1 Generating Mature Beta Cells from Patient-Specific Pluripotent Stem CellsStephanie Micucci

2 Introduction Type 1 diabetes: Destruction of insulin- producing cells of the pancreas No cure Insulin treatment

3 Vocabulary Pluripotent: Able to be differentiated into any cell type (Example: stem cells). Somatic Cell: Body cells, not gametes. Transcription Factor: Proteins that control which genes are turned on/off in the genome. Teratoma: Benign tumor formation.

4 Introduction Cell replacement therapy Human embryonic stem cells

5 Human Embryonic Stem Cells

6 Introduction Cell replacement therapy Somatic cell nuclear transferHuman embryonic stem cells Somatic cell nuclear transfer Polyhormonal, immature Teratoma formation

7 Review of Literature “Human embryonic stem cells (hESCs) have the potential to replicate into a countless number of cell types.” Stanekzai, J., Isenovic, E. R., & Mousa, S. A. (2012, December). Treatment options for diabetes: Potential role of stem cells. Diabetes Research and Clinical Practice, 98(3), Retrieved from /j.diabres

8 Review of Literature “To generate genetically identical stem cells, nuclear transfer embryonic stem cells have been created.” Tachibana, M., Amato, P., Sparman, M., Gutierrez, N. M., Tippner-Hedges, R., Ma, H., & Kang, E. (2013, June 6). Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 153(6), doi: /j.cell

9 Review of Literature “The differentiated cells are often polyhormonal, expressing other hormones such as glucagon and somatostatin, and therefore resemble immature beta cells.” Basford, C. L., Prentice, K. J., Hardy, A. B., Sarangi, F., Micallef, S. J., Li, X., & Guo, Q. (2012, February). The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells. Diabetologia, 55(2), doi: /s x ls.”

10 Purpose Test efficiency of differentiation on human somatic cell nuclear transfer cells Observe differentiated cells after transplantation Analyze teratomas from mice with transplanted beta cells

11 Methodology Overview Somatic Cell Nuclear Transfer

12 Methodology: Somatic Cell Nuclear TransferNoggle, Scott, Ho-Lim Fung, Athurva Gore, Hector Martinez, and Kathleen C. Satriani. "Human oocytes reprogram somatic cells to a pluripotent state." Nature 478 (2011): Print.

13 Methodology Overview Somatic Cell Nuclear Transfer Cell Passage

14 Methodology: Cell Passage

15 Methodology Overview Cell Passage Cell DifferentiationSomatic Cell Nuclear Transfer Cell Passage Cell Differentiation

16 Methodology: Cell DifferentiationStage 1 Stage 2 Stage 3 Stage 4 Stage 5 Endoderm Diff Kit RPMI-1640 +PS +Glutamax DMEM-HG +PS CMRL 2% FBS B27 A B KGF (50 ng/ml) KAAD-cyclopamine (0.25 uM) exendin-4 (50 ng/ml) RA (2uM) ALK5 inh (1uM) LDN (250 nM) Day 1 Day 2-3 Day 4-5 Day 6-8 Day 9-12 Day 13-15

17 Methodology Overview Cell Passage Cell DifferentiationSomatic Cell Nuclear Transfer Cell Passage Cell Differentiation Immunofluorescence Analysis

18 Methodology: Immunofluorescence AnalysisFixed in 4% PFA 20 minutes Sealed with parafilm at 4°C Overnight Blocking Solution 30 minutes Primary Antibodies in Blocking Solution Secondary Antibodies in Blocking Solution 1 Hour DAPI Solution during wash 15 minutes Pdx1 and Nkx6.1 Day Day 13 Day Day 13 C-Peptide and Insulin

19 Methodology Overview Cell Passage Cell DifferentiationSomatic Cell Nuclear Transfer Cell Passage Cell Differentiation Immunofluorescence Analysis Transplantation Teratoma Analysis

20 Methodology: Teratoma Analysis

21 Methodology: Teratoma Analysis

22 Methodology: Teratoma AnalysisSlide De-paraffinization and Rehydration Antigen Revival Blocking Solution Primary Antibodies TABLE 1: Primary Antibody Solution 2T - 1 2T - 4 2T - 8 Total 200 µl PBST 600 µl PBST 4 µl Donkey Serum (5%) 12 µl 1 µl rabbit x insulin 3 µl rabbit x insulin 1 µl mouse x glucagon sheep x ngn3 goat x pdx1

23 Methodology: Teratoma AnalysisSlide De-paraffinization and Rehydration Antigen Revival Blocking Solution Primary Antibodies Secondary Antibodies TABLE 2: Secondary Antibody Solution 2T - 1 2T - 4 2T - 8 Total 200 µl PBST 600 µl PBST 4 µl Donkey Serum (4%) 12 µl 0.2 µl 488 x mouse 0.2 µl 488 x rabbit 0.2 µl 488 x goat 0.2 µl 555 x rabbit 0.2 µl 555 x sheep

24 Methodology: Teratoma AnalysisSlide De-paraffinization and Rehydration Antigen Revival Blocking Solution Primary Antibodies Secondary Antibodies DAPI Solution Coverslips

25 Results: Immunofluorescence AnalysisDay 9 Differentiation Pdx1 Nkx6.1 Pdx1, Nkx6.1, DAPI Day 13 Differentiation Pdx1 Nkx6.1 Pdx1, Nkx6.1, DAPI Day 13 Differentiation C-Peptide, Glucagon, DAPI C-Peptide Glucagon

26 Results: Transplantation2 months after transplantation  Teratomas Teratomas in transplantation site In vivo functional tests could not be performed

27 Results: Teratoma AnalysisMouse 2T - 1 Glucagon, Insulin, DAPI Glucagon Insulin Mouse 2T - 4 Ngn3, Insulin, DAPI Mouse 2T - 8 Pdx1 Insulin Pdx1, Insulin, DAPI

28 Conclusion: DifferentiationIn vitro differentiation moderately successful Day 9: Minimal nkx6.1 Day 13: High concentration of pdx1 and nkx6.1 Day 13: Isolated expression of C-Peptide C-Peptide + Glucagon = Immature

29 Conclusion Transplantation Teratoma Analysis Teratomas developedProblem with: Transplantation? Differentiation? Some pancreatic factors present All: moderate insulin Still largely unknown

30 Discussion Moderate differentiation  improved for futurePrevention of teratoma formation Teratomas possess some pancreatic factors

31 Acknowledgements

32 Bibliography Basford, C. L., Prentice, K. J., Hardy, A. B., Sarangi, F., Micallef, S. J., Li, X., & Guo, Q. (2012, February). The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells. Diabetologia, 55(2), doi: /s x Hrvatin, S., O'Donnell, C. W., Deng, F., Millman, J. R., Pagliuca, F. W., Dilorio, P., & Rezania, A. (2014, February 25). Differentiated human stem cells resemble fetal, not adult, β cells. Proceedings of the National Academy of Sciences, 111(8), doi: /pnas Micallef, S. J., Li, X., Schiesser, J. V., Hirst, C. E., Yu, Q. C., Lim, S. M., & Nostro, M. C. (2011, November 26). INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells. Diabetologia, 55(3), doi: /s y Rong, Z., Fu, X., Wang, M., & Xu, Y. (2012, September 21). A scalable approach to prevent teratoma formation of human embryonic stem cells. The Journal Biological Chemistry, 287(39), doi: /jbc.M Stanekzai, J., Isenovic, E. R., & Mousa, S. A. (2012, December). Treatment options for diabetes: Potential role of stem cells.Diabetes Research and Clinical Practice, 98(3), Retrieved from /j.diabres Statistics: JDRF and Diabetes (n.d.). In JDRF. Retrieved November 14, 2014, from Tachibana, M., Amato, P., Sparman, M., Gutierrez, N. M., Tippner-Hedges, R., Ma, H., & Kang, E. (2013, June 6). Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 153(6), doi: /j.cell

33 Generating Mature Beta Cells from Patient-Specific Pluripotent Stem CellsStephanie Micucci