Sam Rhine - Genetic Update Conferences - www.samrhine.com
The State of the Genome…..Gene Control…..X-Inactivation
Telomeres…..Prenatal Genome Analysis…..
Human Cloning and Stem Cells…..New Medical Applications
The State of the Human Genome - September 2013:
I.
INTRODUCTION: Genomes and Genes…..
A. References:
1. Text: Human Genetics - Concepts and Applications (10th ed.) by Ricki Lewis
2. Blog: DNA Science Blog by Ricki Lewis: http://blogs.plos.org/dnascience/author/rlewis/
B. Genome: the sum total of all the genetic information for any biologic organism
1. a genome may be DNA - double strand
2. a genome may be RNA - single strand
3. a genome is expressed as the total number of nucleotides
a. Human Genome: ~3,000,000,000 nucleotides pairs, ~34,500 genes
b. HIV Genome
9,749 nucleotides
9 genes
C. Human Genome Project (HGP) - Historical Perspective
1.
2.
3.
4.
5.
6.
Largest scientific project in world history
Projected to be 15 year international cooperative effort
20 countries involved - main contribution from Britain and U.S.
Began: October 1, 1990 / End: April 25, 2003
Dr. Francis Collins - Director of HGP
1990: Human Complexity - organism as complex as a human needs ~100,000 proteins
since genes make proteins…..humans must have ~100,000 genes
7. 1990: most of the DNA in the genome must carry the genetic code for proteins
D. 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
3. Coding DNA Gene - Transcription followed by Translation
a.
b.
c.
d.
DNA double strand with Promoter (on/off switch) in the nucleus
Promoter is a ‘Docking Site’ for control molecules to turn gene on or off
Transcription factor docks on the promoter - initiates transcription
DNA transcribed into mRNA (transcript) in nucleus
transcript with genetic code, plus 5' and 3' UTRs - UnTranslated Regions
e. Transcript translated into protein at ribosome - on endoplasmic reticulum
TRANSLATION ay the RIBOSOME
f. Coding DNA segments within the gene are actually in separate units - Exons
Exons - Coding DNA
- part of the 1.5% Coding DNA
Introns - Non-Coding DNA - part of the 28.5% Non Coding DNA
g. RNA Processing - occurs in the nucleus
Transcript: Remove the Introns / Splice the Exons together
at the Spliceosome – structure with 5 RNAs and > 100 proteins
PROCESSING AT THE SPLICEOSOME
h. ALTERNATIVE SPLICING - Special Processing Activity
Make a protein with Exons 1, 2, 3, 4
1, 3, 4
1, 2, 4
1 Gene > 5 Proteins
1, 2, 3
2, 3, 4
95% of human genes are involved with alternative splicing
this is how we can make >100,000 Proteins from only 21,000 genes
i.
EXOME - sum total of all the Coding DNA in all the Exons
- 1.5%
INTROME - sum total of all the Non Coding DNA in all the Introns - 98.5%
Exome plus Introme = ~30% of the genome
4.
Non-Coding DNA Genes - Transcription followed by shRNA Dicing
a.
5.
produce sncRNAs - small non-coding RNAs
microRNA = miRNA = miR
lncRNAS - long non-coding RNAs
b. Non-Coding Gene DNA is transcribed > miR Transcript
miR Transcript is a linear nucleotide ‘mirror image’
folds over on itself forming a Stem Loop or hairpin structure
referred to as ‘short hairpin DNA or shRNA = double strand RNA
double strand hairpin, shRNA moves from nucleus to cytoplasm
shRNA induces production of ‘Dicer’ enzyme in the cytoplasm
Dicer dices the shRNA into small fragments of RNA = miRNA
miRNA attaches to the 3’ UTR of a coding DNA gene transcript
miRNA guides RISC (RNA Induced Silencing Complex) to 3’ UTR
RISC protein complex - ‘turns off’ the gene = ‘Gene Silencing’
Summary
a. Coding DNA Genes: Coding DNA in Exons; Non-Coding DNA in Introns
b. Non-Coding DNA Genes: Non-Coding DNA - only
E. Human Genome Project and ENCODE - 2013 Update
1.
April 25, 2003 - HGC Final Completion date…..National DNA Day
April 25, 1953 - Watson & Crick paper - DNA is a double helix
Final Cost ~$3,000,000,000 / Finished ahead of schedule and under budget!
Every $1 invested in HGP has already returned $140 to US economy
2.
ENCODE - ENCyclopedia Of Dna Elements…..3,000,000,000 nucleotides
International Consortium / 32 Research Institutions / 442 Investigators
10 years of genome research - all published on one date - September 5, 2012
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
September 2013: ~13,000 lncRNA genes + ~500 sncRNA = ~13,500
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
Genome / Transcriptome / Proteome / Interactome
II. GENE CONTROL = GENE REGULATION - ON / OFF MECHASNISMS
A. Transcription Factors - turn transcription ON and OFF
a. attaches to the Promoter - Promoter acts as 'Docking Site'
b. TF Activators - bind to promoter and turns genes ON
c. TF Repressors - bind to promoter and turns gene OFF
d. Enhancers - can also bind to DNA and enhance protein output
B. microRNA = miRNA - negative regulators - turn genes OFF
a. microRNA, ~22 nts, complementary to and binds to 3' UTR of transcript
b. helps usher RISC (RNA Induced Silencing Complex) to the 3' UTR
c. RISC blocks the ribosome > blocks translation > turns gene off
d. referred to as miRNA 'Gene Silencing'
C. Epigenetics - 'Epi' means upon / on top of / above and beyond DNA sequence
1. Chromatin - epigenetic control mechanism acts on chromatin
DNA (Genome) interacting with Histone Proteins
DNA / Histone Complex
2. Histone Proteins:
Nucleosome - DNA wrapped around the NCP
Nucleosome Fiber - strand of multiple nucleosomes
DNA…..'Never Acts Alone'
c. Gene Control: the Degree of Chromatin Compaction
i. Methylation of Cytosine in the DNA of Chromatin at CpG Islands
add methyl groups - compact = 'Closed' chromatin = OFF
lose methyl groups - loose
= 'Open' chromatin = ON
ii. Acetylation of Histone Proteins
= 'Open' chromatin = ON
4. Methylation and Acetylation
Epigenetic Marks on the Chromatin
Remodels the Chromatin Architecture
5. Enzyme Controlled Mechanism:
Methylation Enzymes: DNMT - DNA Methyl Transferase
de novo
DNMT-3
maintenance - DNMT-1
Acetylation Enzymes:
HAT Histone Acetylase
HDAC Histone DeAcetylase
6. Epigenome
Chromatin modified - DNA sequence does not change - 'above & beyond'
Genome
- 3,000,000,000 nucleotides of DNA
Epigenome - Chromatin modifications - determines all of our phenotypes
Modification is an ongoing, life-long process, affected by the environment
GENOME
- stable - does not change
EPIGENOME - fluid - constantly changing
Epigenetic modifications can be passed through the germ line!!
7. TURN OFF a CHROMOSOME - Chromosome Inactivation - turn off hundreds of genes
Some interesting information about females
Neurons - have an interesting pot in their nuclei - paranuclear body
White Blood Cells - have a ‘drum stick’
Squamous Epithelium from inside the cheek - Barr Body = Inactive X
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
X Inactivation - mechanism for ‘Dosage Compensation’
Active X
- 1717 genes working = ON
Inactive X - 1717 genes inactive = OFF - condensed to form the Barr Body
Barr Body - condensed Inactive X in an RNA capsule or cage
How does inactivation occur?
XIST gene (X Inactivation Specific Transcript) on X chromosome - Xq13.2
XIST has a counting mechanism - counts the X chromosomes
Counts 1 X chromosome to be active / any others Xs become inactive
XIST is a non-coding DNA gene - makes RNA Transcript - becomes RNA cage
All females are Mosaics for the genes on the X chromosome
Lyonization - Mary Lyon - 1961
X-linked Recessive Inheritance - Males affected / Females carriers who are OK
X Inactivation is a random process…..most females will be 50 / 50
Lyonization:
X maternal - 50 / 60 / 70 / 80 / 90
X paternal - 50 / 40 / 30 / 20 / 10
If a female carries a harmful X-Linked Recessive allele (a), and Lyonizes 90:10
that female can manifest characteristics of that X-linked trait
III. Telomeres: the Amazing Tips of our Chromosomes - Non-Coding DNA
January 1, 1900 - Human life expectancy = 47 years…..Jan 1, 2000 = 77 years
January 1, 2100 - 107? Can Human Aging be Postponed?
Jean Calment - b. Feb 21, 1875, d. Sept 29, 1997 = 122+
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)
Cell Cycle - S phase DNA Replication - DNA Polymerase enzyme
DNA Polymerase always make a mistake at the end of the DNA molecule
Lose ~300 nucleotides with every mitotic division from end of chromosome
15,000 divided by 300 = 50
Cell can divide about ~50 times before the telomeres erode away
ALT - another way to elongate your telomeres - swap with another chromosome
Telomerase Enzyme…..Bad News / Good News
Bad - every time a cell divides - lose ~300 nucleotides from the telomeres
Good - we have an enzyme that will fix and replace the telomere DNA that is lost
Telomere Erosion - ~50 cell divisions > Replicative Senescence – Mitosis Stops!
Telomerase - inhibits telomere erosion - maintains telomeres - avoids senescence
Bad - Telomerase is only working in Ovary, Testis and Stem Cells
those cells can grow forever = Biological Immortality
Good - we may one day be able to activate telomerase in somatic cells
How could that work?
After ~49 cell divisions, the shortened telomere send a signal to the cell cycle
Mitosis will cease - replicative senescence
As we get older…..more and more cells use up their 49 cell divisions
…..more and more cells become senescent
Question - How does the human body replace worn out cells? Answer - mitosis!
As we age, more cells become senescent…..Fewer cells are going through mitosis!
Fewer cells available to replace worn out cells…..Fewer worn out cells replaced!
Worn out cells, that are not being replaced, accumulate…..the result…..
Slow, down hill, physical and mental deterioration…..process called AGING
Progeria - highly accelerated aging / Down Syndrome (DS) - moderate acccelerated
DS with triple USP16 - antagonizes self-renewal…..fewer cells to renew
SPECULATION: What if we could activate the Telomere Promoter in cells all
over the body and maintain their telomeres at normal length.
Continue to replace worn out cells and slow down aging!
“We may soon be able to extend maximum human life span and postpone
or prevent the onset of diseases associated with aging” JAMA
Telomerase - the Immortality Enzyme…...the Telomerase Booth!
Healthy Life Styles - maintain telomere length: diet, exercise, stress management
World Population…..7,181,122,620…..Clock: http://www.poodwaddle.com/Stats/
Test to available to measure telomere length:
http://lifelength.com/index-eng.html?gclid=CKa-vpCe1bkCFa1FMgodB3EAbQ
How Does Telomerase work - Reverse Transcription
Telomerase is a Reverse Transcriptase enzyme…..RNA > DNA
Two Main units: TERT - TElomerase Reverse Transcriptase
TERC – Telomerase RNA Component
A A U C C C - RNA template
Cancer Cells and Telomerase…..Biologic Immortality
Over 90% of cancer cells - telomerase promoter is activated - grow forever
Conundrum: If we turn on telomerase all over the body to increase life expectancy
Will that increase our chances of getting cancer
Question:
What is the #1 recognized carcinogenic factor in humans? Aging!
Why? Cancer begins when one cell in your body mutates to a cancel cell.
The older we become - the more time for that mutation to occur
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
of other wise healthy individuals
Human Cloning and Stem Cells…..New Medical Applications
I. HUMAN CLONING and STEM CELLS - INTRODUCTION
A. Scientific Literacy…..Well Informed - GTFS - Informed Decisions!
B. Cloning: Two Procedures…..
1. Reproductive Cloning
2. Therapeutic Cloning
How do those procedures work?
How are they similar?
How are they different?
What is the end point?
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
Nobel Prize in Physiology or Medicine - 2012
What are those cells?
When are they available for study and to use?
Where would we fine them?
Why would we want them?
How would we use them?
D. Somatic Cells: ~220 Types
1. Normal Body Cells - Specialized for Normal Body Functions
2. Examples: Skin, Nerve, Gland, Muscle, Liver etc.
E. STEM?
1. Word is ‘borrowed’ from the Plants
2. The stem of a plant gives rise to all the branches
3. ESCs give rise to 220 branches…..220 Somatic Cells
4. When and Where…..in the developing human embryo
5. ESCs give rise to 220 highly specialized somatic cells
6. Stem Cells do not come from the brain stem
F. 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
G. Non-Biologic Issues:
1. Controversial / Bioethical Issues:
2. ethical, moral, religious, political issues
H. Biologic Basis of Cloning & Stem Cell Technologies:
1. Human Embryology - Applied Embryology
a. Fetus at 7 weeks - ~14 grams, will rest on a quarter
b. Fetus at 8 weeks - size of a silver dollar with 200 specialized cells
2. HOW? DIFFERENTIATION - cells become different 220 ways
a. What does Differentiation do?
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
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220 Differentiated (Specialized) Human Somatic Cells
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
They can Activate or Repress dozens to hundreds of genes at one time
Cells and Genes - during differentiation of somatic cells:
Liver: ON Liver Genes OFF Kidney, Nerve, and Spleen Genes
Kidney: ON Kidney Genes OFF Liver, Nerve, and Spleen Genes
Nerve: ON Nerve Genes OFF Liver, Kidney and Spleen Genes
Spleen: ON Spleen Genes OFF Liver, Kidney, and Nerve Genes
e. Differentiation Pathway for Pancreatic Beta (insulin) cells: 10 signals
f. Stem Cell Lineage and Identity – unique markers for every step
g. Human Embryome Project - International effort
find all 220 sets of signals
we will then have the signals to make any human cell
with a pluripotent stem cell and the appropriate signals…..
we will be able to make any human cell in a laboratory
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
'Ten Day Rule' - ESCs only exist 10 days under natural conditions
ESCs first grown successfully in a lab – 1998 - James Thomson - U. Wisconsin
Muscle, Bone
Cartilage, Tendon,
Liver, Stomach
Heart, Blood Vessels Lungs, Pancreas
Blood
Cells
HSC
MSC
Brain
Spinal Cord
Epidermis
NSC
ESC
ESCs
HSC
MSC
ESC
NSC
Beginning of Adult Stem Cells:
g. Embryonic Stem Cells…..
From the ESCs stem immediately come four major branches:
HSCs - Hematopoetic SCs > forms all blood cells
MSCs - Mesenchymal SCs > forms muscle, bone, cartilage, tendon
heart, blood vessels
ESCs - Endodermal SCs > forms liver, stomach, lungs, pancreas
NSCs - Neural SCs
> brain, spinal cord, epidermis
These four major branches: beginnings of Adult Stem Cells
220 branches
220 one way streets
220 sets of signals
220 unique sets of epigenetic remodeling marks
h. Adult Stem Cells:
Initial function
- differentiate 220 somatic cells in the developing fetus
Continued function - produce replacement cells the rest of your life
Where are they today?
HSCs - in bone marrow (and in umbilical cord)
producing replacement blood cells
produce ~15,000,000 RBC per second
MSCs - in bone marrow, adipose, amniotic fluid
produce new muscle, bone, cartilage, tendon,
heart, blood vessels
produce paracrine signals for tissue repair
ESCs - in lining of stomach and lining of intestine
making replacement cells
NSCs - in hippocampus and sub-ventricular zones of the brain
i. HSCs and MSCs are easily obtained from bone marrow or adipose
used therapeutically for over 50 years - 1st BM transplant was 1959
j. Differentiation begins at around 10-11 days…..and continues forever
k. Adult Stem Cells - term might be misleading
ESCs - only in the early blastocyst embryo - gone by 10 days
ASCs - in early embryo, fetus, newborn, toddler, child, teen, adult
Know and identify individual somatic cell types by cellular markers
l. Ectoderm = NSCs, Endoderm = ESCs, Mesoderm = MSC and HSCs
m. Adult Stem Cells - two categories:
1.
HSCs, MSCs, ESCs, NSCs
normal, routine, daily differentiation for cell replacement
2.
Tissue Specific - Localized Somatic Stem / Progenitor Cells
small subpopulation of stem cells in all adult tissues
exist in a state of quiescence - mitotically inactive state
rapidly activated in response to injury or trauma
differentiate to replace cells lost in trauma
for Emergency / Catastrophic cell replacement
n. Stem Cells - unique characteristic:
“Stemness”
Self Renewal - Biologic Immortality / Telomerase
Differentiate - produce any of 220 specialized cells
Migrate
- through the developing embryo as the fetus forms
DNA Repair - fix lethal DNA damage from radiation/harmful chemicals
Quiescent - some may be inactive under normal conditions
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…..they 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: 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
E. Dolly born July 5, 1996 / died February 14, 2003
death from virus infection - not due to cloning
1. Ian Wilmut's lab in Roslin, Scotland
2. Dolly today: on display in 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, 'Miracle' Pig in China, Wooly Mammoth?
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
Why?…..Imprints/Epigenetics
H. Serial Cloning:
Normal differentiating somatic cells > 1 trip
Dolly’s (a clone’s) differentiated somatic cells > 3 trips
Serial Cloning - Cloned Clone - 2nd generation > 5 trips
Cloned Clones - 26th generation > 53 trips
I. 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 with Spinal Cord Injury
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
Clone of my friend’s embryo
Remove ECSs from the embryo and grow the cells in the lab
Establish a cell line from ESCs, let them grow until you have
~10,000,000,000 ESCs - Self Renewal / Pluripotent
Directed Differentiation:
a. give those cells the signals for Spinal Motor Neurons
b. result: > ~10,000,000,000 Spinal Motor Neurons
genetically identical to my friend
c. the 'DREAM' - use those cells to repair his spinal cord
- perhaps one day he walks again!
I.
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
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
IV. 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 neurons
Type I Diabetes donor > pancreatic beta cells
Down Syndrome donor > neurons
3. Drug Therapy Screening:
Test thousands of small molecules at one time
Find one of 1000 that has a beneficial therapeutic effect
4. Regenerative Medicine / Tissue Engineering
Produce cells in the laboratory for use for production
human tissues and organs in vitro
5. Nuclear Transfer Cloning Complications:
Oocyte Retrieval Procedure Difficult / Oocyte Shortage
Tedious / Costly
Tumors / Teratomas - ESCs form Teratoma Tumors
both in vivo and in vitro
Bioethical Issues - Ethical, Moral, Religious, Political
Therapeutic Cloning - must remove pluripotent ESCs
from the blastocyst
Removal procedure destroys the embryo
Blastocyst under the microscope - 'Clump of Cells' or
'Unborn Baby'
Who has the Theological / Philosophical wisdom make that determination?
Therapeutic Cloning Success - reported 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
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
1.
2.
3.
4.
5.
6.
Biopsy 8-cell embryo
ANT - Altered Nuclear Transfer,
Living cells from 'Dead Embryo'
Parthenogenesis
Reprogramming
REPROGRAMMING
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
'Yamanaka Factors'
4 signals: Wisconsin - OCT4, SOX2, NANOG, LIN28
'Thomson Factors'
Yamanaka & Gurdon - Nobel Prize – 2012
OSMK Signals: 0.1% efficient / OSMK + disabled Mbd3: ~100% efficient
Nature - OL - September 18, 2013
iPSCs ARE NOT: Adult Stem Cells (HSCs / MSCs / ESCs/ NSCs)
iPSCs ARE:
Adult Somatic Cells Converted into Stem Cells
“Greatest Advancement ever in the History of Stem Cell Technology”
S. Rhine, November 20. 2007
4. iPSCs Applications:
Skin Biopsy > Reprogram Skin cells to iPSCs
Directed Differentiation of iPS > spinal motor neurons
> pancreatic beta cells
> dopamine producing neurons
Use for Cell Replacement / Disease Modeling
No Rejection - Autologous - your own cells
No Embryos are Destroyed
5. iPSCs Tests for Pluripotency:
a.
b.
c.
d.
e.
Cellular Morphology under the microscope
Cellular Biomarkers for pluripotency
Cells induces teratoma in SCID mouse
Cells with GFP followed in chimera embryos > egg and sperm
Cells form an embryo in 'Tetraploid Complementation' Test
…..the Definitive Test
Nature, September 3, 2009
V. iPSCs Applications:
1. Correct Human sickle cell in mice with iPSCs + genetic engineering
December 27, 2007
2. Fibroblast iPSCs for Parkinson's in rat model
GFP cell sorting to stop tumors
April 15, 2008
3. iPSCs from ALS patients (Lou Gerhig's) to study in vitro
July 31, 2008
4. iPSCs: Huntington Dx, Parkinson Dx, Muscular Dystrophy, Type 1
Diabetes, ADA Deficient SCID, Gaucher Dx III, ShwachmanBodian-Diamond Syndrome, Lesch-Nyhan Carrier and
Down Syndrome – study disease model cells in vitro August 6, 2008
5. iPSCs from patients with type 1 diabetes = DiPS
August 31, 2009
6. Acinar > Beta - Direct Cellular Reprogramming in vivo
October 2, 2008
7. Direct Reprogramming: Somatic Cell > Desired Somatic Cell
No iPSC stage = No Teratoma Risk!
Fibroblasts > Functional Hepatocytes
July 21, 2011
Fibroblasts > Functional Neurons
Fibroblasts > Dopaminergic Neurons
August 5, 2011
August 11, 2011
8. Other Applications
Deafness - Generation of Inner Ear sensory epithelia via 3-D Culture
‘Organoid’ - IU
Nature, August 8, 2013
Blindness - 25,000,000 World Wide - visually impaired or blind
>190 Genes Mutated to cause blindness
AMD - Age Related Macular Degeneration
Dry Form - Photoreceptor Loss
Wet Form - Neovascularization
Stargardt's - Photoreceptor loss - pediatric form
huESCs from donated blastocysts for Dry AMD and Stargardt's
low risk of rejection - retina is immunoprivileged site
ESC > RPE - Retina Pigment Epithelium - 99% pure
First Successful Human Trials
Lancet - January 23, 2012
RP - Retinitis Pigmentosa - Most Common Inherited Vision Loss
AD, AR, XLR - Rod Cell Loss
iPSCs cells become Retinal Rod Cells Organoids
Nature Biotechnology August, 2013
Gene Therapy - Fix One Gene
Leber Congenital Amaurosis Type 2 - LCA2
injected 'RPE65' gene therapy AAV vector Lancet, Nov 7, 2009
‘The Forever Fix’ - Gene Therapy book by Ricki Lewis
Chromosome Therapy - Fix Hundreds of Genes
Down Syndrome - Trisomy 21 - 1/750
correct 477 Genes or Inactivate on of the #21s
How? a normal chromosome inactivation mechanism
normal mechanism for Dosage Compensation
in every cell, of every female - one X is inactive
inactivates 1717 genes on X = off
inactive X becomes the Barr Body in the interphase nucleus
confined in an RNA cage
inactivation due to XIST gene on X chromosome
(X Inactivation Specific Transcript)
transfer XIST from X chromosome to #21 in Down Syn
inactivates one of the three #21s > #21 Barr Body
trisomy 21 cell > disomy 21 cell - in vitro Nature, August 15, 2013
Down Syndrome Therapy - Hedgehog Therapy corrects deficits
in DS mouse model in vivo Science Translational Med, Sept 4, 2013
Brain Cells…..100 billion neurons
100 trillion synapses
100 billion astrocytes
Human Astrocytes - Special for LTP (Long-Term Potentiation) Function
Glia cells - billions of non-electrical cells in the brain
astrocytes: active at all synaptic junctions
microglia
oligodendrocytes: myelinating cells
insulates axons in a protective myelin sheath
Neural SC Engraftment and Re-Myelination in the Human Brain
Pelizaeus-Merzbacher Dx - 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
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 into newborn mice >
‘Smarter’ mice with human astrocytes in their brains
Now huiPSCs > huNSCs > therapy for ASD, Schizo?
Cell Stem Cell - March 7, 2013
Stem Cell Transplant Restores Memory and Learning in Mice
huESCs > huMGE-like Progenitors
(MGE = Medial Ganglionic Eminence)
Connected to BFCN - Basal Forebrain Cholinergic Neurons
Synaptically Connected to Endogenous Neurons
Nature Biotechnology - April 2013
Most Common Cause of Intellectual Disability…..1/100
FASD - Fetal Alcohol Spectrum Disorder
Stem Cell Therapy: ‘Social Recognition Recovery in a Fetal
Alcohol Spectrum Disorder Model
Intravenous Injection of NSCs’
Translational Psychiatry - OL - November 2102
Huntington Disease - AD - CAG Triplet Repeat Mutation
Gene elongates every generation
Classic ‘Late Onset’ Condition
MIM = 143100 / 4p16.3
Huntingtin elongated gene > Huntingtin elongated protein
Huntingtin Protein > Gain of Function
Loss of Medium Spiny Neurons in Striatum of Brain
http://en.wikipedia.org/wiki/Huntington's_disease
HUNTINGTON THERAPY:
1. Genetic Correction of HD Phenotypes in iPSCs
HD Patient Fibroblasts > HD iPSCs
HD iPSCs Genetically Corrected via Homologous
Recombination CAG72 > CAG21
Corrrected HD iPSCs > Medium Spiny Striatal Neurons
Patient specific, genetically corrected, HD patient iPSCs
Critical step for the eventual use of these cells in
Replacement Therapy
Cell Stem Cell - August 3, 2013
2. siRNA Gene Silencing - turn off the Huntingtin Gene
Man-made microRNA attaches to 3’ UTR
turns gene Huntingtin OFF
3. Localized Somatic Stem Cells
Adult Stem Cells - for Emergency Cell Replacement
Induce Normal, Pre-existing, Quiescent brain stem cells
to form new medium spiny neurons
Two Induction Signals: BDNF (Brain Derived Neurotrophic Factor)
Noggin
Activates Quiescent Localized Brain Stem Cells and they
differentiate to form new Medium Spiny Neurons which
migrate to the striatum, connect and function normally!
Cell Stem Cell - June 6, 2013
ALS - Amyotrophic Lateral Sclerosis = Lou Gerhig’s Disease
Slow loss of motor neurons
1.
ALS fibroblasts > iPSCs > ALS motor neurons
‘Disease in a Dish’ screened over 5,000 potential molecules
Kenpaullone - 1 of 5,000 - greatly enhanced motor cell survival
Cell Stem Cell - April 18, 2013
2.
‘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 - Degeneration of Dopaminergic Neurons in
in Substantia Nigra of Midbrain
Dopaminergic Neurons: Key Regulators of
Emotional Behavior and Motor Coordination
‘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 - February 2013
Stem Cells for Cancer Therapy - T Lymphocytes & B Lymphocytes
‘Generation of tumor-targeted human T Lymphocytes from
iPSCs for cancer therapy
1.
2.
3.
T Cells > iPSCs
iPSCs - Genetically Engineered > CAR
(Chimeric Antigen Receptor)
iPSCs with CAR > T Lymphocytes
Nature Biotechnology - August 2013
Also iPSCs for:
Alpha-1-Antitrypsin Deficiency
Hutchison Guilford Progeria
Friedreich's Ataxia
Long Q T Syndrome - heart condition
Fragile X Syndrome
Rett Syndrome - Autism Spectrum Model
Schizophrenia
Creating Spermatids from Skin - male infertility
Creating Oocytes from Skin
- female infertility
‘Egg Engineers’ - Nature, August 22, 2013, p.392
Oocyte Stem Cells - Women can procreate for ever!
Nature Medicine – March 2012
Reprogramming to Somatic Cells into iPS in vivo using four stemness
genes under the control of a doxycycline (antibiotic) switch
in vivo iPSCs more closely resemble ESCs in morula
iPOD: http://www.nature.com/news/stem-cells-created-in-living-mice-1.13725#/ref-link-1
9. "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
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
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
Nature - April 7, 2011
Liver Organoids:
Vascularized and Functional Human Liver from
iPSCs-Derived Organ bud Transplant
Nature - July 23, 2013
The Brain Maker: mESCs > mNSCs
>
>
>
>
Primitive Optic Cup Retina
Cerebral Cortex
Primitive Hypothalamus with hormone production
Primitive Cerebellum
Nature - August 23, 2012
‘BRAIN’ - The Human Brainome Project:
‘Brain Research through Advancing Innovative Technologies’
Ten Year Project to Develop Advanced Tools for
Tracking Human Brain Activity Science News - May 4, 2013
Human Brain Organoids - Minibrains - size of an apple seed
Resembles the Fetal Brain at ~9 Weeks
Microcephaly Brain Organoid - smaller than normal
“…..information for generating a brain is intrinsic”
‘Cerebral organoids model human brain
development and microcephaly’ Nature, August 28, 2013
What if we could control human memory? Optogenetics
‘Creting a False Memory in the Hippocampus’
Created a False Memory in mice by optogenetically
manipulating engram-bearing cells in the Dentate
Gyrus of the Hippocmpus!
Science - July 26, 2013
Cardiac Repair after MI (Myocardial Infarction)
Cardiac Fibroblasts > Cardiomyocytes – 3 signals
Cell - August 6, 2010
Cardiac ‘Emergency’ Stem Cells for Functional Cardiac
Regeneration and Repair
Ellison, et. al., Cell 154, pp.827-842, August 15, 2013
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 and
finger nail stem cells
Meat Lab - make burger and sausage from muscle stem cells
The first Test-Tube burger made from 3,000 tiny strips of meat
grown from stem cells taken from a cow’s muscle tissue - cost…..$383,875.
Make a Heart in the Lab? Cannot be done…..Scaffold is too Complex!
BUT…..there is a normal, natural heart scaffold - inside every heart
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
Video: http://www.nature.com/news/tissue-engineering-how-to-build-a-heart-1.13327
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!
“WHEN IT GETS HERE…..WHEN IT ARRIVES…..
SCIENCE FACT IS ALWYS MORE AMAZING THAN SCIENCE FICTION”
S. Rhine - September 2013
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!!
"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
UPDATED: Sept 20, 2013