Human pluripotent stem cells in understanding genetic cardiovascular disease and effects of drugs


MY NAME IS PAM ROBEY AND I’M A SENIOR INVESTIGATOR IN THE NIDCR AND ON BEHALF OF DR. COLLINS, WHO COULD NOT BE HERE WITH US TODAY DUE IT TO A MEETING DOWNTOWN, GOOD LUCK TO HIM — IT IS A GREAT PLEASURE FOR ME TO INTRODUCE PROFESSOR CRISTINE MUM MUMRY AS TODAY’S SPEAKER. SO PROFESSOR MUMRY HAS AN OUT OUT-OF-THE-ORDINARY BACKGROUND COMPARED WITH MANY OF US IN THE FIELD, HAVING RECEIVED HER UNDER UNDERGRADUATE TRAINING IN FIZZ PHYSICS AT THE UNIVERSITY /OF KNOTTINOTTINGHAM IN THE UK. AND HER PH.D TRAINING IN BIOFIZZ BIOPHYSICS AT THE UNIVERSITY OF LONDON, WHERE SHE STUDIED THE EFFECTS OF ULTRASOUND ON WOUND HEALING. SHE THEN DID A POST DOCTOR FELLOWSHIP AT THE HUBER IN INSTITUTE, WHICH IS A PART OF THE ACADEMY OFATO ARTS AND
SCIENCES SCIENCES, AND THIS INSTITUTE HAS — HAD A LONG TERM HISTORY IN THE STUDY OF DEVELOPMENT AND STEM CELLS. AND THERE SHE STUDIED THE DIFFERENTIATION OF BLOOD STOLMA CELLS AND REGULATION OF THE CELL CYCLE. BUT DURING THE COURSE OF HER STUDIES HERE, SHE BECAME REALLY FASCINATING — FASCINATED BY THE DIFFERENTIATION POTENTIAL OF MOUSE AND HUMAN EMBRYONOCARCINOM EMBRYONOCARCINOMA CELLS, WHICH THAT THE TIME WERE THE MOST PERIMETRIMITIVE CELLS THAT WE
HAD AVAILABLE FOR STUDYING OF VARIOUS DIFFERENTIATION PATTERNS PATTERNS. AND THESE CELLS ALSO WERE FOUND TO BE E/HRELECICALLY ACTIVE CELL TYPES, WHICH IS WHERE HER BACKGROUND IN BIOFIPHYSICS AND QUANTITATIVE ANALYSIS REALLY SERVED HER WELL. AND SHE WAS TENURED AT THE IN INSTITUTE FOR WORK AT /TTHAT
TIME. SUBSEQUENTLY IN WORKING WITH A NUMBER OF LEADERS IN THE FIELD OF PLEURIPOETENT STEM CELL
BIOLOGY AROUND /THE WORLD SUCH AS CHRIS GRAHAM, MARTIN PAR /KWRA AND
ELLEN THOMPSON, SHE ESTABLISHED THE USE OF MOUSE AND HUMAN EMBRYONIC STEM CELLS IN THE NETHERLANDS AND BECAME A FULL-/TPHREPLGD FULL BIOLOGIST AND DEVELOPMENTAL BIOLOGIST AND WE’RE GLAD SHE DID DID. SHE HAS HAD A MAJOR FOCUS ON THE FUNCTION /AAL DIFFERENTIATION OF CARD /KWIOMI / MEIOCYTES IN
VASCULAR CELLS AND AFTER A SABBATICAL AT THE HARVARD UNIVERSITY STEM CELL INSTITUTE AS NOTAN /EUPINSTITUTE FELLOW, WHERE SHE WAS FOCUSING ON INDUCED PLEURIPOETENT STEM
CELL CELLS, SHE RETURNED TO THE NETH NETHERLANDS IN 2008, AS /AA PRO PROFESSOR OF DEVELOPMENTAL BIOLOGY AND CHAIR OF THE DEPARTMENT /OF A/TPHANATOMY AND
EMBRY EMBRYOLOGY AT LIDEN UNIVERSITY MEDICAL CENTER. AND THESE ARE POSITIONS THAT SHE STILL HOLDS. IT REALLY WOULD BE IMPOSSIBLE TO LIST ALL OF PROFESSOR’S ACCOMPLISHMENTS IN SUCH A SHORT PERIOD OF TIME. BUT PERHAPS MOST NOTABLE IS THAT SHE WAS REALLY ONE OF THE FIRST TO DIFFERENTIATE PLURIPOTENT STEM CELLS INTO FUNCTION /AAL
MYO MYOCARDIAL CYTES AND EMPHASIZE THE WORD FUNCTIONAL BECAUSE THESE CELLS DID HAVE ALL OF THE E/HRELECTRO/TPEUPHYSIO/HROPBLLOG
ICAL CHARA
CTE CHARACTERISTICS OF IMMATURE BUT CARD /KWRO /PHAOIOMY MEIOCYTES
AS /OPPOSED
TO EXPRESSION OF JUST /AA FEW
MARKERS MARKERS, WHICH FOR THOSE OF YOU THAT KNOW ME, THIS IS ONE /OF MY PET PEEVES. SHE ALSO HAS WORKED
EX/TEPTENSIVELY ON DIFFERENTIATION OF VASCULAR ELEMENTS. HER WORK IS REALLY NOTED IN THE RIGOR THAT SHE USES TO CHARACTER CHARACTERIZE THE FUNCTION /AAL ASPECT OF THESE CELLS AND SHE REALLY IS A TRUE LARRY IN THE FIELD. IN ADDITION TO HER SCIENTIFIC ACCOMPLISHMENTS, PROFESSOR MUMRY IS AN ACTIVE MEMBER OF THE STEM CELL COMMUNITY, NOT ONLY IN THE NETHERLANDS BUT ALSO INTERNATIONALLY. SHE HAS SERVED ON NUMEROUS BOARDS IN THE NETHERLANDS AND WAS A FORMER BOARD MEMBER OF THE INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH. SHE HAS BEEN ACTIVELY ENGAGED IN THE PROPER CONDUCT OF STEM CELL RESEARCH AND ASSOCIATED DEVELOPMENTS OF CLINICAL THERAPIES. SHE WAS ONE OF THE FOUNDING HE HAD TORS OF THE JOURNAL OF STEM CELL RESEARCH, AND THEN MOVED ON TO BE THE FOUNDING HE HEDITOR IN CHIEF OF THE JOURNAL STEM CELL REPORTS. UNDER HER INDIVIDUVIGILANT
GUIDANCE, THE JOURNAL IS ALREADY ONE OF THE LEADING STEM CELL RESEARCH JOURNALS AFTER ONLY ABOUT FIVE YEARS /TIN PUBLISCATION. SO TODAY SHE’S GOING TO BRING US UP TO DATE ON HER CURRENT STUDIES ON THE PATH /OJEGENIC
MECH MECHANISMS OF CARD /KWRO
/SRAFIOVASCULAR DISEASE THROUGH THE USE OF NORMAL AND MUTE TATEED PLURIPOET PLURIPOTENT STEM CELL THAT’S RIGHT DIFFERENTIATED INTO CARDIO VASCULAR FEPHENOTYPES AND SHE’S GOING TO PARTICULARLY EMPHASIZE THE NEED FOR RIGOROUS ASSAYS TO PROBE THE FUNCTIONALITY OF THESE CELLS AND HOW THE CELLS THEMSELVES CAN BE USED AS /AA PATIENT FOR DRUG DISCOVERY. SO PLEASE JOIN ME IN WELCOMING PROFESSOR MUMRY TO THE PODIUM. TO PRESENT TODAY’SUAL — LECTURE LECTURE. /PHRA [APPLAUSE]>> THANK YOU. SO THANK YOU. AND THANK YOU, PAM, FOR THE INITIATION IN/SREVITATION TO
COME. I AM EXTREMELY HONORED TO BE SPEAKING HERE. THE NIH SANE PLACE I VISITED, BUT IT’S BEEN A DELIGHT TO BE HERE AND SEE ALL THE WONDERFUL RESEARCH THAT’S GOING ON. SO YOU ARE VERY ACTIVE IN STEM CELL RESEARCH. AND WHAT I AM GOING TO TALK ABOUT TODAY IS /THE USE AND UNDERSTANDING CARD /KWRO
/SRAFIOVASCULAR DISEASE AND THE EFFECTS OF DRUGS DRUGS. I WILL ALSO TOUCH ON A LITTLE ON TRANSPLANTATION STUDIES, WHICH WE’VE DONE IN THE PAST, BUT MAY BE A LITTLE — MAYBE A LITTLE UPDATE IS IN PLACE. BUT I UNDERSTOOD THE AUDIENCE WAS FAIRLY /SPWROD I AM JUST GOING TO START WITH A SLIDE WHICH DIVIDES STEM CELLS, HUMAN STEM CELLS INTO BASICALLY THREE FUNCTION /AAL CATEGORIES. SO WE STARTED IN 1978 — SORRY, 1998 WITH PLURIPOTENT STEM CELLS FROM HUMAN EMBRYOS. NOW AS YOU WILL SKNOW SHE’S ARE SENSITIVE BECAUSE IT INVOLVES DESTROYING THE EMBRYO WHEN YOU TAKE OUT THE INNER CELL IN CULTURE. THE ADVANTAGE OF THESE CELLS WAS IMMEDIATELY OBVIOUS IN THAT THEY WERE SIMILAR TO MOUSE EMBRY ONC ONCIC STEM CELLS AND THEY COMPLETELY FORMED ALL CELLS OF THE BROID /* BODY IN PRINCIPLE. SO THAT BROADLY IS ABOUT 200 DIFFERENT STEM CELL TYPES. NOW THEIR ETHICAL SENSITIVITY DROVE A REINVESTIGATION OF ANOTHER TYPE OF STEM CELL, ADULT STEM CELLS. AND WE LEARNED FOR MANY YEARS OF COURSE THAT THESE CAN BE DESTROYED FROM — DERIVED FROM BONE MARROW. BUT PARTICULARLY THROUGH THE WORK IN THE INSTITUTE ACTUALLY WHERE I WAS, HE DISCOVERED THAT MANY IN/TETESTINAL ORGANIZS ALSO
HAD A STEM CELL POPULATION ADULT EMPHASIS /* STEM CELLS, AND HE CHARACTERIZED THE MOST FROM THE IN/TETESTINE BUT HE ALSO
DISCOVERED THEM IN THE MIDDLE OF THE PAN PANCREAS AND PROCESS /STAESTATE,
MANY OTHER ORGANIS. NOW THE DIFFERENCE WITH THESE CELLS IS THEY CONFIRM THE EP EP/THAOITHELIAL COMPONENTS OF
THE TISSUES IN WHICH THEY RESIDE. SO FROM THE DOTS, THEY FORMED THE LINING OF THE IN/TETESTINE,
THE EP/THAOITHELIAL LAYER, ALL THE NOT FORM THE STROMA. THEY WILL NOT FORM THE /EUPENDO ENDO/THAOTHELIAL CELLS. SO THEY HAVE A DIFFERENT SORT OF DIFFERENTIATION POTENTIAL THAN PLURIPOTENT STEM CELLS. THE ADVANTAGE IS THEY ARE GENERALLY MORE MATURE SO THEY DO SHOW ADULT FEPHENOTYPES, WHEREAS PLURIPOTENT STEM CELLS SHOW MOSTLY IMMATURE PHENOTYPES. NOW THESE TWO THINGS WERE BALANCED AGAINST ANOTHER WHEN WE ALSO DISCOVERED THAT WE COULD IN INDUCE PLURIPOTENCY AND. OF COURSE JAIME THOMPSON DISCOVERED THAT YOU COULD RE REPROGRAM ADULT CELLS TO A STATE WHICH WAS ESSENTIALLY IN INDISTINGUISHABLE FROM EMBRYONIC STEM CELLS SIMPLY BY FOUR TRANSCRIPTION FACTORS. NOW, THERE ARE VARIOUS VARIANTS ON THIS PROGRAM RIGHT NOW. BUT NEVERTHELESS, THEY PROVIDED A SOURCE OF CELLS, WHICH WE COULD DERIVE MANY DIFFERENT TYPE — CELL TYPES FROM. SO WHERE ARE WE IN THE PLURIPOET PLURIPOTENT STEM CELLS THESE DAYS? SO WE HAVE THESE FROM EMBRYOS. WHEN THEY WERE FIRST DERIVED, THEY WERE VERY DIFFERENT TO CULTURE. THEY REQUIRED WHAT WE CALLED MECHANICAL PASSAGE. THEY EASILY DIED. THEY WERE VERY DIFFICULT TO CULTURE, AND IT WAS ALMOST IM IMPOSSIBLE TO GENETICALLY MAN MANIPULATE THEM. THAT’S ESSENTIALLY BEIEN SOLVED NOW. IT’S LARGELY THE DRIVE FROM COMMERCIAL COMPANIES WHO PRODUCE EXCELLENT REAGENTS TO DO THIS NOW. YOU CAN GROW THESE CELLS FAIRLY EASILY. YOU CAN INTRODUCE MUTATIONS INTO THESE CELLS, AND IN /THIS WAY, CREATE GENETIC LINES CARRYING HUMAN RELEVANCE DISEASES. YOU CAN ALSO GET DISEASE MODELS, FUR INTERESTED IN STUDYING HUMAN DISEASE, FROM EMBRYOS THAT HAVE BEEN REJECTED AFTER
PRE/TPHAENATAL PRE PREIMPLANTATION GENETIC DIAGNOSIS. AND THIS IS USUALLY FOR VERY RARE DISEASES, SOMETHING LIKE HUNTINGSON DISEASE IN THESE FAMILIES. THE EMBRYOS WILL BE MADE BY IVF. ONE CELL WILL BE TAKEN OFF /THE EIGHT CELL STAGE OF DIAGNOSIS AND IF IT’S NOT AFFECTED, THE EM EMBRYO WILL BE PUT BACK IN THE MOTHER. IF AFFECTED IT WILL BE USED TO DERIVE AN EMBRYONIC STEM CELL LINE WITH DISEASE. THERE IS A VERY LARGE COLLECTION OF THESE CELLS THAT I STEM OUTSIDE PARIS SOME 200 OF THESE GENETIC DISEASE MODELS. NOW THE DISADVANTAGE OF THESE CELLS IS /THE INDIVIDUAL, WITH THESE MUTATIONS WOULD NEVER HAVE BEEN ALIVE AS AN ADULT. SO WE WILL NOT KNOW THE AGE OF ONE SET OF THE DISEASE. WE WILL NOT KNOW ITS SEVERITY. WE WILL NOT KNOW TO WHICH DRUGS THE PATIENT WOULD HAVE RESPONDED RESPONDED. THAT’S THE ADVANTAGE OF IPS CELLS. YOU CAN DERIVE THEM FROM ANY INDIVIDUAL. YOU WILL NOTE PATIENT HISTORY. YOU WILL KNOW THE /TPAEFAMILY
HISTORY HISTORY. AND YOU WILL ALSO KNOW WHICH DRUGS THEY MAY OR MAY HAVE NOT RESPONDD TO. BUT THE PROBLEM HERE IS WHAT DO YOU TAKE AS /AA CONTROL? AND IN THE BEGINNING, VERY MANY STUDIES USED A HEALTHY CONTROL. WE CAN MAKE IPS CELLS FROM I OF YOU. BUT YOU ACTUALLY HAVE — AND THAT WAS A PROBLEM, BUT THE ISSUE WAS SOLVED BY ALL TECHNOLOGY THAT WENT INTO GENT GENETIC MANIPULATION OF HUMAN ES CELLS. IT TURNED OUT THAT TECHNOLOGY A ALLOWED US TO /REPAIR GENES IN
IP IPS CELL. SO IT’S NOW POSSIBLE TO GENERATE GENERATEITOJGENIC PAIRS, ONLY DIFFERING IN MUTATION OF INTEREST. AND OF COURSE THIS IS INTERESTING. NOT ONLY IN THE CONTEXT OF POTENTIAL OT THE OLGUS TRANS TRANSPORTATION, BUT PARTICULARLY IN MAKING DISEASE MODELS OF GENT GENETIC CONDITIONS, FAPHARMACO PHARMACOLODGICAL SCREENING AND ALSO FOR TOXICITY TESTING. SO THIS IS WHERE WE ARE BASICALLY TODAY. NOW, JUST TO GIVE AN IDEA OF HOW THIS FIELD IS — HAS EXPLODED, IF WE LOOK HERE, IN 2008, THE FIRST DISEASE LINE /OF IPS CELLS WAS PUBLISHED, AND THAT BECAME FROM — CAME FROM GEORGE DAILY’S LAB. AND YOU CAN SEE HERE THAT EVERY YEAR MORE AND MORE PAPERS HAVE A AAPPEARED WITH A DISEASE — DISEASE MODELS. AND THIS PARTICULAR PREVIEW IN MY LAB AND I MADE A SUMMARY OF ALL THE DIFFERENT LINEAGES, THE DIFFERENT TYPES OF TISSUE FOR WHICH CELL LINES HAVE BEEN DERIVED. AND YOU CAN SEE IN 2015 AT LEAST THERE IS A HUGE INCREASE. THERE IS A LARGE NUMBER OF THESE EVERY YEAR AND IT’S EVEN IN INCREASING MORE. SO THERE IS A HUGE INTEREST IN CREATING THESE DISEASE MODELS. AND WHEN WE ARE TRYING TO MAKE DISEASE MODELS, THIS ALSO E EVOLVED. SO AROUND 2007, WE WERE LOOKING BASICALLY AT WHAT WE CALL SPONTANEOUS DIFFERENTIATION IN STRUCTURES AGGREGATES OF CELLS WE CALLED EMBRYO BODIES. AND WITHIN THOSE BODIES, THE CELLS WERE /* WILL SPONTANEOUSLY DIFFERENTIATE AND COULD BE SLIGHTLY DIRECTED DEPENDING ON WHICH GROSS /TPAOURBGTS ADD. ALTHOUGH THIS TIME IT WAS OFTEN TO GET MON /OTIYPIC CULTURES. SO PURE POPULATIONS OF LIVER AND HEART CELLS. PURE POPULATIONS OF NEWURONS. BUT THEN IT TURNED OUT IT MIGHT BE ACTUALLY MORE INTERESTING TO MAKE MORE COMPLEX STRUCTURES. AND THIS WAS BY ANALOGY OF THE ORGANIEGOGANOID THAT’S, FOR
EXAMPLE, THE ADULT STEM CELLS MADE, TO MAKE MORE COMPLEX TISSUES, CONTAINING MULTIPLE DIFFERENT CELL TYPES AS THEY WOULD BE PRESENT IN /AA NORMAL ADULT
TISSUE TISSUE. AND GOING ON BEYOND THAT THAT, MORE RECENTLY, WE STARTED TO MOVE INTO 3D CULTURES EVEN TO WHAT WE CALL ORGANI ON CHIP DEVICES WHICH PUT SMALL PIECES OF STEM CELL-DERIVED CELLS INTO ORGANI-ON CHIP DEVICES WHICH YOU CAN MEASURE THINGS IN THEM. THERE ARE SENSOR S IS IN THEM
AND THESE ARE NOW WHERE THIS FIELD IS GOING, AND I THINK IN THE U.S. THERE HAS BEEN A LARGE INVESTMENT IN /THIS TYPE OF CHIP MODELS. BUT THIS IS HOW THEY HAVE E EVOLVED OVER TIME. AND YOU CAN SEE HERE IT’S SUM SUMMARIZED IN ALL THE LITERATURE IN /THIS AREA. WHILST IN THE EARLY DAYS WE WERE LOOKING TOWARDS MON /OTIYPIC CULTURES. YOU SIEE HERE /TPIN MORE RECENT TIMES YOU HAVE GONE OVER TO HETERORITIC CULTURES WITH MULTI MULTIPLE CELL TYPES AND NOW INTO DEVICE S S IN 2 AND 3 DIMENSIONS
IN MI/KRCRO TISSUES AND
ORGANIZEGOGANOIDS. SO IT’S A GAME THAT’S CONTINUAL CONTINUALLY EVOLVING. SO WHAT DO WE HAVE? MY FOCUS IS ON CARDIAC AND VASC VASCULAR CELLS. BASICALLY WE USE THE PRINCIPLES OF DEVELOPMENT TO GET THESE CELLS. I WON’T GO INTO DETAILS, BUT THIS IS ALL SO FREE. THIS MAKES IT MUCH MORE DEFINED AND WE ADD FACTORS THAT ARE IMPORTANT FOR EARLY FORCE, PASSENGER CARDIAC AND WE GET THESE OF BEATING CARD /KWRIO
MIOCYTES MIOCYTES. IF YOU WANT TO MAKE
ENDO/THAOTHELIAL CELLS, WE BASICALLY FOLLOW THE SAME PRINCIPLES BECAUSE WE ALSO WANT /TO MAKE EARLY
MESODETERMINE AND THAT DMESODETERMINE IS
PATENT PATENTD IN DIFFERENT WAY BY ADDING A GROSS FACTOR INHIBITING INHIBITING. THERE IS OTHER SIGNALS, AND FINALLY WE GET ENDO/THAOTHELIAL
CELLS EXPRESS THINGS LIKE P CAM OR CD 31. AND LOOKING ESSENTIALLY LIKE END ENDO/THAOTHELIAL CELLS. I’LL COME BACK TO /TTHAT IN A MINUTE. FROM THIS SAME DIFFERENTIATION PROTOCOL, WE CAN ALSO GET SOMETHING THAT LOOKS A LITTLE LIKE A PARASITE I’LL COME BACK TO /TTHAT LATER ON. BUT THIS IS BASICALLY THE MESSAGE WE USED THE PRINCIPLES OF DEVELOPMENT TO GET THESE CELL TYPES. AND IT’S TRUE WHETHER YOU DO ANY DIFFERENTIATION PROTOCOL, THE NEWUROPROTOCOLS, THE PANCREAS PROTOCOLS, THE LIVER PROTOCOLS, ALL OF THEM USE THE PRINCIPLES OF DEVELOPMENT. SO, WHEN WE ARE TALKING ABOUT THE HEART, HOW DO WE KNOW THAT THESE ARE FUNCTION /AAL CARD
/KWROIO MIO MIOCYTES? NOW, IF YOU LOOK AT THE HEART, YOU HAVE HERE THE DIFFERENT CHAMBERS OF THE HEART. AND CHARACTERIZED
E/HRELECTRO/TPEUZ ELECTROPHYSIOLOGY BY DIFFERENT E E/HRELECTRICAL ACTIVITIES THAT
WE CALL ACTION POTENTIAL OUT. PUT A SHARP E/HRELECTRODE IN THESE /SKPR*EULZ GET SOMETHING CALLED THE ACTION POTENTIAL LOOKING LIKE THIS. IF IT’S A VENTRICULAR CELL, WILL HAVE THIS SHAPE. IT’S AN TRIIAL CELL, WILL HAVE THIS SHAPE. AND OUR VERY FIRST PROTOCOLS, VERY EARLY ON, WE HAVE MIXTURES OF CELLS. WE HAD 85% VENTRICULAR-LIKE CELLS LOOKING LIKE THIS. ABOUT 15% A/TRTRIAL CELLS. AND 1% — WE’RE GOING TO MODEL DISEASE OF DIFFERENT TISSUE, WE WOULD NEED PURE POPULATIONS OF EACH CELL TYPE. IN ADDITION, IT WOULD BE VERY HELPFUL IF WE COULD EXPAND THE COMMITTED PRO GENESIS OF THESE LINEAGES. SO WE WANTED TO FIRST TO KNOW HOW DO WE DIRECT DIFFERENTIATION TO THESE DIFFERENT CELL TYPES? AND CAN WE MAKE CARDIAC PRO GENT GENTORS GROW? SO NEN YOU WORKED ON
NEWUROCELLS, YOU ARE IN THE ADVANTAGEOUS POSITION IF YOU START THE DIFFERENTIATION PROTOCOL, YOU CAN STOP IT AT A PARTICULAR POINT AT THE NEWUROPRO
/SKWREPBGENITOR STAGE, EXPAND THOSE CELLS, FREES /* FREEZE THEM DOWN AND GET NEWURONS AT ANY MOMENT YOU
CHOOSE CHOOSE. WE’VE NEVER BEEN ABLE TO DO THAT WITH CARDIAC PRO
/SKWREPGENITORS. SO THIS JUST BASICALLY SUMMARIZE SUMMARIZES RATHER A LOT /OF WORK IN WHICH WE USED A TRICK. SO WE PUT IN AN /EUINDUCIBLE
NECK NECKTINE INTO THE UN/TKEUF UNDIFFERENTIATED CELLS AND SIMPLY BY ADDING DOXICYCLIN JUST EARLY IN THE DIFFERENTIATION PROCESS, WE WERE ABLE TO MAKE THESE CELLS GROW IF WE ADDED A SET /OF GROSS FACTORS. WHAT WAS CRUCIAL TO THIS WAS THIS PARTICULAR REPORTER LINE HERE /TIN WHICH GFP HAS BEEN IN INSERTED INTO THE 2.5. THIS IS A TRANSCRIPTION FACTOR, IMPORTANT IN HEART DEVELOPMENT. AND IF WE ADDED THESE FACTORS JUST BEFORE NK X 2.5 WAS WAS /TKWEUFPD ON, WE COULD EXPAND THESE CELLS ENORMOUSLY. WE CAN PUT THEM THROUGH AT LEAST 70 PASSAGES AND CRYOPRESERVE THEM. AND ENABLE UPON THAWING IN COMBINATION DIFFERENT GROSS FACTORS TO FORM CARD /KWRIO
MIOCYTES BUT ALSO CARDIAC-DERIVED VASC VASCULAR CELLS. SO WHAT DOES THIS LOOK LIKE? THIS IS WHAT IT LOOKS LIKE IN PRACTICE. THIS IS A DISH CELLS GROWING A SMALL AGGREGATES IN THESE DISHES DISHES. AND NOTHING MUCH HAPPENS. IF WE ADD GROSS FACTORS, NOT A LOT HAPPENS EITHER /TKPWR-FPLT WE ADD DOXICYCLIN, WE SEE THESE EXPANDING. AND IF WE ADD IT IN COMBINATION WITH IGF AND HEDGEHOG INHIBITOR JUX SEE THESE VERY LARGE COL COLONIES FORMING. SO WE WANTED TO CHARACTERIZE THESE CELLS. SO WE HAD THE GFP MARKER IN HERE AND THESE ARE DEFINITELY CARDIO CARDIOMI CITES. BUT WE HAD A VERY STRANGE POPULATION OF CELLS, AT LEAST MY POST DOC FOUND THEM STRANGE. HE /SAESAID I’VE GOT THESE CELLS THAT ARE BEATING BUT THEY’RE NOT GFP-POSITIVE. HOW IS THAT POSSIBLE? AND IF WE KNOW A LITTLE BIT ABOUT HEART DEVELOPMENT, WE KNOW THERE IS ONE REGION OF THE HEART WHICH HAS CARD /KWRO /PHAOIOMY
MEIOCYTES B
UT IS NKX 2.5-NEGATIVE AND THAT’S THE REGION OF THE HEART THAT FORMS THE PACEMAKER. AND THOSE CELLS SHOULD EXPRESS A PROTEIN CALLED POTOPLANIN ON THE CELL SURFACE AND THAT’S INDEED WHAT THEY DID. SO THESE CELLS, WE PREDICTED, SHOULD BE A/TRTRIAL —
PACEMAKER- PACEMAKER-LIKE CELLS. AND THEY DID THE
E/HRELECTRO/TPEUZ ELECTROPHYSIOLOGY, WE FOUND THAT INDEED WAS THE CASE. SO ON THE BASIS OF THIS CELL SURFACE MARKER FOR FMIXED POPULATIONS, WE CAN SORT OUT THE BASIS OF THE PACEMAKER-LIKE CELLS. THAT ALLOWS US TO /EBEXPAND
CARDIAC PRO FENT SHALL — PRO
/SKWREPGENITORS AND FOR SOME /SPHAOEPL, NOT VERY USED TO USING PLURIPOTENT STEM CELLS, THIS CABE VERY USEFUL /STKPWREUFPBLGT WHAT WE WANTED TO DO, TO ADDRESS A VERY SIMPLE QUESTION. IN THE CONTEXT OF CELL TRANS TRANSPLANTATION FOR HEART REPAIR REPAIR. NOW WHAT’S NORMALLY DONE IN TEST TESTING CARD /KWRO /PHAOIOMY
MEIOCYTES FOR
HEART REPAIR. IF YOU DIFFERENTIATE THE CELLS INTO CARD /KWRO /PHAOIOMY
MEIOCYTES, HERE AGAIN THEY ARE GREEN BECAUSE OF A PROMOTER THAT ALLOWS THEM TO BE MARKED. AND YOU INJECT THE CELLS INTO THE HEART OF /AA MOUSE. THAT’S THE USUAL PROCEDURE. NOW IF PEOPLE ARE TESTING THE EFFECT OF LET’S SAY BONE MARROW, STROMA CELLS, WHICH SOME PEOPLE THINK MIGHT HELP HEART REPAIR, THEY GIVE THE MOUSE A MYOCARDIAL INFARCTION AND INJECT THEM INTO THE BLOOD STREAM. BUT THE PRINCIPLE IS /THE SAME WHATEVER PEOPLE ARE TESTING. BASICALLY YOU /HRAOLOOK FOR
FUNCTION FUNCTIONAL ANALYSIS. YOU LOOK WITH MRI OR SOME KIND OF FUNCTION /AAL ANALYSIS OF THE HEART AND YOU CARRY OUT HIST HISTOLOGY. SO THIS WAS SOME OF OUR WORK IN OUR 2006-2007. THIS IS A MOUSE HEART. IT’S UNDERGONE A MYOCARDIAL IN INFARCTION BECAUSE AS YOU CAN SEE HERE, WE HAVE TIED OFF A BLOOD VESSEL. AND WHAT YOU SEE THESE GREEN CELLS HERE ARE CARD /KWRIO
MIOCYTES IN THE MOUSE HEART. NOW THEY COULD STAY THERE FOR A VERY LONG TIME. WE HAVE SEEN THESE GRAFTS UP TO NINE MONTHS OF TRANSPLANTATION AND WE WERE FIRST VERY EN ENCOURAGED. BUT THERE WAS NO ALIGNMENT OF THESE CELLS ALONG THE NORMAL HEART MUSCLE. THERE THERE IS NO COUPLING WITH THE HOST. THE GRAPHS VERY SMALL AND THERE WAS A SHORTTIME EFFECT BENEFIT ON CARDIAC FUNCTION IN THE FIRST FOUR WEEKS, BUT NO LONG TERM IM IMPROVEMENT AT ALL. SO LOOKING AT THREE MONTHS, YOU COULD NOT SEE ANY DIFFERENCE BETWEEN THE HEART THAT RECEIVED CARD /KWRO /PHAOIOMY MEIOCYTES
AND THOSE T
HAT ECEIVED A NON-CARD /KWRIO
MIOCYTE CONTROL. YOU ALWAYS SEE A DIFFERENCE IF YOU’D PUT ANOTHER CONTROL, WHICH IS JUST THE CULTURE
IMMEDIAMEDIUM. SO YOU HAVE TO TAKE CARE OF YOUR CONTROLS. BUT WHAT WE WANTED TO DO WITH THESE NEW PRO /SKWREPGENITOR
CELLS WE WOULD EXPAND /SKWRAOFPLT WE ADDRESS THE TISSISSUE OF SMALL GRAFTS SO, WHAT WE DID. WE REVISITED TRANSPLANTATION IN OUR FIRST ASSAYS. WE PUT THESE CARD /KWIO PRO
GENTORS UNDER /THE SKIN OF NON-SKID MICE AND GAVE THE MICE DOXICYCLN IN THE DRINKING WATER. AND YOU CAN SEE HERE UNDER /THE SKIN THESE CARD /KWIO PRO
/SKWREPBGENITORS
E EXPANDED SUBSTANTIALLY. THEY HAVE KI 667 INVESTIGATION AND THEY EXPANDED ENORMOUSLY. SO THEN WE TRANSPLANTED THEM ALSO INTO THE HEART. AND THIS IS A REGULAR HEART. THIS IS WITHOUT MYO /KARCARDIAL
IN INFARCTION. AND YOU CAN SEE THESE GRAFTS HERE ARE VERY SUBSTANTIAL. SO IN PRACTICE WHAT WE DO IS FOR THE FIRST COUPLE /OF WEEKS, WE GAVE MICE DOXICYCLIN IN THE DRINKING WATER AND TAKE IT AWAY AND WE INJECT THEM WITH MGF AND B /PH-FPMP AND IN/TKAOUDEUCES
THE FORMATIO
N OF ENDO/THAOTHELIAL CELLS SO WE
GET THESE VERY LARGE GRAFTS AND WE ARE FOLLOWING THIS UP TO ASK THE VERY SIMPLE QUESTION. DOES GRAFT SCIENCE MATTER? SO IT’S NOT THAT I THINK THIS IS IN /TPHANY WAY APPLICABLE TO PATIENTS IN THE CLINIC BUT IT WILL HELP US TO ADDRESS THAT. SO WHAT I’VE TOLD YOU SO /TPFAR
IS — AND THIS SUMMARIZES IT, THAT WE FOLLOW THE PRINCIPLES OF DEVELOPMENT AND WE CAN GET VARIOUS CELL TYPES OUT /OF DIFFERENTIATION. WE START HERE WITH A PLURIPOTENT STEM CELL. THEY UNDERGO FAST DIFFERENT DIFFERENTIATION INTO A MESOODERM GENESIS AND CARDIAC MESO MESODETERMINE INTO HEART-FILLED SPECIFIC PRO /SKWREPGENITORS AND
FINALLY CARDIAC MYEIOCYTES. THE SIGNALS WE USE ALL DERIVE FROM DEVELOPMENTS. THEY INVOLVE ADDITION OF SIGNALS SIGNALS, SIGNALS — SIGNALS, BLOCKING PARTICULAR SIGNALS AND EACH OF THESE STEPS IS MARKED BY SPECIFIC COMBINATIONS OF TRANSCRIPTION FACTORS. NOW WE’VE MADE REPORTS OF CELL LINES FOR MANY OF THESE STAGES, WHEREAS PARTICULARLY THIS AKS 2. 2.5 LINE AND THIS ALLOWED TO US SORT THE CELLS OUT AT DIFFERENT STAGES. DO MICROARRAY ANALYSIS AND TRY TO FIGURE OUT WHICH CELLS SERVE AS PROTEINS AND ENABLE SORTING AND WE FOUND THAT A AND /SKR-FPLS CAM CORRESPONDED LARGE LARGELY 95% WITH NKX 2.5. SO THAT MEANS WE CAN USE THESE CELLS SURFACE MARKERS TO SORT OUT THE CARD /KWRO /PHAOIOMY
MEIOCYTES. AND THIS IS A WIDELY APPLIED PRINCIPLES. DOESN’T MATTER WHICH LINEAGE YOU ARE LOOKING FOR. YOU CAN SUUSE THIS REPORTER
LINES TO FIGURE OUT WHICH COMBINATION OF SURFACE PROTEINS ANTIBODIES WOULD HELP YOU. WHAT /I ALSO SHOWED YOU IS THAT THE CARDIAC PRO /SKWREPGENITORS
WILL GROW UP TO 70 PASSAGES UNDER INFLUENCE OF DOGS. BUT IT’S AN INDEPENDENT STUDY. WE WE ADD RECEITIN OIC ACID WE
CAN TURN THE CELLS LARGELY INTO MYO CITES AS /*. AND WE CAN AS LSO ADD ANOTHER COMBINATION OF GROWTH FACTORS AND GET PACE MARKER CELLS. SO WE HAVE THIS SYSTEM FAIRLY WELL UNDER CONTROL, I’D LIKE TO SAY. SO WHY WOULD WE USE HUMAN CARDIO MYEIOCYTES TO DO THESE
MODELINGS? WELL, THE MOST IMPORTANT REASON IS THAT MOUSE AND HUMAN HEARTS DIFFER. YOU CAN SEE WITHOUT BEING A CARD CARDIOLOGIST, YOU CAN SEE HERE. THIS IS AN /AE/HREBLECTRO/KARD
/KWROCARDIO
GRAM, WHICH A CARDIOLOGIST WOULD GET AND YOU CAN SEE HERE THESE DIFFERENT PEAKS. THIS LARGE PEAK HERE CORRESPONDS TO WHEN THE VENN CRITTRICLE
CONTRACTS AND PUMPS THE BLOOD AROUND /THE BODY. THIS IS HERE. THESE TWO PEAKS ARE VERY IMPORTANT AND I’LL COME BACK TO IT IN A MINUTE. IT’S CALLED THE Q /T-FPT
INTERVAL. IT’S THE DIFFERENT BETWEEN THESE TWO PEAKS JUST IF YOU LOOK AT THE E/HRELECTRO/KARD
/KWROCARDIOGRAM OF A
MOUSE, IT LOOKS QUITE A DIFFERENT SHAPE. THE MOUSE HEART BEATS AT 500 TIMES A MINUTE. THE HUMAN HEART IS ABOUT 6 /0. SO THERE IS ALMOST A FACTOR OF 10 DIFFERENCE. SO THE COURSE OF FIPHYSIOLOGY IS DIFFERENT. IF WE LOOK AT THESE IMCHANNELS FOR THE LENGTH /OF THESE ARROWS GIVES THE SIZE OF THE CURRENT GOING ACROSS THE PLASMA AND RATE OF CARD /KWRO /PHAOIOMY
MEIOCYTES. AND WE CAN SEE HERE. THE I IS /THE CURRENT. THE SODIUM SECURITY. IN THE HUMAN AND MOUSE, THE LENGTH /OF THESE ARROW IS /-THE SAME. SO IF YOU HAVE A /TKRAOEURG GENE MUTATION THAT AFFECTS THIS CHANNEL, THE RESPONSE WILL BE EV EVIDENT IN BOTH HUMANS AND MICE. BUT IF YOU LOOK AT /THIS
CHANNEL, WHICH IS VERY IMPORTANT IN DETERMINING THE Q /T-FPLT
INTERVAL PO PO/TTASSIUM CHANNEL, IT’S VERY LARGE IN HUMANS AND TINY IN MICE MICE. THIS IS WHY WE SOMETIMES MISS PROBLEMS OF IRON CHANNEL MUTE MUTATION S S IN MICE. SO A DRUG AFFECTING THIS CHANNEL AND MUTATION WILL HAVE NO EFFECT ON MICE. SO WHEN WE ARE LOOKING FOR CARD CARDIAC ERYTH /KPWHRAEUL /*S, AB ABNORMAL HEART RHYTHMS, WE HAVE VARIOUS TYPES. WE HAVE THE CON/SKWREPGENITAL
FORM, WHICH IS USUALLY CAUSED BY MUTE MUTATION S S IN IRON CHANNELS. AND WE HAVE ACQUIRED OR DRUG IN INDUCED FORMS. AND THEN MAYBE A COMBINATION OF THE TWO. BUT BASICALLY IF WE HAVE THESE KIND OF FAMILY TREES, WE MIGHT HAVE TO O– IN A FAMILY WHO DIED DIED. WE MIGHT HAVE INDIVIDUALS IN THAT FAMILY WHO HAVE ABNORMAL HEART RHYTHMS AND IT MIGHT TURNED /* TURN OUT THAT ONE OF THE FOUNDERS WAS ONE /OF THESE PARENTS. SO WHAT WE’RE TRYING TO FIGURE OUT IS WHO IS LIKELY TO DIE. WHO ARE AFFECTED CARRIERS? AND WHO ARE UN/TPAEBGAFFECTED
CARRIERS? WHAT WILL HAPPEN TO THEIR CHILDREN? BUT AS IMPORTANTLY, WHO WILL DIE AS A CONSEQUENCE OF DRUGS THEY TAKE OR OTHER
PREVIO/TKEUDISPOSITION FACTORS? SO THIS IS WHERE THE FAMILIAL ANALYSIS CAN BE VERY IMPORTANT IN LOOKING FOR IPS MODELS. SO HUMAN HEART CELLS IN DRUG RESEARCH ARE IMPORTANT FOR VARIOUS REASONS. HEART DISEASE HAS HIGH MORTALITY AND MORBIDITY. IT’S A HUGE MARKET. SO BILLIONS OF DOLLARS GO INTO HEART DRUGS. THERE IS AN /EUINCREASING MARKET DUE TO THE AGE POPULATION BUT NOT ONLY THAT. AFTER CANCER TREATMENT, DOXIRUB DOXIRUBISIN, WHICH IS OFTEN USED TO TREAT BREAST CANCER, IS CAUSING AN EP/TKEIDEMIC OF HEART FAILURE, PARTICULARLY IN YOUNG WOMEN. TEN YEARS AFTER SURVIVAL OF THEIR TREATMENT, OF THEIR CANCER CANCER. AND THEY ARE ARE NO NEW DRUGS IN THE /PHRAO*EUPB TO TREAT HEART FAILURE. THE PHARMACEUTICAL INDUSTRY IS INVESTING MORE AND MORE MONEY IN NEW DRUGS, BUT FEWER AND FEWER ACTUALLY ARE ENTERING THE MARKET MARKET. 20% OF THEM ARE NOT SUITABLE FOR CLINICAL USE BECAUSE THE SIDE EFFECTS ON THE HEART. IT MEANS THAT WE ARE UNABLE TO DETECT THEM USING THE REGULAR MODELS. SO WHAT WE ASK. CAN THESE BIOASSAYS BASED ON STEM CELLS DERIVE THE RELATIVE CARD /KWRO /PHAOIOMY MEIOCYTES
FOR SAFETY
FAR PHARMACOLOGY, DRUG REPURPOSING, AND LOOKING FOR NEW COMPOUNDS? SO THIS IS A VERY SIMPLE ASSAY, WHICH WE ARE USING MI/KCROE MICROELECTRODE CHIPS. SO THIS IS A SINGLE CHIP, AND IF YOU LOOK AT /THIS, IT’S EATING WAY ON THESE EXTERNAL
E/HRELECTRODES ELECTRODES. IT’S A KIND OF E /KR-FPC G OF
CARDIO MI MEIOCYTES. AND WHAT YOU SEE UNDER NORMAL CONDITIONS OF THESE — ARE THESE THESE I REGULAR PEAKS. BUT IF YOU ADD A DRUG WHICH CAN CAUSE ABNORMAL HEART RHYTHM YOU SEE HERE THE INTERVALS BETWEEN THE PEAKS ARE VERY ABNORMAL. YOU HAVE WHAT ARE CALLED EARLY AFTER DE/PORLGSZ. THESE ABNORMAL PEAKS HERE. AND IT LOOKS EXTREMELY ODD. AND YOU CAN SEE THESE CELLS HERE COULD BE FROM A PATIENT OR THEY COULD BE FROM CONTROLS. BUT WHAT WE WANTED TO DO IS TO FIGURE OUT WHETHER WE CAN DO SAFETY FAPHARMACOLOGY ON THESE CELLS. BUT BECAUSE OF THESE PROMISING RESULTS, THE FDA IS ALREADY PLANNING TO ADOPT THIS KIND AF SAY FOR SAFETY FAPHARMACOLOGY,
AND THEY EXPECT NOT ONLY TO WANT /TO LOOK AT THE EFFECT OF WHAT WE CALL THE HERB CHANNELS, WHICH I SHOWED YOU WAS VERY IMPORTANT FOR Q /T-FPT INTERVAL. UNTIL RECENTLY, ALL YOU WIHAD TO DO AS /AA PHARMACEUTICAL COMPANY WAS EXPRESS THAT CHANNEL IN /AA HECK CELL. A NON-CARD /KWRO /PHAOIOMYOCITE
AND LOOK A
T THE /* OF THE EFFECTS ON /- THAT HAVE CHANNEL. BUT NOW THE FDA WANTS TO YOU LOOK AT ALL OF THE EFFECT ON ALL CHANNELS AND FOR THIS THEY WILL BE USING IPS DEVICE CARD /KWRIO
MIO MIOCYTES. SO WHAT KIND OF THINGS CAN WE SEE? SO IN THAT VERY SIMPLE ASSAY I SHOWED YOU WITH THE MI/KCROE MICROELECTROARRAYS. YOU CAN SEE HOW IT CHANGES, THE DURATION OF FIELD POTENTIAL. AND WE’VE TAKEN TWO DRUGS HERE, WHICH WE KNOW AFFECT THE Q T INTERVAL AND WHICH CAN HAVE SIDE EFFECTS ON THE HEART. SO THIS ONE HERE QUINOSCIENCE USED TO TREAT WHAT WE CALL A A/TRTRIAL ARHYTHMIA AND EXTENDS
THE Q F /* Q /T-FPT TO A LITTLE BIT
IN PATIENTS. IT’S SUPPOSED TO DO THAT. SO WE DROPPED THE CONCENTRATION OF QUINODINE AGAINST /-THE LONG LONGATION OF THE FAILED POTENTIAL. SEE A FLAT CURVE UNTIL WE GET HERE AND THEN IT TURNS UP. SO THIS IS TELLING US SOMETHING IS HAPPENING ON THE Q /T-FPT
INTERVAL INTERVAL. THIS GRAY BAR HERE IS /THE RANGE OF CONCENTRATIONS IN THE PATIENTS. SO WE SEE AN OVERLAP. SO WE WOULD SAY THIS COMPOUND IS AT RISK OF CAUSING A CHANGE IN Q T INTERVAL. VERY SIMPLE-MIND ADD SAY. THIS IS ANOTHER COMPOUND HERE, AND AGAIN WE SEE IN THE RANGE IN THE PATIENTS IT ALSO HAS A POTENTIAL TO ALTER Q /T-FPT
INTERVAL. SO WHAT WE DID WAS FIRST TESTED 12 COMPOUNDS WE /* WHICH WE KNEW THE OUTCOME AND WE FOUND THIS VERY GOOD PREDICTION. BUT THE PHARMACEUTICAL INDUSTRY SAID THAT YOU KNEW THE OUTCOME. THAT WAS CHEATING. WE KNOW THE OUTCOME AND YOU DON’T. SEE WHAT YOU COME UP WITH. NOW OVF THOSE 30 COMPOUNDS, WE GOT 28 CORRECT AS HAVING A RISK OR NO RISK. BUT OUR CONCERN IS WHY DID WE MISS TWO? WE MISSED TWO COMPOUNDS THAT SHOULD HAVE HAY SIDE EFFECT ON THE HEART AND THEY DIDN’T AND THESE ARE COMPOUNDS THAT HAVE BEEN REMOVED FROM THE MARKET BECAUSE THEY CAUSED SUDDEN CARD CARDIAC DEATH AFTER THEY HAD BEEN INTRODUCED. AND THIS IS ONE OF THE COMPOUNDS COMPOUNDS. IT’S CALLED J AND J 303. IT’S A BLOCKER OF THE SLOW PO PO/TTASSIUM COUNT. NOW, VERY RECENTLY THE HEART HAS WHAT WE CAIL REPOLARIZATION RESERVE. THAT IKR CHANNEL MAY BE IMPORTANT IN CONTROLLING WHAT WE CALL THIS REPOLARIZATION RESERVE RESERVE. SO THIS IS WHAT WE NORMALLY SAW WITH THIS J AND J 303. COMPLETELY FLAT CURVE APPARENTLY NO RISK. BUT IF WE ADD IS/OPRO TINE ERROL WHICH BLOCKS THE I CANR CHANNEL, THIS IS WHAT WE SEE. WE SEE THIS REVEALING OF AN IN INCREASED SENSITIVITY. SO THIS, BY REDUCING THE RE REPOLARIZATION RESERVE INCREASES DRUG SENSITIVITY. SO THE PREDICTION WOULD BE, IF WE HAD A PATIENT IPS CELL OR A PATIENT WITH A MUTATION IN THE I IKR CHANNEL, THEN THEY SHOULD BE EXTRAORDINARILY SENSITIVE TO THIS DRUG AND THAT’S EXACTLY WHAT WE FOUND. SO THIS AGAIN IS THAT SAME A ASSUAY — ASSAY ON THE Y J 303, THE DOSE RESPONSE CURVE IN THE HEALTHY CONTROL LINE. THIS IS THE PATIENT LINE. YOU SEE AN EXTRAORDINARY IN INCREASE IN THE SENSITIVITY, IN THE IPS CELLS FROM THIS INDIVIDUAL WITH. AND SAME WAS TRUE FOR THE SECOND DRUG THAT WE MISSED. WE SAW THIS DIFFERENCE AGAIN. IF IT’S CLINICALLY RELEVANT, YES YES, IT IS, THE PATIENT HAS SOMETHING CALLED LONG Q /T-FPT SYNDROME TYPE-2 DIABETES. AND IT WAS KNOWN THAT A DRUG LIKE COT OROL EXTENDS THE Q T INTERRAL. IT’S A BETA BLOCK ER AER AND
THIS IS A CONTROLLED GROUP OF PATIENT THAT WAS TAKING SO THEOOL HAS A DRUG AND YOU CAN SEE IN /THIS GROUP, THERE IS NO CHANGE OF THEIR REAL Q /T-FPT INTERVAL. BUT IN /THIS GROUP OF PATIENTS WHO HAVE MUTATIONS THAT COULD LEAD TO LUNG Q /T-FPT, YOU CAN
SEE THEY ARE HIGH-RISK OF THIS PARTICULAR DRUG. NOW THIS IS REALLY IMPORTANT BECAUSE THERE IS LOTS OF THINGS THAT CAN CHANGE YOUR RE REPOLARIZATION RESERVE. IT CAN BE JUST /AA FEVER. IT CAN BE ANTIBIOTICICS. AND IN /THIS WAY, WE MAY BE ABLE TO SCREEN COMPOUNDS FOR THEIR ABILITY TO INTERACT WITH OTHER COMPOUNDS OR GENETIC MUTATIONS, AND WE CAN IDENTIFY POTENTIAL RISK GROUPS. SO I SAID EARLIER
TAKINGITOJGENIC PAIRS OF CARDIAC — OF IPS LINES IS VERY IMPORTANT TO FIGURE OUT WHETHER THE GENETIC MUTATION IS ACTUALLY CAUSING YOUR
FEPHENOTYPE. AND WE DID THIS AGAIN FOR THAT LONG Q /T-FPT SYNDROME. WE HAD THE PATIENT IPS LINES. WE REPAIRED THE MUTATION USING HOMO/LOGOUS RECOMBINATION AND IT GAVE US A PAIR OF IPS LINES WITH LONG Q /T-FPT INTERVAL. AT THE SAME TIME, WE TOOK THIS 2 2.5 TARGETED HUMAN LINE AND INTRODUCED THE SAME MUTATION /SKWRAO*EFPLGTS IT GAVE US A SECOND PAIR OFITOJGENIC CELLS. SO THESE ARE GENETICALLY MATCHED CONTROLS. WE CAN EXAMINE WHETHER THE SPECIFICITY OF THE MUTATION IN CAUSING THE DISEASE FEPHENOTYPE AND WE CAN MEASURE THE EFFECT OF GENETIC BACKGROUND ON THE DISEASE FEPHENOTYPE. SO JUST TO SUMMARIZE RATHER A COMPLICATED AND LONG PAPER, BASICALLY THIS IS WHAT WE SEE. SO THIS IS THE LONG Q /T-FPT IPS
LINE LINE. YOU CAN SEE HERE, THIS IS THE ACTION POTENTIAL. AND WHEN WE CORRECT THE LINE, IT GETS SHORTER. SO THAT’S THE GENETIC CORRECTION CORRECTION. AND THIS IS THE HUMAN ES CELL IN WHICH THIS IS THE CONTROL ACTION POTENTIAL INTRODUCED TO MUTATION IT GETS LONGER. SO WE SEE IN THE ACTION POTENTIAL LONG Q /T-FPT
QUANTITFIED HERE. BUT WHAT WE NOTICED WAS THE CONTROL VALUES. SO THE DURATION OF THE ACTION POTENTIAL WAS DIFFERENT IN THESE DIFFERENT KINDS /OF CARD /KWROIO
MIO MIOCYTES. I’LL COME BACK TO THAT LATER. BUT WHAT THIS ALLOWED US TO DO, PARTICULARLY THIS GFP LINE A ALLOWED US TO SORT THE CARDIO MI MYOCYTES FROM THE DISEASE LINE, VERSUS THE HEALTHY MATCHED CONTROL. AND WHAT WE SEE IS THIS IS THE HERB CHANNEL. WOULD NORMALLY BE ON THE CELL SURFACE AND WHAT WE SEE IN THE MUTANT LINE IS THAT THE HERB CHANNEL HAS A SHORTER PROTEIN IN THE CIYTOPLASM. SO FOR THE FIRST TIME WE DISCOVERED THAT THIS IS A PROTEIN TRAFFICKING PROBLEM. SO IT REVEALED TO US THE UNDER UNDERLYING CAUSE OF THE DISEASE. SO MAYBE DRUGS THAT AFFECT PROTEIN TRAFFICKING COULD POSSIBLY BE CANDIDATE. BUT THIS IS AN EXAMPLE OF HOW WE CAN REVEAL UNDERLYING DISEASE MECHANISMS. I KNOW I MENTIONED A DIFFERENCE BETWEEN THE CELL LINES THAT WAS NOTICEABLE IN THAT ASSAY. SO I WAS KIND OF CONCERNED ABOUT THIS, AND I ASKED ONE OF OUR POSTDOCS, LUCAS TO HAV A LOOK AT ALL THE LITERATURE AND FIND ALL THE STUDIES IN WHICH THE E E/HRELECTRO/TPEUPHYSIOLOGY OF
THE DIFFERENT CARD /KWRO
/PHAOIOMYOCYTES FROM HUMAN IPS CELLS HAD BEEN DONE IN EXACTLY THE SAME WAY. AND THIS IS THE RANGE HE FOUND. NOW THIS IS QUITE SUBSTANTIAL. AMONG THESE ARE SEVERAL COMMERCIAL LINES. SO IF YOU BUY CD I CELLS, THEY ARE SOMEWHERE ON THIS LINE BUT THEY’RE DIFFERENT. AND IF YOU TAKE THE IS/OJEGENIC MATCH CONTROLS, YOU FIND EXACTLY WHAT YOU EXPECT. SO THEY ARE CORRECT RELATIVE TO ONE ANOTHER. BUT IF YOU TAKE ANY OF THESE SO SO-CALLED HEALTHY CONTROLS, YOU WILL BE COMPLETELY CONFUSED. SO YOU HAVE TO TAKE THE GENETIC GENETICICALLY MATCHED CONTROLS. THIS WAS ALSO EVIDENT IN ANOTHER STUDY. SO WE WERE LOOKING FOR AL STERIC COMPOUNDS THAT COULD CORRECT THE FEPHENOTYPES. SO INSTEAD OF
JUST HAVING A GENETIC CONNECTION, WE WANTED TO HAVE A DRUG THAT CORRECTED THE Q /T-FPT INTERVAL. AND WE FOUND — WE WORKED WITH A MEDICINAL CHEMIST WHO HAD A COUPLE /OF CANDIDATES. AND YOU CAN SEE HERE THIS IS THE LONG Q /T-FPT-TYPE ONE AND TYPE
TWO. AND IN EACH CASE WE GOT A SHORT SHOTENING OF THE Q /T-FPT
INTERVAL. SO THIS IS A POTENTIAL DRUG. THIS IS QUITE NICE. AND IF WE LOOKED AT THE DOSE RESPONSE CURVES IN THIS CASE, THIS ALSO LOOKED QUITE IS NICE AND EACH ONE IS COMPARED WITH ITS IS/OJEGENIC MATCH CONTROL. BUT IF YOU LOOK HERE, ONLY AT THE ABSOLUTE VALUES, YOU SEE FOR THE CORRECTION AND THE DISEASE, THEY ARE VERY SIMILAR. SO THIS WOULD BE VERY CONFUSING. SO AGAIN, YOU NEED TO HAVE A GENETICALLY MATCHED CONTROLS. BUT WHEN YOU LOOK AT THE LITERATURE AGAIN, THIS IS WHAT YOU SEE. SO OVER TIME THE PROBLEM OF PUBLICATIONS USING A HEALTHY CONTROL ARE VERY LARGE. SOME STUDIES USE A FAMILY MEMBER AS /AA CONTROL, BUT VERY FEW STUDIES TO DATE USE THE
IS/OJEGENIC PAIRS. AND I AM GETTING THIS EXAMPLE PARTICULARLY FOR THE HEART BUT THE SAME IS TRUE FOR NERVE, ANY KIND OF CELL. VERY FEW PEOPLE ARE USING THE IS IS/OJEGENETICALLY MATCHED PAIRS. SO WITH SHORTCOMING OF IPS CELLS FOR HEART DISEASE MODELS. IT COULD BE THE REPROGRAMMING METHOD, THE DIFFERENTIATION PROT PROTOCOL, THE IMMEDIAMEDIA, THE
GENETIC BACKGROUND, TISSUE SOURCE MIGHT ALL PLAY A ROLE. THE MATURE OF THE DERIVATIVES ARE STILL AN ISSUE. WE ARE ABOUT 16 WEEKS GESTATION COMPARED TO NORMAL HEART STU THAT’S VERY DIFFERENT. THIS IS TRUE OF ALL PLURIPOTENT STEM CELL DERIVATIVES. /* WHEN WE TRY TO MATURE THEM, OF COURSE WHAT’S NOTABLE IN THE HEART. UNDERGOES STRAIN FOR YEARS ON END. DOES NOT AFFECT MATURATION? SHOULD WE TRY OPTICAL PIERCING? — PACING? SHOULD WE USE DIFFERENT ENERGY SOURCES? FATTY ACIDS INSTEAD OF GLUCOSE? YOU GO FROM USING HIGH GLUCOSE INTO LIPIDS AND FATTY ACIDS. MAYBE THAT’S A WAY TO GO. T 3 RIN CREASED AT BIRTH. MAYBE IT’S COCANCER WITH OTHER HEART CELLS AND ENDO/THAOTHELIAL CELLS, EPCARD /KWIO CELLS AND
PAR PARASITES. IT’S IMPORTANT. AND JUST AS AN EXAMPLE OF WHY MATURATION IS IMPORTANT. I’LL JUST SHOW A SHORT STUDY IN WHICH WE LOOKED AT VARIOUS FACTORS THAT MAY AFFECT MATURATION WITH /SW-FPT SO WE USED A VERY SIMPLE AW SAY HERE. WE USED A DYE THAT IS TAKEN UP BY MITOCHONDRION. WE KNEW THAT DURING MATURATION THE NUMBER OF MITOCHONDRION IN THE CARD /KWRO /PHAOIOMYOCITE GO
UP AND WE
USED A GFP DRIVEN BY THE LOCUS AS A VERY ROUGH RATIO OF THE MIGHT /* MITOCHONDRIAL TO CIYTO CYTOPLASMIC VOLUME. AND WHAT WE SHOWED WAS THAT THE COMBINATION OF T 3 IGF 1 AND FECTION ANDTA METHOZONE IN INCREASED THE RATIO OF MITOCHONDRI MITOCHONDRION TO SITE CYTOPLASMIC VOLUME, WHICH MEANT WE HAD MORE MITOCHONDRION. IT AFFECTED THE ACTION POTENTIAL SO WE HAD MUCH LARGER ACTION POTENTIALS AND LOWER RESTING RATE POTENTIALS ON — UNDER THESE CONDITIONS. AND WE ALSO COULD MEASURE VARIOUS PROPERTIES OF THESE CELLS. THIS IS JUST AN EXAMPLE OF THE TYPE OF THING WE DO WITH THESE CELLS NOW. SO WE PUT THEM ON A /PPOLYMER
SUB SUB/STKPRA*EUT WHAT YOU SEE HERE IS CASTLCIUM TRANSIENTS. YOU SEE AND YOU CAN SEE THESE FLUORESCENT BEADS EM/PWBED IN
THE P PD MS WHICH YOU CAN USE AS A MEASURE OF FORCE OR CONTRACTION. SO WHAT WE DID WAS TO LOOK AT WHETHER THE CELLS MATURED UNDER THESE CONDITIONS IN TERMS OF FORCE OR CONTRACTION. SUSPECT SEE VERY CLEARLY THE CELLS POLE MUCH MORE STRONGLY UNDER THESE MATURATION CONDITION CONDITIONS. IN ADDITION, WHEN WE LOOKED AT A MUTATION IN /AEA PROTEIN C 3,
WHAT WE FOUND WAS THAT WE COULD SEE THE CONTRACTION VERY EASILY ONLY UNDER /THE MATURIZATION
CONDITION CONDITIONS. SO HERE WE HAVE TWO HEALTHY CONTROLS. THIS IS THE FORCE OF CONTRACTION CONTRACTION. UNDER A MATURIZATION CONDITIONS. THIS IS AN IPS LINE FROM A BOY WHO COLLAPSED ON THE FOOTBALL FIELD, NOW A HOSPITAL, AND WHICH CAME INTO THE HOSPITAL AND TURNED OUT HE HAD A MUTATON THAT HE DID NOT KNOW IN THE MYO MYOSIN BINDING PROTEIN C 3. WE HAD CELLS FROM /SPHEUPL HIS FATHER AND FROM AN UNRELATED PATIENT WITH THE SAME MUTATION. AND WHAT YOU SEE HERE, THE CON CONTRACTION IS REDUCED. WE CONFIRMED IT IN /THIS CASE BY A /TPHOKNOCKDOWN. SO WITHOUT THE MATURIZATION COMMISSIONS, WE COULD NOT REVEAL THIS FEPHENOTYPE. WE GO FROM WEEK 6 /0 TO ABOUT
WEEK 20. SO IT’S STILL FETAL BUT IT DOES REVEAL FEPHENOTYPES. I MAKE A SWITCH NOW TO GO TO SOME ENDO/THAOTHELIAL CELLS. I KNOW YOU ARE INTERESTED IN MORE ENDO/THAOTHELIAL CELLS /SKWROEUFPL SHOWED YOU EARLIER THE DIFFERENTIATION PROTOCOL. WE WITH HE TOOK THE CELLS AND WHEN WE LOOKED AT FACT-TO-CELL PROTEINS THEY BASICALLY EXPRESS THE PROTEINS WE MIGHT EXPECT. WE LOOKED AT THEM FUNCTION
/AALLY AS WELL. WE INJECTED THEM INTO SEB ARE YOU FISH, AND THESE ARE MARKED WITH GFP, AND WHAT YOU SEE HERE. IT IS BEAUTIFUL INTEGRATION OF BOTH IN /THIS CASE IT’S THE IPS CELLS. BUT ALSO WITH THE ES DERIVED END ENDO/THAOTHELIAL CELLS THAT
INTEGRATE BEAUTIFULLY INTO THIS. YOU MIGHT ASK HOW WE DO IT. ABOUT 30 DEGREES AND HUMAN CELLS NEED ABOUT 37 DEGREES. SO WE COOKED THE FISH SLIGHTLY AND GOT THIS INTEGRATION. BUT IF YOU LOOK AT FOR EXAMPLE — WHICH IS EVERYBODY’S FAVORITE EASY TO CULTURE END OOTHELIAL CELL /KWRURGS SEE THEY TO CANNOT INTEGRATE AT ALL SO THEY HAARE
NOT BEHAVI BEHAVING FUNCTION /AALLY IN
/TTHIS WAY. DIFFERENTIATION PROTOCOL. WE ALSO GET A CD 31-NEGATIVE POPULATION, WHICH EXPRESSES THIS SET /OF SURFACE MARKERS. , WHICH HAVE BEEN ASSOCIATED — THEY LOOK LIKE PARASITES. SO THEY EXPRESS THINGS LIKE CD 146. AND UNDER NORMAL CIRCUMSTANCES THEY DO NOT EXPRESS 22. BUT IF WE ADD TGF /PW-FPB, THEY
UP UPREGULATE AND THEY UPREGULATE 2 22, WHICH SUGGESTS THEY ARE TURNING INTO SMOOTH MUSCLE CELLS CELLS. NOW WHY WOULD THEY DO HA? NORMALLY THEY GET SECRETED TGF AND P /TK-FPD GF FROM
ENDO/THAOETHELIAL CE
LL CELLS, WHICH ARE OF COURSE CLOSE TO /PPER /*ICITES AND WE CAN SEE
THAT IF WE PUT THESE CELLS IN HETERO HETEROTYPIC CULTURES. SO THE ENDO/THAOTHELIAL CELLS
HERE ARE IN GREEN. AND WE MIX THESE CELLS, THE CD 3 31 POSITIVE AND NEGATIVE CELLS TOGETHER.^ AND THE PER /KWIO
CITES, ALL THE CELLS ARE STAINED IN BLUE. AND THE PER /KWIO CITES ADJACENT
TO THE ENDO/THAOTHELIAL CELLS HAVE TURNED RED FOR FM 22. SO THOSE INTERACTING WITH THE ENDO/THAOTHELIAL CELLS ARE
SWITCHING THEIR STATE. NOW, WE KNOW THAT, FOR EXAMPLE, NOT SIGNALING IS VERY IMPORTANT IN THE INTER/AOBACTION BETWEEN
PER SITES, SMOOTH MUSCLE CELLS AND ENDO/THAOTHELIAL CELLS. AND IF WE ADD AN /EUINHIBITOR OF NOT NALG, SO — THIS IS WHAT YOU SEE IN THE SAME COCULTURES. SO WE BLOCKED THIS POSSIBILITY OF INTER/AOBACTION, AND VERY FEW
OF THE PER /KWIO CITES ASSOCIATED
WITH THE ENDO/THAOTHELIAL CELLS. AND THIS IS IMPORTANT. GENETIC DISEASE, WHICH IS CAUSED BY DEFECTIVE SIGNALING. SO WE’RE QUITE OPTIMISTIC WE WILL BE ABLE TO MIMIC THIS GENT GENETIC DISEASE IN /THIS MODEL. BUT THE IMPORTANCE OF HETEROTIP HETEROTYPIC CULTURES BECAUSE IF WE LOOK AT THE ENDO/THAOTHELIAL
CELLS ON THEIR OWN OR SMOOTH MUSCLE CELLS ON THEIR OWN, WE WILL SEE NOTHING. WE NEED BOTH CELL TYPES PRESENT TO BE ABLE TO SEE THE
FEPHENOTYPE. AND THIS IS MAYBE TRANSFUSE OOM GENETIC CAUSES OF VASCULAR RIGHT FORMATIONS, WHICH RELY NOT ONLY ON VHAVING VASCULAR SMOOTH
MUSCLE CELLS BUT IN THE BRAIN IT CAN BE THE GLIAL CELLS AND MULTIPLE OTHER CELL TYPES. AND MANY OF THE GENES ASSOCIATED WITH INTER/AOBACTIONS BETWEEN
THESE CELL /TKWRAOEUFPLTZ AND YOU WILL SEE MUTATION S IS IN MANY OF
THEM CAUSED VASCULAR DISEASE. SO THERE IS PLENTY /OF ROOM FOR EVERYBODY TO WORK IN /THIS AREA. AND WE WORK ON A DISEASE CALLED HETEROTRY CAUSED BY MUTATIONS IN THE TGF /PW-FPB SIGNALING PATH
WAWAY BETWEEN THE ENDO/THAOTHELIAL
CELLS AND THE SMOOTH MUSCLE CELLS THAT ARE IMPORTANT. SO I AM NOT GOING TO GO INTO THAT INTO ANY DETAIL. BUT JUST TO SHOW YOU THE KIND OF THINGS YOU CAN MODEL WITH PLURI PLURIPOTENT STEM CELL ENDO ENDO/THAOTHELIAL CELLS. THIS IS A SYSTEM MANUFACTURED AND SOLD BY SELLEX. AND WHAT /TKDO YOU IS IN THESE,
IT CHANNELS THE ENDO/THAOTHELIAL
CELLS AND THESE NICE MINUI PUMPS CAN PUMP FLUID THROUGH THESE CHANNELS. AND WE CAN USE IT TO LOOK AT THROMBOFORMATIONS. SO IF YOU HAVE AN /EUINDENDATION
OF THIS CHANNEL, YOU CAN GET THROMB THROMBOFORMATION. IF YOU PUT IN BLOOD, YOU CAN USE IT TO LOOK AT ENDO/THAOTHELIAL
SOLID SOLIDATION. I’LL JUST SHOW A VERY SIMPLE EXAMPLE OF MANY MORE WE HAVE IN OUR LAB. SO THE ENDO/THAOTHELIAL CELLS
ARE PLATED IN THE BOTTOM OF THIS CHANNEL. THESE ARE LUEUCKOCYTES, IN
/TTHIS CASE PRIMARY, FLOWING OVER /-THE TOP. BUT WE CAN NOW GET THEM FROM IPS CELLS. AND WHAT YOU SEE THESE CELLS HERE HAVE ATTACHED TO THE IPS- IPS-DERIVED ENDO/THAOTHELIAL
CELLS. NOW IF WE ADD TNEF INTO THIS, WHICH IS AN /EUINFLAMMATORY
CITYTO CYTOKINE, BASICALLY WHAT YOU SEE IS /THE SAME ASSAY. MORE OF THESE CELLS ATTACH SUSPECT.^ SEE VERY CLEARLY, IF YOU FLUORESCENTLY LABEL A LUEUCO LEUCOITE. SO YOU CAN QUANTIFY THIS AND YOU SEE THERE ARE MANY, MANY LUEUCO LEUKOCYTES ATTACHED HERE. SO YOU DON’T HAVE TO — WE NEED TOIL HOW EXCITING THE OPPORTUNITIES ARE HERE. WE CAN LOOK AND SEE WHICH THE CULPRIT IS IN DIFFERENT DISEASE, ESPECIALLY IF WE CAN USE LUEUCO LEUKOCYTES DERIVED FROM IPS CELLS. SO I AM GOING TO CHANGE TACT IMMEDIATELY BECAUSE I AM NOT A NEUROLOGIST. BUT I THINK THIS IS A FAS FASCINATING EXAMPLE FROM KEVIN EGGINS LAB ON WHAT PLURIPOTENT IPS STEM CELLS CAN MEAN. AND WHAT KEVIN DID WAS TO LOOK AT ALS I HAVE TO EXPLAIN THAT MOST FORMS OF ALS ARE
/SPRSPORADIC. NOBODY KNOWS WHY THEY ARE CAUSED BUT THERE ARE SOME GENETIC IN INHERITED FORMS CAUSED BY MUTE MUTENATION GENE CALLED SOD 1. THE SOD 1 MUTANT MICE DONE REALLY HAVE A FEPHENOTYPE. BUT THERE ARE A FEW PATIENTS WHO HAVE SOD 1 MUTATIONS, FAMILIES, AND KEVIN WAS LUCKY ENOUGH TO FIND AN SOD 1 MUTANT PATIENT YES.^ MADE THE IPS CELLS. HE REPAIRED THE GENE, AND HE LOOKED AT THE
E/HRELECTRO/TPEUPHYSIOLOGY USING EXACTLY THE SAME TECHNOLOGY AS /I SHOWED YOU AND BASICALLY WHAT HE FOUND WAS AN ABNORMAL SODIUM — RATIO OF SODE SODIUM-TO-PO/TTASSIUM CURRENT,
THE USE OF THE CHANNELS. AND HE KNEW THERE WAS A DRUG CALLED RETIGABIN, WHICH IS AN ANTI-EP/HREILEPTIC. AND THIS HAS A NUMBER OF PROPERTIES. IT ALTERS THE
SODIUM-PO/TATASSIUM BALANCE, AND IT PASSES THROUGH THE BLOOD-BRAIN BARRIERS. THROWS TWO IMPORTANT CRITERIA. WHAT KEVIN ALSO DID WAS HE TOOK A SECOND INHERITED FORM OF ALS, NOT CAUSED BY SOD 1 BUT ENTIRELY INDEPENDENT GENE. HE FOUND EXACTLY THE SAME FENE PHENOTYPE, NOT WITH THE SAME GENETIC BASIS. AND WHAT HE FOUND WITH THIS ANTI-EP/HREILEPTIC IS A COUPLE
/-OF THINGS. FIRSTLY, IT CORRECTED THE SODIUM SODIUM-PO/TTASSIUM BALANCE. AND IN ADDITION, IT CORRECTED THE EXCITEABILITY OF THE
NEWURONS NEURONS. SO THIS IS THE NEWURONS OF ALS /PRAEURBT HYPEREXCITED. YOU CAN SEE THIS HERE IN THE IPS IPS-DERIVED NEWURONS. AND THIS DRUG COMPLETELY FLATTEN FLATTENED IT OUT. BECAUSE IT HAD WORKED IN TWO INDEPENDENT GENE MUTATIONS, IT HAD THE CHANCE THAT IT WOULD WORK IN /SPRSPORADIC MUTATIONS. SO I WANT YEAR OF SHOWING THIS AND PUBLISHING THIS PAPER, KEVIN GOT PERMISSION TO TAKE 100 ALS PATIENTS INTO THE CLINIC WITH THIS ANTI-EP/HRELEPT
/SKWREUFPBLGT IT’S ALREADY APPROVED AND ON THE MARKET, FROM EACH OF THESE PATIENTS, HE’S MAKING IPS CELLS AND WILL TEST THAT DRUG. AND FIVE YEARS DOWN THE LINE HE WILL BE ABLE TO TELL US WHEN NORMAL CONTROL ALS PATIENTS ARE — WHETHER ANY OF THEM HAVE SURVIVED, WHETHER ANY OF THEM HAVE REDUCED SYMPTOMS AND WHETHER THERE IS ANY CORRELATION WITH THIS IPS NEWURON
FENPHENOTYPE. AND THE ABILITY TO BE CORRECTED. SO THIS IS A VERY IMPORTANT STUDY AND IT SHOWS YOU THAT YOU CAN GO VERY FAST, PARTICULARLY BY DRUG REPURPOSING INTO CLINIC APPLICATION WITHOUT A SINGLE EX EXTRA ANIMAL EXPERIMENT. SO I AM GOING TO ROUND OFF NOW WITH A FEW CONCLUSIONS, REALLY LOOKING TOWARDS THE FUTURE IN THIS DISEASE MODELING. I THINKITOJGENIC PLURIPOTENT
STEM CELL LINES WITH DISEASE-RELEVANT MUTATIONS WILL BE WIDELY AVAILABLE WITH APPROPRIATELY IN INFORMED CONSENTS. WE WILL NOT BE MAKING C AND I IP IPS CELLS IN THE FUTURE UNLESS THERE ARE UNUSUAL PATIENTS. AN-AND-APPROPRIATELY INFORMED CONSENTS MEANS THAT THE PATIENTS HAVE DOUNATED THEIR TISSUE KNOWING THEY COULD BE USED BY COMMERCIAL COMPANIES. NOBODY WANTS ANOTHER HALO INCIDENT. THESE IPS LINES WILL HAVE GENOME GENOMIC DATA AVAILABLE. THE MEDICAL HISTORY AND THE DRUG RESPONSES AS FAR AS THEY KNOW. AND WHAT’S REALLY IMPORTANT THAT THIS DATA COULD PROVIDE THE MISSING LINK FOR /SKWHRAOEPBL WIDE ASSOCIATION STUDIES, PARTICULARLY THROUGH PRECISION ENGINEERING. WE CAN NOW ENGINEER ANY MUTATION OF INTEREST, INCLUDING UP STREAM SNIPS VARIANTS. IF OUR BIOASSAYS DOWNSTREAMS ARE SENSITIVE ENOUGH TO PICK /UUP DIFFERENCES, WE WILL BE ABLE TO SEE WHETHER A SNIP,
PRE/TKEUDISPOSE S TO A FEPHENOTYPE. I THINK THIS IS A REALLY EXCITING AREA. IT WILL BE POSSIBLE TO TEST COMBINATIONS OF DRUGS OF DIFFERENT DOSES FOR THE EFFECT EFFECTIVE NESS AND TOXICITY IN PATIENTS, PARTICULARLY COMBINATIONS OF DRUGS. PATIENTS ALWAYS TAKE ONE DRUG. THEY TAKE SEVERAL AND WE DON’T KNOW ALL THE DIFFERENT COMBINATIONS THAT COULD HAVE AN EFFECT. WE WILL BE ABLE TO DO THAT IN THE FUTURE. AND IT WILL BE LIKELY, I THINK, TO DEVELOP DRUG TREATMENTS FOR SPECIFIC DISEASES, PARTICULARLY THAT DELAY REVERSE SYMPTOMS. BUT DELAYING SYMPTOMS IS AN ENORMOUS GAIN IN HEALTH. ALL OF IT WILL REQUIRE NEW AND ROBUST TYPES OF BIOASSAYS. AND THAT’S WHY WE NEED MULTIDIS MULTIDISCIPLINARY RESEARCH. AND I AM EN/SRVIOUS OF YOU HERE WITH THOSE OPPORTUNITIES AT HAND HND. SO I WILL JUST ACKNOWLEDGE THE PEOPLE IN MY GROUP WHO HAVE DONE THIS WORK OVER /THE YEARS. PEES /THAO*EZ ARE PEOPLE WHO HAVE SUPPLIED US WITH GRANTS. THESE ARE OUR COLLABORATORS. I’D BE HAPPY TO ANSWER ANY QUESTIONS. /PHRA [APPLAUSE]>> THANK YOU. WE DO HAVE TIME FOR QUESTIONS. WHILE PEOPLE ARE COMING TO THE MICROPHONES, I’D LIKE TO TELL YOU THAT THE CM ACTIVITY CODE FOR TODAY’S IS 7913. THAT’S 7913. ALSO, I’D LIKE TO REMIND YOU THAT THE SAES IS SPONSORING THE RECEPTION THAT WE WILL HAVE FOLLOWING THE QUESTION-AND- QUESTION-AND-ANSWER PERIOD OUT IN THE A/TRTRIUM. THANK YOU.>> CONGRATULATIONS FOR OUTLINES OUTSTANDING WORK /SAND /AEA
SUPERB PRESENTATION. SO CONSIDERING ALL THE POSSIBILITIES, DO YOU THINK WE WILL BE ABLE TO MAKE CONOMYO CONOMEIOCYTES FOR MYO
/KARCARDIAC RE REPAIRS? I KNOW TANCE ISSUE. HOW MANY GENES ARE THERE? DO YOU THINK WE CAN CORRECT THEM TO TAKE CARE OF THE PROBLEM?>> I MEAN, — SO MYOCARDIAL IN INFARCTION IS NOT ACTUALLY SUCH AN ISSUE AS IT WAS A FEW YEARS AGO. SO /-MANY PEOPLE RECOVER. IN FACT, THERE IS A VERY LARGE TRIAL ONGOING IN EUROPE, WHICH I AM ABLE TO FIND SUFFICIENT MYO MYOCARDIAL INFARCTION IN PATIENTS TO INCLUDE IN THE TRIAL TRIAL. THE PROBLEM IS REALLY LONGER LONGER-TERM HEART FAILURE AND THAT WILL BE THE PATIENT GROUP. I THINK IT WILL BE VERY DIFFICULT TO TREAT THOSE PATIENT PATIENT. FENE WE CAN GET MILLIONS AND MILLIONS OF CARD /KWRO
/PHAOIOMYOCYTES IN THE RIGHT STATE. I THINK IT WILL BE VERY DIFFICULT TO DISTRIBUTE THEM IN THE HEART TISSUE IN SUCH A WAY THAT THEY ARE ALIGNED PROPERLY AND INTEGRATE. BUT BECAUSE OF ALL THE
FI/PWROBROSIS IN HE HEART. BUT I BEG TO BE PROVEN WRONG. PERSONALLY, I’VE GIVEN UP.>> YES. THERE WAS A PAPER A WHILE BACK BY EITHER ORLAND OR ORKIN WHERE HE DID BONE MARROW TRANSPLANT /TKPHRAO*ELTZ MICE AND ACTUALLY HAD A RESPONSE. THIS WAS SEVERAL YEARS BACK. AND IT’S KIND OF A FOLLOW-UP TOOT GENTLEMAN’S QUESTION, I GUESS. THERE HAVE BEEN SINCE THAT TIME SEVERAL CLINICAL /TRAOEUTRIALS
WITH BONE MARROW CELLS AND MANY CD WHATEVER PLUS THIS, THAT AND THE OTHER. AND FIBROBLAST CELLS AND WHATEVER AND NONE OF THEM. IF YOU GO THROUGH THE LITERATURE VERY CAREFULLY, HAVE SHOWN THAT THE CELLS ACTUALLY TAKE. IT’S SUPPOSEDLY WHAT THEY SECRETE THAT CAUSES THE OTHER CELLS TO BECOME ACTIVE AND DO THINGS. AND THESE HAVE BEEN — THERE IS COUNTLESS NUMBERS NOW IN PROGRESS AND THEY ALSO /TKHRAEUPL THEY ARE MEASURING VENTRICULAR SMORG ANOTHER. BUT NONE OF THEM HAVE ANY REAL CONCRETE RESULTS. >> EXACTLY. AND THERE ARE HUNDREDS OF MILLIONS OF DOLLARS GOING MOO THAT TYPE OF RESEARCH AND EXACTLY WHAT YOU SAY. THERE IS NO CLEAR OUTCOME. THERE SEEMS TO BE /AA TRANSIENT
IM IM/TPHROFLT HEART FUNCTION. AND THAT’S PROBABLY DUE TO IN THE FIRST PLACE SOME AMEL /KWROR AMELIORATION OF CARDIAC DAMAGE BUT IT’S NOT SUSTAINEDIZE LONG PERIOD IN THE SENSE THERE IS NO LONG TERM BENEFIT X THERE IS SOME NEWUROVASCULARIZATION IN
THE INFARCTS. BUT OTHER THAN THAT, THERE IS NO SUSTAINED IMPROVEMENT. IN MANY OF THE STUDIES, THERE ARE ERRORS. IN MANY OF THE STUDIES, THERE ARE INAPPROPRIATE CONTROLS. SO IF YOU TAKE DISH MADE THAT POINT — IF YOU TAKE A PROPER CELLULAR CONTROL RATHER THAN JUST CULTURE FLUID OR WHATEVER, YOU DO NOT SEE A BENEFIT. IN THE STUDIES IN MICE, THE MOUSE — SURVIVAL OF THE MOUSE IS OFTEN INFARCT IS COMPROMISED BY CARDIAC RUPTURE. ANY CELL TYPE, EXCEPT THE DETERMIDERMAL FIB OR BLAST, WILL CAUSE A CARD CARDIAL RUPTURE. IT HAS NOTHING TO DO WITH A REL RELEVANCE OF HUMANS. SO WE CAN DISCOUNT THE MOUSE AS BEING RELEVANT. THE QUESTION DOES IT WORK? THERE IS WHAT’S SUPPOSED TO BE THE DEFINITIVE TRIAL IN THAT STUDY, AS SAID GOING ON IN EUROPE. THEY WERE SUPPOSED TO INCLUDE 3 3,000 PATIENTS WITH AN
/EUINJECTION FRACTION OF — WHAT’S THE AMOUNT OF BLOOD PUMPED OUT /-OF THE
HEART OF 35% OR LESS. THEY WERE UNABLE TO INCLUDE SUFFICIENT PATIENTS AS FAR AS I KNOW TO DATE THEY’VE ONLY INCLUDED 350. BECAUSE REGULAR STANDARD OF CARE IS ACTUALLY HELPING THESE PATIENTS. SO I YOU — AT THE MOMENT I’M A AFRAID I CALL IT EXPENSIVE HOMEO PATHY. THERE IS NO SOLID EVIDENCE. IN FACT, MORE DISAPPOINTING IS SOME OF THE EARLY STUDIES IN WHICH IT’S BASED APPARENTLY — SO IT’S BEING INVESTIGATED SERIOUSLY AT THE MOMENT FOR POTENTIAL WITHDRAWAL FROM THE LITERATURE, WHICH I THINK IS CORRECT. SO /-MANY OF THE IMAGINING WAS
IN INCORRECT, TO PUT IT MILDLY.>> BUT THE LITERATURE IS UN UNSATISFYING, IF YOU WANT TO MAKE A CASE FOR STEM CELL TRANS TRANSPLANTATION OF THE HEART. BUT THE LITERATURE DOES SEEM TO POINT TOWARDS SOME TYPE OF A CIYTOKINE OR SOMETHING BOOST IN THE SHORT RUN, AS YOU SAID, FOR VASCULAR PROLIFERATION, NO?>> WELL, IT’S HARD TO TELL. WHEN PEOPLE JUST DO A LOT /OF HAND /SKPWAEUFG IT’S — SAY IT’S — I ALWAYS WANT /TO SAY WHAT MOLL /SKPAOUL WHAT ARE THE RE RECEPTORS AND HOW DOES IT WORKING? WHAT SEEMS TO HAVE BEEN EXCLUDED BY SOME VERY NICE WORK OF RICH LEE, THERE WAS THIS SPECULATION IT WOULD RECRUIT CARDIAC PRO GENTORS IN THE ADULT HEART TO THE INFARCT ZONE. FIRSTLY, IT’S CLEAR THEY DON’T. SECONDLY, IT’S REALLY QUESTIONABLE HE WHETHER OR NOT THERE ARE CARDIAC PRO
/SKWREPGENITORS AT ALL IN THE ADULT HEART. SO I THINK WE CAN CROSS THAT ONE OUT. ANOTHER QUESTION WAS WHETHER IT PREVENTS CARD /KWRO
/PHAOIOMYOCITE DEATH. BUT POSSIBLY A MECHANISM AND I THINK THAT’S HOW THE VASCULAR VASCULARIZATION MIGHT WORK. THE THIRD OPTION WAS IT AS YOUS CARD /KWRO /PHAOIO MICITES IN
THE HEART TO
PROLIFERATE. THAT LITERATURE IS ALSO VERY HIGHLY QUESTIONED OF WHETHER THE METHODOLOGY WAS CORRECT TO DO THAT. SO THERE ARE, AGAIN, IMAGING DATA WHICH DOES NOT SEEM TO BE CORRECT IN THAT AREA. SO EVERYTHING HAS TO HAVE A MECH MECHANISM. AND IF PEOPLE HAVE LOOKED AND FIND OUT MECHANISM, THERE DOESN’T SEEM TO BE A LOT LEFT. WHAT YOU WOULD BE LOOKING FOR IS ALWAYS SUSTAINED IMPROVEMENT IN HEART FUNCTION, NOT SOMETHING TRANSIENT. SO IF IT’S ONLY TRANSIENT, — >> WE HAVE ONE LAST QUESTION HERE. YOU CAN COME DOWN AND ASK — >> I’D LIKE TO COME BACK TO WHAT YOU WERE PRESENTING PARTICULAR, THE CONCEPT OF MAKING THE CELLS MATURE FURTHER. IT WAS IMPRESSIVE THAT YOU WERE ABLE TO GO FROM 16 WEEKS TO 20 WEEKS. I’D LIKE TO ASK YOU IF YOU COULD ELABORATE ON WHAT CAN BE USED TO KEEP GOING FARTHER? YOU MENTION MENTIONED CELL TYPES. WHAT?>> IT’S A BIT OF GUESSWORK AND EM/PPIRICAL STUDIES AT THE
MOMENT. WE DON’T MO. BUT I CAN SAY THAT THERE ARE ELLEN /KRAEUPBCOURAGEING WORK
FROM THOMAS, WHO MAKES THESE ENGINEER ENGINEERED HEART TISSUES. SO THEY ARE IN 3D AND MIXED CELLS POPULATIONS AND HE HAS THEM WITH KIND OF ELASTIC BANDS THAT STRETCHED ON LITTLE POLES. SO THEY ARE A VERY IMPORTANT STRUCTURE BUT ONLY APPEAR AROUND BIRTH AND THEY ARE VERY IMPORTANT FOR
CASTLCIUM-HANDLING. YOU CAN SEE THESE /STRAURZ PEAR. SO WHY IT’S ENCOURAGING. TELLS US THAT THESE CELLS ACTUALLY CAN MAKE THESE STRUCTURES. SO THEY CAN BE THERE. IT’S A QUESTION OF HOW WE CAN DO IT. NOW HE CAN DO IT, BUT NOT EVERYBODY WANTS TO BE ABLE TO MAKE THESE COMPLEX STRUCTURES. YOU’D LIKE TO BE ABLE TO DO IT IN /AA SIMPLIFIED CELL CULTURE MODEL AND THAT’S WHAT MANY GROUPS ARE WORKING ON RIGHT NOW. I THOSE YOU — SHOWED YOU ONE EXPECT /* ASPECT OF THINGS WE CAN OPTIMIZE AND WE CAN
IM/PROPROVE THE POTENTIAL. BUT CAN WE IM/PROPROVE
CASTLLCIUM HANDLING? CAN WE IM/PROPROVE MANY OTHER ASPECTS? ALIGNMENT LE IP, THE SHAPE, THE DISTRIBUTION OF CONNECTIONS? SO EVERYTHING EVERYBODY IS LOOKING AT RIGHT NOW TO /TRAOTRY
AND FIGURE OUT. I AM QUITE OPTIMISTIC WE’LL GET THERE IN THE END.>> OKAY. THANK YOU, CRIST /KWRAO*EUINE,
VERY MUCH FOR /AA VERY INTERESTING
LECTURE. /PHRA [APPLAUSE] AND AGAIN, WE DO HAVE A RE RECEPTION OUTSIDE THAT IS SPONSORED BY THE FAES. AND CME ACTIVITY CODE IS 7913.

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