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Download Derivation of pluripotent epiblast stem cells from mammalian embryos
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Culture and differentiation of embryonic stem cells Hong-Lin Su Department of Life Sciences, National Chung-Hsing University Topics-ES cell maintenance • Establishment • Culture condition, including the serum, LIF and the feeder cells • Mouse strains • Non-rodent ES cells • Why ICM? 4 2 8 8 16 64 compaction compaction Morula blastocyst h 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). James Thomso Human ES cell maintenance • Establishment • Culture condition: LIF-independent • Serum: FGF, nodal/activin and Wnt and KSR; LIF • Feeder still important • Why ICM Derivation of pluripotent epiblast stem cells from mammalian embryos New cell lines from mouse epiblast share defining features with human embryonic stem cells The differences between the mouse and human ES cells • • • • • Morphology LIF or FGF dependence or independence Growth condition BMP responsibility Activin/nodal responsibility EpiSC cell culture Pleuripotency markers of EpiSC Pleuripotency in vivo Pleuripotency in vitro Oct4 methylation status in mouse epiSC and ES cells EpiSC is more similar to hESC Fail of integration into inner cell mass EpiSC cells are AP negative Neural Induction 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? Spemann’s demonstration of nuclear equivalence in newt cleavage Asymmetry in the amphibian egg Determination of ectoderm during newt gastrulation Organization of a secondary axis by dorsal blastopore lip tissue (Part 1) Organization of a secondary axis by dorsal blastopore lip tissue (Part 2) Model of organizer function and axis specification in the Xenopus gastrula Neural Induction （Xenopus ） Uncommitted Neural Precursors Ectodermal Cells Neural Inducers Chordin, Noggin BMP4 Blocking BMP Signals is essential and sufficient to induce neural differentiation. Neural Induction （Mouse） Neural precursors ES cells ? BMP4 Blocking BMP signals is essential but not sufficient. Additional signaling is required. Stromal cell-Derived Inducing Activity (SDIA) PA6 cells as feeder cells dissociated ES cells 8 days in serum-free medium PA6 cell cell line derived from mouse calvaria OP9 cell cell line derived from mouse calvaria NIH3T3 cell embryonic fibroblast cell line in vitro neural differentiation of ES cells induced by SDIA nestin (neural precursor) TuJ (mature neuron) Quantification of neural marker expression NCAM (pan-neural) colony (%) 92 ± 5 nestin (neural precursors) cell (%) 47 ± 10 GFAP (glial) mesoderm markers 2±2 <2 TuJ (mature neurons) 52 ± 9 Efficient induction of dopaminergic neurons TH day10 Diencephalon Mesencephalon Metencephalon r1 Telencephalon Midbrain dopaminergic neurons r2 r3 r4 5-HT neurons Locus coeruleus neurons r5 Neural Patterning factors dorsal Activities of BMPs BMP gastrula inhibition of neural induction neurula promotion of dorsal CSN & neural crest differentiation ventral dorsal BMP motor neurons ventral floor plate SFEB: Serum-Free Embryoid Body-like culture v.s. SDIA: Stromal cell-Derived Inducing Activity Knock-out replacement serum PA6 cells as feeder cells dissociated ES cells dissociated ES cells 8 days in serum-free KSR 8 days in serum-free KSR floating adhesive 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). Generation of Cerebellar Neuron Precursors from Mouse Embryonic Stem Cells Patterning factors for upper rhombic lips dorsal BMP ventral EGL precursor neurons are induced from ES cells by using SFEB method Generation of Cerebellar Purkinje cells from ES cells 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.
