Differentiation strategies and differentiation analysis Hessah Alshammari

Cellular differentiation The early steps in the development of a mammal that end with fully-differentiated cell types that eventually form in the adult.

Cellular differentiation Cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of as the organism changes from a single zygote to a complex system of tissue and cell types. Differentiation is a common process in adults as well: adult stem cell divide and create fully-differentiated daughter cell during tissue repair and during normal cell turnover. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly-controlled modifications in genes expression.

Differentiation potential ((cell potency)) The potency ,its potential to differentiate into different cell types. Totipotent stem cells: Cells able to give rise to all embryonic somatic cells and germ cells. In other words, they can build a whole animal. The zygote and a few early cells of the morula are totipotent. Pluripotent stem cells: These cells are descendants of totipotent stem cells and can give rise to cells of the three germ layers: endoderm, mesoderm, and ectoderm. They have no contribution to extraembryonic membranes or the placenta. Multipotent stem cells: These produce cells of a particular lineage or closely related family.

Stem Cell Plasticity The ability of adult stem cells to cross lineage barriers adopt the expression profile and functional phenotypes of cells that are unique to other tissues. Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells. Bailey AS, Jiang S, Afentoulis M, Baumann CI, Schroeder DA, Olson SB, Wong MH, Fleming WH (2004). Blood 103:13. Conversion of myoblasts to physiologically active neuronal phenotype. Watanabe Y, Kameoka S, Gopalakrishnan V, Aldape KD, Pan ZZ, Lang FF, and Majumder S (2004). Genes Dev. published 12 April 2004, 10.1101/gad.1179004.

Strategies of stem cell differentiation Culture Stimulatory factors Co-culture

Culture methods of differentiation Differentiation could be started in: Monolayer. 3Dimension e.g. Scaffold. Embryoid body.

monolayer 3D Embryoid body Mimic early embryogenesis. Homogeneous differentiation equal exposure to media component. 2D Small size. 3D Media transport. Cell to cell contact. High surface area. Mechanical properties. Morphological similar to nature ECM and promote cell adhesion , proliferation and differentiation. Drug delivery. Embryoid body Mimic early embryogenesis. Heterogeneous differentiation. Promote cell to cell contact.

Factors Osteocytes Cardiocytes Neuron   Factors promotes differentiation to specific lineages e.g. Osteocytes Basic Fibroblast Growth Factor Bone Morphogenetic Protein-2 simvastatin dexamethazone Cardiocytes Vascular endothelial growth factor . 5-azacytidine .. Neuron Retinoic acid. BDNF

Co-culture Co culture of stem cell with differentiated cell promotes differentiation of stem cells into specific cells lineages.

Results: This initiated differentiation to beating muscle. Differentiation of Human Embryonic Stem Cells to Cardiomyocytes. Role of Coculture With Visceral Endoderm-Like Cells Christine and Leon Tertoolen ,Monash Institute of Reproduction and Development, Australia 2003. Methods: hES cells were cocultured with visceral-endoderm (VE)-like cells from the mouse. Results: This initiated differentiation to beating muscle. Induction of differentiation of hES cells by coculture with VE-like mEND-2 cells. A, Undifferentiated hES cell colony on MEF “feeder cells.” B, Nuclear oct-4 staining of undifferentiated cells in area of colony indicated in A. C, hES cells after 11 days of coculture with END-2 cells with beating aggregate (arrow). D, Various morphologies of beating muscle aggregates. E, Phase-contrast image showing beating muscle areas in hES/ END-2 coculture.

Differentiation analysis Stem cell differentiation could be detected via several way: cell morphology. Neural stem cell differentiated into : 1-Neuros : bipolar elongated cell very tiny cell with thin projection shiny and attached to plastic culture. 2- Astrocytes : star-shaped ,larger cells with longer projection , a dense, flat gray and like shade. 3-Oligodendrocytes: Network-like cell , like fork and very thin fingers. bIII-tubulin (neurons, red), GFAP (astrocytes, blue), and O4 (oligodendrocytes, green)

deposition of proteoglycans Biological marker. Osteocytes chondrocytes adipocyte Alacian green minarlization Oil Red Alizarin red that assess miarlization via detect calcium Lipid droplets deposition of proteoglycans

Ryan M. Fryer et al. originally published online Mar 31, 2003; Genetic analysis : Ascorbic Acid Enhances Differentiation of Embryonic Stem Cells Into Cardiac Myocytes. Ryan M. Fryer et al. originally published online Mar 31, 2003; Ascorbic acid-induced cardiac genes in ES cells. Cells were treated with or without 104 mol/L ascorbic acid for 12 days (A) or for the indicated periods of time (B). A, Semiquantitative RT-PCR was performed with the indicated specific primers. B, The expression of GATA4, -MHC, -MHC, and –tubulin was examined with kinetic PCR, and the results were expressed as relative expression to -tubulin and plotted as percentages of the maximum (circles, control; squares, ascorbic acid). *P0.05 versus control of respective time points.

Immunocytochemistry Immunohistochemistry Proteomic