>>GOOD AFTERNOON EVERYONE I'M DELIGHTED TO JOIN YOU AND INTRODUCE TODAY'S SPEAKER FOR THE WEDNESDAY AFTERNOON LECTURE SERIES ALBEIT DONE VIRTUALLY. THIS IS LEONARD ZON. MY FRIEND WHO I'VE KNOWN FOR QUITE A FEW DECADES DOING REMARKABLE WORK ON HEMATOPOIETIC AND CANCER AND THE TITLE OF HIS PRESENTATION, BLOOD STEM CELL CLONALITY AND THE NICHE AND WHAT NICHE HE'S TALKING ABOUT WE'LL FIND OUT. SO LET ME SAY A WORD ABOUT LEN. HE GOT HIS UNDERGRADUATE DEGREE AT A COLLEGE NOT TOO MANY KNOW ABOUT AND GOT HIM OFF TO A GOOD START AND HIS M.D. AT THOMAS JEFFERSON AND A FELLOW IN ONCOLOGY AT DANA FARBER AND AS RESEARCH FELLOW IN CHILDREN'S HOSPITAL. BY 1991 HE BECAME ASSISTANT PROFESSOR OF PEDIATRICS. NOT LONG AFTER THAT ASSISTANT INVESTIGATOR OF HHMI AND OVER THE COURSE OF COMING YEARS FULL PROFESSOR OF PEDIATRICS NOW GROUSBECK PROFESSOR AND CONTINUES TO BE SUPPORTED BY THE HOWARD HUGHES INSTITUTE AND ASSOCIATE MEMBER OF THE BROAD INSTITUTE AND GOT A PHYSICIAN SIN SCIENTIST AWARD AND IS A MEMBER OF THE NATIONAL ACADEMY OF MEDICINE AND NATIONAL ACADEMY OF ARTS AND SCIENCES AND RECEIVE THE PRIZE FOR THE AMERICAN SOCIETY OF HEMATOLOGY AND THE DONALD METCALF AWARD FOR EXPERIMENTAL HEMATOLOGY AND HE GOT THE ALFRED KNUDSEN AWARD FROM THE NCI IN 2016. HIS WORK OVER THESE YEARS HAS BEEN GROUNDBREAKING IN MANY WAYS BUT I THINK PERHAPS MANY PEOPLE KNOW HIM PERHAPS PROMINENTLY AS USING THE ZEBRAFISH AS A REMARKABLE SYSTEM FOR UNDERSTANDING A LOT ABOUT STEM CELL BIOLOGY IN CANCER UNDERSTANDING VERTEBRATE DEVELOPMENT AND TREATMENTS FOR PATIENTS WITH MELANOMA AND BLOOD DISORDERS. OVER THE YEARS, PROGRESSIVELY MOVING THIS MORE AND MORE TO SOPHISTICATED UNDERSTAND OF THE NATURE OF THOSE MALIGNANCIES THAT ARISE IN THE HEMATOPOIETIC SYSTEM BRINGS US TO TODAY WHERE I THINK HE'LL TELL US QUITE A BIT ABOUT HOW BLOOD CELL CLONALALITY OCCURS AND WHAT WE MAY START THINKING ABOUT DOING IT IN NOVEL WAYS THAT WOULD NOT HAVE BEEN APPARENT WITHOUT THIS KIND OF RESEARCH. LEN AND I HAD BEEN FRIENDS AS MENTIONED AND IT'S WONDERFUL TO HAVE HIM COME TO NIH TO GIVE THE WEDNESDAY AFTERNOON LECTURE. I APPRECIATE THE PEOPLE WHO LINKED UP TO HEAR THIS. I APOLOGIZE I WON'T BE AT THE END FOR THE Q&A BECAUSE OF A CONGRESSIONAL MEETING AND I'LL BE HERE UNTIL I HAVE TO JUMP INTO THAT ROLE AND LET ME WELCOME PROFESSOR LEONARD ZON TEACHING US ABOUT BLOOD CELL CLONALITY AND THE NICHE. >> THIS ISN'T A MEDICAL MEETING BUT MANY ON THE CALL PROBABLY KNOW THOSE FOLKS. THIS WAS OUR BAND AND THE DOC ROCK BAND AND WE PLAYED NINE YEARS AGO AT THE CELEBRATION OF SCIENCE WHERE WE WERE TRYING TO SAVE THE NIH FROM SEQUESTRATION IF YOU REMEMBER, FRANCES. >> DO I, YES. >> I TOLD FRANCIS NOW THAT HE'S TAKING A DIFFERENT POSITION WE NEED TO TAKE THE BAND ON THE ROAD. HOPEFULLY THAT WORKS. >> I'M THERE. >> I'VE STARTED A NUMBER OF BIOTECH COMPANIES AND WANTED TO PUT THEM IN AS DISCLOSURES THEY DON'T RELATE TO MY TALK TODAY. I FOCUSSED MY ENTIRE LIFE ON STUDYING THE BLOOD SYSTEM AND PARTICULARLY UNDERSTANDING HOW STEM CELLS SELF-RENEW. SO WHAT HAPPENS HERE IS YOU HAVE A STEM CELL AND IT MY GRIGRATES INTO THE MARROW AND CAN SELF-RENEW OR DIFFERENTIATE INTO THE LINEAGES OF STEM CELLS AND WE KNOW THIS PROCESS WORKS INCREDIBLY WELL BECAUSE WE CAN DO A BONE MARROW TRANSPLANT ON A PATIENT AND RES CURE THEIR SYSTEM FOR A LONG TIME AND YOU ONLY NEED ONE TRANSPLANT FOR A LIFE TIME. THIS PROCESS IS FACILITATED BY THE NICHE. THIS INCLUDES THE VASCULAR ENDOTHELIAL CELLS AND THE STROMAL CELLS THE FIBROBLASTS TO ALLOWING THE STEM CELL TO SELF-RENEW. THOUGH WE DO BONE MARROW TRANSPLANT EVERY DAY STILL 25% OF PATIENTS DIE FROM THEIR TRANSPLANTS AND UNDERSTANDING THIS NICHE WOULD BE HELP UNTIL THERAPEUTICALLY IN THE FUTURE AND WE USE THE ZEBRAFISH TO STUDY THIS PROCESS AND I'D LIKE TO SHOW YOU HOW BLOOD STEM CELLS ARE BORN. THIS IS A 36 HOUR EMBRYO AND WE'RE GOING TO DIVE IN THE STEM CELL AORTA. WHEN WE DIVE IN WE SEE AN ENDOTHELIAL CELL THAT IS GOING TO MICK A BLOOD STEM CELL AND FORMS A HAMMOCK AND ROUNDS UP AND THAT'S A STEM CELL AND HIS OCCURS AND IT WILL GO OFF IN CIRCULATION AND COLONIZE THE SITES OF HEMATOPOIESIS AND THE STEM CELL STOPS ON THE ENDOTHELIAL CELL AND TRANS MIGRATES THAT THE NICHE ENVIRONMENT, DOUBLES AND THEN SOME OF THESE STEM CELLS CAN THEN GO OFF IN CIRCULATION AND COLONIZE THE MARROW. NOW, THE MARROW IN THE ZEBRAFISH IS IN ITS KIDNEY. IN HUMANS IT'S INSIDE THE BONES AND MAKES THE KIDNEY A HEMATOPOIETIC ORGAN IN THE FISH AND THIS STARTS THE IMMUNE SYSTEM OF THE FISH FROM THE THYMUS AND LETS THE ANIMAL HAVE BLOOD DURING THE LIFE TIME AND THE POWER OF THE ZEBRAFISH IS THE GREAT ABILITY TO WATCH ALL THESE THINGS LIVE. SEVERAL YEARS AGO WE PUBLISHED A FISH WITH GREEN STEM CELLS. WE USED AN ENHANCER A GENE SPECIFICALLY EXPRESSING IN THE STEM CELLS BORN IN THE AORTA. THESE THEN GO OFF IN CIRCULATION AND THEN LAND IN THE HEMATOPOIETIC TISSUE. I ALWAYS WONDERED WHAT IT LOOKED LIKE WHEN A STEM CELL LAND AND WE'RE OPAQUE BUT IN THE FISH IF YOU FOLLOW THE CIRCLE HERE, YOU'LL SEE THE STEM CELL TRANS MIGRATES OUTSIDE THE BLOOD VESSEL SYSTEM AND THEN WE SAW SOMETHING STRANGE, THE BLOOD VESSELS WRAP THEMSELVES BACK AROUND THE STEM CELL IN A PROCESS WE CALL ENDOTHELIAL CUDDLING, THERE'S A DIVISION AND TURN AND ONE STEM CELL LEAVES AND THIS IS STEREOTYPIC BEHAVIOR. WE'RE ABLE TO SEND THE ANIMAL FOR CORRELATIVE ELECTRON MICROSCOPY SHOWING THE VIEW AND WE GOT SURPRISES. HERE'S THE STEM PROGENITOR CELL SURROUNDED BY FIVE ENDOTHELIAL CELLS FORMING A POCKET AND THE STEM CELL TRAVELLING AROUND LANDS AND ATTACHES TO A STROMAL CELL AND IT'S ON THE SURFACE OF THE STROMAL CELL THE STEM CELL WILL DIVIDE. THIS NICHE BECAME INTERESTING TO US AND WANTED TO UNDERSTAND AT A BETTER LEVEL. AN ASSISTANT PROFESSOR AT BOSTON UNIVERSITY DID THIS SET OF EXPERIMENTS. HE DID SOMETHING CALLED RNA TOMOGRAPHY. HE TAKES THIS REGION WHICH IS GOING TO BE THE BLOOD NICHE AND SECTIONED ACROSS THE NICHE. THEN EVERY SECTION WENT INTO A TUBE FOR RNA SEQ. THIS ALLOWS A SPECIAL EVALUATION AND YOU'RE ABLE TO SEE GENES LIKE THAT ARE EPIDERMAL AGAINST ON THE OUTSIDE OF THE TAIL TOP AND BOTTOM AND FOR US WHAT WAS EXCITING THERE WERE GENES EXPRESSED BY THE NICHE NOT STEM CELLS. THIS MADE YOU WONDER WHAT'S SO SPECIAL ABOUT THESE GENES AND TISSUES IN THE NICHE? IT TURNED OUT 29 OF THE GENES WERE EXPRESSED IN THE BLOOD VESSELS. WE KNEW THIS BECAUSE WHEN WE HAD DONE OUR OWN RNA SEQ EXPERTS A NUMBER SHOWED UP IN THE KDRL RECEPTORS AND SORT THE CELLS AND SHOW THEY WERE THERE AND THEN WE GOT INTERESTING INFORMING. THIS IS AGENE EXPRESSED BY ALL ENDOTHELIAL CELLS THEY'RE EXAMPLES OF THE 29 WE SAW IN THE BLOOD VESSELS. THESE ARE ONLY EXPRESSED YOU CAN LOOK AT THE FISH HERE. THERE'S NO EXPRESSION EXCEPT IN THE NICHE WHERE THE BLOOD CELLS ARE AND GPR182. THESE TURN OUT TO BE THE SPECIALIZED PART OF THE MARROW. WE'RE ABLE TO FIND A MOLECULAR SIGNATURE BASED ON THE ANALYSIS. I WONDERED HOW DO YOU GET THIS KIND OF EXPRESSION PATTERN. WE TOOK THE PROMOTER OF TWO OF THE GENES AND WE DROVE GREEN FLUORESCENT PROTEIN IN THE RED MARKER AND SEE HOW BEAUTIFUL THIS IS. IN THIS PARTICULAR TRANSGENIC OF 1.3 KILO BASES OME -- ONLY LIGHTS UP AND THERE'S SOMETHING THAT TELLS THE CELLS TO ACQUIRE THE FATE AND WE DECIDE TO GO AFTER THE CODE. FIRST WE DID ATAX SEQ AND YOU CAN SEE THE NICHE SPECIFIC ENDOTHELIAL CELLS HAVE AN OPEN CHROMATIN BUT NOWHERE ELSE ON ANY OF THE OTHER ENDOTHELIAL CELLS. WE TOOK THIS LITTLE PEAK AND COUPLED IT TO GREEN FLUORESCENT PROTEIN AND INJECTED FISH AND THE ONLY PLACE WE SAW THE GREEN WAS IN THE NICHE ENDOTHELIAL CELLS. THIS IS A SPECIFIC AFFECT AN STARTED STUDYING OTHER PICS AND THEY DRIVE IN THE TISSUE. 12 OUT OF 15 CLONED ONES DROVE THE SINUSOIDS AND AGAIN SUGGESTING A CODE RESPONSIBLE FOR MAKING THE SPECIALIZED VENOUS CELLS. WE FOUND THE SITES THAT IS PARTICULARLY SITE. WE DECIDED TO LOOK AT WHAT TRANSCRIPTION FACTORS WERE SPECIFICALLY ADDRESSED AND WE SEE SEVEN TRANSCRIPTION FACTORS, THREE RELATED TRANSCRIPTION FACTORS AND FAMILY MEMBERS AND TWO NUCLEAR HORMONE RECEPTOR FAMILY MEMBERS. WE DECIDED TO SEE IF THEY HAD THE ABILITY TO REPROGRAM SINUSOID AND WE DROVE THE GENES BY OVER EXPRESSION AND LOOKED AT MRC 1 OR E SELECTIN AND COMPARED TO CONTROLS, IF YOU LOOK AT THE SEVEN FACTOR INJECTION WE START GETTING EXTRA VEINS ESSENTIALLY AND IF YOU HOOK AT THE VEIN SPECIFIC REPORTER YOU CAN SE AN EXTRA VESSEL NORMALLY NOT THIS BUT IT IS A VEINOUS SINUSOID AND IF YOU EXPRESS THE MEMBERS OF THE FAMILY YOU COULD GET A HIGH RATE OF THE EXTRA VESSELS. SO ELLIOTT CAME TO ME ONE DAY AND SAID I'VE DONE THIS EXPERIMENT AND I THINK IT'S THE BEST DAY I'VE DONE AS A POTT DOCK. HE SAID I'VE DONE THIS IN THE BACKGROUND OF RED STEM CELLS. THIS IS ECTOPIC NICHE FORMED AND I HOPE YOU CAN SEE THE RED STEM CELLS LEFT THEIR NORMAL NICHE AND WENT TO THE NEW NICHE. I THOUGHT THAT WAS SUPER EXCITED. I SAID DO THEY CUDDLE BECAUSE I FEEL CUDDLING WAS A FUNCTION THAT WAS IMPORTANT AND HE SAID YES AND I SAID DO THEY DIVIDE AND HE SAID YEP, THEY DIVIDE. THIS IS THE ANIMAL AND NORMAL NICHE AND WE'VE BEEN LUCKY ENOUGH TO CAPTURE A LITTLE ECTOPIC NICHE. IT'S ATTRACTED THE STEM CELLS THERE AND IF YOU FOCUS ON THE TOP WHAT YOU'LL SEE IS THOSE STEM CELLS GO ON TO DIVIDE. THEY'RE EVEN RELEASED. THIS IS A FULLY FUNCTIONAL NICHE REPROGRAMMED AND I THINK THAT'S VERY EXCITING. WE WANTED TO SEE IF THIS WAS TRUE IN THE ADULTHOOD AND ENDED UP DOING SINGLE CELL ANALYSIS ON THE ADULT NICHE AND MANY GENES WERE PRESENT THERE AND THEN WE COLLABORATED WITH A LAB WHO HAD TAKEN ENDOTHELIAL CELLS FROM MANY DIFFERENT ORGANS THROUGH DEVELOPMENT AND WHAT WAS INTERESTING IS OURING IS TURE GENES WAS VERY HIGH IN THE FETAL LIVER IN THE MOUSE AND KIND OF WENT DOWN IN THE LIVER AS THE ANIMALS WERE BORN AND MANY KNOW THE LIVER IS NOT A HEMATOPOIETIC ORGAN IN THE MOUSE LATER IN DEVELOPMENT AND WHERE WE SEE IT COME BACK IN THE BONE MARROW OF THE ADULT. IT SEEMS THE VEINOUS SINUSOIDS ARE IN THE SITES HEMATOPOIETIC. IN THIS PARTICULAR STORY WERE ABLE TO FIND RNA TOMOGRAPHY WERE FIND VEIN WERE SEN -- SINUSOIDAL SPECIFIC AND WERE ABLE TO REPROGRAM THE CELLS TO IN VIVO IN A NICHE AND THIS IS IMPORTANT FOR SOME PATIENTS ONCE THE MARROW FIBROSIS AND THEY DON'T DO WELL IF WE CAN TAKE IPS CELLS AND REPROGRAM THEM INTO THESE VEINOUS SINUSOIDS MAYBE WE CAN INJECT THEM INTO THE ARM OF A PATIENT WITH MBS AND MAKE THE PATIENTS HAVE A NEW NICHE WHERE THE STEM CELLS WOULD GO AND IT WOULD RESCUE THE. WE'VE BEEN PLAYING WITH IPS DERIVED VEINS AND SEEING IF WE CAN PROGRAM THEM AND HAVE INTERESTING PRELIMINARY DATA HOPEFULLY THE NEXT TIME I DO A WALS LECTURE I'LL BE BACK TO TALK ABOUT THOSE STUDIES. NOW I'D LIKE TO THINK ABOUT CLONALITY. MOST OF US HAVE POLY CLONAL BLOOD. THAT MEANS WHEN YOU HAVE STEM CELLS BORN IN AORTA AND HAD A CERTAIN NUMBER OF STEM CELLS AND AS PEOPLE AGE INVESTIGATORS HAVE FOUND THERE'S MUTATIONS IN THE PERIPHERAL BLOOD OF AGED INDIVIDUALS AND THE MUTATIONS OFTEN ARE IN EPIGENETIC REGULATOR AGAINST. THIS IS A THEME GOING ON FOR MANY TISSUES AND IF YOU THINK OF EYELIDS. THE WRINKLES HAVE MUTANT B53 CLONES AND THINK OF THE ESOPHAGUS OF OLD FOLKS IT HAS CLONES OF NOTCH MUTATIONS. THE INTESTINE THERE'S CLONES OF CELLS THAT ARE PRECANCEROUS BUT THEY EXIST AND IN THESE PATIENTS WHO HAVE CLONAL HEMATOPOIESIS POTENTIAL AND THE BLOOD OF THE PATIENTS 20% TO 30% OF THE BLOOD COMES FROM A SINGLE STEM CELL AND THIS POSITIONS YOU TO MAYBE LIEUKEMIA AND WOULD LIKE TO UNDERSTAND THIS BETTER AND WE NEEDED A METHOD TO CLONALLY TRACK THE STEM CELLS AND WE COLORED EACH STEM CELL A DIFFERENT COLOR. WHAT WE DO IS WE HAVE THIS RAINBOW TYPE OF FISH AND HAVE RED, BLUE AND GREEN PLASMIDED AND IF YOU NOW COME IN WITH A BLOOD SPECIFIC CREE2 YOU START REARRANGING THE PLASMID AND WE HAVE AN INDUCIBLE FACTION BY GIVING TAMOXIFEN AND CAN GROW THEM UP TO ADULTHOOD AND ALL THE EM BRIE -- EMBRYONIC COLOR IS GONE AND WHAT YOU'LL SEE IS THE NUMBER PRODUCED. AND THIS IS AN EMBRYO AND HAS MULTI-COLORED BLOOD. IF YOU FOCUS ON THIS PARTICULAR CELL HERE, THAT'S IN AN EXTRA VASCULAR LOCATION. IT'S PROBABLY A BLUE-GREEN STEM CELL. WHEN THAT BLUE-GREEN STEM CELL GROWS UP TO ADULTHOOD IT WILL MAKE BLUE-GREEN BLOOD AND GROW IS THREE TO FOUR MONTHS AND IT'S AN ADULT AND DO A PERIPHERAL BLOOD SMEAR. AS FRANCIS CAN TELL YOU I'M A HEMATOLOGIST AND I THINK THAT'S THE PRETTIEST BLOOD SMEAR I'VE EVER SEEN. THE NUMBER OF COLORS IF YOU DO IT AT 25 HOURS OF DEVELOPMENT IN A FISH LABELLING, THAT'S BEFORE THE BIRTH OF BLOOD STEM CELLS THERE'S 21 COLORS HERE. AND SO THAT WOULD MEAN THERE'S 21 CLONES OF STEM CELLS THAT START THE BLOOD PROGRAM. YOU NEVER HAVE MORE CELLS BEING BORN. THEY'RE ALWAYS AMPLIFYING FROM THOSE POMPS. -- POPULATIONS. THAT'S 24 HOURS THERE'S 21 CLONES OF STEM CELLS. NOW WE CAN START TO THINK OF MODELLING THE HEMATOPOIESIS DISORDER AND IF YOU GO TO THE HUMAN YOU NORMALLY HAVE POLY CLONAL HE MAT OWE POIESIS AND WE CAN TARGET GENES AND LOOK FOR CLONAL DOMINANCE IN OUR ADULT FISH THERE BE MODELLING THE DISEASE. HOW DO YOU GO ABOUT DOING THIS? FIRST, WHAT IS THE MECHANISM SO FAR FOR CLONAL HEMATOPOIESIS. YOU GET A MUTATION AND THE MUTANT STEM CELL IS PRETTY NORMAL THOUGH IT'S CARRYING A MUTATION AND EPIGENETIC REGULATOR AND THEY'LL GO ON TO MAKE MUTANT MONOCYTES AND MACROPHAGES AND THEY'RE PUMPING OUT INFLAMMATORY CITYTOKINES AND THIS LEADS TO THE MARROW BEING INFLAMED AND THERE'S CLONAL DOMINANCE IF AN UNKNOWN MECHANISM AND SECOND MUTATIONS CAN HAPPEN SO YOU END UP PROGRESSING TO LEUKEMIA OR MBS. AND THIS IS TISSUE EDITING WITH INDUCIBLE STEM CELL TAGGING VIA RECOMBINATION. SHE INJECTED 20 GUIDE RNAs FOR MOST THE MUTATION THAT CAUSE HUMAN CLONAL HEMATOPOIESIS AND INJECTED IT WITH KAS 9 AND NOT PROTEINS AND WE WERE ABLE TO MEASURE ALLELE AND IT'S NOT AS EFFICIENT BY THE PROTEIN AND ALLOWS 50% OF THE STEM CELL TO BE WILD TYPE AND 50% MUTANT TO COMPETE IN VIVO TWO STEM CELL POPULATIONS AGAINST EACH OTHER WITHOUT A TRANSPLANT AND THIS IS A VERY BIG VANCE FOR THE FIELD. WE CAN THEN DO THE COLOR TRICKS AND MARK THE CLONES AND LATER GROW THE FISH UP TO ADULTHOOD AND LEAD THEM TO SEE WHAT CLONALITY IS OVER TIME AND ULTIMATELY SACRIFICE THE FISH TO GET THEIR MARROW. WHAT'S NICE ABOUT THIS IS BECAUSE THE DOMINANT CLONE WILL BE IN A CERTAIN COLOR WE CAN FACT SORT THAT COLOR AND THIS ALLOWS US TO LOOK AT WHAT PATHWAYS ARE FOUND IN DOMINANT VERSUS NON-DOMINANT CLONES WHICH I THINK CAN HAVE AN IMPACT THERAPEUTICALLY. THIS IS A LIST OF GENES AND YOU MAY HAVE HEARD OF THE ONES THAT MOST COMMONLY CAUSE CHIP BUT A FEW OTHERS AND THERE WERE SOME CONTROL GENES AND PSEUDO GENES PUT IN TO MAKE SURE EVERYTHING WAS BEHAVING. OVER HERE IS A CONTROLLED FISH. YOU SEE 20 COLORS OF CELLS AND NOTHING DOMINANT HERE BUT LOCK AT THIS FISH. IT'S GOT A MASSIVELY DOMINANT CLONE AND 60% OF THE BLOOD IS COMING LITERALLY FROM ONE STEM CELL. SO WHEN WE LOOK AT THIS, IF YOU LOOK AT CLUSTER SIZE IN THIS POOL YOU CAN SEE THERE'S A LOT OF FISH WITH CLONAL DOMINANCE AN THERE'S ANOTHER WAY OF LOOKING AT THIS TO SEE HOW DIVERSE THE POPULATIONS ARE AND SEE AN INCREASE COEFFICIENT WITH THE MUTANTS. CLEARLY WE'VE BEEN ABLE TO MODEL CHIP. NOW IN OUR SYSTEM THE GENE THAT POPPED OUT MOST IS THIS EPIGENETIC REGULATOR AND FREQUENTLY IN HEMATOPOIESIS AND WHEN WE KNOCK IT OUT WE DON'T GET CLONAL DOMINANCE BUT IT'S ONLY XON 12 THAT GET DOMINANCE AN THERE'S SOMETHING ABOUT BEING A MORPHIC ALLELE AND WE GET OTHER MUTATIONS AND MANY MUTATIONS CAN HAPPEN IN THE FISH AND CAN READ OUT. SO NOW WE GO AHEAD AND SORT THE DOMINANT CLONES AND HERE'S A FISH WITH 48% COMING FROM ONE COLOR. AGAIN, IT'S STRONG IN THE DOMINANT COMPARED TO THE NOM-DOMINANT CLUSTERS. THE THIS ONE IN OUR SYSTEM IS EXTREMELY STRONG. SO WE WENT AHEAD AND DID SINGLE CELL RNA SEQ TO SEE WHAT WOULD HAPPEN AND THIS IS WHAT YOU GET FROM A MARROW OF A ZEBRAFISH AND IT LOOKS SIMILAR TO WHAT YOU'D SEE IN THE SINGLE CELL SEQ OF A HUMAN MARROW AND ALL THE POPULATIONS ARE THIS AND WE'RE ABLE TO LOOK AT THE MUTANT POOL AND CONTROL POOL AND SEE IF THERE'S ANYTHING INTERESTING IN TERM OF GENE EXPRESSION. THE FIRST THING THAT CAME OUT WHICH REALLY WAS BLAZING IS THE NEUTROPHILS AND MACROPHAGES THE DOMINANT CLONE ARE EXPRESSING IL1 AND TFNL AND THAT IS MODELLING THE DISEASE AND THESE MAY MARROW S ARE INFLAMED AND MODULATORS KIND OF ANTI-INFLAMMATORY FACTORS WERE PRESENT IN THE STEM CELLS. THAT. THIS MADE US THINK THE STEM CELLS ARE PERHAPS RESISTANT TO THE INFLAMMATION AND MAYBE WHY IT OCCURS IN AGED INDIVIDUALS. AND THIS MAKES INFLAMMATORY CYTOKINES AND SUPPRESS THE NORMAL CELLS BUT THOSE STEM CELLS HAVE ANTI-INFLAMMATORY FACTORS AND WILL MAKE THEM RESISTA RESISTANT. THIS ALSO OFFERS THE OPPORTUNITY IF YOU CAN DISRUPT THE ANTI-INFLAMMATORY FACTORS THIS COULD TREAT THE DISEASE. CERTAINLY IT'S ALSO POSSIBLE TO BLOCK THE INFLAMMATORY CYTOKINES BUT THERE'S A LOT OF INFLAMMATORY CYTOKINES BEING MADE AND YOU NEED A COUPLE THERAPIES. PERHAPS GETTING AT THE SEED OF THE BAD STEM CELLS MAY BE USEFUL. SERENE DID THIS EXPERIMENT WHICH I THOUGHT WAS EXCITING. WE TARGETED ASXL1 AND YOU WOULD EXPECT WITH INFLAMMATORY CYTOKINES THIS IT WOULD LEAD TO CLONAL DOMINANCE AND WE ALSO TARGETED ASXL1 AND BECAUSE IT'S A MOSAIC MUTO GENESIS PROTOCOL AND THEY WOULD CONTINUE TO EXPAND BUT IT WOULD ALLOW US TO TEST OUR MODEL TO SEE IF INHIBITING WOULD BLOCK STEM CELL EXPANSION OF THE MUTANT CLONES. IN FISH YOU GET A LOT OF FISH AND YOU CAN DO A LOT OF STATISTIC AND IT WORK REALLY WELL. YOU CAN SEE A DOMINANT CLONE IN THE FISH AND HERE'S ONE THAT HAS ASXL1 KNOCKED OUT AND SEE IT'S A SMALL CLONE. THAT'S THE RESULT WHEN WE LOCK AT ASXH1 MUTATION YOU CAN SEE THE CLUSTER SIZE AND WHEN YOU ARE A HOMOZYGOUS MUTANT BUT ASXL1 MUTATION YOU SEE A REDUCTION IN CLONALITY. THIS WOULD SUGGEST INHIBITING NR4A1 BLOCKS THE DOMINANCE. IF YOU LOOK AT THE THE GUIDE RNA HAS NO EFFECT ON CLONE OR CLUSTER SIZE. THIS WOULD INDICATE NO EFFECT ON THE NORMAL HEMATOPOIESIS BUT IT WOULD BE THE PERFECT THING FOR THERAPY. THIS TWISTER SYSTEM ALLOWS THE STUDY OF BLOWN ZONE CLONALITY IN THE NATIVE NICHE AND THIS MOSAIC MUTOGENESIS ALLOWS YOU TO DO THIS IN VIVO AND TRANSPLANT ARTIFICIAL FOR MEASURING THESE BUT AS THE A TREMENDOUS BODY OF WORK FOR TRANSPLANT. WE SEE THIS IN INFLAMMATORY SIGNATURE IN THE MUTANT MYELOID CELLS WE THINK THEY CREATE A SITUATION FOR CLONAL FITNESS BUT IF YOU HAVE A BIALL EELIC NR4A1 IT WOULD BE DELETERIOUS AN ID LOOKS LIKE WE MAY BE ABLE TO SUPPRESS CLONAL DOMINANCE AND WOULD END UP BEING SOME TYPE OF THERAPY BUT IF YOU'RE GOING TO TREAT A PATIENT WITH HEMATOPOIESIS YOU HAVE TO MAKE SURE IT'S INCREDIBLY SAFE. NOW I'D LIKE TO TELL YOU ANOTHER NEW STORY ON THE INFLUENCE OF MACROPHAGES ON CLONALITY. THIS IS THE WORK OF A GRADUATE STUDENT. IN THIS STORY, WE WERE IMAGING MACROPHAGE AND STEM CELLS AND SAW BIZARRE BEHAVIORS. HERE'S A MACROPHAGE AND YOU CAN SEE IT INTIMATELY INTERACTING WITH THE STEM CELL. IT'S ALSO REMOVING SOME CITO PLASMIC MATERIAL AND THIS WAS A SURPRISE. I'LL MAY THAT MOVIE AGAIN. WHAT IS THIS MACROPHAGE DOING? WE THOUGHT IT WAS PRETTY FREQUENT MACROPHAGES WERE INTERACTING WITH THE STEM CELL IN THIS FETAL LIVER EQUIVALENT AREA. SO SAM DECIDED TO DO SOME MORE INTENSE ANALYSIS. WHAT HE DID WAS TO LOOK AND REMEMBER THE STEM CELLS WERE JUST BORN AND HAVE TRAVELED TO THIS FETAL LIVER EQUIVALENT. WHAT HE SAW FIRST WAS ABOUT 20% OF ALL STEM CELLS THAT HAD JUST BEEN BORN ARE DOOMED. THEY'RE COMPLETELY EATEN BY MACROPHAGE. THAT WAS A SURPRISE AND MADE US THINK THERE WAS A QUALITY ASSURANCE MACROPHAGE FOR STEM CELLS AND SAW OTHER BEHAVIOR WHICH WAS REMARKABLE AND THIS IS A GROOMING. YOU'LL SEE A STEM CELL ABOUT TO BE GROOMED AND THE MACROPHAGE COMES AND TAKES A CHUNK OF THE STEM CELL AND THEN THAT STEM CELL ALWAYS GOES ON TO DIVIDE. THIS IS ALMOST AS IF AS THE A LICENSING EVENT. YOU NEED THE MACROPHAGE INTERACTION TO GET THE CELL DIVISION. THIS WAS VERY REMARKABLE AND MAKES YOU THINK ABOUT A NUMBER OF QUESTIONS. WHAT'S THE FREQUENCY OF THESE EVENTS? I'LL TELL YOU IT'S PRETTY FREQUENT. WHAT DETERMINES THE INTERACTION BETWEEN THE MACROPHAGE AND STEM CELLS. IS THIS ANYTHING SPECIAL ABOUT THESE PARTICULAR MACROPHAGES VERSUS OTHER MACROPHAGES. AND WHAT IS IT THAT'S DETERMINING WHETHER YOU'RE DOOMED OR YOU'RE GROOMED? AND THEN ALSO THIS WOULD BE A QUALITY ASSURANCE MECHANISM FOR STEM CELLS I DON'T THINK HAS BEEN DESCRIBED BEFORE AND THE QUESTION IS WHAT'S BEING MONITORED AND WHAT'S WRONG WITH THE STEM CELLS THAT GOT COMPLETELY EATEN AND WHY DOES THIS EVEN HAPPEN IN THE FIRST PLACE? SO ONE OF THE THINGS WE LOOKED AT EARLY ON WAS IS IT SPECIFIC FOR THE STEM CELLS AND IT SEEMS TO BE. WE CAN SEE THE INTERACTIONS WITH THE POSITIVE CELLS AN DON'T SEE THE MACROPHAGES INTERACTING A LOT WITH THE RED BLOOD CELLS AND WERE ABLE TO ASSEMBLE THIS IN IN VITRO AND SAW MORE INTERACT WITH THE STEM CELLS THAN THE RED BLOOD CELLS. THE PERCENTAGE OF CELLS GROOMED AND DOOMED ARE ROUGHLY EQUAL. SO DEVELOPMENTALLY THE DIFFERENCE BETWEEN DAY TWO AND DAY THREE ARE BASICALLY THE SAME. SO AGAIN THIS PROCESS IS VERY FREQUENT UNTIL THE COLONIZATION OF THE LIVER EQUIVALENT. WE WANTED TO SEE IF THERE WAS AN IMPACT ON STEM CELL CLONES WE GIVE THIS AND SEE WHAT HAPPENS TO THE NUMBER OF STEM CELL CLONES WHEN THE FISH BECOMES AN ADULT. AND HERE WE GOT A PRETTY INTERESTING RESULT. IN THE SIBLING CONTROLS YOU HAVE THOSE 20 COLORS I WAS TALKING ABOUT. IN THE CLODRONATE AND IT'S REMARKABLE AT SELECTING CLONALITY FOR THE ADULT AND IT'S DERMED BY THE EARLY PERIOD OF DEVELOPMENT. WE CAN GIVE THE CLODRONATE AN THIS IS CLOSE TO WHEN THE STEM CELLS WERE BORN AND WE DID IT LATER AND STILL GET A REDUCTION IN THE NUMBER OF CLONES AND ALSO WE HAVE A MUTANT AND AMORPHENT FISH AND HAVE A MIGRATION PROBLEM OF STEM CELLS BUT THEY'RE CLONALITY IS NORMAL. SAM WAS TRYING TO FIGURE THIS OUT AND WHAT HE SAID WAS IT MUST BE POSSIBLE TO FAX PURIFY THE MACROPHAGES THAT INTERACTED BECAUSE THEY STILL HAVE A CHUNK OF RED IN THEM AND SURE ENOUGH YOU CAN FAX THE MACROPHAGES AND SEE WHAT'S DIFFERENCE THAN ARE INTERACTING WITH THE STEM CELLS. NOW, THE OTHER THING HE CAN DO BESIDES SINGLE CELL RNA SEQ ARE PROTEOMICS AND THERE'S A CORE AT HARVARD THAT CAN DO PROTEOMICS AND THEY LOOKED AT WHAT HAD EATEN THE STEM CELLS. AND WHAT HE FOUND WAS THIS SET OF PROTEINS THE RETICULIN A AND B AND THE REASON IT WAS KNOWN ABOUT IS IT'S AN E.R. PROTEN. IT HAS A RETENTION SEQUENCE KDEL. BUT IT CAN GET CLEAVED. THE KDL CAN GET CLEAVED AND CAN MOVE TO THE SURFACE AND DECORATE FLIKKO PROTEINS ON THE SURFACE AND -- GLYCOPROTEINS AND IT'S BEEN SHOWN IN CANCER CELLS THAT CAL RETTICULIN CAN INTERACT WITH LRP1, THE RECEPTOR, THE LIPID RECEPTOR AND WE CAP ACTUALLY SEE THIS INTERACTION WILL CAUSE SOME TYPE OF EATING SIGNAL. FIRST OF ALL, IF WE LOOK AT THE STEM CELL WE THINK IT HAS CAL RITICULIN AND THEY'RE EXPRESS THE RECEPTOR AND IT'S ALSO KNOWN THERE'S A BRIDGE MOLECULE BETWEEN CAL RETICULIN AND IT'S A COMPLIMENT C1Q AND OUR MACRO FATHER -- MACROPHAGES ARE CLOSE TO M2 LIKE ARE POSITIVE. CAN WE LOOK AT THE MACROPHAGE INTERACTIONS BY KNOCKING DOWN AND YOU SEE LESS STEM CELL MACROPHAGE INTERACTIONS PARTICULARLY WITH 3A AND 3B. WE CAN ALSO OVER EXPRESS A CAL R3A WITH AN E.R. RETENTION SIGNAL. IF YOU LOOK AT THE INTER ACTS -- INTERACTIONS OVER EXPRESSING AND OVEREXPRESS ANY OF THE MUTANTS AS THE KDL MUTANTS WILL LEAD TO MORE INTERACTIONS. THEN WE DID THE EXPERIMENT OF KNOCKING THEM DOWN AND EVALUATING STEM CELL CLONALITY AND THERE'S AN AN IMPACT OF REDUCED CLONALITY. KNOW, ONE OF -- NOW, IS THERE A SPECIFIC TIME PERIOD WHERE THIS INTERACTION IS ACTUALLY HAPPENING? WHAT WE SAW ACTUALLY IS USING OUR FUCCI FISH YOU CAN SEE THE MACROPHAGES ARE INTERACTING WITH THE STEM CELLS AS THEY'RE IN THE G2M PHASE. AND THAT STEM CELL WILL ULTIMATE HI DIVIDE. WE ALSO CAN QUANTIFY THIS AND YOU CAN SEE AGAIN IF THEY'VE INTERACTED WOMAN A MACROPHAGE THEY'RE MORE LIKELY TO BE FUCCI POSITIVE AND DIVIDE AND THE TIME IS WITHIN THE FIRST 45 MINUTES AFTER THE INTERACTION. NOW, WE WANTED TO KNOW MORE ABOUT THE CELL DIVISION AND INTERACT INTERACTED AND INJECTED AND DID E.D.U. LABELLING. WE HOPED WE WOULD SEE REDUCED CELL PROLIFERATION AS A RESULT OF INACTIVATING THE STEM CELL INTERACTIONS AND THAT'S WHAT WE SAW. IF YOU INACTIVATE YOU CAN SEE A REDUCTION IN THE NUMBER OF CYCLING. MOST THE FIELD FELT CLONALITY MAY BE DETERMINED AT THE TIME OF BIRTH OF THE STEM CELLS. IN OTHER WORDS, THERE'S A NUMBER OF COLORED STEM CELLS AND THAT'S WHAT YOU HAVE FOR YOUR LIFE TIME AND THERE'S PROBABLY A DETERMINATE OF AN ADULT CLONALITY BETWEEN WHEN THEY'RE BORN AND WHEN THE COLONIZE OF THE FETAL LIVER WOULD HAPPEN. ONE THING WE WOULD SAY IS YOU START OFF WITH THE COLORING WE'RE LABELLING AT THE TIME OF STEM CELL BERTH AND NORMALLY YOU GET SOME CLONES THAT DOUBLE AND SOME THAT DON'T BUT EVERYTHING IS PRETTY SYMMETRIC. YOU END UP WITH ALL THE COLORS IN THE ADULTHOOD. IN THE CASE WHERE YOU HAVE FEWER DIVISIONS, PERHAPS WHAT HAPPENS IS YOU SEND UP WITH SELECTIVE AMPLIFICATION OF CERTAIN COLORS AND WHEN THEY GO TO COLONIZE THEY HAVE A HIGHER CHANCE OF CONTRIBUTING TO ADULTHOOD AND END UP WITH A CLONAL DOMINANCE WHICH MEANS LESS COLORS IN THE PERIPHERAL BLOOD FOR OUR EXPERIMENTS. THIS IS A CLONAL COMPETITION MODEL AND WE'RE SEEING IT IN VIVO WITHOUT HAVING TO DO THE TRANSPLANT. WE WANTED TO KNOW WHERE THE CALRETICULIN HAS TO BE AND IN AN EMBRYO CARING A CELL SPECIFIC MARKER WE CAN ACTIVATE CALRETICK CALRETICK -- CALRETICULIN AND THE MACROPHAGES AND THE STEM CELL S ALLOW IT TO BE SEEN WHERE IT'S REQUIRED. I LOVE THESE EXPERIMENTS. THIS IS CRAZY YOU CAN MAKE THESE PARABIOTIC ANIMAL THAT HAVE JOINED CIRCULATION. EVERY SO OFTEN YOU GET THESE FUSED AT THE HEAD AND YOU GET THE MACROPHAGES AND STEM CELLS IN DIFFERENT COLORS. THE RESULT IS VERY INTERESTING. THAT IS THAT THAT CALRETICULIN IS REQUIRED ON THE STEM CELL. THIS IS THE CONTROL INJECTED ANIMAL AND M PEG INJECTED ANIMAL AND IF YOU INACTIVATED CALRETICULIN YOU GET A REDUCTION IF IT'S ON THE RUNT POSITIVE CELLS BUT FOR THE TRUE ON THE MACROPHAGES. YOU NEED THE CALRETICULIN ON THE STEM CELLS. THE STEM CELLS WILL BE COLONIZING THE FETAL LIVER EQUIV EQUIVALENT AND THE STEM CELLS GET READY TO INTERACT WITH MACROPHAGES. THE MACROPHAGES HAVE TWO BEHAVIORS. WHERE THE SURFACE OF THE STEM CELL HAS A LOT OF CALRETICULIN IT THE EAT THE WHOLE STEM CELL AND GOOD-BYE TO THE STEM CELL. AND ANOTHER BEHAVIOR IS THE GROOMING BEHAVIOR AND IN THIS PARTICULAR SITUATION WHAT HAPPENS IS THE MACROPHAGE COMES IN AND TAKES A CHUNK OF THE STEM CELL IN THIS SITUATION THE STEM CELL WILL GOOD ON AND DIVIDE AND IN A VERY CHARACTERISTIC MANNER IT GOES IN THE CUDDLED POCKETS AND ATTACHES TO THE STROMAL CELL AND IT GOES PERPENDICULAR TO THE STROMAL CELL. THIS RAISES INTERESTING QUESTIONS THAT WE'RE STILL WORKING ON. IT ALLOWS A MODEL WHERE THE MACROPHAGE ARE LOOKING FOR STEM CELLS WITH CALRETICULIN ON THEM AND THINK IT LEADS TO THE PARTIALLY EATING BEHAVIOR AND ULTIMATELY TO THE DIVISION OF THE STEM CELLS. IN THIS SITUATION WHERE YOU HAVE A LOT OF CALRETICULIN WE THINK NEEDS TO BE EATEN. I SHOULD POINT OUT CALRETICULIN IS KNOWN TO BE IN HUMANS AND IT BINDS TO THE RECEPTOR AND STIMULATES THE PROLIFERATION OF THE STEM CELLS. PERHAPS THIS INTERACTION IS ALSO CAUSED BY SOME CYTOKINE INTERACTION WITH CALRETICULIN AND UNDERSTANDING HOW THE DIVISION HAPPENS IS SOMETHING WE'RE STILL WORKING ON. THE OTHER ASPECT IS WHY IS THIS HAPPENING AND WHAT'S IN THE STEM CELLS TO CAUSE THIS EFFECT AND AND WE THINK THERE'S THINGS THE CELLS HAVE GONE THROUGH, STRESS, TRAFFICKING OR MITOSIS AND MAB AS THE A SENSING OF DAMAGE COULD BE DNA OR REACTIVE OXIDANT IN THE STEM CELLS BEING MONITORED AND STEM CELLS THAT HAVE TOO MUCH OF THAT ARE TAKEN OUT BECAUSE IT COULD LEAD TO PROBLEMS LATER AND MAYBE A DEFICIT IN RESPONDING TO INFLAMMATION, FOR INSTANCE. THESE ARE THINGS WE'RE TRYING TO UNDERSTAND ABOUT PRESENTING ON THE SURFACE AND ALLOWING THE QUALITY ASSURANCE MECHANISM TO WORK. THAT WOULD BE THE END OF MY TALK AND A WANT TO THANK THE PEOPLE WHO DID THE WORK. WE HAVE A GREAT BAR CODING THEME TEAM WITH INVESTIGATORS AND WITH THAT I'M HAPPY TO TAKE QUESTIONS.HEME TEAM WITH INVESTIGATORS AND WITH THAT I'M HAPPY TO TAKE QUESTIONS.EME TEAM WITH INVESTIGATORS AND WITH THAT I'M HAPPY TO TAKE QUESTIONS.ME TEAM WITH INVESTIGATORS AND WITH THAT I'M HAPPY TO TAKE QUESTIONS.E TEAM WITH INVESTIGATORS AND WITH THAT I'M HAPPY TO TAKE QUESTIONS. TEAM WITH INVESTIGATORS AND WITH THAT I'M HAPPY TO TAKE QUESTIONS. >> THANK YOU FOR A GREAT TALK AND AS YOU CAN SEE FRANCIS HAS TRANSFORMED INTO ME AND THE DIRECTOR OF THE NIDDK AND I TOO HAVE BEEN A LONG TIME FRIEND OF LEN'S. AND PLEASED TO HAVE HIM BE OUR WALS LECTURE. IF YOU WANT IT SEND A QUESTION IN DO SO BY CLICKING ON THE BUTTON BELOW YOUR VIDEOCAST WINDOW THAT SAYS SEND LIVE FEEDBACK. IT WILL RELAY THAT TO DR. ZON. YOU CAN SUBMIT A QUESTION AT ANY TIME. RON'S QUESTION IS THE CORRELATIVE IMAGING SHOWS THE STEM CELL ADHERENT TO STROMAL CELLS AND DIVIDING ON THE STROMAL CELLS. ARE THESE STROMAL CELLS UBIQUITOUSLY PRESENT AND ARE THEY NOT IN THE END NON ESSENTIAL. >> I WOULD SAY THE STROMAL CELLS ARE ESSENTIAL AND WHAT WE SEE NOW WHICH IS EXCITING IS THERE ARE IN THE NEW NICHES FORMED, THERE ARE FIBROBLASTS, PROBABLY STROME CELLS EXPRESSING CXCL12 AND HAVE AN ATTRACTIVE MECHANISM TO COME IN THE NICHE. I THINK IT'S MER -- MORE ACTIVE AND WE SEE THE SAME BEHAVIOR IN THE HEMATOPOIETIC TISSUE AND WE'RE TRYING TO UNDERSTAND THAT WHETHER THERE'S INTEGRINS OR A FORCE THERE AND WE THINK IT'S ACTIVE AND I BET THE STROMAL CELLS ARE REQUIRED IN SOME WAY AS MANY OF YOU KNOW, KIT LIGAND IS MADE BY THOSE STROMAL CELLS AND OTHER MECHANISMS MAY BE AT PLAY. >> ONE OTHER POINT I THINK DR. COLLINS MADE BUT TO REPEAT IT, FOR THOSE INTERESTED IN GETTING CME CREDITS THE CODE IS 37921. I KNOW IT SOUNDS LIKE A WORD FROM OUR SPONSOR. WE'LL DO OUR BEST TO MAKE SURE YOU GET YOUR CME CREDIT. WE HAVE ANOTHER QUESTION UP FROM DR. PEREZ. THE QUESTION IS DO YOU KNOW WHAT HAPPENS IF YOU DEPLETE COMPLEMENT C1A. DO YOU STILL FIND THE SAME CLONALITY IN THE STEM CELLS AND DO YOU STILL GET GROOMING BEHAVIOR ON THE MONOCYTES? >> WE'RE DOING THAT EXPERIMENT RIGHT NOW BUT WE DON'T KNOW THE RESULT. ONE OF THE THINGS ABOUT COMPLEMENT THAT'S INTERESTING IS THE WORK OF BETH STEVENS WHERE SHE'S LOOKED AT MICROGLIAL CELLS AND HOW THEY'RE ABLE TO INTERACT WITH SYNAPSES AND THE KIND OF APPROACH IS SIMILAR AND SO YOU WONDER IF THE COMPLIMENT WILL HAVE AN EFFECT ON CLONALITY ALSO. IT'S SOMETHING WE'RE LOOKING AT BUT DON'T KNOW RESULT YET. >> FANTASTIC. >> AS WE'RE WAITING FOR THE NEXT QUESTION, I KNOW YOUR LAB HAS BEEN AT THE FOREFRONT IN DETERMINING AND DISSECTING THE MECHANISMS OF MELANOMA. I KNOW YOU DEVELOPED THE FIRST ANIMAL MODEL AND IS ANY OF THE WORK YOU PRESENTED TODAY IN ANY WAY RELATED TO THE WORK ON MELANOMA? >> ONE OF THE THINGS WE'VE BEEN DOING RECENTLY AND IT'S VERY EXCITING IS TO STUDY THE CANCER NICHE AND COMPARE IT TO THE HEMATOPOIETIC NICHE. DURING MY DAY I WAS SHOWING VIDEOS AND WE'VE BEEN ABLE TO CD8 POSITIVE T CELLS WITH A TRANS GENE AND WATCH THE T CELLS GO LIVE INTO A MELANOMA AND CAN IMAGE FOR 18 HOURS AND WHEN YOU DO THAT YOU SEE WHERE THE T CELLS AND DENDRITIC CELLS COMPANY TO THE SURFACE OF THE TUMOR AND WE SEE INTERESTING INTERACTIONS AND IF WE GIVE A THERAPEUTIC SUCH AS WHAT IS USED IN THE CLINIC WE SEE MASSIVE CHANGES IN THE TUMOR NICHE AND STROMAL CELLS AND ENDOTHELIAL CELLS AND THE COLLAGEN PRESENT AROUND THE TUMOR. WHEN WE COMPARE HOW THE TRAFFICKING OF CELLS IS TO THIS LITTLE STEM CELL NICHE AND THE ENDOTHELIAL CELLS ARE INTERESTING AND THE STROMAL CELLS AND I'M LOOKING FORWARD TO DOING THE COMPARATIVE ANALYSIS AND TARGETING THE STROMAL AND ENDOTHELIAL CELLS CAN HAVE INTERESTING OPPORTUNITIES IN THE FUTURE. WE'RE INTERESTED IN THOSE. >> FANTASTIC. A COUPLE MORE QUESTIONS HAVE COME IN AND I'M GOING TO BE SENSITIVE TO YOUR TIME BECAUSE I KNOW WE'RE GETTING THE MONEY'S WORTH OF YOU. HAVE YOU A NUMBER OF ZOOM MEETINGS LINED UP. THIS QUESTION ASKS DO HEMATOPOIETIC STEM CELLS AND MACROPHAGE MAINTAIN THE SAME INTERACTION YOU NICELY SHOWED US IN THE ADULT MARROW? >> WE DON'T KNOW THE RESULT YET BUT I THINK THEY'RE GOING TO HAVE A SIMILAR INTERACTION. THE PLACE I THINK IS MOST SIMILAR WHEN WE DO A BONE MAYOREE TRANSPLANT WE PUT IN A LOT OF CELLS AND WE KNOW YOU DON'T ENGRAFT WITH ALL THOSE CELLS. QUESTION IS IS THERE A RECOGNITION OF CERTAIN STEM CELLS PRONE TO ENGRAFT BASED ON THEIR CALRETICULIN AND TO SELECT THE BEST CELLS TO LAST FOR A LIFE TIME AND I THINK THE INTERACTIONS WILL BE SIMILAR PROBABLY DURING THE COLONIZATION AND WE DON'T KNOW THE RESULT YET. >> A QUESTION FROM MICHAEL DURANT. AT WHAT TIME DOES CLONAL DOMINANCE OCCUR RELATIVE TO THE DISEASE SYMPTOMS? THIS IS A QUESTION OF YOU AS A HEMATOLOGIST AND AT THE TIME OF DISEASE SYMPTOMS IS IT TOO LATE TO ATTACK THOSE DOMINANT CLONES YOU SHOWED. WHAT'S THE STATE OF LESS DOMINANT CLONES AT THE TIME OF THE DISEASE SYMPTOMS. HE'S HOPING THE QUESTION IS CLEAR. >> THAT'S SUPER USEFUL. WHAT HAPPENS IS IT'S 10% OF 70-YEAR-OLDS AND 50% OF 90-YEAR-OLDS HAVE CLONAL HEMATOPOIESIS LEADING TO 20% TO 30% OF THEIR BLOOD BEING DOMINANT. TWO OR THREE WEEKS AGO A FRIEND CALLED AND HER FATHER HAD MDS DOING VERY WELL AND WHAT HAPPENED WAS THE PATIENT HAD TATTOO MUTATIONS BUT JUST DEVELOPED AN ASXL1 MUTANT CLONE. CLEARLY THE PERSON IS ON THE WAY OF HAVING MORE SYMPTOMS. THAT'S PROBABLY THE PLACE TO INTERVENE IN THE PERIOD OF TIME AND A NUMBER OF US ARE THINKING ABOUT USING CELLULAR BAR CODING TECHNOLOGY AND BEARING ALLELE FRACTIONS TO FIGURE OUT THE PATIENT POPULATIONS AN ULTIMATELY DO CLINICAL TRIAL, PERHAPS SHORT TERM TRIALS TO MAKE CLONES OF CELLS NOT BE DOMINANT. I THINK THAT'S THE AREA TO LOOK IN. TO BE FAIR YOU'D WANT TO BASICALLY USE AN INHIBITER IN THIS PARTICULAR PATIENT WITH THAT NEW ASXL1 MUTANT CLONE. >> ONE FINAL QUESTION, IT'S A VERY NICE TALK AND THERE'S A GENERAL QUESTION, CAN YOU MODEL IN THE ZEBRAFISH. I THINK MOST YOUR ANSWERS DEALT WITH THAT BUT YOU MAY WANT TO RECAP. >> I THINK WE'RE ACCURATELY MOLDING THE SITUATION AND WE EVEN IS FISH WITH LEUKEMIA AND HAVE DONE CLONAL BAR CODING AND IT'S BEEN VERY INTERESTING. I THINK WE CAN MODEL IT. THE ONLY THING I WOULD SAY THAT'S A CAVEAT AND DON'T WANT TO OVER SELL EVERYTHING WITH THE ZEBRAFISH, WE MUTATE THE STEM CELLS AT THE BEGINNING OF THEIR DEVELOPMENT. THEY BECOME DOMINANT OVER TIME AND IT DOES TAKE MONTHS FOR THEM TO BE DOMINANT. I DO THINK IN A HUMAN YOU HAVE AN ADULT WHEN THE MUTATION HAPPENS SO IT'S A LITTLE BIT DIFFERENT. THE OTHER THING I'D SAY WHICH IS INTERESTING WE STARTED TO MODEL PEDIATRIC SYNDROMES AND PEDIATRIC PREDISPOSITIONS TO LEUKEMIA AND EARLY IN A FISH'S LIFE IF YOU HAVE A MUTATION YOU'RE CLONALLY DOMINANT WHEN YOUR BORN. SO THAT STARTS TO MAKE YOU THINK OF PREDISPOSITION SYNDROMES AND ALREADY BEING DOMINANT AND. SO THE MECHANISM OF AGING IS IDENTICAL EXCEPT THE FACT YOU'RE BORN DOMINANT IN THE MUTATION VERSUS ACQUIRED MUTATIONS LATER IN LIFE. IT'S PRETTY INTERESTING. >> EXCELLENT. THERE ARE A FEW MORE QUESTIONS BUT I SEE YOUR NEXT HANDLERS ARE HERE TO WHISK YOU OFF TO YOUR NEXT MEETING. AND I KNOW HAVE YOU A TEE AT 4:15. THAT WAS A GREAT TALK. IT COVERED A LOT OF AND IT'S A BEAUTIFUL MODEL TO VISUALIZE WHAT'S GOING ON IN VIVO. ON BEHALF OF DR. COLLINS AND I'M SURE HE WOULD DO THAT IF HE WAS HERE, LET'S GIVE DR. ZON OUR VIRTUAL CONGRATULATIONS ON THE PERFECT TALK. WITH THAT THANKS VERY MUCH AND I'LL TELL THE AUDIENCE THAT WE'RE NOW ADJOURNED. I WANT YOU TO STAY ON. >> THANKS. I APPRECIATE IT.