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1.
采用大鼠心肌条件培养基(RH CM)培养ICR小鼠的桑椹胚和囊胚,发现由囊胚分离的ES细胞传代后ES集落的出现率显著高于桑椹胚(P<0.05),囊胚更适合作为ES细胞分离克隆的材料。以RH CM为培养基的试验组ES细胞传代的平均时间间隔为38 h,对照组传代的时间间隔平均为78 h,两者差异显著(P<0.05)。表明RH CM能够促进ES细胞贴壁增殖和ES集落的形成,有效地维持ES细胞未分化状态。试验中设计的3 种培养条件对原代ES集落的形成影响不显著,但对传代后的ES集落的形成和传代的代次有显著差异。其中以MEF作饲养层,添加RH CM培养基的效果最好。  相似文献   

2.
Pigs serve as a better research model for human beings than other species. The Chinese laboratory miniature pig is a new laboratory animal and is expected to be applicable in many medical research fields. This study was to establish effective technologies to isolate and culture ES cells in Chinese miniature pigs. For isolation of the inner cell mass from blastocysts, an enzyme-digestive method was compared with the traditional immunosurgery. Isolated ICM were cultured in three feeder cell layers: mouse embryonic fibroblasts (MEF), porcine embryonic fibroblasts (PEF) and a continuous cell line of mouse embryonic fibroblasts (STO). Microtubule activity of the three feeder cells was further examined by immunofluorescence. ICM were successfully isolated from 85% of blastocysts by the enzyme-digestive method, compared to only 40% by immunosurgery. When ICM were cultured in three feeder layers for two to three days, 75%, 65% and 20% of ICMs formed primary cell colonies in MEF, PEF and STO, respectively. Colonies were also formed during subcultures after 9, 5 and 1 passage in MEF, PEF and STO, respectively. Microtubules in STO cells were significantly fewer than those in MEF and PEF. When the ES-like cells were cultured in a differentiation medium, they differentiated to neuron-like cells and other types of cells. These results indicate that healthier ICM can be obtained with the enzyme-digestive method. Successful culture of ICM to ES-like cells has been achieved not only in MEF, but also in homologous (pig) feeder layer. The ES cells obtained in the present study were pluripotent.  相似文献   

3.
Summary

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400g). The use of 3% bovine leucocyte‐conditioned medium, produced by stimulation of blood lymphocytes with 4 pg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte‐conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose‐based medium requires different culture conditions, which are different from the culture conditions for human cells.  相似文献   

4.
Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels.  相似文献   

5.
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6.
The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.  相似文献   

7.
We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca2+ mobilization of the cells was evaluated using the Ca2+ indicator Fluo3 to monitor Ca2+ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca2+ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.  相似文献   

8.
The objective of this study was to establish the long-term in vitro culture system for chicken gonadal primordial germ cells (gPGCs). Primitive gonads collected from 5.5-day-old chicken embryos were dissociated and explanted onto plates pre-coated with 0.1% gelatin. Each of the four different conditioned media from proliferating and mitotically inactivated chicken embryonic fibroblast (CEF) cells and murine embryonic fibroblasts (STO cells, CRL-1053, ATCC, USA), respectively, was supplemented with growth factors and used to support the growth of gPGCs. The result showed that all the conditioned media could promote the growth and colony formation of gPGCs in vitro , in particular the medium conditioned by inactivated CEF cells. The gPGC-derived colonies maintained in inactivated CEF cells-conditioned medium up to 281 days were positively stained by periodic acid Schiff reaction and antibodies specific to anti-SSEA-1, SSEA-3, SSEA-4, integrin α6 and integrin β1. Their capacities of migration via vascular system and taking up residence in the primary gonadal ridge were further demonstrated by transferring to the dorsal aorta of stage 17 recipient embryos. These results suggested that our culture system is able to maintain chicken gPGCs for long-term in vitro culture without losing their capacity to express pluripotent markers and to integrate into the gonads.  相似文献   

9.
The aim of this study was to test the effect of insulin–transferrin–selenium (ITS) and L‐ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre‐pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus–oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post‐warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre‐pubertal goat small oocytes in GM would be useful to improve the quality of in vitro‐produced blastocysts.  相似文献   

10.
BackgroundCanine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking.ObjectivesIn this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation.MethodsTo induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested.ResultsMDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780.ConclusionsTaken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.  相似文献   

11.
小鼠皮肤成纤维细胞的体细胞核移植   总被引：1，自引：1，他引：0  
取成年小鼠唇部皮肤进行培养，分离成纤维细胞并血清饥饿培养1周，用作核供体。对成年小鼠进行超排，取卵母细胞用作核受体，核移植重构胚经SrCl2激活处理6h后，同mM16培养液和小鼠输卵管上皮细胞共培养，把发育到早期囊胚的重构胚转移至小鼠胎儿成纤维细胞饲养层上，添加ES细胞条件培养液，消化分离ICM，然后接种培养，对孵出的ES细胞样集落进行鉴定培养。结果显示，小鼠唇部皮肤成纤维细胞为核供体，核移植重构胚2-细胞率为54．05％，桑椹胚率17．14％，囊胚率6．90％，对照组卵丘细胞的核移植重构胚2-细胞率为60．00％，桑椹胚率21．85％，囊胚率11．69％，但2种供体细胞在支持核移植重构胚发育能力上差异不显著。成纤维细胞重构囊胚中6个囊胚分离出ES细胞样集落，3个ES细胞样集落可稳定传代；对照组卵丘细胞重构囊胚中9个囊胚中分离出ES细胞样集落，5个ES细胞样集落可稳定传代。从核移植重构胚中分离出的ES细胞样集落具有岛状或巢状群体生长形态，生长旺盛的集落可自发分化成单个散在或片状存在的上皮样或梭形细胞，碱性磷酸酶检测为阳性，常规冻存复苏，仍显示ES细胞特征。  相似文献   

12.
Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.  相似文献   

13.
Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.  相似文献   

14.
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15.
The generation of multinucleated giant cells (MGC) from cells of the bovine monocyte-macrophage lineage was investigated. Freshly isolated monocytes were incubated with the conditioned medium (CM) of peripheral blood mononuclear cell cultures treated with Concanavalin A for 1-4 days (CM1 to CM4). Only CM1 generated MGC despite similar concentrations of IFNgamma in all CMs. Nevertheless, MGC formation from monocytes was enhanced by adding either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), MGC formations from macrophages were observed only when macrophages were cultured with GM-CSF plus CM. These results indicate that several mechanisms to generate MGC from bovine monocytes-macrophage lineage cells exist, and that GM-CSF is a major mediator of MGC formation in cattle.  相似文献   

16.
Techniques for the development of ovine bone marrow-derived haemopoietic progenitor cells and in situ identification of colony morphology are described. Both mitogen stimulated lymphoid cells and antigen stimulated helper T-cells generated potent colony-stimulating activity in conditioned medium. Monocyte/macrophage, neutrophil, eosinophil, basophil/mast cell, neutrophil/monocyte and mixed phenotype colonies developed in stimulated bone marrow cultures in a conditioned medium dose-dependent manner. Neutrophil, monocyte/macrophage and eosinophil colonies were detected in greater numbers than the other types, with mixed colonies representing only around 1% of the total. Eosinophil colonies were particularly abundant when compared to published reports of the numbers obtained with similar cultures of 'normal' mouse or human bone marrow cells. This culture technique will allow a detailed analysis of both ovine colony-stimulating factors and of the distribution of haemopoietic progenitor cells in vivo.  相似文献   

17.
Calf bone marrow cells cultured in a semi-solid medium of 0.8% methyl cellulose produced colonies of granulocytic cells and macrophages by seven days. A prerequisite for colony growth was the presence of serum obtained from a calf three hours after intravenous injection of endotoxin. Three morphological types of colonies were seen but cell types within these types of colonies did not differ. Cultured cells were identified by morphological and cytochemical characteristics.

Optimum growth occurred when serum from endotoxin stimulated calves and fetal calf serum were present in a volumetric ratio of 7:3. Inhibition of colony growth occurred when endotoxin-stimulated serum was present at greater than optimum concentration. Normal calf serum, fetal calf serum, mouse L-cell conditioned medium and bovine urine did not stimulate significant colony growth when 8.0 x 104 marrow cells were cultured.

There was a linear relationhip between the number of marrow cells in the cultures and the number of colonies produced. Colony forming efficiency ranged from 13 to 59 colonies per 105 cells plated.

The behaviour of calf colony forming units in suspension culture was similar to that reported for mouse colony forming units.

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18.
This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague‐Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co‐cultured with autologous cumulus cells. We also experimented with co‐culture in 100 µL drops. Drop co‐culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co‐cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post‐transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co‐culture in Cook medium with cumulus cells compared to in vivo‐derived blastocysts. The advantage of the co‐culture system is in generating more blastocysts available for transfer.  相似文献   

19.
Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.  相似文献   

20.
《Veterinary microbiology》1998,61(3):191-197
The attachment to fully characterized primary rumen epithelial cell cultures of Escherichia coli strains isolated from different animal species and expressing F1–F4 or F17 fimbriae was examined. As the cell cultures contained stratified (keratinized) and non-stratified (non-keratinized) cells which grew either confluently or non-confluently, the strength of attachment of the different bacterial strains was assessed in relation to the differentiation state of the cells. Thus, strains having F1 fimbriae attached to all types of cultured cells, while strains with F2 and F3 fimbriae did not bind at all. E. coli strains having F4 or F17 fimbrae attached only to non-keratinized cells, particularly to confluent areas. As membrane glycosylation is known to change with differentiation (keratinization), our results suggest that the attachment of fimbriated E. coli strains which were capable of binding to rumen cells was more likely to be dependent on differentiation than the host specificity of the bacteria.  相似文献   

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