Talk:2009 Lecture 21

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ANAT3231 Lecture 13�Stem Cells/Development Dr Mark Hill Room G20 Email: UNSW Copyright Notice Textbook/Online References Essential Cell Biology Chapter19 Tissues p622-627 Molecular Biology of the Cell (3rd ed) Chapter 19 Cellular Mechanisms of Development p1037-1039 Online References UNSW Embryology Molecular Biology of the Cell online Molecular Cell Biology online. 4th ed. Lodish etal Lecture- Development/Stem Cells Establishment of order from 1 cell clonal stem cells Developmental Models Signals, position, time differentiation Patterns, programs 3 Adult Cell Types (Proliferation) Differentiated cells no longer capable of cell division Cardiac muscle cells, neurons produced during embryonic development, differentiate, then retained throughout life of organism Cells in G0 stage of cell cycle resume proliferation when needed to replace cells skin fibroblasts, smooth muscle cells, endothelial cells, epithelial cells of internal organs, such as the liver, pancreas, kidney, lung, prostate and breast Stem Cells differentiated cells have short life spans continually replaced blood cells, epithelial cells of skin and digestive tract fully differentiated cells do not proliferate proliferation of less differentiated- stem cells produce daughter cells that either differentiate or remain as stem cells Blood Cells All different types of blood cells develop from a pluripotent stem cell in bone marrow Precursors of differentiated cells undergo several rounds of cell division as they mature proliferation ceases at terminal stages of differentiation Movie: Neural Stem Cell PubMed- Stem Cells Medline Search “stem cell” 2002 - 110,920 2004 - 128,485 2005 - 140,966 2006 - 154,176

NIH - Stem Cells National Institute of Health (USA) Stem Cells Report (2001)

Stem cells in Development Blastocyst

Why are they in the News? Scientific and Ethical Therapeutic uses Issues relating to human cloning Use of excess human eggs/sperm for research purposes Availability of human stem cell lines What can they be used for? Generation of “knock out” mice Studying regulation of cell differentiation in development Therapeutic uses? Genetic disease Neurodegenerative Injury

FourHumanESCell Lines Long Term ES Cell Culture Human ES Cell Differentiation Stem Cells in the Adult Connective Tissue Bone marrow Blood Cells, Osteoclasts, blasts Epithelia Gut Skin Epidermis: Immortal Stem Cell Stem cell each generation at least 1 "immortal" stem cell descendants present in patch in future Other basal cells leave basal layer and differentiate Committed, born different or may be stem cells equivalent to immortal stem cell in character mortal in sense that their progeny jostled out of basal layer and shed from skin Amplifying Cells Stem cells in many tissues divide only rarely give rise to transit amplifying cells daughters committed to differentiation that go through a limited series of more rapid divisions before completing the process. each stem cell division gives rise in this way to eight terminally differentiated progeny Stem Cell Production Stem Cell Daughter Fates Environmental asymmetry daughters are initially similar different pathways according to environmental influences that act on them after they are born number of stem cells can be increased or reduced to fit niche available Divisional asymmetry stem cell has an internal asymmetry divides in such a way two daughters are already have different determinants at time of their birth Neural Stem Cells: Development Neural Stem Cells: Adult Neural Stem Cells: Adult Purification of a pluripotent neural stem cell from the adult mouse brain. Rietze et al. The Walter and Eliza Hall Institute of Medical Research purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties Nature. 2001 Aug 16;412(6848):736-9. Current research on stem cells How to: Isolate Grow Maintain, store Differentiate Therapeutic uses Growth of Embryonic Stem Cells Mouse blastocyst-derived ES cell line D3 from American Type Culture Collection (ATCC) Undifferentiated ES cells maintained on gelatin-coated dishes earlier studies, feeder layer DMEM (dulbecco’s modified essential media) with

2 mM glutamine (essential amino acid)
0.001% beta-mercaptoethanol (reducing agent)

1x nonessential amino acids (amino acids for growth) 10% donor horse serum (source of growth factors etc)

human recombinant leukemia inhibitory factor (LIF) 2,000 units/ml

Stem Cell Markers Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands) Different types of receptors differ in structure and affinity for signalling molecules Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body Covered in Lecture 11, 12 Same cell surface receptors are stem cell markers Stage-Specific Embryonic Antigen-1,4 Tumor Rejection Antigen (TRA-1-60) Stem Cell Antigen 1 (Sca-1) Marker Examples Stage-Specific Embryonic Antigen-1 (SSEA-1) role in cell adhesion, migration and differentiation often differentially expressed during development Stage-Specific Embryonic Antigen-4 (SSEA-4) surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES) down-regulated following differentiation of human EC cells Antigen not expressed on undifferentiated murine EC, ES and EG cells but up-regulated on differentiation of murine EC and ES cells ES Therapeutics Possible Therapeutic Uses Neural Parkinson’s, ALS, spinal cord injury…….. Cell Replacement cell death, loss of function Grafting where host-graft rejection normally requires substantial ongoing immunosuppression Repair Spinal cord and brain injury Other Diseases Diabetes, muscular dystrophies, cardiac, vital organs…… Stem Cells and Grafting Nature paper showed possible therapeutic use of ES cells in grafting host-graft rejection normally requires substantial ongoing immunosuppression Pre-implantation stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning Aug02 FANDRICH etal. Stem Cell Experiments Human 2 labs in 1998 human embryonic stem cells Gearhart 1998, Thomson et al. 1998 inner cell masses of embryos not implanted into infertility patients germ cells derived from spontaneously aborted fetuses Mouse ES cells cultured in conditions to form glial stem cells glial stem cells were transplanted into mice that had a genetic deficiency of glial function, and cured defect (Brüstle et al. 1999) Neural stem cells derived from mouse ES cells divide and differentiate into functional neurons when injected into a damaged rodent nervous system (McDonald et al. 1999) Human Stem Cells Human neural stem cells improve sensorimotor deficits in adult rat brain with experimental focal ischemia Ischemic stroke caused by interruption of cerebral blood flow leads to brain damage with long-term sensorimotor deficits intravenous injection determine migration, differentiation and long-term viabilities of human NSCs in rat brain Human NSCs were detected in lesion side and labelled with marker for neurons or astrocytes Post-ischemic hemispheric atrophy noted but reduced in NSCs-ischemia group Neural Therapeutic uses? Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model Implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients current methods are rudimentary lacking a reliable donor cell source Transplanted ES cells can develop spontaneously into dopamine (DA) neurons Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002 Parkinson Rat Model Embryonic stem cell Transplant transplanting low doses of undifferentiated mouse embryonic stem (ES) cells into rat striatum results in a proliferation of ES cells into fully differentiated DA neurons ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002 Staining of a Graft 16 weeks after implantation of D3 ES cells into adult 6-OHDA lesioned striatum TH-positive neurons were found within the graft (A and B, green) All TH-positive profiles coexpressed the neuronal marker NeuN (A, red) TH (B) also was coexpressed with DAT (C, red) and AADC (D, blue), shown by white triple labelling (E) Rotation response to Amphetamine 6-OHDA-lesioned animals were selected for transplantation by quantification of rotational behaviour in response to amphetamine response was examined post-transplantation at 5, 7, and 9 weeks Animals with ES cell-derived DA neurons showed recovery over time from amphetamine-induced turning behavior Developmental Models What makes one cell different from the next? Developmental Models drosophila- fruit fly c. elegans- worm xenopus- frog zebrafish- fish chicken- bird mouse- mammal Background to Patterning All cells contain the same genetic material and are initially equipotent Development is about pattern formation axis and tissue formation Regulators of pattern - signaling Contact (cell-cell, cell-ECM, surfaces) Morphogen (soluble, modified) Time “window”, undifferentiated/differentiated, stem Genome modification, regulators Drosophila Development Model Position-Specific Patterning Cascade of short range inductions Secretion of long range signal Long range secondary signal from primary inductive spinal cord /neural crest, examples of complex structure generated by such a signalling process

Short Range Inductive Cascade Long Range Signal Long Range Secondary Signal from Primary Inductive