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1.
The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Bone marrow, umbilical cord and amniotic fluid samples were harvested from different horses. Cells were analyzed for proliferation, immunocytochemical, stem cell gene expression and multilineage plasticity. BM- and AF-MSCs took similar time to reach confluence and showed comparable plating efficiency. All cell lines expressed identical stem cell markers and capability to differentiate towards osteogenic lineage. Almost all cell lines differentiated into the adipogenic lineage as demonstrated by cytochemical staining, even if no adipose gene expression was detectable for AF-MSCs. AF- and EUC-MSCs showed a limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. These findings suggest that AF-MSCs appeared to be a readily obtainable and highly proliferative cell line from an uninvasive source that may represent a good model system for stem cell biology. More studies are needed to investigate their multilineage potential. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation.  相似文献   

2.
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3.
Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.  相似文献   

4.
The use of adult stem cells in tissue regeneration appears to be a powerful research tool, due to the intrinsic characteristics of these cells, i.e., self-renewal and unlimited capacity for proliferation. In particular, mesenchymal stem cells (MSCs) obtained from bone marrow or peripheral blood can be easily isolated, cultivated, propagated and can be differentiated into several specialized cell types thanks to their plasticity. Among these cells, MSCs can evolve into cardiac cell lineages. Since heart damage leads to the irreversible loss of cardiac function, cell transplantation could be a potential therapy for heart injury. Our laboratory has focused on the purification and expansion of rat and sheep MSCs, their differentiation into cardiomyocytes and their characterisation. Numerous results indicate that MSCs could be promising for therapy, however we need to better understand the biology of stem cells to improve methods for delivery and/or pharmacological activation. These techniques can indeed track engrafted cells and systems to guarantee their safe use.  相似文献   

5.
Embryonic stem (ES) cells, derived from the inner cell mass of a blastocyst, are believed to pluripotent cells and give rise to embryonic, but not extraembryonic, tissues. In mice, totipotent 2-cell stage embryo-like (2-cell-like) cells, which are identified by reactivation of murine endogenous retrovirus with leucin transfer RNA primer (MuERV-L), arise at a very few frequencies in ES cell cultures. Here, we found that a lipid droplet forms during the transition from ES cells to 2-cell-like cells, and we propose that 2-cell-like cells utilize a unique energy storage and production pathway.  相似文献   

6.
Stem cells have an intrinsic capacity to self-renew and can differentiate to at least one specialized cell type. Different types of stem cells exist that can be cultured in vitro. The identity of the stem cells is marked by their origin and differentiation potential. Germ cells have similarities with pluripotent stem cells but are of a special order: They do not self-renew and are already differentiated, but they have the capacity to form a complete new organism after fertilization. This review focuses on pluripotent stem cells and discusses possibilities of generating pluripotent stem cells from germ cell precursors and possibilities of generating germ cells from stem cells. As it accompanies a plenary lecture at the 15th annual ESDAR Conference 2011, the overview is focused on stem cells from farm animal species and on results from my own research group.  相似文献   

7.
Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. Murine ES cells are able to reintegrate fully into embryogenesis when returned into an early embryo, even after extensive genetic manipulation. In the resulting chimeric offspring produced by blastocyst injection or morula aggregation, ES cell descendants are represented among all cell types, including functional gametes. Therefore, mouse ES cells represent an important tool for genetic engineering, in particular via homologous recombination, to introduce gene knock-outs and other precise genomic modifications into the mouse germ line. Because of these properties ES cell technology is of high interest for other model organisms and for livestock species like cattle and pigs. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have yet been established for vertebrate species other than the mouse (Evans and Kaufman, 1981; Martin, 1981) and chicken (Pain et al., 1996). The in vitro differentiation capacity of ES cells provides unique opportunities for experimental analysis of gene regulation and function during cell commitment and differentiation in early embryogenesis. Recently, pluripotent stem cells were established from human embryos (Thomson et al., 1998) and early fetuses (Shamblott et al., 1998), opening new scenarios both for research in human developmental biology and for medical applications, i.e. cell replacement strategies. At about the same time, research activities focused on characteristics and differentiation potential of somatic stem cells, unravelling an unexpected plasticity of these cell types. Somatic stem cells are found in differentiated tissues and can renew themselves in addition to generating the specialized cell types of the tissue from which they originate. Additional to discoveries of somatic stem cells in tissues that were previously not thought to contain these kinds of cells, they also appear to be capable of developing into cell types of other tissues, but have a reduced differentiation potential as compared to embryo-derived stem cells. Therefore, somatic stem cells are referred to as multipotent rather than pluripotent. This review summarizes characteristics of pluripotent stem cells in the mouse and in selected livestock species, explains their use for genetic engineering and basic research on embryonic development, and evaluates their potential for cell therapy as compared to somatic stem cells.  相似文献   

8.
The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.  相似文献   

9.
Reasons for performing study: Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood‐derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. Objectives: To optimise cell culture conditions for isolation, propagation and differentiation of adult stem cells from peripheral blood and to assess the effect of hyperbaric oxygen treatment on adult stem cell concentrations. Methods: Peripheral blood was collected from the jugular vein of 6 research mares, and mononuclear cells were isolated. They were subjected to cell culture conditions that promote the adherence and proliferation of adult stem cells. The cells were characterised by their adherence, expression of cellular antigen markers, and trans‐differentiation. Each horse was subjected to 3 hyperbaric oxygen treatments, and stem cells were compared before and after treatments. Stem cells derived from adipose tissue were used as controls. Results: One‐third of the horses yielded viable stem cells from peripheral blood, positive for CD51, CD90 and CD105, and demonstrated osteocyte, chondrocyte and adipocyte differentiation. Hyperbaric oxygen treatment resulted in a significant increase in CD90‐positive cells. Horses that did not yield any cells pretreatment did so only after 3 hyperbaric oxygen treatments. Conclusions and potential relevance: Peripheral blood can be a valuable source of adult stem cells, if one can identify reliable equine‐specific markers, provide methods to increase the number of circulating progenitor cells and optimise cell culture conditions for growth and viability. Our findings are important for further studies towards technological advances in basic and clinical equine regenerative medicine.  相似文献   

10.
In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.  相似文献   

11.
Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells, which can differentiation into cells of connective tissue and neural lineages. This study investigated the potential for neuronal differentiation of red fluorescent protein (RFP)-transgenic cat UCB-derived MSCs. The cells were cultured in pre-induction medium for 24 hr and in neuronal-induction medium for 72 hr. Immunofluorescent staining showed that 6.85% of the total cells were beta III-tubulin-positive, 3.37% were neurofilament light (NF-L)-positive and 7.04% were neurofilament medium (NF-M)-positive. A beta III-tubulin band was detected by western blot analysis. Our results demonstrate that RFP-transgenic UCB-derived MSCs can be differentiated into neuronal cells in vitro. Thus, RFP-transgenic MSCs could provide alternative tracing material for stem cell transplantation.  相似文献   

12.
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may have a wide range of applications in cell and gene therapy. However, the safety issues and the low efficiency associated with generating human iPSCs have limited their usage in clinical settings. The cell type used to create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid cells from the prenatal diagnosis of a β-thalassemia patient can be efficiently reprogrammed using a doxycycline (DOX)-inducible humanized version of the single lentiviral "stem cell cassette" vector flanked by loxP sites, which can be excised with Cre recombinase. We also demonstrated that the patient-derived iPSCs can be characterized based on the expression of pluripotency markers, and they can be differentiated into various somatic cell types in vitro and in vivo. Moreover, microarray analysis demonstrates a high correlation coefficient between human β-thalassemia iPS cells and human embryonic stem (hES) cells but a low correlation coefficient between human β-thalassemia amniotic fluid cells and human β-thalassemia iPS cells. Our data suggest that amniotic fluid cells may be an ideal human somatic cell resource for rapid and efficient generation of patient-specific iPS cells.  相似文献   

13.
Somatic cell nuclear transfer, the first established technique for producing patient-specific autologous stem cells, inevitably requires the sacrifice of viable embryos. To circumvent the serious ethical issues associated with this use of embryos, researchers have developed several alternative methods for the production of histocompatible stem cells. In our research, we have used two methods to derive histocompatible stem cells from murine ovarian tissue. First, we have established autologous stem cells by culturing degeneration-fated preantral follicles to produce developmentally competent, mature oocytes and then parthenogenetically activating these mature oocytes to acquire genetic homogeneity. Second, we have used cell-to-cell interactions to derive stem cells from ovarian stromal cells without undertaking genetic modification. We have successfully derived autologous murine stem cells by manipulating primary and early secondary follicles in vitro, and this method has proved successful even for follicles retrieved from aged ovaries. Furthermore, we believe that it will be possible to isolate stem cells directly from non-germline ovarian tissue or to derive stem cells by culturing the ovarian cells with other somatic cells. If achieved, these aims will greatly advance the development of induced pluripotent stem cell technology, as well as tissue-specific stem cell research. In this review, we introduce the relevant technologies for establishing histocompatible stem cells from ovarian tissue cells without undertaking genetic manipulation and review the current limitations of, and future research directions in, stem cell biology.  相似文献   

14.
滋养层干细胞(TSc)是形成胎盘组织细胞的前体细胞,与胚胎着床和胎盘形成密切相关。体外分离滋养层干细胞为研究滋养层的发育与功能提供了研究工具和基础。滋养层干细胞已经成功从小鼠附植前囊胚中获得,在猪上也有相关报道,但并没有关于6 d囊胚中分离到猪滋养层干细胞(pTSc)的报道。本试验通过对6 d孤雌囊胚透明带进行划口处理, 饲养层和基础培养液中附加碱性成纤维生长因子(bFGF)和人白血病抑制因子(hLIF)分离猪滋养层干细胞样细胞。同时,通过采用形态学和基因表达分析的方法对获得的猪滋养层干细胞样细胞进行了初步鉴定。结果显示,本试验分离得到的细胞呈现上皮样细胞形态,细胞间结合紧密,边缘光滑,核质比较大,RT-PCR结果显示表达滋养层干细胞标记基因Cdx2,体现了滋养层干细胞特征。结果表明,本试验成功在6 d孤雌囊胚分离得到猪滋养层干细胞样细胞,为后续研究猪滋养层发育提供了试验基础。  相似文献   

15.
乳腺是一个高度活跃的器官,在青春期和生殖周期中,其上皮细胞发生了极大的变化。这些变化是由专门的干细胞和祖细胞推动的。研究乳腺干细胞对动物乳腺发育、哺乳和乳腺癌等方面都有着重大意义。如今,在乳腺干细胞生物学研究方面已经取得了显著进步。乳腺干细胞是组织学上未分化的上皮细胞,可以对称分裂产生两个相同的干细胞,或不对称地产生一个干细胞和一个腔上皮祖细胞或基底/肌上皮祖细胞。通过标记滞留细胞、染料排斥法、干细胞抗原-1标记、细胞表面标记物标记以及谱系示踪等方法可以鉴定出多种不同类型的乳腺干/祖细胞,应用多种不同方法分离鉴定乳腺干细胞有助于深入了解其异质性。乳腺的研究主要分为6个阶段：胚胎期、青春期、性成熟期、妊娠期、泌乳期和退化期。在乳腺不同发育阶段,乳腺干/祖细胞的类型及特性不同。有研究表明,在胚胎期就有基底和管腔谱系的祖细胞产生,静止的乳腺干细胞也可能来自于胚胎期,出生后,静止干细胞可以在卵巢激素的刺激作用下重新进入细胞周期,产生乳腺细胞谱系和自我更新。作者介绍了乳腺干细胞和祖细胞的存在、形态特征、鉴定方法,简述了乳腺干细胞在胚胎期、青春期和妊娠期的特征,分析了目前该领域研究的不足之处以及对未来应用的展望。  相似文献   

16.
Accumulation of intramuscular adipose tissue (IMAT) and development of fibrous tissues due to accumulation of collagen both affect meat quality such as tenderness, texture, and flavor. Thus, it is important for the production of high‐quality meat to regulate the amount of adipose and fibrous tissues in skeletal muscle. IMAT is comprised of adipocytes, while collagens included in fibrous tissues are mainly produced by activated fibroblasts. Both adipocytes and fibroblasts are differentiated from their common ancestors, called mesenchymal progenitor cells (MPC). We previously established rat MPC clone, 2G11 cells. As several reports implicated the plasticity of fibroblast differentiation, in the present study, using 2G11 cells, we asked whether myofibroblasts differentiated from MPC are capable of re‐gaining adipogenic potential in vitro. By treating with bFGF, their αSMA expression was reduced and adipogenic potential was restored partially. Furthermore, by lowering cell density together with bFGF treatment, 2G11 cell‐derived myofibroblasts lost αSMA expression and showed the highest adipogenic potential, and this was along with their morphological change from flattened‐ to spindle‐like shape, which is typically observed with MPC. These results indicated that MPC‐derived myofibroblasts could re‐acquire adipogenic potential, possibly mediated through returning to an undifferentiated MPC‐like state.  相似文献   

17.
The present study was performed to develop a culture system for feather keratinocyte stem cells to enable the genetic manipulation of endangered avian species. The feather follicle cells were isolated from growing feathers of adult White Leghorn chicken. Leukemia inhibitory factor (LIF) was used to maintain the characterization of the keratinocyte colony-forming cells (KCFCs). The EGFPN1 plasmid DNA retroviral vector was used to deliver Green Fluorescent Protein (GFP) gene, which was introduced to the KCFCs by lipofection. After removal of the fibroblast-like cells, the feather KCFCs attached to the substrate within 24 h of seeding. The cells continued to proliferate for at least 30 days in the presence of LIF. The cell-adhesion molecules such as integrin beta1 and CD49c were immunocytochemically positive in the cells. The KCFCs differentiated into barbular cells and pennaceous feather vane in the LIF-free medium. The GFP gene-transfected KCFCs stably expressed GFP. The present results indicate that the KCFCs derived from feather follicles are closely related to multipotent stem cells. In addition, gene manipulation of such stem cells may be useful for the production of chimera in avian species.  相似文献   

18.
Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC‐like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram both mouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additional studies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC‐driven bovine technologies can be realized.  相似文献   

19.
采用全骨髓培养法对野猪股骨中骨髓间充质干细胞（Bone mesenchymal stem cells,BMSC）进行分离、培养并传代,建立野猪骨髓间充质干细胞体外培养方法及对其生物学特性进行观察研究。结果显示,细胞形态呈梭形,漩涡状生长,生长曲线为典型S型,在诱导液作用下,可分化为成脂肪样细胞。结果表明,通过本方法能够分离到较纯的BMSC,为野猪资源保存和体细胞核移植提供技术支持。  相似文献   

20.
Adipose tissue‐derived stem cells (ASCs) can be obtained from different adipose tissue sources within the body. It is an abundant cell pool, easily accessible, suitable for cultivation and expansion in vitro and preparation for therapeutic approaches. Amongst these therapeutic approaches are tissue engineering and nervous system disorders such as spinal cord injuries. For such treatment, ASCs have to be reliably differentiated in to the neuronal direction. Therefore, we investigated the neural differentiation potential of ASCs using protocols with neurogenic inductors such as valproic acid and forskolin, while dog brain tissue served as control. Morphological changes could already be noticed 1 h after neuronal induction. Gene expression analysis revealed that the neuronal markers nestin and βIII‐tubulin as well as MAP2 were expressed after induction of neuronal differentiation. Additionally, the expression of the neurotrophic factors NGF, BDNF and GDNF was determined. Some of the neuronal markers and neurotrophic factors were already expressed in undifferentiated cells. Our findings point out that ASCs can reliably be differentiated into the neuronal lineage; therefore, these cells are a suitable cell source for cell transplantation in disorders of the central nervous system. Follow‐up studies would show the clinical benefit of these cells after transplantation.  相似文献   

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