Sam Rhine - Genetic Update Conference - 2013-14

Video in 4 parts. Run time: 3 hours: 50 minutes Part 1: Genome, Coding and Non-coding Genes and Gene Control (1 hr. 10 min.) Part 2: X inactivation and Lyonization; Telomeres (40 min.) Part 3: Stem Cells and Reproductive Cloning (55 min.) Part 4: Therapeutic Cloning, iPSC’s and Medical Applications (1 hr. 5 min.)

Outline Part One I. Human Genome (Part1 00:06:30 to 00:11:19)

A. 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 B. Human Genome Project (HGP) - Historical Perspective

1. Largest scientific project in world history 2. Projected to be 15 year international cooperative effort 3. 20 countries involved - main contributions from Britain and U.S. 4. Began: October 1, 1990 / End: April 25, 2003 5. Dr. Francis Collins - Director of HGP 6. 1990 geneticists thought: a. An organism as complex as a human needs ~100,000 proteins and,

since genes make proteins…..humans must have ~100,000 genes b. Most of the DNA in the genome must carry the genetic code for proteins

7. Now, the HGP has shown that humans have approximately 34,500 genes.

II. Coding and Non-Coding Genes (Part1 00:11:20 to 00: 32:35) A. Coding vs. Non-coding Genes

1. Two Types of DNA….. ~1.5% - 'Coding' DNA………..GENETIC CODE (triplet code) for PROTEINS

~98.5% - 'Non Coding' DNA…..NO GENETIC CODE for PROTEINS

2. Two Types of Genes….. a. Coding DNA Genes Sequence of DNA responsible for production of a specific Protein molecule Genes produce ~21,000 protein molecules – Transcription & Translation b. Non-Coding DNA Genes Sequence of DNA responsible for production of a specific RNA molecule Genes produce ~13,500 RNA molecules - Transcription & shRNA Dicing 1

B. Coding DNA Gene - Transcription followed by Translation 1. DNA double strand with Promoter (on/off switch) in the nucleus 2. Promoter is a ‘Docking Site’ for control molecules to attach - turn gene on or off 3. Transcription factor docks on the promoter - initiates transcription 4. DNA transcribed into mRNA (transcript) in nucleus transcript with genetic code, plus 5' and 3' UTRs - UnTranslated Regions 5. Transcript translated into protein at ribosome - on endoplasmic reticulum TRANSLATION at the RIBOSOME 6. Coding DNA Genes are Discontinuous - in separate units called - Exons Exons - Coding DNA - part of the 1.5% Coding DNA Introns - Non-Coding DNA - part of the 98.5% Non Coding DNA 7. 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 8. ALTERNATIVE SPLICING - Special Activity - occurs during Processing 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

9. 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 - 28.5% Exome plus Introme = ~30% of the genome

C. Non-Coding DNA Genes - Transcription followed by shRNA Dicing

1. produce sncRNAs - small non-coding RNAs

microRNA = miRNA = miR lncRNAS - long non-coding RNAs

2. Non-Coding Gene DNA is transcribed > microRNA 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 = microRNA microRNA attaches to the 3’ UTR of a Coding DNA gene transcript micro RNA guides RISC (RNA Induced Silencing Complex) to 3’ UTR RISC protein complex - ‘turns off’ the gene = ‘Gene Silencing’

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D. Human Genome Project and ENCODE - Review and 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: a. Docking Sites: Many non-coding parts of the genome, the Junk, contain ‘Docking Sites.’ Docking Sites are where control proteins bind to affect gene expression. How Many? Already knew of ~21,000…..2,890,000 new Docking Sites b. New genes: ~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

3. 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

III. Gene Control—On/Off Mechanisms (Part1 00:47:28 to 01:10:00) A. Transcription Factors - turn transcription ON and OFF 1. attaches to the Promoter - Promoter acts as 'Docking Site' 2. TF Activators - bind to promoter and turns genes ON 3. TF Repressors - bind to promoter and turns gene OFF 4. Enhancers - can also bind to DNA and enhance protein output B. microRNA = miRNA - negative regulators - turn genes OFF 1. microRNA, ~22 nts, complementary to and binds to 3' UTR of transcript 2. helps usher RISC (RNA Induced Silencing Complex) to the 3' UTR 3. RISC blocks the ribosome > blocks translation > turns gene off 4. referred to as miRNA 'Gene Silencing' 3

C. Epigenetics - 'Epi' means…..upon / on top of / above and beyond (52:10 to 1:10) 1. Components a. Chromatin - epigenetic control mechanism acts on chromatin DNA (Genome) interacting with Histone Proteins DNA / Histone Complex b. Histone Proteins: Nucleosome - DNA wrapped around the NCP Nucleosome Fiber - strand of multiple nucleosomes DNA…..'Never Acts Alone'

2. Mechanisms: Gene Control: the Degree of Chromatin Compaction a. 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 b. Acetylation of Histone Proteins = 'Open' chromatin = ON c. Methylation and Acetylation Epigenetic Marks on the Chromatin Remodels the Chromatin Architecture d. Enzyme Controlled Mechanism: Methylation Enzymes: DNMT - DNA Methyl Transferase de novo DNMT-3 maintenance - DNMT-1 Acetylation Enzymes: HAT Histone Acetylase HDAC Histone DeAcetylase 3. 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 220 Cellular Phenotypes GENOME - stable - does not change EPIGENOME - fluid - constantly changing Epigenetic modifications can be passed through the germ line! Part 2 IV. X Inactivation and Lyonization ( Part2 00:00:00 to 00:23:30) A. Some interesting information about females

Neurons - have an interesting spot in their nuclei - paranuclear body White Blood Cells - have a ‘drum stick’ Squamous Epithelium from inside the cheek - Barr Body = Inactive X

B. 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 4

C. 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

D. Mechanism (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

E. Lyonization - Mary Lyon - 1961

X-linked Recessive Inheritance - usually Males affected / Females carriers OK X Inactivation is a random process…..most females will be 50 / 50

How it works:

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 Identical Twin girls who carry Muscular Dystrophy: Lyonize 50/50 and 90/10 Mother who carries Anhidrotic Ectodermal Dysplasia: Lyonization skin map

F. Why it is important? The mechanism might be the key to treating chromosome anomalies such as Trisomy 21 (Down Syndrome)

V. Telomeres: the Amazing Tips of our Chromosomes (Part2 00:24:48 to 00:40:08)

A. Human life span: 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+

B. Form and Function 1. Telomeres - protective cap on the end of all of our chromosomes 2. 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

C. What are telomeres made of? 1. ~15,000 Non-Coding DNA nucleotides - on the tips of each chromosome 2. Repeating Hexanucleotide unit - T T A G G G - x2500 (6 x 2,500 = 15,000)

D. 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 5

E. Telomere fun facts: 1. ALT – another way to elongate your telomeres - swap with another chromosome

2. 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 3. Telomere Erosion - ~50 cell divisions > Replicative Senescence – Mitosis Stops! 4. 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

a. 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? Stem Cell 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 6ccelerated DS with triple USP16 - antagonizes stem cell mitosis

b. 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

5. World Population…..7,181,122,620…..Clock: http://www.poodwaddle.com/Stats/ 6. Test to available to measure telomere length:

http://lifelength.com/index-eng.html?gclid=CKa-vpCe1bkCFa1FMgodB3EAbQ

F. 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 G. Telomerase NOT fun fact: Cancer Cells and Telomerase…..Biologic Immortality

Part 3 [cut the first 2:22] VI. Stem Cells (Part3 00:00:00 to 00:37:40)

A. Definitions:

1. Stem Cells: Four Main Types a. Embryonic Stem Cells (ESCs) - first grown in lab - 1998 - natural

b. Adult Stem Cells (ASCs) - first recognized - 1961 - natural c Cancer Stem Cells (CSCs) – first recognized - 1997 - natural d. iPSCs (induced Pluripotent Stem Cells) - 2006 / 2007 - man-made Nobel Prize in Physiology or Medicine – 2012 2. Somatic Cells: ~220 Types 6

3. Why are they called stem cells?

a. Word is ‘borrowed’ from the Plants b. The stem of a plant gives rise to all the branches c. ESCs give rise to 220 branches…..220 Somatic Cells d. When and Where…..in the developing human embryo e. ESCs give rise to 220 highly specialized somatic cells

4. Special Quality of Human Stem Cells – Potency a. Pluripotent - a stem cell with the potential to become

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

MANY of the 220 specialized human cells: 2. Adult Stem Cells c. Totipotent – a stem cell with the Total Potential to form the

Fetus, Placenta and Membranes: zygote / early blastomeres Blastomeres from 8 cell embryo > Identical Octuplets

B. Differentiation: 1. The process by which stem cells become 220 different types of somatic cells.

2. How does it work? (see diagram)

O O S N E E T S W A of Y S S I T G R N E A E L T S S 220 Differentiated (Specialized) Human Somatic Cells

7 3. Molecular Signal Proteins direct differentiation. (“signals”)

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

4. Pancreatic Beta (insulin) cell example: set of 10 signals 5. Embryology (The beginning of differentiation.)

ESCs

HSC MSC ESC NSC

Blood Cells

Cartilage, Tendon, Liver, Stomach Lungs, Pancreas Brain

Spinal Cord

Beginning of Adult Stem Cells

HSC MSC ESC NSC

Muscle, Bone

Epidermis

Heart, Blood Vessels

VII. REPRODUCTIVE CLONING (Part3 00:37:40 to 00:54:51)

A. Definitions: 1. Clone: A genetically identical copy of…..anything you choose

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

2. Reproductive Cloning: Reproducing an individual who is a genetically identical copy of another individual. a. Reproductive clones are born as babies.

b. First Mammalian Clone: Animal - Sheep [Names - Megan & Morag - cloned from donated embryo nucleus] c. Second Mammalian Clone: DOLLY

[unique because she was the first clone from donated adult somatic donor nucleus]

B. 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 6. Success rate for Dolly: 0.36% 7: Dolly: a. Dolly born July 5, 1996 / died February 14, 2003] b. created in Ian Wilmut's lab in Roslin, Scotland c. today Dolly is stuffed and on display in Royal Museum in Edinburgh C. Reproductive Cloning in humans: Not done because it is too risky. [imprints, epigenetics] D. 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? E. 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 - 25th generation > 51 trips F. Most important lesson from Dolly:

1. Highly specialized, terminally differentiated, somatic donor cell nucleus, when placed into an enucleated egg, is reprogrammed from specialized reverts 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? 9

Part 4 VIII. THERAPEUTIC CLONING (Part4 00:00:00 to 00:20:55)

A. Definition: Creating a genetically identical copy of an individual’s embryo for the purpose of using the embryonic stem cells for medical therapy.

B. Procedure: 1. Human Oocyte Retrieval.

2. Enucleation of the egg - surgery on the human egg to remove nucleus 3. Somatic Cell Nuclear Transfer (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

6. Remove Embryonic Stem Cells (ESCs) from the embryo 7. Grow the cells in the lab to establish a cell line

C. Why? They are pluripotent and with the appropriate signals can make all 220 types of human cells D. Medical Applications for Pluripotent Stem Cells (PPSCS) from BOTH SOURCES: PPSCs from Nuclear Transfer (Therapeutic) Cloning or PPSCs from induced Pluripotent Stem Cells (iPS) made from human somatic cells (skin)

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

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

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 human tissues and organs in vitro E. Therapeutic Cloning is being replaced by iPSCs because: 1. Therapeutic cloning is difficult and costly. 2. Threat of teratomas remains 3. Bioethical Issues - Ethical, Moral, Religious, Political 10

IX. Induced Pluripotent Stem Cells (iPSCs): The new frontier (00:19:20 to 00:37:57)

A. iPSCs are "Ethical" Pluripotent Stem Cells because no embryos are destroyed

B. Definition of iPSCs: Stem Cells created by reprogramming somatic cells back to a pluripotent state.

1. iPSCs ARE NOT adult stem cells (HSCs / MSCs / ESCs/ NSCs) 2. iPSCs ARE adult somatic cells converted into stem cells

C. The Big Discovery: How to reprogram a cell. [Reprogramming = DeDifferentiation]

1. Who? Shinya Yamanaka - Kyoto University in Japan / UCSF Mouse iPSCs - July 7, 2006 Human iPSCs - November 20, 2007 2. How? 4 signals: Japan – OCT ¾, 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] 3. Process: Take a skin Biopsy > Reprogram Skin cells to iPSCs > direct differentiation using signals > create new cells i.e. spinal motor neurons, pancreatic beta cells, or dopamine producing neurons. 4. Application (See Section D. above.) Use for Cell Replacement / Disease Modeling / Drug Therapy Screening

D. iPSCs Tests for Pluripotency:

1. Cellular Morphology under the microscope 2. Cellular Biomarkers for pluripotency 3. Cells induces teratoma in SCID mouse 4. Cells with GFP followed in chimera embryos > egg and sperm 5. Cells form an embryo in 'Tetraploid Complementation' Test [the Definitive Test: Nature, September 3, 2009]

E. Medical advances using iPSCs:

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 11

4. iPSCs: Huntington Dx, Parkinson Dx, Muscular Dystrophy, Type 1 Diabetes, ADA Deficient SCID, Gaucher Dx III, Shwachman- Bodian-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!

a. Fibroblasts > Functional Hepatocytes July 21, 2011 b. Fibroblasts > Functional Neurons August 5, 2011 c. Fibroblasts > Dopaminergic Neurons August 11, 2011

F. Ongoing Research: 1. Deafness - Generation of Inner Ear sensory epithelia via 3-D Culture

‘Organoid’ - IU Nature, August 8, 2013 2. Blindness - 25,000,000 World Wide - visually impaired or blind >190 Genes Mutated to cause blindness

3. 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 4. 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 5. 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] 6. Chromosome Therapy - Fix Hundreds of Genes Down Syndrome - Trisomy 21 - 1/750 How? a. Study a normal chromosome inactivation system. (X inactivation). Inactivation in females is due to XIST (X Inactivation Specific Transcript) gene on X chromosome. Transfer XIST from X chromosome to #21 in

Down syndrome which inactivates one of the three #21s. Done in vitro Nature, August 15, 2013

b. Down Syndrome Therapy - Hedgehog Therapy corrects deficits in DS mouse model in vivo Science Translational Med, Sept 4, 2013 12

X. OTHER MEDICAL RESEARCH: (Part4 00:37:58 to 01:05:37) A. Cell Replacement Therapy: 1. Huntington Disease: AD – CAG triplet repeat Mutation – the 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

2. Parkinson Disease: The degeneration of dopaminergic neurons in substantia nigra of midbrain.

Dopaminergic neurons are the 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

B. iPSCs for: 1. Cancer Therapy - T Lymphocytes & B Lymphocytes

‘Generation of tumor-targeted human T Lymphocytes from iPSCs for cancer therapy

a. T Cells > iPSCs b. iPSCs - Genetically Engineered > CAR

(Chimeric Antigen Receptor) c. iPSCs with CAR > T Lymphocytes

Nature Biotechnology - August 2013 2. Also 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 3. Fertility issues: 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 13

4. Tissue Engineering (man-made tissues and organs): a. Teeth Organoids b. Optic Cup Retina - from Eye Organoids Nature - April 7, 2011 c. Liver Organoids: Vascularized and Functional Human Liver from iPSCs-Derived Organ bud Transplant. Nature - July 23, 2013 d. The Brain Maker: mESCs > mNSCs > Primitive Optic Cup Retina

> Cerebral Cortex > Primitive Hypothalamus with hormone production > Primitive Cerebellum Nature - August 23, 2012 References: ‘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

The “JOIN THE TEAM” segment is inserted here. Students are encouraged to imagine how they might help in the advancement of medical genetics.

[Part 4 00:46:45 to 00:52:20] See final page of outline for specifics. e. Heart: 1. Cardiac Repair after MI (Myocardial Infarction) Cell - August 6, 2010

2. Cardiac ‘Emergency’ Stem Cells for Functional Cardiac Regeneration and Repair Ellison, et. al., Cell 154, pp.827-842, August 15, 2013 3. Heart Vein for newborn baby made in vitro - MSCs on man-made tubular scaffold

4. How to make a heart in the lab: Decellularization - remove all heart cells with detergent / pressure

1. detergent is pumped into the aorta filling the arteries that feed the heart 2. 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. endothelial precursor cells are pumped into the blood vessels 2. 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 14

f. Source of meat: make burger from muscle stem cells! g. Pancreas: How to 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 How to 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! 15

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. Graduate School: You have choices depending on what you would like to do. A medical degree will provide you with the most options but you can also get a master degree (for teaching and counseling) or a Ph.D (for research and teaching at the college level). Some ambitious folks also get a combined M.D. and Ph.D.

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. Then comes Residency which is 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.

Ph.D. Most people who choose the Ph.D. route first get a Masters degree. Generally it takes 2 years to earn a Masters (MA or MS) degree and another 2 to 3 to earn a Ph.D. 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

Masters Degree in Teaching: Many people will make a major contribution to all these careers in the future by majoring in Education in college and 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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