饲养层条件下昆明小鼠胰岛来源胰腺干细胞的建系及鉴定

doi:10.3969/j.issn.2095-4344.2017.13.020

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (13): 2087-2093.doi: 10.3969/j.issn.2095-4344.2017.13.020

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇下一篇

饲养层条件下昆明小鼠胰岛来源胰腺干细胞的建系及鉴定

岑妍慧^1，2，杨瑞¹，贾微¹，李中华¹，钟振国¹，钟静¹，包鹃¹，何国珍¹，吴晓君¹，李熠毅¹

¹广西中医药大学基础医学院，广西壮族自治区南宁市 530222；²广西中医药大学第一附属医院，广西壮族自治区南宁市 530023

修回日期:2017-03-22 出版日期:2017-05-08 发布日期:2017-06-09
通讯作者: 贾微，硕士，副教授。广西中医药大学基础医学院，广西壮族自治区南宁市 530222;李中华，教授。广西中医药大学基础医学院，广西壮族自治区南宁市 530222
作者简介:岑妍慧，女，1980年生，广西壮族自治区北海市人，汉族，2012年广西医科大学毕业，博士，副教授。并列第一作者：杨瑞，男，湖北省潜江市人，汉族，1981年，硕士，2008年广西中医药大学毕业。
基金资助:
国家自然科学基金项目(81503406)；广西高校科研课题(ZD2014068)；广西高校青年教师能力提升计划(KY2016YB218，KY2016YB224)；广西壮族自治区卫生厅中医药科技专项课题(GZPT13-04，GZBZ16-07，GZLC16-23)，广西中医药大学学生科研训练课题(2014DXS02)

Establishment and identification of pancreatic stem cell strain derived from islets of Kunming mice under feeder layer conditions

Cen Yan-hui^{1, 2}, Yang Rui¹, Jia Wei¹, Li Zhong-hua¹, Zhong Zhen-guo¹, Zhong Jing¹, Bao Juan¹, He Guo-zhen¹, Wu Xiao-jun¹, Li Yi-yi¹

¹Basic Medical College, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China; ²the First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, Guangxi Zhuang Autonomous Region, China

Revised:2017-03-22 Online:2017-05-08 Published:2017-06-09
Contact: Jia Wei, Master, Associate professor, Basic Medical College, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China； Li Zhong-hua, Professor, Basic Medical College, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China
About author:Cen Yan-hui, M.D., Associate professor, Basic Medical College, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China; the First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, Guangxi Zhuang Autonomous Region, China Yang Rui, Master, Basic Medical College, Guangxi University of Chinese Medicine, Nanning 530222, Guangxi Zhuang Autonomous Region, China Cen Yan-hui and Yang Rui contributed equally to this work.
Supported by:
the National Natural Science Foundation of China, No. 81503406; University Research Projects in Guangxi, No. ZD2014068; Project of Improving the Basic Ability of Young Teachers in Universities in Guangxi, No. KY2016YB218, KY2016YB224; Special Scientific Project of Traditional Chinese Medicine supported by Health Department of Guangxi Zhuang Autonomous Region, No. GZPT13-04, GZBZ16-07, GZLC16-23; and Student Research Training Project of Guangxi Medical University, No. 2014DXS02

摘要/Abstract

摘要：

文题释义：
胰腺干细胞的标志分子：巢蛋白是神经干细胞和胰腺干细胞的标志物，是中间纤维家族的成员，被列为第Ⅵ类中间纤维，其结构和分子量与其他的中间纤维成员有差异。
胰腺：分为外分泌腺和内分泌腺两部分。外分泌腺由腺泡和腺管组成，腺泡分泌胰液，腺管是胰液排出的通道。胰液中含有碳酸氢钠、胰蛋白酶原、脂肪酶、淀粉酶等。胰液通过胰腺管排入十二指肠，有消化蛋白质、脂肪和糖的作用。

摘要
背景：到目前为止关于胰腺干细胞能建株甚至建系的报道极少，而且建系率较低，纯化出胰腺干细胞仍有一定的困难。
目的：探索一套更合理、更有效的胰腺干细胞在体外分离及连续传代的技术和方法，使其有望能建立成细胞株甚至细胞系，为后续胰腺干细胞治疗糖尿病的相关研究奠定基础。
方法：实验首先通过Percoll不连续密度梯度离心的方法，使小鼠胰腺的内、外分泌部的细胞分布在不同的密度界面，然后获得来源于胰腺内分泌部即胰岛的干细胞，使用经丝裂霉素C作用的小鼠胚胎成纤维细胞作为饲养层，在饲养层条件下对来源于胰岛的胰腺干细胞进行连续传代，使其连续传至第30代，并通过生长特性检测、形态学观察、相关分子标志物鉴定以及分化特性鉴定等一系列的检测完善对该胰岛来源的胰腺干细胞株检测和鉴定。
结果与结论：在连续传代过程中胰腺干细胞均体现了活跃的分裂增殖能力，并保持典型的干细胞形态学特征、表达胰腺干细胞分子标志物Nestin，经诱导分化后表达胰岛素基因，体现出了分化潜能。结果说明，实验饲养层条件下成功建立并鉴定了昆明小鼠胰岛来源的胰腺干细胞株。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0001-8751-7245 (Jia Wei)

关键词: 干细胞, 分化, 饲养层, 胰岛, 胰腺干细胞株, 连续传代, 鉴定, 生长特性, 形态学, 分子标志物, 国家自然科学基金

Abstract:

BACKGROUND: Until now, little has been reported on establishment of pancreatic stem cell strains and lines, and the purification of pancreatic stem cells is difficult since the cell line establish rate is low.
OBJECTIVE: To explore a more rational and effective technique of in vitro separation and continuous passage of pancreatic stem cells, with the hope to establish cell strains and even cell lines and to lay the foundation for the follow-up study of pancreatic stem cells in the treatment of diabetes.
METHODS: Firstly, Percoll discontinuous density gradient centrifugation method was applied to separate the mouse pancreatic endocrine portion from the exocrine portion, then to obtain cell strains with highly proliferative ability and low differentiation from pancreatic endocrine portion-the islet. We used mouse embryonic fibroblasts treated with mitomycin C as a feeder layer, for in vitro continuous culture of islet-derived pancreatic stem cells under feeder layer conditions until they were transferred to the 30th passage to establish cell lines. Then pancreatic stem cell line derived from pancreatic islet was detected and identified by a series of tests including growth characteristic test, morphological observation, related molecular marker identification and differentiation characteristic identification.
RESULTS AND CONCLUSION: In the continuous process of passage, pancreatic stem cells showed active proliferative ability, and maintained the typical morphological characteristics of stem cells and expression of pancreatic stem cell marker-Nestin. After induction, pancreatic stem cells showed insulin gene expression, reflecting their differentiation potential. Therefore, under the condition of feeder layer, the pancreatic stem cell line derived from Kunming mice was successfully established and the related identification was completed, which lays the foundation for the following research.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Diabetes Mellitus

中图分类号:

R394.2

岑妍慧，杨瑞，贾微，李中华，钟振国，钟静，包鹃，何国珍，吴晓君，李熠毅. 饲养层条件下昆明小鼠胰岛来源胰腺干细胞的建系及鉴定[J]. 中国组织工程研究, 2017, 21(13): 2087-2093.

Cen Yan-hui, Yang Rui, Jia Wei, Li Zhong-hua, Zhong Zhen-guo, Zhong Jing, Bao Juan, He Guo-zhen, Wu Xiao-jun, Li Yi-yi. Establishment and identification of pancreatic stem cell strain derived from islets of Kunming mice under feeder layer conditions[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 2087-2093.

图/表（结果） 2

Identification of cell source after Percoll discontinuous density centrifugation

fter Percoll discontinuous density centrifugation, the pancreatic tissue cells were distributed at three different densities (D-Hank’s/1.068, 1.068/1.096, 1.096/1.118) on the first, second and third density interfaces. Dithizone staining results showed that 80%-90% of the cells collected from the first and second density interfaces were iron red (Figure 1), indicating that the cells were derived from pancreatic endocrine portion (i.e., pancreas islet). Cells collected from the third density interface were non-stained with dithizone, indicating that the cells were derived from the exocrine portion of the pancreas.

Primary culture and identification of pancreatic stem cells derived from pancreatic islets

After 48 hours primary culture, from the pancreatic endocrine there were some large, round, and single-nucleated cells surrounding islet-like cell masses. These cells had strong cytoplasmic refraction, a large proportion of caryoplasm and grew adherently to the wall (Figure 2). These cells reflected the morphology characteristics of pancreatic stem cells, which were confirmed as pancreatic stem-like cells.

The expression of Nestin in pancreatic stem cells was detected by immunocytochemical staining and got positive result as the cytoplasm was stained claybank. These confirmed the pancreatic stem-like cells were confirmed as islet-derived pancreatic stem cells (Figure 3).

The pancreatic stem cells derived from pancreatic islets were continuously passaged to establish cell lines under feeder layer conditions

The feeder layer was prepared from mouse embryonic desmocytes. Then, the pancreatic stem cells were subcultured in the feeder layer. After 48 hours of subculture, the pancreatic stem cells began to differentiate and proliferate and form large colonies (Figure 4A). After 96 hours of passage, the pancreatic stem cells could grow to more than 80% of the orifice area (Figure 4B). Then, the cells could be passaged continuously. Under stable in vitro conditions and with mature operation technique, pancreatic stem cells were passaged till the 30^th generation to established the cell strain.

Islet-derived pancreatic stem cell line detection

Pancreatic stem cells in the process of continuous passages showed an active ability of division and proliferation (Figure 5). Pancreatic stem cell lines still maintained the original morphological features and expressed Nestin during the continuous passage. Under the microscope, the 15^th and the 30^th generations of pancreatic stem cells had large, round, single nucleus, large ratio of cytoplasm, and strong proliferating ability (Figure 6). Nestin staining showed positive results of all generations of pancreatic stem cells, while negative cells were found in the surrounding feeder layers (Figure 7).

After continuous induction and differentiation, a part of cells began to gather, but no significant changes were found in cell morphology. These cells were divided to produce more cells budding to grow upward, forming a spherical islet-like cell structure after about 1 month, having spindly pedicles connected with the bottom of the bottle, and floating in the culture medium. Dithizone staining was iron red (Figure 8).

The P53 gene of synthetic cDNA was amplified by RT-PCR, and the amplified band was about 763 bp by 1.0% agarose gel electrophoresis. This result indicated that the reverse transcription product cDNA had good quality. PCR was carried out using the specific primers of the insulin gene and the cDNA of the islet-like cell cluster as the template. The target band was found by 1.0% agarose gel electrophoresis. The molecular weight of PCR products was 50 bp in consistence with the expected molecular weight of the insulin gene (Figure 9). However, before induction, the cells did not express insulin mRNA.

参考文献

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引言

Diabetes is an endocrine dysfunction syndrome characterized by persistent hyperglycemia, which leads to systemic metabolic disorders and chronic progressive lesions in the eye, kidney, nerve, cardiovascular and other organs, eventually causing functional damage and failure. Therefore, it serve as one of the major diseases affecting human health^[1]. China now has reported the highest rate of diabetes in the world, and there are 92.4 million adults suffering from diabetes, among whom, 148.2 million people are being in the early stage of diabetes^[2-3]. The injection of insulin can control hyperglycemia in diabetic patients^[4], but to cure diabetes can only depend on the pancreas or islet transplantation. However, there are many problems such as the lack of pancreatic donors, surgical trauma defects and long-term immunosuppressive therapy after transplantation that leads to exclusion, infection, cancer and other sequelae^[4]. It is necessary to find a new source of islet cells for pancreas or islet transplantation in the treatment of diabetes.

With the rise of stem cell research and the progress of its technology and methods, people began to focus on the pancreatic stem cells, and carried out a series of studies. In 1996, Rosenberg et al.^[5]reported that a part of pancreatic duct epithelial cells from hamster pancreas have the potential of stem cells and can proliferate and differentiate into islet cells under certain conditions, thus confirming these

cells are pancreatic stem cells. In 2000, Bonner-Weir et al.^[6] found similar results that some cells of pancreatic ductal tissue isolated from human pancreatic tissue can be induced to differentiate into islet-like tissue under in vitro culture conditions. Song et al.^[7] also reported the use of an improved simple method to isolate a large number of adult pancreatic stem cells followed by cell expansion observation and differentiation into islet-like tissue under in vitro culture conditions.

Thus, the existence of pancreatic stem cells in pancreatic tissue is an indisputable fact. However, the research on pancreatic stem cells still faces many problems, as the pancreatic stem cell isolation, purification and amplification methods are still under exploration. So far, the reports about the establishment of pancreatic stem cell strain and line are rare. There was only one case reported in a Ph.D. thesis by Xiao^[8] in 2005. But the purification of pancreatic stem cells is still difficult since the cell line establish rate is low. Therefore, it is necessary to explore a more rational and effective technique and method of in vitro separation and continuous passage of pancreatic stem cells. It is expected to establish cell strains and even cell lines.

Our research group has carried out a series of studies on pancreatic stem cells, including in vitro isolation, culture and identification, and has achieved some results. Firstly, Percoll discontinuous density gradient centrifugation method was applied to separate the mouse pancreatic endocrine portion and exocrine portion so that they could be distributed in different densities. Then cell strains with highly proliferative ability and low differentiation were obtained from pancreatic endocrine portion and exocrine portion respectively. Then these cells were confirmed to be pancreatic stem cells by observing the morphological characteristics and growth pattern of cells and immunohistochemical detection of Nestin. Therefore, a more effective method of in vitro separation, culture and identification of pancreatic stem cells was established. Moreover, pancreatic stem cells from different sources showed different potentialities of differentiation: stem cells derived from the pancreatic exocrine portion express CK-19 that is a pancreatic duct epithelial cell marker, showing the potential to differentiate into pancreatic duct epithelial cells; stem cells derived from the pancreatic endocrine portion express PDX-1 that is another pancreatic stem cell marker, showing the potential to differentiate into pancreatic β-cells.

PDX-1, also known as insulin promoter factor-1 (IPF-1) and somatostain transcription factor-1 (STF-1), is considered to be an important transcription factor regulating pancreas development. It is responsible for β-cell maturation and function maintenance^[9-11]. The expression of PDX-1 in pancreatic stem cells indicates that the specific gene is activated to induce cells to differentiate into insulin-secreting cells (β-cells). By observing the natural differentiation potential of pancreatic stem cells from different sources, it is rational to believe that pancreatic stem cells derived from pancreatic islets can be obtained and effectively expanded in vitro until a cell line is established. Then, it is easier to induce these cells (including pancreatic stem cells derived from the exocrine pancreas) than other stem cells to differentiate into β-cells, as the cells have such natural differentiation potential and can significantly increase the differentiation rate of pancreatic stem cells into β-cells. This method leads to a broad application prospect of pancreatic stem cells transplantation in the treatment of diabetes.

However, long-term culture and continuous passage in vitro are adverse factors to induce natural differentiation of stem cells. The main problem of this study was how to maintain the characteristics of stem cells during continuous passage in vitro. Therefore, we intended to use mouse embryonic fibroblasts treated with mitomycin C as a feeder layer, and carried out in vitro continuous culture of islet-derived pancreatic stem cells under feeder layer conditions until they were transferred to the 30th passage to establish cell lines. Then pancreatic stem cell line derived from the pancreatic islet was detected and identified by a series of tests including growth characteristic test, morphological observation, related molecular marker identification and differentiation characteristic identification. To date, there is no report on the use of feeder layer for the expansion of stem cells until they are constructed.

材料方法

Design

Experimental study on cell culture in vitro.

Time and setting

The study was conducted at the Experimental Center of Basic Medical School in Guangxi University of Chinese Medicine from January 2016 to December 2016.

Materials

Animals

Kunming mice, SPF, newly born with 24 hours, both genders, were purchased from the Experimental Animal Center of Guangxi University of Chinese Medicine (license No. SCXK (Hu) 2007-2005).

Reagents

DMEM medium and trypsin were purchased from Hyclon; fetal bovine serum purchased from Shanghai Sheng Gong Limited of Biological Engineering, China; penicillin and streptomycin purchased from Sigma. Human lymphocyte separation fluid (Ficoll) and dithizone dye solution were purchased from Sigma.

In vitro isolation of neonatal Kunming mouse pancreatic stem cells

Referring to the literature reported^[7], we found out the effective method of cell separation. The neonatal Kunming mice were executed by beheaded method and the surface membrane of pancreas was removed. The pancreatic tissue was cut into approximately 1 mm³ slices by ophthalmic bends. 1 g/L collagenase type V was added for digestion and gentle pipetting. After 20 minutes, DMEM containing 10% fetal bovine serum was added to stop digestion. Keeping in room temperature for several minutes, the upper cell-containing dispersion was collected, and centrifuged at 500 r/min for 5 minutes, the supernatant was discarded, serum-free DMEM medium was added to gently blow and re-suspend cells, followed by centrifugation and supernatant removal. This process was repeated twice. Afterwards, a small amount of serum-free DMEM was added to re-suspend cells. The obtained cell suspensions were transferred to a gradient centrifuge tube (D-Hank’s/1.068, 1.068/1.096, 1.096/1.118) centrifuged at a discontinuous density gradient of

1 000 r/min for 15 minutes, and cells finally were collected from three density interfaces.

Cell source detection

Cells collected from the three density interfaces were stained with dithizone for 10 minutes. After that, the results were observed and photographed under electron microscope. The principle of dyeing is dithizone, which is a kind of complex, can be combined with Zn⁺ to form red complex^[15]. Pancreatic β-cells are rich in Zn⁺, so dithizone staining can be used to distinguish the pancreatic exocrine portion and endocrine portion cells.

Primary culture and identification of islet-derived pancreatic stem cells

After identification by dithizone staining, islet-derived cells were collected for primary culture with low sugar DMEM medium containing 10% fetal bovine serum. After primary culture for 48 hours, the cells were in accordance with the steps of immunocytochemical staining for Nestin detection.

Culture and preparation of feed layer cells

Uterus of Kunming mice at 14 pregnant days were removed and split to take the fetal mice out. Then, their heads and organs were removed and the trunks and limbs were moved into a small vial with D-Hank’s buffer, and cut into 3 cm size tissue pieces. And the tissue pieces were digested by 0.25% trypsin at 37 ℃ constant temperature for about 15 minutes prior to the termination of digestion. Undigested tissue blocks were removed using sterile mercery, then the filtrate was collected and centrifuged at 800 r/min for 5 minutes, and the supernatant was discarded. This step was repeated twice. Then DMEM was added to make cell suspension. The cell density was adjusted to 5×10⁸ cells/L. Finally, the cells were seeded into 24-well plates. When the cells were subcultured to the third passage, the cells were collected until covering over 90% of the bottom. Mitomycin C was used to inhibit cell lysis and proliferation. Then cells were incubated with DMEM containing 10% fetal bovine serum.

Capillary pipetting method for hand-picked pancreatic stem cell passage until the establishment of strains

On the 5^th day of primary culture, the cells were incubated with 0.02% ethylene diamine tetraacetic acid to release the connection of cells and the bottom. Then, the pancreatic stem cells were sucked by capillary pipettes, put into the plate holes with the feeder layer of 30 cells/cm², and incubated with serum-free DMEM medium containing 10 μg/mL keratinocyte growth factor and 20 ng/mL basic fibroblast growth factor. Pancreatic stem cells covered 80% area of the bottom of the hole, and the passage continued until the 30^th generation to establish Kunming mouse pancreatic stem cell strain.

Islet-derived pancreatic stem cell lines identification

Pancreatic stem cell growth observation and morphological and expression characteristics identification

The pancreatic stem cells were passaged to the 15^th and 30^th generations, respectively. The absorbance (A) value at 450 nm was measured by cell counting kit-8 (CCK-8) and the growth curve was drafted to observe cell growth conditions. From the 2^nd day, three wells were set with 10 μL CCK-8 in each well, and incubated 4 hours at 37 ℃ after mixing. The, the A₄₅₀ value was read on the microplate reader, and the A value was read for 15 days. The well only containing culture medium and the well only containing CCK-8 served as blank controls, the mean A value minus the A value of blank control acted as the analysis value. The cell growth curve was plotted with the A₄₅₀ value as the ordinate and the time (day) as the abscissa. The morphological characteristics of the 15^th and 30^th generations of pancreatic stem cells were also observed under microscope to confirm whether the morphological characteristics of pancreatic stem cells changed during passage. Nestin was also detected in pancreatic stem cells to determine whether pancreatic stem cells consistently expressed Nestin during passage.

Identification of pancreatic stem cell differentiation characteristics

The cells were passaged to the 30^th generation in vitro and seeded into 24-well plates. After the cells covered 80% area of the bottom, the culture medium was changed to serum-free DMEM/F12 containing 18 mmol/L glucose and 10 mmol/L nicotinamide followed by induced culture for 7 days. Then, the induced medium was discarded, and serum-free medium containing 10 μg/L hepatocyte cell growth factor and 5 mmol/L glucose was added for 5 hours continuous cultivation. The cells were washed with serum-free medium followed by a 3-hour continuous of 23 mmol/L glucose. Finally, to the cells were induced in the medium containing 18 mmol/L glucose and 10 mmol/L nicotinamide for 1 month. Identification of islet-like cell mass formed after the induction: The pancreatic islet-like cell mass was stained with dithizone as previously reported^[15]. Positive results of dithizone staining serve as an evidence for in vitro differentiation of pancreatic stem cells into pancreatic β-cells.

RT-PCR detection of mouse insulin gene expression: Islet-like cell clumps were picked out under a dissecting microscope. The total RNA was extracted according to the procedure. RT-PCR was performed after total RNA was reversely transferred into cDNA. Primers and PCR reaction conditions were in accordance with the repast by to Yang et al.^[13]. 5 μL of PCR product was mixed with 2 μL of loading buffer. The samples were loaded on 1.0% agarose gel electrophoresis, at 100 V, for about 20 minutes. Samples were observed and photographed under UV light, p53 was set as an internal reference^[14].

讨论

Pancreatic stem cells are a class of adult stem cells present in fetal and adult pancreatic tissues that have self-renewal ability and multi-directional differentiation potential. They are characterized by differentiation into various cells of pancreatic tissue during the process of natural differentiation^[8]. Studies have confirmed that pancreatic stem cells are present in both the endocrine and exocrine portions of the pancreas^[5-7]. However, our study group found that pancreatic stem cells were derived from pancreatic islets and passaged in vitro until the cell lines were established. The cells that are induced to differentiate into β-cells or stem cells from other sources, including pancreatic stem cells derived from the exocrine portion, should be simpler and more straightforward because it has such natural differentiation potential.

In this study, the pancreatic endocrine and exocrine cells were isolated by Percoll discontinuous density gradient centrifugation base on their difference. After a period of culture, a kind of large, round, mononuclear, colonies like growth cell was obtained, and the cells proliferated rapidly and expressed the pancreatic stem cell-specific marker, Nestin.

Nestin is a type VI intermediate filament protein expressed mainly in undifferentiated and highly proliferative cells. It participates not only in cytoskeleton formation but also in cell rearrangement and migration and mitosis of undifferentiated cells^[15]. Although Nestin has long been shown to be a marker of neural stem cells^[16-17], Hunziken et al.^[18] first reported the presence of nestin-positive cells in pancreatic islets, and presumed they were pancreatic precursor/stem cells in 2000. In 2001, Zulewski et al.^[19] reported that a class of nestin-positive cells can be obtained from pancreatic islets and be induced to differentiate into pancreatic endocrine and exocrine portion cells, claiming that nestin-positive cells in the pancreas are pancreatic precursors/stem cells. Lumelsky, Abraham and Blyszczuk et al.^[20-22] also confirm that. So Nestin has become the recognized specific marker of pancreatic stem-like cells.

To establish a cell line in vitro, cell culture for more than 6 months, stable growth, and continuous passage are required. Therefore, in order to maintain the undifferentiated nature of pancreatic stem cells during continuous passage, murine embryonic fibroblasts treated with mitomycin C are used as feeder layers. Pancreatic stem-like cells are maintained in feeder layer conditions for continuous passage in vitro. In 1981, Evans et al.^[23] used fibroblasts as the feeder layer to first successfully establish mouse embryonic stem cell line. Embryo fibroblasts feeder layer was extensively used for clonal culture of embryonic stem cells study, as feeder layer cells can synthesize a variety of factors. Leukemia inhibitory factoras cell differentiation inhibitor can effectively inhibit stem cell differentiation, and fibroblast growth factor can promote stem cell division and proliferation. Some stem cell lines have also started to use embryonic fibroblasts as feeder layers, such as the culture of human embryonic germ cells and the expansion of rabbit limbal stem cells. The feeder layer should be pre-treated before use to maintain the excretive function and eliminate proliferation capability. To avoid competition with stem cell growth, the popular method is to use mitomycin C and r-ray irradiation. In this study, mitomycin C was applied to treat feeder layer cells, mitomycin C alkylation and cell DNA cross-linking, so that their double-stranded depolymerization inhibited DNA replication.

After the proliferation is terminated on the mitomycin C treated feeder layer, the secretion of growth factors funding the growth of they pancreatic stem cells is triggered and these factors also can inhibit the spontaneous differentiation of pancreatic stem cells. In this study, under stable in vitro conditions and with mature operation technique, the pancreatic stem cells were passaged to the 30^th generation and maintain good growth characteristics, high proliferation and undifferentiated state. However, the use of feeder layer makes the system of stem cell culture more complicated. Feeder layer cells can maintain the undifferentiated state of pancreatic stem cells and promote proliferation and differentiation of stem cells, but whether they have adverse effects to pancreatic stem cells is not clear.

We used the induction method established by our research group to test cell proliferation ability. The results show that the obtained pancreatic stem cells could be induced to differentiate into islet-like cell clusters. The dithizone staining of these cell clusters were iron red. The disulfide hydrazone could specifically bind to the zinc of the β-cell insulin granules to make them iron-red. Therefore, the cell mass contained β-cells. In addition, RT-PCR results also confirmed this. These results suggest that the pancreatic stem cell strain derived from islets can be induced to differentiate into pancreatic endocrine precursor cells. In conclusion, this study established a mouse pancreatic stem cell strain derived from Kunming mice under feeder layer conditions and identified it by a series of tests. More importantly, it provides an effective method for the further study on the establishment of human and mammalian pancreatic stem cell lines.

饲养层条件下昆明小鼠胰岛来源胰腺干细胞的建系及鉴定

Establishment and identification of pancreatic stem cell strain derived from islets of Kunming mice under feeder layer conditions

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