>> GOOD AFTERNOON. I'M PLEASED TO INTRODUCE MATTHEW HOFFMAN WHO WILL GIVE TODAY'S NIH DIRECTOR'S LECTURE. HE GOT HIS DEN TALL DEGREE AT THE UNIVERSITY OF OTAGO SCHOOL OF DENTISTRY, DUNEDIN, NEW ZEALAND HE DECIDED TO GET A MASTERS AND THEN THE Ph.D. AT THE UNIVERSITY OF ROCHESTER IN MICROBIOLOGY AND THEN IMMUNOLOGY. THEN HE CAME TO THE NIH AS A VISITING FELLOW, THEN BECAME A STAFF SCIENTIST, THEN WAS COMPETED FOR A TENURE TRACK POSITION AND WAS RECENT HLY TENURED AS A SENIOR INVESTIGATOR AT THE NIH. HE'S BEEN INTERESTED IN ISSUES ARE REHALATED TO DEVELOPMENTAL BIOLOGY, PARTICULARLY FOCUSED ON DEVELOPMENT OF SALIVARY GLANDS THE INTERACTION OF THE VARIOUS CELL TYPES WITH THE INTERCELLULAR MATRIX AND TODAY HE'S GOING TO TELL US ABOUT "EPITHELIAL-PROGENITOR CELL-NEURONAL COMMUNICATION: IMPLICATIONS FOR TISSUE REGENERATION" THANK YOU. >> THANKS FOR THE OPPORTUNITY TO COME ALONG AND TALK TODAY. FIRST I WANT TO GIVE YOU A BACKGROUND TO WHAT I'M GOING TALK ABOUT. BOTH ABOUT SALIVARY GLANDS AND PROGENITOR CELLS AND THEN TALK ABOUT THE ROLL OF NERVES IN EMBRYONIC ORGAN DEVELOPMENT AS WELL AS -- AND THEN FINALLY THERE'S A NUMBER OF PROJECTS THAT I'M GOING TO DISCUSS AND THEY SORT OF COME TOGETHER AND WE'RE NOW LOOKING AT STIMULATING THOSE TO GET PROGENITOR CELLS TO REGENERATE TISSUES. JUST TO BEGIN WITH SOME BACKGROUND, UM, MY LAB STUDIED SALIVARY GLAND DEVELOPMENT RIGHT FROM PROGENITOR CELL COMMITMENT WHICH IS AT THE EARL YEGS -- EARLIEST STAGE OF DEVELOPMENT RIGHT THROUGH TO FORMATION OF SALIVARY GLAND. I HAVE A GREAT GROUP WORKING WITH ME. MOST RECENTLY, SARAH KNOX LEFT THE LAB TO SET UP HER OWN LAB MO LEFT TO GO BACK TO SCHOOL. WHAT I WANT TO DO TODAY IS TALK ABOUT PROJECTS OF MANY PEOPLE; PARTICUL PARTICULARLY, SARAH --- THE MOST RECENT MEMBER TO JOIN THE LAB IS FERARA, A CLINICAL FELLOW CAR I -- CARRY ON SOME OF THE WORK. SALIVARY PROGENITOR CELLS ARE TISSUE-SPECIFIC CELLS AND THEY'RE ESSENTIAL FOR THE DEVELOPMENT OF THE GLAND FOR THE HOMEOSTASIS OF THE TISSUE AND FOR REPAIR OF THE ORGAN. JUST TO SORT OF SIMPLIFY DOWN SOME OF THE DECISIONS THAT A PROGENITOR CELL HAS TO MAKE, WHAT I LIKE YOU TO THINK ABOUT IS THAT WHEN A PROGENITOR CELL DIVIDES, IT HAS THREE OPTIONS. IT CAN UNDERGO A SE ME TRI CALL DIVISION TO GET TWO COPIES. IT EXPANDS THE PROGENITOR CELL POOL. THIS IS IMPORTANT EARLY IN DEVELOPMENT WHERE YOU NEED A POOL FOR FURTHER DEVELOPMENT OF THE GLAND. AS IT DIVIDES IT CAN DO AN ASYMMETRIC DIVISION. IN THIS TYPE OF DIVISION, WE GET MAINTENANCE OF THE PROGENITOR POOL AS WELL AS TISSUE EXPANSION OF OTHER CELL TYPES. WE CAN GET A A CELL DIVISION WHERE IT DIVIDES INTO BOTH DIFFERENTIATED CELLS. THE POOL WOULD BE LOST IN ALL THE CELLS DID THAT AND WE WOULD DEPLETE THE PROGENITOR POOL. WHY ARE WE INTERESTED IN SALIVARY GLAND PROGENITOR CELLS? THE INSTITUTE'S MISSION IS TO IMPROVE THE ORAL HEALTH OF THE NATION, AND LOSS OF SALIVARY GLAND FUNCTION HAS A SEVERE IMPACT ON THE ORAL HEALTH OF PATIENTS. ALL OF US HAVE THREE PAIRS OF SALIVARY GLANDS. WHEN THE FUNCTION OF THESE GLANDS IS COMPROMISED, THERE'S A LOSS OF SALIVA FUNCTION AND THE PATIENTS HAVE PROBLEMS WITH EATING, DRINKING, TALKING AS WELL AS INCREASES IN DENTAL CARRIES AND PARRY OWE DONE TALL DISEASE. -- PARIODONTAL DISEASE. A VERY IMPORTANT CASE OF SALIVARY HYPER FUNCTION IS POST THERAPEUTIC RADIATION. THIS IS MOST COMMONLY FOR HEAD AND NECK RADIATION. THE SALIVARY GLANDS ARE IN THE FIELD OF THE RADIATION DURING THIS AND THEY'RE SENSITIVE TO RADIATION AND AS A RESULT THEY LOSE FUNCTION, PERMANENTLY USING SALIVARY FUNCTIONS. THERE'S ALSO RARE GENETIC DISEASES THAT AFFECT THE DEVELOPMENT OF THE SALIVARY GLANDS RESULTING IN PATIENTS WHO DON'T HAVE MAJOR SALIVARY GLANDS A AND LOSS OF FUNCTION OFF OCCURS AS A SIDE EFFECT OF MEDICATION. OUR LAB IS INTERESTED IN THINKING ABOUT HOW WE WOULD GO ABOUT REGENERATING SALIVARY GLANDS WHEN THE TISSUE UH HAS BEEN PERMANENTLY DAMAGED. THERE'S A NUMBER OF OPTIONS UKTDZ TAKE. YOU COULD REENGINEER THE REMAINING SALIVARY GLAND TISSUE. WE HAVE GENE THERAPY TRIALS GOING ON IN HUMANS WHERE THEY'RE INTRODUCE THE [INDISCERNIBLE] IN ONE GENE TO PROMOTE WATER CHANNEL PRODUCTION IN THE REMAINING TISSUE AFTER A RADIATION. YOU COULD ALSO LOOK AT USING BIOSCAFFOLDS AND MAYBE TRANSPLANTING AN ARTIFICIAL BACK INTO A PATIENT. THE OTHER IS USING IPS CELLS TO REGENERATE THE GLAND. THIS IS THE RESEARCH OUR LAB IS LOOKING AT DOING TO TRANSLATING SOME OF OUR BASIC SCIENCE FINDINGS. HOW WOULD WE GO ABOUT DING THIS? AS THE CLINIC SCENARIO T SALIVARY GLAND PROGENITOR CELL THERAPY COULD BE USED TO REGENERATE A RADIATED SALIVARY GLAND. IN TERMS OF RADIATION TREATMENT PLANNING, UM, A BIOPSY OF A NORMAL SALIVARY GLAND WOULD BE TAKEN BEFORE THE PATIENT EVEN STARTS TREATMENT. THAT COULD WITH B THEN PUT IN CULTURE AND EXPANDED AND IN THIS PICTURE I'M SHOWING YOU SALS FEARS WHICH ARE FER ROADS OF THE PROGENITOR CELLS FROM TISSUE BIOPSY THAT ARE GROWN. THESE WOULD BE THEN FROZEN DOWN AND STORED BY THE PATIENT UNDERWENT THEIR RADIO THERAPY AND CHEMOTHERAPY IF THEY NEEDED IT FOR THEIR TUMOR TREATMENT, AND THEN THESE WOULD BE RETRONS PLANTED BACK INTO THE SAME PATIENT EITHER THROUGH THE DUCTS OR INJECTING INTO THE GLANDS TO REGENERATE AND RESTORE FUNCTION IN THE GLAND. HOWEVER, WE'VE GOT A COUPLE OF CRITICAL QUESTIONS WE NEED TO ANSWER BEFORE WE CAN ACTUALLY DO THIS THERAPY. WHICH PROGENITOR CELL POPULATION AND UH HOW WOULD WE MAIN TAND EXCEL THEM IN CULTURE SO WE COULD USE THEM TO REGENERATE THE TISSUE? THESE ARE ISSUES WE'RE TRYING TO DEAL WITH PARTICULARLY UNDERSTANDING HOW WE WOULD MAINTAIN AND EXPAND THESE CELLS BECAUSE AT THE MOMENT, WE DON'T REALLY UNDERSTAND, FULLY, WHAT ARE ALL THE PROGENITOR CELLS IN THE SALIVARY GLAND. SO DO HUMAN GENETIC DISEASES INFORM US ABOUT PROGENITOR CELLS IN SALIVARY GLANDS? LOOKING AT THE LITERATURE, THEY DO. THERE'S A COUPLE OF RARE HUMAN GENETIC SYNDROMES THAT ARE RESULT OF LOSS OF FIBERGLASS GROWTH FACTOR SIGNALING, WHICH SHOWS THAT THE SALIVARY MROJ MROJ CELL REQUIRE SIGNALING TO PERFO PERFORM -- THESE RESULT IN ALSG WHICH STANDS FOR APPELLATION FOR [INDISCERNIBLE] AND LAD SYNDROME WHICH IS A BROADER SYNDROME WHERE PATIENTS NOT ONLY HAVE PROBLEMS WITH SALIVARY AND LACK MARRY GLAND BUT ALSO PROBLEM WITH HEARING AND DIGITS. SO THE IF WE LOOK AT MICE MODELS, AND OBVIOUSLY OUR LAB'S USING MOUSE MODELS TO UNDERSTAND THESE, WE SEE THAT IN AN FGF 2 OR TWO B KNOCKOUT MICE T MICE DWONT DEVELOP AND THEY DON'T HAVE SALIVARY GLANDS. WHAT'S ALSO IMPORTANT IS THAT WITH THE HETERO ZYGOTES OF BOTH, THE MICE HAVE TWEER SALIVARY GLAND HYPERPLASIA. THESE THIS HIGH LIGHTS THAT THESE TWO MOLECULES ARE IMPORTANT TO THE DEVELOPMENT OF THOSE PROGENITOR CELLS. WHAT I'M DOING IS INTRODUCING THE MOUSE MAN DIB LAR GLAND. AT EMBRYONIC DAY 12 IN THE MOUSE, A PRIMAL EP FEEL Y'ALL INBUD GROWS INTO A MESENCHYME. BY E 13, THAT STARTS TO CLEFT AND UNDERGO A PROCESS CALLED BRANCH MORPHOGENESIS SO BY E 14 THE IT LOOKS LIKE A BUNCH OF LITTLE GRAPES. FGF RECEPTOR 2 B IS PRESENT IN THE EPITHELIAL OF THE GLABD AND FGF 10 IS IN THE CONSENTED GLAND. SO HOW DO WE ACTUALLY STUDY THIS FGF RECEPTOR SIGNALLING IN THE LAB? WHAT WE DO IS USE EXVI OWE SUB MAN DIB LAR -- IN THE TIME LAPSE VIDEO I'M SHOWING, YOU CAN SEE A GLAND GROWING. -- WE CAN ACTUALLY DO A DOSE RESPONSE OF SGF RECEPTOR 2 B AND COMPLETELY INHIBIT GROWTH. BRANCHING INVOLVES SUCK SUCCESSFULIVE -- WILL INVOLVE REGENERATING EP THEEL YAM BUDS AND INDUCTS. WHAT OTHER MODELS DO WE DO TO LOOK AT THESE STRUCTURES? ISOLATING THE SUB MANDIBULAR GLAND EPITHELIUM INTO AN ISOKIND. WE'RE GROWING THIS ISOLATED PRIME MARRY EPITHELIUM IN A GEL. IF WE JUST PUT IT IN THE GEL AND [INDISCERNIBLE] IT DOESN'T GROW AT ALL. -- THE EPITHELIUM GROWS AND BRANCHS. IF WE ADD FGF 10 WE SEE IT GROWS OUT AND GET DUCT ELONGATION. QUESTION REALLY THINK IN THIS SORT OF MODEL THAT WE'VE GOT DIFFERENT GROWTH FACTORS REGULATING BOTH THE INBUD AND THE DUCT DEVELOPMENT. SO REALLY WE AIM TO UNDERSTAND HOW THE EPITHELIAL PROGENITOR CELLS FORM THESE TWO STRUCTURES. THIS WILL PROVIDE US FOR A TEMPLATE TO REGENERATE ADULT TISSUE. OBVIOUS THINK PROGENITOR CELL INTERACTS WITH ITS LOCAL ENVIRONMENT. WHAT I WANT TO DO HERE IS INTRODUCE THIS CONCEPT OF THE NICHE. THE PROGENITOR NICHE THAT PROVIDES SIGNALS AND CUES TO THE PROGENITOR CELL TO HELP IT MAKE ITS DECISIONS TOOZ WHETHER IT'S GOING TO GO SELF-RENEW OR DIFFERENTIATE INTO AN INBUD OR DUCT STRUCTURE. SO WE KNOW THAT OTHER CELL TYPES IN THE SALIVARY GLAND AND THEY CONTRIBUTE TO THIS IN EACH OF THE EP THEEL YAM PROGENITOR CELLS AN INFLUENCE THESE DECISIONS. SO A FEW YEARS AGO, NOW, WE DID SOME IMMUNOSTAINING LOOKING AT THE EPITHELIUM T DUCTS AND THE BLOOD VESSELS IN THE ISOLATED E 13 GLAND AND THE IMAGES I'M SHOWING ARE PROJECTS OF OPTICAL SECTIONS THROUGHOUT A WHOLE GLAND. YOU CAN SEE THE ANTIBODY IN THE TOP OUTLINES THE EPITHELIUM. THE NRS ARE STAINED WITH A ANTIBODY AND THERE'S A BRANCH STRUCTURE OF NERVES. IF WE PROJECT THE PROJECTIONS OF THESE AND ROTATE THEM, THE NERVES LOOK LIKE THEY'RE CRAWLING ALONG THE EPITHELIUM. THEY'RE CLOSELY ASSOCIATED WITH THE EPITHELIUM DURING DEVELOPMENT WHEREAS THE BLOOD VESSELS ARE MORE OUT AROUND THE INBUDS. THIS LED SARAH KNOX WHO CAME TO THE LAB AS A POST DOC TO ASK THE QUESTION; DO THE PARASYMPATHETIC NERVE INFLUENCE THE PROGENITOR CELLS DURING EARLY EMBRYONIC DEVELOPMENT? WHAT SHE DID WAS DEVELOP A MOD WHERE WILL SHE SEPARATED THE EPITHELIUM, THE MESENCHYME AND THE NERVES AND DID RECOMBINATION CULTURES. IN THE TOP IMAGES I'M TOE SHOWING YOU HERE IS WHERE SHE SEPARATED THE THREE TISSUES, RECOMBINED THEM AND THE GLAND GROWS AND BRANCHS LIKE A NORMAL CONTROL AND YOU CAN SEE THAT THE NERVES ASSOCIATE WITH THE EPITHELIUM AND GROW OUT. WHEN WE REMOVE THE PARASYMPATHETIC GANGLIA, IT UNDERGOES A LITTLE BIT OF BRANCHING AND THEN IT STOPS. SHE SCREENED GENE EXPRESSION, AND WHAT JUMPED OUT EARLY ON WAS THAT A COUPLE OF THE CAROTINS, 5 AND 15, AND WATER CHANNEL WERE BOTH DOWN REGULATED IN THE EPITHELIUM WHEN THE THE PARASYMPATHETIC GANGLIA WAS REMOVED. THIS LED US TO THINKING THAT THE NERVES WERE SOMEHOW CONTROLLING PROGENITOR CELLS BECAUSE CAROTIN 5 AND 15 ARE WELL ESTABLISHED MARKERS OF BASIL PROGENITOR CELLS IN OTHER SYSTEMS. WE THEN ASKED WHERE ARE THE CAROTIN 5 EXPRESSING CELLS IN THE SUB MANDIBULAR GLAND? DOING SOME IMMUNOSTAINING OF A SALIVARY GLAND YOU CAN SEE THESE ARE ARE THE GREEN CELLS SHOWING UP HERE AND THE RED CELLS ARE STAINED WITH AN ANTIBODY. YOU CAN SEE THERE'S CELLS PRESENT IN THE INBUDS AND THE DUCTS OF THE INTACT STRUCTURE. ISABEL HAD JOINEDED THE LAB AND SHE DID FAX ANALYSIS OF THESE GLANDS LOOKING AT K 5 EXPRESSION AND WHAT WE COULD MEASURE WAS THAT ABOUT 10% OF THE CELLS IN THE GLAND WERE K 5 EXPRESSING AND THEN WE WENT ON AND DID GENETIC LINEAGE TRACING TO CONFIRM THEY'RE TWALLY PROGENITORS. WE USED [INDISCERNIBLE]. I WON'T GO INTO THE DETAILS, BUT BASICALLY WHAT WE DO IS LABEL THE CELLS GENETICALLY VERY EARLY. WE POST THEM. THEY GET LABELLED WITH THE BLUE STAINING, THEN WE LET THE MOUSE CONTINUE TO DEVELOP AND POST NATALLY, WE LOOK AT THE BETA GENE EXPRESSION IN THE GLAND. WHEN WE INDUCED AT VERY EARLY AGE E 8-11 POST NATELY THE GLAND IS BLUE. THIS PROVES THAT PROGENITOR CELLS ARE DERIVED FROM THE K 5 CELLS. ALL THE EPITHELIAL OF THE GLAND IS DEVELOPED FROM HERE. WHAT ELSE DID WE LEARN? WE ALSO DID A LOT OF OTHER FAX ANALYSIS LOOKING AT OTHER MARKERS. WHEY WANT TO MIELIGHT IS THAT WE IDENTIFIED THREE CELL TYPES EXPRESSING THE [INDISCERNIBLE] RECEPTOR M 1 AND THE EGF RECEPTOR, EGFR. WE IDENTIFIED THREE POPULATIONS. THIS IS SUMMARIZING A LOT OF FAX DATA HERE. THE K 5 POPULATION THAT EXPRESSES BOTH RECREPE ACCEPT TOR -- RECEPTORS AND A POPULATION THAT'S JUST K 19 POSITIVE. WE KNOW FROM THE LOCALIZATION STUDIES THAT K 1 IS ASSOCIATED WITH THE DUCTS OF THE SALIVARY GLAND. WHEN WE TREATED THESE THREE SETS -- WHEN WE TREATED THE GLANDS WITH EITHER CAVACO OR HBGF WE GOT SOME INTERESTING RESULTS. PARTICULAR, WE IDENTIFIED THAT CAVACO STIMULATING THE PROLIFERATION OF THE K 5 AND WE GET A DOUBLING OF THAT NUMBER. WHEREAS HBEF AND CAVACO BOTH STIMULATE THE DEVELOPMENT OF THESE OTHER POPULATIONS. THIS LED US TO CONCLUDE THE IMPORTANT RESULT HERE REALLY IS THAT CAVACO IS DOUBLING THE BASIL K 5 POPULATION AND THIS LED US TO PROPOSE A MOLL MODEL WHICH I WANTED TO START BUILDING INNED TODAY'S TALK. OUR WORKING MODEL IS THAT WE HAVE THE P PARASYMPATHETIC GANGLIA -- THEN CAVACO AND HBGF ALSO DRIVE -- SO THAT -- DURING DEVELOPMENT. SO REALLY WE WERE INTERESTED IN LOOKING AT DOES THIS SIMILAR MECHANISM OCCUR IN OTHER BRANCHING ORGANS. I P PUT IN A SLIDE OF ANOTHER BRANCHING ORGAN WHICH IS THE PROSTATE. A SIMILAR MECHANISM OCCURS IN THE P PROSTATE. WHEN WE LOOK AT K 5 AND A 19 STAINING AND TREAT, WHAT WE SEE IS WE GET A LOSS OF THE K 5 PROGENITOR CELLS WHICH ARE THE BASAL CELLS STAINED IN GREEN. WHEN WE LOOK AT GENE EXPRESSION HERE, WE ALSO SEE LOSS O OF CAROTIN 5 AND ATP 3 AS WELL. THIS IS SUGGESTING THAT A SIMILAR MECHANISM IS OCCURRING IN THE PROS THE TAT. O SO NOW WE'RE ALSO INTERESTED IN WHAT OTHER PROGENITOR CELLS WERE PRESENT IN THE SALIVARY GLAND, AND A FEW YEARS AGO ISABEL IN A DUTCH LAB DURING H HER P H HD, I PUBLISHED A PAPER THAT SHOWS THAT THE KIT RECEPTOR IS A STEM CELL MARKER AND THAT KIT POSITIVE CELLS COULD BE ISOLATEDED FROM ADULT SL VARY GLANDS AND USED TO RESTORE FUNCTION AFTER A RAID YAEGS. THIS WAS A REALLY LANDMARK PAPER AT THE TIME BECAUSE IT SHOWED IN A MOUSE MODEL THAT YOU COULD REGENERATE AN ADULT TISSUE USING ADULT-DERIVED TISSUES. HOWEVER THEY USED HETERO GENE NOUS POPULATION OF KIT POSITIVE CELLS AND DIDN'T REALLY UNDERSTAND -- WE DON'T REALLY UNDERSTAND -- WHAT KIT WAS DOING IN THESE CELLS. IT WAS JUST USED AS A MARKER. ISABEL CAME TO OUR LAB TO START STUDYING THE ROLE OF KIT DURING EMBRYONIC GLAND DEVELOPMENT. WHAT WHERE IS KIT EXPRESSED IN THE SUB MAN DIB HA LAR GLAND. IF WE LOOK AT E 13 AND [INDISCERNIBLE] WHICH IS A BASEMENT MEMBRANE PROTEIN, IT'S OBVIOUS YOU CAN SEE THAT THE KIT POSITIVE CELLS ARE IN THE INBUD OF THE GLAND. WE ALSO KNEW FROM THE PREVIOUS WORK THAT FGF RECEPTOR 2 B IS ESSENTIAL FOR THE DEVELOPMENT OF THE GLAND AND WHEN WE LOOKED AT THE KIT POZ THETIVE CELLS WE COULD SEE 90% WERE EXPRESSING FGF REACCEPT SOR -- RECEPTOR 2 B. WE COULD SEE THE KIT CELLS AND INBUD WERE UNDERGOING PROLIFERATION. WE WERE INTERESTED IN ASKING WHERE THE FGF RECEPTOR 2 -- WE KNEW THAT WITHOUT FGF RECEPTOR 2 B SIGNALING, THERE'S NO SALIVARY GLAND. TOOK ISOLATED EPITHELIUM AND TREATED WITH GROWTH FACTORS. THESE ARE ARE SHORT EXPERIMENTS USING PRIMARY TISSUE AND THEN ASSAYS BY QPCR GENE EXPRESSION DOWNSTREAM OF GROWTH FACTOR STIMULATION. WHAT CAME OUT OF THE SCREENING, WE NOTICED THAT THERE WERE LIE GRANDS FOR FGF RECEPTOR 2 B INCREASED EXPRESSION OF STEM CELL FACTOR. THATTED LED US TO ASK THE QUESTION, DOES STEM CELL FACTOR AFFECT FGF RECEPTOR 2 B SIGNALLING? WHAT SHAWN ABRAMS HAS DONE IS EXPERIMENTS WHERE HE'S LOOKING AT TIME COURSES OF STEM CELL FACTOR AND FGF 10 TREATMENT OF THE PRIMARY EPITHELIUM TO GET A HANDLE ON WHAT'S GOING ON WITH THE SIGNALING. IN THE TOP PANEL THERE'S A WESTERN BLOT. THIS IS A WESTERN BLOT STAINING AND YOU CAN SEE THAT WITHIN FIVE MINUTES WE GET A INDUCTION OF PHOSPHORYLATION OF AKTAKT AND IRK SIGNALING ARE DOWNSTREAM. WE CAN SEE THAT WITH FGF 10 THERE'S A LITTLE BIT OF AKT PHOSPHORYLATION. WHEN WE ADD BOTH STEM CELL FACTOR AND FGF 10 TOGETHER WE GET AN INDUCTION AND ENHANCEMENT OF PHOSPHORYLATION AND THE EXTENT OF THE DURATION OF THE SIGNAL INCREASES AND THIS IS QUANTITATED ON THE GRAPH TO THE RIGHT. WHEN WE LOOK AT IRK PHOSPHORYLATION, WE SEE A SIMILAR THING BUT IN THIS CASE IT'S SGF 10 THAT INDUCES IRK. WHEN WE COMBINE THEM, WE SEE FURTHER INDUKS OF IRK AND THE DURATION OF SIGNALING. SO THIS LED US TO THINK THAT BOTH SIGNALING PATHWAYS WERE WORKING TOGETHER AND IS IER JIEDZING NOT THAT BUT HAVINGED A TI EFFECTS. WHAT IS DOWNSTREAM OF THEM AND ARE THEY BOTH HAVING SIMILAR EFFEC EFFECTS DOWNSTREAM? IF WE JUST TREAT THIS WITH STEM CELL FACTOR IT DOESN'T SURVIVE. IT'S NOT REQUIRED FOR ITS SURVIVAL. I SHOWED YOU IN THOSE VIDEOS THAT IF WE JUST FGF 10, THE GLAND GROWS AND SURVIVES. IT'S SUGGESTED TO US STEM CELL FACTOR IN KIT SIGNALING IS NOT REQUIRED BUT THAT IT'S INFLUENCING THE SIGNALING DOWNSTREAM OF FGF RECEPTOR 2 B. WHAT CHANGES IN GENE EXPRESSION DO WE MEASURE? WE'VE LOOK AT NUMBER OF TRANSCRIPTION FACTORS AND SIGNALING MOLECULES THAT WE KNOW ARE DOWNSTREAM AND WE NOTICE -- AND I'M SHOWING YOU A FEW EXAMPLES HERE, ONE IS DOCKS 10. WHAT I'D LIKE YOU TO NOTICE IS THAT THE CONTROL AND STEM CELL FACTOR WHEN WE ADD THOSE, THERE'S NO DIFFERENCE IN GENE EXPRESSION. STEM CELL FACTOR IS NOT DIRECT HLY CONTROLLING THE EXPRESSION OF THESE GENES. THE GREEN SHOW FGF 10 DREAM WHERE WE GET AN INDUCTION IN GENE TREATMENT. WHEN WE ADD GFG 10 AND STEM CELL FACTOR WHERE WE'RE STIMULATING BOTH RECEPTORS, WE GATE FURTHER REDUCTION IN GENE EXPRESSION AND THIS CAN BE INHIBIT BID ADDING A CHEMICAL INHIBITOR OF SIGNALING. WE KNOW THAT THE DOWNSTREAM TRANSCRIPTION FACTORS ARE BEING AFFECTED. TO ADDRESS QUESTIONS, WE WENT BACK TO THE INTACT GLAND AND CULTURE AND DECIDED TO OVERSTIMULATE THAT WITH BOTH FGF 10 AND FGN 10 IN STEM CELL FACTOR. THERE IS NO DIFFERENCE IN TERMS OF THE MORPHOGENESIS. THE GLANDS LOOK IDENTICAL. HOWEVER, WHEN WE LOOKED AT THE DIFFERENT MARKERS OF PROGENITOR CELLS WITHIN THE GLAND, WE COULD SEE STRIKING DIFFERENCES. IN THIS STAINING HERE, THIS IS AN INBUD WITH FGF 10 TREATMENT, WE CAN SEE KER TAN 14 IS EXPRESSED AROUND THE PERIPHERY OF THE INBUD AND AREA 19, AND SOX TEN IS ALSO IN THE KER THE TIN 14 CELL. WHEN WE ADD STEM CELL FACTOR WE SEE AN INDUCTION IN SOX TEN AND -- WHEN WE DO FAX ANALYSIS OF THESE GLANDS ON THE RIGHT-HAND SIDE, WHAT I WANT TO HIGHLIGHT IS THAT THERE'S AN INCREASE IN THE NUMBER OF KERATIN FIVE EXPRESSING CELLS, THEY DOUBLE, KIT EXPRESSING CELLS, K # AND SOX TEN. WHAT'S IMPORTANT SHEER PROLIFERATION IS NOT CHANGING. THIS IS SUGGESTING THAT STEM CELL FACTOR IS WORKING WITH FGF RECEPTOR 2 B SIGNALLING TO ALTER THE IDENTITY OF THE PROGENITOR CELLS. INCREASING THE EXPRESSION OF MARKERS OF KIT, PROGENITORS AND K 5 PROGENITOR CELLS. THIS REALLY LED US TO ASK A SIMILAR QUESTION WE HAD LOOKED AT BEFORE WITH CAROTIN FIVE WHICH SAY BOUT KERATIN 14; ARE THESE PROGENITOR CELLS IN THE GLAND? IN A LOT OF OTHER TISSUES, KERATIN 5 AND 14 ALWAYS EXPRESSED IN THE SAME CELL. HOWEVER, IN THE SALIVARY GLAND THAT'S NOT THE CASE. KERATIN 14 LABELS A DIFFERENT POPULATION OF CELLS THAN K 5. SO WHAT WE'VE DONE HERE IS LINEAGE TRAINING USING A K 14 K MOUSE CROSSED WITH A REPORTER MOUSE. WHERE THE KREE IS TURNED ON IN THE CELLS T THE GLAND GOES FROM BEING RED TO GREEN. YOU CAN SEE JUST AT A H LOW-POWER IMAGE, BOTH THE SUB LINK GAL AND THE SUB MANDIBULAR GLAND ARE BRIGHT GREEN. WHEN WE DO CON SECTIONS OF THESE WE CAN SEE THE GFP EXPRESSING CELLS ARE THROUGHOUT THE ENTIRE CELL. THIS SUGGESTS ALSO THAT K 14 MARKS PROGENITOR CELLS. THE RED STAINING CELLS ARE THE IND FELL Y'ALL CELLS IN NERVES AND MESENCHYME. WE'VE GOT THIS MODEL THAT KIT IS AFFECTING OR THAT STEM CELL FACTOR IS AFFECTING PROGENITOR CELL FUNCTION. THESE ARE A ALL DONE WITH GAINER PRODUCTS WHERE WE'RE ADDING. WE ALSO WANTED TO LOOK AT LOSS FUNCTION EXPERIMENTS BY REDUCING KIT FUNCTION. ONE OF THE WAYS TO DO THIS IS USING A CHEMICAL INHIBITOR THAT INHIBITS THE PHOSPHORYLATION OF THE KIT RECEPTOR, AND WE DID THIS, THE INHIBITOR IS ICK WHEN WE ADD THAT TO A CONTROL GLAND OR IS CONTROL GLAND AND WE'RE STAINING FOR KERATIN 14, 5 AND 19. WE X SEE THAT KERATIN FIVE IS EXPRESSED ALONG SOME OF THE DUCTAL CELLS AND BASAL CELLS, KERATIN 19 IN THE DUCT. WE CAN SEE THE GLAND DOESN'T GROW AS WELL THERE'S AN ENHANCEMENT IN THE -- THERE'S A BIG INCREASE IN K 19 SO O THE CELLS ARE ALL GOING ALONG THE K 19 LINEAGE AND IT'S SO CLEAR HERE BUT THERE IS SOME ACTUALLY K 14 STAINING HERE. WHEN WE DO FAX ANALYSIS, WHEN E WE INHIBIT KIT EXPRESSION OR SIGNALING, WE GET A REDMUKS THE NUMBER OF KIT CELLS, K 1, K 5 AND SOX TEN. WE ALSO GET DOUBLING OF THE KERATIN 19 CELLS. WHAT ABOUT AN IN VIVO MODEL? WE USE A KIT MUTANT MICE. THESE MICE HAVE BEEN AROUND FAR LONG TIME. THEY'RE THE WHITE SPOT MUTATION MICE. THEY HAVE A POINT MUTATION IN THE KIT RECEPTOR, SO THE PROTEIN IS ACTUALLY THERE, BUT THE RECEPTOR CAN'T SIGNAL. THE MICE ARE EMBRYONIC LETHAL AT DAY 14 BECAUSE OF MAJOR PROBLEMS WITH THEIR HEART AND CARDIOVASCULAR SYSTEM. FORTUNATELY FOR OUR PURPOSES WE COULD USE THEM TO GET SAL VARY GLAND AT E 12 AND 13 AND CONTINUE TO GROW THEM. WHEN WE LOOKED AT KIT WILD TYPE MOUSE, AGAIN, LOOKING AT K 14, 5, AND 19, WE SEE A SIMILAR PATTERN AS I SHOWED PREVIOUSLY. WHEN WE LOOKED AT THE KITS WW MICE WE E SEE EXPRESSION OF K 19 AND REDUCTION IN K 14. BOTH THESE LOSS OF FUNCTION MODELS WE NOW INCLUDE AND MAKE A WORKING MODEL WHERE WE HAVE THE GANGLIA PRODUCING [INDISCERNIBLE] AFFECTING THE K 5 CELLS WITH THE ADEGS OF FGF 10 STEM CELL SECTOR WE NOW HAVE A K 5, K 14 POPULATION THAT INCLUDES RECEPTOR 2 B IN THE SAME CELL AND WE GET INBUD DIFFERENTIATION WHEN WE HAVE K 14 CELLS WITH 2 B AND KIT. SO ANOTHER THING THAT WE WERE VERY INTERESTED IN IS IN THE KIT WW MUTANT SUB MANDIBULAR GLANDS THERE WAS A REDUCTION IN THE K 5 CELLS. THIS SUGGESTED THAT SOMEHOW KIT IS SOMEHOW SIGNALING BACK TO THESE KER 5 CELLS. WE HYPOTHESIZE THAT MAYBE THERE'S A PROBLEM WITH THE INNOVATION AND THAT'S VOOI VIA THE PARA SYMPATHETIC GANGLIA. THAT LED US TO ASK A BASIC LE: IS THAT EPITHELIUM AT VERY EARLY STAGES PRODUCE NEUROTROPIC FACTORS? I'M GOING TO DO A SIDESTEP AND SHOW YOU DATA THAT WE USED MICRORAY ANALYSIS TO COMPARE GENE EXPRESSION OF THE EARLY EMBRYONIC EPITHELIUM WITH THE MESENCHYME. WE FILTERED THE RESULTS. THIS IS NOT SOMETHING YOU WOULD OFTEN DO IN AN EPITHELIAL POPULATION, BUT WE WERE HYPOTHESIZING THAT EPITHELIUM BUZZ GOING TO PRODUCE SOMETHING THAT WOULD BE AFFECTING NERVOUS SYSTEM DEVELOPMENT. USING FILTERING CRITERIA WHERE WE LOOKED AT GENES THAT WERE MORE THAN FIVE FOLD EXPRESSED -- WE NARROWED IT DOWN TO A LIST OF ABOUT 36 GENES. THERE WAS ONLY ONE OR TWO SECRETEDED NEUROTROE IF I CAN PRACTICE. I WANT TO TALK ABOUT NUTURON. IT'S IMPORTANT FOR PARASYMPATHETIC TARGET INNOVATION AND THE KNOCKOUT MICE WHICH HAVE BEEN STUDIED EXTENSIVELY LOOKING AT NERVOUS SYSTEM DEVELOPMENT AND HAVE ALWAYS NOTED THAT THIS IMPAIRED SALIVARY GLAND FUNCTION. IT MIGHT BE A SECRETEDED MOLECULE FROM THE EPITHELIUM THAT WAS AFFECTED THE PARA -- SO THEN WHAT HAPPENS TO THE NERVES IN NEW TU RON DEAF DF NEW -- WHEN WE LOOK AT THESE GLAND WHERE IS WE'VE LOST OUR KIT K 14 CELLS WE SEE A REDUCTION IN NERVES AND THE AXON EXTENSIONS ARE REDUCED. LOOKING AT QPCR AT WHAT'S GOING ON WITH EXPRESSION OF NEURONAL GENES, WE GET REDUCTIONS IN NEW WHERE ARE THEY EXPRESSED IN THE SALIVARY GLAND? NEW TU RON IS EXPRESSED IN THE N BUD EPITHELIUM. NUTURON'S THE GREEN STAINING. THIS IS NO SURPRISE. THIS IS WHAT WE WOULD EXPECTED BASED ON THE ARRAY DATA AND OTHER DATA. WHAT WE DID WAS FAX SORTED THE KIT POSITIVE CELLS AND LOOKED AT NEUTRON EXPRESSION. DOES NEUTRON INFLUENCE [INDISCERNIBLE]. WE COULD DO THIS USING FUNCTION BLOCKING ANTIBODIES. WHAT WE WERE INTERESTED IN LOOKING AT AUZ THE DEVELOPMENT OF THE PARASYMPATHETIC GANGLIA AND THE INNOVATION OF THE SALIVARY GLAND. IN THE LEFT WHEN WE ADD THE ANTIBODY THAT THE GLAND DOES NOT GROW AS WELL AND DOES NOT UNDERGO AS MUCH BRANCH AS THE CONTROLLED GLAND. WHEN WE LOOK AT THE NERVE STAINING, WE CAN SEE THAT THERE'S A REDUCTION IN THE NEURON OUTGROWTH AND WE CAN QUANTI QUANTITATE THIS. WHAT SHOW UP ON THE IMMUNOSTAINING ADDS LITTLE SPECKS, WE SEE THERE'S A REDUCTION IN [INDISCERNIBLE] NUMBER AS WELL. SO THEN WE WERE INTERESTED, COULD WE USE NEUTRON TO INFLUENCE PARASYMPATHETIC FUNCTION. WHAT WE DID HERE WAS DIE EKTED IS OUT THE SUB MANDIBULAR GANGLIA AND ADDS BEADS SOCIETIEDED WITH NEWT TRON AND BSA. WHAT WE COULD REALLY CLEARLY SEE AFTER 24 HOURS AFTER GROWTH IS THAT THAT GANGLIA EXTENDS NEW RITES. WHEN WE LOOK AT HIGH POWER OF THESE NEW RITES, WE SEE THERE'S A LOT OF [INDISCERNIBLE] SUGGESTING THEY'RE FUNCTIONAL AS WELL. THIS STARTEDED TO LEAD THE LAB REALLY TO START THINKING ABOUT THE BIG QUESTION; COULD NEW TU RONUTURON NUTURON -- ALL OUR WORK HAS BEEN DONE IN DEVELOPMENTAL MODELS SO FAR TRYING TO UNDERSTAND NORMAL DEVELOPMENT AND WHAT'S GOING ON AS THE GLAND GROWS AND BRANCHS. WHAT WE DID WAS DEVELOPED A MODEL USING RADIATION OF EMBRYONIC TISSUE BECAUSE WE WANTED TO SEE COULD WE TRANSLATE WHAT WE'RE FINDING DURING EARLY DEVELOPMENT DIRECTLY. WE RADIATED THE GLANDS WITH INCREASING DOSES OF RADIATION, AND NOW WHAT WE WERE ABLE TO DO WAS USE A MOUSE MODEL THAT EXPRESSES KERATIN 5 CONVENIENCE CELLS. WE COULD LOOK IN THE CONVENIENCE CELLS ARE GREEN. WE COULD LOOK DIRECTLY AT THE K 5 PROGENITOR CELLS AFTER RADIATION. WE'VE ALSO STAINED HERE FOR THE NERVES. SO WHAT I'D LIKE TO YOU NOTICE IS THAT IN THE CONTROL GLAND THE K 5 CELLS, ONLY 10% OF THE GLANDS, PRESENT THROUGHOUT THE GLAND ADDS I SHOWED BEFORE IN BOTH INBUD AND DUCTAL STRUCTURES. WHEN WE RADIATE WITH FIVE GRAY, WE CAN STILL SEE THE K FIVE CELL THERE IS. GET A REDMUKS THE -- WITH ZEN GRAY WE GET A MAJOR REDUCTION IN EXXONAL OUTGROWTH. THE GANGLIA ARE STILL HEALTHY AND ALIVE BUT THE K 5 CELLS ARE STILL THERE. A LOT OF WORK HAS BEEN DONE TO SHOW THAT PROGENITOR CELLS ARE -- RADIATION. THE PROBLEM IN -- AFTER PEOPLE HAVE BEEN RADIATED FOR HEAD AND NECK CANCER, THEY DON'T REPAIR THEIR OWN GLANDS. MAYBE THE PROBLEM WAS DUE TO A LOSS OF PARA SYMPATHETIC NERVE. WHEN WE LOOK AT QPCR, WE SEE BETWEEN FIVE AND SEVEN GRAYS, WE ALSO GET DECREASE IN NUTURON EXPRESSION. FOR THESE EXPERIMENTS, WE RADIATED WITH FIVE [INDISCERNIBLE] RADIATION SO WE STILL HAD SOME PARASYMPATHETIC GANG RI YEAH SURVIVING AND ADD NUTURON AFTER RADIATION. WE GOT A DOUBLING IN THE AMOUNT OF BRANCHING FOFR MORPHOGENESIS. YOU CAN SEE THE NERVES AND GANGLIA ARE PRESENT IN THE GLANDS. THERE'S A GLAND AS A CONTROL. WHAT CHANGES IN GENE EXPRESSION ARE OCCURRING WHEN WE'RE ADDING NUTURON TO A GLAND? RADIATION REDUCES GENES INVOLVED IN NEURONAL FUNCTION. THIS IS A BIT OF A COMPLICATED GRAPH COMPARING THE RADIATION WITH THE RADIATION PLUS NUTURON. DOESN'T LOOK ANY DIFFERENT AFTER A RADIATION SO MOST OF THE APOPTOSIS WAS JUST BEGINNING TO OCCUR. THE TISSUES LOOK IDENTICAL BUT WE COULD SEE THERE WAS A MAJOR REDUCTION IN THE WHITE BUDS ON THIS SIDE A REDUCED EXPRESSION OF GENES INVOLVED IN NEURONAL FUNCTION. -- CHROME ONE WHICH IS A [INDISCERNIBLE] RECEPTOR ON THE EPITHELIUM THAT'S STIMULATED BY [INDISCERNIBLE]. WE COULD SEE AN INCREASE IN DUCTAL MARKER. ALL THESE GENES THAT WE KNOW WILL CONTINUE ALONG THE DUCTAL LINEAGE WITH THE INDUCTION OF HBDGF AN IT'S INCREASING. WE ALSO SAW A REDUCTION IN FGF RECEPTOR SIGNALING. WHEN WE ADD NUTURON, WHAT WE SAW WAS A RESTORATION OF A LOT OF NEURONAL FUNCTION. NOT MF CHANGE ON THE DUCTAL MARKER, AND WE SAW SOME RESTORATION OF FGF 1 AND INCREASE IN FGF RECEPTOR TWO SIGNALING. THIS SUGGESTING TO US THAT THE NUTURON WAS STIMULATING THE NEURONS. THEY WERE BECOMING FUNCTIONAL AGAIN AND STIMULATING THE EPITHELIUM TO REGROW. LET ME PUT ALL THAT INTO THE MODEL. THIS IS REALLY SUMMERING UP THE SUPPLE OF DIFFERENT STORIES I'VE BEEN TALKING TO YOU ABOUT TODAY. PARA -- WITH THE ADDITION OF HBGF WE START TO GET -- WITH THE ADDITION OF FGF 10 AND STEM CELL FACTOR, WE'RE GETTING INBUD DIFFERENTIATION WITH K 5 AND K 14. THEN MOST RECENT WILLY WITH THE NUTURON STORY, WE'VE SHOWN THAT THE KIT POSITIVE CELLS ARE PRODUCING THE NUTURON, BINDING THE GFR ALPHA TWO OF THE PARASYMPATHETIC GANGLIA. THIS SETTING UP A LOOP FOR HOW THE TISSUE GROWS AND MAINTAINS ITSELF. WHAT HAPPENS WITH THE RADIATION? WE COME IN HERE T MAJOR POPULATION THAT'S AFFECTED IN THE RADIATION IS THE INBUD OF THE SALIVARY GLAND, WHICH IS THE KIT EXPRESSING CELLS. THEY UNDERGO APOPTOSIS, WE E LOSE NEW UH TRUE RON EXPRESSION, GET A REDUCTION IN PARASYMPATHETIC GANGLIA FUNCTION. WE GET LOSS OF AXON EXTENSIONS, WE LOSE THE ABILITY TO PRODUCE THE INBUD NPS FITS IN WITH WHAT'S BEING SHOWN FOR WHAT HAPPENS IN HUMAN SALIVARY GLANDS. SO THEN THAT LED US TO THINK, WELL COULD NUTURON BE USEFUL FOR HUMAN PATIENTS? WHAT WE WANTED TO DO WAS START LOOKING AT BIOPSIES OF PATIENTSED THAT BEEN RADIATED. WE WERE FORTUNATE IN THE ST INSTITUTE TO HAVE COLLABORATORS THAT HAPPENED TO HAVE BIOPSIES OF RADIATED TISSUES SITTING IN THEIR FREEZER AND WE WERE ABLE TO START LOOKING AT THE PLAYERS I'VE BEEN TALKING ABOUT IN THE TALK TODAY. SO WE ANALYZED BIOPSIES FROM PATIENTS ONE MONTH AFTER A RADIATION. NOW THE REASON THIS HAPPENS IS THAT PATIENTS WITH HEAD AND NECK CANCER GO IN FOR TREATMENT AND IF THEIR TREATMENT IS GOING TO INVOLVE RADIATION, THEY'RE OFTEN START A RADIATION FIRST AND SEE IF THE TUMOR RESPONDS. IF IT DOESN'T, SURGERY HAPPENS, THEY GO BACK IN TO REMOVE THE PRIMARY TUMOR. THIS IS OFTEN WHERE THEY WOULD TAKE A BIOPSY OF THE SURROUNDING TISSUES TO SEE IF THERE'S BEEN ANY SPREAD. THIS IS WHERE WE CAN GET BIOPSIES OF PATIENTS THAT HAVE HAD RADIATION FOR A MONTH. A REAL LOSS OF [INDISCERNIBLE] AND THE PATIENTS ALREADY HAVE A LOT OF SALIVARY FUNCTION BY THIS STAGE. HOWEVER, WHEN WE LOOK AT K FIVE CELLS, WE SEE THERE'S STILL A LOT OF K 5 FIVE CELLS PRESENT IN THE HUMAN GLAND. THIS WOULD FIT IN WITH OUR MODEL. WE SEE THE K FIVE CELLS SURVIVED THE RADIATION. WHEN WE LOOK AT NUTURON EXPRESSION, WE SEE A REDUCTION IN NUTURON EXPRESSION. WE FOUND A FEW END PIECES THAT HAVE GOT A LITTLE BIT OF NUTURON EXPRESSION, BUT MOST OF THE CELLS PRODUCING NUTURON ARE LOST IN THE GLAND OF THE SALIVARY EPITHELIAL CELLS. WHEN WE LOOK BY QPCR AND WHAT WE DID HERE WAS TOOK FROZEN BIOPSY SECTIONS AND USED THEM FOR QPCR, WE COULD SEE THERE WAS A REDUCTION IN NUTURON, [INDISCERNIBLE] CHROME ONE, CHROME THREE AND FGF RECEPTOR 2 B IN THE HUMAN GLAND. THIS IS SUGGESTING THAT SIMILAR THINGS ARE CHANGING IN OUR MAST MOUSE MODEL AS THE HUMAN TISSUE. ALSO AN INCREASE THIS K 19, EGF RECEPTOR AND K 5 WHICH FITS IN WITH OUR MODEL FROM THE MICE. AND THEN WE WERE INTERESTED IN LOOKING AT WHAT'S GOING ON IN TERMS OF NEURONAL STAINING. THIS IS DIFFERENT IN BIOPSIES BECAUSE IF UH YOU TAKE A SPIN SECTION BIOPSY, YOU CAN'T SEE THE NERVES. WHAT SARAH DID WAS TOOK VERY THICK BIOPSY SECTIONS AND STAINED THEM IN A SIMILAR WAY AS WE DO WHOLE MOUSE STAINING OF THE GLAND. IN THIS WAY WE COULD SEE NERVES GROWING THROUGH THE TISSUE. WHAT WAS REALLY TRISTRIKING WITH THE HUMAN TISSUED THAT BEEN NON-RADIATED IS WE COULD SEE THE RED STAINING. THE BLUE IS WITH THE [INDISCERNIBLE] ANTIBODY THAT'S OUTLINING ALL THE BASEMENT MEMBRANES. WITH THE RADIATION, WE GOT LOSS OF THE PARASYMPATHETIC NERVE. THEY WERE LESS PARA SYMPATHETIC NERVES. THERE WAS AN INCREASE IN [INDISCERNIBLE] STAINING. THIS IS A MARKER THAT IS USED FOR SYMPATHETIC NERVES. ONE THING I SHOULD HAVE MENTIONED IS THAT IN THE EMBRYONIC DEVELOPMENT OF THE GLAND, WE ONLY HAVE THE PARASYMPATHETIC NERVOUS SYSTEM. ALL OF OUR EARLY EMBRYONIC WORK IS JUST STUDYING THE PARASYMPATHETIC. AN ADULT TISSUE WE HAVE BOTH SYMPATHETIC AND PARASYMPATHETIC. WHAT THIS DATA IS SUGGESTING HERE IS THAT AFTER A RADIATION, WE HAVE AN IMBALANCE IN THE NERVOUS SYSTEM WITHIN THE GLAND. THIS IS SORT OF WHERE WE ARE AT THE MOMENT. WE WERE VERY PLEASED WITH THESE HUMAN BIOPSIES SEEING A LOT OF OUR MOUSE WORK IS FITTING WITH HUMAN SITUATION. REALLY WHAT THE LAB IS FOCUSED ON DOING NOW IS LOOKING AT THE TESTING VECTORS THAT MIGHT EXPRESS NUTURON IN BOTH EXVI OWE AND VIVO MODELS. NOW I'D ALSO TO HIGHLIGHT THAT NUTURON VECTORS ARE BEING USED IN HUMAN CLINICAL TRIALS AT THE THE MOMENT TO LOOK AT PARKINSON'S'S DISEASE INJECTING THE VIRUSES INTO BRAINS OF PATIENTS WITH PARKIN' SONS TO SEE IF THEY ARE CAN REGENERATE PARASYMPATHETIC. IT'S LOOKING LIKE IT MIGHT BE SOMETHING THAT COULD BE USEFUL APPROACH TO TAKE. SO THANK YOU VERY MUCH FOR YOUR ATTENTION. I'D LIKE TO THANK, AGAIN T FOLKS IN MY LAB MOST OF WHOM I THINK I'VE BEEN ACKNOWLEDGING AS I GO ALONG, PARTICULARLY ISABEL, SHAWN, WENDY AND CAN DICE, SARAH,DICE, SARAH, IVAN, OUR COLLABORATORS AT UCSF AND INTRAMURAL COLLABORATORS WHO HAVE BEEN HELPING WITH THE K FIVE PROJECT AND WITH THE HUMAN BIOPSIES. THANKS VERY MUCH AND I'M HAPPY TO ANSWER ANY QUESTIONS. [APPLAUSE] >> QUESTIONS? PLEASE GO TO THE MICROPHONE. >> SO WHAT IS THAT [INDISCERNIBLE] YOU USED FOR GROWING SOME OF THESE CULTURE CELLS? WE >> WE STARTED OFF USING [INDISCERNIBLE]. WHAT WE'VE CON IS USING PER FIED LAMB NIN ONE. THAT SUPPORTS THE EPITHELIUM TO GROW AND THE PARASYMPATHETIC GANGLIA TO GROW. INVI OWE, I SHOULD SAY, THE MEASURE INCAME CELLS PRODUCING A LOT OF MAY TRICKS. WE NEED TO ADD A MATRIX COMPONENT AND GROWTH FACTOR TO GET GROWTH OF THE EPITHELIAL TISSUE. >> SO AFTER [INDISCERNIBLE]. >>. >> THE NUTURON WILL BE BINDING THE GSR -- IT SIGNALS WITH RED RECEPTOR. WE ALSO KNOW FROM SOME WORK I HAVEN'T SHOWN USING BIOCORE THAT NUTURON IS [INDISCERNIBLE] BINDING GROWTH FACTOR. ADDING IT TO THE TISSUE, WE WOULD EXPECT IT TO BIND TO ALL [INDISCERNIBLE] SULFATE COMPONENTS IN THE EXTRA CELLAR MATRIX. I WILL ALSO SAY THAT WITH THE NUTURON, WHEN WE ADD THAT TO THE DEVELOPING SALIVARY TISSUES, IF WE KEEP ADDING A LOT OF NUTURON, WE UPSET THE FOFR JE TICK GRADIENT FOR THE SERVES. IN OURED MODEL THE INBUD IS PRODUCING THE NUTURON AND THE BUD IS GROWING ALONG TOWARD THE NUTURON. IF WE PUT A LOT OF NUTURON OUT INTO THE MEDIA, NERVES LET GO AND GROW OUT INTO THE MEDIA. THAT'S WHERE I SEE THIS BEING PARTICULARLY HOPEFUL FOR USING VIRAL VECTORS TO INFECT THE REMAINING EPITHELIUM AFTER A RADIATION BECAUSE THEN THE LOCALIZED SOURCE OF NUTURON WILL BE ACTUALLY THE CELLS WE WANT THE TO GET THE IF NERVES GROWING TOWARDS. >> THERE ARE THREE GLANDS, THE FUNCTIONS ARE SIMILAR? >> NO. THEY'RE NOT SIMILAR, AND THEIR INNOVATION IS DIFFERENT TOO. WE'RE FOCUSED MAINLY ON TWO OF THEM AND LOOKING AT THE THIRD AS WELL. BUT THERE ARE DIFFERENCES. >> NICE TALK. ANY PLAN TO STUDY THE EFFECTS OF [INDISCERNIBLE] IN RECOVERING THE FUNCTION OF THE SALIVARY GLAND SUCH AS FLOW RATE OR EVEN COMPOSITION? >> SO WE'RE ACTUALLY AT THE MOMENT WORKING WITH COLLABORATORS HERE AT NIDCR TO START LOOKING IN MOUSE MODELS FOR THAT. WHAT WE WOULD DO IS START WITH DAMAGED MOUSE MODEL THAT HAVE BEEN RADIATED AND USE VIRAL VECTORS AND THERE WE CAN LOOK AT MEASURE FUNCTION OF THE GLAND AS IT RECOVERS. OBVIOUSLY IN OUR MODEL SYSTEM WE DON'T HAVE AN EASY WAY OF DOING THAT BECAUSE WE'RE VERY EARL HLY IN DEVELOPMENT AND A LOT OF THE SECRETED DIFFERENTIATION HASN'T OCCURRED SO WE CAN'T MEASURE THAT EASILY IN THE EX VIVO MODEL. >> THAT WAS BEAUTIFUL. I WAS WONDERING SO YOUR K 14 POSITIVE PROGENITOR CELLS LOOKED TO ME IN A LOT OF THOSE IMMUNOSTAIN LIKE THEY WERE EITHER THE OUTER LAYER OR MAYBE THE ONE RIGHT BELOW IT. HAVE YOU GUYED LOOKED AT -- IS THERE A SPECIFIC ROLE FOR THE BASIC MEMBRANE ITSELF OTHER THAN BINDING PROPERTIES OF NUTURON BASICALLY TO MAINTAIN THE LOCALIZATION OF THOSE K 14 POSITIVE CELLS? >> GOOD OBSERVATION AND YOU'RE PROBABLY RIGHT. WE HAVEN'T LOOKED SPECIFICALLY, BUT IT'S KNOWN THAT THE BASEMENT MEMBRANE THROUGH INTEGRIN RECEPTORS IS PROVIDING A LOT OF QUEUES FOR POLARIZATION OF THOSE CELLS -- THE BASAL LAYER OF CELLS THAT ARE THE K 14 CELLS OFTEN WITH THE [INDISCERNIBLE] SECTIONING IT LOOKS LIKE SOME OF THE INTERNAL CELLS ARE K 14. THEY WILL P LOW K 14 AND MAYBE TRANSITIONING TO ANOTHER PHENOTYPE. SO IT'S PREDOMINANTLY THE BASAL CELLS OF THE GLAND THAT ARE K 14. I'M SURE THERE ARE SOME INTERACTIONS. WE HAVEN'T LOOKED DIRECTLY AT THE ATOP KAL POLARIZATION AND THE EFFECTS OF BASAL MEMBRANE ON THE KERATINS. >> GIVEN A LOT OF THESE PATIENTS KNOW THEY'RE GOING TO BE RADIATED BEFORE THEY GO INTO TREATMENT, HAVE YOU CONSIDERED USING YOUR TERM AS A PROTECTIVE AGENT AND DOING IT BEFORE? >> WE THOUGHT ABOUT THAT AND SO OUR MODELS THAT WOULD SUGGEST THAT'S NOT GOING TO WORK. IF WE PRETREAT THE GLANDS WITH NUTURON BEFORE WE IR RAID YEAH, WE KILL A LOT MORE OF THE NEURONS. THEY'RE MORE SENSE TIFF TO RADIATION. WHAT'S QUITE INTERESTING ABOUT THE WORK WE'VE DONE IS THAT -- AND I SORT OF MENTIONED WE TREAT THEM ONCE FOR FOUR HOURS WITH NUTURON. IT SEEMS LIKE WE ONLY NEED A BOWL OF STIMULUS AFTER A RADIATION, BUT THE WORK WE'VE DONE SO FAR TRYING TO PRETREAT WAS NOT SUCCESSFUL WHEN IT SEEMS TO SENSITIZE THEM TO RADIATION. THAT ALSO FITS WITH OTHER WORK THAT'S BEEN DONE ON THE TIMING OF NEURON PROTECTIVE FACTORS IS OFTEN CRITICAL DEPENDING ON THE MECHANISM AS WHICH WE'RE GOING TO WORK BECAUSE PEOPLE HAVE USED FGF 7 AS WELL AND OBVIOUSLY NOW WE WOULD PREDICT IT'S GOING STIMULATE THE KIT POSITIVE CELLS AND OBVIOUS THINK TIMING OF THAT IS GOING TO BE IMPORTANT TO WHETHER YOU ABLATE THEM IF YOU STIMULATE THEM BEFORE THEIR RADIATION. >> DOES THE ALTERATION IMBALANCE THAT YOU'RE SEEING IN THE HUMAN GLANDS IN THE PARASYMPATHETIC VERSUS SYMPATHETIC SYSTEM IS INTERESTING. ARE YOU SEEING THAT ALSO IN THE MOUSE, AND IS IT POSSIBLE THAT THE SYMPATHETIC SYSTEM IS PRODUCING A NEGATIVE FACTOR OR JUST NOT [INDISCERNIBLE] YOU NEED TO GET THE WHOLE SYSTEM TO GO? >> SO WE HAVEN'T ACTUALLY DIRECTLY LOOKED AT THAT IN THE ADULT MOUSE. >> RIGHT. >> BUT WHAT I WOULD PREDICT BASED ON THE FUNCTIONS OF THE SYMPATHETIC AND PARASYMPATHETIC, THERE'S A NUMBER OF TISSUES AND NUMBER OF SITUATIONS WHERE THEY'RE THOUGHT TO HAVE OPPOSING FUNCTIONS. THE SALIVARY GLAND SOMEONE OF THOSE ODD ONES WHERE THEY BOTH HAVE THE SAME FUNCTION FOR SOME OF THE SECRETE RI FUNCTIONS OF THE GLAND, BUT IN TERMS OF LOOKING AT PROGENITOR CELL MAINTENANCE AND PROLIFERATION AND DIFFERENTIATION, IT'S NOT CLEAR AND IT COULD BE VERY WELL LIKELY, THERE'S LITERATURE I WANT TO SAY IN [INDISCERNIBLE] STEM CELL FIELD WHERE THEY SHOW THAT THE SYMPATHETIC SYSTEM IS PRODUCING NEGATIVE FACTORS, SO IT'S LIKELY THAT COULD BE HAPG. OBVIOUSLY WE HAVEN'T LOOKED IN DETAIL AT WHAT THAT IMBALANCE IS ACTUALLY DOING. >> THANK YOU. >> JUST A A QUICK FOLLOW-UP QUESTION ABOUT THAT. THE IMBALANCE SUGGESTS THAT YOU MIGHT BE ABLE TO FARM LOGICALLY INTERVENE BY BLOCKING SYMPATHETIC ACTIVATION. HAS ANYONE TRIED THAT? >> NO ONE HAS BECAUSE THERE'S BEEN NO RATIONALE BUT THIS IS SUGGESTING THAT IT MIGHT. THAT COULD BE A USEFUL WAY OF GOING. WHAT PEOPLE HAVE TRIED TO DO IS PUT PARASIFRP THE TICK ANALOGS TO STIMULATE THE PARASYMPATHETIC SYSTEM, PARTICULARLY WITH [INDISCERNIBLE] AS ONE OF THEM AND THERE IS SOME LITERATURE THAT THAT DOES IMPROVE THE FUNCTION OF PATIENTS, UM, BUT AT THE TIME, THE IT WAS DONE AS AN EXPERIMENT THAT WASN'T CLEAR HOW IT WAS GOING BE WORKING, SO THERE IS ARE LITERATURE -- THAT'S FROM DUTCH GROUP THAT HAVE TRIED THAT -- BUT NOT FOR THE SYMPATHETIC. >> IF IF NO FURTHER QUESTIONS, LET'S THANK MATT FOR A VERY STIMULATING LECTURE.