In Vitro Analog of the Primitive Streak (ANIMATED)

Turovets Nikolay, PhDDirector 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

Efficient derivation of highly enriched populations of hepatocytes

from various types of human pluripotent stem cells

In vitro Analog of the Primitive Streak:In vitro Analog of the Primitive Streak:

www.turovets.com

hpSC Definitive endoderm (DE) derivation

DE-specification toward hepatocyte fate

Hepatocyte development

HepatocyteMaturation

0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex

Differentiation days

Developmental signaling

Derivation of hepatocytesfrom human pluripotent stem cells: the first step is a key of purity

Nikolay Turovets, PhD www.turovets.com

Definitive endoderm formation in vivo: transition through primitive streakDefinitive endoderm formation in vivo: transition through primitive streak


Extracellular matrix

Definitive endodermPluripotent stem cells

Differentiation signaling

Derivation of high-purity definitive endoderm using an in vitro analog of the primitive streakDerivation of high-purity definitive endoderm using an in vitro analog of the primitive streak


Epithelial-to-Mesenchymal Transition is engine of cell migration in the

3D-differentiation system

Epithelial-to-Mesenchymal Transition is engine of cell migration in the





Hepatocyte-like cells maturation

0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex




Epithelial-to-Mesenchymal TransitionEpithelial-to-Mesenchymal Transition

hallmarks:cytoskeleton rearrangements



hallmarks:cytoskeleton rearrangements

cadherin switch: E→N



hallmarks:cadherin switch: E→Ncytoskeleton rearrangementsacquisition of ability to migrate


Human pluripotent stem cells undergoing differentiation toward definitive endoderm in the 3D-differentiation system demonstrate appropriate gene expression dynamics

Human pluripotent stem cells undergoing differentiation toward definitive endoderm in the 3D-differentiation system demonstrate appropriate gene expression dynamics


Human pluripotent stem cells undergoing differentiation toward definitive endoderm in the 3D-differentiation system demonstrate appropriate developmental transitions

Human pluripotent stem cells undergoing differentiation toward definitive endoderm in the 3D-differentiation system demonstrate appropriate developmental transitions


Differentiated cultures located in 3D-ECM do not demonstrate consistent increases in transcription levels of genes associated with other differentiation directions

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)


Flow cytometric analysis demonstrates absence of undifferentiated (OCT4-positive) cells in the DE cultures collected from the 3D-ECM of the differentiation system

Flow cytometric analysis demonstrates absence of undifferentiated (OCT4-positive) cells in the DE cultures collected from the 3D-ECM of the differentiation system


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 DEfrom human pluripotent stem cells

3D- differentiation system enables the derivation of high-purity DEfrom human pluripotent stem cells

CXCR4 CXCR4






0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex



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

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


days of differentiation

hpSC Definitive endoderm

Early AFP-expressing hepatocyte progenitors

+ HGF/hepatocyte maturation factor0 3 8

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


flat plastic dish

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Marker expression by Marker expression by early hepatocyte progenitors derived in 3D-differentiation systemin 3D-differentiation system






0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex




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

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 testPAS test

glycogen storage


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


Hepatocyte-like cellsderived 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


3D-differentiation system:3D-differentiation system:

•demonstrate universal properties: works well with various types of pluripotent stem cells;

•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;

•based on a natural physiological process: no sorting procedures (FACS, magnetic beads etc ) are required;


•based on a three dimensional extracellular matrix: 3D environment;

•based on a three dimensional extracellular matrix: 3D environment;

Cell Transplantation, 2011doi: 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.


TeamTeam

University of California, San Diego, CAMarie Csete, M.D., Ph.D.

Cedars-Sinai Medical Center, Los Angeles, CA

Jeffrey Fair, M.D.

Organovo,San Diego, CALi Cui, Ph.D.

West Labs Scientific,Kalamazoo, MIRichard West

International Stem Cell Corporation,Oceanside, CANikolay Turovets, Ph.D.Larissa Agapova, Ph.D.Alina Ostrowska, Ph.D.Jeffrey JanusIrina TurovetsVladimir AgapovOlga Kochetkova


In Vitro Analog of the Primitive Streak (ANIMATED)

Health & Medicine

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