Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, thepotential of pax-4-nucleofected cells in medical treatment is promising.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting

瞄准：用没有浆液的、调节媒介从胰管的人的成年纸巾孤立通常认为的胰腺的干细胞(PSC ) 。这些 PSC 的表面显型的描述被流动血细胞计数分析。为胰腺的系和在这些 PSC 的贝它房间区别的能力的潜力也被评估。方法：由使用与必要生长因素补充的没有浆液的媒介，我们试图孤立被报导了表示巢在的通常认为的 PSC 和 pdx-1。Matrigel (TM ) 被采用评估孤立的房间的微分能力。染色的染色的双硫腙，胰岛素内容 / 分泌物测量，和免疫组织化学被用来监视区别。荧光(FACS ) 激活的房间排序被用来检测通常认为的 PSC 的表型的标记。结果：像锭子的房间的单层被栽培。通常认为的 PSC 表示了 pdx-1 和巢在里面。他们也能区分进胰岛素 -- ， glucagon- ，和 somatostatin 积极的房间。在 PSC 的表型的标记的光谱被调查；当用人的骨头导出髓的干细胞作为比较级试验时，类似被揭示，例如 CD29， CD44， CD49，半数治愈量， CD51， CD62E， PDGFR-alpha， CD73 (SH2 ) ， CD81， CD105 (SH3 ) 。结论：在这研究，我们成功地由使用没有浆液的媒介从成年的人的胰腺的管孤立 PSC。这些 PSC 不仅表示的巢在和 pdx-1 而且对间充质的干细胞可归因的展出标记。尽管工作被需要阐明这些房间的角色，这些 PSC 的申请可能是为糖尿病的治疗学的策略。