ANNOUNCEMENTS. HW1 is due Thursday 1/28 by 12:00 pm. Office hours: Monday 12:50 1:20 (ECCH 134) Wednesday 9:45 10:15 (ECCH 134)

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1 ANNOUNCEMENTS HW1 is due Thursday 1/28 by 12:00 pm. Office hours: Monday 12:50 1:20 (ECCH 134) Wednesday 9:45 10:15 (ECCH 134)

2 Lecture 5: Kinetics of Cell Growth in Culture Phenomenological mathematical descriptions of cell growth - Internal control - External control - Cell density limited cell growth Experimental methods for quantifying cell number in cultures - Hemocytometer - Coulter counter - Biochemical assays

3 Biochemical assay for cell number: DNA Assay Strands of DNA Remove cells from culture dish Lyse cells Add a dye that associates with DNA Intercalator Quantify emitted light (fluorescence) using a fluorimeter Compare to standards made from samples with known cell number

4 Standard curve for DNA assay of cell number 4,000,000 y = 0.359x + 123,000 Fluorescence output R 2 = Cell number 1.1 x 10 7

5 Lectures 6 and 7 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 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 IPSCs FDA regulations surrounding stem cell therapeutics

6 What is a stem cell? Stem cells are unspecialized cells that can reproduce themselves (by symmetric division) and generate more specialized cells (by asymmetric division), indefinitely. Symmetric Division (Increases Cell #) Homeostasis Wound Healing Asymmetric Division (Maintains # of differentiated cell types) (1) Proteins and mrna distribute asymmetrically throughout the cytoplasm. (2) Loss of contact with the stem cell niche. Limited renewal 2 identical daughter cells Stem Cell (Self-renewal) Multipotent stem cell Terminally Differentiated Cells They are present at all stages of development and are likely in all adult tissues as well.

7 2 reporter tags are used in the movie: Asymmetric division in action! Red = Histone-2B-red fluorescent protein (RFP) Protein associated with chromosomes in the nucleus Green = Partner of Numb (PON) green fluorescent protein (GFP) Protein associated with the development of a more differentiated cell type Asymmetric Division C. elegans worm

8 The stem cell niche Symmetric Division (Increases Cell #) Homeostasis Wound Healing Asymmetric Division (Maintains # of differentiated cell types) ECM = extracellular matrix: Identified in: Laminin Collagen IV Nidogen Heparan Sulfate Proteoglycan (bfgf) A B Intestine Bone marrow Muscle Corneal Epithelium Brain (SVZ) (A) Population mechanism Both cells remain in contact with the niche and remain stem cells. Stem cell number in the niche will increase. Symmetric division. (B) Lineage mechanism A cell in contact with the niche remains a stem cell; the other cell loses contact with the niche and differentiates. Stem cell number in the niche remains fairly constant. Asymmetric division.

9 Stem Cells with Different Lineage Potential A Pluripotent Stem Cell, can form all cells of the body. Embryonic stem cells A Multipotent Stem Cell, displays a more limited differentiation potential (e.g. precursors, progenitors). Hematopoetic stem cells, Neural stem cells A Unipotent Stem Cell, can form only one differentiated cell type. Hepatocytes of the liver

10 Lectures 6 and 7 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 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 IPSCs FDA regulations surrounding stem cell therapeutics

11 VIDEO