Ivan Zamorano

Acupunture Treatment

2018 Demystifying Medicine: Use of induced pluripotent stem cells (iPSC) for regenerative medicine

2018 Demystifying Medicine: Use of induced pluripotent stem cells (iPSC) for regenerative medicine


WHAT IS THIS A PICTURE OF? NOW, IS THERE ANYBODY HERE THAT THINKS THIS IS THE GOLDEN GATE BRIDGE OVER SAN FRANCISCO BAY? WHAT IS THE PICTURE OF, DO YOU KNOW? WHAT?>>[INDISCERNIBLE]>>WELL, I CAN’T HEAR YOU BUT IF YOU SAY SAID THE BROOKLYN BRIDGE, YOOURE RIGHT. SO THIS IS THE MOST FAMOUS BRIDGE IN BRIDGE HISTORY. AND IT’S LIKE WE’RE ALL BRIDGE BUILDERS. AT LEAST THAT’S THE INTENT OF IT SO MAKE BELIEVE THAT ON THE BROKLYN SIDE ARE ALL THE ADVANCES IN CONTEMPORARY BIOLOGICAL AND ENGINEERING SCIENCES AND ON THE NEW YORK SIDE ARE ALL THE NEEDS AND MEDICINES IN IDENTIFYING AND TREATING DISEASE. SO THE PROBLEM IS THERE’S A HUGE GAP BETWEEN THESE 2 DISCIPLINES WHICH TO SOME EXTENT HAVE BEEN MOVE NOTHING OPPOSITE DIRECTIONS OVER THE PAST 30 OR 40 YEARS. SO THIS COURSE IS A MODEST EFFORT TO TRY AND BRIDGE THAT GAP AND YOU CAN VISUALIZE YOURSELF AS THE 2 MEN ON THE CAT WALK. AND SO WHAT WE HAVE DONE OVER THE PAST IS TO BRING TOGETHER AND PROCESS FROM A SOMEWHAT CLINICAL PERSPECTIVE AND OTHERS FROM A MORE REDUCTIONIST SORT OF, CALL IT BASIC SCIENCE PERSPECTIVE. AND THEY PRESENT BRIEFLY, AND WE ENCOURAGE QUESTIONS AND DON’T HESITATE TO ASK. NOW, TODAY’S SUBJECT IS LIKE ALMOST ALL THE 1S WE HAVE IDEALLY SUITED TO BE CONSIDERED AS AN ELEGANT BRIDGE. I THINK THIS IS PERHAPS–THIS IS SOME SUBJECTIVE BUT I THINK TODAY’S SUBJECT IS THE MOST ELEGANT BRIDGE THAT’S EVOLVED IN THE PAST 10 YEARS. AND THIS STORY REALLY IS BEST ILLUSTRATED BY THE 2 SCIENTISTS WHO WON THE NOBEL PRIZE FOR THIS. SO I DIVIDED THIS INTO 2 PARTS BECAUSE EACH 1 HAS A BRIEF STORY WHICH IS PARTICULARLY RELEVANT TO YOUNG SCIENTISTS HERE AND ELSEWHERE. PART 1 IS SIR JOHN GIRDEN, WHO WHEN HE WAS A STUDENT AT EATON, HE WRITED AND LINKED TO CAME BRIDGE IS PROFESSORS WROTE: THERE’S NO FUTURE HERE IN BIOLOGY GO BE A LAWYER OR SOMETHING, I DON’T KNOW WHAT. AT ANY RATE, 64 YEARS LATER, HE WAS WIDELY RECOGNIZED AS 1 OF THE FINEST MINES OF HIS GENERATION. AND HOW DID THAT HAPPEN? HE SHARED THE NOBEL PRIZE IN 2012. SO 1 OF HIS EXPERIMENTS WHICH THE NOBEL PRIZE, GI–GURDON, AND HE IS A CELL BIOLOGIST AND NEUROECTODERMAL CLOLOGY FORT AND HE WONDERED WHAT WOULD HAPPEN IF HE MOVED THE NEUROECTODERMAL CLE US OF AN INTESTINAL CELL OF A FROG AND PUT THAT NUCLEUS INTO A FRAG EGG WHICH HAD BEEN DENUCLEATED AND WHEN HE WRITES ABOUT IT IN HIS NOBLE LECTURE, THERE’S VARIOUS IDEAS HE EXPRESSES AS TO WHAT MIGHT HAPPEN, BUT HE WASN’T CONVINCED THAT HE KNEW UNTIL HE DID THE EXPERIMENT AND WHAT HAPPENED WAS THAT A TADPOLE EMERGED AND THIS WAS 1 OF THE FIRST EXPERIMENTS TO INDICATE–IT IS A LONG TIME AGO, DIFFERENTIAL FUNCTIONS OF A NUCLEAR NATURE AND OTHERWISE CELLULAR NATURE. WELL, NOW WE JUMP TO MORE RECENT TIMES WITH SHINYUN ON AND HE’S TRAINED AS AN ORTHOPEDIC SURGEON AND HE WAS VERY MUCH INTO MARSHAL ARTS AND HAD MANY ACCIDENTS AND HAD A FEW BROKEN BONES AND THAT’S–HE SAYS THAT’S WHAT LED HIM INTO ORGT O PEDIC SURGERY. NOW WHAT’S INTERESTING, AND WHAT HE TOLD US WHEN HE WAS VISITING HERE, WHEN HE WON THE NOBEL PRIZE, AFTER BEING A SURGEON AND REPAIRING INJURY, PARTICULARLY IN INHERITABLE DISEASES OF BONE, HE WAS CURIOUS ABOUT WACAUSED THE DISEASE–ABOUT WHAT CAUSED THE DISEASE IN THE FIRST PLACE AND THAT’S WHAT LED HIM MORE INTO A BIOLOGICAL APPROACH AND HE THEN SPENT ABOUT 14 YEARS AT THE UNIVERSITY OF CALIFORNIA IN SAN FRANCISCO, WHERE HE GOT A Ph.D. IN MOLECULAR BIOLOGY. RETURN TO JAPAN FROM SAN FRANCISCO, HIS WIFE WANTED TO GO BACK HOME WITH THE CHILDREN AND SO HE APPLIED FOR A JOB AT HIS UNIVERSITY WHICH WAS THEN KYOTO, AND SOME WISE COMMITTEE SAID WE WILL ACCEPT YOU BUT YOU HAVE TO PROPOSE SOMETHING THAT’S REALLY EXCITING NOT JUST A CONTINUATION OF SOMETHING THAT’S GOING ON OR SOMETHING THAT’S ALMOST REPETITIVE OR PROVE SOMETHING THAT SEEMS TO BE PRETTY SOLID BUT REALLY OUT OF THE BOX. AND HE TELLS THE STORY OF READING ABOUT EARLIER WORK BY THE EXPERIMENTAL EMBRYOLOGYST AND HE CAME UP WITH THE IDEA THAT MAYBE IT’S POSSIBLE TO TAKE A DIFFERENTIATED CELL AND HAVE IT GO BACKWARDS INTO AN UNDIFFERENTIATED STEM CELL. AND 1 OF HIS PROFESSORS CONVINCED HIM AND THE W STANDS FOR HARD WORK AND HIS WHOLE CAREER ILLUSTRATES THAT BECAUSE THESE EXPERIMENTS THAT HE PERFORMED DID NOT HAPPEN OVER NIGHT. THEY HAPPEN BECAUSE OF DILIGENCE. NOW WHAT DID THEY DO? HE TOOK A DIFFERENTERATED CELL AND AN EMBRYONIC STEM CELL AND THEY DID COMPLETE SEQUENCING ON BOTH OF THEM AND IDENTIFIED VARIOUS GENE CANDIDATES THAT WERE EXPRESSED IN THE HUMAN EMBRYONIC STEM CELLS, BUT NOT úSO WITH A GROUP OF VERY
DILIGENT GRADUATE STUDENTS WHO SPENT 3 YEARS DOING THIS AND FOR THE FIRST 2 AND HALF YEARS GETTING NOWHERE, THEY WERE REPLACING 1 GENE AT A TIME AND TO THE UNDIFFERENTIATED–INTO THE DIFFERENTIATED CELL TO SEE IF THERE WAS REVERSION. IT DIDN’T WORK. THEY PUT 2 GENES AT THE TIME, 3 GENES AT A TIME AND THEN FINALLY AS HE TELLS THE STORY, THE STUDENT SAID WE’RE DOING THIS BACKWARDS. WE SHOULD PUT THEM ALL IN AND THEN SUBTRACT AND THAT’S WHAT THEY DID. THEY EVENTUALLY NARROWED IT DOWN TO 4 TRANSCRIPTION FACTORS INDICATED ON THE SLIDE AND THOSE TRANSCRIPTION FACTORS WHEN INTRODUCED TO A DIFFERENTIATED CELL CAUSE ITS REVERSION BACK TO ESSENTIALLY AN EMBRYONIC TYPE STEM CELL. AND INDUCED PLURIPOTENT CELL, IPS. WELL, ONCE THAT HAPPENED THIS WAS LIKE A BRAVE NEW WORLD AND 1 OF THESE, IT BECAME AN AREA OF INTENSE RESEARCH AND AN AREA OF INTENSE HEIGHTENED. PEOPLE WERE THINKING IN TERMS OF REGENERATION, WE CAN MAKE TISSUES AND REPLACE WHAT’S OLD. WE CAN USE TISSUES TO TEST DRUGS, WE CAN DO ALL KINDS OF THINGS, SOME OF WHICH IS IN THE PIPELINE. BUT IT’S BEEN QUITE A LONG PIPELINE AND IT HASN’T BEEN AN EASY PROCESS, AT ANY RATE, YAMA, A KA SHARED THE NOBEL PRIZE IN 2012 AND I PUT HERE THE TITLE OF THEIR 2 NOBEL LECTURES WHICH IF YOU READ ANYTHING IN THE REFERENCE MATERIAL THAT WE POST ALL THE TIME, I WOULD URGE YOU TO READ THESE BECAUSE THEY’RE NOT ONLY ELEGANT LITERATURE BUT THEY TELL THE STORY OF THE HUMAN BEING WHO ACTUALLY DID THE WORK AND WHY AND HOW IT HAPPENED AND IT’S MOTIVATEING. NOW EVERYBODY WHO USES VISION AND HARD WORK DOESN’T COME OUT WITH A NOBEL PRIZE AND PROBABLY MORE OFTEN THAN NOT, YOU HAVE TO KNOW WHEN ENOUGH IS ENOUGH AND GO ON TO SOMETHING ELSE. BUT WHEN IT WORKS, IT WORKS IN A WAY THAT IS MOST DRAMATIC AND CERTAINLY THIS DISCOVERY OF THE IPS CELL IS PROFOUNDLY CHANGING THE WAY IN WHICH WE THINK ABOUT BIOLOGY AND ALSO ABOUT MEDICINE. AND THAT’S THE NATURE OF THE BRIDGE. SO, TODAY’S PARTICIPANTS ON THE BRIDGE ARE 2 NIH EXPERTS IN THIS FIELD. THE FIRST IS PAM ROBEY WHO IS A SENIOR INVESTIGATOR HERE. PAM GOT HER Ph.D. FROM CATHOLIC UNIVERSITY AND CAME TO THE NIH IN 1979 AND HAS PROGRESSIVELY WORKED PRIMARILY IN DISEASES IN BIOLOGY OF BONE. PARTICULARLY THE ROLE OF MISENK MALCELLS IN BONES LEADING TO CELL TYPES AND BECAUSE OF THIS WORK AND HER REALLY, HER EXPERTISE, SHE WAS APPOINTED AS THE ACTING DIRECTOR OF THE NIH COMBINED STEM CELL UNIT WHICH SHE IS AT THE PRESENT. SHE RECEIVED MANY AWARDS AND IS WIDELY PUBLISHED IN THE AREA OF MANY INTEREST. AND OUR SECOND SPEAKER IS KAPIL BHARTI, IN THE NATIONAL EYE INSTITUTE. HE’S THE DIRECTOR OF THE OCULAR AND STEM CELL TRANSLATION RESEARCH UNIT WHICH IS DESIGNED TO DO TRANSLATIONAL RESEARCH INVOLVING DEGENERATIVE EYE DISEASES BASED ON THE USE OF IPS CELLS. DR. BHARTI, TRAINED IN BIOPHYSICS IN INDIA AND THEN RECEIVED HIS Ph.D. FROM Mc GURGTY UNIVERSITY IN GERMANY. HE CAME TO THE NIH AND THE NINDS AS A POST DOCTORAL FELLOW AND HIS MAJOR EFFORTS IN WORK HAVE BEEN IN THE AREA OF TRANSCRIPTIONAL REGULATION OF PIGMENT CELL BIOLOGY AND THE EYE AND THE USE OF THE EYE AS A ORGAN FOR THERAPEUTIC USE OF IPS CELLS. SO WE’RE GRATEFUL TO BOTH OF YOU TO BE WITH US AND I GUESS, PAM ARE YOU GOING TO SPEAK FIRST?>>WELL THANK YOU, I APPRECIATE THE INVITATION TO TALK ABOUT STEM CELL BIOLOGY TODAY. I APOLOGIZE TO THOSE WHO ARE EXPERTS IN THIS PARTICULAR AREA, BUT I HAVE TO TELL YOU THAT IN MY TRAVELS AROUND THE COUNTRY AND AROUND THE WORLD THAT THERE IS STILL A GREAT DEAL OF CONFUSION ABOUT WHAT DIFFERENT KINDS OF STEM CELLS ARE AND WHAT THEY CAN DO AND CANNOT DO I’LL START WITH STEM CELL
HISTORY AND DEFINITIONS, I WILL TALK ABOUT THE DEVELOPMENT AND TYPES OF STEM CELLS SOME OF THEIR POTENTIAL USES AND I WILL USE EXAMPLES OF SKELETAL TISSUES BECAUSE THAT IS MY PARTICULAR AREA OF RESEARCH AND I WILL TALK ABOUT SUCCESS STORIES IN TERMS OF STEM CELLS IN CLINICAL APPLICATIONS AND THEN KAPIL WILL TELL YOU ABOUT HIS VERY EXCITING WORK IN TERMS OF RETINAL PIGMENT EPITHELIAL CELLS. SO TO START OFF WITH THE STEM CELL HYPOTHESIS IS NOT REALLY A NEW THING. IT WAS PUT TOGETHER BOY A BUNCH OF REAL LEE ASTUTE HEMATOLOGISTS AND DEVELOPMENT BIOLOGISTS WORKING IN THE LATE 1800S AND AROUND THE TURN OF THE CENTURY& IN THE 1900S. IN PARTICULAR, RIGOUGH, AND OTHERS, I WOULD LIKE TO POINT OUT THAT DACIN HCHIKOFF, WAS 1 OF THE FIRST WOMEN PROFESSORS IN ALL OF EUROPE BASED ON THE WORK SHE DID IN DEVELOPMENTAL AND STEM CELL BIOLOGY. AND WHAT THESE PEOPLE NOTICED WERE THAT DURING EMBRYONIC DEVELOPMENT AND THE GROWTH OF AN ORGANISM AFTER BIRTH, TISSUES ARE CONTINUOUSLY REMODELING AND REGENERATING AND THE OHM BE WAY THAT THIS COULD HAPPEN–ONLY WAY THIS COULD HAPPEN IS IF THERE IS A STEM CELL THAT IS FUELING THE FIRE SO TO SPEAK. WITHOUT HAVING A STEM CELL, ONCE THE CELL DIES THEN THE TISSUE WOULD LANGUISH AND YOU WOULD NO LONGER HAVE FUNCTIONAL TISSUE. BUT WHAT THEY DIDN’T REALLY NOTICE AT THE TIME AND HAS COME ALONG SINCE THEN, IS THAT THERE REALLY IS A HIERARCHY OF TISSUE TURNOVER AND TISSUE SPECIFIC STEM AND PROGENITOR CELLS. THERE ARE SOME TISSUES THAT ARE REPLACED AT A VERY, VERY HIGH RATE SUCH AS BLOOD, SKIN AND THE GASTROINTESTINAL TRACT. THERE ARE TISSUES THAT HAVE A VERY SLOW RATE OF REPLENISHMENT SUCH AS BONE, THERE ARE TISSUES WITH LIMITED ABILITY TO REPAIR SUCH AS MUSCLE AND ALSO THE INNER MOST HARD SURFACE OF THE TOOTH, THE DENTIN AND THEN THERE ARE TISSUES THAT WERE NEVER THOUGHT TO REPAIR, SUCH AS NERVOUS TISSUE BUT ALL OF THESE TISSUES DO IN FACT HAVE A STEM OR PROGENITOR CELL THAT WILL FUEL THE FIRE AND REPLACE SMALL AMOUNTS OF TISSUE AT LEAST. HOWEVER, IT’S NOT ONE-SIZE-FITS-ALL. STEM CELLS ARE A VARIATION ON A THEME AND THAT VARIATION IS BASED ON THE RATE OF TISSUE TURNOVER AND THE SPECIFICS OF VARIOUS DIFFERENT TISSUES THAT CONTAIN STEM CELLS. SO IF YOU WERE TO ASK A ROOM FULL OF STEM CELL BIOLOGISTS WHAT THE DEFINITION OF A STEM CELL IS, YOU WOULD GET ABOUT 10 DIFFERENT DEFINITIONS BASED ON THEIR TISSUE OF INTEREST BUT THERE ARE 2 PROPERTIES OF STEM CELLS THAT EVERYONE CAN AGREE ON AND THE FIRST 1 IS THAT THE OFFSPRING OF A SINGLE CELL ARE ABLE TO REBUILD THE FUNCTIONAL PATHWAY GIVES RENK MA OF A TISSUE, IN OTHER WORDS THE WORKING CELL TYPE, NOT THE STROAMA OR CAP SUEULAR KINDS OF TISSUES BUT THE FUNCTIONAL PART OF THE TISSUE AND THAT A STEM CELL HAS THE ABILITY TO SELF-RENEW. SO WHAT EXACTLY IS SELF-RENEWAL BECAUSE THIS TERM IS ABUSED MORE FREQUENTLY PROBABLY IN STEM CELL BIOLOGY THAN ANY OTHER TERM. AND THAT IS HOW A STEM CELL KEEPS ITSELF REPLENISHED SO IT’S THOUGHT THATPOSTNATAL STEM CELLS THAT IT UNDERGOES AN ASYMMETRIC DIVISION WHERE 1 DAUGHTER CELL IS A MORE COMMITTED CELL AND GOES ON TO TRANSIENTLY AMPLIFY AND THEN TO DIFFERENTIATE INTO A POST FUNCTIONAL CELL, BUT THEN THE SECOND DAUGHTER CELL REMAINS A STEM CELL. IT TURNS OUT THAT IT’S A BIT MORE COMPLICATED THAN THAT, SOMETIMES THE STEM CELL WILL UNDERGO A SYMMETRIC DIVISION, MEANING THAT THE STEM CELL IS NOT BEING REPLENISHED AND YOU HAVE STEM CELL LOSS AND THEN THERE ARE OTHER SITUATIONS WHERE THE STEM CELL DIVIDES AND BOTH DAUGHTERS ARE STEM CELLS ANOTHER FORM OF SYMMETRIC DIVISION. THIS MAY BE SOMETHING THAT IS HAPPENING WHEN YOU HAVE A TUMOR FORMED. THIS IS WHAT YOU HAVE HAPPENING WHEN YOU HAVE IPS CELL DIVISION AND IN FACT, IN A TISSUE, THE STEM CELL MAY TOGGLE BACK AND FORTH BETWEEN SYMMETRIC DIVISIONS AND ASYMMETRIC DIVISIONS DEPENDING ON THE MICRO ENVIRONMENT AND WE TRULY DO NOT HAVE A GOOD HANDLE ON HOW STEM CELLS IN EACH INDIVIDUAL TISSUE SELF-RENEW. IT MAY BE MANY DIFFERENT PROCESSES, DEPENDING ON THE MICRO ENVIRONMENT BUT THE 1 TECHNOLOGY TRANSFER THAT WE DO KNOW IS THAT SELF-RENEWAL DOES NOT MEAN EXTENSIVE PROLIFERATION. TIME AND TIME AGAIN, YOU WILL HEAR PEOPLE SAY OR READ IN PAPERS THAT THEY HAVE A STEM CELL BECAUSE THOSE CELLS PROLIFERATE EXTENTIVELY AND THAT DOES NOT MAKE THE CELL A STEM CELL. THERE ARE OTHER CRITERIA, MEANING THEY HAVE THE ABILITY TO DIFFERENTIATE INTO A FUNCTIONAL TISSUE. THE OTHER PROPERTIES OF STEM CELLS ARE REALLY ASSUMPTIONS WE’VE GIVEN TO THESE CELLS BASED ON WHAT THEY DO IN A CULTURE DISH. IT’S THOUGHT THAT STEM CELLS ARE CLONE O GENIC, AS A MENTIONED, THAT SELF-RENEWAL MEANS UNLIMITED PROLIFERATION AND IT’S ALSO BEEN THOUGHT THAT STEMNESS MEANS THAT THE CELL TYPE IS UNDIFFERENTIATED BUT THE FACT OF THE MATTER IS, THIS IS NOT NECESSARILY TRUE FOR ALL STEM CELLS AND THAT WE HAVE TO FIND OUT WHAT THE DYNAMICS OF EACH INDIVIDUAL STEM CELL IS, AND NOT TRY TO LUMP THEM ALL IN THE SAME BOX. SO IF WE START OFF IN DEVELOPMENT, WE HAVE THE FERTILIZED EGG WHICH IS TOTI-POTENT WHICH IS THERAPIST IS WHAT ELIZABETH HAY, A DEVELOPMENTAL BIOLOGIST WHO DIED A FEW YEARS BACK WOULD CALL THE MOTHER OF ALL STEM CELLS AND IT’S THE MOTHER OF ALL STEM CELLS BECAUSE NOT ONLY DOES IT CREATE OVER CELL IN THE BODY, IT ALSO CREATES THE PLACENTA MEMBRANES. SO THIS IS WHAT IS CALLED A TOT-EYE POTENT CELL WHICH GOES ON TO DIVIDE TO FORM THE BLASTOCYST WHICH HAS THE INNER CELL MASS AND THE INNER CELL MASS CONTAINS PLURIPOTENT CELLS, AND THESE CELLS REMEMBER FORM EVERY CELL IN THE BODY, THIS IS THE SOURCE OF EMBRYONIC STEM CELLS, BUT IT’S NOT ABLE TO FORM THE PLACENTA MEMBRANES. AND IT BY ITSELF CANNOT FORM AN EMBRYO, IT NEEDS THE EXTRA EMBRYONIC MEMBRANES TO DO THAT. BUT IN TERMS OF WHAT THESE PLURIPOTENT CELLS CAN DO, IF YOU WERE TO TAKE A MOUSE ESO, OR A PLURIPOTENT CELL AND PUT IT INTO A PLAOF THE O SIS, THE EMBRYO WILL FORM. THIS WAS DONE MANY YEARS AGO BY MANY DIFFERENT PEOPLE. INVITRO, THEY WILL FORM VIRTUALLY EVERY CELL TYPE IN THE BODY. IF YOU DON’T ALLOW THEM TO ATTACH THEY FORM AN ‘EM BROID BODY WHICH CONTAINS TISSUE TYPES OF ALL DIFFERENT KINDS THAT ARE FOUND IN THE BODY AND IF YOU PUT IT INTO THE MOUSE IT WILL FORM A TERATOMA WHICH HAS ALL THE DIFFERENTIATED CELL TYPES. SO BASED ON THESE TYPES OF ASSAYS, WE CAN DETERMINE THE POTENCY OF A GIVEN POPULATION OF CELLS. AS DEVELOPMENT CONTINUES, THE EMBRYO UNDERGOES GASTROALATION TO FORM THE 3 LAYERS ECTODERM, MESODERM AND ENDODERM. THESE ARE MULTIPOTENT. A SINGLE CELL FROM 1 OF THESE GERM LAYERS CANNOT REFORM AN EMBRYO, THEY’VE LOST SOME OF THEIR POTENCY BUT THEY’VE BECOME LINEAGE COMMITTED AND THEY ARE MULTIPOTENT STEM CELLS. AND THEN AFTER BIRTH AS I MENTIONED, WE FIND IN MANY DIFFERENT TISSUES, IF NOT ALL TISSUES, SOME FORM OF ADULT STEM PROGENITOR CELL THAT IS ABLE TO REFUEL FORMATION OF TISSUES AND PARTICIPATE IN TISSUE TURNOVER. SO AGAIN DURING THE SPECIFICATION, THEY BECOME SEGMENTED INTO THE PRAXAMEL, AND EKD O DERM AND MESODERM AND ENDODERM AND THEN AS IT CONTINUES MORE SEGMENTATION OCCURS FOR EXAMPLE, THE THESE GO ON TO CELL MIGHTS AND THESE FORM MORE TISSUES SO THERE IS CONTINUAL SPECIFICATION AS THE EMBRYO IS GROWING. SO WHEN DEVELOPMENT IS FINISHED, APPROXIMATELY 220 DIFFERENT CELL TYPES ARE FORMED JUST FROM THAT 1 SINGLE FERTILIZED EGG THAT HAS UNDERGONE THAT PARTICULAR PROCESS WHICH IS REALLY A REMARKABLE THING WHEN YOU THINK ABOUT IT THAT EMBRYONIC DEVELOPMENT OCCURS NORMALLY IN VIRTUALLY, THE VAST MAJORITY OF TIMES. SO AS WYNN MENTIONED WE URPD WENT A REVOLUTION STARTING IN 1998, WHEN JAMIE THOMPSON FIRST CREATED HUMAN EMBRYONIC STEM CELLS, USING THE TERATOMA ASSAY WHICH IS REALLY THE GOLD STANDARD BY WHICH YOU JUST PLURI-POTENCY HE HAD REPRESENTATIVES OF ALL 3 GERM LAYERS AND STROAMA, MUSCLE, CARTILAGE, GLUMERRIAL I, WHICH IS ENDODERM AND THEN AS YOU HEARD YAMANAKA, USING THE 3 FACTORS, THOSE ALSO USING THE TERATOMA ASSAY, AGAIN SHOWED PLURI-POTENCY IN THESE REPROGRAMMED CELLS AND THEN JAMIE THOMPSON USING DIFFERENT FACTORS ALSO DERIVED HUMAN PLURIPOTENT STEM CELLS, USING HUMAN SOMATIC CELLS AND I’D LIKE TO HILIGHT THAT IN ADDITION TO THE TERATOMA FORMATION, THE EXPRESSION, SURFACE EXPRESSION OF THESE MARKERS SSEA3 AND 4, TRIAL 160 AND TRIAL 181, AND OCT 4 AND SOX 2 AND LIN 28 ARE MARKERS THAT ARE TYPICALLY USED TO DETERMINE THE PLURI-POTENCY OF A POPULATION THAT HAS BEEN GENERATED AND ARE QUITE USEFUL. SO AFTER THE FIRST DISCOVERY OF THE REPROGRAMMING USING VIRAL TRANSDUCTION, A NUMBER OF OTHER DIFFERENT METHODS HAVE BEEN DEVELOPED FOR CREATING PLURIPOTENT STEM CELLS, INCLUDING VIRAL TRANSDUCTION WITH MANY DIFFERENT TYPES OF VECTORS AND VIRUSES, AND PLAZ MIDS MIDS, MINICIRCLES, PROTEIN, TRANSDUCTION, RNA TRANSDUCTION AND I THINK RIGHT NOW, BASICALLY THE 2 THAT SEEM TO BE WORKING THE BEST ACCORDING TO BARBARA AND THE STEM CELL CHARACTERIZATION FACILITY ARE USING SENDI VIRUS WITH THE 4 FACTORS AND RNA TRANSFECTION, BUT THERE ARE REAL ISSUES IN TERMS OF REPROGRAMMING, NOT EVERYTHING THAT YOU GENERATE IN THE DISH IS ACTUALLY PLURIPOTENT SO THERE’S QUALITY OF REPROGRAMMING THAT HAS TO BE EVALUATED. IT TURNS OUT THERE ARE DIFFERENT SUBSTATES OF PLURIPOTENT CELLS THAT GIVE THEM A PARTICULAR PREFERENCE FOR 1 EMBRYONIC GERM LAYER THAN ANOTHER. THERE ARE TIMES WHERE YOU HAVE NOT COMPLETELY WIPED OUT THE MEMORY OF THE CELL, SO IT REMEMBERS A LITTLE BIT ABOUT WHERE IT CAME FROM AND THEN THERE WAS THE ISSUE OF NAIVE VERSUS PRIMED. NAIVE ARE CELLS THAT ARE A LITTLE BIT MORE PRIME ARDIAL AND–PRIMORDIAL AND BETTER MATCHED CELLS THAN THE MOUSE EMBRYO. PRIMED APPEARED TO BE READY TO GO INTO 1 PARTICULAR GERM LAYER OR ANOTHER AND THERE ARE A LOT OF PAPERS ABOUT THIS FROM THESE 2 WORKERS. AND THEN THERE’S ALSO WHAT I CALL DIRECT REPROGRAMMING AND THIS IS EXEMPLIFIED BY THIS PAPER FROM NORIYUI, IN JAPAN WHERE HE TOOK SKIN 1 BLAST AND TRANSFECTED THEM WITH CMYC AND KLF 4 AND SOX9 AND THAT’S THE MASTER GENE FOR CARTILAGE AND THEN HE DEVELOPED WHAT HE CALLED THESE ICON CELL WHICH IS IF YOU PUT THEM INTO A PELLET CULTURE, THEY FORM BEAUTIFUL CARTILAGE AND THIS IS IN COMPARISON TO THE–THE PARENTAL SKIN 1 BLASTS AND ALSO IN COMPARISON TO OTHER CELLS THAT FORM CARTILAGE. THE CARTILAGE LOOKS REALLY GOOD. THE PROS OF THIS APPROACH IS THAT THERE’S LESS RISK OF TERATOMA FORMATION AND YOU CAN ALSO SORT THESE CELLS FOR MARKERS OF CHOND ROW SIGHTS AND PURIFY THEM, THE CONS IS THAT UNFORTUNATELY MANY OF THESE CELLS FORMED CHOND ROMAS WHICH ARE WORSE THAN TERATOMAS, THEY’RE NASTY TUMORS AND THE OTHER PROBLEM IS THAT DEPENDING ON THE CELL TYPE, YOU HAVE VERY, VERY LIMITED ECPANGZ AFTER YOU DIRECTED THEM USING THESE TRANSCRIPTION FACTORS. AND SO GENERATING ENOUGH CELLS COULD BE A PROBLEM WITH THIS TYPE OF MESSAGE. SO IN TERMS OF THE VARIOUS DIFFERENT TYPES OF STEM CELLS, I’VE KIND OF HIGHLIGHTED HOW THEY COME ABOUT DURING DEVELOPMENT AND HOW WE CREATE THEM IN A DISH. WHAT I WOULD LIKE TO HIGHLIGHT NOW IS THAT WE NEED TO KNOW ABOUT ALL KINDS OF STEM CELLS, IF WE ARE GOING TO USE THEM SUCCESSFULLY IN TISSUE ENGINEERING AND REGENERATION FOR EXAMPLE, WE NEED TO KNOW FROM EMBRYONIC STEM CELLS AND IPS CELLS, WHAT GIVES THEM THE CAPACITY TO PROLIFERATE CONTINUOUSLY AND WHAT MAKES THEM PLURIPOTENT, WHAT ARE THE FACTORS AND I THINK THAT WE ONLY ARE SCRATCHING THE SURFACE ON OUR KNOWLEDGE IN TERMS OF FETAL STEM CELLS, WE KNOW THEY CAN PROLIFERATE EXTENSIVELY, THEY ARE MULTIPTENT BUT THEY’RE COMMITTED TO A PARTICULAR LINEAGE SO WHAT ARE THOSE FACTORS AND THEN FINALLY, THERE ARE THE POSTNATAL STEM CELLS. WE KNOW THAT THAT THEY HAVE VERY CONTROLLED AND PREDICTABLE DIFFERENTIATION. SO WHAT BETTER CELL TYPE THAN A POSTNATAL CELL TO EDUCATE AN EMBRYONIC OR A PLURIPOTENT CELL ON HOW TO DIFFERENTIATE AND WHAT BETTER CELL TYPE THAN AN EMBRYONIC OR AN IPS CELL TO TELL A POSTNATAL CELL HOW THEY CAN PROLIFERATE MORE EBS TENSIVELY. SO, 1 CELL TYPE IS NOT GOING TO GET US TO WHERE WE WANT TO BE. WE WANT TO BE LOOKING AT ALL OF THESE DIFFERENT KINDS OF STEM CELLS. WHAT ARE WE USING THESE FOR? WELL WE CAN USE THEM FOR DEVELOPMENT. FOR EXAMPLE, WE CAN DETERMINE HOW A COMPLEX ORGANISM CAN EMANATE FROM A SINGLE FERTILIZED EGG. WE CAN FOLLOW THE STEM CELLS AS THEY DIVIDE AND BECOME MORE SPECIFIED. WE CAN USE THEM TO IDENTIFY THE SIGNALS AND THE MECHANISMS THAT DETERMINE STEM CELL, SELF-RENEWAL VERSUS DIFFERENTIATION, THIS IS REALLY IMPORTANT IN TERMS OF TISSUE HOMEOSTASIS, AND WE ALSO CAN USE THEM TO DETERMINE HOW THEY MAKE THEIR LINEAGE COMMITMENT TO CHOICES. SO FOR EXAMPLE, IN BONE BIOLOGY, CELLS CAN BECOME EITHER BONE OR CARTILAGE, HOW DO THEY DECIDE WHICH WAY THEY’RE GOING TO GO AND WE CAN USE IPS CELLS TO TRY AND UNDERSTAND THESE. AND SO, DEVIN EAGAN HAS ACTUALLY PUT TOGETHER THIS ROADMAP ON HOW YOU CAN GET PLURIPOTENT CELLS TO DIFFERENTIATE INTO ALL THE CELLS IN THE BODY, YOU WILL HEAR ABOUT THIS RETINAL PIGMENTED EPITHELIUM FROM KAPIL, IN TERMS OF THE DIFFERENTIATION, I HAVE SOME MISGIVINGS ABOUT HIS MAP HERE BUT YOU KNOW IT’S A START. YOU HAVE TO START SOMEWHERE. THIS IS A WORK IN PROGRESS AND THIS IS REALLY THE GOAL TO BE ABLE TO GET THESE CELLS TO NOT ONLY DIFFERENTIATE INTO A SPECIFIC CELL TYPE BUT ALSO TO GET THEM TO DIFFERENTIATE INTO A MATURE CELL TYPE, THIS HAS BEEN A REAL PROBLEM IN THE FIELD THAT WE CAN GET HEMATOPOIETIC LIKE CELLS BUT THEY’RE NOT MATURE. YOU CAN GET MUSCLE LIKE CELLS BUT THEY’RE NOT MATURE. DOES IT MATTER IF WE PUT THEM IN THE BODY, WILL THE BODY TELL THEM HOW TO MATURE? OR DO WE NEED TO TELL THEM HOW TO MATURE IN THE DISH? IN TERMS OF OFTIO GENIC AND CHONDROGENNIC DIFFERENTIATION, WE STARTED OFF TREATING THE CELLS LIKE THEY WERE BONA FIDE OFTIO GENIC PRECURSOR CELLS AND WE WERE ABLE TO GET SMALL AMOUNTS OF BONE AND CARTILAGE. ONE LINE FORMED BOTH BONE AND CARTILAGE BUT THIS WAS NOT ROBUST, WE WERE NOT GETTING THE AMOUNT OF BONE AND CARTILAGE THAT WE WOULD GET FROM BONAIFIED CHOND RASIGHTS AND BONE. SO THE NEXT APPROACH WAS TO FOLLOW A MORE DEVELOPMENTAL LINE AND THAT’S BEING DONE BY FAHAD KID WAY IN MY LAB. IT TURNS OUT, WE KNEW FOR A LONG TIME, BUT OTHERS DIDN’T KNOW THAT THERE ARE AT LEAST 3 DIFFERENT SOURCES OF BONE. THERE’S THE NEURAL CREST WHICH GIVES RISE TO FACIAL BONE AND CARTILAGE, THERE’S THE DORSAL PART OF THE MIGHTS AND THE SCLER TELOMERE THAT GIVES RISE TO THE AXIAL SKELETON AND THEN THERE’S THE SOPHISTICATED SOMATIC
LATERAL PLATE WH
ICH GIVES RISE TO THE APPENDICULAR SKELETON. SO 3 DIFFERENT EMBRYONIC SOURCES AND IT MAKES A DIFFERENCE IN HOW YOU TREAT THEM TO GET THESE DIFFERENT EMBRYONIC SOURCES, AND SO THIS IS KIND OF THE ROADMAP THAT WE’RE USING AT THE MOMENT TO TRY AND GET THEM TO DIFFERENTIATE IN BONE AND CARTILAGE FROM THE 3 DIFFERENT GERM LAYERS BECAUSE IT’S OUR HUNCH THAT IT MATTERS. IF THE CARTILAGE OR BONE IS NEUROCREST IN ORIGIN, VERSUS LATERAL PLATE OR PATHWAY GIVES RACKSIAL, THERE ARE SOME CLINICAL INDICATIONS THAT IT REALLY DOES MATTER WHERE IT COMES FROM. YOU CAN USE STEM CELLS TO STUDY DISEASE SO FOR EXAMPLE, IT’S VERY DIFFICULT TO OFTEN TO GET SUFFICIENT CELLS THAT ARE FROM A PATIENT TO STUDY THEM IN DETAIL. IF WE CAN CREATE STEM CELLS WITH A DISEASE CAUSING MUTATION OR ENGINEER THEM, THAT OFFERS A VIABLE ALTERNATIVE TO STUDYING THE PATHOGENETIC MECHANISMS AND WE CAN ALSO USE THEM FOR INVIVO TRANSPLANTATION INTO HUMANIZED OR IMMUNE O SUPPRESSED MICE TO DEVELOP A DISEASE TISSUE IN ORDER TO TRY AND COME UP WITH A MECHANISM AND IN FACT, WE’VE DONE THIS. CELLS WHICH ARE THE CELLS THAT I WORK ON, WE HYPOTHESIZE THAT BECAUSE THESE CELLS ARE VERY IMPORTANT IN SKELETAL HOMEOSTASIS THAT ANY GENETIC MUTATION OR ANY CHANGE IN THE MICRO ENVIRONMENT THAT CHANGES THEIR ACTIVITY WILL RESULT IN A DISEASE. AND OUR EXAMPLE WAS FIBROUS DISPLACIA BONE AND WHAT WE DID WAS WE ISOLATED SKELETAL STEM CELLS FROM NORMAL BONE AND FROM FIEB ROW SIS PLASTIC BONE. YOU CAN SEE THIS IS VERY ABNORMAL, THERE’S NO MARROW WOVEN BONE, VERY WEAK WOVEN BONE. IF WE TAKE THOSE CELLS AND PUT THEM ON TO A SCAFFOLD AND TRANSPLANT THEM INTO A MOUSE, THE NORMAL SKELETAL STEM CELLS MAKE THE NORMAL BONE AND THE 1S FROM THE ABNORMAL MARROW, MAKE WHAT WE CALL AN FD-OTHICALL, IT’S RECAPITULATE THE FORMATION OF A ABNORMAL BONE IN A SMALL ANIMAL. SO THIS IS A MODEL THAT WE CAN USE FOR STUDYING THE PATHOGENETIC MECHANISMS AND ALSO TO COME UP WITH DIFFERENT WAYS OF TREATING THESE CELLS TO REDUCE THE EFFECT OF THE MUTATION. THIS THERE HAVE BEEN A NUMBER OF ARTICLES WRITTEN ABOUT USING PLURIPOTENT STEM CELLS IN TERMS OF MODELING DISEASE AND THIS IS 1 OF THEM TALKING ABOUT USING THE CELLS FOR 2 D MODELS, 3D MODELS AND ALSO 4 D BY ADDING IN THE ELEMENT OF TIME, WATCHING HOW A TISSUE EVOLVES WHETHER IT BE IN AN ORGANOID OR ON A PLATE, TISSUE ON A CHIP IS BEING ENGINEERED IN MANY DIFFERENT LABS AROUND THE WORLD. AND ALSO IN TERMS OF RPE, THERE ARE STUDIES WHERE THERE WAS A MUTATION IN MITOCHONDRIAIAL DNA THAT WAS USED TO STUDY THE DYSFUNCTIONAL RPE WHERE YOU HAD VERY ABNORMAL SWOLLEN MITOCHONDRIA AND ABNORMAL MELANO STUDIES OF MULTIPLE ENDOCRINES SO THESE CELLS CAN BE VERY USEFUL IN TERMS OF COMING UP WITH MODELS OF DISEASE.& AND IN ORDER TO DO THIS, WE CAN START OFF WITH NORMAL CELLS AND USE THE EMERGING TECHNOLOGIES FOR GENOME EDITING WHERE WE CAN MODULATE, CREATE OR CORRECT MUTATION AND THERE ARE A NUMBER OF DIFFERENT METHODS USING PRELOCKS FOR MODULATING THE LEVEL OF A MUTANT PROTEIN, CRSPR AND ZINC AND TALON HAVE BEEN USED TO CREATE MUTATIONS OR CORRECT MUTATIONS IN IPS CELLS. WE CAN USE STEM CELLS TO REPLACE DAMAGED TISSUES, SO THIS OFFERS A WAY TO CORRECT A SEGMENTAL DEFECT THAT WOULD NEVER HEAL ON ITS OWN. THIS IS BEING DONE WITH A LIMITED NUMBER OF POSTNATAL STEM CELLS SUCH AS HEMEAT O POETEC STEM CELLS WHICH ARE USED IN BONE MARROW TRANSPLANTATION FOR HEMEATOLOGICAL DISORDERS, AND WE CAN REALLY LOOK AT THEM AS A VERY EXCITING OPPORTUNITY, BUT THERE ARE A LOT OF HURDLES WE HAVE TO JUMP OVER BEFORE IPS CELLS WILL BE READY FOR PRIME TIME AND I THINK KAPIL’S PROJECT IS 1 OF THE PRIME EXAMPLES THAT LOOKS VERY, VERY PROMISING. SO IN TERMS OF OTHER STEM CELL BASED THERAPIES FOR EXAMPLE, THERE’S PARKINSON’S DISEASE WHICH INITIALLY WAS TREATED WITH FETAL NEURAL TISSUE. THOSE STUDIES DID NOT WORK VERY WELL BUT IT’S NOW ACTUALLY BEING REVISITED BECAUSE THEY LEARNED A LOT OF LESSONS ABOUT HOW TO TRANSPLANT THESE CELLS WHETHER FETAL TISSUE WILL REALLY BE USEFUL FOR TREATING PARKINSONS, WE DON’T KNOW BUT THIS REVIEW ARTICLE TALKS ABOUT WHAT THEY LEARNED FROM THE EARLY TRIALS AND WHAT THEY’RE CHANGING NOW IN THE LATER TRIALS AND IT’S A EUROPEAN CONSORTIUM THAT IS NOW DOING THIS. IN TERMS OF BONE REGENERATION, WE KNOW THAT WE CAN TAKE BONE MARROW STROAMAL CELLS AND SKELETAL STEM CELLS, WE CAN ATTACH THEM TO A SCAFFOLD AND PUT THEM INTO A SEGMENTAL INJURY ASK WE CAN DIRECT THEM INTO AN INJURY WITH AN INJECTABLE CARRIER AND REGENERATE BONE AND THIS IS ACTUALLY BEEN DONE IT’S ALONG WITH HYDROXY APPLICATIONS APPETITE AND YOU CAN SEE THERE IS BONE REGENERATION AND WE ARE WORKING VERY MUCH ON TRYING TO RECONSTRUCT CRANEIO FACIAL DEFECTS SUCH AS CALFARRIAL DEFECTS AND JAW DEFECTS USING THIS TECHNOLOGY AND HERE CAN YOU SEE THAT AND I HOPE THAT I WILL GET TO DO THIS PROTOCOL SKIN DISEASES, THIS IS A TERRIBLE DISEASE CALLED EPIDERMAL ICEIS BELOSA AND IT’S CAUSED BY MUTATIONS, LAM BETA 3, THIS MARKS THE AREAS WHERE THERE WAS EITHER BLISTERING OR COMPLETE DENEWTING OF THE SKIN FROM THIS BOY WITH THIS DISEASE. YOU CAN SEE THAT THESE COMPLETELY MISSING LIMB 332 WHICH IS LAMIN AND 5. THEY TOOK HIS EPIDERMAL CELLS, STEM CELLS, THEY TRANSFECTED WITH THE DEFECTIVE GENE, EXPANDED AND THEY HAVE PRETTY MUCH SAVED THIS PATIENT. HE IS COMPLETELY COVERED WITH NORMAL SKIN AT THIS POINT AND IT REALLY HAS A BIG BREAK THROUGH. YOU WILL HEAR FROM KAPIL ABOUT REGENERATION OF RPE, AND ALSO WE CAN USE STEM CELLS FOR TESTING NEW MEDICAL TREATMENTS. THIS IS 1 OF THE THINGS THAT THEY ARE DOING OUT AT NCATS, TRYING TO DEVELOP WAYS OF USING VARIOUS DIFFERENT–DIFFERENTIATED PROGENY OF THE STEM CELLS, USING THEIR LIBRARIES AND THEIR ROBOTICS TO SCREEN FOR NEW DRUGS TO TREAT VARIOUS DIFFERENT DISEASES. SO I JUST WANT TO BRIEFLY MENTION THE RESOURCES THAT WE HAVE HERE AT NIH TO DO THIS KIND OF WORK. THE FIRST IS THE NIH STEM CELL UNIT THAT BARBARA IS THE TECHNICAL DIRECTOR AND SHE BASICALLY HAS DONE A HUGE AMOUNT OF WORK IN OPTIMIZING THE GROWTH AND THE GENERATION OF IPS CELLS AND ESTABLISHING STANDARDS, SHE HAS A DATABASE THAT’S ON THE NIH WEBSITE WHERE YOU CAN PLUG IN COMPARE IT TO THE IPS CELLS AND EMBRYONIC CELLS AND SHE PROVIDES A TREMENDOUS AMOUNT OF SUPPORT TO THE INTRAMURAL INVESTIGATORS, AND THIS IS AN EXAMPLE HERE OF DRIVING STEM CELLS FROM EITHER SKIN 1 BLASTS, CD34 POSITIVE CELLS, OR FROM MY FAVORITE CELL BONE MARROW STROAMAL CELLS, AND USING EITHER EMBRYONIC 1 BLASTS AS A FEEDER OR MATRIGEL AND DOING MONOLAYER CULTURES AND SHE AND KEVIN AND KIYUN AND HAVE WORKED OUT ALL THESE METHODS. AND THEN THE OTHER THING YOU HAVE TO THINK ABOUT IN TERMS OF DIFFERENTIATION IS WHETHER OR NOT YOU WILL USE EMB ROID BODIES WHICH CONSTAIN THE DIFFERENTIATED CELL TYPES AND MONOLAYER CULTURES TO DO YOUR DIFFERENTIATION. THE PROS ARE FOR ‘EM BROID BODIES IS THAT YOU CAN TAKE YOUR ‘EM BROID BODY AND DIGEST IT WITH AN ENZYME AND DO FACTS TO ISOLATE THE CELLS WITH THE CELL SURFACE CHARACTER THAT YOU WANT TO ISOLATE BUT THE CONIS THAT THE ‘EM BROID BODIES ARE EXTREMELY VARIABLE AND THEN THE PROBLEM IS THAT THE NUMBER OF CELLS THAT YOU CAN GET AND THEIR PROLIFERATIVE CAPACITY MAY BE QUITE REDUCED. THE PROS FOR THE MONOLAYER IS THAT YOU CAN GENERATE LARGE NUMBERS OF CELLS. IT’S VERY EASY TO SCALE UP BUT THE CONIS THAT, UNLESS YOU REALLY KNOW WHAT YOU’RE DEVELOPMENTAL SEQUENCE IS, YOU CAN END UP WITH A REALLY MIXED POPULATION THAT REALLY WILL NOT PERFORM THE WAY YOU WANT TO. THE OTHER RESOURCE SYSTEM THE DEPARTMENT OF TRANSFUSION MEDICINE, THE CELL PROCESSING SECTION, HEADED UP BY DAVID STRAMSIC, AND THEIR MISSIO IS TO COLLECT MANUFACTURE AND STORE CELLULAR THERAPIES. THEY DO A LOT OF DONOR EVALUATION, LABORATORY, TESTING, AND THEY HAVE A LOT OF MEDICAL CONSULTS WITH PEOPLE THAT ARE USING CAR T-CELLS TO TREAT VARIOUS DIFFERENT TYPES OF BLOOD DISORDERS. AS YOU CAN IMAGINE, AND KAPIL WILL TALK ABOUT THIS, THERE ARE A HUGE NUMBER OF ISSUES THAT YOU HAVE TO DEAL WITH BEFORE THE FDA WILL APPROVE YOUR CELL PRODUCT FOR USE IN HUMANS. IDENTITY AND PURITY ARE REALLY THE MAIN 1S. AND EFFICACY, STABILITY, STERILITY, THESE ARE ALL THINGS THAT KAPIL WILL TALK TO YOU ABOUT. SO THIS IS MY LAST SLIDE, I AM BASICALLY, IT LISTS THE PROS AND CONS OF DIFFERENT KINDS OF STEM CELLS. BASIC–BASICALLY IT’S JUST DEPENDS ON YOUR PARTICULAR APPLICATION. IN TERMS OF THE PURITY OF THE POPULATION AND WHAT YOU ARE GOING TO USE IT FOR YOU REALLY NEED TO TAILOR YOUR CELL PRODUCT TO THE APPLICATION IN TERMS OF HUMAN ESLs, THEY ARE THE PUREST POPULATION BECAUSE THEY HAVE NOT BEEN GENETICALLY MANIPULATED BUT THEY HAVE ETHICAL ISSUES AND THERE ARE PROBLEMS WITH TERATOMAS. IPS CELLS DON’T HAVE THE ETHICAL ISSUES BUT WE REALLY DON’T KNOW HOW TO DIFFERENT THEM VERY WELL. FETAL CELLS HAVE ETHICAL ISSUES, WE DON’T HAVE LARGE NUMBERS OF THESE CELLS, POSTNATAL CELLS ARE VERY GOOD AT DIFFERENTIATING INTO THEIR SPECIFIC TISSUE BUT WE CAN’T GET ENOUGH OF THEM AND YOU CAN GO ON AND ON AND YOU CAN END UP WITH A REALLY LONG LIST OF PROS AND CONS AND SO IT’S A MATTER OF REALLY TRYING TO TAILOR IT TO WHAT YOUR APPLICATION IS. SO AT THIS POINT, I WANT TO ACKNOWLEDGE MANY PEOPLE HERE, ALEXANDER FRIEDEN STEIN AND BIANCA, WHO STUDIED THE SKELETAL CELL AND WHAT IT COULD DO IN THE ENVIRONMENT, MY FELLOWS LISTED HERE AND THOSE WHO WORKED ON THESE VARIOUS DIFFERENT CELLS. THIS IS THE CURRENT GROUP HERE WORKING ON SINGLE CELL ANALYSIS OF VARIOUS STEM CELLS, HERE’S THE STEM CELL CHARACTERIZATION FACILITY WITH BARBARA, KEVIN AND KYUN, DAVID WITH THE CELL PROCESSING, SECTION AND MIKE WHO’S IN THE CROWD WHO IS MY PARTNER IN CRIME IN TERMS OF FIBROUS DISPLACIA OF BONE AND SOME OF THE WORK WE DID EARLY IN THAT GAME. SO, AT THIS POINT, I WILL STOP AND ASK IF YOU HAVE ANY QUESTIONS. FEEL FREE TO CONTACT ME, THIS IS MY E-MAIL ADDRESS, AM MORE THAN HAPPY TO SHARE MY SLIDES WITH YOU IF THEY WOULD BE USEFUL TO YOU OR HELPFUL TO YOU IN ANY WAY SHAPE OR FORM.>>THANK YOU VERY MUCH. [ APPLAUSE ]>>WE HAVE TIME FOR A FEW QUESTIONS IF PEOPLE WILL USE THE MICROPHONES.>>THE SPEAKER WOULD LIKE TO HEAR THE QUESTION AS WELL.>>I AM INTERESTED IN THE MODULATORS AND YOU TOUCHED ON IT WITH NCATS, BUT IS THIS A RATIONAL STRATEGY OR IS IT JUST SCREENING RANDOMLY THROUGH LIBRARIES, BECAUSE YOU MENTIONED AND WYNN MENTIONED THE 4 GENES THAT CAN PERFORM THIS MODULATION, ARE THERE OTHER GENES THAT CAN BE MODULATED CHEMICALLY FOR THE OTHER TYPE OF TRANSFORMATION.>>THE FACT THAT JAMIE THOMPSON AND YANANA KA CAME UP WITH 4 DIFFERENT COMBINATIONS MEANS THERE IS REDUNDANCY AND WE HAD [INDISCERNIBLE]–THAT’S HOW YOU SAY IT GIVE A SEMINAR IN THE NIDCR AND HE BASICALLY HAS BEEN DOING SOME SCREENING WITH A LARGE LIBRARY, BUT HE’S ALSO BEEN DOING SCREENING WITH SPECIFIC PATHWAYS. SO HE’S HEDGING HIS BETS.>>SO I’M 1 OF THOSE PEOPLE WHO LEARN THEY ARE DIFFERENT ORIGINS IN THE BONES OF THE BODY, THANK YOU AND USING THAT KNOWLEDGE, THE CUSHION IS THE BONE MARROW, STEM CELLS TO TREAT LET’S SAY A FEMALE VERSUS WHAT YOU ARE TRYING TO DO FOR THE FACIAL BONES DO YOU HAVE TO USE DIFFERENT TYPE OF BONE MARROW STEM CELLS OR CAN YOU USE HETEROGENEOUSROLOGIOUS CELLS?>>THERE’S AN ANECDOTAL IN A FEW PAPERS FROM THE DENTAL FIELD SAYING IF YOU TAKE ILIAC CREST AND PACK IT INTO THE JAW IT DISAPPEARS SO FAHAD IS REALLY GOING TO TRY AND TEST THAT HYPOTHESIS BY TAKING CELLS FROM–THAT HAVE GONE THROUGH LATERAL PLATE AND PUTTING THEM INTO THE JAW AND VICE VERSA TO SEE WHAT THE INTERACTIONS ARE. PERHAPS WE WILL HAVE TO TEACH ILIAC CREST HOW TO BE MORE LIKE NEURAL CRESTS.>>SO I WONDER AND 1 OF THE PROBLEMS IS THE IPS CELLS, MANY TAKE A LONG, LONG TIME, WEEKS, MONTHS, BEFORE THE CONVERSION TAKES PLACE AND SO, THERE AREN’T A LARGE NUMBER OF CELLS, BUT THE PHENOMENON IS TAKING PLACE. SO IS THE RATE OF CONVERSION RELATED IN ANY WAY TO THE BIOLOGICAL TURNOVER OF THOSE CELLS AND THE IMPACT ORGANISM?>>INTUITIVELY THAT’S WHAT PEOPLE HAVE THOUGHT BUT I’M NOT SURE THAT THERE’S ANY DATA THAT SUPPORTS THAT. CERTAINLY, THEIR MEMORY WHETHER THEY’RE VERY DIFFERENTIATED OR A PROGENITOR COULD ALSO BE IMPACKING ON HOW FAST ABOUT IF YOU THINK ABOUT IT THE EPIGENETIC LANDSCAPE HAS TO BE CHANGED SO DRAMATICALLY AND I THINK THAT JUST TAKES TIME.>>SO HAVE YOU A BONE GROWING IN A PET RIDISH, OTHER FACTORS KNOWN THAT DETERMINE THE FINAL SHAPE OF THAT BONE OCILE, OR FEMUR WITHOUT HAVING IT IN PLACE–>>YEAH, MECHANICAL FORCES ARE EVERYTHING AND YOU KNOW IN OUR SUBCUTANEOUS OSSICLE, THEY LITERALLY LOOK LIKE LITTLE PANCAKES AND IT’S BECAUSE OF THE FORCINGS OF THE MUSCLE–FORCES OF THE MUSCLE ON THE IMPLANT. IN TERMS OF DEVELOPMENT, BOY THAT’S LIKE REALLY THE $60,000 QUESTION. YOU KNOW, HOW DO YOU GET THE SHAPE AND THE TRUTH AND EVEN MORE AMAZING WHEN YOU THINK ABOUT ALL THE CUSPS AND THE ROOTS AND THINGS LIKE THAT, WHAT DICTATES THE SHAPE IS REALLY, REALLY NOT KNOWN.>>BY THE WAY, ALL OF THESE IMAGES ARE ON THE WEBSITE, FOR THIS AND ANY OF THE PREVIOUS LECTURES. SO IF YOU WANT TO CONTEMPLATE SOME OF THE DETAILED INFORMATION, CAN YOU GO THERE AND SEE IT. SO YOU IN PASSING TALKED ABOUT THE PUTATIVE CANCER STEM CELLS AND YOU SORT OF PUT IT ON THE LIST AS 1 OF THE SOMEWHERE IN BETWEEN, BUT IT SEEMS TO ME, THERE’S A HUGE DEBATE AS TO WHETHER THESE ARE TRULY STEM CELLS OR JUST CELLS THAT ARE PROGENITORS IN A SEQUENCE. DID YOU WANT TO COMMENT–>>I THINK I COME DOWN ON THE SIDE THAT THESE ARE JUST PROGENITORS IN THE SEQUENCE THAT FOR SOME REASON CAN’T GO ON TO THE NEXT STEP OF DIFFERENTIATION. AND YOU KNOW IN TERMS OF WHAT THE CANCER INITIATING CELL, I’M MORE IN FAVOR OF THAT TERM THAN A CANCER STEM CELL. IT’S WHAT INITIATES AND PROPAGATES AND WHERE IT COMES UP IS A BIG QUESTION. FOR EXAMPLE, IN OFTIO SARCOMA, MY GUT FEELING IS THAT IT’S A BONE MARROW STROAMAL CELL THAT STARTS TO PROLIFERATE HAS ITS VARIOUS DIFFERENT MUTATIONS OR A NUMBER THAT ARE KNOWN AND IT JUST CANNOT GO ON TO THE NEXT STEP. IT HASN’T GONE BACK TO THE MOST–AS FAR AS I CAN SEE, IT HAS NOT GONE BACK TO THE MOST PRIMORDIAL CELL IN THE LINEAGE IS THERE A ROLL FOR FIBROUS DISPLACIA?>>WELL THE SOMATIC MOSAIC DISEASE SO WE COULD REGENERATE BONE FROM THEIR NORMAL CELLS, MICHAEL IS DEVELOPING SMALL MOLECULES TO TRY AND KNOCK DOWN THE ACTIVITY OF THE MUTATED GENE. I THINK THERE ARE OTHER DISEASES THAT ARE PROBABLY–THE FACT THAT WE CAN GET NORMAL CELLS FROM THESE PATIENTS IS A GOOD THING. THERE ARE OTHER DISEASES THAT–WHERE WE WOULD HAVE TO CORRECT THE MUTATION BEFORE WE COULD USE STEM CELLS.>>OKAY, WELL, THANK YOU. THERE WILL BE TIME FOR MORE QUESTIONS AND NOW WE’LL GO ON TO DR. BARTI.>>I WANT TO THANK WIN FOR TALKING ABOUT OUR WORK AND I WANT TO THANK PAM FOR GIVING SUCH A BROAD OVERVIEW ON STEM CELLS SO THAT GIVES ME THE OPPORTUNITY TO DIRECTLY FOCUS ON REALLY THE WORK THAT WE ARE DOING IN THE LAB AND DEVELOPING CELL THERAPY USING IPS CELLS. SO NOW I WILL FOCUS ON MACULAR DEGENERATION, TO FOCUS WHAT THIS ENTAILS, ON THE RIGHT SIDE WE SEE A NORMAL VISION OF A PERSON AND ON THE OTHER SIDE, YOU SEE HOW A PERSON WITH AGE RELATED MACKULAR DEGENERATION AMD SEES AND THE CENTRAL PART WILL BE COMPLETELY BLIND WHENEVER THEY LOOK, THIS IS A DISEASE THAT EFFECTS PEOPLE THERE’S SEVERAL MILLION PEOPLE WORLD WIDE THAT ARE EFFECTED BY THIS DISEASE AND CURRENTLY FOR 1 SPECIFIC FORM OF THIS DISEASE CALLED THE DRY FORM THAT EFFECTS 90% OF THE PEOPLE WITH THE MD THERE’S NO TREATMENT SO THERE IS A BIG UNMET MEDICAL NEED IN THE FIELD. SO IF WE LOOK INTO THE BACK OF THE EYE AND TRY TO UNDERSTAND HOW THIS DISEASE HAPPENS, THE NEXTLE OF SLIDES THAT HAPPEN, SHOW A SCHEMATIC OF HOW WE CURRENTLY BEST UNDERSTAND THE KIND OF PATHOGENESIS OF THIS DISEASE. SO THIS WOULD BE THE EYE, ON TOP YOU SEE THE RETINAL NEURONS WHICH ARE CONNECTED TO THE PHOTO RECEPTORS, REALLY THE CELL TYPE OF THE RETINAL WHICH SENDS SIGNATS TO THE BRAIN SO WE COULD ALL SEE AND RIGHT NEXT TO PHOTEE RECEPTORS IS A LAYER OF CELLS CALLED THE RPE, HA IS THE TOPIC OF DISCUSSION FOR TODAY’S TALK AND WHAT YOU WILL NOTICE IS A COUPLE OF INTERESTING FEATURES OF THIS CELL TYPE WHICH FIRST OF ALL IT HAS TRANSLATIONAL RESEARCH STIGIC LOCATION, PRESENT BETWEEN THE FOCUSED ONY ONY RECEPTORS AND THE BLEW SUPPLY, THESE ARE HIGHLY POLARIZED AND THEY HAVE TIGHT JUNCTIONS BETWEEN NEIGHBORING CELLS, AND THAT MEANS THAT PLOOD IS NOTHING LIKE–PLOOD IS NOT IN
DIRECT AN
D THOSE THAT HAVE TO GO TO THE RETINA HAVE TO PASS TO THE RB CELLS AND ALL THE NUTRIENT VS TO PASS THROUGH THE RB CELLS CELLS
AND THOSE HAVE TO PASS BACK AGAIN FROM RP INTO THE CURRENT BROOD SUPPLY AND THE OTHER TECHNOLOGY TRANSFER YOU NOTICE IS THEY HAVE THIS HAIR LIKE STRUCTURE WE CALL THIS APICKAL PROCESSES OF THIS THESE CELLS AND THEY SIT ON THIS MEMBRANE AND THESE AIR LIKE STRUCTURES IS THE BUSINESS END OF THE RPE CELLS AND THEY INTERACT WITH SEGMENTS AND HOW TO PERFORM THE FUNCTIONS RPE CELLS DO THROUGHOUT THEIR LIFE TO MAINTAIN HEALTH AND INTEGRITY OF THE PHOTO RECEPTORS SO, THE PHOT PIGMENT FOR RECEPTORS AND THEY HAVE THE PHOTO RECEPTORS SO ALL OF THESE TISSUES, REALLY THERE TO HELP THE PHOTO RECEPTORS SURVIVE. SO OBVIOUSLY IN A DISEASE LIKE AMD, WHERE RPE CELLS DIE OFF, PHOTO RECEPTORS START TO DIE AND THAT’S THE REASON WHY PEOPLE REALLY GO BLIND AND SO THIS IS WHAT WOULD BE KIND OF THE CENTRAL PART OF A PERSON WITH AMD, YOU CAN SEE THE PHOTO RECEPTORS ARE GONE AND THE CELLS ARE DEAD AND THE CAP ILLEGALSARYS ARE STARTING TO PAN OUT. BUT IF YOU MOVE AWAY FROM THE AREA, THE CENTRAL AREA WHERE THE THOSE PEOPLE ARE BLIND TOWARDS THE HEALTHY PART OF THE EYE, THIS IS AN AREA CALLED THE TRANSITION ZONE. IT’S CALLED THE TRANSITION ZONE BECAUSE IF YOU NOTICE HERE THE RPE CELLS ARE MISSING BUT THE FOALT O RECEPTORS ARE STILL HANGING IN THERE AND IT’S NOT THAT THESE AREN’T DYSFUNCTIONAL, THEY’RE LIKELY DESTINED TO DIE BECAUSE THEY DON’T HAVE RPE SUPPORT, BUT WHAT SURGEONS HAVE TRIED FOR A WHILE AND THOUGHT FOR A WHILE IS IF YOU ARE IN THIS RPE, CAN YOU PRO TECT THIS FROM THE LINE, AND THIS HAS BEEN TESTED SURGICALLY SO IF YOU THINK ABOUT THE WHOLE EYE AND YOU HAVE DAMAGE IN THE CENTER PART OF THE EYE, THEY WILL CUT OUT A PIECE OF RPE, FROM THE PERIPHERY OF THE SAME EYE, TRANSLOCATE IT INTO THE CENTER OF THE ZONE AND LEAVE THE TRANSPLANT THERE. AND IN SMALL NUMBER OF CASES, WHERE THE STUDIES WERE SUCCESSFUL THEY SAW THAT PATIENTS PHOTO RECEPTORS WERE SURVIVING FOR SEVERAL YEARS TO COME AND PATIENTS VISION WAS RESCUED AND STABILIZED FOR YEARS TO COME AND THESE ARE SOME OF THE PAPERS THAT HAVE PREVIOUSRY DEMONSTRATED THAT AND FOR ME, THIS IS A CRITICAL PROOF OF PRINCIPLE EXPERIMENT AND IF YOU WERE TO MAKE AN AHATOLOGIOUS PATCH FROM THE MD PATIENTS AND DELIVER IT TO THE RIGHT PLACE, YOU COULD ACTUALLY SAVE THE VISION FROM GETTING WORSE. AND THAT IS REALLY THE DRIVING PRINCIPAL FOR US TO MAKE RPE PATCH FOR THE PATIENTS. SO WE START WITH THE PATIENT’S OWN CELLS AS PAM EXPLAINED THE IPS CELL TECHNOLOGY MAKE, REPROGRAM THE SOMATIC CELLS INTO THE IPS CELLS AND DIFFERENTIATE THOSE CELLS INTO AN RPE PATCH, THOSE CELLS MAKE A PATCH AND DELIVER IT BACK TO THE PATIENTS. SO THERE SLIDE SHOWS YOU OF WHAT THE PATCH LOOKS LIKE, IT’S A 4 BY 2-MILLIMETER PATCH THAT CURRENTLY THE CENTER OF THE EYE, AND WHAT YOU SEE IN HERE IS RPCELLS ARE ON TOP, THE PIGMENTED CELLS AND WHEN WE FLIP THE PATCH, YOU WILL SEE THEY ARE SITTING ON A SCAFFOLD ON THE BIODEGRADABLE SCAFFOLD WHICH IS COLORED WHITE HERE AND ADDED THE IDEA IS THAT THE CELLS THAT MAKE THAT ARE NOT ECM, AND DON’T INTEGRATE AND DEGRADE AND THE CELLS SIT CONTINUE TO SIT ON THEIR ACM AND EPITHELIAL GREAT INTO THE BACK OF THE EYE. SO WHAT I’M GOING TO SHOW AND DUE TODAY IS GO WHERE THE WHOLE PROCESS OF HOW WE ACTUALLY MAKE THIS PATCH FOR TRANSPLANTATION, THE 3 PARTS OF IND APPLICATION THAT WE NEED TO START A PHASE CLINICAL TRIAL WITH THESE CELLS AND HOW WE MANUFACTURE WITH IPS CELLS AND HOW WE MANUFACTURE THESE PATCHES FROM RAMIFICATIONS WHAT’S CALL THE CMKRR PART OF THE CHEMISTRY MARVIN MUSCANNING AND CONTROLS AND HOW DO WE TEST THESE PATCH AND PRECLINICAL ANIMAL MODELS TO DEMONSTRATE THAT THE CELLS ARE SAFE, THEY ARE NOT TUMOR GENIC AND NOT TOXIC AND THEY DO HAVE SOME EFFICACY IN THESE ANIMAL MODELS AND THEN HOW DO WE DEMONSTRATE THAT THE TOOL AND THE SCAFFOLD WE HAVE MADE TO DELIVER THIS PATCH IS ALSO BY COMPATIBLE AND OF COURSE EVENTUALLY WOULD HAVE A CLINICAL PROTOCOL HOW WE DELIVER IT TO THE PATIENTS. SO JUST A BRIEF OVERVIEW OF THE MANUFACTURING PROCESS OR THE DEVELOPMENTAL PROCESS OF HOW WE MAKE RPE CELLS. SO THIS LECTURE IS FROM IPS CELLS ALL THE WAY TO MATURE RPE ISSUES THE PROCESS TAKES ABOUT 10 WEEKS SO IT’S A FAIRLY LONG PROCESS BUT IT’S WELL OPTIMIZED BUT IMPORTANT POINT TO REMEMBER HERE OR TO REALIZE HERE IS THAT IPS CELLS ARE COAXED INTO DEVELOPMENTAL STAGES, KIND OF THAT BIG OVERVIEW YOU SAW IN THE PREVIOUS TALK, THE CELLS GO FROM A NEUROECTODERM STAGE TO THE PROGENITORS ALL THE WAY TO MATURE RPE, AND WE DO THAT MY MANIPULATING GROWTH FACTORS IN THE TIME POINT AND THIS IS JUST REALLY REPRODUCING DEVELOPMENT OF BIOLOGY FOR RPE CELLS IN A DISH, ALL THE WORK WE HAVE LEARNED FROM OVER DECADES IN VARIOUS ANIMAL MODELS HOW WE MAKE RPE CELLS. WE TRY TO REPRODUCE THAT ON IPS CELLS ON A DISH AND INTERESTINGLY IN THE CASE OF RPE CELLS WE ACTUALLY CAN GET FUNCTIONALLY RPE CELLS, 1 OF THOSE VARIOUS CELL TYPES WE RECENTLY PUBLISHED WE CAN MAKE IT INTO MATURE PIECES AND THAT BECOMES RELEVANT WHEN YOU’RE TRANSPLANTING IT INTO ALL THE PEOPLE, YOU WANT TO TRANSPLANT LIKELY MATURE RPE CELLS, THAT ARE FUNCTIONING RIGHT AWAY. SO THE BOTTOM PANEL OF THIS SLIDE SHOWS THAT WE CAN GET PURE CELLS, THIS IS A DISH FULL OF PIGMENTED RPE CELLS AND WE CAN SHOW THAT THESE CELLS ARE POSITIVE FOR A NUMBER OF DIFFERENT RPE MARKERS AND THIS ALSO DEMONSTRATES THAT THE CELLS ARE FAIRLY HOMOGENIUS, IN TERMS OF EXPRESSION OF RPE MARKER. AN IMPORTANT POINT THAT WE HAVE TO CONSIDER IS THAT WE ARE TRYING TO DEVELOP AN AHATOLOGIOUS THERAPY, SO IF YOU HAVE TO DO IT HAS TO BE HIGHLY REPRODUCIBLE AND HERE WE HAVE TESTED THIS OVER 34 DIFFERENT DONOR IPS CELL LINES AND INDIVIDUALS WITH DIFFERENT TYPES OF DISEASES, CLINICAL GRADE CELLS, RESEARCH GRADE CELLS AND PRETTY MUCH IN EVERY SINGLE CASE, WE WERE SUCCESSFUL IN MAKING RPE CELLS AND MATURE RPE CELLS FROM IPS CELLS SO THAT’S AN IMPORTANT POINT GOING FORWARD THAT THIS PROCESS IS REPRODUCIBLE IN MAKING RPE CELLS. ONCE WE HAVE OUR RELEVANT CELL TYPE, WE PUT THEM ON THIS SCAFFOLD, THE 1 THAT I SHOWED BIODEGRADABLE SCAFFOLD, THESE WERE DESIGNED SUCH THAT AS WE CAN SEE, THESE ARE NANO FIBERS ABOUT 300 FOCUSED ON 400 NANO METER DIAMETER, FIBERS OF THE GLYCOLLIC ACID THAT ARE FUSED SO THAT THE CELLS WOULD FORM A MONOLAYER ON TOP AND THEN YET, THE SCAFFOLD WOULD HAVE SMALL HOLES TO ALLOW NUTRIENT FLOW AND CELLS CAN DEPOSIT IT ON ACM SO THEY CAN FALL ON BROOKS MEMBRANE AND THIS NICELY ILLUSTRATE THAT THESE CELLS FORM A NICE MEMBRANE STRUCTURE. SO THIS IS SCANNING EM OF RP PE CELLS GROWN ON A PLGS SCAFFOLD, IN THIS CASE IT’S PSEUDOCOLLORRED TO SHOW YOU THE SCAFFOLD IN BLUE AND SINCE YOU’RE LOOKING AT THE STRUCTURE FROM A SIDE LAYER, YOU SEE 3 CELLS, 1 CELL, ANOTHER 1 AND ANOTHER CELL BUT YOU SEE THIS IN GREEN APICKAL STRUCTURE OF ALL THE CELLS ON THE MONOLAYER AND THESE WERE A MEMBER OF MAIN BUSINESS END OF THE CELLS AND I HOPE YOU CAN APPRECIATE THAT PRETTY MUCH ALL THE CELLS HAVE EXTENSIVE STRUCTURES AND PRETTY HOMEIO GENIUS THROUGHOUT THE TISSUE. SUGGESTING THAT REALLY WE CAN MAKE POLARIZED CELLS USING IPS CELLS. AND THIS PATCH AND THIS IS 1 EXAMPLE OF THE CLEAR FUNCTION OF THE CELLS CAN PERFORM SO THESE ARE EPITHELIAL CELLS AND THEY HAVE TIGHT JUNCTIONS THAT FORM THE AUTOBLOOD-BRAIN BARRIER AND BECAUSE OF THE TYPE JUNCTIONS THEY HAVE WHAT IS ELECTRICAL RESISTANCE AND EPITHELIAL RESISTANCE SO WHAT I MEAN BY THAT IS IF I PUT AN 1 ON TOP, 1 ON BOTTOM, YOU PASS CURRENT SHOWS RESISTANCE TO THE FLOW OF CURRENT AND THAT RESISTANCE IS AN INDICATION OF MATURITY OF CELLS SO WE CAN MEASURE THAT RESISTERRANCE AS IN THIS CASE, SEVERAL PER CENTIMETER SHOWING THESE ARE MATURE. AND BECAUSE OFLET SAME TIME THE 2 MEMBRANES, APICKAL MEMBRANE AND ISOLATED MEMBRANES CAN BE MEASURED OF THE APICKAL MEMBRANES AGAIN A FEATURE OF POLARIZED CELLS AND THEN WE CAN PERFORM PROTOBATIONS LIKE CHANGING POTASSIUMOT SIDE OR ATP ON THE APICKAL SIDE, PHYSIOLOGICAL CHANGES IN THE CELLS RESPOND BY HYPER POLARIZING AND DEPOLARIZING AND SHOW THE RESPONSE IS SIMILAR TO WHAT RPE CELLS DO. SO TO DEMONSTRATE AND PROVE THESE CELLS ARE NOT ONLY STRUCTURALLY MATURE, THESE CELLS ALSO FUNCTIONALLY MATURE MADE FROM IPS CELLS ON A SCAFFOLD. SO THEN WE SPENT QUITE SOMETIME OPTIMIZING AND VALIDATING HOW WE MAKE THESE CELLS IN THE GMP GRADE SETTINGS BECAUSE EVEN AS WE KNOW WELL, IF YOU HAVE TO TRANSPLANT A PRODUCT IN PATIENTS IT HAS TO BE DONE UNDER THE CURRENT MANUFACTURING PRACTICES AS FDA REQUIRES. SO YOU TALK ABOUT THE PROCESS THIS LONG, YES IT IS LONG, IT TAKES ABOUT 6 MONTHS TO MAKE A START OF THE PATIENT ON DAY 0 AND TO HAVE A FINAL PRODUCT READY FOR THE PATIENT. BUT SO WE GO FROM IN THIS PROCESS IN THIS CASE, ISOLATING CD34 POSITIVE PATIENTS AND MAKING–REPROGRAMMING THEM INTO IPS CELLS THAT WE CAN EXPAND TO A SMALL–WHAT WE CALL A WORKING BANK AT PASSAGE 10 AND THIS ITSELF TAKES ROUGHLY 3 MONTHS AND ANOTHER 3 MONTHS FROM THIS IPS CELL BANK TO MAKING A MATURE RPE PRODUCT SIMILAR TO WHAT I JUST SHOWED YOU, WE GO TO THE PROGENITOR STAGE, SEATING CELLS ON THERE THAT WE CAN RELEASE FOR TRANSPLANTATION. NOW THE PROCESS IS, IF THE PROCESS WASN’T SO ALONG, WE HAVE TO HAVE QUALITY CONTROLS DURING THE MANUFACTURE MANUFACTURING PROCESS AT VARIOUS STEPS TO SEE IF IT’S GOING IN THE RIGHT DIRECTION, SO FOR INSTANCE WE CONNECT OVER IPS CELL BANK, QUALITY OF IPS CELLS, AND THEN WE–WHEN YOU START DIFFERENTIATING THEM INTO RPE, WE AGAIN MAKE SURE THAT THE DIFFERENTIATION IS GOING IN THE RIGHT DIRECTION AND FINALLY VALIDATE THE PRODUCT BEFORE WE TRANSPLANT. AND THEN AT THE SAME TIME WE HAVE PUT IN PLACE, SEVERAL STEPS WHERE WE CAN KRIST O PRESERVE OUR INTERMEDIATE PRODUCTS SO THAT IN CASE THE PROCESS FAILS AT ANY OF THE STEPS WE CAN GO BACK TO THE PATIENT STARTING CD34 POSITIVE CELLS, OR 1 OF OUR IPSC BANKS OR THE PROGENITOR IPE BANK AND NOT HAVE TO GO THROUGH THE WHOLE 6 MONTHS OF MANUFACTURING AGAIN. SO THIS BOX IS NOW BEING DONE AT THE DTM, DEPARTMENT OF TRANSPORMATION MEDICINE, PAM MENTIONED IN THE UNIT AND HAS BEEN TESTED ON A COUPLE OF DIFFERENT DONORS AND THE MD DONORS AND IT WORKS REPRODUCIBLE IN THE GMP CONDITIONS. SO, AGAIN, 1 OF THE CRITICAL PARAMETERS IS WHEN YOU’RE MAKING AHATOLOGIOUS CELL THERAPY, THAT YOU WANT TO HAVE A WAY OF VALIDATING WHAT IS A TRANSPLANT TABLE TISSUE BECAUSE THERE WILL BE SOME HETEROGENEITY IN TISSUES MADE FROM DIFFERENT PEOPLE. SO TO REALLY UNDERSTAND HOW MUCH IS THE HETEROGENEITY AND HOW CAN WE ACTUALLY SAY THAT THIS RP PATCH IS TRANSPLANTABLE, WHEREAS THIS 1 MAY NOT BE. SO TO DO THAT, WE MADE CELLS FROM 3 DIFFERENT AMD PATIENTS
PATIENTS AND IPS CELLS FROM 3 CLONES SO WE HAD 9 CLONES, ALL OF THOSE DIFFERENTIATED INTO AN RP PATCH AND A GMP GRADE CONDITIONS AND WE CHARACTERIZE THOSE PATCHES EXTENSIVELY FOR THOSE DIFFERENT ASSETS THAT HAVE LISTED HERE. INCLUDING SO THE GOLD WAS TO LOOK FOR IF THERE’S GENETIC DIFFERENCES BETWEEN DIFFERENT PEOPLE EFFECT THE QUALITY OF THE END PRODUCT. AND DETECTING DIFFERENCES THEY EFFECT THE END PRODUCT AND WHAT ARE THE DIFFERENT ASSETS WE CAN ACTUALLY USE TO CHARACTERIZE THIS END PRODUCT. SO THERE ARE 2 ASSETS THAT STANDUTE, 1 IS TRANSEPITHELIAL RESISTANCE MEASURING THE ELECTRICAL RESISTANCE OF THE MONOLAYER THAT REALLY FOR US IS A KEY PARAMETER TO TELL HOW MATURE THE CELLS ARE AND THE SECOND I WANT TO BRIEFLY MENTION IS QUANTITATIVE SHIFT METRICS, SO WHAT WE DO IN THIS CASE IS WE USE NEURAL NETWORKS SO WE HAVE DEVELOPING NEURAL NETWORK THAT CAN JUST LOOK AT THE IMAGE OF RPE CELLS AND TELL US HOW GOOD THE CELLS ARE, SO WHAT DEVELOP THAT NETWORK, WE ROAD THE CORD AND WE FED IT WITH IMAGES OF RPE CELLS AS THE CELLS WERE MATURING AND WE FED THIS FUNCTIONAL PARAMETER WITH THE TR-RESISTANCE AND THEN AFTER WE TRAIN THE NETWORK, WE ASK THE NETWORK TO PREDICT THE FUNCTION AND CAN YOU NOW LOOK AT A PICTURE OF CELLS AND TELL ARE THEY FUNCTIONAL OR NOT. AND THESE ARE REALLY 1 OF THE PRELIMINARY RESULTS WE HAVE BECAUSE THIS IS JUST USING 1 TRAINING SET SO FAR. BUT THE ASSAY, THE OUTCOME LOOKS REALLY INTERESTING. SO WHAT YOU HAVE ON THIS GRAPH ON THE Y-AXIS IS THE PREDICTED TR-TRANSREPLICATED RESISTANCE THAT THE NETWORK PREDICTED ANDOT X-AXIS WE HAVE THE MEASURED TR SO THE MEASURE FROM THE PATCH SO WHAT WE DID IS WE CLEAN THE NETWORK ON 3 DIFFERENT TYPES OF RPE CELLS, CELLS THAT WERE FULLY MATURED, THAT ARE PARTIALLY MATURED HERE IN GREAN AND RPE CELLS THAT ARE IN A FREE LIKE STATE. SO THE FIRST THING YOU NOTICE IS THAT THE NETWORK IS–THE PREDICTION OF NETWORK IS ALMOST AS GOOD AS OUR MEASUREMENT THAT WE PERFORMED EXPERIMENTALLY, THEY ARE LINEAR IN THE OUTCOME. THERE’S ONLY 1 MISTAKE, OTHER THAN THAT THEY ARE BOTH VERY SIMILAR IN THEIR OUTCOME. THE SECOND MORE IMPORTANT THING IS THAT THE NETWORK IS ABLE TO JUST BY LOOKING AT RPE IMAGE IT IS–IMAGES IT CAN TELL THE MATURE FROM THE PARTIAL CELLS AND THESE ARE BASED ON EXPERIMENT RESULTS [INDISCERNIBLE] BENEFIT THAT WE CALL THESE CELLS TRANSPLANTABLE. SO WE’RE VERY EXCITE WE THINK THAT THIS MACHINE LEARNING BASED APPROACH CAN BECAUSE IT’S SO EASY TO IMPLEMENT ON THE GMP GRADE SETTINGS CAN TELL US AS WE MOVE FORWARD, WITH THE LARGE SCALE FROM THE GOOD OR A BAD RPE PATCH TO BE TRANSPLANTED OR NOT NOW IN THE SECOND PART OF MY TALK, I WILL MOVE AWAY FROM MANUFACTURING TO NOW TESTING THESE CELLS IN PRECLINICAL ANIMAL MODELS. SO THE FIRST TEST IS, 1S WE TRANSPLANT THESE CELLS AND TRANSPLANT AN RPE PATCH DOES IT FORM TUMOR OR KERRA TOMA IN ANIMALS AND THE STUDIES ARE DONE IN IMMUNE O COMPROMISED ANIMALS BECAUSE YOU WANT THESE XENO SETTINGS AND YOU WANT THE CELLS TO SURVIVE LONG ENOUGH THAT THEY HAVE THE POSSIBILITY OF FORMING A TUMOR OR NOT. SO THE POSITIVE CONTROL, WHERE WE TRANSPLANTING PURE IPS CELLS, IN THE SUBRETINAL SPACE ALL FOR IMMUNE O COMPROMISED, THIS IS THE LENS OF THE RRED EYE AND YOU CAN IMAGE NOTICE IS A BIG MASSIVE TERATOMA THAT THESE CELLS FORM WHICH IS WHAT THIS IS SUPPOSED TO DO. IPS CELLS WHEN LEFT UNDER UNCONTROLLED CONDITIONS CAN LEAD TO THIS FORMATION, BUT IN CONTRAST TO PURE IPS CELLS WHEN WE INJECT PURE IPS DERIVED CELLS IN THE SAME RATS WE DO NOT SEE ANY TUMOR OR TERATOMA IN ANY OF THESE RATS, 2 EXAMPLES, MOST OF THE TIMES THESE CELLS JUST SIT IN THE SUBRETINAL SPACE UNDER THE RETINA AND THEY DON’T DO ANYTHING, AND BECAUSE HERE WE INJECT CELLS IN SUSPENSION THIS RED IS A MARKER FOR 1 OF THE IPSC PROTEIN FEMALE 17 AND HUMAN SPECIFIC SO WE CAN ONLY SEE HUMAN CELLS IN THIS CASE. AND HERE ON THIS SIDE, YOU WILL SEE MARKER FOR STEM 121 WHICH IS HUMAN SPECIFIC ANTIGEN. EVERY ONCE IN A WHILE WE SEE SUSPENSION CELLS INTEGRATE INTO THE RAT EYE, WE DON’T KNOW WHY. WE DON’T KNOW HOW. BUT A VERY SMALL NUMBER OF CELLS WILL INTEGRATE INTO THE RED EYE BUT THEY DON’T LEAD TO ANY TUMOR FORMATION. BUT INTERESTINGLY WHEN WE TRANSPLANT THE RPE PATCHOT BIODEGRADABLE SCAFFOLD, THE ENTIRE SCAFFOLD INTEGRATE INTO THE BACK OF EYE INTO THE BACK OF THE RAT EYE AS WE HAD HOPED FOR AND IN THIS CASE WHAT YOU SEE IN RED IS STEM 121 AGAIN, THE HUMAN SPECIFIC ANTIGEN, WE TRANSPLANTED HALF MILLION WITH THE DIAMETER PATCH IN THE RED EYE AND IF YOU MEASURE THIS SCALE BAR FROM 1 END TO THE OTHER END IT’S EXACTLY HALF MILLIMETER. THIS IS 10 WEEKS POST TRANSPLANTATION, SO THE SCAFFOLD HAS ALREADY COMPLETELY DEGRADED AND THE CELLS USE THE ECM TO INTEGRATE HUMAN CELLS INTEGRATED INTO THE RAT EYE AND YOU SEE THE RAT EYE, BECAUSE THEY’RE NOT POSITIVE FOR THIS STAIN AND RIGHT HERE, THE RAT EYE ENDS AND THE HUMAN RAT RPE-ENDS AND THE HUMAN BEGINNINGS. THIS IS REALLY WHATEE HOPED FOR THAT TRANSPLANTING THESE ON A PATCH WOULD INCREASE THE INTEGRATION, INCREASE SURVIVAL IN THE EYE AND THAT’S EXACTLY WHAT WE SAW. SO THIS IS–THIS IS REALLY EXCITING. NOW BOTH THE SUSPENSION RESULT AND THE PATCH RESULT LED US TO PERFORM A MASSIVE GLP GRADE PRECLINICAL STUDY AT A VENDOR IN WHICH WE HAVE TESTED A .2-MILLIMETER DIAMETER OR .5, OR 2-POINT-MILLIMETER AREA, OR .5-MILLIMETER SQUARE RPE PATCH AND A HUNDRED THOUSAND CELLS IN SUSPENSION FOR 4 DIFFERENT TIME POINTS, 2 WEEKS, 13 WEEKS, 26 WEEKS AND 9 MONTHS AND ANIMALS WERE SACRIFICED AND WE LOOKED FOR WHETHER THESE CELLS WERE TOXIC OR DISTRIBUTING TO OTHER ORGANS AND TUMOR GENIC AND I’M HAPPY TO REPORT THAT AFTER PERFORMING THIS ON 450 RATS, WE DIDN’T FIND ANY SIGNS OF ANY OF THIS TOXIC EVENTS FROM ANY OF THESE CELLS SO THESE CELLS ARE AT LEAST UNDER THIS IMMUNE O SUPPRESSED SETTINGS ARE COMPLETELY SAFE IN RATS. SO THEN WE WENT OUT TO TEST WHETHER THESE CELLS OF THIS PATCH HAS ANY EFFICACY AT LEAST IN AN ANIMAL MODEL SETTING AND SO IN THIS CASE, BECAUSE WE WANTED TO TEST THAT RETIRED PATCH, A 4 BY 2-MILLIMETER PATCH THAT WE WANT TO TRANSPLANT IN PATIENTS WE DECIDED TO USE PIGs AS AN ANIMAL MODEL AND THE OTHER ADVANTAGE WAS THAT USING PIGS EYE AND ANATOMY WAS SIMILAR TO HUMAN EYE AMAT ME SO WE COULD OPTIMIZE THE TOOL FOR DELIVER FOR TRANSPLANTATION AND A SURGICAL PROCEDURE, HOW DO WE DELIVER IT INTO THE RETINA. BUT THE PROBLEM WITH PIG SYSTEM THAT THERE’S NO GENETIC MODEL THAT LOOKS SIMILAR TO WHAT HAPPENS IN DRY AMD IN PATIENTS. SOPHISTICATEDY WOO CREATED AN INJURY MODEL IN PIGS. WE USED MICROPULSE LASER TO LACER A PLATE RPE CELLS IN THE BACK OF A BIG EYE AND THE REASON MICROPULSE LASER SPECIFICALLY KNOCKS OUT RPE CELLS IS THAT WE USE A SPECIFIC WAVE LENGTH OF LASER 532, 532 NANO METER, AND THAT IS ABSORBED BY THE PIGMENT OF RPE CELLS BECAUSE THESE CELLS ARE PIGMENTED AND THEY–THEY–OVER TIME THEY JUST HEAT UP AND THEN BURST AND DIE OFF. SO THAT–AS A RESULT, PHOTO RECEPTORS, SO IT’S A SIMILAR SITUATION IN THE AMD PATIENTS BUT WE USE TODAY BY INJURYING RPE CELLS. THE BOTTOM PANEL HERE SHOWS YOU THE RESULTS OF THE LASER MODEL AND BY INFRARED IMAGING AND THAT’S THE ADVANTAGE OF WORKING WITH THE EYES IS THAT WE CAN DO SO MUCH ANALYSIS BY IMAGING THE EYE AND KNOWING WE WILL SEE EVERYTHING IN THE BACK OF THE EYE. SO IN THIS CASE, THIS IS AN INFRARED IMAGE OFF THE BACK OF THE PIG EYE WHERE THE RPE HAS BEEN LASER DAMAGED AND AS CAN YOU SEE THE PIGMENTED CELLS ARE GONE IN 7 BY 4-MILLIMETER AREA, THE INFRARED LINE LIGHT SHINES THROUGH NORMALLY, THEY WILL ABSORB THE INFRARED LIGHT AND THEY WILL NOT SHINE THROUGH AND THEY CALL THIS AS I RENDERED DEFECT AND THESE ARE GONE IN THIS AREA. AND YOU CAN ALSO PROVE THAT BY JUST PERFORMING HISTOLOGY, WHERE THESE–YOU CAN SEE RPE MONOLAYER IS GONE, THEY HAVE CLUMPED UP RPE CELLS AND AS A RESULT THE PHOTO RECEPTORS ARE DYING BECAUSE THE RPE SUPPORT IS MISSING. THE IDEA IS THAT AS WE TRANSPLANT IN THIS AREA, HUMAN RPE PATCH AND ASK IT TO PROTECT OUTER LINEUPING RPE PATCH TO TRANSPLANT IN A TRANSPLANTED PIG TO PROTECT THE O-LINE PHOTO RECEPTORS. SO FIRST OF ALL TO DELIVER THE PATCH WE NEED TO DEVELOP A TOOL. A TOOL WE DEVELOPED HAS A 1 THAT FITS THE BACK OF THE EYE, IT TAKES THE TRANSPLANT, THIS IS THE SIZE OF OUR TRANSPLANT, IT’S LIKE A BULLET SHAPE AND IT GOES INSIDE THE TUBE IN 1 ORIENTATION, SO THE SURGEON WOULD NOT BE DELIVERING IT UPSIDE DOWN AND THEN IT IS CONNECTED THROUGH A FLUIDIC SYSTEM FOR SO THE SURGEON CAN CONTROL THE DELIVERY OF THE PATCH BACK AND FORTH EASILY JUST WITH 1 HAND HOLDING THE TOOL AND DELIVER THE PATCH IN THE BACK OF THE EYE. SO IN THE NEXT SLIDE I’M GOING TO SHOW YOU IS A SHORT VIDEO HOW WE TRANSPLANT THIS PATCH AND THE PIG EYE. SO AS CAN YOU SEE, THE EYE HAS BEEN PREPPED. THE SURGEON IS MAKING 1 BIGGER SO CAN YOU MAKE THE TRANSPLANT TOOL INSIDE. AND THE NEXT SHOT YOU WILL SEE THE PATCH–IT’S A TIP OF THE TUBE, YOU CAN SEE THE CELLS ON TOP. THE CUT IN THE RETINA, IT HAS BEEN LIFTED HERE AND THE SURGEON IS DELIVERING THE PATCH RIGHT UNDER THE RETINA AND THEN HE WILL FLATTEN THE RETINA ON TOP SO THE PATCH STAYS RIGHT IN THAT PLACE. AND AGAIN BY IMAGING WE CAN SEE THE PATCH ONCE IT’S TRAN PLANTED WE USE THIS TECHNIQUE BUT IT’S OPTICAL IMAGESERAL TISSUE AND MACROPHAGESSOGRAPHY WHERE WE CAN SEE FROM THE [INDISCERNIBLE] VIEW, THERE’S A PATCH, CAN YOU SEE THE BULLET SHAPE OF THE PATCH, CUT IN THE RETINA TO WHICH THE PATCH WAS DELIVERED AND NOW AS WE SEE THE SIDE VIEW RIGHT HERE, CAN YOU SEE THIS IS–I’VE OUTLINED THE PATCH HERE, CAN YOU SEE THIS WOULD BE THE PATCH RIGHT HERE, AND ACTUALLY, THE BRIGHT SIGNAL ON TOP OF THE PATCH SHOWS YOU THAT IT HAS CELLS ON TOP. SO NOW WE SEE IT SITTING AT THE RIGHT PLACE, RETINA ON TOP OF THE PATCH AND FLATTENED ON TOP OF THE PATCH AND THE NEXT STEP IS IT’S STRUCTURALLY THE RIGHT PLACE BUT IS IT FUNCTIONING? SO TO DO THAT, WE USE ANOTHER TECHNIQUE CALLED MULTIFOCAL, THE RETINA HAS NEURONS SO IT HAS ELECTRICAL POTENTIAL HERE, IT RESPONDS TO LIGHT SO IF YOU SHINE LIGHT TO THE RETINA, WE CAN MEASURE ELECTRICAL SIGNALS FROM THE RETINA BUT IN THIS CASE WE USE A TECHNIQUE WHERE WE SHINE MULTIPLE LIGHT PULSES, WE GET A SMALL AREA OF THE RETINA AND THIS CAN BE AS SMALL AS HALF A MILL MITER TO 1 MILLIMETER AREA OF THE RETINA AND THAT GIVES US ADVANTAGE FROM THE SAME EYE MEASURE SIGNAL FROM HOW THE AREA, WE GET THIS NICE AND BIG ROBUST RESPONSE AND THEN WHEN WE SHINE THE LIGHT ON TO A LACER INJURED PART WHICH WOULD BE SOMEWHERE RIGHT HERE WHERE WE LASERRED THE EYE BUT DID NOT DELIVER THE PATCH, WE GET ALMOST NO RESPONSE BECAUSE IT’S ALMOST DEAD IN THIS AREA, BUT IF WE SHINE THE LIGHT ON THIS SPOT WHERE WE’RE DELIVERED THE PATCH, WE LASERRED THE RETINAL LOCATION MA AND DELIVERED THE PATCH, WE GET A RESPONSE THAT IS IN BETWEEN. SUGGESTING THAT HUMAN PATCH IS ABLE TO RESCUE THE PIG RETINA FROM DIEING AND WE CAN MEASURE THAT ELECTRICALLY. WE CAN CHECK THAT BY HISTOLOGY. IF YOU DO IPT GREATER IMMUNE O STAINING FROM THE BACK OF THE EYE AND THIS IS NOT PIG EYE AFTER THE ANIMAL WAS EUTHANIZED WE CAN SEE HUMAN RP-PATCH JUST LIKE IN RATS IT’S INTEGRATED INTO THE BACK OF THE PIG EYE. IN THIS CASE, THE RED IS A STAINED FOR HUMAN–OFF A MARKER THAT STAINED BOTH THE PIG RPE, AND THE HUMAN RPE. AND THE GREEN IS A STAINED AT ONLY STAINS HUMAN CELLS, STEM 121. SO CAN YOU SEE THIS WOULD BE–WOULD BE RPE, AND WOULD END úRIGHT HERE AND THE PIG RPE STARTS RIGHT HERE AND YOU CAN SEE HERE THE HUMAN RPE ALMOST STARTS INTERACTING WITH THE PIG RPE, WE CAN ALSO SHOW THAT RETINA IS BEING RESCUED ON TOP OF THE PATCH. THIS IS JUST ANOTHER HISTOLOGY FROM THE SAME EYE, AREA THAT WAS LASERRED BUT NO TRANSPLANT WAS DELIVERED, COMPARED TO AREA THAT WAS LASERRED, THE TRANSPLANT HIGHLIGHT INDEED YELLOW HERE, NOTICE THE THICKNESS OF NUCLEI, HERE STAINED BLUE, AND THESE ARE THE ONL IS AUTONUCLEAR LAYER OF PHOTO RECEPTORS, AND NOTICE THE THICKNESS OF PHOTO RECEPTORS ON THE–ON ABOVE THE TRANSPLANT WAS AWAY FROM THE TRANSPLANT, IT’S ALMOST DOUBLE THE THICKNESS, SUGGESTING THE HUMAN TRANSPLANT HAS RESCUED THEM FROM DYING AND WE HAVE TO AT LEAST RETAIN AN ENTIRE PHOTO SEPTORSOR NUCLEAR LAYER IN THIS CASE. SO THIS WORK THAT I SHOWED YOU HAS NOW LED TO A PRECLINICAL STUDY IN PIGS, WHERE WE HAVE TRANSPLANTED NOW, OUR 30 PIGS THAT WHERE WE TESTED JUST A SCAFFOLD, SURGERY, ALL DIFFERENT COMPONENTS OF OUR PATCH TO DREMON STRAIGHT THAT THE–DEMONSTRATE THAT THE PATCH IS STRAIGHT AND EFFICACIOUS SO WE ARE ABOUT TO FINISH THIS STUDY. SO JUST TO SUMMARIZE ALL OF THIS. WHAT I’VE SHOWN YOU IS THAT WE HAVE DEVELOPED A CLEAN GRADE PROTOCOL TO MAKE IPS RPE PATCH AND WE DEMONSTRATED THE QUALITY OF THIS PATCH USING PRECLONAL PATIENTS BY PERFORMING THE VALIDATION STUDIES AND THE RAT SAFETY STUDIES, I DIDN’T HAVE TIME TO GO INTO A RAT EFFICACY STUDIES WE PERFORMED BUT I SHOWED YOU THE DATA FROM THE PIGS EFFICACY STUDIES SO ALTOGETHER THIS WORK IS BEING PUT TOGETHER FOR IND S SUBMISSION IN THE NEXT COUPLE OF MOONTHS, AND WITH THE TRIAL, PERHAPS LATER THIS YEAR WHAT I WANT TO SHOW NOW IN THE OVER THE NEXT STEM CELLS IS HOW WE ARE TAKING AND WHERE WE’RE TAKING THIS WHOLE TECHNOLOGY HOWEVER WE TALKED ABOUT HOW DOES DISEASE HAPPEN, DISEASE HAPPENS. WE THINK THAT RPE CELLS DIE OFF AND AS A RESULTS CAP ILLEGALSARYS BECOME CLEAN AND PHOTO RECEPTORS DIE ON TOP OF THE RPE, SO WHAT WE WANT TO DO IS WE WANT TO RECAPITULATE THE ENTIRE DISEASE IN A DISH, NOT THE DISH BUT THE PHOTO RECEPTORS AND SO TO DO THAT, I’VE ALREADY SHOWN YOU WE CAN MAKE RPE PATCH, BUT NOW WE WANT TO ADD THE CAP ILLEGALSARIES TO THAT PATCH. SO FOR THAT, FIRST THING WE NEED TO DO IS MAKE THE ENDOTHELIAL CELLS OF THE CAP ILLEGALSARYS SO WE USE A PROCESS AGAIN, VERY SIMILAR TO WHAT WE USE FOR MAKING RPE CELLS MATURE ENDOTHELIAL CELLS FROM IPS CELLS USING A PUBLISHED PROTOCOL FROM CHRISTINE [INDISCERNIBLE]’S LAB, WE HAVE DONE IT WITH AT LEAST 8 IPS SELL LINES AND WE CAN GET CD31 POSITIVE CELLS, NOW WE TAKE THESE CELLS ANDEE 3D BIOPRINT THEM TO MAKE THIS NETWORK AND SO THE WAY THIS WORKS IS IT IS A MAJOR OFF A PRINTER IT HAS 2 NEEDLES AND THESE NEEDLES CAN MOVE IN A VERY PRECISE X, Y, Z ORIENTATION AND CAN YOU SEE HOW THESE MOVE, AND 1 EXAMPLE HERE WE’RE MAKING A ZIGZAG PATTERN WITH NEEDLES AND WHAT THEY ARE EXTRUDING IS WHAT IS CALLED A BIOINC. SO BIOINC. CONTAINS HYDRO GEL AND CELLS YOU WANT TO PRINT. IN THIS CASE IT’S ENDOTHELIAL CELLS AND OTHER SUPPORT CELLS PARASITES AND 1 PLAOF THES AND WE WILL MAKE A ZIGZAG PATTERN SO THE ADVANTAGE IS THAT THIS KEEPS THE CELLS TOGETHER FOR A WHILE THAT THEY WOULD START TO INTERACT SUCH THAT THEY COULD MAKE THE VASCULAR NETWORK AND THE EMPTY SPACES BETWEEN THE RAT CELLS IS THE SMALLER FORMS WILL FORM. SO HOW WE DO IT AGAIN AND WE START WITH OUR KAF OLD, WE FLIP IT ON 1 SIDE, BIOINK ON 1 SIDE OF THE SCAFFOLD, WE FLIP IT UPSIDE DOWN, WE SEE RPE CELLS, WE ARE ALREADY SHOWING YOU AND THEN WE LET THIS TISSUE MATURE FOR A FEW WEEKS AND IT TAKES ABOUT 4-5 WEEKS BUT THE SCAFFOLDING IN THIS CASE COMPLETELY DEGRADES AND THE 2 CELL TYPES ON BOTH SIDES RPE CELLS AND 1 SIDE AND 1 BLAST AND THE TOOLS ON THE OTHER SIDE MAKE THE EXTRA CELLULAR MATRIX TO REPLACE THE SCAFFOLD, WITH WHAT LOOKS LIKE REALLY A MEMBRANE OF THE BACK OF THE EYE BROOKS MEMORY RESPONSE BREAN AND THE TISSUE STARTS TO LOOK LIKE THIS. THIS IS A BEO PRINTED—BIOPRIPTED TISSUE WHERE ON TOP YOU SEE THE RPE CELLS IN THE SPACE WHERE YOU DON’T HAVE CELLS WITH THE ECM HAS FILLED IN, THIS IS THE BROOKS HERE AND THEN HERE IN THE BOTTOM, ARE THE CAP ILLEGALSARYINGS LABELED WITH THE ENDOTHELIAL CELLS IN GREEN AND PARASITES IN RED AND YOU CAN SEE THE CAP ILLEGALSARYS THAT GO ACROSS THE TISSUE, AND THE SECTION TO THE BOTTOM PART. THIS IS WHAT YOU WOULD SEE AS THE CAP ILLEGALSARYS WITH THE DIAMETERS AND THIS IS GOING ACROSS THE ENTIRE TISSUE IF YOU NOTICE THE SCALE BAR, THIS CAP ILLEGALSARY IS SIMILAR TO 20-50-MICRON, REALLY WHAT IS THE DIAMETER OF CAP ILLEGALSARYS NATURALLY AND THE OTHER THING IS TD LABEL HERE IS IN GREEN AND ENDOTHELIAL CELLS IN RED IS PARASITES AS CAN YOU SEE, NICELY WRAP THEMSELVES AROUND THE ENDOTHELIAL CAP ILLEGALSARYS TO SUPPORT THEM FUNCTIONALLY AND STRUCTUREALLY. SO WHEN WE PUT THE RPE ON THE OTHER SIDE, THE RPE CELLS NICELY MATURE AND YOU CAN SEE IN GREEN IS APICKAL MARKER FOR RPE CELLS WHICH IS PRESENT IN THE APICKAL STRUCTURES CALLED ASERINE, AND IN THE RED IS THE JUNCTIONS AND ON THE BOTTOM SIDE IS AGAIN MARKED HERE IN RED ON THE VASCULAR NETWORK THAT HAS NICE LOW NOW COVERED THE ENTIRE TISSUE AND IN THAT CASE, THE NETWORK IS STAINED FOR A PROTEIN CALLED PLAB. IT’S A STRUCTURAL PROTEIN FOR WHAT IT CALLED FENNISTERATION, IT IS VERY IMPORTANT WHAT IS FENNISTERATION, IT IS ACTUALLY IN CAP ILLEGALSARYS 7200 NANO METER POST NEGLIGENCE THE MEMBRANE OF ENDOTHELIAL CELLS THAT ALLOW EASY FLOW OF MACROMOLECULES. REMEMBER I SAID RPE CELLS MAINTAIN THE OUTER BLOOD BRAIN BARRIER SO THE NUCLEOTIDES TRI EPTS HAVE TO FLOW FROM CAP ILLEGALSARYS IN THROUGH THE RPE AND INTO THE PHOTO RECEPTORS AND TO ALLOW THE FLOW, THE CAP AILARYS OF HAVE FENNISTERATIONESTRATION IN THEM AND THEY ALLOW IT TO PASS THROUGH MUCH BETTER. SO WE ASK WHEN WE MAKE THIS CAP ILLEGALSARY NETWORK WITH THE RPE DO THEY BECOME [INDISCERNIBLE]. AND THE ANSWER IS YES. ON THIS SIDE, YOU SEE WHAT IS PUBLISHED FENNISTERATIONITRATION IMAGES FROM [INDISCERNIBLE]. PUBLISHED LAST YEAR AND CAN YOU SEE THESE ARROWS MARK THIS KIND OF TINY LITTLE POST IN THE MEMBRANE OF ENDOTHELIAL CELLS, COMPARE THAT TO WHAT WE SEE IN THE IPS DERIVED COLOARIDE AND WE HAV A VERY SIMILAR NETWORK IN OUR CAP ILLEGALSARYS SO WE THINK THESE CAP ILLEGALSARYS JUST LIKE WHAT HAPPENS IN THE CHOROIDIAL BECOME FENNISTERATION STRAIGHTED AND THESE ARE DEPENDENT ON THE RPE CELLS ON THE OTHER SIDE. SO HERE I’M SHOWING 2 EMPLOY ANDS, HERE IS A TISSUE THAT WAS PRINT INDEED THE PRESENCE OF RPE CELLS AND THE TISSUE THAT WAS PRINTED WITHOUT THE RPE CELLS SO CAN YOU NOTICE RPE CELLS ARE PRESENT HERE BUT THEY’RE MISSING HERE, NOW WHAT YOU WILL SEE IS, ON TOP PANELS, NOTICE A THICKNESS OF THESE SESESLES AND HOW MUCH THEY ARE PROLIFERATING IN THE PRESENCE OF RPE, AND THEY’RE MUCH THICKER AND THEY HAVE A MUCH HIGHER FENNISTERATIONISTRATION MARKER AND THIS IS A SMALL PIECE ABOUT A HUNDRED MICROMETER BUT IF YOU LOOK ACROSS THE ENTIRE TISSUE, AND WE CAN BRING TISSUES THAT ARE 6 BY 6 MILL MITER, YOU CAN SEE IN BETWEEN OUR PRINTED AREA, THIS IS A REALLY MINIMAL ANGIO GENESIS THAT HAPPENS IN THE ABSENCE OF RPE. BUT IN THE PRESENCE OF RPE, THE VESSELS PROLIFERATE AND AS A PROLIFERATE, THEY BECOME FENNISTERATION STRAIGHTED, THEY START EXPRESSING THE FENNISTERATIONISTRATION MARKER SO WE THINK IN THIS 3D TISSUE, RPE CELLS REALLY HELP VESSEL PROLIFERATION JUST LIKE HOW THEY SHOULD DO IN THE EYE AND ALSO HOW THESE VESSELS BECOME FENNISTERATION STRAIGHTED. SO RIGHT NOW WE’RE USING THIS TISSUE FOR TODAYING HOW REALLY AMD PATHOGENESIS HAPPENS IN A DISH AT LEAST AND WE HOPE THAT ACTUALLY WE CAN TURN THIS INTO A TISSUE THERAPY 1 DAY, WHETHER WE TRANSPLANT BOTH RPE, OR CHLORIDE STAGES OF MACK LA DEGENERATION, SO WITH THAT, I WANT TO THANK THE TEAM THAT REALLY WORKED TOGETHER FOR THE CELL THERAPY PROJECT BECAUSE AS YOU CAN IMAGINE, THE CELL TYPE OF PROG ELECTRIC LIGHTING, IT TAKES A VILLAGE TO DO SOMETHING LIKE THAT, YOU NEED PEOPLE WITH THESE GMP EXPETTER EASE AND THESE ARE ALL PEOPLE WITH EXPERTISE THAT CAME TOGETHER AND MAKE IT HAPPEN. AND BECAUSE OF THAT, THEY LAST YEAR GOT AN AWARD FROM DR. COLLINS FOR THE EFFORTS TO DEVELOP CELL THERAPYOT NIH CAMPUS SO WITH THAT I AM HAPPY TO TAKE ANY QUESTIONS. [ APPLAUSE ]>>DO WE HAVE QUESTIONS OR COMMENTS? IS THERE A WAY YOU CAN TELL WHETHER THE ANIMAL HAS RESTORED VISION? WE CAN ASK THEM TO READ. KD–SALLY LAWOF [LAUGHTER] IN PICKS IT’S VERY DIFFICULT TO DO, CAN YOU DO SUCH AN EXPERIMENT IN RATTINGS WHERE WE PUT THEM ON A PLATFORM AND WE ROTATE A CYLINDER AROUND THEM THAT HAS PLAQUE AND WHITE STRIPES AND JUST IF THE RAT CAN SEE, TRACKS THE STRIPES BUT IF THE RAT CAN’T SEE IT WILL JUST RANDOMLY LOOK ON BOTH SIDES SO WE PERFORMED THAT EXPERIMENT USING OUR CELLS AND AFTER TRANSPLANTATION WE CAN SEE THAT CLEARLY THOSE RATS START SO THESE ARE DISTROPIC RATS WHERE THE RPE IS DYSFUNCTIONAL AND THE RETINA START TO DEGENERATE SO IF THEY REGENERATE THEY ARE RANDOMLY LOOKING ON BOTH SIDES BUT ONCE THEY TRANSPLANT OR PATCH, THEY START TO TRACK THE STRIPES BETTER SOPHISTICATEDY THAT’S THE ONLY WAY WE CAN SEE THAT WE CAN KNOW THAT THEY SEE. PIG SYSTEM MUCH HARDER TO DO ANY OF SUCH EXPERIMENT SO WE RELY ON THOSE ELECTRICAL MEASUREMENTS OF THE EYE.>>[INDISCERNIBLE].>>IN PATIENTS THERE ARE MANY WAYS TO DO THIS. PATIENTS CAN YOU ASK THEM TO READ.>>[INDISCERNIBLE]>>SAY AGAIN?>>WHAT ABOUT AN ANESTHETIZED PIG?>>THESE EXPERIMENTS AND ELECTRICAL MEASUREMENTS THAT I SHOWED YOU ARE DONE ON ANESTHETIZED PIGS BUT THAT’S WHAT YOU CAN DO LOOKING AT THE BACK OF THE EYE.>>I’M NOT SURE I MISSED THIS OR NOT BUT WHEN YOU MADE THAT INCISION AND PUT THE PATCH IN, DID YOU CLOSE THE INCISION AFTERWARDS?>>SO THE RETINA INCISION IS NOT REALLY CLOSED. IT CLOSES BY ITSELF SO THERE’S A LITTLE SURGICAL GLUE AND WHEN THEY COME TOGETHER, YOU FLATTEN THE RETINA ON TOP AND OVER THE NEXT FEW DAYS IT HEALS BY ITSELF, YOU HAVE TO SUTURE THE EYE WALL INCISION BECAUSE THAT’S A LARGE INCISION OTHERWISE IT STARTS BLEEDING. YEAH.>>THANK YOU.>>IT’S REALLY BEAUTIFUL WORK SO THE QUESTION IS WHETHER OR NOT YOU’VE BEEN WORKING ON TRYING TO SPEED UP THE DIFFERENTIATION? AND WHAT IS THE TIME FRAME OF THE RPE DIFFERENTIATION IN THE DISH COMPARED TO DURING DEVELOPMENT? I MEAN IS THAT JUST THE WAY IT’S GOING TO BE? OR DO YOU THINK YOU CAN MAKE IT FASTER?>>SO THIS WAS THE FASTEST WE COULD GET IT AND THIS IS THE–TRADITIONALLY IT TAKES ABOUT 20 WEEKS SO WE SHORTEN IT TO ABOUT HALF. AND THIS HALF IS RPE, IF YOU THINK ABOUT HUMAN DEVELOPMENT BY DAY 21, ACTUALLY RPE CELLS ALREADY START TO BE THERE, AND THEN THEY MATURE OVER THE NEXT FEW WEEKS SO THE TIMELINE IS PRETTY SIMILAR TO THE YOU HAD HAN TIMELINE, WE MAY HAVE FASCINATE A BIT USING OUR APPROACH BUT WE COULD NOT FASTER IT ANYMORE WITHOUT COMPROMISING THE QUALITY OF CELLS. THE TRADITIONAL METHODS THAT WERE USED IN SOME OF THE OTHER STUDIES WHICH ARE CALLED SPONTANEOUS DIFFERENTIATION, THAT TAKES 20-25 WEEKS THAT’S A SLOWER PROCESS. SLOWER THAN WHAT HAPPENS IN HUMAN DEVELOPMENT.>>WHAT CAUSES THOSE CAP ILLEGALSARYS TO BECOME–CAPEULARYS–ILLEGALSARYS TO BE FENNISTERATION STRAIGHTED?>>THAT’S THE MILLION DOLLAR QUESTION, PREVIOUSLY WE THOUGHT IT WAS VEG F BYOU NO 1 KNOWS SO NOW WE TRY TOKING IF OUT WHAT THEY’RE SECRETING TO MAKE IT FENNISTERATIONISTRATION, SO WE’RE TRYING TO ISOLATE THE CELLS AND SEE WHAT THEY’RE SECRETING ON THE BASAL SIDE.>>I CAN TELL YOU IN HEPATOCYTES THE FENNISTERATION ENDOTHELIAM IS CONTRACTUAL. THE RING WHICH IS AN ACTIN RING IS CAPABLE OF CONTRACTION AND MODULATING THE SIZE OF THE POOR AS LITTLE AS AROUND 20 NANO METERS, TO 150 OR SO.>>THAT’S INTERESTING.>>WELL, ANY OTHER QUESTIONS? OKAY. WELL, THANK YOU BOTH. , THANK YOU VERY MUCH.>>THANK YOU BOTH VERY, VERY MUCH.>>[ APPLAUSE ] CLEAR I WILL POINT OUT THAT NEXT WEEK WE HAVE 2 INSTITUTES

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