Sam Rhine - Genetic Update Conferences - www.samrhine.com

- GWAS and Stem Cells Revisited -Genetics of Common Human Traits and Diseases…..

their Multifactorial / Polygenic Origins A. HISTORICALLY…..TEACHING of HUMAN GENETICS:

BOX 1 - Cytogenetics and BOX 2 - Monogenic ‘Genetic Medicine’ - use our knowledge about chromosomal syndromes (~6100) and single gene disorders (~23,008) to improve the diagnosis

and treatment of our patientsB. CLASSIC GENETIC CLASSROOM: Teaching Genetic Medicine

1. BOX 1 - Cytogenetics: Chromosomes / Karyotypes / Non-Disjunction:a. Down Syndrome Trisomy 21 (1/800)b. Klinefelter Syndrome XXY (1/500 males)c. Edwards Syndrome Trisomy 18 (1/6000)d. Patau Syndrome Trisomy 13 (1/10,000)e. Turner Syndrome Monosomy X (1/5000 females) EASY to Understand - via Karyotype - but most syndromes are very RARE

2. BOX 2 - Mendelian: Monogenic Traits - AD, AR, XLR, Mito - Punnett Squares:a. AR - Sickle Cell Anemia / Cystic Fibrosis / Tay-Sachs Diseaseb. AD - Huntington Disease / Neurofibromatosis / Marfan Syndrome

c. XL - Fragile X Syndrome / Hemophilia / Duchene Muscular Dystrophyd. AR, AD, XLR or MITO - Retinitis Pigmentosa EASY to Understand - via Punnett Square - but most conditions are very RARE

3. Why don't you genetics people study the genetics of…..Common Conditions:Adult Type 2 Diabetes / Macular Degeneration / Hypertension High Cholesterol / Heart Attack & Stroke / Tumors & Cancer Bi-Polar Disorder (Manic/Depressive) / ADHD - Attention Deficit / Alzheimer Alcohol Dependency / Autism Spectrum - ASD / Epilepsy / Asthma / etc. …..COMMON DISEASES…..but COMPLEX GENETIC TRAITS

4. Why don't we study the genetics of …..Autoimmune Diseases:Type 1 Diabetes (insulin dependent) / Rheumatoid Arthritis / Multiple Sclerosis Lupus Erythematosis / Scleroderma / Crohn's Dx / Grave's Dx / Psoriasis

…..COMMON DISEASES…..but COMPLEX GENETIC TRAITS

C. SECRET: There has always been a THIRD BOX…..that was ignored for many decades!BUT BOX 3 WAS PADLOCKED…..FOUND the KEY - April 25, 2003

1. MULTIFACTORIAL - Box 3 - Complex Genetic Traits - COMMON DISEASES the KEY to the New Era of Human Genetics…..the HUMAN GENOME the Most Important Box! - the ‘New Era’ in human Genetics the Most Important Area of Human Genetics

2. GENOME: The sum total of all the genetic material for any biologic organism, may be DNA or RNA, expressed as the total number of nucleotides.

Genetic Update Conferences - 2015-2016

Large genome: Human (DNA) > 3,000,000,000 nts / ~34,500 genes Small genome: HIV virus (RNA) 9,749 nts / 9 genes3. NEW ERA in HUMAN GENETICS: ‘Genomic Medicine’

Use our knowledge of the entire GENOME plus ENVIRONMENTAL factors in POPULATIONS of people…..to understand COMMON MEDICAL conditions

a. Opens Up the ‘Genetics of Common Diseases’

b. NO 'Normal'…..no normal human genome

c. 'Variants' - millions of differences among individuals in a population SNPs - small variants and CNPs - large variants

d. 'Mutation' - DNA variant that is pathologic - causes genetic disease or cancer"We are all Mutants" - ~60 new mutations per generation

Person to Person Genomic Sequence - 99.6% identical DNA Sequences 0.4% different DNA Sequences

0.4% different = ~24,000,000 nucleotides variants in the genome~24,000,000 variants in DNA sequence between any two people.

4. 'COMMON DISEASES / COMMON VARIANTS HYPOTHESIS'

a. Mid 1990s - Hypothesis to try to explain Common Diseases

b. Persons with the same COMMON DISEASE….. would have a unique set of DNA VARIANTS in COMMONc. Variant = DNA change found in 1% to 5% of the population

d. Quantitative Human Traits: Traits that are easily measured – quantified

Continuous Distribution in a PopulationMost Common Diseases are Part of a Quantitative Trait

5. MULTIFACTORIAL HUMAN CONDITIONS - Box 3

a. BIOLOGIC BASIS of COMMON DISEASES

b. QUANTATATIVE HUMAN TRAITS –

easily measurable traits - easy to quantify: continuous distribution in a POPULATION

Height, Weight, Finger Print Ridge Count, Blood Pressure, Blood Glucose Level, IQ, etc.

Human Quantitative Traits in a general population….. normal distribution - bell shaped curve

Low Average High

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*COMMON DISEASES are part of a QUANTATIVE TRAIT*

c. VARIABLE EXPRESSION in DISEASES - from very mild to very severe

d. COMMON DISEASES - known to have a GENETIC COMPONENT run through families

twin studies - compare MZ (identical) v. DZ (fraternal)chromosomes are fine - not part of Box 1no Mendelian inheritance pattern…..AR, AD, XLR - not part of Box 2

e. POLYGENES - POLYGENIC - many pairs of gene involved ie: human height…..predicted to be ~700 polygenes involved

f. ENVIRONMENTAL FACTORS also play a significant role

HERITABILITY: H - the proportion of Phenotype variation for a particular trait that is strictly due to Genetic differences in a certain population at a certain time

Height H = ~80% E = ~20% Cystic Fibrosis H = ~90% E = ~10%Adult II Diabetes H = ~55% E = ~45%HIV / AIDS H = ~5% E = ~95%

g. MULTIFACTORIAL = GENOME + ENVIRONMENTAL EFFECTS

6. HUMAN QUANTATIVE TRAITS…..Locating Polygenes on a Chromosome

IF we could find one of the polygenes for a quantitative trait

a. locate it's QTL - Quantitative Trait Locus - e.g. 6p24.2 (chromosome #6, short arm (p), region 2, subregion 4.2)

b. then check the genes at 6p24.2 from human genome map database

c. relate that polygene to the gene function - what does that gene do?

d. how could that gene contribute to that quantitative trait?7. Flipping Pennies Model…..within a Normal Curve in a Population

a. each penny represents one polygene for a quantitative trait - height

ten polygenes (actually ~700) …..five from Mom and five from Dad

b. Height: Head = Tall gene / Tail = Short gene

c. Polygenes - Additive or Cumulative - NO Dominant & Recessive

d. Reassortment - the mixing of genes and genetic material from the parentsinto new combinations of genes in their offspring

d. This Means: Two parents of average height…..

WILL USUALLY have an average height children - most likely - near the Mean

BUT…..two average height parents can have a very tall child OR……two average height parents can have a very short child

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WHY?…..Genetic Reassortment: that is how polygenes work…..all the polygenes reassort every generation yielding potentially thousands of new and unique possibilities

e. Polygenes and Medical Conditions - Add THRESHOLD effect to the curve:

Blood Pressure: Most people have average blood pressure

High Blood Pressure - cross threshold - Cardiac Risk Low Blood Pressure - cross opposite threshold - BP concerns

Blood Glucose Level: Most people have average blood glucose levels

High Blood Glucose - cross the threshold - DiabetesLow Blood Glucose - cross the opposite threshold

Neuronal Synaptic Pruning in Early Development:

Most people have the average number of synaptic connections Less Pruning = Excessive Connections - cross threshold - Autism (ASD)

Extra Pruning = Fewer Connections - cross opposite threshold

f. TWO perfectly NORMAL parents…..from the middle of the normal curve

Can have an AFFECTED child at one extreme or the other!!

Because of the polygenes Reassortment every generation

D. QUANTATIVE TRAITS and POLYGENES in COMMON DISEASES:

What we do NOT know…..a. How many polygenes for a particular common disease or trait?b. Where are the polygenes located - their QTLs = chromosomal addressc. What do those genes do - what is the function of that one gene?d. Can we one day prevent the medical problems?

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…..Modify the polygenes in such a way so that we could …..Correct a Polygenic Disease

…..Stop people from Crossing the Threshold!

E. What would it take to find the Polygenes for Common Diseases?

1. HGP - Human Genome Project - Director: Dr. Francis Collins

Largest ever Scientific Endeavor!15 Year International Cooperative effort - 20 Countries - mainly US + UKBegan: October 1, 1990 - projected completion date: Sept. 30, 2005Actual Completion Date: April 25, 2003 - Watson / Crick Anniversary 1953How many human genes? 100,000 predicted > 23,000 coding genes found

Plus the addresses of all the gene loci >3,000,000,000 nucleotide pairs

~1.5% Coding DNA / ~98.5% Non-Coding DNA'Rough Draft' - June 26, 2000 - US / British joint announcement

1600 Pennsylvania Avenue in DC / 10 Downing Street in LondonHuman DNA - we are all 99.6% Identical DNA Sequence…..0.4% different

Same among and between all international people groups No DNA basis for the term 'Race'

Rough Draft Published: Nature - February 15, 2001 / Science - February 16, 2001

"To determine our DNA Sequence is to achieve an historic step forward in Human Knowledge"

Genome Magazine - FREE subscription - http://genomemag.com

2. SNPs and CNPs

a. SNPs - Single Nucleotide Polymorphisms = SNVs - Single Nucleotide Variant "snip" - found in >1% of the population = common how many SNPs in the average person? ~3,750,000

b. CNPs - Copy Number Polymorphisms = CNVs - Copy Number Variants "cnip" - found in >1% of the population = common

c. We are all 99.6% identical in our DNA sequences

d. Differences that cause common diseases - must be in 0.4% where we are different

SNPs = SNVs - Small Scale variants - ~80% of the 0.4% Change one nucleotide…..A > T; G > C

CNPs = CNVs - Large Scale variants - ~20% of the 0.4% Add or Lose 1 kilobase up to 5 megabases - add or lose a gene

2 copies of a gene > 1 copy or 2 copies of a gene becomes > 3

SNPs & CNPs - Common - Normal Variants in the population

e. How do we find the SNPs? Search for groups of SNPs - Haplotypes

Haplotype - Sets of nearby SNPs - located close together on a chromosome HAPMAP - Haplotype Map - find the addresses of the SNP loci

Hapmap I 2005 - 1,000,000 most common SNPsHapmap II 2006 - 10,000,000 SNPsHapmap III 2010 - 1,440,616 SNPs for detailed studies

Makes Possible: Routine, Affordable SNP & CNP testingAverage Person: 1 SNP every ~800 bp = ~3,750,000

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f. HYPOTHESIS: COMMON DISEASES / COMMON VARIANTS…..OR

COMMON DISEASES / COMMON SNPs

F. GWAS - GenomeWide Association Studies

1. Evaluate the entire genome of thousands of people, in a population, all at one time

2. See if any unique set of SNPs might be Associated with a particular Common Disease

3. GWAS Example: T2D = Type 2 Diabetes - usually adult onset

a. Experimental Group: 2500 people - medically confirmed DO have T2Db. Control Group: 2500 persons - medically confirmed - DO NOT have T2Dc. Scan their 5,000 genomes and check to see which SNPs variants are presentd. Question? Do the 2500 persons with T2D have a unique set of SNPs

in Common that persons without T2D DO NOT HAVE? YESe. AND…..we know the QTLs of those unique SNPS…..

one unique T2D SNP is located at 10p12.3one of the polygenes for T2D must be located at 10p12.3 - Candidate Gene

f. go to Human Genome Map - to 10p12.3 - see which genes are thereg. attempt to link that SNP to a known gene Function

4. We have TESTED the CD/CV HYPOTHESIS and it is VALID

COMMON DISEASE / COMMON VARIANTS HYPOTHESIS = VALID

G. GENOMICS of COMMMON DISEASES - MIT & Harvard - Sept 6 - 9, 2008 'The Beginning of the New Genetics'

GWAS STUDIES:

1. T2D - Adult Onset Type 2 Diabetesa. 18 SNPs = 18 Polygenes - 18 QTLs / today = 53 b. QTLs connected to glucose metabolism pathway genes…..300 total

2. T1D - Juvenile Onset Type 1 Diabetes - autoimmune diseasea. 18 previous SNPs + 14 new SNPs = 32 Polygenes / QTLs / today = 45b. QTLs connected to HLA - immune system genes

3. Inflammatory Bowel Disease - Crohn's and Ulcerative Colitisa. Crohn's Disease - 32 SNPs - 32 QTLs / today = 71b. Ulcerative Colitis - 10 SNPs - 10 QTLs / today = 47

4. GIANT Studies - Genomic Investigation of ANthropormorphic Traits GWAS for BMI, Obesity, Height, Weight, Adiposity

a. Obesity - 6 SNPs = 6QTLs - BMI and Risk of Obesity Genes / today = 34 9 SNPs = 15 QTLs - Hypothalmic Weight Control Axis in brain

Obesity is a polarizing polygenic trait with thin habitus

b. Height - H = 80% - 180 SNPs = 180 QTLs - only 20% of H - 700 total? Single Gene Mutation - override 700 polygenes effect - achondroplasia

5. Blood Lipids - major indicator of heart disease 100,000 persons tested - 95 distinct SNP and CNP variants 59 new - never found before

6. SNPs and I.Q. - g = general cognitive ability - Scientific American - Oct 2008

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7. SNPs and Human Personality Traits: Neuroticism / Extraversion / Openness / Agreeableness / Conscientiousness / Music Ability / Leadership Skills Leadership Skills……….All human behaviors polygenic/multifactorial??

8. SNPs and Human Facial Appearance - VisiGen - International Visible Trait Geneticsa. 5 polygenes found that affect human facial shapeb. 24 polygenes for eye and hair color - Forensic - facial ID from blood SNPsc. Hope to identify victims / perps face from DNA in blood samples at crime scenes

9. Five major psychiatric disorders with shared risk effects; 60,000 person GWAS a. ASD - Autism Spectrum Disorders; b. Schizophrenia;

c. Bipolar Disorder; d. Major Depression; e. ADHD These five conditions have a group shared SNPs…..

2 SNPs were for genes affecting calcium channels – might lead to therapy?

10. GWAS - Big Questions…..a. ONLY ~12% of GWAS SNPs are located in coding DNA gene regionsb. ~40% of GWAS SNPs are in non-coding intronsc. ~49% of GWAS SNPs are in intergenic regionsd. may indicate intronic or intergenic gene control elements e. "Missing Heritability"

H. CNPs = CNVs - Large Scale variants - ~20% of the 0.4% Add a gene or Lose a Gene: Add or Lose from 1 kilobase up to 5megabases of DNA

1. Normal - 2 copies of a gene / CNP = 1 gene copy or 3 gene copies, etc.2. Large Scale Variation - add or lose a whole gene or genes3. INDELS: INsertions / DELetions or Rearrangements

a. MAY be INHERITED…..passed from the parents , orb. MAY arise DE NOVO…..mistake in meiosis - not passed from parents

‘Genetic but not Inherited’c. Copy Number Variant - three instead of two, or one instead of twod. Most Common Example - Down Syndrome - all genes on chromosome #21 - x3

4. Epilepsy / HIV / Heart / Schizophrenia / Tourette / Obesity / Autism

I. AUTISM EXAMPLE - Autism Spectrum Disorders 1 in 68 children.....1 in 42 males.....Males : Females = 4 : 1

1. Spectrum - from very mild to very severe effects2. may be Autism only - Simplex Family - one child with autism

Multiplex Family - two or more children w/ autism3. maybe a Syndrome: Fragile X, Rett, Tuberous Sclerosis, Angelman4. Many forms of Autism = Many different causes

            Neurodevelopmental disorder characterized by:         Social Impairments, Cognitive Impairments         Communications Difficulties, Repetitive Behaviors              Can occur in any ethnic, socioeconomic and age groups.             some children with autism appear normal before age 1 or 2 then suddenly 'regress' and lose language skills they had previously gained - this is the ‘Regressive’ type of autism.            1 2 3

GWAS RESULTS: GWAS Trios - normal mother and normal father with autistic child

1. Children with autism carry a higher load of rare CNVs 2. Some inherited from parents as polygenes - others having arisen de novo 3. de novo CNVs account for 5-8% of simplex cases - paternal age effect

4. Recent Information - perhaps the main cause of ASD are “Ultra-Rare” mutations of genes that are classified as “Vulnerable” genes which harbor an LGD - “Likely Gene Disruption” - and the LSD mutations can occur de novo – genetic but not inherited.

PNAS - on line, September 23, 2015 5. Similar results Mental Retardation - numerous de novo mutations with Exome Trios

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6. Examples of CNVs and Reciprocal Variants, +1 gene v -1 gene - Phenotypic Opposites Obesity < 1 - Underweight >1Macrocephaly < 1 - Microcephaly > 1

Autism < 1 - Schizophrenia > 1

MUTATION and HUMAN DISEASE - Science (special issue) - September 25, 2015 J. SNPs (SNVs)and CNPs (CNVs) associated with:

Migraines / Fatty Liver Disease / Glaucoma / Narcolepsy / Alzheimer DiseaseScleroderma / Intracranial Aneurysms / Essential Tremor / EpilepsyOsteoarthritis / Osteoporosis / Restless Leg Syndrome / Tobacco DependencyCerebral Palsy / Attention Deficit Hyperactivity Disorder / PsoriasisAsthma / Voter Turnout / Criminal Behavior?.....one Indy family…..50 convictions!

K. All GWAS results available at One Web Site: www.genome.gov/gwastudies

L. Early Genetics…..1800's - 19th Century - Inheritance = BLENDING Blending of Characters of Mother & Father

Beginning in 1900's - 20th Century - Inheritance = PARTICULATE Particles called genes - Mendelian Inheritance

Box 3 - 21st Century - BLENDING of PARTICLES called POLYGENES from Mother and Father reassort every generation

M. DEPENDENCY STUDIED1. SAGE - Study of Addiction Genetics and Environment2. COGA - Collaborative Study of Genetics of Alcoholism3. FSCD - Family Study of Cocaine Dependence4. COGEND - Collaborative Genetic Study of Nicotine Dependence

N. HUMAN MICROBIOME PROJECT - sum total of all the microbes of human body “the Other Genome” - Major Environmental Factors

1. 800,000,000,000,000 - Cells in Human Body…..American Academy Microbiology

80,000,000,000,000 - Human Cells…..~220 different types

720,000,000,000,000 - Human Microbiome - Bacteria, Fungi, Protozoa - Live in Us and On US >2,000 different species - weighs ~2 ½ pounds

2. You think you are ONLY human…..You better think again! Human Microbiome holds the key to many future medical treatments

3. “Instead of declaring war on microbes, we need to think in the context of microbial ecosystems within our bodies. Figuring out how to encourage good microbes to thrive, while eliminating the bad ones will be of increasing future importance.”

“We are all surrounded by our own personal microbial cloud…..24/7”

4. “We are the Sum of Our Parts” http://www.the-scientist.com//?articles.view/articleNo/43379/title/The-Sum-of-Our-Parts/

5. Personal Microbiome Evaluation - $99: * http://humanfoodproject.com/americangut *

6. MULTICACTORIAL = GENOME + ENVIRONMENTAL EFFECTS

O. TCGA - THE CANCER GENOME ATLAS - discover all human predisposing SNPs1. Prostate Cancer - 45 predisposition SNPs2. Breast Cancer - 29 predisposition SNPs…..Hereditary Breast & Ovarian Cancer3. Colorectal Cancer - 20 predisposition SNPs

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4. Lung Cancer - Tumor cells contain up to 50,000 SNPs…..Cancer Causing Mutations!

P. OPEN TREE of LIFE: the first comprehensive genomic tree of life You can check how closely related you are to any other biologic organism http://tree.opentreeoflife.org/

Q. THE '1000 GENOME PROJECT' - began in January 2008 - http://www.1000genomes.org/

1. Collaboration among US, UK, China, Germany and nine other countries 2. Produce an extensive international catalogue of human genetic variation - SNPs & CNPs3. Goal was to sequence at least 1000 persons to provide a resource for almost all human variants4. Evaluated 2,504 total genomes from 26 people groups from six continents - form the basis for

ethnic group comparisons and tracking people groups5. Project Completed - Nature (cover article) October 1, 2015

R. YOUR PERSONAL TOTAL GENOME EVALUATION

1. 'The Language of Life' - book by Dr. Francis Collins - Director of NIH

2. Construct Your Own Detailed Family History / Pedigree Online via HHS www.familyhistory.hhs.gov

3. Faster and Cheaper in the Future…..Whole Genome: 2015 - $1000 / 2020 - $100

4. www.23andMe.com - can check anyone today for ~35 medical conditions for $199!

S. YOUR PERSONAL DNA CHIP

1. 'Genomic Era of Medicine' - 'Personalized Medicine' or ‘Precision Medicine’2. Pharmacogenetics - Personalized / Precision Prescriptions and Cancer Therapy3. GPP - your personal Genetic Predisposition Profile - your Medical Future

a. Do you want to know? b. Who else should know? c. Who pays? Insurance? d. Who else should have access?

e. Where will you keep the information? f. Who will explain it?

4. GINA - Genetic Information Nondiscrimination Act

5. Prenatal Diagnosis and SNP evaluation of the unborn?

Your unborn baby is:a. XY - boy b. height SNPS - he will be about 6'1" c. T2D SNPs - ~4.1% chance for adult onset Type 2 Diabetes d. ~1.9% chance of developing adult rheumatoid arthritis e. ~2.6% chance of developing adult osteoporosis f. ~8.1% chance of childhood attention deficit hyperactivity disorder g. ~4.9% chance of developing hypertension h. ~0.6% chance of becoming alcohol dependent i. ~16.2% chance of developing prostate cancer as an adult j. ~57.7% chance of being gifted musically k. ~5.9% chance of having an agreeable personality l. ~39.4% chance of being very conscientious

What if there was ~75% chance of migraines?What if there was ~80% chance of being autistic?First Non-Invasive Human Prenatal Genome Evaluation - July 2012

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Scientific American - December 2012 Time Magazine - Cover - Dec. 24, 2012

Now - Prenatal Stem Cell Therapy for Osteogenesis Imperfecta

T. SNP / CNP WHOLE GENOME EVALUATIONS - will soon be routine and cheap! Over 2,000,000,000 evaluations in the next ten years

The amount of Data Storage will be greater than Twitter and YouTube together

Human Cloning and Stem Cells….. Current Applications and the Future of Medicine

A. DNA - Genome - sum total of all DNA information

mRNA (transcript) - Transcriptome - sum total of all RNA transcripts Protein - Proteome - sum total of all the human proteins

Proteins Interact - Interactome - sum total of all the protein-protein interactions

B. Scientific Literacy…..Become Well Informed - GTFS - Informed Decisions!

C. Cloning: Two Procedures….. 1. Reproductive Cloning

2. Therapeutic CloningHow do those procedures work? How are they similar?

How are they different? What is the end point? D. Stem Cells: Four Main Types

1. Embryonic Stem Cells (ESCs): 10 - natural existence 10 days - natural2. Adult Stem Cells (ASCs): 4 types; first recognized in BM 1961 - natural 3 Cancer Stem Cells (CSCs): predicted to mutate to cancer in 1997 - natural 4. iPSCs (induced Pluripotent Stem Cells) - 2006 / 2007 - man-made

Nobel Prize in Physiology or Medicine - 2012What are those cells? When are they available for study and to use? Where would we find them? Why would we want them? How would we use them?

E. Somatic Cells: ~220 Types1. Normal Body Cells - Specialized for Normal Body Functions2. Examples: Skin, Nerve, Gland, Muscle, Liver etc.

F. Why are stem cells called ‘stem’1. Word is ‘borrowed’ from the Plants2. The Stem of a plant gives rise to all the branches3. ESCs give rise to all the branches…..220 Somatic Cells4. When and Where? …..initially in the developing human embryo

G. Stem Cell Jargon - Potency

1. Pluripotent - a stem cell with the potential to become

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ANY of the 220 specialized cells: 1. ESCs / 4. iPSCs2. Multipotent - a stem cell with the potential to become

MANY of the 220 specialized human cells: 2. Adult Stem Cells3. Unique characteristics of Stem Cells

Self Renewal - the unique ability to self renew Mitosis - two different mechanisms

Symmetric Mitosis - the two resulting cells are identical mitosis results in cell expansion make identical copies of existing cells

Asymmetric Mitosis - the two resulting cells are different produces one cell - copy of original cell - Self Renewal brings into existence - one new somatic cell - Differentiation

Asymmetric mitosis a million times…..

> 1 stem / > 1,000,000 somatic cells

H. Stem Cell Categories…..

1. Embryonic Stem Cells - gone in 10 days DO NOT disappear in 10 days - they transform into…..

2. Adult Stem Cells - four types:

HSCs - Hematopoietic Stem CellsMSCs - Mesenchymal Stem CellsESCs - Endodermal Stem Cells

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NSCs - Neural Stem Cells

Adult Stem Cells - in the Fetus and Embryo….. Produce all the ORIGINAL somatic cells in you and me today…..

Produce all the REPLACEMENT somatic cellsExamples:

Adult Stem Cells in bone marrow - HSCs make all the replacement blood cells: RBCs, WBCs, etc.

Adult Stem Cells in skin - skin is replaced every 7 days:Epidermal Stem Cells - make new replacement epidermisMelanocyte Stem Cells - make new replacement melanocytesDermal Stem Cells - make new replacements dermis

EMBRYONIC STEM CELLS > > ADULT STEM CELLS…..~10 Days Beginning of Differentiation!

“STEMNESS” - the unique characteristics of Stem Cells

1. Self Renewal - Asymmetric Mitosis2. Differentiate - produce any oy 220 Somatic Cells3. Biologic Immortality - Telomerase enzyme maintains

chromosome telomere tips

II. BIOLOGIC BASIS of CLONING and STEM CELL TECHNOLOGY

A. Human Embryology - Applied Embryology Fetus at 7 weeks - 14 grams, the size of a quarter Fetus at 8 weeks - size of a silver dollar with 200 specialized cells

B. DIFFERENTIATION - cells become Different 220 ways Cells become DIFFERENT one from another.

Cells become SPECIALIZED 220 different ways. Adult Stem Cells Differentiate into 200 Somatic Cells

O SN EE T

S W A of Y

S SS IT GR NE AE LT SS

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Differentiation:Amazing process - 220 fetal somatic cells become 'Specialized'…..220 Ways 220 One Way Streets = 220 Sets of Signals Terminal Differentiation - end point of 220 Streets - 220 Specialized Cells All Embryology - One Way Street - Basic Tenet of Human Embryology

C. What are the signals? Transcription Factors / Epigenetic Chromatin Regulators They can Activate or Repress dozens of genes at one time

Transcription Factors - turn transcription ON and OFFa. attaches to the Promoter - Promoter acts as 'Docking Site'b. TF Activators - bind to promoter and turns genes ONc. TF Repressors - bind to promoter and turns gene OFFd. Enhancers - can also bind to DNA and enhance protein output

microRNA = miRNA - negative regulators - turn genes OFFa. microRNA, ~22 nts, complementary to and binds to 3' UTR of transcriptb. helps usher RISC (RNA Induced Silencing Complex) to the 3' UTRc. RISC blocks the ribosome > blocks translation > turns gene offd. referred to as miRNA 'Gene Silencing'

Epigenetics - 'Epi' means…..upon / on top of / above and beyond a. Chromatin - epigenetic control mechanism acts on chromatin

1. DNA (Genome) interacting with 2. Histone Proteins…..DNA / Histone Complex

b. Histone Proteins: Nucleosome - DNA wrapped around the NCPNucleosome Fiber - strand of multiple nucleosomesDNA…..'Never Acts Alone'

c. Gene Control: the Degree of Chromatin Compactioni. Methylation of Cytosine in the DNA of Chromatin at CpG Islands

add methyl groups - compact = 'Closed' chromatin = OFFlose methyl groups - loose = 'Open' chromatin = ON

ii. Acetylation of Histone Proteins = 'Open' chromatin = ONd. Methylation and Acetylation

Epigenetic Marks on the Chromatin - Remodels the Chromatine. Enzyme Controlled Mechanism:

Methylation Enzymes: DNMT - DNA Methyl Transferasede novo DNMT-3maintenance DNMT-1

Acetylation: HAT - Histone Acetylase / HDAC - Histone DeAcetylase

EPIGENETIC MARKS: Open Chromatin - genes ON Closed Chromatin - genes OFF

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EPIGENOME: Sum total of all the epigenetic marksE. CELLS, GENES and CHROMATIN - EPIGENETIC MARKS

Liver: OPEN Liver Genes CLOSED Kidney, Nerve, and Spleen Genes

Kidney: OPEN Kidney Genes CLOSED Liver, Nerve, and Spleen Genes

Nerve: OPEN Nerve Genes CLOSED Liver, Kidney and Spleen Genes

Spleen: OPEN Spleen Genes CLOSED Liver, Kidney, and Nerve Genes

220 Cellular Phenotypes

Example - Differentiation Pathway for Pancreatic Beta cells

F. Human Embryome Project - International effort

find all 220 sets of signals - know the signals to make any human cell

then…..any pluripotent stem cell and the appropriate signals….. would will be able to make any human cell…..in a laboratory

G. Fertilization to Embryonic Stem Cells…..

Fertilization or Conception - egg and sperm unite

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Zygote = fertilized egg - size = point of a straight pin 2 cell embryo at ~24 hours - 4 cell embryo at ~48 hours - only 32 genes working 8 cell embryo at ~72 hours - only 129 genes working 16 cells > 32 cells > 64 cells Tohonen, et. al. Nature Communications, September, 2015 Blastocyst at ~ 6 days

'basketball' - size of the point of a pin = implantation stage

inside blastocyst - fluid filled - one small cluster of cells = ICMICM - Inner Cell Mass or Epiblast

Embryonic Stem Cells - ESCs - becomes fetus / amnion'Ten Day Rule' - ESCs only exist 10 days under natural conditions ESCs grown successfully in a lab 1998 - James Thomson U. Wis.

HSCs, MSCs, ESCs, NSCs…..beginnings of ADULT STEM CELLS

MULTIPOTENT…..produce many cell types but not all

H. Embryonic Stem Cells…..immediately become four major branches:ADULT STEM CELLS:

HSCs - Hematopoetic SCs > forms all blood cells - RBCs, WBCs etc.MSCs - Mesenchymal SCs > forms muscle, bone, cartilage, etc.

ESCs - Endodermal SCs > forms liver, stomach, lungs, pancreasNSCs - Neural SCs > brain, spinal cord, epidermis

I. Adult Stem Cells:

INITIAL function - differentiate the ORIGINAL 220 somatic cells in the fetus

ADULT function - differentiate REPLACEMENT 220 cells the rest of your life

Where are they found today?

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HSCs - in bone marrow (and in umbilical cord) produce replacement blood cells…..~15,000,000 RBC per second

MSCs - in bone marrow, adipose, amniotic fluid produce replacement muscle, bone, cartilage, tendon, heart, blood vessels, etc. plus produce paracrine signals for tissue repair

ESCs - in lining of stomach and lining of intestine produce replacement cells

NSCs - in hippocampus and sub-ventricular zones of the brain produce replacement neural cells

HSCs and MSCs are easily obtained from bone marrow or adipose used therapeutically for over 50 years

1st BM transplant was 1959

Classic Terms: Ectoderm = NSCs, Endoderm = ESCs, Mesoderm = MSC & HSCs

III. REPRODUCTIVE CLONING

A. Clone – definition: a genetically identically copy of…..anything you choose

B. Robert Briggs - ‘Father of Cloning’ (December 10, 1911 - March 4, 1983) first to clone an animal - frogs in 1952 - IU

C. Reproductive Cloning - Definition reproduce an individual…..create a genetically identical copy of a particular individual…..clone will be born as a new baby

First Mammalian Clone: Animal - Sheep Names - Megan & Morag - cloned from donated embryo nucleus

Second Mammalian Clone: DOLLY First clone from donated adult somatic donor nucleus

D. Reproductive Cloning Procedure: S C N T - Somatic Cell Nuclear Transfer1. Oocyte Retrieval - obtain egg from the sheep ovary2. Enucleation - remove the nucleus from egg - via cell surgery3. SCNT - transfer donor somatic nucleus into the egg

new set of instructions - make a copy of the donor “the DONOR will be CLONED”

4. Artificial Activation of the egg > 2cell, > 4 cell, > 8 cell embryo…..5. ET - Embryo Transfer from the petri dish into the uterus of the

surrogate mother - who carries the clone embryo to term

E. Dolly: born July 5, 1996 / died February 14, 2003 - death from virus infection - not due to cloning1. Ian Wilmut's lab in Roslin, Scotland2. Dolly today: special exhibit - Royal Museum in Edinburgh

F. END POINT for Reproductive Cloning…..

BABY born to surrogate mother - too risky for humans

G. Other Mammalian clones:

Sheep, Mouse, Cattle, Pig, Goat, Gaur & Mouflon (endangered species), Rabbit, Cat, Mule, Rat, African Wildcat, Dog, Water Buffalo, Horse, Ferret, Wolf, Banteng, Camel,

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Wooly Mammoth? - 1. SCNT with Asian elephant as surrogate mother 2. Build a Mammoth / Elephant hybrid with ‘Genome Engineering’

Prometea - first cloned horse - donor was also the surrogate 1/841 = .12% success rate - surrogate delivered her own identical twin

Dolly - 1/277 = .36% success rate - very inefficient process - not epigenetically identical

H. Most important lesson from Dolly:1. A highly specialized, terminally differentiated, somatic donor

nucleus…..when placed into an enucleated egg, is reprogrammed from a highly specialized nucleus - reverts back to a pluripotent nucleus

2. What in the oocyte reprograms the nucleus back to pluripotency?3. Must be a cytoplasmic factor - because the egg nucleus in gone4. Educated guess…..it must be SIGNALS in the cytoplasm - how many?5. WHAT are the SIGNALS - “Stemness Signals”

III. THERAPEUTIC CLONING and STEM CELLS

A. Therapeutic Cloning Procedure: via Somatic Cell Nuclear Transfer 1. Human Oocyte Retrieval - very difficult procedure2. Enucleation of the egg – surgical removal of nucleus from human oocyte3. SCNT - transfer donor somatic nucleus to the egg

4. Artificial Activation of the egg > 2 cell, 4 cell, 8 cell…..5. Embryo continues to grow in lab for 6 days - clone in the petri dish

Clone of my friend’s embryo, remove ECSs from embryo, grow in the labEstablish a cell line from ESCs, grow until you have ~10,000,000,000 ESCs

Directed Differentiation:a. give those ESCs the signals to differentiate into Spinal Motor Neuronsb. result: > ~10,000,000,000 Spinal Motor Neurons

genetically identical to my friendc. the 'DREAM' - those cells to repair his spinal cord - one day he walks again!

B. Therapeutic Cloning = NT (Nuclear Transfer) Cloning Create a genetic clone (copy) of an individual's embryo;grow ECSs from the embryo; then use the appropriate signals;produce any of 220 human somatic cells use for medical therapy

END POINT: a CELL that can be used for medical therapy

REPRODUCTIVE CLONING - END POINT:BABY born to surrogate mother

THERAPEUTIC (N.T.) CLONING - END POINT:CELL used for medical therapy

First Therapeutic Cloning Success - 2004…..Fraud / Paper Retracted

First Validated N.T. Cloning - June 6, 2013‘Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer’

Mitalipov, et.al., Cell 153, pp. 1228-1238, June 6, 2013

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IV. ALTERNATIVE WAYS TO PRODUCE PLURIPOTENT STEMS

“Safe” and "Ethical" Pluripotent Stems: Develop new lab procedures that would yield pluripotent stem cells but, DOES NOT involve destruction of an embryo 1. Biopsy 8-cell embryo, 2. ANT - Altered Nuclear Transfer,

3. Living cells from 'Dead Embryo', 4. Parthenogenesis5. REPROGRAMMING

V. REPROGRAMMING = DeDifferentiation

1. Take one of the 220 Differentiated, Specialized, Somatic Cells backwards to a pluripotent stateDifferentiation: Stem Cell becomes Somatic Cell - Differentiation SignalsDeDifferentiation: Somatic Cell becomes Stem Cell - ‘Stemness’ Signals!

2. How? The lesson learned from Dolly!! Signals in oocyte cytoplasm - 4 signals - ‘Stemness’ Signals

3. Who? Shinya Yamanaka - Kyoto University in Japan / UCSF Mouse iPSCs - July 7, 2006

Human iPSCs - November 20, 2007 iPSCs……induced Pluripotent Stem Cells 4 signals: Japan - OCT 3/4, SOX2, c-MYC, KLF4

'Yamanaka Factors' - OSMK Signals Yamanaka & Gurdon - Nobel Prize – 2012

iPSCs ARE NOT: Adult Stem Cells (HSCs / MSCs / ESCs/ NSCs)iPSCs ARE: Adult Somatic Cells converted into Stem CellsiPSCs and Embryonic Stem Cells are Functionally Equivalent

Nature Biotechnology, On Line, October 26, 2015************************************************************************************************************

CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats Bacterial immunity enzyme system that removes a harmful gene and inserts a good

gene in its place, may lead to therapy for R.P., AIDS, Sickle Cell, Hemophilia “Brave New Genome’, Eric Lander, NEJM, 373, pp. 5-8, July 2, 2015

CRISPR/Cas9 - TARGETED GENE THERAPY: New Era in Molecular Biology

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V. POTENTIAL APPLICATIONS of PLURIPOTENT STEM CELLSs: 1. Cell Replacement Therapy: autologous = no rejection

Spinal Motor Neurons - spinal cord injury Pancreatic Beta Cells to produce insulin - Type I Diabetes Dopamine producing neurons - Parkinson Disease Red Blood Cells - Sickle Cell Anemia…..~216 other possibilities

2. Human Disease Modeling : ‘Disease in a Dish’

Follow the cellular development of any genetic disease in vitro…..Lou Gerhig's Disease (ALS) donor > motor neurons

Huntington Disease donor > medium spiny neuronsType I Diabetes donor > pancreatic beta cells

3. Drug Therapy Screening :

Test thousands of small molecules at one time in a petri plateFind one of 1000s that has a beneficial therapeutic effect

4. Regenerative Medicine / Tissue Engineering:

Produce cells in the laboratory for use for production

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human tissues and organs in vitro

5. Combination: #2 - Disease Modeling & #4 - Regenerative Medicine

Cell Replacement Examples: Cell Stem Cell - July2, 2015 iPSCs from patients with type 1 diabetes - DiPS Diabetic iPS August 31, 2009

Viacyte - human trials underway http://viacyte.com/

VIDEO: https://www.youtube.com/watch?v=Q6U5kf5ByNE Acinar > Beta Differentiation - Direct Cellular Reprogramming in vivo Nature Biotechnology - Vol 32, p. 1223, December 2014

Direct Reprogramming: Somatic Cell > Desired Somatic Cell No iPSC stage = No Teratoma Risk!

Fibroblasts > Functional Neurons August 5, 2011Fibroblasts > Functional Dopaminergic Neurons - August 11, 2011 Fibroblasts > Functional Lung Tissue -

Blindness - 25,000,000 World Wide - visually impaired or blind

>190 Genes Mutated to cause blindness

AMD - Age Related Macular Degeneration

iPSCs > RPE - Retina Pigment Epithelium - 99% pure First Successful Human Trials Lancet - January 23, 2012iPSCs > RPE ‘Patches’ - transplanted to back of retina / or RPE cell injections

Stem Cells Portal - October 5, 2015 RP - Retinitis Pigmentosa - Most Common Inherited Vision Loss

AD, AR, XLR, Mito - Rod Cell LossAchondroplasia - most common cause of short stature

Neural ApplicationsHuman Brain: brain cells…..100 billion neurons

100 trillion synapses 100 billion astrocytes human astrocytes - Special LTP (Long-Term Potentiation) Function

glia cells - billions of non-electrical cells in the brainastrocytes: active at all synaptic junctions

microglia oligodendrocytes: myelin producing cells

myelin - insulates axons in a protective sheath

Neural SC Engraftment and Re-Myelination in the Human Brain

Pelizaeus-Merzbacher Dx - PMD - hypomyelination of axons Oligodendrocyte Deficiency - no myelination

huCNS-SCs - human Central Nervous System Stem Cells huGPCs - human Glial Progenitor Cells

injected into the PMD brains > form new oligodendrocytes

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myelinated the brain axons > functional improvement Therapy for Demyelinating Diseases…..Multiple Sclerosis?

Science Translational Medicine October 2012

Human Astrocytes - special adaptations for LTP

huNSCs > huGPCs > human astrocytes – injected in newborn mice ‘Smarter’ mice with human astrocytes in their brains

Future huiPSCs > huNSCs > therapy for ASD, Schizophrenia ? Cell Stem Cell - March 7, 2013

Huntington Disease - AD - CAG Triplet Repeat Mutation – 4p16.3 Gene elongates every generation - Classic ‘Late Onset’ Condition

Huntingtin elongated gene > Huntingtin elongated proteinHuntingtin Protein > Loss of Medium Spiny Neurons in Striatum

Huntington Stem Cell Therapy: HD Patient Fibroblasts > HD iPSCs

HD iPSCs Genetically Corrected CAG72 > CAG21 Corrected HD iPSCs > Medium Spiny Striatal NeuronsPatient specific, genetically corrected, HD patient iPSCs

Cell Stem Cell - August 3, 2013

ALS - Amyotrophic Lateral Sclerosis = Lou Gerhig’s - Slow loss of motor neurons ‘Neural Progenitors Derived from Human iPSCs Survive and Differentiate upon Transplantation in a Rat Model of ALS

Stem Cell Translational Medicine - March 2013

Parkinson Disease - Loss of Dopaminergic Neurons in Substantia Nigra of midbrain ‘Rapid Generation of Functional Dopaminergic Neurons from

Human iPSCs through a Single-Step Procedure using Cell Lineage Transcription Factors: ASCL1 / NURR1 / LMX1A

Stem Cell Translational Medicine - Vol 4, pp. 31-43, December 2014Reproductive Applications:

Creating Spermatids from Skin - male infertility Creating Oocytes from Skin - female infertility

‘Egg Engineers’ - Nature, p.392, August 22, 2013 Oocyte Stem Cells - Women can procreate for ever!

Nature Medicine - March 2012 iPOD Link: http://www.nature.com/news/stem-cells-created-in-living-mice-1.13725#/ref-

link-1

Disease Modeling Examples:

“Organoids” - primitive organ-like 3-D masses of differentiated cells grown in matrigel Rise of the Organoids – Nature, July 30, 2015

Inner Ear Organoid: sensory epithelia via 3-D Culture - IUMC Nature, August 8, 2013

Retinal Rod Cell Organoids: from iPSCs for Retinitis Pigmentosa Therapy Nature Biotechnology August, 2013

Teeth Organoids: man made from Stem Cells

Eye Organoids: Optic Cup Retina Nature - April 7, 2011

Liver Organoids: Vascularized and Functional Human Liver from iPSCs-Derived Organ bud Transplant Nature - July 23, 2013

Kidney Organoid:

Heart Organoids: Cardiac Repair after MI (Myocardial Infarction) Cardiac Fibroblasts > Cardiomyocytes - 3 signals

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Cell - August 6, 2010Large Intestine Organoid: Study the development stages of colon cancer

Sequential Cancer Mutations, Nature, May 7, 2015 Living Colon Cancer Patient Organoid Biobank - Cell, 161, pp. 933-945, May 7, 2015

The Brain Maker: mouse ESCs > mouse NSCs Primitive Optic Cup Retina / Cerebral Cortex / Primitive Cerebellum Primitive Hypothalamus with hormone production Nature - August 23, 2012

Human Brain Organoids - Minibrains - size of an apple seed Resembles the Fetal Brain at ~9 Weeks / Microcephaly Brain Organoid - smaller ‘Cerebral organoids model human brain development and microcephaly’

Nature - August 28, 2013Model for study of ASD - autism using the skin from a child with ASD diagnosisModel for developing drugs for various brain related conditions

Human 5 Week Fetal Brain in a petri plate made from iPSCs - size of a pencil eraser 99% of normal genes working - contains part of spinal cord and retina

Takes ~12 weeks in vitro to reach the 5 week in vivo stage May be a model to study experimental drugs and causes of brain disease

May be as a model to study: ASD, Parkinsons, Alzheimers - using their skin

https://news.osu.edu/news/2015/08/18/human-brain-model/

“Organs on a Chip” - Multichannel, 3-D, microfluid, cell culture on a plastic, chip….. with living vascular cells to mimic the body’s circulatory system

Simulates the basic anatomy and function of entire organs. can be used to test chemical toxicity, normal cells v. disease

Lung on a Chip

Heart on a Chip

Nephron on a Chip

Artery on a Chip

Bone Marrow on a Chip

Intestine on a Chip > colon Cancer on a Chip

Person on a chip Microfluidic organs-on-chips - Nature Biotechnology, Vol 32, August 2014, pp. 760-770

Regenerative Medicine Applications - produce human tissues and organs in the lab Scaffold = framework - basic shape of that tissue or organ

"IDEAL STEM CELL": 1. Easy Access - from blood or bone marrow

2. Timely Access - obtain cells anytime 3. No Tumor Risks - no teratomas 4. Multipotent or Pluripotent 5. Homing Mechanism - migrates to site of injury or tumor 6. Non-Immunogenic - ImmunoModulatory

7. Paracrine effect - secrete factors to benefit neighboring cells

IMPOSSIBLE! - Adult Stem Cells - Mesenchymal Stem Cells = MSCs

MSCs - Leukemia - Decrease GVHD in BM / UC Transplants Amniocytes to produce new heart valve before birth

Parental adult MSC stems correct O.I. = osteogenesis imperfecta Adipose Derived Stem Cells (ADSC) for heart therapy

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MCSs from bone marrow and cord blood - therapy for E.B. E.B. = Epidermolysis Ballosa

http://www.ahc.umn.edu/eb/home.html

How do they work? ‘Home’ to area of injury of inflammation

‘Home’ to tumor and carry drug - interferon beta - to kill tumor

Heart Vein made in vitro - MSCs on man-made tubular scaffold Science, August 26, 2011

Tracheas made from scratch - MSCs plus man-made scaffold - new scaffolds from 3-D printer 3D Bioprinting of tissues and organs - Nature Biotechnology, August 2014

Cartilage made from scratch - MSCs in seeded on polymer scaffold

Finger Regeneration - cellular matrix powder from a pig bladder and finger nail stem cells

Meat Lab - make burger and sausage from muscle stem cells First Test-Tube burger made from 3,000 tiny strips of meat grown from muscle muscle stem cells taken from a cow’s muscle tissue…..cost…..$383,875

Make a Heart in the Lab? Can it be done?…..heart scaffold is too complex! Video: http://www.nature.com/news/tissue-engineering-how-to-build-a-heart-1.13327 BUT…..there is a normal, natural heart scaffold - inside every heart

Decellularization - remove all heart cells and endothelial cells with detergent, under pressure, left with a ‘deflated’ acellular human heart scaffold

Recellularization – in a Bioreactor: add new heart cells and endothelial cells made ahead of time in the lab, from iPSCs of the recipient

Biomaterials - vol. 52, pp. 103-112, June, 2015 Lungs - Decelluarization and Recellularization

Pig lung formed in a Bioreactor in lab / successfully transplanted back http://tmcnews.tendenciapp.com/articles/team-at-utmb-seeks-to-rebuild-donated-human-lungs/Liver - Decelluarization and Recellularization

Bioengineered transplantable porcine livers – Biomaterials, February, 2015Kidneys - Decelluarization and Recellularization

Place human replacement cells in pig kidney scaffold Regeneration of Bioengineered Kidney - Nature Medicine, OL, April 14, 2013

Limbs - Decelluarization and Recellularization

Detergent removal of all cells, leaving cell free scaffold of blood vessels, tendons and muscle from a rat…..took several weeks…..then in a bioreactor, supplied the progenitor

cells for replacement cells, even outer skin covering - and the new limbs responded normally to electric stimuli and the fingers contracted. http://www.nydailynews.com/life-style/health/watch-mass-researchers-grow-bioengineered

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-rat-limb-article-1.2249891Severed hand grafted to patient’s leg…..then later transferred back to the arm

http://www.cnn.com/2015/07/24/health/severed-hand/index.html?sr=fb072415severedhand1pStoryGalLink

Chimeric Mammalian Embryos - for neo-organ productionChimera - an organism that is made up of a mixture of cells from two or more organisms

Make a rat pancreas in a mouse embryo…..mouse/rat chimera

Inject ‘blue’ rat iPSCs into mutant mouse embryo - can’t make a pancreasAt birth, the newborn mouse survives because it has a ‘blue’ rat pancreasIn vivo organogenesis - make an organ of one animal in the embryo of another

Make a human pancreas in the embryo of a pig…..pig/human chimera Inject human iPSCs into mutant pig embryo - can’t make a pancreas

At birth, new newborn pig survives because it has a human pancreasWhen pig reaches maturity - transplant pancreas from pig to human iPSC donorAnimal Embryo Chimeras used to produce human organs!

Cell Stem Cell - Vol 15, pp. 406-409, October 2, 2014What about using Pig Organs - transplanting them into humans?? Two Problems: 1. PERVs - Porcine Endogenous RetroViruses

2. Pig antigen genes that would cause rejection Both eliminated in pig embryos by CRISPR genome editing - PNAS Conference - October 5, 2015

Future - Yamanaka developing an iPS Cell Bank in Japan ~75 HLA compatible iPS donors would provide iPS for ~80% of the Japanese population

“When it gets here, when it arrives,,,,,Science Fact is always more amazing than Science Fiction” Sam Rhine, Oct. 13, 2015

Sam Rhine - College and Career suggestions:

Check Out: http://www.kumc.edu/gec/prof/career.html

1. Go to you favorite Undergraduate college and obtain your 4-year Bachelor's degree. Major in biology, biochemistry, molecular biology, bio-engineering etc. Make sure you satisfy the Pre-Med requirements so you can apply to medical school if you decide that is the best route for you.

2. Medical School is four years and the curriculum is very similar at all medical schools in the US. The reason for that is that everyone must pass the same national exam after finishing medical school - therefore the schools must cover the basic subject matter. If you pass that exam the summer after finishing medical school, you can then put M.D. behind your name.

3. Residency is then 4 - 8 years of ‘Specialty Training’ to become a pediatrician, obstetrician, orthopedic surgeon, oncologist, neurosurgeon or whichever specialty you choose. If you want to pursue a career in Tissue Engineering then you might want to get a residency with Dr. Anthony Atalla at Wake Forest University. If you want to use CD-47 antibodies to functionally disable cancer stem cells you might want to do your residency in oncology at Stanford University with Dr. Irv Weissman or Dr. Michael Clarke.

Keep your ‘antennae out’ during your four years of medical school – to determine who is doing the research you want to pursue for a career - and go do your residency with that person - he or she.

_________________________________________________________________________

2. For those who are not interested in medical school - they might want to pursue a career in research and they will go on after their undergrad work and get their Masters and Ph.D. which may be 4 to 6 more years.

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3. The Ph.D. is usually followed by Post Doctoral studies, Post Doc, for 2 - 4 years to gain more special expertise for the research career you want to follow. Then you will be ready to join the faculty at a university to do research and teach. Others will opt to get a job doing research in industry for biotech companies. Also, some of these people are getting their Ph.D. in biostatistics or computer

sciences where they will help with the planning and evaluation of research data.

4. M.D. / Ph.D. Most major Medical Schools offer a combined M.D. / Ph.D. for a person who may one day be the chairperson of the Department of Molecular Medicine at some medical college - check that out for each individual medical school.

5. Masters Degree in Genetic Counseling - is another option for some. There are almost 30 places in the US where those programs are available. For more information - check out this web site: http://www.nsgc.org/iframepages/GeneticCounselingTrainingPrograms/tabid/336/Default.aspx

6. Teaching - Also Remember…..many people who will make a major contribution to all These careers in the future will do so by majoring in Education in college and

will be preparing young people in the future…..as your Teachers have been preparing you!! "Teachers Make All Other Professions Happen!"

Also Consider: Physician Assistant (PA): http://www.aapa.org/   Student Academy: http://www.aapa.org/saaapa/ MD/MS Genomic Medicine: http://admissions.med.miami.edu/md-programs/md-ms-in-genomic- medicine

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