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Stem Cell Research A stem cell taken from a human embryo Impact on Dental Tissue Engineering Michele Vasquez

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What are stem cells? 2 types of stem cells How are stem cells obtained? Alternative ways of obtaining stem cells (de-differentiation) Plasticity Applications Lecture outline

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What are stem cells? Cell that are: unspecialised do not have any tissue-specific structures that allow them to perform specialized functions 2. capable of dividing and renewing themselves for long periods of time able to differentiate give rise to specialized cells

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Types of stem cells Embryonic Adult (somatic) Blastocyst Bone marrow,blood, cornea and retina of the eye, dental pulp of the tooth , liver, skin, gastrointestinal tract, pancreas Pluripotent Typically generate the cell types of the tissue in which they reside. Postnatal Umbilical cord blood, deciduous teeth Able to grow much faster and double their populations in culture at a greater rate Typically generate the cell types of the tissue in which they reside.

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How embryonic stem cells are obtained Inner cell mass

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How postnatal stem cells are obtained From deciduous teeth From the umbilical cord

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Wash and filter Culture in presence of lineage-specific differentiation factors Mesodermal cells Subcutaneous fat Liposuction aspirate Liposuction Muscle Bone Cartilage Fat How adult stem cells are obtained

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Obtaining stem cells via de-differentiation Somatic cell nuclear transfer Somatic nucleus Enucleated egg

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Plasticity Plasticity is the ability of an adult stem cell from one tissue to generate the specialized cell type of another tissue.

Embryonic Stem Cell Advantages:

Embryonic Stem Cell Advantages Immortal: One cell line could supply endless amounts of cells with carefully defined characteristics. Like an endless fountain, the cell line itself would remain intact. Flexible: They can make any body cell. Available: Human embryos remaining after in-vitro fertilization are routinely destroyed by fertility clinics. http://whyfiles.org/127stem_cell/index.html

Embryonic Stem Cell Disadvantages:

Embryonic Stem Cell Disadvantages Hard to control Can form a variety of tissues Could form teratomas Formation of different tissues is controlled by chemical cues that are only partially understood Ethically controversial Rejected by the immune system Therapeutic cloning – generation of blastocysts by nuclear transplantation using patient’s nuclei and donor oocytes – could be used to address this problem http://whyfiles.org/127stem_cell/index.html

Adult Stem Cell Advantages:

Adult Stem Cell Advantages Immune to immune attack: If patients receive the products of their own stem cells, they will not mount an immune response. May be readily available: Some types, like blood stem cells, are easy to find. Partly specialized: That reduces the amount of outside direction needed to create specialized cells. Flexible: Adult stem cells may form other tissue types. Plasticity demonstrated in several studies

Adult Stem Cell Disadvantages:

Adult Stem Cell Disadvantages Scarce: Not all types of adult stem cells have been found yet. Unavailable. They can be dangerous to extract (you wouldn't want to poke around in someone's brain for neural stem cells). Vanishing: They don't live as long as embryonic cells in culture. Rare Adult stem cells are never very common, and grow more scarce as we age, when the cells might be needed most. Like the following problem, this is relevant for self-transplants of a patient's stem cells. Insufficient adult stem cells for transplantation Current techniques do not allow expansion of adult stem cell populations in culture http://whyfiles.org/127stem_cell/index.html

How Could Stem Cells Be Used?:

How Could Stem Cells Be Used? Basic research How do unspecialized cells turn into specialized tissues like liver, heart, muscle or nerves? What signals control cell fate? Testing drugs, toxic compounds, and carcinogens Treatment of specific diseases

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How Could Stem Cells Be Used?

Treating Diseases:

Treating Diseases

Heart Muscle Repair With Adult Stem Cells:

Heart Muscle Repair With Adult Stem Cells Congestive heart failure affects 4.8 million people in U.S. Destruction of heart cells due to variety of causes Could stem cells replace damaged heart cells? Some promising results from animal models of heart disease

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Treatment of diabetes Islet of Langerhans

Why all the fuss?:

Why all the fuss? Stem cells may be able to replace damaged cells in the body Today: lymphoma, leukemia Future? Parkinson’s, Alzheimer’s, diabetes... Promising animal studies Reproduced by permission of The Providence Journal Courtesy of The Michael J. Fox Foundation for Parkinson’s Research

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Bone marrow Adenovirus/Interferon ß gene Mesenchymal stem cells with IFN- ß gene Tumor cells Anti-tumor gene therapy Mesenchymal stem cells INF- ß

Cloning:

Cloning

Cloning—History:

Cloning—History 1970s, an adult frog was cloned from a somatic cell (body cell) But, scientist thought it was impossible to clone animals as complex as mammals

History:

History The mammoth task of cloning a mammal, once thought to be impossible has been made possible in 1996 This incredible feat spurred violent media reactions worldwide

Types of cloning:

Types of cloning DNA cloning Reproductive cloning Therapeutic cloning

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DNA cloning

Therapeutic Cloning:

Therapeutic Cloning AKA Embryo cloning

Reproductive cloning:

Somatic cell nuclear transfer Reproductive cloning

Applications:

Applications Insulin production by E. coli for human use Gene therapy by introducing viruses carrying corrected copies of defective genes into person Genetically Modified (GM) plants and animals

Therapeutic cloning ≠ Reproductive cloning:

Therapeutic cloning ≠ Reproductive cloning The use of somatic cell nuclear transfer for technology (SCNT) for replacement therapy That does not lead to creation of entirely new human Reproductive cloning is universally rejected

Bioethics:

Bioethics Ethical implications to stem cell research / therapeutic cloning

Origins of stem cells:

Origins of stem cells The controversy The embryo is being sacrificed for its stem cells The debate When does life begin?

When does life begin?:

When does life begin? For An embryo is only a potential foetus Only accorded moral status 14 days post-fertilisation, when central nervous system develops Under 14 days, embryo is special, but not human Against To treat embryos as de facto humans is to diminish respect for human life No accepted physiological point at which an embryo becomes more “human than before

Do we still need more embryonic stem cells?:

Do we still need more embryonic stem cells? The need for future embryonic stem cells is modest Cell-lines obtained have been serially subcultured over 200 times Available cells can be supplied for researchers around the whole world forever without destroying any additional embryos .

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Adult (somatic)SCs Bone marrow ,blood, cornea and retina of the eye, dental pulp of the tooth , liver, skin, gastrointestinal tract, pancreas Typically generate the cell types of the tissue in which they reside.

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Dental Tissue Engineering Journal of Dental Education  Volume 66, N°.6;June 2002

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Interactions between cells direct promoters Cells differentiation is due to different gene Switches going on and off These cells are different because they express a subset of their 30,000 genes

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Regulation of promoters Gene Promoter Factor 1: sensitive to general type of tissue: e.g. retina

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Regulation of promoters Gene Promoter Factor 1: sensitive to general type of tissue: e.g. retina Factor 2: sensitive to age type of tissue: e.g. not-fetal Factor 3: sensitive to cell type: e.g. cone = color Factor 4: sensitive to cone type: e.g. red Factor 5: sensitive to time of day: e.g. on during the day Expression of gene directed in adult red-sensitive retinal cone cell during day

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Regulation of promoters Gene Promoter Factor 1: sensitive to general type of tissue: e.g. retina Factor 2: sensitive to age type of tissue: e.g. not-fetal Factor 3: sensitive to cell type: e.g. cone = color Factor 4: sensitive to cone type: e.g. red Factor 5: sensitive to time of day: e.g. on during the day Expression of gene directed in adult red-sensitive retinal cone cell during day Neighbors Long term clock Neighbors Neighbors Circadian clock

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Factor 1: sensitive to general type of tissue: e.g. retina Factor 2: sensitive to age type of tissue: e.g. not-fetal Factor 3: sensitive to cell type: e.g. cone = color Factor 4: sensitive to cone type: e.g. red Factor 5: sensitive to time of day: e.g. on during the day Gene Promoter Neighbors Long term clock Neighbors Neighbors Circadian clock

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Gene Promoter To specify a new cell: place it in the right environment or

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Gene Promoter or substitute drugs/proteins for environment Mophogens FGF Hedgehog EGF Wnt Retinoic acid HGF

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How to make a stem cell become what you want it to become FGF Hedgehog EGF Wnt Retinoic acid HGF Use body’s signals Endogenous signals Make inside body Simple and cheap: cells home into right place and do their job Requires that embryonic signals are still there If it goes wrong: developmental defects Use artificial signals: Make outside body More difficult and expensive to emulate embryonic signals in Petri dish Requires exqusit understanding of biology Replacement tissues can be made and pre-tested.

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Tooth morphogenesis Different protein mediated mechanisms, regulating differentiation, proliferation and/or self-renewal: BMPs/TGF- b FGFs Wnt signalling pathway. Hedgehog (Hhs) signalling pathway. Morphogens

SMADs and TGF-Beta:

SMADs and TGF-Beta A large group of transcriptional activators is now known that have an even more direct route to the nucleus They are known as SMADs (named after two representatives, Sma in worms and Mad in flies) TGF-beta style receptors, from which SMADs are activated, behave like TK receptors except that these are Ser-Thr autophosphorylators

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BMPs/TGF- b family Bone morphogenic protein 4 (BMP-4) is a member of the transforming growth factor b (TGF- b ) family. Induces haematopoietic, skeletal & dental tissues commitment. The fate of the cell is dependent on the dose of BMP-4. BMP-4 can also induce ectodermal cells to form blood cells.

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Note the resemblance to the TK receptor pathways. Once Smad 2 or 3 is active, it “dimerizes” with Smad 4

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Once “dimerized”, the Smads migrate to the nucleus and exert their effect, which is to activate the transcription of genes containing the appropriate TGF response element in their promoters

The TGFb Signal Pathway:

The TGF b Signal Pathway Growth Control, Cancer, and Heritable Disorders

Transforming Growth Factor Pathway:

Transforming Growth Factor Pathway Occupies central position in control of growth, differentiation, and final fate of cells Recent progress made on this system---it is interesting and novel because It involves cell growth and fate Defects may result in cancers and/or heritable disorders It is apparently universal Evolutionary homologues are used in a variety of pathways The proteins are receptor ser/thr kinases

Basics of the pathway:

Basics of the pathway Vertebrates may have as many as 30 different TGF-like receptors C. elegans has at least 4 Drosophila has at least 7 Two broad categories are identified; functions differ but the effects are often complementary Often cell or tissue specific

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The basics of the signaling pathway are shown here: Receptors dimerize in response to binding of the ligand; the type II is brought close to type I, thereby activating it. The type I subunit phosphorylates a receptor Smad (R-Smad) of one type or another, releasing it from a SARA protein and exposing a nuclear import signal----once in, the Smad participates in transcription regulation events, usually via a cofactor. The Smad-4 is an accessory factor .

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All of the known TGF-like ligand/receptor interactions and downstream events. Drosophila paralogues shown in red.

Levels of Control:

Levels of Control Control of these pathways can be exerted at almost any level: Inhibition of ligand/receptor interaction can be achieved Enhancement of ligand binding can also occur via betaglycan and endoglin, for example Dimerization of the receptors can be inhibited or interfered with (FKBP inhibits, BAMBI interferes (it is a truncated type I receptor) R-Smads can be targeted for breakdown (by Smurf, an E3 ubiquitin ligase) or prevented from proper interaction with the receptor (Smad 6 and 7 are decoys that do this) Smads can be kept from the nucleus via phosphorylation (Erk does this) Etc.

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A summary of the numerous pathways exerting control over the TGF signaling pathway

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A number of human hereditary disorders and hereditary and sporadic cancers are associated with defects in the TGF signalling pathway, indicating its importance in cell cycle control, growth, differentiation, and developmental fate determination. What are some of these?

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Colon cancers Defects in TGF receptor pathways can lead to early onset of colon polyps and predispose to colorectal cancer A juvenile onset form represents a defect in Smad4, and Smad4 mutations are found in 50% of all pancreatic cancers and 30% of all late-onset colon cancers Smad4 mutation also found in some other cancer types, but there, it is associated with a receptor defect as well Smad2 mutants may be causative in small number of colorectal cancers

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So…What’s the Point? Understanding TGF pathways is critical to understanding causation in many human diseases It allows us to construct animal models for investigating mechanisms and testing potential treatments Gives insight into other diseases that we didn’t know were the result of TGF pathway defects---some forms of hypertension come to mind here It’s just cool to look at the integration of these pathways into other pathways of signalling

Proteolysis Pathways:

Proteolysis Pathways

Proteolysis pathways:

Proteolysis pathways Involve the cleavage of part of the receptor; the cleaved product is itself the second messenger Pathways are used primarily during developmental processes, and are highly conserved in evolution Also primarily used when cells in contact need to adopt different developmental states Wnt signalling pathway Hedgehog signalling

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Wnt signalling pathway (Wingless and int-related proteins) Wnts are regulators of proliferation and differentiation Wnts are secreted glycoproteins Bind to: Frizzled cell-surface receptor Lipoprotein receptor-related proteins 5 and 6 (LRP-5/6) Three different Wnt pathways The b –catenin pathway The planar cell polarity pathway The Wnt/Ca 2+ pathway

b –catenin pathway:

b –catenin pathway The b –catenin pathway is the most important. Wnt signalling leads to stabilization of cytosolic b –catenin through inhibition of Glycogen Synthase Kynase (GSK-3 b). b –catenin associates with Lymphoid Enhancer Factor (LEF/TCF) in the nucleus leading to transcription of target genes.

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Another important pathway is the Wnt/frizzled pathway, involving stabilization or breakdown of a protein called beta-catenin If Wnt is not present, the frizzled receptor remains inactive; as a result, beta –catenin is unstable and becomes targeted for the degradative pathway As a result, Wnt-responsive genes are off (an aside: Groucho is so-named because one of its alleles in flies causes excess bristle growth above the eyes, kind of like Groucho Marx’s eyebrows…)

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If the Wnt signal is present, however, Wnt, LRP, and Frizzled all get together to activate the frizzled receptor It activates dishevelled, allowing beta catenin to to become stable It migrates to the nucleus, displaces groucho, and activates Wnt-responsive genes This pathway is very similar to another one in flies, the Hedgehog pathway…

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Hedgehog signalling (Hhs) Involved in proliferation and growth. Hedgehog proteins (Hh) are glycoproteins expressed on the cell surface. There are three types: Sonic Hh (Shh). Indian Hh (Ihh). Desert Hh (Dhh). Hhs can mediate signalling through: Cell-cell contact between adjacent cells. NH2-terminal cleavage of Hh results in soluble Hh proteins.

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Without the hedgehog signal, patched negatively regulates smoothened receptor protein As a result, the Cubitus Interruptus (CI) protein is cleaved away from its sequestering complex It migratrs to the nucleus and acts as a transcriptional corepressor But if hedgehog is present…

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…then smoothened is active since patched is inactivated The intact CI protein is a transcriptional activator rather than a corepressor

Hedgehog signalling:

Hedgehog signalling Hhs bind to a membrane receptor complex containing Patched (Ptc) and Smoothened (Smo). Without Hh signalling Ptc inhibits Smo. Upon binding Hhs inhibit Ptc, which results in release of Smo.

Hedgehog signalling:

Hedgehog signalling Cytoplasmic signalling events regulate Gli zinc-finger transcription factors. Gli target genes: Wnt2a Noggin (BMP-4) specific inhibitor.

Dental Tissue Engineering:

Dental Tissue Engineering Morphogenesis Althoug four distinct families of morphogens are involved in embryonic craniofacial morphogenesis, BMPs alone appear to be sufficient for regeneration of dental tissues in adults.

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Dental Tissue Engineering Journal of Dental Education  Volume 66, N ° .6;June 2002

Dental Tissue Engineering:

Dental Tissue Engineering

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Dental Tissue Engineering NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 9 SEPTEMBER 2003

Dental Tissue Engineering:

Dental Tissue Engineering Coffee break