ANNOUNCEMENTS. HW2 is due Thursday 2/4 by 12:00 pm. Office hours: Monday 12:50 1:20 (ECCH 134)

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1 ANNOUNCEMENTS HW2 is due Thursday 2/4 by 12:00 pm. Office hours: Monday 12:50 1:20 (ECCH 134)

2 Lectures 6 8 Outline Stem Cell Division - symmetric - asymmetric Stem cell lineage potential - pluripotent - multipotent - unipotent ES Cells - Establishment and expansion of an ES Cell line - Differentiation of ES cells - Clinical application of ES Cells Somatic Cell Nuclear Transfer Technology IPSCs Multipotent stem cells How do we study multipotent stem cells? - Isolation and characterization of multipotent stem cells - Study in culture - Study in tissue - Problems associated with these techniques Adult stem cells - HSC FDA regulations surrounding stem cell therapeutics

3 Induced pluripotent stem cells (mouse, human) Steps 2-4 (black box): re-programming takes ~ 10 days ES cell Differentiate in culture Teratoma Chimeric animals 1. Isolate and culture donor cells (fibroblasts for example) Nerve Skin Bone Muscle Epigenetic Differences 2. Transfect stem cell genes (transcription factors: c-myc, Klf4, Oct4 and Sox2 into donor cells with viral vectors). 3. Culture cells as you would ES cells (feeder layers, etc). 4. A subset of the transfected cells are re-programmed and become ips cells. - Viruses (mechanism for transfection can cause genomic mutations) - In best cases, inefficient ( % of cells become ips cells) - Next goal: no genetic mutations and removal of transcription factors after 10 days

4 Blocking p53 activity as a means to increase ipsc generation efficiency??? p53 is a gene encoding for a tumor suppressor protein in humans (prevents cancer) The effect of blocking p53 in ipsc cultures (presence of ipsc is indicated by the color red): D. Normile Science 325, 803-a (2009) Published by AAAS

5 VIDEO

6 Lectures 6 8 Outline Stem Cell Division - symmetric - asymmetric Stem cell lineage potential - pluripotent - multipotent - unipotent ES Cells - Establishment and expansion of an ES Cell line - Differentiation of ES cells - Clinical application of ES Cells Somatic Cell Nuclear Transfer Technology IPSCs Multipotent stem cells How do we study multipotent stem cells? - Isolation and characterization of multipotent stem cells - Study in culture - Study in tissue - Problems associated with these techniques Adult stem cells - HSC FDA regulations surrounding stem cell therapeutics

7 Adult Human Stem Cells Capable of self-renewal, but limited differentiation potential (relative to ES cells). No pluripotent adult stem cells have been identified to date. All are multipotent. Stem Cell Tissue Differentiated Cell Type Hematopoietic Bone marrow Bone marrow, Immune cells Mesenchymal stem cells Bone marrow Bone, cartilage, tendon, adipose tissue, muscle, neural cells Neural stem cells Ependymal cells, astrocytes Neurons, astrocytes, oligodendrocytes Hepatic stem cells Liver Hepatocytes, ductular cells Pancreatic stem cells Pancreas (Islets, Ducts) Beta cells Skeletal muscle stem cells Muscle fibers Skeletal muscle fibers Skin stem cells Epidermis Epidermal cells Epithelial stem cells Lung, intestine Various specialized cells of the lung and intestine

8 How are multipotent stem cells studied outside of the body? ADULT EMBRYO ADULT EGG EMBRYO EG Dissect out tissue of interest from the body DISSECTION DISSECTION Dissociate the tissue (digest ECM) Grow the cells in the presence of 1 or more protein(s) that promote stem cell survival and proliferation. ENZYME DISSECTION FINELY CHOPPED F IF Differentiate the cells by exposing them to protein(s) that encourage differentiation. CELL CULTURE Count the number of differentiated cells in culture PRIMARY EXPLANTS

9 Differentiated Neurons Derived from Neural Stem Cell Cultures Red, Neurons Green, Nuclei If you observe success, figure out why? For therapeutic applications, need to separate non-neuronal cells from other cells

10 If the culture initially contained the cell types listed below, which of the choices below would be a plausible explanation for the an increase in the number of neurons in your culture dish when they are grown in the presence of a protein called nerve growth factor? Neuronal precursors, a cell type that can self-renew or differentiate into neurons. Glial precursors, a cell type that can self-renew or differentiate into glial cells (a nonneuronal cell type) Neural stem cell, a cell type that can self-renew and differentiate into neurons or glial cells. (a) A decrease in the doubling time of neuronal precursors in culture. (b) A decrease in the doubling time of glial precursors in culture. (c) A decrease in the survival of neuronal precursors in culture. (d) A decrease in the survival of neural stem cells Answer: A

11 Clonal cell culture is used to identify the effect of culture conditions on the lineage potential of a single cell Growth Factor No Growth Factor Neuronal precursor Glial precursor Neural Stem Cell 18 culture wells Another approach is to identify the potential in vivo (in adult tissue, embryonic tissue, injured tissues, local environment is important). Labeled Stem Cell BRDU, e.g. must be distinguishable from Native Host Cell