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Sam Rhine - Genetic Update Conferences - www.samrhine.com "the times, they are aChangin" Bob Dylan - 1964 I. Molecular Biology - let's get the new definitions straight! A. GENOME - sum total of all the genetic information for any biologic organism - DNA or RNA may be expressed as the number of nucleotides pairs human genome - ~3,000,000,000 nts Nucleotides: A T G C - three components 1. Nitrogenous Base 2. Pentose Sugar Molecule 3. Phosphate Complementary Pairs - Base Pairs = bp - Hydrogen Bonds DNA: A - T, G - C RNA: A - U, G - C DNA: (double strand) - 5' to 3' - Double Helix - Deoxyribose Sugar RNA: (single strand) - 5' to 3' - Single Helix - U (Uracil) replaces T - Ribose Sugar B. GENE - 1. sequence of DNA responsible for the production of a specific protein molecule; two step process - transcription & translation 'Coding DNA' gene - carries genetic code for protein production Humans - ~20,000 Coding DNA genes…..Apples - ~57,000! DNA > mRNA (Transcript) > Protein; Transcription and Translation at Ribosome ~20,000 coding genes can produce over 100,000 proteins Gene Expression: DNA sequence with promoter - ON / OFF control region Transcript - CAP beyond 5' end…..poly A tail beyond 3' end 5' UTR and 3'UTR (UnTranslated Region) at the end of each Transcript Ribosome attaches to transcript at end of 5'UTR - beginning of RNA code Human Genome: Coding and NonCoding DNA ~1.5% of the human genome = 'Coding DNA' - carries code for a protein ~98.5% of the human genome = 'Non-Coding DNA' Both Coding and Non Coding DNA found within a single human gene Exons - Coding DNA / Introns - Non-Coding DNA EXons - EXpressed in the Protein Introns - not expressed - must be removed RNA Processing: at the Transcript Remove the Introns - Splice the Exons together - at the Spliceosome Alternative Splicing: 95% of our genes have the capacity to have their exons spliced together in different alternative ways - one gene produces more than one protein! Titin Gene 80,780 bp - 178 Exons - 177 Introns Dystrophin Gene: 2.4 million bp - 79 Exons - 78 Introns Human Genome = 231,667 Exons - average gene has over 11 exons Some Human coding genes: Nested - gene within a gene Bidirectional - coding sequence on both sides of the DNA One coding gene > one protein . cleaved into multiple smaller proteins Neandertal DNA in the genome of modern humans C. GENE CONTROL - how are genes turned ON and OFF ~20,000 coding genes producing > 100,000 proteins…..ON / OFF correct time and place - cell, tissue, organ…..24:7 one mechanism - Transcription Factors - affect the Promoter Activator - attaches to promoter - Positive Gene Control = ON Repressor - attaches to promoter - Negative Gene Control = OFF ON / OFF Errors….. ONCOGENES - ONC Gene Normally Turned OFF - Mutates to Mistakenly Turn ON TUMOR SUPPRESSOR GENES - TSG Gene Normally Turned ON - Mutates to Mistakenly Turn OFF Causes Cancer - >400 known cancer causing mutations D. GENE - 2. sequence of DNA responsible for the production of a specific microRNA molecule; two step process - transcription & shRNA Dicing 'Non Coding' DNA Gene - does not carry the code for a protein does carry the code for RNA only Humans - ~20,000 Coding DNA Genes - located in the 1.5% ~500 NonCoding DNA Genes - located in the 98.5% Total Number of Human Genes: 20,000 + 500 = ~20,500 We can no longer say "Gene" - must designate: 'Coding' or 'Non-Coding' gene RNAs from the Non Coding Genes - sncRNAs sncRNAs = small non coding RNAs 1. rRNA 2. tRNA 3. microRNAs microRNAs = miRNA = miR - ~22 nucleotides stops protein production of a Coding DNA gene negative regulators of Coding DNA genes = OFF controls over 50% of human Coding DNA genes miRNA Gene Silencing coding gene is turned on by transcription factor activator at promoter miRNA attached to the 3' UTR of the transcript leads to blocking the ribosome and blocking translation = OFF Genes have two ON / OFF switches 1. Promoter Switch - affected by Activators and Repressors 2. 3' UTR Switch - affected by miRNA Switch #2 overrides Switch #1 Errors - ONCOmirs - cancer causing mutated Non-Coding genes E. PHENOTYPE - Classic Definition Evaluation of a trait that is observable or testable Hair Color - beware of a Phenocopy! Blood Type - ABO, Rh, MN Cerumen - Ear Wax: Dry or Wet Eye Color F. GENOTYPE - Classic Definition The DNA sequence of the two alleles responsible for one Mendelian trait A A B B A O A O B O B O Blood type A Blood type B Blood type AB Blood type O G. GENOTYPE - New Definition - Genomic Definition The entire DNA sequence of an individual's genome Your 3,000,000,000 base pair nucleotide sequence H. PHENOTYPE - New Definition - Genomic Definition The effect of your Genotype modified to become your EpiGenotype Your Genome is epigenetically modified to become your EpiGenome Your epigenome determines your phenotypes Human Epigenome - special area of genetic study…..Epigenetics Karyotype - banded chromosomes Scanning Electron Micrograph of a chromosome Chromatin - DNA (Genome) + Histone Proteins DNA / Histone Complex Gene Control - ON / OFF / at the Chromatin Level Many times based on Methylation ( -CH3) of C in DNA Methylation of C in C-p-G Island - often at gene promoters Degree of Chromatin Compaction - modification of DNA Highly compacted - add methyl groups - 'OFF' blocks accessibility for transcription enzymes Loosely compacted - lose methyl groups - 'ON' provides accessibility for transcription enzymes Epigenetics - Mat (Maternal) and Pat (Paternal) alleles - both methylated - both "OFF" Genetics v. EpiGenetics - two individuals who are genetically identical - MZ twins identical DNA sequences in their genomes but if they are NOT epigenetically identical….. they can have different phenotypes may become more pronounced over time some epigenetic changes may be Environmental epigenetics can be influenced by the environment Imprinting - special type of epigenetic modification of the DNA - Parent Specific Paternal allele imprinted…..Maternal allele is not - MAT - 'ON' / PAT - 'OFF' Maternal allele imprinted…..Paternal allele is not - PAT - 'OFF' - PAT - 'ON' Most alleles - double dose of the protein Imprinting - single dose of the protein Mammalian imprinted genes - 50? 100? MAT preferential in fetal brain PAT preferential in adult brain >1300! Epigenetic Modifications: Methylation of Chromatin DNA - Methylation of Cytosine Acetlyation of Histone Proteins of chromatin Acetylation of Lysine AA of Histones HAT - Histone Acetylaae - adds acetyl group to histones HDAC - Histone Deacetylase - removes acetyl groups On / Off Errors - Oncogenes and Tumor Suppressor Genes - cause Cancer Epigenetic Errors - On / Off Errors - Hypermethylation of TSG cause Cancer I. -OMES - Genetic Super Suffix Genome - sum total of all the human DNA DNA Sequence - ~20,500 genes - ~3,000,000,000 bp EpiGenome - sum total of all the On / Off modifications methylation and acetylation of chromatin Transcriptome - sum total of all the human transcripts = mRNA - >100,000 Proteome - sum total of all the human proteins - >100,000 Interactome - sum total of all the protein / protein interactions can be used in cancer prognosis Exome - sum total of all the Coding DNA sequences in all the human Exons Exome Sequencing to find mutations in rare Mendelian Syndromes 1.5% of genome Introme - sum total of all the Non-Coding DNA sequences in the human Introns 28.5% of genome Coding Genes make up 30% of Genome…..1.5% + 28.5% J. WHAT'S NEW in MOLECULAR BIOLOGY DNA Replication - DNA Polymerase (DNA Dependent DNA Polymerase) Transcription - RNA Polymerase (DNA Dependent RNA Polymerase) Rule…..all RNA comes from DNA RNA Replication - New Discovery in Humans - Nature, July 20, 2010 RNA Polymerase (RNA Dependent RNA Polymerase) sRNAs - small RNAs - <200 nts New Rule…..RNA comes from DNA and RNA!! K. LIFE - that quality or property of plants, animals and microbes that distinguishes it from inorganic or dead matter ability to metabolize ability to self adapt ability to self reproduce all life comes from pre-existing life - until July 2, 2010 Science - July 2, 2010 J. Craig Venter - 'Synthetic Biology' - Synthesized Life…..'from scratch' Inserted a totally synthetic Mycoplasma mycoides genome into a Mycoplasma capricolum cell "The new cells have all the expected phenotypic properties are capable of self-replication." Synthetic "Life," Ethics, National Security and Public Disclosure - Science, July 2, 2010 II. THE NEW ERA of HUMAN GENETICS A. Introduction - Banded Karyotype / Ideogram Chromosomes Regions and SubRegions with numbering system Locus = address of a gene Alzheimer Gene: 10q24.1 C. Genetic Medicine - the use of knowledge about single genes to improve the diagnosis and treatment of single gene diseases…..~20,000 NEJM - August 26, 2010 D. Genomic Medicine - the use of knowledge about the entire GENOME and NONGENOMIC factors that effect health and disease; new diagnostic and therapeutic approaches to help us understand COMMON MEDICAL conditions There is no 'normal' human genome 'Variants' - differences among individuals 'Normal' or 'Wild Type' - most common variants 'Mutation' - DNA variant that is pathologic / causes disease "We are all Mutants" E. Person to Person Genomic Sequence - 99.6% identical DNA Sequence 0.4% different = 24,000,000 bp in the genome - 24,000,000variants F. Classic Classroom - Genetic Medicine Down Syndrome Patau Syndrome Edwards Syndrome Klinefelter Syndrome Turner Syndrome Trisomy 21 Trisomy 13 Trisomy 18 XXY Monosomy X Easy to Understand - Karyotype…..but most syndromes are RARE Sickle Cell Anemia / Cystic Fibrosis / Tay-Sachs Disease - AR Huntington Disease / Neurofibromatosis / Marfan Syndrome - AD Fragile X Syndrome / Hemophilia / Duchene Muscular Dystrophy - XLR Retinitis Pigmentosa - AR or AD or XLR Easy to Understand - Mendelian - Punnett Square…..but most conditions RARE G. What about the genetics of….. Adult Type 2 Diabetes / Macular Degeneration / Hypertension High Cholesterol / Heart Disease & Stroke / Cancer & Tumors BiPolar Disorder (Manic/Depressive) / ADHD - Attention Deficit / Alzheimer Disease Alcohol Dependency / ASD - Autism / Epilepsy / etc. COMMON DISEASES…..but COMPLEX GENETICS MULTIFACTORIAL - Complex Genetic Traits Autoimmune Diseases Type 1 Diabetes (insulin dependent) / Rheumatoid Arthritis / Multiple Sclerosis Lupus Erythematosis / Scleroderma / Crohn's Disease Graves Disease / Psoriasis COMMON…..but COMPLEX Hypothesis to Explain Complex Diseases: COMMON DISEASES / COMMON VARIANTS HYPOTHESIS Variant = DNA change found in 1% to 5% of the population Persons with the same Common Disease would have a unique set of DNA Variants in Common H. Classic Genetics: Cytogenetics - Chromosomes - Karyotype Monogenic - Mendelian - RARE - Punnett Square - RARE Multifactorial - Complex Traits - COMMON DISEASES I. MULTIFACTORIAL Common Diseases Genetic Component runs through the family twin study evidence - MZ v. DZ no Mendelian inheritance pattern - no AR, AD, or XLR chromosomes are fine Polygenic - many genes involved - 180 polygenes for height Quantitative Traits - Continuous Distribution in a Population Most Common Diseases are part of a quantitative trait Environmental Factors involved H = Heritability - the proportion of Phenotype variation for a particular trait that is due to Genetic differences in a certain population at a certain time Cystic Fibrosis Adult II Diabetes HIV / AIDS FASD / FAS H H H H = 90% = 55% = 5% = 5% E E E E = = = = 10% 45% 95% 95% Human Quantitative Traits - continuous distribution in a population easily measurable traits Height / Finger Print Ridge Count Blood Pressure / Blood Glucose Level / IQ Normal Distribution in a population - bell shaped curve Low / Mean / High 68% of population within one standard deviation of mean if we could find one of the genes for a quantitative trait and locate it's QTL - Quantitative Trait Locus - e.g. 6p24.2 Flipping Pennies Model pennies represent genes for quantitative trait - height normal distribution - bell shaped curve Height: Heads = Tall gene; Tails = Short gene H = 80% Polygenes - Additive / Cumulative - not Dominant and Recessive Medical Conditions - Threshold effect: Blood Pressure > High Blood Pressure = Cardiac Risk Blood Glucose Level > High Blood Glucose = Diabetes Neuron Development > Fewer Connections = ASD - Autism Polygenic / Multiple Gene Model: 1 pair of genes v. 10 pairs of genes falls into a Normal Distribution - each gene with a small effect J. Quantitative Traits and Polygenes in Common Diseases: What we do not know….. 1. How many polygenes for a particular common disease or trait? 2. Where are they located - addresses = QTLs? 3. What do they do - function of that one gene? 4. Can we one day prevent the medical problems? Find a way to keep anyone from crossing the threshold! K. What would it take to find the Polygenes? 1. HGP - Human Genome Project - Director: Dr. Francis Collins Largest ever Scientific Endeavor! 15 Year International Cooperative effort - 20 Countries Mainly US + UK Began: October 1, 1990 projected completion date: Sept. 30, 2005 Actual Completion Date: April 25, 2003 - Watson / Crick Anniversary How many human genes? 100,000 > 30,000 > ~20,000 coding genes Plus the addresses of all the gene loci How many Nucleotides - ~3,000,000,000 'Rough Draft' - June 26, 2000 - US / British joint announcement 1600 Pennsylvania Avenue in DC / 10 Downing Street in London Human DNA - we are all 99.6% Identical DNA Sequence Same among and between racial groups No DNA basis for the term 'Race' Published: Nature - February 15, 2001 / Science - February 16, 2001 "To determine our DNA Sequence is to achieve an historic step forward in Human Knowledge" 2. SNPs - Single Nucleotide Polymorphisms - "snips" - found in >1% of the population >1% = COMMON How many SNPs in me or you? ~3,750,000 If we are all 99.6% identical in our DNA sequences Differences in cause common diseases - must be in 0.4% where we are different SNPs - Small scale variants - ~80% of the 0.4% CNPs - Large Scale variants - ~20% of the 0.4% Mutation - rare nucleotide change - associated with pathology / disease SNPs - Common - normal variant in the population How do we find the SNPs? Search for groups of SNPs - Haplotypes Haplotype - Sets of nearby SNPs HAPMAP - Haplotype Map - find the addresses of the SNP loci Hapmap I 2005 - 1,000,000 most common SNPs Hapmap II 2006 - 10,000,000 SNPs Hapmap III 2010 - 1,440,616 SNPs for detailed studies Average Person: 1 SNP every ~800 bp = ~3,750,000 COMMON DISEASES / COMMON VARIANTS COMMON DISEASES / = COMMON SNPs 3. GWAS - GenomeWide Association Studies - New England Journal Med July 8, 2010 Evaluate the entire genome and see if any SNPs might be Associated with a particular Common Disease GWAS Example: T2D - Type 2 Diabetes - usually adult onset Experimental Group: 2500 people - medically confirmed do have T2D Control Group: 2500 persons - medically confirmed - do not have T2D Scan their 5,000 genomes and check to see which SNPs are present Question? Do the 2500 persons with T2D have a unique set of SNPs in Common that the persons without T2D DO NOT HAVE? YES Tested the Hypothesis: COMMON DISEASE / COMMON VARIANTS AND…..we know the QTLs of those unique SNPS….. one unique T2D SNP is located at 10p12.3 one of the polygenes for T2D must be located at 10p12.3 - Candidate Gene go to Human Genome Map - to 10p12.3 - see which genes are there attempt to link that SNP to a known gene Function GWAS Web Site: www.genome.gov/gwastudies/ GWAS: > 600 GenomeWide Association Studies > 150 Distinct Diseases and Traits > 800 SNP - Trait Associations New England Journal of Medicine - July 8, 2010 Breakthrough of the Year: Science - Dec 21, 2007 How Does it Work? Using CHIPS to find the SNPs CGH Technology - Comparative Genomic Hybridization Microarray Chip = SNP Chip = DNA Chip Glass chip with array of laboratory synthesized DNA fragments Fragments on chip - one side of the DNA molecule - 5' to 3' side = PROBE Location and sequence of all PROBE fragments on array is known Each PROBE fragment on the Chip carries a known SNP sequence Unknown DNA to be tested is chopped into fragments - 3' to 5' side Unknown fragments are tagged with a fluorescent color label Unknown DNA fragments - 3' to 5' - placed on the chip with the PROBES Unknown fragments hybridize (form double helix) with known fragment Laser Scanner 'reads' the fluorescent colored spots on the Chip Presence & Intensity of colored spots identifies the SNPs in unknown DNA Affymetrix 'Gene Chip' - >1.8 million probes = genetic markers 906,600 probes for SNPs + 946,000 probes for CNPs GWAS STUDIES: T2D - Adult Onset Type 2 Diabetes 18 SNPs = 18 Polygenes - 18 QTLs QTLs connected to sugar metabolism pathway genes T1D - Juvenile Onset Type 1 Diabetes - autoimmune disease 18 previous SNPs + 14 newly discovered SNPs = 32 Polygenes / QTLs QTLs connected to HLA - immune system genes Inflammatory Bowel Disease Crohn's Disease 32 SNPs - 32 QTLs Ulcerative Colitis 10 SNPs - 10 QTLs Some QTLS connect to autophagy gene loci GIANT Studies - Genomic Investigation of ANthropormorphic Traits GWAS for BMI, Obesity, Height, Weight, Adiposity 6 SNPs = 6QTLs - BMI and Risk of Obesity Genes 9 SNPs = 15 QTLs - Hypothalmic Weight Control Height - H = 80% - 180 SNPs = 180 QTLs - only 20% of H - 700 total? Malaria Resistance Medication SNPs Blood Lipids - major indicator of heart disease 100,000 persons tested - 95 distinct gene variants 59 never found before SNPs and I.Q. - g = general cognitive ability - Scientific American - Oct 2008 SNPs and Human Personality Traits Neuroticism / Extraversion / Openness / Agreeableness / Conscientiousness / Music Ability Pet Project - Neuropsychiatric Disorders: Nature - August 26, 2010 Doberman Pinscher - narcolepsy / obsessive compulsive Golden Retriever - aggression / dominance / seizures English Cocker Spaniel - epilepsy / sudden onset aggression Dalmation - deafness / kidney stones / aggression GWAS - Big Questions….. ONLY ~12% of GWAS SNPs are located in coding gene regions ~40% of GWAS SNPs are in non-coding introns ~49% of GWAS SNPs are in intergenic regions may indicate intronic or intergenic gene control elements "Missing Heritability" T2D: 18 SNPS - 18 Polygenes - H = 55% - only 6% of H 94% - "Missing Heritability Height: 180 SNPs - only 20% of H ?? Look Deeper - 500,000 persons with SNP evaluations? 'Revolution Postponed' - Scientific American - October 2010 4. CNPs - Copy Number Polymorphisms Normal - 2 copies / CNP = 1 copy or 3 copies Large Scale Variation - ~20% of the 0.4% INDELS - INsertions DELetions Rearrangements Copy Number Variant Most Common Example - Down Syndrome all genes on chromosome #21 - x3 Epilepsy / HIV / Heart / Autism Spectrum / Schizophrenia / Tourette's ASD - Autism: repetitive behaviors severely restricted interests difficulty with social interactions & communications carry a higher load of rare CNPs some inherited from parents some having arisen de novo 5. TCGA - The CANCER GENOME ATLAS Acute Myeloid Leukemia Thyroid Cancer Prostate Cancer - CaP - Prostate Cancer 25 SNPs - predisposition SNPs 1. Increasing Age, 2. African Ancestry, 3. Family History Breast Cancer - 17 predisposition SNPs Monthly self evaluations! Lung Cancer - Tumor cells contain up to 50,000 SNPs….. Cancer causing Mutations! 6. HUMAN MICROBIOME PROJECT 100,000,000,000,000 - cells in the human body 10,000,000,000,000 - human cells 90,000,000,000,000 - Microbes: Bacteria, Fungi, Protozoa Major Environmental Factors - Multifactorial III. Your Personal Genome Evaluation 'The Language of Life' - book by Dr. Francis Collins - Director of NIH 'Your Life in Your Hands' - Nature, November 6, 2008 'Know Your DNA' - Scientific American, January 2006 Today - Check you Whole Genome - 3,000,000,000 bp - $25,000 Partial Genome & Common SNPs - $1,000 Faster and Cheaper Sequencing in the Future…..Whole Genome - 2014 - $1000 2020 - $100 Navigenics / 23andMe / DeCODEme / National Geographic - trace origins HHS - Construct Your Own Detailed Family History / Pedigree Online www.familyhistory.hhs.gov/ Whole Genome Evaluations: Combined for HGP James Watson / J. Craig Venter First African / First Asian / First Cancer Patient - Nature, Nov 6, 2008 The '1000 Genome Project' - began in January 2008 Collaboration among US, UK, China, Germany Produce an extensive catalogue of human genetic variation - SNPs & CNPs Goal is to provide a resource of almost all human variants Will allow more extensive GWAS studies / Hapmap III is a part of it Over 1000 genomes of unidentified individuals from around the world Form the basis for ethnic group comparisons and tracking people groups Three Pilot Studies….. 1. Deep sequencing of Mother-Father-Adult child trios 2. Light sequencing to see how data can be combined across samples 3. Cataloguing Coding DNA Exome regions of the 1000 genome Web Site: http://www.1000genomes.org/page.php Your Personal DNA Chip . the "Genomic Era of Medicine' - Personalized Medicine Pharmacogenetics - Personalized Prescriptions - Personalized Cancer Therapy GPP - your Genetic Predisposition Profile - your Medical Future Do you want to know? / Who else should know? / Who pays? Insurance? Who should have access? / Where will you keep the information? Prenatal Diagnosis and SNP evaluation? GINA - Genetic Information Nondiscrimination Act Who is going to explain it to the average person? IV. Regenerative Medicine and Tissue Engineering Why? >100,000 persons on waiting lists: heart, lung, liver, kidney, pancreas, intestines - too few donors! average person on liver waiting list…..waits 26 months! average person on lung waiting list…..waits 3 years! ~50% of those over 50, waiting for a kidney, will die while waiting! Regenerative Medicine: inducing human tissues that are damaged, worn out or missing….. to return to their normal state persuading the body to heal itself through the delivery, to the appropriate site, of cells, biomolecules and / or supporting structures Tissue Engineering: production of human tissues and / or organs in the laboratory and their subsequent transfer back into the body to compensate for an organ / tissue that was not functioning normally Tissue and Organ Sourcing: Histocompatability: ABO / Rh; HLA (Human Leukocyte Antigens) 1. Autograft (Autologous) - Auto = self use your own tissue 2. Isograft (Isologous) - Iso = equal use tissue from an identical twin First Kidney Transplant - December 23, 1954 - Dr. Joseph Murray - Harvard Medical School Nobel Prize in Medicine - 1990 Herrick Twins - Ronald / Donor…..Richard Recipient 3. Allograft (Allogenic) - Allo = Different use tissue from a different person rejection - may need immunosuppression 4. Xenograft (Xenologous) - Xeno = foreign use tissue from a different species 5. Biocompatible material - inert = not rejected plastic, titanium, aluminum, ceramics What causes rejection? Immune System: Discriminates between 'Self' and 'NonSelf' Host v. Graft - most rejections - Host rejects the transplanted tissue Graft v. Host - some rejections - transplanted tissue rejects the host GVHD - Graft Versus Host Disease Bone marrow transplants / Face transplants Donor Cell or Tissue Sources: Stem Cells - Embryonic Stem Cells Adult Stem Cells iPS - induced Pluripotent Stems - man-made from skin SCAFFOLD - Framework for the engineered tissue or organ Biocompatible / Biodegradable Materials Polymers - PGA: PolyGlycolic Acid Ceramics - Collagen - Natural TISSUE ENGINEERNG EXAMPLES: HSE - Human Skin Equivalent Scaffold = bovine type I collagen + Epidermis = keratinocytes + Dermis = fibroblasts Primary Utilization - skin grafts in burn victims / bed sores / diabetic skin ulcers EB - Epidermolysis Ballosa MIM = #2267000 Genetically Engineered Skin - Tissue Engineering + Genetic Engineering Spray-on Skin Cells: http://www.msnbc.msn.com/id/36232647/ns/health-health_care/ Skin Cell Donors - newborn baby boys…..one donation > 6 football fields Urinary Bladder often too small - due to neural tube defect Urinary Bladder - two layers of cells: inner Uroepithelium / outer Smooth Muscle Recipe: Biopsy existing bladder to obtain cells for new bladder - autologous Separate Epithelial cells from Muscle Cells Expand both cell types in vitro = mitosis Transfer to Biocompatible Biodegradable Scaffold - made of PGA Biopsy to surgical insertion = 7 - 8 weeks! Future - bladder cancer! Nose - Beck Weathers - severe frost bite - PGA scaffold seeded inside and out with patient's own cells - autologous Ear - Treacher-Collins Syndrome - Biocompatible BUT NOT Biodegradable scaffold Grown in petri dish for start - transferred to under the skin of a mouse for completion Chin / Jaw - facial cancer - titanium mesh cage scaffold + bone mineral blocks + BM mixture + recombinant human BMP-7 - grown initially in latissimus dorsi muscle under patient's arm Finger - porous ceramic scaffold, seeded with patients cells Arteries / Veins - made in a flat sheet and then rolled into a tube - like pasta Cornea - grow new cornea cell layer within a plastic boundary ring - 21 days biopsy cornea epithelium of one eye - cells to make new cornea for the other eye biopsy oral epithelium to make new cornea for both eyes - transfer with no suture Teeth - grow an organ in a test tube Encapsulated Cell Therapy - capsule the size of a pencil lead - contains cells making therapeutic protein - protein exits through pores but immune cells cannot enter to induce rejection Retinitis Pigmentosa / Huntington Disease Ligaments / Tendons - Orb Weaver spider web protein - strongest biological molecule Produced by transgenic goats or E. coli Sutures - covered with autologous cells / can be genetically engineered Petroleum Jelly - 'Vaseline' Gene Therapy - for external use on skin Kidney - First Man-made kidney - 'Organoid' - 2004 - made from bovine ESCs Artificial human kidney lined with human renal cells Liver - inject liquid polymer into liver vasculature digest away all the liver cells - leaving 3-D pattern of normal liver vasculature vasculature pattern is programmed into computer program permits making layers of scaffold with holes for blood vessels 'Liver Cell Jet Printer' distributes liver cells across the scaffold layers layers are tiered back together > Liver in vitro Heart - perfectly natural / normal heart scaffold exists within every heart Decelluarization - Detergent Perfusion > Heart Scaffold > Recellularization Rat Lung recelluarized with lung cells from murine ESCs Mouse Liver - decellularization and recellularization Trachea - damage from tuberculosis - stripped and reseeded with MSCs from Bone Marrow Heart Valve - anomaly in unborn baby - amniocentesis to obtain MSCs used to construct a new heart valve before birth - surgical replace valve at birth O.I. - Osteogenesis Imperfecta - very fragile bones prone to multiple fractures picked up in utero - mothers MSCs injected into the umbilical cord before birth - corrects OI Finger Regeneration - use powder from dessicated bladder of a pig - grew back in 4 weeks http://www.cbsnews.com/video/watch/?id=3805459n MaSC - Adult Mammary Stem Cell - regenerate murine mammary gland from one stem cell PSC - Prostate Stem Cell - regenerate murine adult prostate from one stem cell Mouse / Rat Chimeras - Blastocyst Complementation - in vivo Organogenesis produce an organ of one species from the embryo of another species produce pancreas of a rat in a mouse place rat iPSs in the blastocyst of a pancreatic- mouse Future therapy for human Type I diabetes? Hand Transplant Bilateral Arm Transplant Ovary transplant Face transplants Genetic Counseling Programs: http://www.nsgc.org/iframepages/GeneticCounselingTrainingPrograms/tabid/336/Default.aspx