corso di Genomica a.a. 2010-2011 lezione 25-26. laurea magistrale Biotecnologia Industriale. Martedì 11 Gennaio 2011 aula 6A orario : Martedì ore 14.00 - 16.00 Giovedì ore 13.00 - 15.00 . Esami: 24 Febbraio, 3 Marzo, 23 Marzo. D. Frezza. Espressione x staminalità.
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Martedì 11 Gennaio 2011
orario : Martedì ore 14.00 - 16.00
Giovedì ore 13.00 - 15.00
Esami: 24 Febbraio, 3 Marzo, 23 Marzo
4 geni di staminalità Oct 3/4 (Pouf 5f1), Sox2, c-myc, Klf4
Fibroblasti trasfettati e selezionati per espressione di Fbxo15
cellule iPS = induced pluripotent (Fbx15iPS)
Fenotipo come ES, proliferano e danno teratomi;
Diverse per l’espressione del DNA e metilazione, non producono topi chimerici adulti iniettandole in blastocisti
Nuovo esperimento: selezione delle iPS per l’espressione di Nanog
Produzione di fenotipo germline-competent iPS
aumento di espressione: di geni ES-cell-like e del pattern di metilazione rispetto alle Fbx15 iPS
I 4 transgeni Oct3/4, Sox2, c-myc e Klf4 sono silenziati nei cloni di cellule Nanog iPS
Sono stati ottenuti topi chimerici da 7 cloni Nanog iPS ed un clone è stato trasmesso nella generazione successiva di un topo transgenico
Circa il 20% dei topi transgenici sviluppava tumori per la riattivazione di c-myc
c-myc transgenico genera rischio neoplastico nel 20% della progenie dei topi transgenici
Cellule iPS competenti per avere topi chimerici “germline” e poi transgenici possono essere ottenute da fibroblasti
Deve essere evitato l’uso della transgenesi c-myc per le applicazioni cliniche.
Mitsui K, Yamanaka S. et al.
Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.
Cell. 2003 May 30;113(5):631-42
Silva J, Chambers I, Pollard S, Smith A.
Through cell fusion, embryonic stem (ES) cells can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ES cells. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain (~indurre) undifferentiated ES cell propagation. Here we report that in fusions between ES cells and neural stem (NS) cells, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ES cell characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ES cells; however, fewer colonies are obtained than from ES x NS cell fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NS x ES cell fusions elevated Nanog enables primary hybrids to develop into ES cell colonies with identical frequency to homotypic ES x ES fusion products. This means that in hybrids, increased Nanog is sufficient for the NS cell epigenome to be reset completely to a state of pluripotency. We conclude that Nanog can orchestrate ES cell machinery to instate pluripotency with an efficiency of up to 100% depending on the differentiation status of the somatic cell.
SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells.Pan C,
Hicks A, Guan X, Chen H, Bishop CE.
College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A & F University, Yangling 712100, China.
Induced pluripotent stem (iPS) cells can be derived from human somatic cells by cellular reprogramming. This technology provides a potential source of non-controversial therapeutic cells for tissue repair, drug discovery, and opportunities for studying the molecular basis of human disease. Normally, mouse embryonic fibroblasts (MEFs) are used as feeder layers in the initial derivation of iPS lines. The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cells reprogrammed from somatic cells using lentiviral expressed reprogramming factors. In our study, iPS cells expressed common pluripotency markers, displayed human embryonic stem cells (hESCs) morphology and unmethylated promoters of NANOG and OCT4. These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.
J Genet Genomics. 2010 Apr;37(4):241-8.