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Transcript
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