Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions
Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions
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DOI：
10.1038/sj.cr.7310138
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发表时间：
2007-01-01
期刊：
影响因子：
44.1
通讯作者：
Cheng, Linzhao
中科院分区：
文献类型：
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作者：
Cai, Liuhong;Ye, Zhaohui;Cheng, Linzhao
我们先前表明，Wnt3a能够刺激人胚胎干细胞（hES）增殖并影响细胞命运决定。在缺乏饲养层细胞衍生因子的情况下，在无饲养层条件下培养的hES细胞存活且增殖不良。在缺乏饲养层细胞衍生因子的情况下添加重组Wnt3a刺激了hES细胞增殖，但也促进了分化。在本研究中，我们进一步将分析扩展到其他Wnt配体，如Wnt1和Wnt5a。虽然Wnt1对hES细胞显示出与Wnt3a相似的作用，但Wnt5a在该系统中几乎没有作用。当饲养层衍生的自我更新因子和碱性成纤维细胞生长因子（bFGF）也存在时，Wnt3a和Wnt1增强了未分化hES细胞的增殖。为了探索通过激活Wnt信号促进未分化hES细胞增殖的可能性，我们在永生化的人成纤维细胞（HAFT）中过表达Wnt3a或Wnt1基因，这些细胞在支持未分化hES细胞长期生长方面优于原代小鼠胚胎成纤维细胞。含有或不含有Wnt转基因的HAFT细胞都可以无限增殖。Wnt3a基因的过表达显著增强了HAFT饲养层细胞支持我们所测试的3种不同hES细胞系未分化生长的能力。在过表达Wnt3a的HAFT细胞中共表达三种常用的药物选择基因，进一步使我们能够在稳定转染或转导后选择罕见的hES克隆。这些共表达如Wnt3a等生长促进基因和三种药物选择基因的永生化工程饲养层细胞（W3R）应该使我们能够有效地制备用于基础和转化研究的基因修饰hES细胞系。
We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Writ ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Writ signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFT) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFT cells with or without a Writ transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFT feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFT cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.