Kuise Takashi, Noguchi Hirofumi, Tazawa Hiroshi, Kawai Takashi, Iwamuro Masaya, Saitoh Issei, Kataoka Hitomi Usui, Watanabe Masami, Noguchi Yasufumi, Fujiwara Toshiyoshi
Department of Surgery, Chiba-East National Hospital, National Hospital Organization, Chiba 260-8712, Japan.
Biomed Eng Online. 2014 May 27;13:64. doi: 10.1186/1475-925X-13-64.
For cell therapies to treat diabetes, it is important to produce a sufficient number of pancreatic endocrine cells that function similarly to primary islets. Induced pluripotent stem (iPS) cells represent a potentially unlimited source of functional pancreatic endocrine cells. However, the use of iPS cells for laboratory studies and cell-based therapies is hampered by their high tumorigenic potential and limited ability to generate pure populations of differentiated cell types in vitro. The purpose of this study was to establish a pancreatic stem cell line from iPS cells derived from mouse fibroblasts.
Mouse iPS cells were induced to differentiate into insulin-producing cells by a multi-step differentiation protocol, which was conducted as described previously with minor modifications. Selection of the pancreatic stem cell was based on morphology and Pdx1 expression. The pancreatic potential of the pancreatic stem cells was evaluated using a reverse transcription PCR, real-time PCR, immunofluorescence, and a glucose challenge test. To assess potential tumorigenicity of the pancreatic stem cells, the cells were injected into the quadriceps femoris muscle of the left hindlimb of nude mice.
The iPS-derived pancreatic stem cells expressed the transcription factor--Pdx1--a marker of pancreatic development, and continued to divide actively beyond passage 80. Endocrine cells derived from these pancreatic stem cells expressed insulin and pancreatic genes, and they released insulin in response to glucose stimulation. Mice injected with the pancreatic stem cells did not develop tumors, in contrast to mice injected with an equal number of iPS cells.
This strategy provides a new approach for generation of insulin-producing cells that is more efficient and safer than using iPS cells. We believe that this approach will help to develop a patient-specific cell transplantation therapy for diabetes in the near future.
对于治疗糖尿病的细胞疗法而言,产生足够数量且功能与原代胰岛相似的胰腺内分泌细胞至关重要。诱导多能干细胞(iPS细胞)是功能性胰腺内分泌细胞潜在的无限来源。然而,iPS细胞用于实验室研究和基于细胞的治疗受到其高致瘤潜力以及在体外产生纯分化细胞类型群体能力有限的阻碍。本研究的目的是从小鼠成纤维细胞来源的iPS细胞建立胰腺干细胞系。
通过多步分化方案诱导小鼠iPS细胞分化为胰岛素产生细胞,该方案按先前所述进行并略有修改。胰腺干细胞的选择基于形态学和Pdx1表达。使用逆转录PCR、实时PCR、免疫荧光和葡萄糖激发试验评估胰腺干细胞的胰腺潜能。为评估胰腺干细胞的潜在致瘤性,将细胞注射到裸鼠左后肢股四头肌中。
iPS来源的胰腺干细胞表达转录因子——Pdx1——胰腺发育的标志物,并且在传代80代后仍继续活跃分裂。源自这些胰腺干细胞的内分泌细胞表达胰岛素和胰腺基因,并在葡萄糖刺激下释放胰岛素。与注射等量iPS细胞的小鼠相比,注射胰腺干细胞的小鼠未发生肿瘤。
该策略为产生胰岛素产生细胞提供了一种新方法,比使用iPS细胞更高效、更安全。我们相信这种方法将有助于在不久的将来开发针对糖尿病的患者特异性细胞移植疗法。