Controlled induction of human pancreatic progenitors produces functional beta-like cells
Methods are provided for the simple, fast, effective and safe directed differentiation of embryonic stem cells into pancreatic beta-like cells secreting insulin in response to glucose levels. The cells are useful transplant therapeutics for diabetic individuals.
1. A method of generating a PDX1+/NKX6.1+ progenitor cell comprising contacting an embryonic stem cell with an effective amount of a retinoic acid compound to induce a PDX1+ progenitor cell, further comprising contacting the PDX1+ progenitor cell with effective amounts of epidermal growth factor and keratinocyte growth factor, wherein the PDX1+ progenitor cell is not contacted with a bone morphogenic protein (BMP) inhibitor prior to expression of NKX6.1 by the cell, thereby inducing formation of a PDX1+/NKX6.1+ progenitor cell.
2. The method according to claim 1 wherein the embryonic stem cell is a human embryonic stem cell.
3. The method according to claim 1 wherein the embryonic stem cell is contacted with a retinoic acid compound in vitro.
4. The method according to claim 1 further comprising inducing the PDX1+/NKX6.1+ progenitor cell to express NEUROG3, wherein the NEUROG3 expression is induced by contacting the PDX1+/NKX6.1+ progenitor cell with an effective amount of an inhibitor of bone morphogenetic protein, an inhibitor of TGFβ/ALK, or an inhibitor of sonic hedgehog, resulting in production of an INS+/NKX6.1+ cell.
5. The method according to claim 4 wherein the PDX1+/NKX6.1+ progenitor cell is contacted by an effective amount of the inhibitor of bone morphogenetic protein and an effective amount of an inhibitor of TGFβ/ALK.
6. The method according to claim 5 wherein the PDX1+/NKX6.1+ progenitor cell is contacted by an effective amount of an inhibitor of bone morphogenetic protein and an effective amount of an inhibitor of sonic hedgehog.
7. The method according to claim 4 wherein the inhibitor of bone morphogenetic protein is Noggin or the inhibitor of sonic hedgehog is Cyclopamine.
8. The method according to claim 4 wherein the NEUROG3 expression is induced by exposure of the PDX1+/NKX6.1+ progenitor cell to effective amounts of a TATA-Binding Protein, an Activin receptor-Like Kinase inhibitor, Noggin and Keratinocyte Growth Factor.
9. The method according to claim 4 wherein the NEUROG3 expression begins before expression of NKX2.2 is detected.
10. The method according to claim 1 wherein no more than 5% of the PDX1+ progenitor cells are polyhormonal cells.
11. The method according to claim 4 wherein the INS+/NKX6.1+ cell is a beta-like cell responsive to glucose levels.
12. The method according to claim 11 wherein the INS+/NKX6.1+ beta-like cell secretes an increased level of insulin in response to an increased glucose level.
13. The method according to claim 4 wherein the INS+/NKX6.1+ cell does not express a detectable level of a Ki67 marker.
14. A method of using the INS+/NKX6.1+ cell according to claim 8 compromising transplanting the INS+/NKX6.1+ cell into a human.
15. The method according to claim 14 wherein the human is diabetic.