From Stem Cells to Beta Cells: Possible Cure for Diabetes Mellitus

1. From Stem Cells to Beta Cells: Possible Cure for Diabetes Mellitus By Ryan Scavinski

2. Diabetes Mellitus • Type 1 Diabetes is caused by the autoimmune destruction of β-cells within the pancreas. • No β-cells, no insulin • Diabetics need to monitor blood glucose and control it with insulin.

3. Past Treatments • Transplantation of pancreatic islet cells • Problems • Patient requires more than 600 islets/kg body weight = two deceased organ donors • Immunological rejection

4. Using Stem Cells • Two approaches used to differentiate Embryonic Stem Cells into β-cells or Insulin Producing Cells (IPCs) • Embryoid Body Formation • Definitive Endoderm Formation

5. Embryoid Body Formation • An embryoid is the arrangement of stem cells destined to differentiate into the ectoderm, mesoderm and endoderm. • With multiple treatment of growth factors, Embryonic stem cells give rise to Nestin cells, which in turn differentiate into IPCs.

7. Problems • Low efficiency for producing IPCs • Also showed development of tumors in the kidney and spleen in some transplanted mice.

8. Definitive Endoderm Approach • This approach bypasses the Embryoid formation and generates the endoderm, in which produce the cells needed for insulin production.

9. Differentiation Steps • 1. ESCs were placed onto a culture dish with a chemically defined medium (CDM) containing 50 ng/mL Activin A for 4 days

10. With and without Activin A • PCR of expression of endoderm genes • Gapd, sox17, pdx1, hlxb9, hnf4a and insulin gapd sox17 pdx1 hlxb9 hnf4α insulin

11. continued • 2. Then the cells were transferred onto a CDM with 10-6 M Retinoic acid (RA) for another 4 days

12. Without Activin A and RA With Activin A and RA small clusters of differentiated ES

13. PCR • A+/RA+ • A-/RA- • A+/RA- • A-/RA+ gapd sox17 pdx1 hlxb9 hnf4α insulin

14. continued • 3. Then the CDM was changed to modified islet maturation medium containing bFGF- a pancreatic cell maturation factor for 3 days • 4. Finally the differentiated cells were switched to a islet maturation medium containing nicotinamide and the bFGF for another 3 days.

15. In this final stage, many differentiated cells formed spherical clusters • Also expressed the pancreatic β cell markers such as pdx1, INSULIN, glucokinase and glut2 shown in PCR

16. Differentiated Embryonic Stem Cells gapd sox17 pdx1 hlxb9 hnf4α insulin glut2 Amy SST Sur1 GCG GCK Maturation Control

17. To test cells for insulin release, cells were incubated in buffer containing 2.5 mM glucose for 15 min • Then incubated with 27.5 mM glucose for another 15 min • Tested for insulin release with a Rat/Mouse insulin ELISA

18. 2.5 mM 27.5 mM Insulin secretion (ng/mg) Suspension Adhesion

19. Transplantation • Differentiated cells were transplanted under the renal capsule (kidney) of diabetic mice. • 30% showed normal blood glucose levels for 6 weeks • They removed the cell transplanted kidney-mice regained hyperglycemia

20. 30 25 20 15 10 5 0 Blood glucose (mM) 0 7 14 21 28 35 42 49 56 Days after transplantation

21. Results and Discussion • The combination of Activin A and Retinoic Acid is an effective method to induce Embryonic Stem Cells to differentiate into insulin producing cells • Further research in needed to see if the difference between human and mouse will impair the function of transplanted ESC derived cells

22. My Opinion

23. Reference: • Soria, B., Skoudy, A., and Martin, F. 2001. From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 44 407-415 • Raikwar, S. and Zavazava, N. 2009. Insulin producing cells derived from embryonic stem cells: are we there yet?. Journal of Cellular Physiology, 218 256-263 • Jiang, W., Shi, Y., Zhao, D., Chen, S., Youg, J., Zhang, J., Qing, T., Sun, X., Zhang, P., Ding, M., Li, D., and Deng, H. 2007. In vitro derivation of functional insulin producing cells from human embryonic stem cells. Cell Research. 17 333-344