Mouse model to assess putative beta cell progenitor cells

我们建立了灵敏和强大的体内测试系统，以评估假定的干细胞分化为能够生理调节胰岛素分泌的细胞的能力。利用我们的小鼠实验性自身免疫性糖尿病(EAD)模型系统，我们设计了一种体内试验，以测试干细胞是否能够在持续的自身免疫反应中有效地产生和维持β细胞。该模型的具体特征包括：(I)在较长时间内维持正常的宿主代谢，随后(Ii)胰岛功能逐渐下降，需要新的β细胞，(Iii)胰岛内炎症，这可能为干细胞向胰腺的调节迁移和向β细胞分化提供关键线索，以及(Iv)新开发的干细胞来源的β细胞抵抗自身抗原特异性T细胞介导的攻击。使用这个系统，我们测试了一些报道的造血干细胞(HSC)的粗品，其中含有能够转分化为胰岛β细胞的细胞。我们发现HSC在体内没有形成β细胞的功能相关能力。使用该模型，我们彻底评估并实验排除了几个关键问题，如体内未能产生胰岛素产生的β细胞是否可能是由于新形成的β细胞无法承受炎症环境的毒性，这种毒性通常与免疫介导的胰岛破坏有关。我们获得的数据有力地支持了这样的说法，即通过证实在混合胰岛移植排斥反应期间，供者来源的胰岛(干细胞基因型)比宿主类型的胰岛选择性存活，来自骨髓供者来源的新产生的β细胞将被保护而不受免疫介导的攻击。作为这个模型的阳性对照，我们已经证明了从胚胎小鼠胰腺分离的单个细胞，然后注入成年小鼠胰腺，将分化为成熟的胰岛。更具体地说，利用芝加哥大学Manami Hari博士提供的小鼠，在小鼠中，绿色荧光蛋白的β细胞表达是由小鼠胰岛素启动子驱动的，我们已经证明了14.5天的单细胞悬液可以将胰腺芽细胞注射到成年同基因小鼠的胰腺中，并且来自胚胎小鼠胰腺的定向祖细胞将在成年小鼠胰腺分化为成熟的出现的胰岛。此外，与胚胎捐赠者区分开来的胰岛明显是由绿色区分出来的。这些数据正在提交以供发布。在相关工作中，Pechhold博士与合作者使用细胞标记技术证明，胰岛β细胞不能有效地去分化为快速增殖的细胞(即所谓的上皮细胞到间质细胞的转变)，然后在体外可以重新分化为产生胰岛素的细胞。 在去年的工作中，我们证明了被其他人描述为有效促进转分化的基因治疗方法，即将肝细胞转化为胰岛素产生细胞，实际上是非常低效的。我们已发表的研究表明，腺病毒载体驱动的小鼠肝脏PDX-1表达产生的肝脏胰岛素产量低于16个胰岛，这一数量显然不足以维持葡萄糖稳态。

We established sensitive and robust in vivo test systems to assess putative stem cells for their ability to differentiate into cells capable of physiologically regulated insulin secretion. Using our mouse experimental autoimmune diabetes (EAD) model system, we have designed an in vivo assay to test if stem cells can efficiently generate and maintain beta cells during an ongoing autoimmune response. Specific features of the model include: (i) normal host metabolism is maintained for an extended time followed by (ii) gradually declining pancreatic islet function creating a need for new beta cells, (iii) intra-islet inflammation which may provide critical cues for regulated stem cell migration to the pancreas and differentiation into beta cells, and (iv) newly developed, stem cell-derived beta cells are resistant to autoantigen-specific T cell mediated attack. Using this system, we tested crude preparations of hematopoetic stem cells (HSC) reported by some to contain cells capable of transdifferentiating into pancreatic beta cells. We found that the HSC had no functionally relevant capacity to form beta cells in vivo. Using the model, we thoroughly evaluated and experimentally ruled out several critical questions such as whether the failure to generate insulin producing beta cells in vivo might have been due to the inability of newly developed beta cells to withstand the toxicity of an inflammatory environment typically associated with immune-mediated pancreatic islet destruction. Data weve generated strongly support the claim that newly generated beta cells from the bone marrow donor origin would be protected from immune mediated attack, by confirming the selective survival of donor-derived pancreatic islets (stem-cell genotype) over host-type islets during rejection of mixed islet transplants. As a positive control for this model, we've shown that single cells isolated from embryonic mouse pancreata, then injected into the adult mouse pancreas, will differentiate into mature islets. More specifically, using mice provided by Dr. Manami Hari (University of Chicago) in which beta cell expression of green fluorescent protein is driven by the mouse insulin promoter, weve shown that a single cell suspension of day e14.5 pancreatic bud cells can be injected into the pancreas of adult syngeneic mice and that committed progenitor cells from the embryonic mouse pancreas will differentiate in the adult mouse pancreas into mature appearing islets. Moreover, the islets differentiating from the embryonic donors are clearly differentiated by their green color. These data are being submitted for publication. In related work, Dr. Pechhold demonstrated with collaborators using cell labeling techniques that islet beta cells do not efficiently de-differentiate into rapidly proliferating cells (so called epithelial to mesenchymal transition) that can then be re-differentiated into insulin producing cells in vitro. In work from last year, we demonstrated that a gene-therapy approach described by others as efficiently promoting transdifferentiation, i.e. converting liver cells into insulin producing cells, was in fact very inefficient. Our published studies suggested that mouse liver Pdx-1 expression driven by adenoviral vectors generated less hepatic insulin production than 16 islets, an amount clearly insufficient to maintain glucose homeostasis.