体外诱导人羊膜上皮细胞分化为胰岛素分泌细胞的研究

目的:通过体外诱导分化实验,探讨人羊膜上皮细胞(hAECs)向胰岛素分泌细胞(ISCs)分化的能力。方法:采用胰蛋白酶消化法从人羊膜组织分离提取hAECs,用流式细胞仪和免疫细胞化学法进行鉴定。取第3代hAECs在含尼克酰胺和N2补充物的无血清培养基中诱导培养,分别于诱导不同时间采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子-1(PDX-1)mRNA的表达。结果:①hAECs高表达CD29、CD73、CD166和CK19;②hAECs诱导组第7、142、1天胰岛素阳性细胞百分率分别为74.00%±1.73%、75.33%±1.15%和75.67%±0.58%,而对照组未见胰岛素阳性细胞;③hAECs诱导组第7、14、21天培养物上清液中胰岛素含量分别达(328.47±3.22)μIU/ml、(332.26±1.22)μIU/ml和(329.68±2.57)μIU/ml,均显著高于对照组(P均<0.01);④hAECs诱导前后均有PDX-1 mRNA和β2微球蛋白表达,胰岛素mRNA表达仅见于诱导组。结论:hAECs能分化为ISCs,在Ⅰ型糖尿病细胞移植治疗方面具有潜在应用前景。     

Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector

Human amniotic epithelial cells (hAECs) are a recently identified type of stem cell. Thanks to their ready availability and the lower risk of teratoma formation, hAECs have been studied and tested for a variety of human disease treatments and tissue reconstruction efforts. This aim of this study was to establish a stable tracking system to further monitor hAECs in vivo after transplantation. hAECs were isolated from the placentas of patients who visited the Hunan Province Maternity and Child Care Hospitals between Jan 2008 and Jan 2009. Using the classic transfection/infection technique, we successfully introduced green fluorescent protein (GFP) into cultured hAECs with an adeno-associated virus (AAV) vector. The initial preparation of the AAV-GFP virus stock was titrated using HT1081 cells, and further used for the infection of hAECs. GFP⁺ hAECs preserve the capacity of differentiation into hepatocytelike cells with the expression of cytokeratin-18 (CK18) and albumin (ALB). AAV-GFP virus-infected hAECs were transplanted through the spleen into severe combined immune deficiency (SCID) mice via hepatectomy. Four weeks later, the GFP and human albumin expressions were examined in multiple organs through immunoflourence staining. In culture, over 50% of the hAECs were GFP-positive 3 days after infection. Following transplantation, AAV-GFPinfected hAECs survived and continued to express GFP in the host for up to 4 weeks. These cells were primarily found in the spleen and liver, expressing human albumin. This study provides a feasible and stable system to track hAECs. It may prove useful to further identify their biological characteristics after transplantation and to elucidate their beneficial roles for therapeutic purposes.     

牛乳腺上皮细胞的分离培养及其生物学特性

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牛乳腺上皮细胞。形态学观察表明,培养的细胞具有典型的上皮细胞形态特征;染色体分析结果表明,培养的细胞具有正常的染色体数目。通过荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白5和8。该细胞在添加胰岛素、氢化可的松以及羊催乳素的无血清培养液中诱导培养时,用RT-PCR方法检测到了β-酪蛋白基因的转录。这些结果表明,分离培养的细胞是乳腺上皮细胞,这些细胞在诱导培养的条件下能够转录表达β-酪蛋白。     

Human amniotic epithelial cells maintain mouse spermatogonial stem cells in an undifferentiated state due to high leukemia inhibitor factor (LIF) expression

Spermatogonial stem cells (SSCs), like other stem cells, have unique properties: prolonged proliferation, self-renewal, generation of differentiated progeny, and maintenance of developmental potential. Long-term cultivation of normal SSCs into stable cell lines, and maintaining SSCs in an undifferentiated state capable of self-renewal, is a major challenge. Here, we compare the effect of leukemia inhibitory factor (LIF) expression on mouse SSCs isolated from testicular tissue cultured under different conditions. We found that human amniotic epithelial cells (hAECs) with high LIF expression (LIF(high)) feeder cells allowed mouse SSCs to maintain a high level of AP activity when cultured long term. Expression of some important stem cell markers was higher in mouse SSCs cultured on hAECs (LIF(high)) compared to those cultured on hAECs (LIF(low)). Taken together, these results suggest that LIF expression could be a crucial component for feeder cells to maintain mouse SSCs in an undifferentiated, proliferative state capable of self-renewal.     

Use of human amniotic epithelial cells as a feeder layer to support undifferentiated growth of mouse spermatogonial stem cells via epigenetic regulation of the Nanog and Oct-4 promoters

Spermatogonial stem cells (SSCs) are defined by unique properties like other stem cells. However, there are two major challenges: long-term cultivation of normal SSCs into stable cell lines and maintaining the SSCs as undifferentiated and capable of self-renewal. Here, we compared different culture methods for mouse SSCs isolated and cultured from testicular tissue. We found that human amniotic epithelial cells (hAECs) can behave as feeder cells, allowing mouse SSCs to maintain a high level of alkaline phosphatase (AP) activity when cultured long-term. Also, we observed that expression of Nanog, Oct-4 and other important stem cells markers were higher in mouse SSCs cultured on hAECs compared to those cultured on MEF or without any feeder cells. Furthermore, we demonstrated that the CpG islands of the Nanog and Oct-4 promoters were hypomethylated in cells cultured on hAECs. In addition, mouse SSCs cultured on hAECs exhibited higher levels of H3AC and H3K4Me3 in the Nanog and Oct-4 promoters than those cultured on MEF or without feeder cells. Taken together, these results suggest that the hAEC-induced epigenetic modifications at the Nanog and Oct-4 locus could be a key mechanism for maintaining mouse SSCs in an undifferentiated state capable of self-renewal.     

Isolation,Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.     

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth medium supplemented with serum (3T3 system) or without feeder cells in a dedicated serum-free medium (EpiLife). During the culture, the cells were maintained either at ambient oxygen tension (20%) or at different levels of hypoxia (15, 10, 5, and 2% oxygen). The effect of oxygen on cell growth, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET).     

牛肝干细胞的分离培养与鉴定

该实验旨在分离牛肝干细胞,培养观察其生长特性,为比较医学研究及开发牛肝脏体外研究工具提供实验基础。实验采用改良的离体两步胶原酶灌流法,结合密度梯度离心分离牛肝干细胞,观察细胞形态及生长特性,并利用免疫荧光染色和PCR对牛肝干细胞相关标志物进行鉴定。实验分离的细胞接种48 h开始贴壁分裂,随后呈集落样生长,表现出干细胞的特性,低密度下培养504 h后细胞呈肝细胞样分化。正常接种密度的细胞可传至P3代,HE染色可见细胞均为单核,核浆比大,呈幼稚低分化状态;免疫荧光染色和PCR结果均显示,该细胞表达甲胎蛋白AFP、上皮细胞黏附蛋白(EpCAM)、造血干/祖细胞表面标志CD34、干细胞因子受体蛋白(c-kit)、细胞角蛋白CK18、CK19。结果表明,该研究分离了一种具有明显干细胞特征,并表达成熟肝细胞和胆管上皮细胞表面标志物的牛肝干细胞。     

Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype

The embryonal origin of hepatic stellate cells (HSCs), the principal cells in hepatic fibrogenesis, is still intriguing. To explore the origin and the differentiation of HSCs, we studied the expression of cytokeratin 18 (CK18) and 19 (CK19), the standard markers of simple epithelial cells, in cultured human HSCs. Hepatic stellate cells were isolated from five normal human livers. In immunofluorescence staining, both clone C-51 anti-CK18 antibody and clone RCK108 anti-CK19 antibody labeled almost all stellate cells in the primary culture. Double immunofluorescence staining for cytokeratin/vimentin and cytokeratin/alpha-smooth muscle actin detected by confocal laser scanning microscopy clearly demonstrated the localization of cytokeratin immunoreactivity in human HSCs. During subsequent cultivation of human HSCs to the tenth passage, immunocytochemical staining and western blot analysis demonstrated gradually decreasing profiles of CK18 and CK19 expression. The progressive reduction of cytokeratin expression was further confirmed in a culture of clone cells originated from a single HSC. In conclusion, both CK18 and CK19 are expressed in cultured human HSCs, and the extent of their expression decreases gradually during prolonged cultivation. Our results suggest that HSCs may be of epithelial origin, and that they undergo the transdifferentiation from epithelial to mesenchymal phenotype during an activation process in vitro.     

Changes in culture expanded human amniotic epithelial cells: implications for potential therapeutic applications

Human amniotic epithelial cells (hAEC) isolated from term placenta have stem cell-like properties, differentiate into tissue specific cells and reduce lung and liver inflammation and fibrosis following transplantation into disease models established in mice. These features together with their low immunogenicity and immunosuppressive properties make hAEC an attractive source of cells for potential therapeutic applications. However, generation of large cell numbers required for therapies through serial expansion in xenobiotic-free media may be a limiting factor. We investigated if hAEC could be expanded in xenobiotic-free media and if expansion altered their differentiation capacity, immunophenotype, immunosuppressive properties and production of immunomodulatory factors. Serial expansion in xenobiotic-free media was limited with cumulative cell numbers and population doubling times significantly lower than controls maintained in fetal calf serum. The epithelial morphology of primary hAEC changed into mesenchymal-stromal like cells by passage 4-5 (P4-P5) with down regulation of epithelial markers CK7, CD49f, EpCAM and E-cadherin and elevation of mesenchymal-stromal markers CD44, CD105, CD146 and vimentin. The P5 hAEC expanded in xenobiotic-free medium differentiated into osteocyte and alveolar epithelium-like cells, but not chondrocyte, hepatocyte, α- and β-pancreatic-like cells. Expression of HLA Class IA, Class II and co-stimulatory molecules CD80, CD86 and CD40 remained unaltered. The P5 hAEC suppressed mitogen stimulated T cell proliferation, but were less suppressive compared with primary hAEC at higher splenocyte ratios. Primary and P5 hAEC did not secrete the immunosuppressive factors IL-10 and HGF, whereas TGF-β1 and HLA-G were reduced and IL-6 elevated in P5 hAEC. These findings suggest that primary and expanded hAEC may be suitable for different cellular therapeutic applications.     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vero细胞最佳培养体系筛选

为了筛选出适合Vero细胞生长的最佳条件,本研究分别对Vero细胞的培养液、传代比例和接种浓度进行了优化.首先将Vero细胞在不同组合培养液中进行连续3代传代培养,筛选出适宜Vero细胞连续稳定传代的最佳培养液组成.然后分别在不同的传代比例和接种浓度下进行连续3代培养,筛选出适宜Vero细胞连续稳定传代的最佳传代比例和接种浓度.最终结果显示,Vero细胞在1.296%MEM溶液与特级胎牛血清的组合优于其他组合,可满足Vero细胞连续稳定传代.并且在该培养基的培养下,1∶6的传代比例为最适宜Vero细胞的传代比例,3×10^4/cm^2的接种密度为最适宜Vero细胞的接种密度.本研究最终完成了Vero细胞最佳培养体系条件筛选,并证实该条件满足Vero细胞连续稳定传代.     

Amnion Epithelial Cells of Buffalo (Bubalus bubalis) Term Placenta Expressed Embryonic Stem Cells Markers and Differentiated into Cells of Neurogenic Lineage In Vitro

Aim of the present study was the isolation, culture, and characterization of amniotic membrane-derived epithelial cells (AE) from term placenta collected postpartum in buffalo. We found that cultured cells were of polygonal in shape, resistance to trypsin digestion and expressed cytokeratin-18 indicating that they were of epithelial origin. These cells have negative expression of mesenchymal stem cell markers (CD29, CD44, and CD105) and positive for pluripotency marker (OCT4) genes indicated that cultured cells were not contaminated with mesenchymal stem cells. Immunofluorescence staining with pluripotent stem cell surface markers, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 indicated that these cells may retain pluripotent stem cell characteristics even after long period of differentiation. Differentiation potential of these cells was determined by their potential to differentiate into cells of neurogenic lineages using retinoic acid. In conclusion, we demonstrate that AE cells expressed pluripotent stem cell markers and have propensity to differentiate into cells of neurogenic lineage upon directed differentiation in vitro.     

Characterization of cultured human prostatic epithelial cells by cluster designation antigen expression

Cultured prostatic epithelial cells have been extensively studied as a model of prostate biology. What is the lineage relationship of the cultured cells to the epithelial cell types in tissue? How different are cultured cells derived from tumor tissue to those derived from benign tissue? Expression of cluster designation (CD) cell surface molecules has been shown to be useful in characterizing cells according to lineage. A CD profile was therefore generated for cultured human prostatic epithelial cells and compared with those previously established for basal and luminal epithelial cells in the prostate. Presence of CD44, CD49b, CD49f, and CD104 and absence of CD57 suggests that cultured cells were derived from basal cells of prostatic tissues. However, expression of certain CD antigens characteristic of luminal epithelial cells was also observed in subpopulations of cultured cells. The pattern of CD antigens in cultured cells reflects a phenotype similar to that of transit-amplifying cells that have been described in the prostate. Several CD antigens were found expressed by both cultured prostatic epithelial and stromal cells, and are probably associated with cell proliferation. The CD profiles of cultured epithelial cell strains derived from normal compared with malignant tissues were notably similar to each other and to that of the prostate cancer cell line PC-3. We conclude that cells in culture retain expression of certain lineage-characteristic CD antigens. Furthermore, CD antigens can define subpopulations of cells with differential gene expression.     

Isolation of tissue progenitor cells from duct-ligated salivary glands of swine

Tissue stem cells participate in the repopulation of tissue after injury. Tissue injury stimulates the normally quiescent tissue stem cells to differentiate and proliferate, in the process of replacing and/or repairing the damaged cells, and hence effecting tissue regeneration. The salivary glands retain the ability for frequent regeneration. Previously, we isolated progenitor cells from the injured salivary glands of mice and rats that differentiated into hepatic and pancreatic lineages. The isolated progenitors were CD49f-positive and intracellular laminin-positive, and proliferated on type I collagen while maintaining their multipotency. In this study, we analyzed the tissue stem cells induced by ligating the main excretory duct of the salivary gland in swine. After duct ligation of the gland, acinar cells receded due to apoptosis, and epithelial cells subsequently proliferated. We cultured cells obtained from the duct-ligated salivary gland and purified the cells by limited dilution. The isolated cells were positive for CD29, CD49f, intracellular laminin, AFP, CK19, CK18, and Thy-1(CD90), and weakly positive for c-Kit (CD117). After three-dimensional formation, the cells expressed insulin and albumin. We designated the cells as swine salivary gland-derived progenitor cells. Gene expression of insulin and albumin was significantly increased (five-fold) and that of insulin was also increased (3.8-fold) with differentiation medium with nicotinamide and/or GLP-1 treatment in spherical culture. The expressions of albumin and insulin were 1/10-fold and 1/4-fold compared to porcine hepatocytes and pancreatic endocrine cells. The differentiated SGP cells could release insulin, which were stimulated by glucose and potassium. These results indicate that swine SGP cells could differentiate into hepatocytes and beta-cells, functionally. Swine SGP cells were useful tools for therapy and analyzing endodermal regenerative models in large animals.     

体外培养的人牙胚上皮细胞的成釉分化

人表皮干细胞可以作为牙齿再生中上皮源性的种子细胞,但是其成釉分化的效率低下. 本研究分离培养了人牙胚上皮细胞,利用E13.5的小鼠牙间充质与其重组,构建重组牙胚,对其成釉分化的潜能和机制进行研究. 研究结果发现,体外培养的P1代人牙胚上皮细胞成釉率高达50%. 随着传代次数的增加,成釉率明显下降. 通过对牙上皮发育分化相关基因的表达检测和分析表明,重组牙胚成牙分化能力和成釉潜能的下降与牙上皮发育相关基因的表达状态密切相关. 特别是FGF8表达水平的下调以及PITX2不同亚型在人牙胚细胞中表达量的不均衡,可能是导致人牙胚细胞成釉潜能下降并丧失的主要原因. 本研究结果为理解牙齿再生过程中上皮源性的种子细胞的成釉机制提供了新的实验数据,对进一步提高表皮干细胞在牙齿再生过程中的成釉率有指导意义.     

胎儿胰岛样细胞团源上皮样细胞分离、纯化和鉴定

旨在优化胰腺干细胞分离、鉴定的方法和体系,为糖尿病研究和治疗提供种子细胞。采用胶原酶消化法,分离培养出胰岛样细胞团(ICCs),对其进行贴壁培养,从中纯化出上皮样细胞。采用MTT法测定其生长情况并绘制生长曲线。采用免疫组织化学染色检测分离得到细胞的PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin、Insulin表达情况。应用流式细胞仪检测其表面标志。由分离培养的ICCs成功纯化出上皮样细胞;传40代,每代冻存106~108个细胞;生长曲线显示其传代第3天进入对数生长期,第5天进入平台期;免疫组织化学染色显示其表达PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin;不表达Insulin;流式细胞仪分析表明其表达CD29、CD44、CD166,不表达CD11a、CD14、CD34、CD45、CD90、CD105、CD117。由胎儿胰腺能分离出具有自我更新能力的上皮样细胞,为导管来源,具干细胞特性。     

Human amniotic epithelial cells inhibit activation and pro-inflammatory cytokines production of naive CD4+ T cells from women with unexplained recurrent spontaneous abortion

Unexplained recurrent spontaneous abortion (URSA) has been assumed to be caused by a defect in maternal immunological tolerance to the fetus. Human amniotic epithelial cells (hAECs) have stem cell-like features and the ability to modulate the innate and adoptive immune responses. This study aimed to investigate whether hAECs have immunomodulatory effects on naive CD4+ T cells from URSA patients. hAECs were obtained from 15 healthy pregnant women and phenotypic profile of hAECs was determined by flow cytometry. Naive CD4+ T cells were isolated from 25 URSA patients using an immunomagnetic separation method. Naive T cells were stimulated with anti-CD3/anti-CD28 antibodies and co-cultured with different numbers of hAECs for 3 and 6 days. Immunomodulatory effect of hAECs on activation of stimulated T cell was assessed by flow cytometry and Enzyme-linked immunoasorbent assay (ELISA). The hAECs effect on pro-inflammatory cytokines production of activated T cells was also measured by ELISA. Our results indicated that hAECs significantly inhibited the activation of naive T cells in a dose-dependent manner (p?<?0.0001–0.05). They significantly reduced the production of transforming growth factor-beta1 (TGF-β1) of stimulated CD4+T cells (p?<?0.0001–0.05). Moreover, hAECs had potent immunomodulatory effects on the production of interferon-gamma (IFN-γ) and interleukin-17A (IL-17A) of activated T cells (p?<?0.0001–0.01). These findings suggest that hAECs may be a suitable cell source to modulate abnormal immune responses in women with URSA.