differentiation strategies and differentiation analysis hessah alshammari

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Differentiation strategies and differentiation analysis Hessah Alshammari

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Page 1: Differentiation strategies and differentiation analysis Hessah Alshammari

Differentiation strategies and differentiation analysis

Hessah Alshammari

Page 2: 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.

Page 3: Differentiation strategies and differentiation analysis Hessah Alshammari

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.

Page 4: Differentiation strategies and differentiation analysis Hessah Alshammari

Differentiation potential ((cell potency))

The potency ,its potential to differentiate into different cell types.

Totipotent stem cells: Cells able to give rise to allembryonic somatic cells and germ cells. In otherwords, they can build a whole animal. The zygoteand 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.

Page 5: Differentiation strategies and differentiation analysis Hessah Alshammari

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, andMajumder S (2004). Genes Dev. published 12 April 2004, 10.1101/gad.1179004.

Page 6: Differentiation strategies and differentiation analysis Hessah Alshammari

Strategies of stem cell differentiation

Culture Stimulatory

factorsCo-culture

Page 7: Differentiation strategies and differentiation analysis Hessah Alshammari

Culture methods of differentiation

Differentiation could be started in:• Monolayer.•3Dimension e.g. Scaffold.• Embryoid body.

Page 8: Differentiation strategies and differentiation analysis Hessah Alshammari

monolayer

• Homogeneous differentiation

• equal exposure to media component.

• 2D• Small size.

3D

• 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.

• 3D• Heterogeneous

differentiation.• Promote cell to cell

contact.

Page 9: Differentiation strategies and differentiation analysis Hessah Alshammari

Factors

Osteocytes • Basic Fibroblast

Growth Factor• Bone

Morphogenetic Protein-2

• simvastatin• dexamethazone

Cardiocytes • Vascular

endothelial growth factor .

• 5-azacytidine ..

Neuron

• Retinoic acid.

• BDNF

Factors promotes differentiation to specific lineages e.g.

Page 10: Differentiation strategies and differentiation analysis Hessah Alshammari

Co-culture

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

cells lineages.

Page 11: Differentiation strategies and differentiation analysis Hessah Alshammari

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 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.

Page 12: Differentiation strategies and differentiation analysis Hessah Alshammari

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)

Page 13: Differentiation strategies and differentiation analysis Hessah Alshammari

•Biological marker.

Osteocytes chondrocytes

minarlization Alacian green

Alizarin red that assess miarlization via detect calcium

Oil Red

adipocyte

deposition of proteoglycans

Lipid droplets

Page 14: Differentiation strategies and differentiation analysis Hessah Alshammari

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 cardiacgenes 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.

Page 15: Differentiation strategies and differentiation analysis Hessah Alshammari

•Immunocytochemistry

•Immunohistochemistry

•Proteomic