Download Sam Rhine Outline - Spring Branch ISD

Sam Rhine - Genetic Update Conferences - www.samrhine.com
INTRODUCTION: Genomes and Genes…..
Genome: the sum total of all the genetic information for any biologic organism
Human Genome 2013:
1. Two Types of DNA…..
~1.5% - 'Coding' DNA………..GENETIC CODE for PROTEINS
~98.5% - 'Non Coding' DNA…..NO GENETIC CODE for PROTEINS
2. Two Types of Genes…..
Coding DNA Genes
Sequence of DNA responsible for production of a specific Protein molecule
Genes produce ~21,000 protein molecules - Transcription & Translation
Non-Coding DNA Genes
Sequence of DNA responsible for production of a specific RNA molecule
Genes produce ~13,500 RNA molecules - Transcription & shRNA Dicing
Coding DNA Gene - Transcription followed by Translation
Non-Coding DNA Genes - Transcription followed by shRNA Dicing
a. produce sncRNAs - small non-coding RNAs
lncRNAS - long non-coding RNAs
ENCODE - ENCyclopedia Of Dna Elements…..3,000,000,000 nucleotides
Findings: Many non-coding parts of the genome, the Junk - contain ‘Docking Sites’
Docking Sites where control proteins bind to affect gene expression
Some are near the gene they control - others are at a great distance
How Many? Already knew of ~21,000…..2,890,000 new Docking Sites
~200,00 active in any one cell at any one time
Switch genes on and off via a ‘Dimmer’ mechanism
Also found ~9,500 lncRNA - new Non-Coding Genes
10,000
added to ~500 sncRNA - known Non-Coding Genes
State of the Genome - September 2013:
1.5% Coding DNA - Exons = EXOME
98.5% Non-Coding DNA
28.5% - Introns = INTROME
51.5% - 500 sncRNA genes + 13,000 lncRNA genes
plus…..2,890,000 ‘Docking Sites’
20.0 % - Yet to be determined
30% - Exons and Introns of the 21,000 Coding DNA Genes
HGP - Human Genome Project: Find the 3,000,000,000 nucleotides
ENCODE: Find the function of each of the 3,000,000,000 nucleotides
II. GENE CONTROL = GENE REGULATION - ON / OFF MECHASNISMS
A. Transcription Factors - turn transcription ON and OFF
B. microRNA = miRNA - negative regulators - turn genes OFF
C. Epigenetics - 'Epi' means upon / on top of / above and beyond DNA sequence
Gene Control: the Degree of Chromatin Compaction
TURN OFF a CHROMOSOME - Chromosome Inactivation - turn off hundreds of genes
Male sex chromosome - X Y / Female sex chromosomes - X X
Chromosomal Inequity - X Inactivation reconciles the inequity
Males have one X / Females have one X that works and the other is inactive
Average female - ~50% of body cells - paternal X is active / maternal X inactive
~50% of body cells - maternal X is active / paternal X inactive
XIST gene (X Inactivation Specific Transcript) on X chromosome - Xq13.2
III. Telomeres: the Amazing Tips of our Chromosomes - Non-Coding DNA
Telomeres - protective cap on the end of all of our chromosomes
Prevents ‘unraveling’ of the Coding DNA which lies below the Telomere
Act like an ‘Aglet’ - tip of your shoe string
Unraveling of the coding DNA > increase chance for cancer
Telomeres on normal chromosomes
~15,000 Non-Coding DNA nucleotides - on the tips of each chromosome
Repeating Hexanucleotide unit - T T A G G G - x2500 (6 x 2,500 = 15,000)
Telomere Erosion - ~50 cell divisions > Replicative Senescence – Mitosis Stops!
Telomerase - inhibits telomere erosion - maintains telomeres - avoids senescence
Telomerase is a Reverse Transcriptase enzyme…..RNA > DNA
Two Main units: TERT - TElomerase Reverse Transcriptase
TERC – Telomerase RNA Component
IV. Checking the Fetal Genome…..
1. Amniocentesis - ~14 weeks post conception
2. CVS - Chorionic Villus sampling - ~10 weeks post conception
3. PGD - Preimplantation Genetic Diagnosis - 48 hours post conception
4. Blastocoel Cavity DNA Analysis - ~6 days after post conception
5. Fetal DNA in Maternal Serum - ~ 7 weeks post conception
6. GSNSD - Genome Sequencing and Newborn Screening Disorders - at birth
Exome sequencing of 1,500 babies to evaluate clinical utility of sequencing
HUMAN CLONING and STEM CELLS - INTRODUCTION
A. Scientific Literacy…..Well Informed - GTFS - Informed Decisions!
B. Cloning: Two Procedures…..
1. Reproductive Cloning
2. Therapeutic Cloning
C. Stem Cells: Four Main Types
1. Embryonic Stem Cells (ESCs) - first grown in lab - 1998 - natural
2. Adult Stem Cells (ASCs) - first recognized - 1961
- natural
3 Cancer Stem Cells (CSCs) – first recognized - 1997
- natural
4. iPSCs (induced Pluripotent Stem Cells) - 2006 / 2007 - man-made
Somatic Cells: ~220 Types - Normal Body Cells - Specialized for Normal Body Functions
D. Special Quality of Human Stem Cells – Potency
1. Pluripotent - a stem cell with the potential to become
ANY of the 220 specialized cells: 1. ESCs / 4. iPSCs
2. Multipotent - a stem cell with the potential to become
MANY of the 220 specialized human cells: 2. Adult Stem Cells
3. Totipotent – a stem cell with the Total; Potential to form the
Fetus, Placenta and Membranes: zygote / early blastomeres
b. Makes the brain, heart, liver, kidneys, lungs etc.
O
N
E
S
E
T
S
W
A
Y
of
S
I
G
N
A
L
S
S
T
R
E
E
T
S
c. Differentiation:
amazing process - all 220 fetal somatic cells become 'Specialized'…..220 Ways
220 One Way Streets = 220 Sets of Signals
Terminal Differentiation - end of 220 Streets - 220 Specialized Somatic Cells
All Differentiation - One Way Streets - Basic Tenet of Human Embryology
d. What are the signals? - Molecular Signal Proteins
Transcription Factors and Epigenetic Chromatin Regulators
e. Differentiation Pathway for Pancreatic Beta (insulin) cells: 10 signals
g. Human Embryome Project - International effort
find all 220 sets of signals
h. Fertilization to Embryonic Stem Cells…..
Fertilization or Conception - egg and sperm unite
Zygote = fertilized egg - size = point of a straight pin
2 cell embryo ~36 hours…..4 cell ~42 hours…..8 cell ~48 hours > 16 cells > 32 cells > 64s
Blastocyst at ~ 6 days
'basketball' - size of the point of a pin = implantation stage
inside blastocyst - fluid filled - one small cluster of cells = ICM
ICM - Inner Cell Mass or Epiblast = Embryonic Stem Cells - ESCs - fetus/amnion
g. From the ESCs stem immediately come four major branches:
HSCs - Hematopoetic SCs > forms all blood cells
MSCs - Mesenchymal SCs > forms muscle, bone, cartilage, tendon
ESCs - Endodermal SCs > forms liver, stomach, lungs, pancreas
NSCs - Neural SCs
> brain, spinal cord, epidermis
h. Adult Stem Cells:
Initial function
- differentiate 220 somatic cells in the developing fetus
Continued function - produce replacement cells the rest of your life
III. REPRODUCTIVE CLONING
A. Reproductive Cloning Procedure: Somatic Cell Nuclear Transfer - SNCT
1. Oocyte Retrieval - obtain egg from the sheep ovary
2. Enucleation - remove the nucleus from egg - via cell surgery
3. 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…..
5. ET - Embryo Transfer from the petri dish into the uterus
of a surrogate mother - who carries the clone embryo to term
B. END POINT for Reproductive Cloning…..
BABY born to surrogate mother - too risky for humans
C. Most important lesson from Dolly:
1. Highly specialized, terminally differentiated, somatic donor
nucleus, when placed into an enucleated egg, is reprogrammed
from specialized back to a pluripotent state
2. What is in the oocyte > reprograms the nucleus back to pluripotency?
3. Must be a cytoplasmic factor - because the egg nucleus in gone
4. Guess…..it must be SIGNALS in the cytoplasm
5. How long would it take to find those signals?
6. How many signals would it take to reprogram a nucleus backward
to a pluripotent state?
III. THERAPEUTIC CLONING and STEM CELLS
A. Human Blastocyst…..Inner Cell Mass = Epiblast
Embryonic Stem Cells - ‘10 Day Rule’
Stemness - self renewal, telomerase, biologic immortality
Pluripotent – can produce any human cell
ESCs in vivo - natural conditions…..~10 days
in vitro - in a petri dish in a lab…..biologic immortality
ESCs - source of pluripotent human stem cells…..but also
source of moral, ethical, religious and political issues
B. Therapeutic Cloning Procedure: SCNT…..Friend needs Spinal Motor Neurons
1. Human Oocyte Retrieval - very difficult procedure
2. Enucleation of the egg - surgery on the human egg to remove nucleus
3. SCNT - transfer donor somatic nucleus to the egg
new set of instructions - make a copy of my friend
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
Definition: 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
REPRODUCTIVE CLONING
- END POINT: BABY born to surrogate mother
THERAPEUTIC (N.T.) CLONING - END POINT: CELL used for medical therapy
IV. APPLICATIONS OF PLURIPOTENT STEM CELLSs:
1. Cell Replacement Therapy: autologous = no rejection
2. Human Disease Modeling: ‘Disease in a Dish’
Follow the cellular development of any genetic disease in vitro
2. Drug Therapy Screening:
Test thousands of small molecules at one time
Find one of 1000 that has a beneficial therapeutic effect
3. Regenerative Medicine / Tissue Engineering
Produce cells in the laboratory for use for production
human tissues and organs in vitro
V. ALTERNATIVE WAYS TO PRODUCE PLURIPOTENT STEMS
"Ethical" Pluripotent Stem Cells:
Develop new lab procedures that would yield Pluripotent Stem
Cells but…..DOES NOT involve destruction of an embryo
V. REPROGRAMMING = DeDifferentiation
1. Take one of the 220 Differentiated Somatic Cells backward
to a pluripotent state
Differentiation:
Stem Cell becomes Somatic Cell
How? Signals
DeDifferentiation: Somatic Cell becomes Stem Cell
How? Reverse the 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 = OSMK
8. Other Applications
Deafness - Generation of Inner Ear sensory epithelia via 3-D Culture
Blindness - 25,000,000 World Wide - visually impaired or blind
AMD - Age Related Macular Degeneration
RP - Retinitis Pigmentosa - Most Common Inherited Vision Loss
Gene Therapy - Fix One Gene
Leber Congenital Amaurosis Type 2 - LCA2
injected 'RPE65' gene therapy AAV vector
Chromosome Therapy - Fix Hundreds of Genes
Down Syndrome - Trisomy 21 - 1/750
transfer XIST from X chromosome to #21 in Down Syndrome
inactivates one of the three #21s > #21 Barr Body
Brain Cells…..100 billion neurons / 100 billion astrocytes
Pelizaeus-Merzbacher Dx - hypomyelination of axons
Huntington Disease - AD - CAG Triplet Repeat Mutation
1. Genetic Correction of HD Phenotypes in iPSCs
2. siRNA Gene Silencing - turn off the Huntingtin Gene
3. Localized Somatic Stem Cells
Creating Spermatids from Skin - male infertility
Creating Oocytes from Skin
- female infertility
10. Stem Cells and Regenerative Medicine / Tissue Engineering
Man-Made Tissues and Organs…..‘ORGANOID’ Applications:
Man Made Teeth Organoids from Stem Cells
Optic Cup Retina - from Eye Organoids
The Brain Maker: mESCs > mNSCs
‘BRAIN’ - The Human Brainome Project
Human Brain Organoids - Minibrains - size of an apple seed
Resembles the Fetal Brain at ~9 Weeks
Cardiac Repair after MI (Myocardial Infarction)
Cardiac Fibroblasts > Cardiomyocytes – 3 signals
Heart Vein for newborn baby made in vitro - MSCs on man-made tubular scaffold
Tracheas made from scratch - MSCs plus man-made biodegradable scaffold
Knee Joint Cartilage made from scratch - MSCs in seeded on polymer scaffold
Prenatal Diagnosis - heart valve anomaly - AF MSCs > new valve
Osteogenesis Imperfecta corrected before birth with maternal MSCs via cordocentesis
Finger Regeneration - cellular matrix powder from a pig bladder
Meat Lab - make burger and sausage from muscle stem cells
Make a Heart in the Lab? Cannot be done…..Scaffold is too Complex!
Decellularization - remove all heart cells with detergent / pressure
1.
2.
detergent is pumped into the aorta filling the arteries that feed the heart
detergent flowing through existing blood vessels dissolving the heart cells
You are left with a ‘deflated’ acellular human heart scaffold
Recellularization - add heart cells made in the lab made from iPSCs
1.
2.
endothelial precursor cells are pumped into the blood vessels
heart muscle precursor cells are injected into the muscle space
Re-Start the Heart - get the new heart to beat again
1.
pulsing flow of nutrients into the heart forces heart to begin to beat
2. electrical stimulation helps the heart muscles start contracting on their own
Make a rat pancreas in a mouse embryo…..
Inject ‘blue’ rat iPSCs into mutant mouse embryo that cannot make a pancreas
At birth, the newborn mouse survives because it has a ‘blue’ rat pancreas
“In Vivo Organogenesis” - make an organ of one animal in the embryo of another
Make a human pancreas in the embryo of a pig??
Inject human donor iPSCs into mutant pig embryo that cannot make a pancreas
At birth, new newborn pig survives because it has a human pancreas
When pig reaches maturity - transplant pancreas from pig to human iPSC donor
Animal Embryo Chimeras used to produce human organs!
Here are some College and Career 'Contribution' suggestions:
1. Go to you favorite Undergraduate college and obtain your 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 subjects. If you pass that exam the summer after
finishing medical school then you can 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 whatever
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 antibodies to stop leukemia caused by
cancer stem cells you might want to do your residency in oncology at
Stanford University. Keep your ‘antennae out’ during the four years of
medical school - determine who is doing what 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.
3. The Ph.D. is usually followed by Post Doctoral studies for 2 - 4 years to
gain special expertise for the research career you want to follow. Then
you will be ready to job on 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.s in biostatistics or computer science
where they will help with the planning and evaluation of research data being generated.
Many 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
Another option for some will be to get a Masters Degree in Genetic Counseling.
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
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!!