>> GOOD AFTERNOON, AND THANK YOU

FOR COMING AFTER THE LONG SUMMER

BREAK TO THE STEM CELL SEMINAR.

TODAY THE THE TOPIC OF THE

LECTURE IS TO PENTINE--POTENTIAL
USE OF

CARDIOLOGY AND MYOPATHY

TECHNOLOGY.

IT IS MY DISTINGUISHED HONOR TO

INTRODUCE DR. TIMOTHY KAMP.

TIM GRADUATED AS AN MDPh.D.

FROM THE UNIVERSITY'VE CHICAGO

AND CONTINUED HIS CLINICAL

TRAINING IN CARDIOLOGY AT JOHN

HOPKINS HOSPITAL WHERE HE

COMBINED BASIC STUDY IN CARDIAC

ELECTROPHYSIOLOGY AND PATIENT

CARE.

HE THEN TRIED AS A FACULTYY, THE

UNIVERSITY OF WISCONSIN WHERE HE

BECAME A LEADING EXPERT IN HUMAN

AND IPS CELL RELATED TO

CARDIOVASCULAR APPLICATIONS.

TODAY WE WILL TALK ABOUT HUMAN

PLURIPOTENT

PLURIPOTENT STEM CELL AND TOOLS

FOR CARDIOVASCULAR MEDICINE.

PLEASE WELCOME DR. TIM KAMP.

>> THANK YOU VERY MUCH, FOR THE

KIND INTRODUCTION AND THE

INVITATION AND ALL OF YOU FOR

BEING HERE AND WHAT I'D LIKE TO

TO TALK YOU TOED ABOUT IS SOME

OF OUR WORK USING PLURIPOTENT
PLURIPOTENT

CELLSY POTENT STATE EPT

STIMCELLS IN CARDIOVASCULAR

RESEARCH AND MEDICINE.

I KNOW WE HAVE A BIT OF A

DIVERSE AUDIENCE SO I'LL TOUCH

ON SEVERAL DIFFERENT PROJECTS IN

THE LAB AND FINDING THINGS OF

INTEREST TO ALL OF YOU.

BUT I'LL START WITH A LITTLE

BACKGROUND ON WHAT I DEFINED

HUMAN PLURIPOTENT STEM CELLS AS

AND TALK ABOUT IPS CELLS IS

THEIR ABILITY TO FORM MYOCYTES

AND THE CHALLENGES FOR USING IPS

AND ESL'S AND OUR MODELING

INHERITED CARDIAC DISEASES AND

USING IPS CARDIOMYOCYTES AND

SOME THOUGHTS AND PROGRESS USING

STEM CELLS FOR CARDIAC REPAIR.

AND ALSO, SHARE DISCLOSURE THAT

I'M A CO-FOUNDER OF A COMPANY

CALLED CELLULAR DYNAMICS

INTERNATIONAL, THE THE USE OF

STEM CELLS FOR DRUG TESTING.

SO I WON'T BELABOR THIS

INTRODUCTION FOR THIS GROUP

BECAUSE I'M SURE, MOST PEOPLE

ARE FAMILIAR WITH THIS, BUT JUST

TO DEFINE HUMAN PLURIPOTENT STEM

CELLS AS LARGELY IN TWO CLASSES

HERE AND THAT IS EMBRYONIC STEM

CELLS SHOWN AT THE BOTTOM OF THE

SLIDE HERE WHICH ARE DERIVEDDED

FROM SURPLUS INVITRO

FERTILIZATION EMBRYOS, WHEN THE

INNER CELL MASS IS GROWN UNDER

SPECIALIZED CULTURE CONDITIONS

AND SUSTAIN THEM IN AN

UN

DIFFERENTIATED STATE.

AND THEY CAN FORM ALL STEM TYPES

IN THE BODY AND ARE PLURIPOTENT

HERE AND SO HUMAN EMBRYONIC STEM

CELLS ARE WHAT GROT ME

INTERESTED IN STEM CELL RESEARCH

BECAUSE MY COLLEAGUE AND FRIEND

JAMIE THOMPSON BACK IN 1998,

MADE FIRST SEMINOLE DISCOVERY

ALLOWING HIM TO ISOLATE HUMAN

$Z

BEGAN A COLLABORATION TO GET

THOSE CELLS INTO CARDIOMYOCYTES

AT THAT TIME AND THAT STARTED BY

CRASH COURSE IN STEM CELL

BIOLOGY FROM MY TRAINING REALLY

IN CARDIAC CELLULAR

ELECTROPHYSIOLOGY.

MORE RECENTLY, COLLEAGUES HAVE

SUCCEEDED IN PROGRAMMING

SEEMATTIC CELLS AND INITIALLY 69

FIBROBLASTS USING A VARIETY OF

TRANSCRIPTION FACTORS, PRIMEAR

ILLY THAT ARE EXPRESSED IN THE

CELLS TO REPROGRAM THE CELLS

INTO WHAT HAVE BEEN CALLED

PLURIPOTENT STEM CELLS THAT CAN

SELF-REFLEW--RENEW AND ARE PLOTY

POTENT USING STEM CELLS, SO I AM

DESCRIBE THE WORK USE,ING--USING

HUMAN IPS SELLS.

THE THIS HAS ATTRACTED A LOT OF

EXCITEMENT AND IT'S A LITTLE

LESS CONTROVERSIAL THAN ESLs

WITH REGUARD TO THEIR SOURCE OF

DERIFFATION WHICH MADE IT EASIER

TO GAIN ACCESS TO THESE LINES

AND ALSO BECAUSE THEY HOLD

PROMISE FOR THOSE DERIVE FRIDAY

PARTICULAR PATIENTS AND IN

DISEASE SPECIFIC WAYS SO CAN YOU

STUDY DISEASES AND THEY HAVE

ALREADY BEEN PROMINENT

PUBLICATIONS STUDYING LEOPARD

SYNDROME, LONG QT SYNDROMES

SHOWN THERE BY A NUMBER AROUND

THE WORLD, USING DISEASE

MODELING AND THIS IS JUST A THE

BEGIN OF THIS RESEARCH.

AND THIS IS JUST USING THE

PROMISE OF THESE CELLS AS A CELL

SOURCE FOR A THERAPEUTIC

APPLICATIONS.

BUT THE FIRST QUESTION, WE

WANTED TO ADDRESS IS WHETHER

HUMAN IPS CELLS COULD UNDERGO

CARDIO GENESIS AND FORM

CARDIOMYOCYTES LIKE EMBRYONIC

STEM CELLS, OBVIOUSLY, WE WERE

HOPE EMPLOY, THE THE ANSWER TO

THAT WAS YES, BUT THERE WERE

SEVERAL REASONS TO WORRY THAT IT

MIGHT NOT BE YES.

FOR EXAMPLE, THE REPROGRAMMING

GENES IN THE FIRST GENERATION

THAT USE LENTIVIRUS MAY STILL

HAVE SOME EFFECT ON THE CELLS,

EITHER BECAUSE OF PERSISTENT

TRANSGENE EXPRESSION OR BECAUSE

OF INTEGRATION EFFECTS AND FURGT

EARLY MORE, WHAT DOES THIS

REPROGRAM STATE MEAN AS FAR AS

THE FUNCTION OF THE IPS SELLS

AND THEIR ABILITY TO

DIFFERENTIATE AND OBVIOUSLY, WE

CAN'T DO WHAT WE CAN DO IN THE

MOUSE IPS SELL CELLS SYSTEM AND

THAT IS TO PUT NEM IN A

BLASTOSIS AND SEE IF THEY CAN

TRIBUTE TO ALL CELL LINEAGES IN

THE DEVELOPING ORGANISM.

SO WE WENT BACK TO INVITRO

CHARACTERIZATION METHODS WHERE

WE GROW UP THE HUMAN IPS SELLS

AND THEN CREATE AGGREGATES OR

CLUMPS OF THE CELLS AND ALLOW

THEM TO SPONTANEOUSLY

DIFFERENTIATE IN THE SERUM

CONTAINING MEDIA THAT WE COULD

THEN CHARACTERIZE THE CARDIO

MYEE SIGHTS THAT FORM IN THOSE

BODIES USING A VARIETY OF

TECHNIQUES INNERRING--INCLUDING

BY O TECHNICAL TECHNIQUES AND

USING THOSE CELL W'S VIDEO

MICROSCOPY.

AND WHEN WE DID IMMUNOSTAINING

AND LOOKED AT CARDIOMYOCYTES

THAT DIFFERENTIATE FRIDAY THE

IPS CELLS WE SEE THAT FIRST

THESE VIRALLY GENERATED CELLS,

IMR 90, CLONE FOUR GENERATED BY

JAMIE THOMPSON AND HIS POST DOC

CHEN YU, FORMED CARDIO MIGHT O

SIGHTS THAT WERE STRIATED, AND

THEY WERE SHOWN DOWN HERE AND

THEY EXPRESSED ANOTHER

MONOFILAMENT PROTEIN CHAIN

THAT'S NICE INTERDIGITATING

TRIATED PATTERN WHICH YOU WOULD

EXPECT FOR THE CARDIAC MYOCYTES.

SO THEY FORM SARK O MERES SO

THAT'S A GOOD START AND THEY

ALSO EXHIBIT ACTION POTENTIALS

AND HERE'S EXAMPLES OF SHARP

MICROELECTRODE ACTION

PROTENTIALS IN THE IPS SELL

DERIVED MIGHT O SIGHTS AND THE

ONE OF THE THINGS WE OBSERVED

WITH THE ESL CARDIOMYOCYTES IS

THAT THEY FORM A DIFFERENT TYPE

OF CARDIO MIGHT O SIGHTS

SHOWNING--CARDIOMYOCYTES SHOWING

THE ATRIAL MYOSIGHTS WHICH SHOW

ACTION POTENTIAL AND I WON'T GO

OVER ALL THE OTHER DIFFERENCES

IN THE ACTION POTENTIALS BUT

SUFFICE IT TO SAY, WE CAN AT

LEAST GROUP THE OBSERVED

CARDIOMYOCYTES FUNCTIONALLY INTO

THREE CATEGORYS AND EACH

OUTGROWTH THAT WE IMPALE WITH AN

ELECTRODES IF WE REPEATEDLY

IMPALED, WE A CLUSTERING OF THE

THE PROPERTIES.

HERE'S AN EXAMPLE OF THE ACTION

POTENTIAL DIFFERENTIATION WHERE

IT'S SIMILAR THROUGHOUT THE

OUTGROWTH AND IT CONTAINS

SEVERAL CELLS SUGGESTING THAT

EACH OUTGROWTH DEVELOPS

PRIMARILY A SINGLE POPULATION OF

THE SAME TYPE.

WE ALSO WANTED TO SEE IF THESE

BEHAVED LIKE CARDIOMYOCYTES,

SHOULD BEHAVE AS WE KNOW THEY DO

IN THE NATIVE HEART AND ONE OF

THE CHARACTERISTIC PROPERTIES OF

HUMAN CARDIOMYOCYTES AND ANIMAL

CARDIOMYOCYTES FOR THAT MATTER

IS THEY ADAPT TO RATE.

WHEN WE GET UP TO RUN UP THESE

STAIRS TO LEAVE BECAUSE WE'VE

HEARD ENOUGH YOUR HEART RATE

INCREASES AND YOUR ACTION POETIC

TECTIAL GETS SHORTER.

SO WE STIMULATE THEM

ELECTRICALLY AND INCREASING RATE

TBRS ONE TO THREE HURTZ, THE

ACTION POETIC TECTIAL GETS

FASTER AND THE ACTION POTENTIAL

DURATION PROGRESSIVELY GETS

SHORTER AND THAT RATE OF

ADAPTATION HAS SUGGEST TREAD'S

INAPPROPRIATE EXPRESSION OF THE

VARIOUS ION CHANNELS

PARTICULARLY POTASSIUM CHANNELS

THAT ALLOW THAT ESSENTIAL

SHORTENING OF THE ACTION

POTENTIAL DURATION AT HIGHER

RATES SO AGAIN, EVES THAT THESE

ARE PHYSIOLOGICALLY FUNCTIONAL

CARDIOMYOCYTES.

ONE THING WE'RE WORRIED ABOUT

WITH THE GENERATED IPS CELLS IS

WHETHER THEY STILL EXPRESSED ANY

OF THE REPROGRAMMING GENES AND

I'LL SHOW YOU THAT IF WE LOOK

AT--AT PC R STUDIES OF GENE

EXPRESSION LOOKING AT OCT FOUR

AND NANOG, WHICH ARE PLURIPOTENT

GOONS AND GENES USED TO

PREPROGRAM AND THESE ARE JEANS

THAT ARE EXPRESSED IN

CARDIOMYOCYTES, WE SEE THAT OCT

FOUR NANO GO DOWN IN EMBRYONIC

BODIES FORMED FROM THESE IPS

CELLS BUT IN SOME CLONES THEY

DON'T GO DOWN THAT MUCH SO

THEY'RE STILL PERSISTENT OCT

NANOG, IN THESE FOUR SKIN CLONE

ONE EBs EVEN THOUGH WE GET

CARDIAC DIFFERENTIATION.

ALTHOUGH AT THE PROTEIN LEVEL WE

WEREN'T ABLE TO DETECT MUCH

NANOG OR OCT FOUR, AND SHOWN

HERE IN THE UNDIFFERENTIATED

STATE, PLENTY OF NANOG, HERE IN

RED, BUT IN THE CARDIOMYOCYTES

WE DON'T SEE ANY LONGER THAT

EXPRESSION.

SO, WE GARDLESS, THERE STILL MAY

BE SOME TRANSGENE MESSAGE

EXPRESSION WE'RE NOT SO SURE

ABOUT PROTEIN, WE CAN'T DETECT

THAT, BUT AGAIN, POTENTIAL

CONCERN WITH THEULENTY VIRAL

GENERATED IPS CELLS.

SO, AFTER THAT FIRST STUDY, OF

THE FIRST GENERATION OF IPS

CELLS, AS YOU KNOW, THE

TECHNOLOGY IS EVOLVING AT A--AT

A RAPID RATE, A RATE THAT'S HARD

TO KEEP TRACK OF ALL THE

DIFFERENT APPROACHES BUT NOW

THERE ARE PLASMID MEDIATED

TRANSFECTION APPROACHES, THERE

ARE MINICIRCLE VECTORS THAT

ALLOW MUCH GREATER CONSTRUCTS

WHICH DO NOT INTEGRATE INTO THE

GENOME.

THERE SEA THE OREP, NUCLEAR BAR

ANTIGEN SYSTEM THAT ALLOWS

ECTOSOMAL TRANSDUCERS TO

MAINTAIN PROGRAMMING AND, A

TECHNIQUE USED BY MY COLLEAGUE

JAMIE THOMPSON TO REPROGRAM IN A

FOOT PRINT FREE FASHION SO THESE

ARE NOT INTEGRATED INTO THE

GENOME BUT REPROGRAM THE CELLS.

THERE'S TRANSPOSE ON PIGGYBACK

METHOD TO PROVIDE A RELATIVELY

FOOT PRINT FREE APPROACH TO

REPROGRAMMING.

USING PROTEIN MEDIATED FACTORS

HAS SHOWN SUCCESS AT LEAST IN

MOUSE REPROGRAMMING.

SYNTHETIC RNA, AS GENERATE AID

LOT OF POTENTIAL AND MOST

RECENTLY MICRO RNAs HAVE SHOWN

PROMISE BISE ED MORRISY TO SHOW

IPS INTO IPC CELLS AND SMALL

MOLECULES BEING USEDDEDsA AN

ADJUVANT AND HOPE THAT IN THE

LONG-TERM, THE THE COMBINATION

OF SMALL MOLECULES CAN TOTALLY

REPROGRAM.

SO YOU CAN SEE THE LANDSCAPE FAR

GENERATING THESE IPS CELLS HAS

CHANGED DRAMATICALLY, YOU HAVE

TO REMEMBER THAT THE FIRST IPS

CELLS WERE DESCRIBED BY

YAMANAKA IN JUST IN 2006.

IN 2007, THE FIRST HUMAN IPS

CELLS WERE DESCRIBED BY

YAMANAKA, AND THESE ALL HAVE

COME UP IN A SHORT TIME PERIOD.

SO I'LL SHARE WITH YOU OUR WORK

AS SOON AS WE GOT DONE WITH

THEULENTY VIRAL STUDY, BEFORE WE

GOT DOWN WITH THE IPS SELL

STUDY, JAMIE CAME TO ME AND

SAID, WHY ARE YOU WASTING YOUR

TIME USING THOSE CELLS, YOU

SHOULD USE OUR VECTOR FREE IPS

SELLS WHICH WAS NICE.

WE HAD A NEW--WE HAD A CELL

SOURCE TO WORK WITH BUT IT WAS A

LITTLE LESS NICE THAT WE HAD

JUST SPENT ALL OUR TIME AND

EFFORT WITHz

GENERATION, BUT, SUCH IS LIFE,

SO WE TOOK JAMIE'S SELLS THAT

WERE GENERATED USING THESE

VECTORS WHICH ARE AGAIN THE EPNA

SYSTEM WHERE THE TRANSGENES ARE

EXPRESSED EPISOMEALLY AND JAMIE

REPROGRAMMED A VARIETY OF

FIBROBLASTS, SIMATTIC FIE

FIBROBLASTS INTO PLURIPOTENT IPS

CELLS THAT ARE SHOWN OVER TO THE

RIGHT CAN FORM ALL THREE GERM

LAYERS IN TERATOMAS AND WE TOOK

THOSE AND JUST I WON'T BELABOR

IT BUT SUFFICE IT TO SAY, THEY

COULD DIFFERENT FORM AND

CONTRACT CARDIOMYOCYTES AS SHOWN

HERE.

THIS IS WORK DONE BY GEN,

WA ZANG, AND THERE WAS

50 PERCENT OF CONTRACTING EBs

OVER THESE IPS CELL LINES AND

THESE ARE DERIVED FROM FOUR SKIN

GLASS AND COMPARING IT TO THE

WORK WITH HUMAN ES CELLS, HONE,

HNINE AND TWO, THERE'S

EFFICIENCY IN FORMING

CONTRACTING EMBRYO BODIES AND

THEY DEVELOPED OVER ABOUT THE

SAME TIME COURSE AS ES-CELL

DERIVED MIGHT O SIGHTS.

COZ THESE SEEM TO WORK PRETTY

WELL AND WE GET SARK O MERRIC

PATTERNS AND THE ONE THING I'LL

POINT OUT IS THAT WHEN THEY

DIFFERENTIATE THE TRANSGENE

THARS USED TO REPROGRAM AS

ADVERTISED ARE GONE AND THAT'S

EVEN COURAGING WE DON'T SEE THE

OCT FOUR AND NANOG EXPRESSION

UNLIKE THEULENTY VIRAL EXPRESSED

CELLS.

SO WE GET ACTION POTENTIALS

SIMILAR TO THEULENTY--THE

LENT

IVIRALS AND I TOUCH TALK ABOUT

THAT MORE.

I WILL TOUCH ONA ONE OF THESE,

THAT IS CARDIAC DIFFERENTIATION

FROM THE PLEURY POTENT STELLS

AND IT'S INEFFICIENT WITH THE

METHODS AND IT'S POORLY

REPRODUCIBLE AND CELL LINE

DEPENDENT AND THAT'S A BARRIER

FOR THE FIELD.

AS I DESCRIBED WE USED INITIALLY

EMBRYONIC BODY FORMATION WHICH

IS THE SORT OF HISTORIC GOLD

STANDARD TO FORM CARDIO MYEE

SIGHTS AND AS I DESCRIBED IT,

PRIMARILY INVOLVES TAKING A

CLUMP OF THESE CELLS AND

ALLOWING THEM TO SPONTANEOUSLY

DIFFERENTIATE AND IT'S CALLED AN

EMBRYONIC BODY BECAUSE IT'S A

DISORGANIZED EMBRYO DEVELOPED

TYPE PROCESS AND IN SOME CASES

CARDIOMYOCYTES FORM.

AND ANOTHER IS INDUCTIVE CULTURE

AND THAT WAS MY CHRISTINE

MUMMERY AND HER GRAM WHERE THEY

USED AN ENDODERMAL CELL AND TWO

TO HAVE CARD JACK

DIFFERENTIATION AND THERE'S BEEN

A VARIETY OF DIFFERENT

APPROACHES WHERE INVESTIGATORS

APPLY A VARIETY OF GROWTH

FACTORS TO DEVELOP THE

DIFFERENTIATION TO THE IPS OR

ESLs TO THE LINEAGE OF

INTEREST IN OUR CASE

CARDIOMYOCYTES.

SO WE WANTED TO STEP BACK AND

SEE IF WE COULD TAKE ADVANTAGE

OF DIRECT DIFFERENTIATION BUT

ALSO THINK A LITTLE BIT ABOUT

THE BIOLOGY AND THIS IS JUST A

DIAGRAM FROM A PAPER BY ENDOCHIA

IS CHANG LOOKING AT CHICK EMBRYO

AND GASTROALATION AND THE

EPITHELIAL TRANSITION THAT

OCCURS IN DEVELOPMENT.

THE IDEA BEING THAT WHEN THE

EPI-BLAST IN THE BI LAMINAR

EMBRYO UNDERGOES PRIMITIVE

STREAK FORMATION AND IN

GASTROALATION, THESE CELLS

UNDERGO EPITHELIAL TRANSITION

WITH THESE EPI-BLASTY BLASTS

SHOWING THESE THAT ULTIMATELY A

FRACTION GO ON TO FORM

CARDIOMYOCYTES F. WE COULDED

OPTIMIZE THAT FIRST STEP, THAT

MIGHT GIVE US A GOOD STEP AHEAD,

TO FORM CARDIOMYOCYTES.

AND SOME OF THE THINGS THAT

OCCUR, HERE, ARE THAT IN THE BI

LAMINAR EMBRYO SHOWN HERE IS

THAT EARLY ON ON THE BASEMENT

MEMORY RESPONSE BRINE IDENTIFIED

BY PERLACAM HERE BY THE EPIY

BLAST AS THE CELLS UNDERGO EMT.

AND THEY ARE POLARIZED

EPITHELIUM, AND OCCLUDE ENS AND

THEY CAN TIGHTLY LINK THOSE

CELLS AND THAT'S LOST AS THEY

UNDERGO EMT HERE.

SO CAN WE PROMOTE THAT FIRST

STEP?

SO HERE'S OUR WAY TO TRY AND DO

THAT.

WE USED EXTRA CELLULAR MATRIX AS

A TOOL TO PROMOTE THAT INITIAL

EMS, AND IN ORDER TO START OUT

WITH, I'VE GOT TO SHOW YOU

CULTURES, HUMAN ESLs HERE,

THIS IS HNINE, GROWN AS A

MONOLAYER ON MATE RIGEL, WHERE

IF HAVE YOU THESE, THESE ARE

TIGHTLY ATTACHED AND THESE IS

CONSIST WENT THEM HAVING A POLAR

EPITHELIAM TYPE.

THE BASAL SURFACE DOWN HERE AND

IF YOU DO IMMUNOSTAINING AND

DAPY FOR THE NUCLEI, CAN YOU SEE

THESE CELLS ARE TIGHTLY ATTACHED

WITH EKET HERE AND CONTAINING

JUNCTIONS.

AND THESE ARE CONSISTENT WITH

THE CELLS GROW NOTHING AN EPIY

BLASTIC STATE, IT'S A POLARIZED

EPITHEL YOM AND THEN IF WE TAKE

THIS AND THIS IS WORK AGAIN DONE

BY GENERATED WA ZANG AND ADD

MATE RIGEL ON TOP, WE SEE THE

ORGANIZATION OF THIS POLARIZED

EPITHELIUM CHANGES DRAMATICALLY

AND YOU STILL HAVE MICROVILLY

AND OTHER EPITHELIAL PROPERTIES

ON TOP AND YOU HAVE MULTIPLE

LAYERS AND SOME CELLS THAT ARE

NOT ATTACHED AND THESE ARE MORE

ZINC MALAND MORPHOLOGY.

YOU CAN SEE THESE HERE AND LOWER

DOWN YOU'RE LOSING ECAD HERE AND

EXPRESSION ON DIFFERENT LAYERS

OF CONFOCAL IMAGING OF THESE

CULTURES.

SO HERE'S ANOTHER WAY TO LOOK AT

THAT SAME PHENOMENA AND THIS

USES AN ANTIBODY TO LAMINANT

WHICH IS PRES SPENT ONE OF THE

MAJOR PROTEENS IN MATE RIGEL AND

YOU CAN THINK OF THE CELLS

GROWING ON A GREEN LAWN LAM

NANOG HERE, AND NUCLEI ON TOP

AND USE WHEAT GIRL GLUTENIN

WHICH IS--DETECTING

GLYCOPROTEINS PRESENTOT SURFACE

OF CELLS SO A GREEN LAWN OF

CELLS AS WE LOOK DOWN ON THEM,

GROWING HERE, WITH--ON THE

APICAL SURFACE, WGA STAINING

SUGGESTING SURFACE GLYCOPROTEINS

AND IF YOU LOOK AT FROM THE SIDE

VIEW, WGA ON TOP, NUCLEI HERE

AND SAY SLICE VIEW.

WHEN WE DOT MATRIX SAND WISH WE

SEE THEY GET MUCH MORE CROWDED

SO THERE ARE MORE CELLS THAT

PROMOTE CELL GROWTH AND IT

BECOMES MULTIPLAYER AND THEY'RE

ON TOP OF EACH OTHER, THE WGA IS

SORT OF SCATTERED AROUND A BIT

APPROXIMATE IF YOU LOOK HERE AT

A SIDE VIEW, THE NUCLEI ON TOP

OF EACH OTHER, SO AGAIN, LIKE

THE EMI, I SHOWED YOU, WE GET

MULTIPLAYERS FOCI IN THESE

CULTURES AND THESE FOCI NOT TO

BELABOR IT TOO MUCH, BUT ON THE

TOP OF THE CULTURES, ECAD HERE

IS PRESENT AND IN ONE OF THESE

FOCI'S, THE IMPRESSION IS LOST,

BUT INSTEAD, ONE OF THE

HALLMARKS OF EPITHELIAM

TRANSITION CHARGED FROM ECAT

HERE TO M-CAT EXPRESSION AND

HERE YOU CAN SEE IF WE LOOK AT

THE PLURIPOTENT CELLSY POTENT

STATE--PLURIPOTENT GENE, WE SEE

IT GETS LOWER AND DOWN LOWER AND

THEY DON'T EXPRESS AND IF YOU

HAVE GOOD EYES ACCIDENT SOME

GREEN AROUND THEM SUGGESTING

MCAT HERE SO TO US THIS WAS A

SUGGESTION WE COULD START THE

INITIAL DIFFERENTIATION BY A

BURST OF EXTRA CELLULAR MATRIX,

SOPHISTICATEDY WE TOOK THAT

KNOWLEDGE AND TRIED TO ADAPT IT

TO ONE OF THE EXISTING PROTOCOLS

FOR DIFREPRESENTIATING

CARDIOMYOCYTES PUBLISH

INDEEDINATE OUR BIOTECHNOLOGY

AND WHERE THEY USED A

COMBINATION OF ACTIVE NA AND BMP

FOUR, AFTER MONOLAYER EMBRYONIC

STEM CELLS WERE GROWN IN

CULTURE.

AND IN OUR CASE, WE DIDN'T HAVE

MUCH SUCCESS WITH THAT PROTOCOL

INITIALLY AND UNDER CONTROL

CONDITIONS, AGAIN WITH HNINE,

HUMAN EMBRYONIC STEM LINE,

HNINE, WE GOT VERY FEW

CARDIOMYOCYTES.

WHEN THEY DEVELOPED THIS PROTEIN

COMPLEX K08, THEY USED H-SEVEN

SO THERE MAY BE SOME CELL LINE

DEPENDENCE.

SO WHAT WE STARTED DOING WAS

ADDING MATE RIGELS OVERLAYS TO

SEE IF WE COULD JUMP START THE

PROCESS, SO IF WE PUT 12 DAY

OVER THE GROWTH FACTORS WE DID

GET AN INCREASE IN THE ABILITY

TO FORM CARDIAC COMPONENT

T-CARDIO MYEE SIGHTS IF WE PUT

IN A OVERLAY, WE GOT A BETTER

EFFECT, AND WE GOT THE BEST

EFFECT ABOUT 80% OF THE CELLS

BECAME CARDIOMYOCYTES WHEN

SUBJECTED TO THIS PROTOCOL THAT

INCLUDED BOTH ARKS DITION OF

ACTIVE A-FOR A DAY AND BASIC FGF

IN A MEDIA CALLED RPMI B27 AND

INITIALLY WITHOUT INSULIN AND

WITH INSULIN.

SO WE GET IN THIS CASE, WHEN

THIS PROTOCOL WE'RE TOTAL

CONTRACTS SHEETING OF

CARDIOMYOCYTES IN THE DISHs

SHOWN HERE SO THIS IS A LOW

POWER MAGNIFICATION, ABOUT THE

SIZE OF A QUARTER THAT YOU SEE

CONTRACTING.

SO HOW DOES THIS WORK?

AND ONE OF THE WAYS IN WORKS IS

THAT WHEN WE DO THE MATRIX

OVERLAY AND CHANGE THE MEDIA,

ONE THING THAT HAPPENS IN THE

ABSENCE OF THAT THERE'S

SIGNIFICANCE LOSS OF CELLS,

BECAUSE THE MEDIA FOR ONE THING

IS INSULIN FREE AS I DISCUSSED

AND ALSO THE ACT OF AND A

PROMOTES CELL LOSS AND WITH THE

MATRIX SANDWICH, MORE CELLS

SURVIVE AND ROBUSTLY GROW, AND

THE CELLS THAT SURVIVE ARE THE

BREAKINGUEY POSITIVELES AND THE

CUTLERS OF ENT, AND THEY'RE FEW

OF THEM UNDER CONTROL CONDITIONS

BUT THE MATRIX SOUND WITCH THERE

ARE MULTIPLE AREAS OF THESE

CLUSTERS, THAT DEVELOP.

AND SO USING THIS PROTOCOL IN A

VARIETY OF CELL LINES, IMR 90

CHRKS IS THATULENTY VIRAL IPS

SELL LINE, THAT DS-6993, IS THE

EPITELOMERALULENTY IPS CELL

LINE, THE THE OTHER EPISOMAL IPS

CELL LINE WE HAVE GOOD SUCCESS,

WITH HONE, HUCKS NINE WE CAN GET

ANYWHERE FROM 50 TO 90% OF THE

CELLS OF THE CARDIOMYOCYTES.

SO THAT'S BEEN KAY BIG STEP

FORWARD FOR US, FOR OBTAINING

CARDIOMYOCYTES FROM ESN AND IPS

CELLS AND NOW WE EXPANDED THIS,

I HAVEN'T ADDED IT TO ANOTHER

SIX DIFFERENT LINES INCOLLIDING

DISEASE LINES.

--INCLIEWRDING DISEASE LINES.

SO NOW WE HAVE AT LEAST OVERCOME

ONE CHALLENGE OF THE IPS AND

EXPECTATIONS SLs OR AT LEAST

GETTING BETTER AT CARDIOMYOCYTES

SO WHAT CAN WE DO WITH THOSE

IPS CELLS THAT DERIVE THOSE AND

OBVIOUSLY PEOPLE ARE QUITE

INTERESTED IN MODELING INHERITED

DISEASES AND WE'RE INTERESTED IN

CARDIAC DISEASES AND DRUG

EFFECTS POTENTIALLY.

SO I'LL SHOW SOME OF OUR ONGOING

WORK LOOKING AT POMPEY DISEASE,

AND THIS IS A AUTOSOMAL

RECESSIVE DISEASE DUE TO

MUTATIONS IN THE GENE.

IT'S CARDIAC MANIFESTATIONS ARE

HYPOTROPIC CARDIO MYOPATHY.

FOR LONG PERIODS THEY GET

ARRHYTHMIAS, AND THEY GET

RESPIRATORY DISSTRESS AND MUSCLE

WEAKNESS AND HYPER¨s TONIA AND

MOST OF THESE ENAT THATINFANTS
PASS

AWAY BY THE AGE OF ONE IF THERE

IS NO INTERVENTION AND SO, IT'S

A--IT'S A PRETTY BAD DISEASE TO

HAVE.

IT'S DUE TO ACTIVITY IN THE

CARDIAC MUSCLE IT'S RESPONSIBLE

FOR BANKING BON GLIKE O GEN IN

THE ZONES AND IT'S ACID IN THE

Ph AS IS PRESENCE IN THE

LYSOSOMES AND THAT'S GRUICOSE

THAT CAN BE--GLUCOSE THAT BE

UTILIZED BY THE BODY.

IN THE ABSENCE OF THAT, IT'S UP

WITH GLIKE O GEN AND IT
Jj BECOMES

DISRUPTURED AND THE CELLS ARE

DAMAGED AND SHOWN IN THIS

PATIENT'S SKELETAL MUSCLE

BIOPSY, YOU HAVE SOME

DEGENERATING MIRROR MY O FIBERS
HERE

FILL WIDE GLIKE O GEN AND

LIAISON SO--LYSOSOMES AND

GERONTOLOGYSTS GENERATING

FIBERS.

SO THIS IS A SERIES DISEASE WITH

SOME PATHOPNEUMONIC FEATURES

INCLUDING LIES SOMAL A--LIE SO

SO SOMAL GLIKE O GEN.

SO WE WANTED TO TEACH THAT AND

SEE IF WE COULD COMPARE THE IPS

CELLS LINES AND I TALKED ABOUT

THE IPMR 90, IT'S UNRELATED IPSL

LINE MATED FROM THE IMR90 AND

THEN WE TOOK THE HEALTHY TWO,

WHICH IS FROM THE MOTHER OF

POMPEY ONE AND HAS A DELETION IN

EXON 18 IN ONE ALLELE OF THE

ALPHA ACID GLUE COSDACE GENE AND

WILD-TYPE IN THE OTHER ALLELE

AND THEN, THE POMPEY ONE HAS

DELETION IN EXON 18 BOTH

EXPECTATIONSONS AND POMPEY TWO

IS A GENETICALLY DISTINCT FORM

OF INFANTILE DISEASE THAT HAS

TWO DIFFERENT MUTATIONS IN THE

ALPHA ACID GLUE COSIDATE AND WE

GOT THE FIBROBLASTS FROM CORIEL

AND ACTUALLY A FEW YEARS AGO AND

INTERESTINGLY, I SUBSEQUENTLY

LEARNED THAT THIS EXON 18

DELETION IS THE MOST PREVALENT

DELETION IN THE NETHERLANDS AND

THE MOST COMMON CAUSE OF POMPEY

DISEASE THERE WHERE IT AS A

HIGHER INCIDENCE THAN MOST

PLACES.

SO THAT'S WHERE IT'S NOT UNUSUAL

TO SEE DOUBLE DELETION OF EXON

18.

AND IN COLLABORATION WITH JAMIE

THOMPSON REPROGRAM THEM WITH THE

COMBINATION OF BOTH WHAT HAVE

BEEN CALLED NINE THOMPSON AND

YAMANAKAN FACTORS BECAUSE THEY

WERE STUBON TO REPROGRAM.

AND WE PUT IN SIX REPROGRAMMING

GENES

GENES AND WE WERE ABLE TO GET

PLURIPOTENT CELLS OUT.

AND SHOWN BY THE STAINING.

THEY WERE CARIO TYPICALLY NORMAL

AND THEY FORM TERATOMAS OF WITH

THE DERIVATIVES OF ALL THREE

GERM LINES AND WHEN WE GIVE

RENTIATED THEM WE CAN GET

CLUSTERS OF MYOCYTES SUSTAINING

WITH THE CARDIAC SPECIFIC

STAIBING SO WE CAN GET

CARDIOMYOCYTES OUT OF THOSE IPS

CELLS, AND IF WE THEN DO AN

ENZYMATIC ASSAY FOR ALPHA GLUE

COSDACE ACTIVITY, SO

SPECIFICALLY THE ACTIVITY THAT

WE SEE THAT IN THE POMPEY

DISEASE, THERE'S REALLY NO

DETECTABILITY ENZYME ACTIVITY,

WHEREAS IN THE TWO HEALTHY LINES

WE GET VALUES OF ACTIVITY THAT

ARE COMPARABLE TO CONTROL

PUBLISHED IN THE LITERATURE AND

SO,AST LEAST AT FIRST LOOK IN

THE IPS DERIVED CARDIOMYOCYTE

THERE IS IS RULE A LOSS OF THIS

ENZYME ACTIVITY AS YOU WOULD

PREDICT FROM THE GENETIC DEFECT.

WHAT HAPPENS WHEN WE LOOK AT THE

CARDIOMYOCYTES, HIRE'S EXAMPLES

OF THE EELECTRICITY RON

MICROGOVERNS GRAPHS, FIRST

DEREEVED FROM THE POMPEY CELL

LINE AND THIS IS JUST TO ORIENT

YOU IF YOU'RE NOT USED TO

LOOKING AT EMs AND THIS HERE'S

MONOFILAMENTS HERE THAT ARE

COMING IN AND OUT OF THE PLANE,

YOU SEE, Z-LINES HERE, SO TELLS

YOU THAT THESE ARE MONOFILAMENTS

AND MITOCHONDRIA SCATTERED

AROUND BUT THE THING I WANT YOU

TO NOTICE ARE THESE AREAS HERE.

THESE SHOULDN'T BE HERE.

THIS, THIS, THIS, THIS, THOSE

ARE GLIKE O GEN FILLED LIAISON

SO SOMES AND IF YOU LOOK AT AN

IPSO, YOU SEE MITOCHONDRIA,

MONOFILAMENTS BUT YOU DON'T SEE

ANY OF THESE THINGS.

OR IN HIGHER POWER,

MONOFILAMENTS CUT IT SHORT AXIS

BUT THIS IS A LIES SOME CUT WITH

THE PATHOGEN.

A SIGN OF POMPEY DISEASE.

AND WE DON'T SEE THOSE IN

HEALTHY CITES, WE SEE THOSE THIS

THOSE THIS TWEPPING CARDIO HIGH

O SIGHTS HEIGHT OCHONDRIA AND

THIS IS LIES SOMES SO WE'RE

ENCOURAGED THAT WE CAN

RECAPITULATE SOME OF THESE

DISEASE PHENOTYPE IN THESE I, S

DERIVED CARDIO HIGH O SIGHT

SYSTEM THOU WE'RE UVING ON TO

TRY ASK UNDERSTAND THE AT O

PHYSIOLOGY IN MORE DETAIL,

LOOKING AT A VARIETY PARAMETERS

INCLUDING THEIR CALCIUM CYCLING

AND EC COUPLING WHICH WE FOUND

CLUES FOR DIFFERENCES.

LET ME GIVE YOU ANOTHER EXAMPLE

OF AN IPS CELL STUDY THAT IS

ONGOING IN CLBERATION WITH CRAIG

JANUARY, AND COLLEAGUES, WHO

DIRECT THE INHERITED AREG MIA'S

CLINIC OF WISCONSIN, AND HERE'S

A PEDIGREE THAT HAS THE LONG QT

SYNDROME, THIS IS LONG QT TWO

DUE TO MUTATION IN THE THE HEX

RG POTENTIAL CHANNEL THAT'S

RESPONSIBILITY FOR THE RECTIFIER

DELAY CURRENT, THE RAPID

COMPONENT ALSO CALLED IKR, AND

IT'S DUE TO A TRIP TO FAN 1001

TO STOP CODON AND IT'S A

NONSENSE MUTATION.

THIS PEDIGREE HAS BEEN

CHARACTERIZED AND PROBAND IS A

30 YEAR-OLD WOMAN WHO HA SUDDEN

CARDIAC DEATH BUT THE FAMILY,

THERE'S A 64 YEAR-OLD PATIENT

THAT HAS NOT HAD SYMPTOMS BUT

HAS THE GENOTYPE AND CRAIG TELLS

ME THIS IS THE OLDEST GENOTYPE

PATIENT THAT HE'S AWARE OF, I'M

NOT SURE WHY THEY GENOTYPED HER

BUT THE 102 YEAR-OLD GREAT

GRANDMOTHER ALSO HAS THE

DISEASE, IT'S PROGRESSIVELY

EXPRESSED IN A MORE SEVERE

PHENOTYPE AND LATER GENERATIONS

AND THE SIX YEAR-OLD HAS HAD

SOME ARRHYTHMIAS AS WELL AS TACK

TACKY ARRHYTHMIAS AND SO WE'RE

GENERATING IPS CELL LINES FROM

VARIOUS SUBJECTS IN THIS

PEDIGREE, TO TRY AND UNDERSTAND

LONG QT TWO AND ALSO THE

VARIABLE PENETRANCE OF THIS

PARTICULAR MUTATION.

TO GIVE YOU AN EXAMPLE OF THESE

STUDIES WE CAN DO WITH THE

CARDIO MIRROR IMAGEEE SIGHTS

THESE ARE RECORDS DERIVE FRIDAY

VOLTAGE CLAMPING, CARDIOMYOCYTES

USING THE WHOLE CELL,

PATCH CLAMP TECHNIQUE WHERE WE

CONTROL THE MEMBRANE POTENTIAL

BUT GIVE STEPS TO A RANGE OF

DIFFERENT TEST POTENTIALS AND

MEASURE THE RESULTING IONIC

CURRENCE HERE.

AND SHOWING THESE HERE AND

ACTIVATING THE DIFFERENT DEGREES

AND THEN OFFER OFF WHEN WE

REPOLARIZE.

FOR STUDYING IKR WHICH IS WHAT

THESE CURRENTS SHOW,OOSE EASYST

TO LOOK THE THE TAIL KURTENTS

HERE, THE LARGER THE TAIL

KURTENT AND THE LARGER THE THE

IKR CURRENT SO IN CONTROL,

COMPARED TO THE W-1001 X, THOSE

TAIL CURRENTS ARE SMALLER.

IN FACT THEY'RE ABOUT HALF THE

SIZE ON AVERAGE IN THE MUTANT

IPS CARDIO SIGHTS.

AGAIN CONSISTENT WITH THIS

MUTATION CAUSING A HAPPEN LO

INSUFFICIENCY WHICH OTHER

EXPRESSION MODELS HAS BEEN

SUGSUGGESTED TO BE NONSENSE

MEDIATED RNA DECAY AND WE'RE

TRYING TO SEE IF THAT'S THE CASE

IN THIS CARDIAC MODEL AS WELL.

SO THIS IS OUR FIRST HINT

LOOKING AT USING IPS SELLS TO

MEASURE INHERITED ARRHYTHMIAS

AND OTHER GROUP VS GONE DOWN

THIS ROADED AND HAD SUCCESS

USING IPS SELLS NEWS TO MODEL

INHERITED ARRHYTHMIA SYNDROMES.

AND ALTS FOR DETERMINING DRUG

SAFETY, AGAIN THE CLINICIANS IN

AUDIENCE KNOW THAT THE QT

INTERVAL IS USED FOR THE

UNDERLYING ACTION POTENTIAL, SO

THE LONGER THE VENTRICULAR

ACTION POTENTIAL, THE LENGER TO

GET FROM THE BEGINNING TO THE

ENDING OF THE T-WAY AND THE

REASON I SHOW THAT NEXT TO A

HUMAN CARDIO MY CARDIOMYOCYTE
POETIC

TECTIAL, IF WE GIVE THAT

POTASSIUM POTENTIAL WE GET A

ACTION TWO TENTIAL.

THE ACTION WITH THAT IS IT CAN

CAUSE ACTION POTENTIAL CALLED

TORSAD DIP ONE THAT OCCUR INDEED

A PATIENT THAT WAS ADMITTED TO

OUR HOSPITAL IN THE NIEBTS AND

TACETTEN CARE OF BY MY

COLLEAGUES WHO SAW THIS LONG QT

INTERVAL AND TRYING TO FIGURE

OUT IF THERE WAS A--MEDICAL

STUDENT FOUND SHE HAD AN ON AN

ANTIHIST MINE THAT HAD A LONG

ACTING METABOLITE CALL SEMESTER

VOWEL THAT THEY TOOK TO THE

LABORATORY IT WAS A POTENT

BLOCKER IT AND HAS BEEN PULLED

TO THE MARKET.

IT HAS ARE HAS LED TO ALL

PHARMACEUTICAL AGENTS TO BE

TESTED AND IT'S AN IMPORTANT

SAFETY SIGNAL AND I BRING IT UP

OBVIOUSLY BECAUSE I THINK HAVING

HUMAN HEART CELLS IN A DISH TO

TEST INSTEAD OF PATIENTS TO TEST

IS GOING TO BENEFICIAL SO THIS

BRINGS THE IDEA OF USING

PLURIPOTENT STEM CELLS OR IPS

CELLS OR ESLs TO MAKE MY O

CARDIO SIGHTS THAT CAN YOU USE

IN ALL OF THE DRUG DISCOVERY AND

TWEPMENT PROCESS, TO INCREASE

EFFICIENCY OF OF TESTING AND

IDEALLY DECREASE ANIMAL USE AND

REDUCE THE COST OF EVALUATION

AND HOPEFULLY, IF THESE MODELS

PROVE PROBUST, THEY'LL HAVE

IMPROVED PRODUCTIVE VALUE.

AND ALSO HAVING IPS CELLS HAVING

ONE OF THE MOST EXCITING THING

SYSTEM GENETIC VARIANCE

ASSOCIATE WIDE CLEAR DISEASES

BUT GENETIC VARIANCE AMONG ALL

OF US OUT THERE THAT CAUSE US TO

RESPOND DIFFERENTLY TO DIFFERENT

DRUGS AND SO, WE PROMOTEDLET

IDEA OF CLINICAL TRIALS IN A

DISH TO TEST OUT NEW COMPOUNDS

TO SEE THE POPULATION BASEDDED

EFFECTS.

SO I'LL FINISH UP TALKING ABOUT

USING STEM CELLS FOR CARDIAC

REPAIR.  AND I WON'T BELABOR

THIS BUT AS YOU KNOW MANY OF THE

MOST PREVALENT FORMS OF HEART

DISEASE ARE ASSOCIATED WITH LOSS

OF FUNCTIONAL CARDIOMYOCYTES AND

AND ARRHYTHMIAS CAN BE DUE DUE

TO LOSS OF CERTAIN

CARDIOMYOCYTES

CARDIOMYOCYTES AND HEART DISEASE

DUE TO DEGRENERATION OF THE

HEART VALVES AND CELL DISEASE

OCCUR WHEN IS CELL LINEAGES

DON'T DEVELOP APPROPRIATELY.

SO USING STEM CELLS TO TREAT

THESE HAS ATTRACTED A GREAT DEAL

OF ATTENTION AND SUFFICE IT TO

SAY THAT MANY DIFFERENT SOURCES

OF SEMESTER CELLS FOR CARDIAC

REPAIR HAVE BEEN SUGGESTED HERE

AS A POPULAR DIAGRAM AND NOW

FROM A FEW YEARS AGO, BUT

MODIFIED TO ADD MORE AND MORE

CELLS AND

AND CARDIAC SEMESTER CELLS FROM

THE BLOOD, FROM BONE MARROW,

SATELLITE CELS, CELLS FROM FAT,

EMBRYONIC STEM CELLS--THIS GIVES

ONLY RISE TO A SINGLE CELL TYPE,

SOME ARE MORE ACCESSIBLE THAN

OTHERS OBVIOUSLY AHATOLOGIOUS

CELLS HAVE ADVANTAGES OVER ALOE

GENERATED AIC CELLS FOR

APPLICATIONS AND GENETIC

MODIFICATION AND CELLS IS

ULTIMATELY BENEFICIAL FOR THEIR

IMPACT AND ULTIMATELY, THEY MUST

HAVE SOME COMMERCIAL RELEVANCE

TO REACH WIDE SPREAD CLINICAL

USE AND SOME VARIETY IN ETHICAL

AND PUBLIC POLICY QUESTIONS THAT

WE CONTINUE TO WORK THROUGH AND

IT'S A BIT OF A ROAD TO GET

THESE CELL POPULATIONS TO

CLINICAL APPLICATIONS.

FIRST WE HAVE TO ISOLATE THE

CELL POPULATION AND REALLY

UNDERSTAND IT, AND THEN IN SOME

CASES DEVELOP THE MATRIXS AND

TEST OF THE FUNCTIONAL

PROPERTIES OF THOSE CELLS OR

TISSUES TRANSPLANTED IN VIVO AND

ALSO THINK ABOUT TRANSPLANTING

THEM INTO ANIMAL MODEL DISEASE

AND PREVENTING REJECTION AND HOW

BEST TO DELIVER THOSE CELLS IS

ANOTHER IMPORTANT CONSIDERATION.

AND ULTIMATELY, THESE THERAPIES

ARE INTEGRATED AND IMPROVE IN

VIVO FUNCTION AND IDEALLY CAUSE

THE DISEASE TO REGRESS OR IF

YOU'RE A TRUE OPTIMISTIC CURE

THE DISEASE AND EQUALLY

IMPORTANT WE HAVE TO BE SURE

THAT THE CELL THERAPIES ARE SAFE

AND DON'T BRING ALONG INFECTIOUS

AGENTS, DON'T GET TUMORS THAT

FORM AND INDUCE CERTAIN SORT OF

ORGAN SPECIFIC ADVERSE EFFECTS

SO IN THE CASE OF THE HEART, WE

DON'T WANT TO FIX HEART FAILURE

BUT INDUCE LETHAL ARRHYTHMIAS.

BUT ULTIMATELY WHEN WE HAVE MET

A VARIETY OF THESE PRECLINICAL

TESTING HURDLES THEN WE WITH GO

ONG

SO I JUST INCLUDE THAT SLIDE TO

SHOW IT'S A LONG ROAD FROM SOME

OF THESE PLURIPOTENT STEM CELLS

TO HUMAN APPLICATIONS.

WHAT WE'RE FOCUSING OUR EFFORTS

ON IS TO TRY AND GET A CARDIAC

REGENERATEDITTOR POPULATION OUT

OF EPS OR IPS CELLS THAT WILL BE

USEFUL FOR CELL THERAPY.  AND

THE ADVANTAGES OF A PROGENITOR

POPULATION IS THAT IT'S SCALABLE

OR IF WE GET OUT OF EPS OR IPS

CELLS WE GET UNLIMITED NUMBERS

OF THESE CELLS AND THEY CAN

EXPAND AND INTEGRATE WHEN

TRANSPLANTED HOPEFULLY TAKING

ADVANTAGE OF THE INTRINSIC

REPAIR MECHANISMS IN THE HEART,

AND POTENTIALLY A PROGENITOR

WILL ALLOW US TO DEVELOP

MULTIPLE LINEAGES AND PROKED

DUCTS AND HAS THE POTENTIAL FOR

OFF THE SHELVE APPLICATIONS
APPLICATIONS A
ND

OBVIOUSLY WE WORRY ABOUT THE

POTENTIAL FOR TUMOR FOCUSED
FORMATION

AND IF IT'S AN ALOE GENERATED

AIC CELL SOURCE IMMUNE

REJECTION, HAS TO BE ATTEMPT

WITH AND THERE'S CONCERNS WITH

IPS CELL SUCH AS PREMATURE SIN

ESTIMATE THAD ENS, LIMITED

EXPANSION AND IMPAIRED

DIFFERENTIATION THAT SOME

INVESTIGATORS HAVE DETECTED.

SO, THERE ARE A NUMBER OF

ATTRACTIVE FEATURES BUT AS

ALWAYS SOME CAUTION IS NEEDED.

AND THE WAY WE'VE BEEN TRYING TO

ISOLATE CARDIAC PROGENITORS IS

TO USE WHAT WE THINK IS A

RELATIVELY UNIQUE SYSTEM IS THAT

IS TO USE THE SERUM RESPONSE

FACTOR, BROADLY EXPRESSED IN

MUSCLE, BOTH SKELETAL MUSCLE AND

CARDIAC MUSCLE, SMOOTH MUSCLE,

BUT USING A THREE PRIMED

ENHANCER ELEMENT WITH THE SRF

PROMOTER AND A POST DOC IN MY

LABORATORY, IN THE SCHWARTZ

LABORATORY IN THE MID2000S

IDENTIFIED A CARDIAC SPECIFIC

ENHANCER PROMOTER SEQUENCE FOR

SRF, THAT ALLOWS IT TO BE USED

TO DETECT CARDIOMYOCYTES VERY

EARLY ON, IN DEVELOPMENT OR I

SHOULD SAY CARDIO--CARDIAC

PROGENITORS.

AND ALSO POINT OUT THAT

HOMOLOGOUS KNOCK OUT OF SRF IN

THE MOUSE, PREVENTS MESODERM

FORMATION DURING GASTROALATION

SUGGESTING THAT THIS GENE IS

TURNED ON VERY EARLY IN THE

DEVELOPMENTAL PROGRAM.

SO HERE'S OUR EXPERIENCE USING

THIS SRF GFP REPORTER THIS

CARDIAC REPORTENER MOUSE CELL

WHEN IS WE MAKE EMBRYONIC BODIES

AND LOOK FOR THIS PROTEIN AND

REPORTER TO TURN ON GFP

EXPRESSION AND WE SEE IN ESLs

WE DON'T SEE ANY GFP BUT

OVERTIME IS VERY EARLY ON IN

DIFFERENTIATION, WE START TO SEE

GREEN.

AND THEN THE GREEN IS LOCALIZED

AND I'LL SHOW YOU MORE EVIDENCE

FOR THIS IN CONTRACTING AREAS OF

EMBRYONIC BODIES AS DEVELOPMENT

PROCEEDS.

SO, AS EARLY AS EIGHT HOURS WE

CAN START TO SEE GFP POSITIVE

CELLS IN THESE EMBRYONIC BODIES

SO THAT'S VERY EARLY IN THE

DIFFERENTIATION PROTOCOL

SUGGESTING THIS CAN BE A VERY

EARLY PROGENITOR, AND THEN, BY

THE END OF THESE 24 DAYS, THE

CONTRACTING REGIONS OF THE

EMBRYONIC BODIES ARE ALL GFP

POSITIVE.

SO, IT GIVES US AN IDEA THAT WE

HAVE THE POTENTIAL REPORTER THAT

CAN INDGREAT EARLY CARDIAC

PROGENITORS.

FOR EXAMPLE IF WE TAKE TWO DAYS

OF EBs AND SUBJECT THE CELLS

TO FLOW CYTOMETRY AND SORTING WE

TAKE THE GFP POSITIVE CELLS AND

REAG GREEN CELLSIVATE THEM AND

FORM CARDIO MIGHT O SIGHTS WE

FIND THAT THESE CELLS

DEVELOPMENT FROM THE GFP

POSITIVE AND SRF GFP POSITIVE

CELLS AND THE NEGAATIVE CELLS

DON'T FORM FOR THE T-POSITIVE

CELL SPECIALIZATION OF SPECIFIC

ENDOTHELIAL THAT SUGGESTS TO US

THAT THE CARDIAC PROTENTSIAL IS

PRES END IN THOSE GFP POSITIVE

PROGENITORS AND THEY ALSO FORM A

VARIETY OF OTHER LINEAGES

BESIDES THE CARDIOMYOCYTES AND

SEE SMOOTH MUSCLE AND HEAVY

CHAIN EXPRESS AND WE SEE CD31

STAINING ENDOTHELIAL CELLS

SCATTERED IN THE CULTURES AS

WELL.

SO THEY'RE AT LEAST TRI LINEAGE

POTENT.

IF WE LOOK AFTER FOUR DAYS OF

EMBRYONIC BODIES AND LOOK AT THE

GFP EXPRESSION, WE FIND THAT IT

CO SEGREGATES WITH MARKERS FOR A

VARIETY OF DIFFERENT CARDIAC

PROGENITORS AND TBX 18 AND ISLET

ONE AND SO WE THINK THIS OTHER

GFP REPORTER CAN IDEBTIFY A

RANGE DEVELOPING CARDIAC

PROGENITORS THAT MAY BE

IMPORTANT IN THE PRIMARY HEART

FIELD AND SECONDARY HEART FIELD

AND EPICARDIAL HEART FIELD AND

JUST, TO PUT IT IN PERSPECTIVE

WE THINK THIS REPORTER TURNS ON

DURING A SPECIFICATION PROCESS

HERE IN THIS DIAGRAM OF CARDIAC

DEVELOPMENT WITH CHICK STAGES

AND THE MOUSE STAGES HERE SO WE

THINK IT TAKES--IT FIRST TURN

OUTS SPECIFICATION PROCESS AND

STAYS ON IN CARDIAC PROGENITORS

AND MIGHT O SIGHTS THROUGHOUT

THE DIFFERENTIATION PROCESS.

SO WE'RE EXCITED TO TEST THIS

PROGENITOR IN A VARIETY OF

SYSTEMS INCLUDING IN A MOUSE MY

MY MODEL AND THOSEMENT

EXPERIMENTS ARE--EXPERIMENTS ARE

UNDERWAY AND I HOPE TO SHARE

THOSE RESULTS WITH YOU IN THE

NEXT SEVERAL MONTHS BUT WE DON'T

HAVE RESULTS FOR THAT AS OF YET.

SO FINALLY ONE LAST LITTLE STORY

ABOUT CELL TYPES THAT I'LL TELL

AND YOU ONE OF THE OTHER

CHALLENGES FOR HUMAN EF AND IPS

CELLS TO CARDIOVASCULAR

APPLICATIONS FOR THERAPY IS TO

REALLY HAVE GOOD PRECLINICAL IN

VIVO MODELS SO CAN THE CELL

SURVIVE IN VIVO, HOW DO THE

DIFFERENT PREPARATIONS, THE

POTENCY OF THE DIFFERENT

PREPARATIONS COMPARE, HOW CAN WE

LOOK FOR SAFETY SIGNALS, HOW CAN

WE REALLY UNDERSTAND THE ABILITY

TO TREAT PATHOLOGY, AND THIS IS

HARD BECAUSE IF WE'RE USING

HUMAN CELLS WE HAVE TO DEAL WITH

THE IMMUNE REJECTION, IF WE'RE

DOING IT IN ANOTHER ANIMAL

SPECIES AND MOST OF THESE STUDY

VS BEEN DONE IN SMALL ANIMAL

MODELS WHOSE CARDIAC PHYSIOLOGY

IS QUITE DIFFERENT.

MOUSE HEART GOING AT 600 BEATS

PER MINUTE ISN'T SO GOOD FOR A

MUSEUM MAN STEM SO DERIVE SAID

CARDIOMYOCYTE, IT DOESN'T

THE ELECTRICAL EQUIPMENT TO KEEP

UP WITH THAT AND IT'S WAY TO

DEVELOP TACHYCARDIA INDUCED

CARDIO MIRROR IMAGE OPERATING

GLOBALLY AT SCHEOUR POOR HUMAN

CELLS THAT GET INTO A MOUSE

HEART.

SO WE'VE--NOT USED THAT APPROACH

BUT WE USED SOMETHING A LITTLE

DIFFERENT AND THIS IS A DORSAL

SKIN FOLD WINDOW MODEo

IMMUNO COMPROMISED MOUSE, THE

NOG MOUSE WHICH LACKS T-CELLS

AND B-CELLS AND NATURAL KILLER

CELLS

CELLS AND SEAN LEE DING WHO WAS

A POST DOC HERE CAME TO MY LAB

AND LED THIS STUDY IN

COLLABORATION WITH LUCY, THE

CARDIAC SURGEON ON THE

UNIVERSITY OF WISCONSIN AND

BASICALLY WHAT THEY DO IS PULL

UP THE SKIN ON THE CONTROLLER OF
BACK OF A

MOUSE AND CUT OUT ONE SIDE AND

THEN THE BACK SIDE OF THE SKIN

HERE, IS WHAT PROVIDES THE

MATRIX THAT YOU SEE YOU'RE

GRAPHED ON AND PUT A COVER SLIP

OVER AND CLAMP IT THERE, SO CAN

YOU DO IMAGING OF YOUR CELLS IN

VIVO HERE.

AND TO GIVE YOU AN IDEA OF WHAT

THAT LOOKS LIKE, HERE'S A SKIN

WINDOW, YOU CAN SEE THE

VASCULATURE, FOR THE DERMIS

HERE, AND IF YOU PUT THE CELLS,

CELL GRAFT ON TOP, SHOWN HERE IN

THE OPAQUE GRAY AREAS HERE,

THOSE ARE CELLS, IF YOU DO

HISTOLOGY WITHOUT A GRAFT, YOU

CAN SEE THIS LOOKS LIKE

EPIDERMIS HERE AND DERMIS AND

OUR GRAFT GOES ON TOP HERE.

IT'S ON TOP OF THE DERMIS BELOW

THE GLASS COVER SLIP SO WE CAN

LOOK AT THE CELL THERE IS AND

WHEN WE LOOK AT THEM, WE CAN SEE

THAT THEY CONTRACT AND THERE'S

CONTRACTING, GRAFT, FIVE DAYS,

AFTER THEY WERE TRANSPLANTED,

THESE ARE CARDIOMYOCYTES

DIFREPRESENTIATED BY THE MATRIX

SANDWICH PROTOCOL I TOLD YOU

ABOUT AND ONE OF THE INTERESTING

THINGS ABOUT THIS MODEL, IS THAT

AFTER 15 DAYS THEY UNDERGO

VASCULARIZATION AND SO WE CAN

SEE, BLOOD FLOW AND VASCULATURE,

FORMING IN THESE GOVERNMENTS

THAT ALLOW THEM

ALLOW THEM TO SURVIVE UP TO FOUR

WEEKS IN THIS PARTICULAR MODEL.

THE GRAFTS GET BIGGER AND HERE

IS WITHOUT PUTTING IN CELLS, YOU

CAN SEE THE SKIN WINDOW HERE AND

OVERFOUR WEEKS AND VARIOUS CELL

LINE WHEN IS WE PUT THE CELLS

IN, IT SORT OF GETS MESSY

LOOKING BECAUSE THEY GET

VASCULARIZED OVER TIME AND TURN

INTO CONTRACTING AREAS OF

TISSUE.

AND IF WE DO IMMUNOSTAINING ON

THOSE GRAFTS AFTER WE SACRIFICE

THE ANIMALS WE CAN SEE A VARIETY

OF CARDIAC PROPONENT T, CARDIO

MIGHTEE SIGHTS THERE BUT OTHER

NUCLEI SUGGEST OTHER CELLS ARE

PRESENT AS WELL, AND WE DETECT

ENDOTHELIAL CELLS, AS IT TURNS

OUT IT'S SPECIFIC FOR CAT HERIN

AND THAT IS PRESENT THROUGHOUT

THE GRAFT AND IS IN THE CARDIO

MYEE SIGHTS HERE SO WE CAN GET

GRAFT AND USING HUMAN NUCLEAR

ANTIGEN WE CAN SHOW THAT SOME OF

THESE CELLS AS I SUGGESTED ARE

OF HUMAN ORGANIZATIONS

INTERFERON AND THEY ARE MOUSE

ORIGIN SO WE GET A CHIMERIC

VASCULATURE SUPPLYING THESE

CONTRACTING GRAFFS THAT CAN BE

MAINTAINED IN THE DORSAL SKIN

FOLD WINDOW.

SHOW IS PROVIDING US A TOOL TO

STUDY THE CARDIAC BIOLOGY OF

THESE PREPARATIONS AND TO

UNDERSTAND HOW THEY BEHAVED IN

VIVO AND ALSO, WE'RE WORKING TO

DISSECT WAYS TO OPTIMIZE THEIR

REVASCULARRIZATION WHICH WE

THINK WILL BE CLINICALLY

RELEVANT AS WE TRANSITION TO

LARGER ANIMAL MODELS AND PATIENT

STUDIES.

SO LET ME STOP THERE AND

SUMMARIZE SOME OF THE POINTS

THAT I HOPEFULLY MADE TODAY.

THAT IS THAT WE BELIEVE

BOTHULENTY VIRAL AND VECTOR FREE

CELLS DIFFERENTIATE INTO

CARDIOMYOCYTES THAT ARE LIKE THE

EMBRYONIC STEM CELL DERIVED

CARDIOMYOCYTES WE'VE SEEN

EARLIER AND WE'VE SEEN NO MAJOR

DIFFERENCES ALTHOUGH AS YOU

KNOW, THERE ARE MANY CELL LINES

OUT THERE AND WE'VE SAMPLED A

VAST MINORITY OF THEM AND SO,

THERE'S NO DOUBT THAT AS TIME

PROGRESSES THERE MAY BE SOME

LINES THAT BEHAVE DIFFERENTLY

AND WE'VE NOT SEEN A BIG

SIGNATURES NATURE YET AND ESL

MYOCYTES.

THE FOUND THAT THEY CAN WORK IN

THE TRANSITION OF THE

PLURIPOTENT STEM CELLS WHICH

ENABLES US TO HAVE THE PROTOCOL

FOR CARDIOMYOCYTES AND ALLOWING

US TO HAVE IEFFICIENCY TO

GENERATE PURE POPULATIONS OF

CARDIOMYOCYTES AND I SHOULD ALSO

NOTE THAT WE GET HIGH YIELDS OF

CARDIOMYOCYTES, FIVE TO 10

CARDIOMYOCYTES FOR IPS OR SIC

CELLS WE'VE-SUCCESS USING OTHERS

TO MODEL INHERITED ARRHYTHMIA

AND POMPEY DISEASE, AND SO THIS

IS A WHOLE NEW FIELD THAT IS

OPENING UP AND HAS A LOT OF

POTENTIAL FOR UNMASKING NEW

MECHANISMS FOR OUR UNDERSTANDING

AND ALSO DEVELOPING NEW

TREATMENTS WHICH DEVELOP TO

DEVELOP A NOVEL PROGENITOR

POPULATION USING A PROMOTER

ENHANCER SEQUENCE, AND WE

BELIEVE IT'S IDENTIFYING A VERY

EARLY CARDIAC PROGENITOR THAT

HAS MULTILINEAGE POTENTIAL AND

WE'RE WORKING FORWARD AND

TESTING THAT AND ALSO

DEPRIVATIONING IT IN HUMAN CELLS

AND I FINALLY SHARED WITH YOU

SOME OF OUR WORK ON THE DORSAL

SKIN FOLD WINDOW FOR PRECLINICAL

TESTING OF THE HUMAN ES, AND IPS

CELLS TO UNDERSTAND THE ABILITY

TO PERFORM IN VIVO AND GET

VASCULARIZED.

AND THEN, AISLE END WITH A

LITTLE IDOL SPECULATION ABOUT

THE FUTURE OF CARDIO REGENRATIVE

MEDICINE AND WHERE I SEE SOME OF

THE CHALLENGES AND SOME OF THE

THE FUTURE EFFORTS GOING.

I'D LIKE TO THINK THAT BEDDING

THE BASIC BIOLOGY OF CARDIAC

DEVELOPMENT AND DIFFERENTIATION

IS IMPORTANT BECAUSE IT'LL

CONTINUE TO BENEFIT OUR EFFORTS

AT CARDIAC REPAIR, I THINK THEY

CLEARLY FEED OFF ONE ANOTHER AND

I'M OPTIMISTIC THAT BOTH LINES

OF RESEARCH WILL CONTINUE TO BE

QUITE VALUABLE.

I STILL DON'T THINK WE KNOW THE

RIGHT CELL OR RIGHT INTERVENTION

FOR THE VARIOUS CARDIAC

PATHOLOGIES, I THINK THERE'S

GREAT DEAL OF WORK TO BE DONE

THERE, TO OPTIMIZE OUR CELL

PREPARATIONS AND OUR APPROACHES

THERE.

INCLUDING THE BEST WAY TO

DELIVER THE CELLS AND HOW TO

ENGINEER CONSTRUCTS IN SOME

CASES.

D. WE'RE STILL LEARNING ABOUT

HOW TO DESIGN CLINICAL TRIALS.

FOR CELLULAR THERAPIES AND

ESPECIALLY WITH REGUARD TO THE

QUESTION OF UNDERSTANDING

LONG-TERM SAFETY.

WHICH I THINK IS GOING TO BE

QUITE IMPORTANT.

AND THERE HAS BEEN GROWING

ENGAGEMENT IN THE FIELD AND

BECAUSE WITHOUT THAT AND AND IT

WOULD MEAN IT THEIR HAS TO BE

COST EFFECTIVEPPROACHES THAT

WILL DEVELOP OUT OF THIS WHICH

IS LIKELY IN THE LONG-TERM.

SO WITH THAT I WILL ACKNOWLEDGE

THIS IS THE WORK OF MANY FOLKS

IN MY LAB, JIM WA ZANG DID A LOT

OF WORK I MENTIONED AND DR. DING

AND OTHERS DID THE WORK AND

SEVERAL COLLEAGUE VS BEEN

WORKING WITH THE POMPEY CELLS

AND MY LONG-TERM COLLABORATION

WITH JAMIE THOMPSON FOR THIS

WORK.

THANK YOU VERY MUCH.

[ APPLAUSE ]

>> [INAUDIBLE QUESTION FROM

AUDIENCE ]

>> YES, SO THE QUESTION IS WHAT

IS THE VIABILITY OF THE CARDIAC

GRASP TRANSPLANTED INTO THE SKIN

WINDOW.

HOW MANY SURVIVE AND HOW MANY

ARE LOST?

>> WE KNOW HOW MANY CELLS WE PUT

IN.

WE CAN TRY AND MEASURE HOW MANY

CELLS ARE PRESENT AT THE END OF

THE DAY ALTHOUGH IT'S TOUGH TO

DO THAT CELL COUNTING IN THE BIG

GRAFTS SO I'M--THE BEST

OBSERVATION IS ACTUALLYPˇYkZ
LOOKING

DAILY TO SEE IF AREAS OF THE

CELLS ARE CLEARLY DIEING AND

LOST, AND THERE'S AN INTERESTING

STORY THERE.

IT TURNS OUT YOU HAVE TO PUT A

CERTAIN SIZE OF A GRAFT IN TO

GET GOOD SURVIVAL IF IT'S TOO

SMALL, THE GRAFTS DON'T GET

VASCULARIZED AND SO, IF THEY

DON'T GET VASC ARRHYTHMIAIZED

AND THEY TEND TO GET SMALLER AND

SMALLER AND IF YOU PUT IN LESS

THAN 5000 CELLS FYOU GET OVER A

MILLION CELLS OR TWO MILLION

CELLS IS WHAT WE TYPICALLY USE,

THAT GETS OVER A CRITICAL THRESH

HOLD TO STIMULATE THE

VASCULARIZATION WHICH ALLOWS THE

CELLS TO STICK AROUND FOR THE

FOUR WEEKS WE CAN OBSERVE.

THE LIMITATION OF THE MODEL IS

THAT IT'S RAY SURGICAL MODEL

WITH THE BIG WINDOW ON THE BACK

OF THE MOUSE AND THE WIND O

TENDS TO BREAK DOWN AFTER FOUR

WEEKS AND SO WE LOSE YOU KNOW

STERILITY AND SO, WE CAN'T STUDY

LONGER THAN THAT.

ALTHOUGH, THERE ARE APPROACHES,

CRANIAL WIND OS GUT WINDOWS YOU

COULD DO LONGER.

>> [INAUDIBLE RESPONSE FROM

AUDIENCE ]

>> THE ISSUE OF ARRHYTHMIAS?

IT'S SOMETHING THAT'S WORRIED

MANY PEOPLE IN THE CARDIAC CELL

THERAPY FIELD, THE GOOD NEWS IS

THATC$S

REALLY HAVEN'T PICKED UP A LARGE

SIGNAL OF THE ARRHYTHMIAS, YOU

COULD SAY THEY HAVEN'T DONE MUCH

EITHER AS FAR AS REGENERATING

THE HEART IN MANY OF THE

CLINICAL TRIALS, MY OWN PERSONAL

BIAS IS THAT PUTTING IN

DIFFERENTIATED CARDIOMYOCYTE

SYSTEM POTENTIALLY PROBLEMATIC

FOR GENERATING HETEROGENEITY IN

ARRHYTHMIAS AND BECAUSE OF POOR

COUPLING DIFFERENCES IN AUTOMA

TISSITY BUT PUTTING IN

PROGENITORS THAT WILL TAKE

ADVANTAGE OF THE HEART'S NATIVE

WATO REGENERATE MUSCLE WILL

HOPEFULLY AVOID OR MINIMIZE THE

RISK OF ARRHYTHMIAS AND IT

REMAINS A SIGNIFICANT CONCERN.

>> [INAUDIBLE RESPONSE FROM

AUDIENCE ]

>> SO MAN FRED ASKED A COUPLE

GOOD QUESTIONS, THE FIRST ONE IS

THE POMPEY IPS CELLS HAVE

ALTERED GLUCOSE METABOLISM

BECAUSE OF LIE SOMA GLIA COGEN

ACCUMULATION SO DOES THAT AFFECT

THEIR TERATOMA FORMATION.

DOES THAT VARY AMONG CONTROL AND

IPS CELL LINES?

>> THE ANSWER THAT THAT QUESTION

IS, WE WERE HAPPY TO SEE

TERATOMAS WITH THE POMPEY IPS

CELLS TO SEE THE THREE PRIMARY

GERM LAYERS, WE DIDN'T DO ENOUGH

TERATOMAS TO RIGOROUSLY TEST

DIFFERENCES AS YOU KNOW,

TERATOMAS ARE A BIT EXPENSIVE TO

DO.

IT TAKES TWO MONTHS TYPICALLY

AND WE JUST DO A COUPLE OF MICE

BECAUSE THE ASSAY IS WELL ENOUGH

DEVELOPED WITH TWO DIFFERENT

INJECTION SITES.

SO WE'VE GOTTEN FOUR TERATOMAS

FOR EACH LINE.

TO PROVE THAT THEY'RE

PLURIPOTENT, BUT IN ORDER TO

RIGOROUSLY ADDRESS THE QUESTION

YOU'RE RAISING WE DON'T HAVE

ENOUGH DATA.

THERE WAS NOTHING OBVIOUS, THE

SECOND QUESTION IS, HAVE WE USED

ENZYME REPLACEMENT THERAPY FOR

THE POMPEY IPS CELLS, ENZYME

REPLACEMENT IN FIRST IN THE FORM

OF MY O ZYME HAS BEEN SHOWN TO

BE BENEFICIAL IN INFANTILE

POMPEY DISEASE, INTERESTINGLY IT

TREATS THE CARDIAC BETTER THAN

THE SKELETAL PHENOTYPE,

PARENTALLY BECAUSE THE ENZYME

GETS INTO CARDIOMYOCYTES BETTER

THAN SKELETAL MYOCYTES AND WE

HAVE BEEN TRYING TO GET THAT

ENZYME AND I THINK WE'RE ABOUT

TO GET IT FROM THE COMPANY TO

TRY THAT.

SO, THAT'S AN EXAMPLE WE VERY

MUCH LIKE TO DO BUT THOSE WHO

KNOW THAT ENZYME, IT'S A COSTLY

REAGENT.

[* MUSIC ]

>> SO AGAIN I APOLOGIZE I DIDN'T

GO INTO DETAIL VIS REGARDING

THAT IF WE TAKE EMBRYONIC BODIES

THAT ARE TWO DAYS IN CULTURE AND

ISOLATE THOSE GFP, POSITIVE

CELLS AND PLATE THEM DOWN ON

STOVE CELL WHICH IS ARE A FORM

OF FEEDER CELLS, WE CAN EXPANNED

THEM FOR--EXPAND THEM FOR TWEBT

PASSAGESS SO THEY CAN BE GREATLY

EXPANDED, FROZEN DOWN, WE CAN

THAT YOU OUT THOSE FROZEN

PROGENITORS AND DIFFERENTIATE

THEM INTO CARDIOMYOCYTES AND

SMOOTH MUSCLE CELLS SO, YES, WE

THINK THIS IS AN EXPANDABLE

POPULATION OF PROGENITORS.

>> INAUDIBLE RESPONSE FROM

AUDIENCE BRACHIAL ]

>> WHAT WE THINK ISNIQUE ABOUT

OUR--UNIQUE ABOUT OUR

PROGENITOR, IT'S A EARLY

PROGENITOR.

SO IN THE DEVELOPMENTAL

HIERARCHY IT'S IT IS EARLIEST

KNOWN BATTLE, BUT THAT'S OUR

WORKING HYPOTHESIS AND SO, WE

THINK IT'LL FORM MANY OF THE

OTHER PROGENITOR POPULATIONS

THAT HAVE BEEN DESCRIBED IN THE

LITERATURE, SO WE THEN IS AN

EARLY PROGENITORS ON THE

DIFFERENCE.

>> INAUDIBLE RESPONSE FROM

AUDIENCE ].

>> OH THAT'S A GOOD QUESTION, NO

WE HAVEN'T DONE THAT YET.

WE--YOU KNOW OUR REASON TO USE

THE WINDOW IS MORE TO LOOK AT

HUMAN CELLS IN A MOUSE MODEL,

WE'RE GOING TO USE THE--THE

PROGENITOR AND I THEN IS

CONFUSING IS OUR WORK HAS BEEN

IN MOUSE ESC CELLS, SO THEY'RE

WE'RE HAPPY TO PUT IT IN A MOUSE

HEART BECAUSE THAT'S THE RIGHT

BIOLOGY.f
–

>> [INDISCERNIBLE].

>> [INDISCERNIBLE].

>> OKAY, I'M SORRY I DIDN'T

UNDERSTAND YOUR QUESTION

INITIALLY.

SO, I MUST NOT HAVE STATED THAT

CLEARLY.

THE SRFGFP REPORTER WAS IN MOUSE

EMBRYONIC STEM CELLS SO WE

REALLY PURIFIED IT FROM

DIFFERENTIATING EMBRYONIC BODIES

NOT FROM MOUSE HEARTS.

SO WE HAVEN'T ISOLATE FRIDAY

MOUSE HEARTS.

AGO WE ARE USING THAT REPORTER

SYSTEM AND ENGINEERING

TRANSGENIC MICE BUT WE HAVEN'T

YET DONE THE EXPERIMENT THAT YOU

SUGGEST OR LOOKED IN THE BONE

MARROW, ABOUT BUT THOSE ARE

EXPERIMENTS THAT WE MUCH LIKE TO

DO.

>> [INDISCERNIBLE]

>> PEOPLE HAVE IN THE IPS CELLS

AND THE TELOMERE LINK SEEMS TO

EXPAND AS THEY ARE REPROGRAMMED.

DEPEBBLED O GBA THE

IPS--DEPENDING ON THE IPS CELL

THAT YOU USE, THE SIZE OF THE

TELOMERES AND THE POLYMER SEEMS

TO BE VARIABLE.

BUT MOST INVESTIGATORS SEEM TO

THINK THAT THERE'S A FAIRLY

ROBUST ACTIVATION TELOMERE ACE

WITH REPROGRAMMING.

>> IF THERE ARE NO FURTHER

QUESTIONS, THANK YOU VERY MUCH

DR. KAMP.

[ APPLAUSE ]