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作者：Neta Lavon, Ofra Yanuka, Nissim Benvenisty作者单位：Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel & T3 k, S) W0 E: M- k) G
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【摘要】
. w8 A- l2 c. e" | Human embryonic stem cells (HESCs) are pluripotent cells that may serve as a source of cells for transplantation medicine and as a tool to study human embryogenesis. Using genetic manipulation methodologies, we have investigated the potential of HESCs to differentiate into the various pancreatic cell types. We initially created various HESCs carrying the enhanced green fluorescent protein (eGFP) reporter gene under the control of either the insulin promoter or the pancreatic and duodenal homeobox factor-1 (Pdx1) promoter. Our analysis revealed that during the differentiation of HESCs into embryoid bodies (EBs), we could detect green fluorescent cells when eGFP is regulated by Pdx1 promoter but not by insulin promoter. To examine whether we can induce differentiation into pancreatic cells, we have established human embryonic stem cell lines that constitutively express either Pdx1 or the endodermal transcription factor Foxa2. Following differentiation into EBs, the constitutive expression of Pdx1 enhanced the differentiation of HESCs toward pancreatic endocrine and exocrine cell types. Thus, we have demonstrated expression of several transcription factors that are downstream of Pdx1 and various molecular markers for the different pancreatic cell types. However, the expression of the insulin gene could be demonstrated only when the cells differentiated in vivo into teratomas. We conclude that although overexpression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo. 2 j% j/ i# B) m9 O( @ y' z
【关键词】 Embryonic stem cells Endoderm Pancreas Development Genetic manipulation
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0 p% ^/ K f& u4 X( W; j# QMaintaining blood glucose at normal physiological levels is essential for normal body function. Controlled insulin secretion by the ß cells of the pancreas is the mechanism that enables glucose homeostasis. The pancreas arises as dorsal and ventral buds that emanate from the embryonic endoderm. In the mouse, at embryonic day 9.5 (E9.5), signaling from the notochord induces the patterning of these endodermal cells that are destined to form the pancreatic buds (reviewed in .! s7 q, ]3 d) ]+ ], {& g* q
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Human embryonic stem cells (HESCs) are pluripotent cells capable of differentiating into the three embryonic germ layers, that is, ectoderm, mesoderm, and endoderm. The pluripotency of HESCs has been proven both in vivo and in vitro .4 V, Y! l& j. [- {# k; z
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Differentiation of HESCs into insulin-producing cells was demonstrated by spontaneous differentiation in adherent or suspension culture conditions and by a stepwise protocol of various culture conditions .
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In this study, by using genetic manipulation methodologies, we have analyzed the potential of HESCs to differentiate into the various cell lineages comprising the pancreas. By introducing a reporter gene under the control of the pancreatic and duodenal homeobox factor-1 (Pdx1) promoter, we could trace putative pancreatic precursor cells during the differentiation of HESCs into EBs. Next, we constitutively expressed either the endodermal transcription factor forkhead box A2 (Foxa2) or Pdx1 in HESCs and examined their pancreatic differentiation. In culture, HESCs overexpressing Pdx1 differentiate into cells expressing pancreatic markers earlier than the control cells. Moreover, we show that further differentiation can be achieved in teratomas derived from the Pdx1- or Foxa2-expressing cells.9 O1 l5 u. I& _& V4 {' H( }7 B
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MATERIALS AND METHODS
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Cell Culture/ N! n# E$ {2 }6 x
: E4 w8 s6 n* E: @% X3 f# A; HHESCs and their differentiated derivatives were cultured as previously described . EBs were generated by aggregation of the HESCs for different time points: early EBs (2¨C4 days), mid-EBs (10¨C14 days), and late EBs (21¨C30 days). For teratoma formation, 5 x 106 HESCs were injected into the kidney capsule of 4-week-old mice with severe combined immunodeficiency. One month later, the mice were sacrificed, the teratomas were re; {; l7 ?/ P- z1 r* _! I
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发表于 2009-3-5 00:03
发表于 2015-6-3 08:00
