Future of Stem Cells in Science & Medicine 2024Fall Lecture PDF

5 The Future of Embryonic Stem Cells in Science Regenerative Biology and Disease Dr. Vijendra Sharma Faculty, Department of Biomedical Sciences, University of Windsor Today’s Objectives Alternative mechanisms for deriving ESCs Induced pluripotent stem (iPS) cells Using E...

5 The Future of Embryonic Stem Cells in Science Regenerative Biology and Disease Dr. Vijendra Sharma Faculty, Department of Biomedical Sciences, University of Windsor Today’s Objectives Alternative mechanisms for deriving ESCs Induced pluripotent stem (iPS) cells Using ESCs to clone whole organisms Using ESCs to alter organisms Ethics surrounding the use of ESCs Parthenogenesis Eggs activated without sperm are termed “parthenotes” Electric Manipulate Genes Impuls es Parthenogenesis Critical Events in ‘Activation of the Egg’ Fertilization triggers: Depolarization Polar body extrusion Mos degradation and completion of meiosis Mos Regulation of MII in Egg Activation /Spy1 Electrical Stimulus Parthenogenesis in Mammals Spontaneous egg activation Can occur in nature - dermoid cysts - failure of the follicle to release the egg at met II Can result in teratotoma formation Parthenogenesis in Mammals Haploid parthenotes (haploid gynegenote) early cleavage divisions can occur the zygotic cell cycle does not depend on the full complement of chromosomes. Can become diploid gynegenotes – has generated SCs in some species. Diploid Gynegenote Artificial egg activation before extrusion of the second polar body. It can give rise to stem cells but undergo developmental and cannot give rise to a mammalian embryo. www.newscientist.com Haploid and Diploid Parthenotes Haploid gynegenote Diploid gynegenote Parthenogenesis in Mammals Diploid male fertilization or androgenesis Egg is fertilized by sperm, which then duplicates its own chromosomes because the chromosomes of the egg are either absent or inactive Result in a molar pregnancy (Hydatidiform mole) is an anomalous growth that implants and grows in the uterus. Parthenogenesis in Mammals: What’s the Problem???? Not an ‘in vitro’ problem – the first test tube baby was born in 1978 Androgenesis experiments suggested imprinting Both male and female pronuclei are important Genes of Importance Igf2 – Insulin-like growth factor 2 – Necessary for the blastocyst to implant and develop into the amniotic sac and other tissues that support the growth of the fetus. H19 - Cai and Cullen (2007) a noncoding RNA that functions as a primary miRNA. Critical for the growth of the fetus. In the egg, H19 is turned on but Igf2 is turned off. In sperm, Igf2 is turned on and H19 is turned off.  - (IRC) Imprinting control region (also controls H19 - (DMR1) Differentially- methylated Region-1 - (MAR3) Matrix Attachment Region -3 miRNA is Imprinted H19 - miRNAs that act to downregulate developmentally relevant mRNA species! Figure 7-112 Molecular Biology of the Cell (© Garland Science 2008)  It is possible to fool a mammalian egg to start dividing, but, without Igf2, the blastocyst tends to stop growing and will not support further fetal development…. Transgenic Igf2 female mouse Headlines “Mouse Created Without Father“ “The Mouse that Roared: Virgin Birth"... HUGE significance for stem cell research, cloning, and medicine in general. Demonstrated the mechanism for mammalian parthenogenesis. Efficiency: Started with a total of 598 combined eggs 78% of these activated eggs started to divide 91.2% produced blastulas Transferred into 26 female mice (14-15 eggs per female) 24 of the mice became pregnant Killed the mice and removed the embryos at 19.5 days of gestation - 10 live and 19 dead pups Successfully restored two of the live pups. One of these grew to adulthood and appeared normal and fertile.  One living adult mouse from 598 eggs 91% of the original eggs to produce blastocysts that contain stem cells. Parthenogenesis in Mammals Not technically somatic cell nuclear transfer or cloning  the parthenote had genes from two female mice Theoretical possibility for women to have stem cells that contain their own genes by donating a reasonably small number of eggs and combining them. Can We ‘MAKE’ ESCs from Differentiated Cells? Thomas Graf & Tariq Enver Nature 462, 587-594 (2009) doi:10.1038/nature08533 Induced Pluripotent Stem Cells (iPSC) Takahashi et al 2006, 2007 Cell (Won the Nobel prize) Kazutoshi Takahashi and Shinya Yamanaka, 2006 Generation of iPSC from Fibroblasts Selected 24 candidate genes as “factors” to use to induce pluripotency in somatic cells Developed a system in which the induction of the pluripotent state was detected by resistance to a drug Introduced each of the 24 genes into fibroblasts No single factor produced stem cell colony Examined the effects of removing individual genes one at a time Generation of iPSC from Fibroblasts Identified 10 factors whose individual withdrawal from the pool of 24 resulted in no stem cell formation Used this pool of 10 factors to withdraw individual factors again Colonies did not form when Oct4 or Klf4 were removed When c-Myc was removed, colonies were not stem like Removal of Sox2 results in very few stem cell colonies Removal of any other of the 10 factors had no significant change REPROGRAMMING FACTORS: OCT4 (O), SOX2 (S), KLF4 (K), cMYC (M) (Referred to as: OSKM) Are iPSC TRUE Stem Cells Proof of marker genes – oct 4, sox2 Proof of embryoid body formation – 3D aggregates of pluripotent stem cells that can undergo spontaneous differentiation into all 3 germ lineages Proof of cell differentiation – give factors to induce differentiation and prove functionality Proof of teratoma formation – iPSC injected into mice should develop a teratoma (growth containing all three germ layers) Proof of chimera formation – iPSC injected into mouse embryos should produce an entire viable organism Reprogramming is Extremely Inefficient Less than 1% of cells become iPS cells Cheung et al, 2013 Tumour Suppressor Gene Barrier CycD Cdk4,6 p16 p53 CycE p21 Cdk2 Hydbring and Larsson (2010) Tumour Suppressor Gene Barrier cMyc aids in overriding CycD reprogramming p16 Cdk4,6 induced senescence cMy p53 c CycE p21 Cdk2 Other ideas (inhibit p53/p21?) **Override epigenetic barrier < 1% Hydbring and Larsson (2010) Applications of Human iPSC Chun et al. (2010) Significance Problems with iPSC Search for less carcinogenic gene set Search for non-genetic factors that can facilitate reprogramming Gene substitutes for cMyc Increasing production efficiency Search to decrease mutation rates of iPSC Nuclear Transfer = Whole Organism Cloning Roadblocks technical problems decreasing efficiency politics/ethics Whole Organism Cloning Projects Dolly (1997) used genetic material from an adult mammary gland cell. Lived to 6 and had babies (was she older due to the age of the sheep she was cloned from?) Whole Organism Cloning Projects Human embryonic clone - Newcastle University, UK. Clones survived for only a few days and did not produce any stem cells. November 2007 – cloning of primate embryos using novel method for extracting the DNA. Jan. 2009, scientists from Spain cloned a Pyrenean ibex, an extinct form of goat. Died shortly after birth due to physical defects in its lungs. Human Cloning 2004, S. Korean Group - cloned human embryos and extracted stem cells. Retracted when it emerged that the lead author, Dr Hwang Woo-suk, had fabricated his work. Update on Cloning Advances Not a big business – IVF and pharma have established that there is no monetary advantage to cloning entire humans. Pets and animal cloning are very expensive and do not yield high pay. Horses have been cloned but are now banned from competition. Cloning to make tissues for diseases is a very hot area! (therapeutic cloning) Therapeutic Cloning Extract the nucleus from an egg Nucleus extracted from the somatic cell of the patient. – implant into an egg Stimulated to divide and shortly thereafter forms a cluster of cells known as a blastocyst Embryonic stem cells extracted and formed into lines ESC line infused into the patient - - or differentiated into other tissue before infusion.  Strategies for Making Pluripotent SCs Human Reproduction Open, Volume 2019, Issue 1, 2019, hoy024, https://doi.org/10.1093/hropen/hoy024 ‘Manipulating’ the Genome Can we alter the genome to correct mistakes in disease? (in cells or in whole organisms?) At the very least, manipulating the genome can be used to understand the importance of specific genes in regeneration. Limitations to Transgenic Modification 1) Can’t control where the gene inserts dangerous insertion sites Epigenetic modification of promoter over time. 2) Limited to driving genes with a specific promoter. Clinical Limitations to Targeted Modifications (Crispr/Cas) Technical issues with CRISPR/Cas remaining (off- target effects; efficiency) Targeting modifications in ESCs – ethical barriers Viral Gene Therapy Some viruses bind to the host and introduce their genetic material as part of their replication cycle. Strategy for changing an individual’s DNA Adenovirus was the first to be tested in the clinic Retrovirus (lentivirus) has been among the most successful Viral Gene Transfer Adenovirus – DNA db strand viruses that do not integrate into the human genome Retroviral expression vectors - RNA viruses use RT to copy their RNA into DNA – integrase incorporates into the host. Envelope virus Lentivirus - one of the retroviridae family (eg. HIV) - can infect non-dividing cells! Gene Transduction via Lentiviral Vectors ‘vector’ gag/pol envelope (VSV-G) Retrovirus needs: Gag – processed to matrix and 293T packaging cell line other proteins needed for retroviral core/capsid. Non-replicating Lentivirus Pol – reverse transcriptase, RNaseH, integrase Env – envelope protein Target Cell Infection Gene Transduction via Lentiviral Vectors ‘vector’ gag/pol envelope (VSV-G) 1. Transfect “packaging cells” with plasmids that create non- replicating lentivirus. 293T packaging cell 2. line Concentrate virus ~100x, to Non-replicating obtain 10 8 -10 9 IU/ml Lentivirus 3. Infect target cells Target Cell Infection 4. Assess transduction by fluorescence/flow. Gene Therapy Problems Viral methods pose worry with toxicity and immune and inflammatory responses. Problems with the site of integration! Ectopic expression may become silenced over time. Liposome-mediated therapies may eliminate the worry of using viral backbones. ** Optimal expression is a worry. Ex of Problems! Jesse Gelsinger (June 18, 1981 - September 17, 1999) had a genetic mutation leading to a less severe form of ornithine transcarbamylase deficiency (usually fatal at birth). Jesse’s form was controllable with diet and medication. Trial to use adenovirus (18 yrs) to correct the gene Died four days later due to immune reaction to the virus. SCID Trials Deficiency in B/T cells – different forms In 1990, four-year-old Ashanthi DeSilva became the first patient to undergo successful gene therapy. In 2000, a SCID trial resulted in patients with a functional immune system. 10 patients in one trial developed leukemia – a gene inserted near an oncogene. No leukemia cases have yet been seen in trials of ADA-SCID, which does not involve a gene that may be oncogenic Since 1999, gene therapy has restored the immune systems of at least 17 children with two forms (ADA-SCID and X-SCID) of the disorder. Technologies to Manipulate Gene Expression: HURDLES Delivery mechanism of the DNA Efficiency Stability Safety of stable integration Current State of SC Therapies Importance (clinical, research, testing drugs) is recognized at many levels in the public and government. Science is aware of the levels of rigor that must be followed.  First Approved hESC Clinical Trial Jan. 24, 2009 Economics, not science, thwarts embryonic Nov. 11, 2011 stem cell therapy Dr. Tom Okarma, Geron Inc. Menlo Park CA https://www.nature.com/articles/d41586-018-03268-4 Current hESC trials – US The ACT trial - testing the safety of hESC-derived retinal cells to treat patients with an eye disease called Stargardt Macular Dystrophy (SMD). Second ACT trial - testing the safety of hESC-derived retinal cells to treat patients with age related macular degeneration. Current hESC trials – US MD trials stopped in 2017 because a patient developed a membrane over the eye. Phase I/II clinical trials involving retinal pigment epithelial (RPE) cells, derived from hESC, for severe myopia – approved February 2013 Diabetes Trial Phase I clinical trial - ViaCyte beta cells from hESCs - approved August 2014. Safe and tolerable Animal models show regeneration within months. Getting it Right … Scientific Concerns Manufacture Feeder cells; Risk of Transmissible Spongiform Encephalopathy (TSE); genetic stability; Good manufacturing practice (GMP) Issues with directed differentiation Reproducible production of desired cell type with appropriate functionality Making the First Step.. Safety issues for SC use in vivo Uncontrolled cell proliferation; reprogramming; immune rejection Unexpected side effects (e.g., fetal neurons in Parkinson’s) Efficacy Dose, delivery, and functionality Cardiomyocytes derived from human ES cells can restore some myocardial function in a pig model of cardiac disease Ethics 67 LOGISTICS Who is footing the bill?  Pluripotent stem cells, source here