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Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all

Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all trans retinoic acid in vitro. Electrophoresis 2001, 22, 3067-3075. Presented by Shin jina . Origin of human pluripotent stem cells.

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Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all

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  1. Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all trans retinoic acid in vitro Electrophoresis 2001, 22, 3067-3075 Presented by Shin jina

  2. Origin of human pluripotent stem cells Stem cell : undifferentiated (unspecialized) cellsthat can renew themselves and also give rise to one or more specialized cell types with specific functions in the body

  3. Differentiation of pluripotent stem cells into differentiated derivatives

  4. Differentiation of pluripotent stem cells into differentiated derivatives

  5. cDNA array images of the expression pattern of genes in ES cells and differentiation cells - 18 down regulation - 61 up regulation Molecular Reproduction and Development 56: 113-123 (2000)

  6. Purpose • To gain more insight about the molecular mechanism of early neurogenesis and neural differentiation, we combined the advantages of ES cell in vitro differentiation system and proteomic analysis

  7. morula Mouse embryo genesis and time course of ES cell differentiation In vivo blastocyst ATRA treatment ICM -LIF SEB CEB ESC In vitro 4 days 4 days 10 days Differentiation cells Control(ES cell) RA4 EB4 EB1

  8. Differentiation of ES cells into neural cells at a high efficiency induced by ATRA in vitro A B neurofilament Neural cell from the brain of new-born rat Differentiated cells

  9. ATRA treatment -LIF SEB CEB ESC In vitro 4 days 4 days 10 days Differentiation cells Control(ES cell) RA4 EB4 EB1 Mouse embryo genesis and time course of ES cell differentiation

  10. Comparison of proteomic patterns between parent ES cells and ES-differentiared cells by silver staining 2-DE A B Control (856 spots) EB1 (703 spots) D C EB4 (767 spots) RA4 (668 spots)

  11. Isolation of proteins associated with early-stage differentiation of ES cells into neural cells by coomassie brilliant blue R-250 staining 2-DE B A Control RA4 1-10: down regulation 11-24: up regulation

  12. Identification of proteins associated with early-stage differentiation of ES cells into neural cells by PMF

  13. 24 protein spots - protein match: 12 (table3) - unidentified protein: 12

  14. Vimentin expression alteration during ES cell differentiation into neural cells Control ES cells EB1 EB4 RA4 Vimentin: cytoskeleton protein,beginning marker of neurogenesis while neurofilament proteins begin to be expressed, vimentin expression is gradually lost

  15. Follistatin-relative protein expression alteration during ES cells differentiation into neural cells Control ES cells EB4 EB1 RA4 (Silver stain) Follistatin-relative protein: neural proliferation and survival factor negative regulation of cellular growth, take part in triggering of ES cells into differentiation into neural cells induced by RA

  16. Other proteins expression alteration during ES cells differentiation into neural cells • Tubulin a-3/7: cytoskeleton protein, different tubulin isoforms might play different roles in neurogenesis • Cytokeratin 8/18: typical marker of neural cells • NEL-like protein: contains several EGF-like repeats, it might function as a ligand by stimulating other molecules such as EGF receptor in a neuron-specific signaling pathway • Unknown proteins: further investigation and which identification might shed more light about the detail of molecular mechanisms of neurogenesis

  17. Discussion • Identified some important regulators in neurogenesis by comparing proteome of 4 stage cells during in vitro neurogenesis • The strategy provides a model to analyze effects of cytokines, growth factors or hormones on early neuronal development • Favorable for investigating complex mechanisms regulating neuronal differentiation • A further challenge will be to determine how these factors involved in the process of neuronal cell differentiation or brain development

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