Stem Cell Exam 1 Study Guide
Stem Cell Exam 1 Study Guide 3260
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This 8 page Study Guide was uploaded by AnnaCiara on Friday February 19, 2016. The Study Guide belongs to 3260 at University of Connecticut taught by Dr. Conover in Spring 2016. Since its upload, it has received 65 views. For similar materials see Stem Cell Biology in Physiology at University of Connecticut.
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Date Created: 02/19/16
PNB 3260 Stem Cell Biology Exam 1 Study Guide Questions 1. What are the 2 requirements for a stem cell to be considered pluripotent? 2. What are the main differences between adult/somatic stem cells and embryonic stem cells(ESCs)? 3. When is a cell considered totipotent? 4. What are iPSCs? How are they generated? What are they meant to resemble? 5. What are 2 types of adult/somatic stem cells being considered for research/therapeutic potentials? 6. What is the zona pellucida? What is its function/role? 7. True/False: Spatial organization in the blastula influences a cell's future function. 8. What are the three germ layers and what does each give rise to? 9. What is the difference between the trophoblast and the embryoblast? 10.Give five examples of where pluripotent stem cells can be found. 11.What are three methods of experimentally acquiring pluripotency? 12.What are the functional criteria for pluripotent stem cells? 13.List the stages and corresponding transcriptional factors associated with development from the morula to the post-implantation egg cylinder. 14.Explain the process of gene targeting in chimera formation. 15.What is tetraploid complementation? 16.What was Gurdon's discovery? 17.What was Yamanka's discovery? 18.Who was Hans Spermann and what discovery did he make? 19.Describe the original protocol of nuclear transfer. 20.Describe the "resistance" found in differentiated cells. 21.What is the importance of the cell cycle and what stage does the donor cell and oocyte need to be in for a successful reprogramming? 22.What is one of the major limitations of SCNT? 23.What are two ways to distinguish iPS cells? 24.Define an embryonic stem cell line. 25.What is "gastrulation"? 26.What cell types are in the central nervous system? 27.Name 2 locations of adult neuronal stem cells in rats. 28.What is the neural plate? 29.Define neurulation. 30.Describe the process of neural differentiation starting at the ESC. 31.What are the 2 classes of cortical neurons? 32.In what order are the layer specific neurons generated from the human embryonic stem cells? 33.What is an organoid? 34.What are the requirements for a stem cell to be useful in cell therapy? 35.What is the function of Doublecortin (Dcx)? 36.How can you test the functionality of transplanted neuronal donor cells? 37.Define epigenetics. 38.What variations can be found between iPS cells and ES cells? 39.What might cause a donor cell to retain it's differentiated characteristics? 40.List four possible outcomes of the iPSC model: 41.What are the "master regulators"? Are they sufficient in guaranteeing pluripotency? 42.How is the state of a cell defined? 43.What is the fundamental unit of chromatin? 44.Name two different chromatin structure variations and list their similarities/differences. 45.Where is a common site of DNA methylation? 46.What are passive and active demethylation? 47.What is the function of post-translational histone modifications? 48.Is cellular differentiation more associated with gene activation or gene repression? 49.What molecules are associated with modifying DNA? 50.What molecules are associated with unmodifying DNA? 51.What are chromatin remodelers? 52.Name one possible outcome of a mutation of a chromatin remodeler and it's effects. 53.What is the Polycomb group (PcG) of proteins? 54.What happens if the PcG is not present? 55.What are the activating and repressive roles of Oct4, Klf4 and Sox2? 56.What is the function of cMyc? 57.What are 3 additional factors affecting the genome? 58.What are the 3 ways to validate iPS cells? 59.What is valproic acid (VPA)? 60.What models explain low efficacy of iPSCs? 61.Describe the waves of molecular events underlie cellular reprogramming. 62.What is "direct conversion"? Answers 1. (1) ability to self-renew: division to make copies of themselves and (2) ability to differentiate: able to give rise to mature cell types. Can make the entire embryo i.e. cells from all 3 germ layers 2. Adult/somatic stem cells are multipotent: can generate MANY (but not all) organ-specific cell types - usually only one germ layer. They are already partially specialized - can produce some or all of the mature cell types found in their respective tissue/organ ESCs are pluripotent: able to generate ANY cell types in the body. can make the entire embryo i.e. cells from all 3 germ layers. Experimentally they are obtained during the blastocyst stage of development from an oocyte created by in vitro fertilization 3. A cell is totipotent for a few days after fertilization of the oocyte by a sperm (prior to blastocyst formation). After this time period the cell becomes pluripotent. A totipotent cell has the ability to form ANY and ALL cells in an organism (fertilization to birth to death). Can make the entire organism - reserved for fertilized egg (zygote) or a cell from up to the 8 cell stage. 4. iPSCs are induced pluripotent stem cells. They are generated by exposing adult/somatic (differentiated) cells to transcription factors that are important in ESCs (Yamanaka factors: Oct4, Sox2, c-Myc, Klf4). They are meant to resemble ESCs as much as possible. 5. (1) Blood stem cells: transferred via bone marrow transplants (umbilical cord blood may be viable alternative) (2) Mesenchymal stem cells: ubiquitous cell in body that can become bone, cartilage, fat and possibly muscle 6. The zona pellucida is an extracellular matrix material that surrounds an oocyte and is activated by sperm penetrating the oocyte. Functions: 1-restricts additional sperm from penetrating an already fertilized oocyte 2- maintains distinct formation of dividing cells 3-Prevents ectopic pregnancy by containing the cells until they reach the uterus at which point 'hatching' occurs (embryo breaks out of zona pellucida and implants in uterine wall). 7. TRUE: Spatial organization in the blastula influences a cell's future function. 8. The 3 germ layers and structures they give rise to: a. Ectoderm - epidermis and nervous system i. Outer surface: skin cells of epidermis ii. Central nervous system (CNS): neurons iii. Neural crest: pigment cell (melanocyte) a. Mesoderm - muscle, connective tissue, blood, bone, kidney, heart i. Dorsal: notochord ii. Paraxial: skeletal muscle cells iii. Intermediate: tubule cell of the kidney iv. Lateral: red blood cells v. Head: facial muscle b. Endoderm - epithelium of gut and respiratory system (lungs), relies on mesoderm i. Digestive tube: pancreatic cell ii. Pharynx: thyroid cell iii. Respiratory tube: lung cell (alveolar cell) Note: each layer influences the other layers **Remember order: Zygote-->blastula-->gastrula(inserts into uterine wall)-->3 germ layers AND both germ cell types 9. The trophoblast and the embryoblast are the first two derivations from the blastocyst. The trophoblast is the outermost cell layer that will become the placenta and umbilical cord. The embryoblast (also known as the inner cell mass -ICM) is the inner portion that gives rise to both embryonic tissues (epiblast) and extraembryonic tissues (hypoblast). 10.Pluripotentcy/pluripotent stem cells are found in: the morula(8 cell stage), the inner cell mass, the epiblast, primordial germ cells, germline stem cells. 11.Three methods of experimentally acquiring pluripotency: I. Nuclear transfer (cloning): NT-ESC mouse, non human primate II. Viral transduction with reprogramming factors: iPS cell mouse and human III. Fusion with ES cells (electro- or PEG-mediatied): pluripotent hybrid cell mouse and human 12.Functional criteria for pluripotent stem cells: a. Ability to remain as a stem cell or differentiate into all 3 germ layers b. Presence of active transciptional regulators: demethylated OCT4 and NANOG promoters c. Ability to form a teratoma: all 3 germ layers when injected in vivo *must have this requirement for human embryonic stem cells (hESCs) d. Formation of chimera and ability to trasmit germlines (only in mouse) e. Tetraploid complementation assay (only in mouse) 13.Morula + Oct4--> early blastocyst early blastocyst + Oct4&Nanog --> late blastocyst late blastocyst + Oct4, Sox2, FoxD3 --> post-implantation egg cylinder 14.Gene targeting in chimera mice: 1) targeted ES cells are injected into blastocysts which are implanted into foster mothers 2) foster mothers give birth to chimeric mice 3) mating between chimeric mouse and normal mouse 4) result in some gene targeted mice and some normal mice 15.Tetraploid complementation: the electrofusion at the 2 cell stage to form a 4N blastocyst which, cannot form a viable embryo so, a 2N stem cell is injected into the blastocoel. If there is embryonic formation it will be because of the 2N injected cells that are now further confirmed as pluripotent. a. IVF: 50-60% success rate for forming embryo b. fESC, tetraploid: 2N nucleus-->electrofusion with 2nd 2N embryo-->injection of fESCs (2N). pup is born 2N with 4N placenta 20-30% effective c. ntESC tetraploid: 2N nucleus-->electrofusion with 2nd 2N embryo-->injection of ntESCs (2N). pup is born 2N with 4N placenta 20-30% effective d. Nuclear transfer: enucleated oocyte injected with donor cell 1-2% effective 16.Gurdon successfully preformed somatic cell nuclear transfer (SCNT). transferred an intestinal cell into enucleated egg and it could have normal embryonic development and become a tadpole. Used a mature intestinal cell nuclei to clone Xenopus laevis frogs. (conflicts with Briggs and King) a. limitations: damaging/mechanical manipulation of cell, accessibility to human oocyte is difficult 17. Yamanaka discovered how to successfully induce pluripotency of an already differentiated cell (iPSCs) using transcription factors (Yamanaka's factors: KLF4, Sox2, Myc, Oct4). 18.Hans Spermann determined that the genome must be pluripotent or totipotent if there is normal development. 19.Briggs and King performed the original protocol of nuclear transfer. They enucleated a 1N egg from Rana pipiens and replaced it with a 2N (diploid) nuclei taken from the blastula. Ends with the donor somatic cell nucleus in an activated egg. They later determined that tadpoles could not be generated if they used a more mature donor somatic cell. 20.Resistance to differentiation and reprogramming techniques often occurs in more mature or adult stem cells. It may occur due to epigenetic landscape having changed a significant amount since it was in a pluripotent state. Other possible reasons for resistance to reprogramming: shortened telomeres, oxidative damage, changes in histone modifications (i.e. methylation). 21.The cell cycles of the somatic donor cell and the oocyte must be synchronized so they most closely resemble what happens during normal early stages of development. Somatic donor cell must be in G0 and oocyte must be in metaphase II. **common challenge: it is difficult to determine if the somatic cell donor came from a fully differentiated cell because many adult stem cells exist and do not have many clear markers. 22.SCNT generates a blastula comprised of cells from the somatic cell donor EXCEPT the DNA in the mitochondria is from the oocyte. 23.iPS cells can be distinguished by specific gene expression and patterns of DNA methylation. 24.An embryonic cell line is embryonic stem cells cultured in vivo that divide but remain undifferentiated. 25.Gastrulation is when the embryo differentiates into the three germ layers: the endoderm, mesoderm and ectoderm. 26.CNS cell types: neurons and glia (includes astrocytes, oligodendrocytes and microglia). 27.Adult neuronal stem cells in rats: around lateral ventricles and in the hippocampus. 28.The neural plate is the embryonic structure from which the nervous system develops. 29.Neurulation is the process of forming the neural tube. The neural tube then forms the entire CNS (including the spinal cord, hindbrain, midbrain and forebrain). 30.Embryonic stem cell primitive neural stem cell neural stem cell neuronal restricted precursor (becomes a neuron) and glial restricted precursor (becomes astrocyte and oligodendrocyte) 31.The 2 classes of cortical neurons a. Projection neurons: mostly in deep layers and send long distance projections to other parts of the brain ex: glutamatergic pyramidal neuron b. Interneurons: mostly local connections, ex: GABAergic neurons 32.Order of generation (1st to last) 1. subplate 2. corticothalamic/layers VI callosal 3. subcerebral/layer V callosal 4. layer IV pyramidal 5. upper layer callosal 33.Organoid a. contains organ-specific cells b. able to mimic some organ function c. similar spatial arragements 34.Cells used in cell therapy: a. proliferative b. specificity c. proper migration d. proper/functional integration 35.Doublecortin (Dcx) aids in neuronal migration. 36.Functionality of neuronal donor cells is tested via optogenetic targeting. Light pulses are projected onto ChR2-expressing neurons and tested for whether or not they fire an action potential. 37.Epigenetics: study of heritable changes in gene expression or phenotype NOT resulting from change(s) in actual DNA sequencing. 38.Variation can be found in the trascription of genes, the epigenetic landscape (varying complexities), potential for differentiation (ability to form teratoma) and degree of mutation (caused by the reprogramming method). 39.A donor somatic cell may retain its differentiated characteristics if a. the somatic genes were incompletely silenced b. the ESC-pluripotency genes were not activated strongly enough c. additional marks arise that do not resemble the donor parent cell or ES cells 40.Possible outcomes of the iPSC model: a. Return to totipotency b. Return to semi-reprogrammed state: returns to pluripotency then may differentiate again c. Trans-differentiation into another cell type d. Apoptosis 41.The "master regulators" refers to the Oct4/Sox2/Nanog Triad. They are NOT sufficient in guaranteeing pluripotency. Additional regulators are needed (chromatin modifiers). 42.The state of a cell is defined by: a. genes transcribed by the cell b. epigenetic landscape that regulates expression 43.The fundamental unit of chromatin is the nucleosome, which consists of 2 of each of the 4 (8 total) core histones (H2A, H2B, H3, H4) wrapped with147 base pairs of DNA. Linker histones: H1 and H5 connect the nucleosomes. 44.Chromatin structure: Heterochromatin Euchromatin Typically silent (no transcription occurring) Expressed Tight packaging Loosely packaged Closed form Open form 45.DNA is often methylated on CpG islands- dinucleotides on the 5-carbon position on cytosines. ~60% of all human genes are transcribed from a CG-rich promoter sequences (called CpG islands). 46.Passive demethylation: Transcription factors already bound at promoter region on DNA block the binding of DNMT1 (DNA methyltransferase 1), allowing transcriptional activation. Active demethylation: requires a demethylase to attach opposite to the transcription factors. The demethylase removes methyl groups and allows transcriptional activation. 47.Post-translational histone modifications can recruit covalent repressors/activators and can mediate interactions between adjacent nucleosomes to open or close chromatin. 48.Cellular differentiation is associated with large amounts of gene repression. 49.Modifying: a. Histone acetyltransferase b. Histone methyltransferase c. Histone kinase d. Histone ubiquitin ligase 50.Unmodifying: a. Histone deacetylase (HDAC) b. Histone demethylase c. Histone phosphatase d. Histone deubiquitinase 51. Chromatin remodelers are ATP-dependent complexes that mediate interaction between DNA helix and histones and regulate DNA accessibility by transcription factors. 52.A mutation in CHD7 - the chromodomain helicase DNA binding protein in chromatin remodelers - results in CHARGE: a genetic disorder characterized by severe defects in many cell types at birth. CHDs are important in creating chromatin landscape with active (self-renewal and pluripotency genes) and quiescent (cell lineage-specific genes) segments. 53.Polycomb group (PcG): a group of proteins found in pluripotent cells that can repress differentiation and catalyze post-translational modifications of histones to control developmental gene expression patterns. 54. A lack of polycomb group will result in incorrect expression of developmental genes. 55.Activating role: activate transcriptional regulators found in the pluripotent state. Repressive role: repress genes associated with differentiation and lineage commitment. 56.cMyc is a regulator gene that codes for a transcriptional factor. It targets metabolism and proliferation regulators and is also involved in reprogramming (activates basic energy, proliferation and metabolism of the embryonic state). 57.Additional factors affecting the genome: mechanical forces, biological stimuli, physical material properties 58.3 ways to validate iPS cells: a. Teratoma formation b. Formation of 3 germ layers in vivo and in vitro c. iPS injection into blastocyst and see if generated chimera can pass it's germline to offspring when mated with normal mouse 59.Valproic acid (VPA): a histone deacetylase (HDAC) inhibitor used to make reprogramming more efficient. 60.Models that explain low efficacy of iPSCs a. Elite model i. predetermined: small number of cells are competent for reprogramming ii. induced: only cells with specific viral integration sites are competent for reprogramming b. Stochastic model: all cells are competent for reprogramming i. most supported model 61.Waves of molecular events underlie cellular reprogramming a. Early phase: mesenchymal to epithelial transition, i. MET, Cell cycle, decrease cell contact, decrease cell adhesion, microRNA expression, decreased differentiated cell markers, chromatin modifications b. Intermediate phase: partially reprogrammed cells, resistant cells c. Late phase: increase in pluripotency markers, DNA methylation, microRNA expression 62.Direct conversion refers to the reprogramming of a differentiated cell into another cell type without bringing the cell first to its pluripotent state. Extra study material: Developmental Potential Epigenetics Totipotent Zygote Global DNA demethylation Pluripotent ICM/ES cells -Only active X chromosomes Embryonic germ cell (EGC) -Global repression of Spermatogonial germ stem cell (maGSC) differentiation genes by Polycomb proteins Hypomethylation of promoter Multipotent Adult stem cells (partially reprogrammed cells) -X inactivation -Repression of lineage-specific genes by Polycomb proteins -Hypomethylation of promoter Unipotent Differentiated cell types -X inactivation -Depression of Polycomb silenced lineage genes -Hypomethylation of promoter
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