The document outlines a study by Nikolay Turovets on the efficient derivation of high-purity hepatocyte populations from human pluripotent stem cells through an in vitro analog of the primitive streak. It discusses the developmental signaling pathways and the success of a 3D differentiation system in producing definitive endoderm and hepatocyte-like cells with relevant functionalities. The findings highlight the potential for these derived cells to integrate and function in vivo, contributing to regenerative medicine applications.

1. Turovets Nikolay, PhD Director of Research and Therapeutic Development International Stem Cell Corporation May, 2011 Efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells In vitro Analog of the Primitive Streak: www.turovets.com

2. Differentiation days Developmental signaling Derivation of hepatocytes from human pluripotent stem cells: the first step is a key of purity Nikolay Turovets, PhD www.turovets.com hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte Maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex

3. Definitive endoderm formation in vivo : transition through primitive streak Nikolay Turovets, PhD www.turovets.com

4. Differentiation signaling Derivation of high-purity definitive endoderm using an in vitro analog of the primitive streak Nikolay Turovets, PhD www.turovets.com Extracellular matrix Definitive endoderm Pluripotent stem cells

5. Epithelial-to-Mesenchymal Transition is engine of cell migration in the 3D-differentiation system Differentiation days Developmental signaling Nikolay Turovets, PhD www.turovets.com hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex

6. Epithelial-to-Mesenchymal Transition hallmarks: cytoskeleton rearrangements Nikolay Turovets, PhD www.turovets.com

7. Epithelial-to-Mesenchymal Transition hallmarks: cytoskeleton rearrangements cadherin switch: E->N Nikolay Turovets, PhD www.turovets.com

8. Epithelial-to-Mesenchymal Transition hallmarks: cadherin switch: E->N cytoskeleton rearrangements acquisition of ability to migrate Nikolay Turovets, PhD www.turovets.com

9. Human pluripotent stem cells undergoing differentiation toward definitive endoderm in the 3D-differentiation system demonstrate appropriate gene expression dynamics Nikolay Turovets, PhD www.turovets.com

10. Human pluripotent stem cells undergoing differentiation toward definitive endoderm in the 3D-differentiation system demonstrate appropriate developmental transitions Nikolay Turovets, PhD www.turovets.com

11. Differentiated cultures located in 3D-ECM do not demonstrate consistent increases in transcription levels of genes associated with other differentiation directions NO extraembryonic endoderm (SOX7, AFP) mesoderm (FOXF1, BMP4, MEOX1, FLK1) ectoderm (SOX1, SOX2) trofectoderm (HCG, CDX2) Nikolay Turovets, PhD www.turovets.com

12. Flow cytometric analysis demonstrates absence of undifferentiated (OCT4-positive) cells in the DE cultures collected from the 3D-ECM of the differentiation system Nikolay Turovets, PhD www.turovets.com

13. flat plastic dish 3D-differentiation system Other cell types in population Definitive endoderm Definitive endoderm Other cell types in population 92% 51% 49% 8% 3D- differentiation system enables the derivation of high-purity DE from human pluripotent stem cells CXCR4 CXCR4 Nikolay Turovets, PhD www.turovets.com

14. Differentiation days Developmental signaling Early hepatocyte progenitors derived in the 3D-differentiation system have appropriate hepatocyte morphology and start expression of AFP and albumin on day 6 of differentiation Nikolay Turovets, PhD www.turovets.com hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex

15. Upon addition of HGF to the culture medium differentiated cultures derived in the 3D-differentition system exhibited AFP gene expression 5 times higher than cultures derived in flat plastic dish days of differentiation 3D-differentiation system flat plastic dish Nikolay Turovets, PhD www.turovets.com hpSC Definitive endoderm Early AFP-expressing hepatocyte progenitors + HGF/ hepatocyte maturation factor 0 3 8

16. Marker expression by early hepatocyte progenitors derived in 3D-differentiation system Nikolay Turovets, PhD www.turovets.com

17. Differentiation days Developmental signaling Nikolay Turovets, PhD www.turovets.com hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex

18. Highly enriched cultures of hepatocyte-like cells derived in 3D-differentiation system possessed functions associated with human hepatocytes: glycogen storage (PAS test) and ICG uptake and release ICG test PAS test glycogen storage Nikolay Turovets, PhD www.turovets.com

19. Highly enriched cultures of hepatocyte-like cells (HLC) derived in 3D-differentiation system demonstrate P450 activity Pentoxyresorufin O-Dealkylase (PROD) assay CYP7A1 CYP7A1 HLC HepG2 Nikolay Turovets, PhD www.turovets.com

20. Hepatocyte-like cells derived in 3D- differentiation system are engrafted in vivo Produce liver proteins in vivo Engrafted into mouse liver Perform at least 3 cell divisions in vivo CFSE Nikolay Turovets, PhD www.turovets.com

21. 3D-differentiation system: demonstrate universal properties: works well with various types of pluripotent stem cells; based on a natural physiological process: no sorting procedures (FACS, magnetic beads etc ) are required; Nikolay Turovets, PhD www.turovets.com based on a three dimensional extracellular matrix: 3D environment;

22. Cell Transplantation , 2011 doi: 10.3727/096368911X582723 Derivation of High-Purity Definitive Endoderm from Human Parthenogenetic Stem Cells Using an in vitro Analog of the Primitive Streak Turovets N., Fair J., West R., Ostrowska A., Semechkin R., Janus J., Li Cui, Agapov V., Turovets I., Semechkin A., Csete M., Agapova L. Nikolay Turovets, PhD www.turovets.com

23. Team University of California, San Diego, CA Marie Csete, M.D., Ph.D. Cedars-Sinai Medical Center, Los Angeles, CA Jeffrey Fair, M.D. Organovo, San Diego, CA Li Cui, Ph.D. West Labs Scientific, Kalamazoo, MI Richard West International Stem Cell Corporation, Oceanside, CA Nikolay Turovets, Ph.D. Larissa Agapova, Ph.D. Alina Ostrowska, Ph.D. Jeffrey Janus Irina Turovets Vladimir Agapov Olga Kochetkova Nikolay Turovets, PhD www.turovets.com