Culture and differentiation of embryonic stem cells. Hong-Lin Su Department of Life Sciences, National Chung-Hsing University

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1 Culture and differentiation of embryonic stem cells Hong-Lin Su Department of Life Sciences, National Chung-Hsing University

2 Topics-ES cell maintenance Establishment Culture condition, including the serum, LIF and the feeder cells Mouse strains Non-rodent ES cells Why ICM?

3 compaction Morula blastocyst compaction

4 h

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7 Teratoma and teratocarcinoma Definition: a tumor containing an array of somatic cells usually occur in germ cells The most common form: ovarian dermoid cyst; which are parthenogenetically activated and usually benign. Testicular carcinoma: rarely occur, usually malignant; the cell morphology resembles the primordial germ cells (PGC).

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12 James Thomso

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16 Human ES cell maintenance Establishment Culture condition: LIF-independent Serum: FGF, nodal/activin and Wnt and KSR; LIF Feeder still important Why ICM

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18 Derivation of pluripotent epiblast stem cells from mammalian embryos New cell lines from mouse epiblast share defining features with human embryonic stem cells

19 The differences between the mouse and human ES cells Morphology LIF or FGF dependence or independence Growth condition BMP responsibility Activin/nodal responsibility

20 EpiSC cell culture

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22 Pleuripotency markers of EpiSC

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24 Pleuripotency in vivo

25 Pleuripotency in vitro

26 Oct4 methylation status in mouse episc and ES cells

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28 EpiSC is more similar to hesc

29 Fail of integration into inner cell mass

30 EpiSC cells are AP negative

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32

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34 Neural Induction

35 Topics for neural induction BMP- and Wnt antagonists are essential Culture condition: RA+serum, SDIA, neurobasal/n2, SFEB ES to neuron: relief LIF, detaching and proper cell density Mouse ES: tend to become neuron; Human ES: tend to become mesoendoderm FGF is a determining factor for neurogenesis Is the ES-derived neural cells the same as embryonic neuroepithelial cells?

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38 Spemann s demonstration of nuclear equivalence in newt cleavage

39 Asymmetry in the amphibian egg

40 Determination of ectoderm during newt gastrulation

41 Organization of a secondary axis by dorsal blastopore lip tissue (Part 1)

42 Organization of a secondary axis by dorsal blastopore lip tissue (Part 2)

43 Model of organizer function and axis specification in the Xenopus gastrula

44 Neural Induction ( Xenopus ) Uncommitted Ectodermal Cells Neural Precursors Neural Inducers BMP4 Chordin, Noggin Blocking BMP Signals is essential and sufficient to induce neural differentiation.

45 Neural Induction ( Mouse ) ES cells Neural precursors? BMP4 Blocking BMP signals is essential but not sufficient. Additional signaling is required.

46 Stromal cell-derived Inducing Activity (SDIA) PA6 cells as feeder cells dissociated ES cells 8 days in serum-free medium PA6 cell OP9 cell NIH3T3 cell cell line derived from mouse calvaria cell line derived from mouse calvaria embryonic fibroblast cell line

47 in vitro neural differentiation of ES cells induced by SDIA nestin (neural precursor) TuJ (mature neuron)

48 Quantification of neural marker expression NCAM GFAP mesoderm (glial) markers (pan-neural) colony (%) 92 ± 5 2 ± 2 < 2 nestin (neural precursors) TuJ (mature neurons) cell (%) 47 ± ± 9

49 Efficient induction of dopaminergic neurons TH day10

50 Diencephalon Mesencephalon Metencephalon Telencephalon Midbrain dopaminergic neurons 5-HT neurons r1 r2 r3 r4 r5 Locus coeruleus neurons

51 Neural Patterning factors dorsal BMP gastrula Activities of BMPs inhibition of neural induction neurula promotion of dorsal CSN & neural crest differentiation ventral

52 dorsal BMP motor neurons ventral floor plate

53 SFEB: Serum-Free Embryoid Body-like culture v.s. SDIA: Stromal cell-derived Inducing Activity Knock-out replacement serum dissociated ES cells PA6 cells as feeder cells dissociated ES cells 8 days in serum-free KSR 8 days in serum-free KSR floating adhesive

54 Different neurons are induced by using the SFEB and SDIA SFEB method: suitable for the induction of Forebrain ( Nat. Neurosci. 2005), cerebellum and retinal ganglion cells (Dev. Biol, 2006; PNAS, 2005). SDIA method: midbrain dopaminergic neurons (Neuron, 2000; PNAS, 2002), neural crest derived peripheral neurons (PNAS, 2004).

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56 Generation of Cerebellar Neuron Precursors from Mouse Embryonic Stem Cells

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59 Patterning factors for upper rhombic lips dorsal BMP ventral

60 EGL precursor neurons are induced from ES cells by using SFEB method

61 Generation of Cerebellar Purkinje cells from ES cells

62 Conclusion Math1 positive EGL precursor cells are generated by using the BMP4+Wnt3a/SFEB method. Math1-ES cells faithfully express the specific EGL markers and functional ion channels of IGL cells. Cerebellar Purkinje cells are successfully generated from ES cells under the similar induction condition of EGL-ES cells.

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