Culture and Differentiation of Human Embryonic Stem Cells to Form Specific, Functional Cell Types. NAS June 3, 2010

Transcription

1 Culture and Differentiation of Human Embryonic Stem Cells to Form Specific, Functional Cell Types NAS June 3, 2010

2 hescs: Specific Cell Types Available On Demand for Numerous Applications Human Embryonic Stem Cells Renewable Source of Undifferentiated Cells Cultured & Differentiated Efficiently Produce Functional Cells Cells Can Be Produced & Cryopreserved Use in Multiple Applications 2

3 Today s Presentation Overview of Properties of hescs Examples of Specific Cell Types Representative of the Major Lineages of the Body Produced from hescs Methodology to Produce and Store hesc-derived Cells

4 Human Embryonic Stem Cells Large Characterized Cell Banks Blastocyst Human Embryonic Stem Cells Hepatocytes Chondrocytes Osteoblasts Dendritic Cells Islets Neural Cells Cardiomyocytes Functional Cells of All Lineages Representative Fetal and Adult Stages of Development Research, Screening & Toxicity Testing

5 Cell Differentiation Recapitulates Developmental Biology hescs Progenitors Neural Cells Cardiomyocytes Hepatocytes Islet Cells Endothelial Cells Hematopoietic Cells Retinal Cells Chondrocytes Cardiomyocytes Hepatocytes Neural Cells Hematopoietic Cells Islet Cells Osteoblasts Chondrocytes

6 hesc Derived Neural Progenitor Cells, Neurons, & Glia Representatives of the CNS

7 Neural Progenitor Cell Lines Derived from hescs Progenitors: Nestin positive Over 20 passages (35 doublings) 40 Population Doubling Days in Culture Nestin

8 hescs Differentiate Into All Major Lineages of the CNS PS-NCAM A2B5 Progenitors -tubulin III GFAP Neural Cells neurons astrocytes oligodendrocytes MAP-2 GalC

9 hesc Derived Neurons Develop into Specific Neurotransmitter Releasing Subtypes GABA/ß-tubulin III Synaptophysin Synapses Synaptophysin Glutamate Glycine Neurotransmitters GABA Glutamate Glycine Tyrosine hydroxylase / Dopamine TH

10 hesc-derived Neurons Are Electrophysiologically Active In Vitro 80 mv -100 mv Electrophysiology 200 pa 5 ms 100% of cells tested (n=6) expressed Na + and K + currents I Na I K Current (pa) I Na I K Current (pa) % of cells (n=6) fired action potentials Voltage (mv) Voltage (mv) Voltage (mv) Time(ms) Voltage (mv) Current (pa) Time(ms) Current (pa)

11 hesc Derived Cardiomyocytes

12 Cardiomyocytes Can Be Produced from hescs - 9 actinin/ NKx2.5

13 Cardiomyocytes Express Cell Type Specific Markers Expressed Markers: cardiac troponin I cardiac troponin C myosin heavy chain myosin heavy chain atrial natriuretic factor tropomyosin -actinin desmin N-cadherin connexin 43 myoglobin CK-MB

14 Cardiomyocytes Are the Predominant Cell Type in Differentiated Cultures α-actinin Positive Cells Composition Characterized Using Markers for : Cardiomyocytes Epithelial Cells Skeletal Muscle Cells Smooth Muscle Cells Fibroblasts Mesenchymal Stem Cells Neural Cells Undifferentiated hescs

15 AR: adrenoceptor G: G protein Isoprenaline : 1 + Phenylephrine : Clenbuterol : 2 + AC: adenylate cyclase SR: sarcoplasmic reticulum SERCA: SR calcium ATPase RyR: ryanodine receptor PLB: phosphoamban Pharmacological Responses of Cardiomyocytes AR GTP G GDP +Pi forskolin : + ATP Phosphodiesterase IBMX : - AC Sarcomere camp PPi Bay K 8644 : + camp-dependent protein kinase A Ca ++ AMP Ca ++ ctnc RyR Calsequestrin diltiazem : - gallopamil : - nisddipin : - L-type Ca ++ channel SERCA PLB Ca2+ SR

16 Cardiomyocytes Have EP Profiles Similar to Tissue Cardiomyocytes and Exhibit Relatively Mature EC Coupling >80% Ventricular Cardiomyocytes Ventricular (82%) Mature EC Coupling Atrial (18%) Influx of external Ca 2+ through L-type calcium channels is required for EC coupling Nodal (<1%) Ca 2+ release from the SR amplifies Ca 2+ influx Peng et al. J Pharmacol Toxicol Methods 2010 Feb 11 Mike Laflamme, Wei-Zhong Zhu University of Washington

17 Cardiomyocytes: In Vitro Toxicology Screening Potential More Sensitive than Canine or Rabbit Purkinje Cells More Stable Action Potential Delayed Action Potential Duration with 5 herg Channel Blockers Terfenadine (herg blocker) Chromanol 293B (I Ks blocker) Cisapride Verapamil (I Kr, L-type Ca ++ channel blocker) Ca Channel Blocker Shortens Action Potential Nifedipine (L-type Ca ++ channel blocker) Drug that Blocks Both Channels Shows Same Effects as Seen in Intact Heart Verapamil Study Performed by ChanTest

18 Cardiomyocytes Can Be Cryopreserved and Form Cardiac Grafts Pre-freeze: HNA -MHC 40X 57% -actinin+ ctni/hna R2 87% viable Post-thaw: 69% -actinin+ R2 Frozen Fresh 90% viable 68% recovery

19 hesc-derived Hepatocytes

20 Fig Hepatocyte Differentiation from hesc-derived Endoderm

21 Expression of Endoderm and Hepatocyte Markers During Differentiation RT-PCR Western Blot

22 Expression of Stage Specific Markers During Hepatocyte Differentiation

23 Expression of Hepatocyte Proteins in hesc-derived Hepatocytes

24 Expression of Phase 1 Cytochrome P450 Enzymes by hesc Derived Hepatocytes

25 Manufacturing of hesc-derived Cells

26 Production Process for hescs and Differentiated Cells Provides Sufficient Cells for Multiple Applications hesc Cell Banks (Starting Material) hesc Cell Expansion hesc Differentiation Harvest, Vialing and Cryopreservation 2 Tiered Cell Banking System Enables Large Scale Production Defines Lot Size Defines Cell Type Cryopreserved Product Enables Stable Storage & Broad Distribution Large Batch Production Enables Large Banks of Cells to Be Available as a Uniform Source of Cells for Repetitive or Serial Studies

27 The Extended Proliferative Lifespan of hescs Enables Scalable Manufacture to Meet Cell Needs Projections A Single MCB Can Supply Entire Cell Product Needs 7 9 passages 5 7 passages Single MCB of 200+ vials 200+ WCBs of 200+ vials 5 20 passages >10 4 product lots at >10 14 cells per lot

28 Manufacturing of hesc-derived Cells is Scalable Small Scale Scale-Up Suitable for Differentiated Cell Banks of Up to Cells Suitable for Differentiated Cell Banks of Up to Cells

29 Proof-of-Concept Established for Production Scale to Support Commercialization Needs hescs on Microcarriers Cardiomyocytes on Microcarriers 46% -Actinin Positive

30 Summary hesc are Immortal and Pluripotent Differentiate into Representatives of All Cell Lineages Representative Functional Properties Combinations of Cell Types are Possible Production Can Be Scaled to Provide Large Uniform Banks of Cells for Multiple Applications