells positively immunostained for insulin and for a cocktail of non-beta cell hormones. Mice from both groups had cells positive for non-beta cell hormones but no insulin. Based on these results,O2 combined with directed differentiation protocols is a potentially straightforward method that could be applied to future hESC therapy protocols for improved differentiation and maturation to beta cells.
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