mTert-GFP and Pancreatic Progenitor Cells

描述（由申请人提供）：成体干细胞在再生医学、组织修复和早期发育事件的研究方面具有很大的前景。新的胰岛素产生细胞来源的鉴定可能会为所有形式的糖尿病患者带来新的治疗选择。作为对部分胰腺切除术或生理刺激的反应，胰腺细胞可以通过现有细胞的复制和细胞新生来再生。胰腺祖细胞/干细胞（PPSC）被认为是新生的细胞来源，尽管它们的存在仍然存在争议。我们假设（1）存在PPSC群体，其表达端粒酶（mTert）作为其干细胞表型的标志物，（2）PPSC可以使用mTert-GFP转基因小鼠分离和扩增，以及（3）通过移植，其分化将重现正常的胰岛细胞发育。我们使用了一种创新的方法来产生转基因小鼠与内源性干细胞标记的GFP。我们目前有两个独立的mTert-GFP转基因小鼠系，其中GFP+细胞正在评估其干细胞潜力。在骨髓和睾丸中的验证研究已经证明了GFP在两种成体干细胞群体中表达：造血干细胞和雄性生殖细胞。此外，该模型允许鉴定和分离推定的肠干细胞群，如通过GFP+肠隐窝细胞在胚泡注射后特异性植入肠干细胞龛并在成年嵌合动物中长期存在的能力所定义的。在短期Exendin-4刺激后，最近在胰腺中的研究使用FACS分析和免疫组织化学（IHC）在两种小鼠系中证明了GFP+细胞的可诱导群体。GFP表达定位于导管结构内的细胞，而在胰岛和腺泡细胞中不存在。该提议旨在验证从mTert-GFP转基因小鼠分离的GFP+推定胰腺祖细胞/干细胞的身份和发育潜力。具体而言，我们将使用体外和体内模型系统，例如，细胞培养、移植和胚泡注射，以确定这些细胞发育成胰岛素分泌细胞的潜力。此外，我们提出了谱系追踪研究，使用mTert CreERT 2转基因小鼠，进一步研究mTert表达细胞在成年胰腺中的发育潜力。I型糖尿病是人类痛苦的主要原因，并且由胰腺β细胞的破坏和胰岛素产生的损失引起。这种情况的最终治疗将涉及（1）逆转对β细胞的破坏性自身免疫攻击和（2）正常β细胞的再生。新的胰岛素产生细胞来源的鉴定将对这种疾病患者的未来和医疗保健系统产生巨大影响。我们的建议使用一种创新的新模型来寻找小鼠中的胰腺祖细胞，目的是识别这些难以捉摸的细胞。

DESCRIPTION (provided by applicant): Adult stem cells hold great promise for regenerative medicine, tissue repair and for the study of early developmental events. The identification of new sources of insulin-producing cells would potentially give rise to novel treatment options for patients suffering from diabetes in all its forms. In response to partial pancreatectomy or physiologic stimuli, pancreatic ¿ cells can regenerate by replication of existing ¿ cells and by ¿ cell neogenesis. Pancreatic progenitor/stem cells (PPSC) have been postulated to be the cellular origin of neogenesis, though their existence remains controversial. We hypothesize (1) that a population of PPSC exists, which express telomerase (mTert) as a marker of their stem cell phenotype, (2) that PPSC can be isolated and expanded using mTert-GFP transgenic mice and (3) that with transplantation their differentiation will recapitulate normal islet cell development. We have used an innovative method to generate transgenic mice with endogenous stem cells labeled with GFP. We currently have two independent mTert-GFP transgenic mouse lines, from which GFP+ cells are being evaluated for their stem cell potential. Validation studies in bone marrow and testis have demonstrated GFP expression within two adult stem cell populations: hematopoietic stem cells and male germ cells. In addition, this model has allowed for the identification and isolation of a putative intestinal stem cell population, as defined by the ability of GFP+ intestinal crypt cells to specifically engraft the intestinal stem cell niche following blastocyst injection and to persist long-term in adult chimeric animals. Recent studies in pancreas, following short-term Exendin-4 stimulation, demonstrate an inducible population of GFP+ cells using both FACS analysis and immunohistochemistry (IHC) in both lines of mice. GFP expression is localized to cells within ductal structures and is absent from islets and acinar cells. This proposal aims to validate the identity and developmental potential of GFP+ putative pancreatic progenitor/stem cells isolated from mTert-GFP transgenic mice. Specifically, we will use in vitro and in vivo model systems, e.g., cell culture, transplantation and blastocyst injection, to establish the potential of these cells to develop into insulin secreting cells. In addition, we propose lineage-tracing studies, using mTert-CreERT2 transgenic mice, to further study the developmental potential of mTert expressing cells in adult pancreas. Type I Diabetes is a major cause of human suffering and results from the destruction of pancreatic beta cells and the loss of insulin production. The ultimate treatment for this condition will involve (1) reversal of the destructive autoimmune attack on beta cells and (2) regeneration of normal beta cells. The identification of new sources of insulin producing cells would have an enormous impact on the future of patients with this disease and on the health care system. Our proposal uses an innovative new model to search for pancreatic progenitor cells in mice with the goal to identify these elusive cells.