胎猪胰岛间充质干细胞分离鉴定及向胰岛素分泌细胞分化的研究
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摘要
糖尿病(Diabetes mellitus, DM)及其并发症严重危害着人类的健康。传统的药物治疗和胰岛素注射治疗不能从根本上治疗糖尿病。胰岛移植能够有效的控制血糖变化,避免并发症的发生,然而却受到供体细胞不足的限制。猪的胰岛素结构和功能与人十分相似,并且物种资源丰富,因此可作为有效的异种胰岛细胞供体来源。越来越多的证据表明胰腺组织中存在着具有多向分化潜能的干细胞,这些细胞在特定条件下更容易分化成功能性的胰岛素分泌细胞。本研究旨在从2月龄以上胎猪胰腺组织中分离出胰岛间充质干细胞(Islet mesenchynal stem cells, IMSCs),通过体外诱导将其分化为胰岛素分泌细胞,并将其移植到糖尿病模型裸鼠体内进行糖尿病的治疗,探讨胎猪IMSCs作为异种供体细胞来源的可行性。
1.胎猪胰岛间充质干细胞的分离培养及生物学特性鉴定
由于来源于胰岛组织,胰岛间充质干细胞(IMSCs)在向胰岛素分泌细胞分化方面具有更大的潜力。与成体组织相比,胎儿体内可能含有更多的干细胞。本研究采取先悬浮后贴壁的培养方法,从2月龄以上胎猪胰腺组织中分离出胰岛细胞团,并由胰岛细胞团分离出IMSCs,通过细胞生长曲线、流式细胞表面抗原分析、免疫组化染色、RT-PCR检测、核型分析、成瘤性检测以及体外诱导分化等方法,对该细胞的生物学特性进行了鉴定。结果表明,胰腺组织消化后,采用先悬浮后贴壁的培养方法可有效分离得到胎猪IMSCs。通过检测发现,胎猪IMSCs体外培养过程中,胰岛内分泌细胞标记(胰岛素和胰高血糖素)和上皮细胞标记(CK7)的表达逐渐消失,而间充质干细胞标记(波形蛋白)的表达迅速增强,初步证实胎猪IMSCs可能来源于去分化的p细胞。第14代和第37代胎猪IMSCs的群体倍增时间分别为27.05±1.05 h和28.35±1.02 h,表明该细胞在体外多次传代后增殖能力未发生明显的改变。细胞流式表面抗原分析、免疫组化和RT-PCR检测结果表明,胎猪IMSCs除了表达胰腺干细胞标记胰腺十二指肠同源盒因子1 (Pdx1)和神经元素3(Ngn3)外,还表达Oct4、Sox2、Nanog等胚胎干细胞的一些多能性标记,其表面抗原表达特征与骨髓间充质干细胞十分相似。并且该细胞具有定向分化为神经和心肌细胞的能力,分化后细胞形态发生相应的变化,并表达神经和心肌细胞的一些相关标记。显示该细胞具有多向分化的潜能。46代胎猪IMSCs的核型和致瘤性分析结果表明,体外多次传代后细胞仍具有正常的二倍体核型,移植体内未见致瘤。上述结果表明,胎猪IMSCs具有很强的体外增殖能力和干细胞表达特征,体外具有向其他类型细胞分化的能力,可为组织工程和再生医学研究提供充足的细胞资源。
2.体外定向诱导胎猪IMSCs向胰岛素分泌细胞分化及其移植治疗裸鼠糖尿病
(1)体外向胰岛素分泌细胞分化：与L-DMEM培养基相比,以RPMI1640和H-DMEM作为基础诱导培养基,可显著提高诱导后细胞的胰岛素和C-肽分泌量(P<0.01),然而H-DMEM诱导组诱导后的细胞不具有葡萄糖反应性,不能够根据葡萄糖浓度的变化调节胰岛素和C-肽的释放量,因此不具有实用性。本研究最终确定以RPMI1640作为基础诱导培养基。通过对比发现,添加活化素A (Activin-A)可显著促进胎猪MSCs分泌胰岛素和C-肽(P<0.05或P<0.01)。本研究以无血清RPMI1640为基础诱导液,设计出一个两步法诱导方案。第一步,采用贴壁培养诱导,并添加10 mmol/L尼克酰胺和100 ng/mL Activin-A的方法诱导细胞1周；第二步,通过添加10 nmol/L exendin-4和4 nmol/Lβ-细胞素(BTC),并采用悬浮培养的方法继续诱导1周。结果显示,通过悬浮诱导胎猪IMSCs迅速聚集形成胰岛样细胞团,该细胞团双硫腙(DTZ)染色呈阳性。免疫荧光及RT-PCR结果显示,该细胞团表达胰岛p细胞特征性标记胰岛素(Insulin)和葡萄糖转运子2 (Glut-2)。并且诱导过程中内分泌前体细胞标记Ngn3表达减弱。25 mmol/L葡萄糖刺激2h后,第一步诱导后(诱导1周组)细胞的胰岛素和C-肽分泌量分别为34.92±6.54μIU/mL/106细胞和0.23±0.06 ng/mL/106细胞,第二步诱导后(诱导2周组)细胞的胰岛素和C-肽分泌量分别为287.65±42.24μIU/mL/106细胞和0.37±0.11 ng/mL/106细胞,均显著高于未诱导对照组(胰岛素分泌量2.16±0.37μIU/mL/106细胞,C-肽分泌量为0.11±0.03 ng/mL/106细胞)(P<0.05或P<0.01)；并且诱导后细胞仍具有很高的葡萄糖反应性。上述结果表明胎猪IMSCs具有向胰岛素分泌细胞分化的潜能。
(2)细胞移植治疗小鼠糖尿病：通过小剂量多次注射STZ的方法成功建立了糖尿病裸鼠模型,制模率达到了100%(17/17)。使用CM-DiL荧光染料对移植细胞进行标记,细胞标记率可达90%以上。将第二步诱导后的胎猪IMSCs移植到糖尿病裸鼠左侧睾丸内,观察血糖和体重变化。分别摘取移植1周和移植1个月时诱导移植组裸鼠的左侧睾丸组织进行切片免疫荧光染色,观察细胞体内存活状况。同时设立PBS注射对照组、未诱导细胞移植对照组和未制模正常小鼠对照组。体重检测结果表明,所有模型小鼠的体重均逐渐下降,包括诱导细胞移植组,而正常对照组裸鼠的体重却逐渐增加,表明诱导细胞移植后未改善糖尿病小鼠体重下降的状况。移植后,PBS注射组和未诱导细胞移植组裸鼠血糖水平一直维持在较高水平(>16.7 mmol/L),诱导细胞移植组裸鼠的血糖水平移植后迅速开始下降,第6d时接近正常水平,随后其血糖水平逐渐升高,19 d左右上升到移植前水平,随后一直维持在较高水平(>16.7 mmol/L),正常对照组裸鼠的血糖水平一直维持在正常范围内(<8mmol/L)。上述结果表明诱导细胞移植后短期内可缓解糖尿病小鼠的高血糖状况。睾丸组织切片染色分析发现,移植1周时诱导组移植侧睾丸组织内存在大量的CM-DiL和胰岛素共表达细胞,而移植1个月后该细胞数量大量减少,提示移植细胞在糖尿病裸鼠体内未能够长期存活。
3.猪β细胞特异性启动子系统构建与检测细胞向胰岛素分泌细胞分化的研究
通过使用猪胰岛素启动子和绿色荧光蛋白报告基因构建出一种p细胞特异性检测系统。转染后,观察到胎猪原代胰岛细胞表达绿色荧光,而胎猪IMSCs则不表达绿色荧光,表明该系统具有表达特异性。转染后的胎猪IMSCs经体外诱导分化,表达绿色荧光,并且这些细胞共表达胰岛素。RT-PCR和(?)Vestern blotting检测结果显示,诱导后胰岛素表达增强的同时,绿色荧光蛋白表达也显著增强。表明该系统可用于检测猪胰腺干细胞体外诱导分化过程中生成的胰岛素分泌细胞,具有筛选体外诱导分化体系和分离纯化胰岛素分泌细胞的能力。因此,具有重要的应用价值。
Diabetes mellitus is a devastating disease that has been heavily threatening the health of human beings. Traditional treatment with medicine or insulin injection often does not provide sufficient control of blood glucose and prevent complications of this disease. Islet transplantation offer potential therapeutic options for diabetic patients because this therapy can restore not only the insulin-secreting unit, but also the precise fine tuning of insulin release in response to multiple signals within and outside the islets. However, this therapy has been hampered by the shortage of donor islets. The usage of porcine islet cells is currently viewed as the most promising alternative, as there is a plentiful supply of porcine islet cells; moreover, porcine and human insulins are highly conserved, and porcine normal physiological glucose levels are similar to those in humans. Many studies showed that there are multipotential stem cells exist in pancreas. Under specific conditions, these cells can easily differentiate into functional insulin-producing cells. This study aims to isolate islet mesenchymal stem cells (IMSCs) from porcine fetus older than the age of 2 months, in vitro induce these cells to differentiate into insulin-producing cells, and transplant them into STZ-induced nude mice model for treatment of diabetes to explore the prospects of IMSCs as heterogeneous donor cells.
1 Study on isolation and biological properties of fetal porcine pancreatic islet mesenchymal stem cells
Derived from islets, especially fetal islets, IMSCs has greater potential to differentiate into insulin-producing cells. This study was to isolate IMSCs from porcine fetal pancreas older than the age of 2 months by a suspend-to-adhere culture method. Isolated cells were identified by cell growth curve, cell surface antigen analysis, immunohistochemical staining, RT-PCR, karyotype and tumorigenicity analysis, and of in vitro differentiation potential analysis. Results show that, porcine fetal IMSCs can effectively isolate by suspend-to-adhere culture method。Results by detection the genes expression of different passages porcine fetal IMSCs showed that the expression of markers of islet endocrine cells(such as insulin and glucogan)and epithelial cells(such as CK7) of porcine fetal IMSCs was declined gradually, but the expression of mesenchymal cell marker vimentin was enhanced. These results preliminary demonstrated that the porcine fetal IMSCs may derived fromβcells that undergone dedifferentiation process. The population doubling time of porcine fetal IMSCs of passage 14 and 37 were 27.05±1.05 h and 28.35±1.02 h respectively, which showed that the proliferative capacity of porcine fetal IMSCs was not changed after passages. Cell antigen analysis showed that porcine fetal IMSCs express not only the markers of pancreatic stem cells, but also the multipotent markers of embryonic stem cells, and the expression of cell surface antigens of porcine fetal IMSCs was similar to that of bone marrow mesenchymal stem cells. In vitro differentiation potential analysis showed that porcine fetal IMSCs possessed the ability of differentiation into neurocytes and cardiomyocytes. The passage 46 porcine fetal IMSCs showed normal karyotype and formed no tumor in the nude mice. These results indicated that porcine fetal IMSCs has strong proliferation and differentiation capacity in vitro, the characteristics of stem cells, and can provide ample cell resources for tissue engineering and regenerative medicine.
2. Differentiation of porcine fetal IMSCs into insulin producing cells in vitro and treatment of diabetes
(1) Differentiation of porcine fetal IMSCs into insulin producing cells in vitro: Compared with L-DMEM medium, RPMI 1640 and H-DMEM medium can significantly enhance the secretion of insulin and C-peptide of porcine fetal IMSCs(P<0.01). However, the cells after induction with H-DMEM medium can't regulate the secretion of insulin and C-peptide according to the changes of glucose level, so the RPMI1640 medium was finally chosen as basic inductive medium. Compared with the control group, adding Activin-A could significantly enhance the secretion of insulin and C-peptide of porcine fetal IMSCs (P<0.05 or P<0.01). A two-step induction protocol was devised for the differentiation of porcine fetal IMSCs into insulin-producing cells, in which the serum-free RPMI 1640 medium was used as the basic inductive medium. In the first step, the porcine fetal IMSCs were treated with Activin-A and nicotinamide for 1 week on stick petri dishes. Then the cells were collected by trypsin digestion and seeded onto non-stick petri dishes for another 1 week induction. In this step, exendin-4 and betacellulin were added. In the second step, porcine fetal IMSCs quickly gathered and formed islet-like cell clusters (ICCs), and these ICCs were stained into crimson with DTZ. After 2 weeks induction, ICCs expressed the specific markers of isletβcells (Insulin and Glut2), and the expression of Ngn3, a marker of pancreatic endocrine cells, decreased detected by RT-PCR and immunofluorescent staining. Under stimulation of 25 mmol/L glucose for 2 h, the insulin and C-peptide secretion of porcine fetal IMSCs after 1 week induction were 34.92±6.54μIU/mL/106 cells and 0.23±0.06 ng/mL/106 cells respectively, and that of porcine fetal IMSCs after 2 weeks induction were 287.65±42.24 μIU/mL/106 cells and 0.37±0.11 ng/mL/106 cells respectively, which significantly higher than the uninduced porcine fetal IMSCs(secretion of insulin and C-peptide of this group were 2.16±0.37μIU/mL/106 cells and 0.11±0.03 ng/mL/106 cells respectively) (P<0.05 or P<0.01). These results indicated that porcine fetal IMSCs have the ability to differentiate into insulin producing cells.
(2) Transplantation of induced cells for treatment of diabetes:The diabetic nude mice model was successfully established by intraperitoneal STZ injection, and the rate of molding was 100%(17/17). The transplanted cells were labeled with CM-DiL, a fluorescent dye, and the labeling rate of the cells exceeded 90%. After labeling with CM-DiL, the induced porcine fetal IMSCs were transplanted into the left testes of diabetic mice. The body weight and blood glucose level of mice were detected regularly. The left testes of diabetic mice transplanted with induced cells were removed after one week and one month respectively to observe the survival situation of transplanted cells by slice fluorescent staining. The normal mice and diabetic mice injected with PBS or uninduced cells were used as control. The body weight of diabetic mice, including the mice transplanted with induced cells, decreased. However, that of normal mice increased gradually. These results indicated that the body weight of diabetic nude mice was not increased after cell transplantation. After transplantation, the blood glucose levels of diabetic mice injected with PBS or uninduced cells maintained at a high level (>16.7 mmol/L), but that of diabetic mice transplanted with induced cells quickly declined, and it approach to normal level on the sixth day, then it increased gradually and maintained at a high level(>16.7 mmol/L). The glucose level of normal mice maintained at a low level (<8mmol/L). These results indicated that porcine fetal IMSCs has the ability to ameliorate the hyperglycemia of diabetic nude mice。Analysis of slice fluorescent staining showed that there are large amounts of cells, which coexpressed Insulin and CM-DiL, existed in testes after one week transplantation of induced cells. However, the number of these cells declined after one month. These indicated that the transplanted cells could not survive a long time in vivo.
3. Construction of a porcineβcell specific promoter system and their detection of insulin producing cells derived from porcine fetal islet-derived mesenchymal stem cells
A porcineβcell specific detection system was established by using the porcine insulin promoter (IP) and pEGFP-1 vector. After transfection, primary fetal porcine islet cells expressed the green fluorescent protein (GFP), while porcine fetal IMSCs did not; this indicated that the expression of GFP has specificity. After induction, the porcine fetal IMSCs transfected with IP-pEGFP vector expressed GFP, and it coexpressed with insulin. RT-PCR and Western blotting analysis showed that after induction, the expression of insulin and GFP increased simultaneously, which indicated that this system can be used for identification of insulin-producing cells derived from porcine stem cells, and has important application value on selection of induction protocols and sorting the insulin-producing cells from induced cells.
1.胎猪胰岛间充质干细胞的分离培养及生物学特性鉴定
由于来源于胰岛组织,胰岛间充质干细胞(IMSCs)在向胰岛素分泌细胞分化方面具有更大的潜力。与成体组织相比,胎儿体内可能含有更多的干细胞。本研究采取先悬浮后贴壁的培养方法,从2月龄以上胎猪胰腺组织中分离出胰岛细胞团,并由胰岛细胞团分离出IMSCs,通过细胞生长曲线、流式细胞表面抗原分析、免疫组化染色、RT-PCR检测、核型分析、成瘤性检测以及体外诱导分化等方法,对该细胞的生物学特性进行了鉴定。结果表明,胰腺组织消化后,采用先悬浮后贴壁的培养方法可有效分离得到胎猪IMSCs。通过检测发现,胎猪IMSCs体外培养过程中,胰岛内分泌细胞标记(胰岛素和胰高血糖素)和上皮细胞标记(CK7)的表达逐渐消失,而间充质干细胞标记(波形蛋白)的表达迅速增强,初步证实胎猪IMSCs可能来源于去分化的p细胞。第14代和第37代胎猪IMSCs的群体倍增时间分别为27.05±1.05 h和28.35±1.02 h,表明该细胞在体外多次传代后增殖能力未发生明显的改变。细胞流式表面抗原分析、免疫组化和RT-PCR检测结果表明,胎猪IMSCs除了表达胰腺干细胞标记胰腺十二指肠同源盒因子1 (Pdx1)和神经元素3(Ngn3)外,还表达Oct4、Sox2、Nanog等胚胎干细胞的一些多能性标记,其表面抗原表达特征与骨髓间充质干细胞十分相似。并且该细胞具有定向分化为神经和心肌细胞的能力,分化后细胞形态发生相应的变化,并表达神经和心肌细胞的一些相关标记。显示该细胞具有多向分化的潜能。46代胎猪IMSCs的核型和致瘤性分析结果表明,体外多次传代后细胞仍具有正常的二倍体核型,移植体内未见致瘤。上述结果表明,胎猪IMSCs具有很强的体外增殖能力和干细胞表达特征,体外具有向其他类型细胞分化的能力,可为组织工程和再生医学研究提供充足的细胞资源。
2.体外定向诱导胎猪IMSCs向胰岛素分泌细胞分化及其移植治疗裸鼠糖尿病
(1)体外向胰岛素分泌细胞分化：与L-DMEM培养基相比,以RPMI1640和H-DMEM作为基础诱导培养基,可显著提高诱导后细胞的胰岛素和C-肽分泌量(P<0.01),然而H-DMEM诱导组诱导后的细胞不具有葡萄糖反应性,不能够根据葡萄糖浓度的变化调节胰岛素和C-肽的释放量,因此不具有实用性。本研究最终确定以RPMI1640作为基础诱导培养基。通过对比发现,添加活化素A (Activin-A)可显著促进胎猪MSCs分泌胰岛素和C-肽(P<0.05或P<0.01)。本研究以无血清RPMI1640为基础诱导液,设计出一个两步法诱导方案。第一步,采用贴壁培养诱导,并添加10 mmol/L尼克酰胺和100 ng/mL Activin-A的方法诱导细胞1周；第二步,通过添加10 nmol/L exendin-4和4 nmol/Lβ-细胞素(BTC),并采用悬浮培养的方法继续诱导1周。结果显示,通过悬浮诱导胎猪IMSCs迅速聚集形成胰岛样细胞团,该细胞团双硫腙(DTZ)染色呈阳性。免疫荧光及RT-PCR结果显示,该细胞团表达胰岛p细胞特征性标记胰岛素(Insulin)和葡萄糖转运子2 (Glut-2)。并且诱导过程中内分泌前体细胞标记Ngn3表达减弱。25 mmol/L葡萄糖刺激2h后,第一步诱导后(诱导1周组)细胞的胰岛素和C-肽分泌量分别为34.92±6.54μIU/mL/106细胞和0.23±0.06 ng/mL/106细胞,第二步诱导后(诱导2周组)细胞的胰岛素和C-肽分泌量分别为287.65±42.24μIU/mL/106细胞和0.37±0.11 ng/mL/106细胞,均显著高于未诱导对照组(胰岛素分泌量2.16±0.37μIU/mL/106细胞,C-肽分泌量为0.11±0.03 ng/mL/106细胞)(P<0.05或P<0.01)；并且诱导后细胞仍具有很高的葡萄糖反应性。上述结果表明胎猪IMSCs具有向胰岛素分泌细胞分化的潜能。
(2)细胞移植治疗小鼠糖尿病：通过小剂量多次注射STZ的方法成功建立了糖尿病裸鼠模型,制模率达到了100%(17/17)。使用CM-DiL荧光染料对移植细胞进行标记,细胞标记率可达90%以上。将第二步诱导后的胎猪IMSCs移植到糖尿病裸鼠左侧睾丸内,观察血糖和体重变化。分别摘取移植1周和移植1个月时诱导移植组裸鼠的左侧睾丸组织进行切片免疫荧光染色,观察细胞体内存活状况。同时设立PBS注射对照组、未诱导细胞移植对照组和未制模正常小鼠对照组。体重检测结果表明,所有模型小鼠的体重均逐渐下降,包括诱导细胞移植组,而正常对照组裸鼠的体重却逐渐增加,表明诱导细胞移植后未改善糖尿病小鼠体重下降的状况。移植后,PBS注射组和未诱导细胞移植组裸鼠血糖水平一直维持在较高水平(>16.7 mmol/L),诱导细胞移植组裸鼠的血糖水平移植后迅速开始下降,第6d时接近正常水平,随后其血糖水平逐渐升高,19 d左右上升到移植前水平,随后一直维持在较高水平(>16.7 mmol/L),正常对照组裸鼠的血糖水平一直维持在正常范围内(<8mmol/L)。上述结果表明诱导细胞移植后短期内可缓解糖尿病小鼠的高血糖状况。睾丸组织切片染色分析发现,移植1周时诱导组移植侧睾丸组织内存在大量的CM-DiL和胰岛素共表达细胞,而移植1个月后该细胞数量大量减少,提示移植细胞在糖尿病裸鼠体内未能够长期存活。
3.猪β细胞特异性启动子系统构建与检测细胞向胰岛素分泌细胞分化的研究
通过使用猪胰岛素启动子和绿色荧光蛋白报告基因构建出一种p细胞特异性检测系统。转染后,观察到胎猪原代胰岛细胞表达绿色荧光,而胎猪IMSCs则不表达绿色荧光,表明该系统具有表达特异性。转染后的胎猪IMSCs经体外诱导分化,表达绿色荧光,并且这些细胞共表达胰岛素。RT-PCR和(?)Vestern blotting检测结果显示,诱导后胰岛素表达增强的同时,绿色荧光蛋白表达也显著增强。表明该系统可用于检测猪胰腺干细胞体外诱导分化过程中生成的胰岛素分泌细胞,具有筛选体外诱导分化体系和分离纯化胰岛素分泌细胞的能力。因此,具有重要的应用价值。
Diabetes mellitus is a devastating disease that has been heavily threatening the health of human beings. Traditional treatment with medicine or insulin injection often does not provide sufficient control of blood glucose and prevent complications of this disease. Islet transplantation offer potential therapeutic options for diabetic patients because this therapy can restore not only the insulin-secreting unit, but also the precise fine tuning of insulin release in response to multiple signals within and outside the islets. However, this therapy has been hampered by the shortage of donor islets. The usage of porcine islet cells is currently viewed as the most promising alternative, as there is a plentiful supply of porcine islet cells; moreover, porcine and human insulins are highly conserved, and porcine normal physiological glucose levels are similar to those in humans. Many studies showed that there are multipotential stem cells exist in pancreas. Under specific conditions, these cells can easily differentiate into functional insulin-producing cells. This study aims to isolate islet mesenchymal stem cells (IMSCs) from porcine fetus older than the age of 2 months, in vitro induce these cells to differentiate into insulin-producing cells, and transplant them into STZ-induced nude mice model for treatment of diabetes to explore the prospects of IMSCs as heterogeneous donor cells.
1 Study on isolation and biological properties of fetal porcine pancreatic islet mesenchymal stem cells
Derived from islets, especially fetal islets, IMSCs has greater potential to differentiate into insulin-producing cells. This study was to isolate IMSCs from porcine fetal pancreas older than the age of 2 months by a suspend-to-adhere culture method. Isolated cells were identified by cell growth curve, cell surface antigen analysis, immunohistochemical staining, RT-PCR, karyotype and tumorigenicity analysis, and of in vitro differentiation potential analysis. Results show that, porcine fetal IMSCs can effectively isolate by suspend-to-adhere culture method。Results by detection the genes expression of different passages porcine fetal IMSCs showed that the expression of markers of islet endocrine cells(such as insulin and glucogan)and epithelial cells(such as CK7) of porcine fetal IMSCs was declined gradually, but the expression of mesenchymal cell marker vimentin was enhanced. These results preliminary demonstrated that the porcine fetal IMSCs may derived fromβcells that undergone dedifferentiation process. The population doubling time of porcine fetal IMSCs of passage 14 and 37 were 27.05±1.05 h and 28.35±1.02 h respectively, which showed that the proliferative capacity of porcine fetal IMSCs was not changed after passages. Cell antigen analysis showed that porcine fetal IMSCs express not only the markers of pancreatic stem cells, but also the multipotent markers of embryonic stem cells, and the expression of cell surface antigens of porcine fetal IMSCs was similar to that of bone marrow mesenchymal stem cells. In vitro differentiation potential analysis showed that porcine fetal IMSCs possessed the ability of differentiation into neurocytes and cardiomyocytes. The passage 46 porcine fetal IMSCs showed normal karyotype and formed no tumor in the nude mice. These results indicated that porcine fetal IMSCs has strong proliferation and differentiation capacity in vitro, the characteristics of stem cells, and can provide ample cell resources for tissue engineering and regenerative medicine.
2. Differentiation of porcine fetal IMSCs into insulin producing cells in vitro and treatment of diabetes
(1) Differentiation of porcine fetal IMSCs into insulin producing cells in vitro: Compared with L-DMEM medium, RPMI 1640 and H-DMEM medium can significantly enhance the secretion of insulin and C-peptide of porcine fetal IMSCs(P<0.01). However, the cells after induction with H-DMEM medium can't regulate the secretion of insulin and C-peptide according to the changes of glucose level, so the RPMI1640 medium was finally chosen as basic inductive medium. Compared with the control group, adding Activin-A could significantly enhance the secretion of insulin and C-peptide of porcine fetal IMSCs (P<0.05 or P<0.01). A two-step induction protocol was devised for the differentiation of porcine fetal IMSCs into insulin-producing cells, in which the serum-free RPMI 1640 medium was used as the basic inductive medium. In the first step, the porcine fetal IMSCs were treated with Activin-A and nicotinamide for 1 week on stick petri dishes. Then the cells were collected by trypsin digestion and seeded onto non-stick petri dishes for another 1 week induction. In this step, exendin-4 and betacellulin were added. In the second step, porcine fetal IMSCs quickly gathered and formed islet-like cell clusters (ICCs), and these ICCs were stained into crimson with DTZ. After 2 weeks induction, ICCs expressed the specific markers of isletβcells (Insulin and Glut2), and the expression of Ngn3, a marker of pancreatic endocrine cells, decreased detected by RT-PCR and immunofluorescent staining. Under stimulation of 25 mmol/L glucose for 2 h, the insulin and C-peptide secretion of porcine fetal IMSCs after 1 week induction were 34.92±6.54μIU/mL/106 cells and 0.23±0.06 ng/mL/106 cells respectively, and that of porcine fetal IMSCs after 2 weeks induction were 287.65±42.24 μIU/mL/106 cells and 0.37±0.11 ng/mL/106 cells respectively, which significantly higher than the uninduced porcine fetal IMSCs(secretion of insulin and C-peptide of this group were 2.16±0.37μIU/mL/106 cells and 0.11±0.03 ng/mL/106 cells respectively) (P<0.05 or P<0.01). These results indicated that porcine fetal IMSCs have the ability to differentiate into insulin producing cells.
(2) Transplantation of induced cells for treatment of diabetes:The diabetic nude mice model was successfully established by intraperitoneal STZ injection, and the rate of molding was 100%(17/17). The transplanted cells were labeled with CM-DiL, a fluorescent dye, and the labeling rate of the cells exceeded 90%. After labeling with CM-DiL, the induced porcine fetal IMSCs were transplanted into the left testes of diabetic mice. The body weight and blood glucose level of mice were detected regularly. The left testes of diabetic mice transplanted with induced cells were removed after one week and one month respectively to observe the survival situation of transplanted cells by slice fluorescent staining. The normal mice and diabetic mice injected with PBS or uninduced cells were used as control. The body weight of diabetic mice, including the mice transplanted with induced cells, decreased. However, that of normal mice increased gradually. These results indicated that the body weight of diabetic nude mice was not increased after cell transplantation. After transplantation, the blood glucose levels of diabetic mice injected with PBS or uninduced cells maintained at a high level (>16.7 mmol/L), but that of diabetic mice transplanted with induced cells quickly declined, and it approach to normal level on the sixth day, then it increased gradually and maintained at a high level(>16.7 mmol/L). The glucose level of normal mice maintained at a low level (<8mmol/L). These results indicated that porcine fetal IMSCs has the ability to ameliorate the hyperglycemia of diabetic nude mice。Analysis of slice fluorescent staining showed that there are large amounts of cells, which coexpressed Insulin and CM-DiL, existed in testes after one week transplantation of induced cells. However, the number of these cells declined after one month. These indicated that the transplanted cells could not survive a long time in vivo.
3. Construction of a porcineβcell specific promoter system and their detection of insulin producing cells derived from porcine fetal islet-derived mesenchymal stem cells
A porcineβcell specific detection system was established by using the porcine insulin promoter (IP) and pEGFP-1 vector. After transfection, primary fetal porcine islet cells expressed the green fluorescent protein (GFP), while porcine fetal IMSCs did not; this indicated that the expression of GFP has specificity. After induction, the porcine fetal IMSCs transfected with IP-pEGFP vector expressed GFP, and it coexpressed with insulin. RT-PCR and Western blotting analysis showed that after induction, the expression of insulin and GFP increased simultaneously, which indicated that this system can be used for identification of insulin-producing cells derived from porcine stem cells, and has important application value on selection of induction protocols and sorting the insulin-producing cells from induced cells.
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