Bursal and postbursal cells in chicken. Separation by gradient centrifugation

Lymphoid cells of bursa of Fabricius taken from 2 1/2-week old chickens were separated in a discontinuous albumin gradient into six fractions. At this age, the bursa contains bursal stem cells and early postbursal cells; only the bursal stem cells are capable of restoring the bursal morphology of cyclophosphamide-treated recipients. Cells from each fraction of the albumin gradient were injected into 4-day old cyclophosphamide-treated, histocompatible recipients. Forty days after the cell transplantation, microscopic morphology of bursa and spleen, and antibody formation to sheep red blood cells and Brucella abortus were studied. The low density bursa cells were found capable of a significant restoration of the bursal morphology, while those with a higher density had significantly less effect on the bursal morphology. When bursa cells from 10-week old donors were used, no clear fractionation was obtained according to the capacity to restore the bursal morphology of cyclophosphamide-treated recipients. These findings indicate that the bursal stem cells capable of restoring bursal structure of cyclophosphamide-treated chicks are, in average, lighter in density than the postbursal cells.     

Bursal and postbursal cells in chicken. Occurrence of postbursal cells in bone marrow, thymus and spleen

To study the postbursal nature of lymphoid cells found in the bone marrow, thymus and spleen, their capacity for a long-term restoration of bursa-dependent immune functions and of bursal and splenic morphology was evaluated using cyclophosphamide-treated chicks as cell recipients. The results reveal that postbursal cells appear in significant numbers, first in the spleen and then in the bone marrow and thymus. This observation is interpreted to indicate that migration of postbursal cells from the bursa to bone marrow and thymus most probably occurs through the spleen. The findings also indicate that in the development of the B cell line an early postbursal cell and a mature postbursal cell can be distinguished. Both of these cell types are capable of further function without the influence of a bursal microenvironment. The mature postbursal cell does not have a capacity for formation of germinal centers in significant numbers in the spleen, but the early postbursal cell still shares this potential with its precursor, the bursal stem cell. They are also distinguishable by a slight proliferation of the early postbursal cells in the bursa when transferred into cyclophosphamide-treated newly-hatched recipients. Both of these cell types represent distinct entities in the stepwise development of the B cell line.     

Bursal and thymic reticular epithelial cells in the chicken: preparation of in vitro monolayer cultures

To study the nature of reticular epithelial (REp) cells and their role in the specific microenvironments of the chicken bursa and thymus, a method was developed for the in vitro culture of purified preparations of these cells. For comparison, similar cultures of splenic adherent cells were also performed. REp cell-rich bursa medullary follicles and mildly trypsinized thymic fragments were X-irradiated (850 rad) to eliminate remaining lymphocytes and transferred to culture flasks. In bursal cultures, after 2-4 days incubation the basement membrane (BM) encapsulating the follicles disrupted and the immediately underlying epithelial cells grew out as a monolayer. By 10 days, REp cells at the periphery developed cytoplasmic processes; occasionally these cells appeared to "bud-off" and grow as isolated dendritic cells. Thymic REp cells were generally slower to proliferate but formed a monolayer by 10-14 days. Splenic adherent cells developed extensive growth within 4 days. REp cells were distinguished from fibroblasts, when present, morphologically and by their limited phagocytic ability. The former were also periodic acid-Sciffs reagent (PAS)-positive and produced reticulin granules. Bursal REp cells were also positive for a gut-associated mucin, but this may have been bound in vivo prior to culture. Neither T nor B lymphocyte-specific antigens were detectable on the cultured REp cells or splenic adherent cells, but they were all rich in cytoplasmic actin. A major feature of REp cells to emerge in this study was the obvious presence of subpopulations of these cells, which raises important questions as to their exact nature and lineage. The accompanying paper details the ability of the bursal and thymic REp cell cultures to induce B-or T-lymphocyte differentiation, respectively, in vitro.     

表皮干细胞与毛囊干细胞生物学特性的比较

背景：获取更合适的组织工程皮肤种子细胞可使皮肤功能得到更好的修复。 目的：分离、培养表皮干细胞和毛囊干细胞,并比较两种细胞的生物学特性。 方法：体外分离培养2月龄新西兰兔表皮干细胞和毛囊干细胞,取生长良好的第2,3,6代细胞观察其生物学特性。 结果与结论：毛囊干细胞较表皮干细胞贴壁快,具有更高的增殖活性。免疫组化及荧光定量PCR检测显示毛囊干细胞β1整合素、角蛋白19蛋白及mRNA表达均明显高于表皮干细胞。提示作为皮肤组织工程的种子细胞,毛囊干细胞较表皮干细胞更具优势。     

Functional nicotinic and muscarinic receptors on mesenchymal stem cells

Mesenchymal stem cells (MSCs) are under the control of a large number of signaling systems. In this study, the presence and functionality of the acetylcholine (ACh) signaling system in MSCs was examined. We detected the expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the presence of ACh in MSCs. MSCs also expressed the nicotinic acetylcholine receptor subunits alpha 3, alpha 5, alpha 7, and the muscarinic acetylcholine receptor 2 (M2-receptor). The M2-receptor and the nicotinic alpha 7 receptor subunits were expressed on distinct subpopulations of cells, indicating differential regulation of cholinergic signaling between MSCs. Stimulation of MSCs with the nicotinic receptor agonist nicotine and the muscarinic receptor agonist muscarine induced immediate and transient increases in intracellular Ca(2+) concentration. Furthermore, muscarine had an inhibiting effect on the production of the intracellular signaling molecule cyclic adenosine 3',5'-monophosphate (cAMP). The AChE inhibitor chlorpyrifos, which is widely used as an agricultural insecticide, had similar effects on intracellular Ca(2+) and cAMP in MSCs. Nicotine, muscarine, and chlorpyrifos induced the phosphorylation of extracellular signal-regulated kinases 1 and 2. This study demonstrates that several components of a cholinergic signaling system are present and functional in MSCs. Environmental compounds such as nicotine and agricultural insecticides can interfere with this system and may affect cellular processes in the MSC.     

表皮干细胞的生物学特性及应用

背景：尽早去除创面坏死组织、重建皮肤结构和功能,是大面积深度烧伤治疗的关键和最终目标。表皮干细胞作为皮肤组织特异性干细胞,拥有无可置疑的潜能。 目的：总结表皮干细胞的生物学特性及应用现状。 方法：应用计算机检索2000-01/2010-10 PubMed数据库相关文章,检索词“epidermal stem cells,basic study”,并限定文章语言种类为English。同时计算机检索2005-01/2010-10万方数据库相关文章,检索词为“表皮干细胞”,并限定文章语言种类为中文。共检索到文献271篇,最终纳入符合标准的文献38篇。 结果与结论：表皮干细胞是具有多向分化潜能、自我更新能力和高度增殖能力的细胞,具有慢周期性和对基底膜的黏附能力,其增殖分化受到壁龛及Wnt信号通路、MAPK、c-Myc、Notch信号传导通路、细胞因子等的调控。目前尚无公认的特异性标志物,可用于创面修复、基因治疗、组织工程领域。随着表皮干细胞研究的深入,将为皮肤基础研究、创面功能修复和皮肤遗传病的治疗提供新途径。     

Spermatogonial stem cells: characteristics and experimental possibilities

The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells. They are the only cell type that can repopulate and restore fertility to congenitally infertile recipient mice following transplantation. Although numerous expression markers have been helpful in isolating and enriching spermatogonial stem cells, such as expression of THY-1 and GFRalpha-1 and absence of c-kit, no specific marker for this cell type has yet been identified. Much effort has been put into developing a protocol for the maintenance of spermatogonial cells in vitro. Recently, coculture systems of testicular cells on various feeder cells have made it possible to culture spermatogonial stem cells for a long period of time, as was demonstrated by the transplantation assay. Even expansion of testicular cells, including the spermatogonial stem cells, has been achieved. In these culture systems, hormones and growth factors are investigated for their role in the process of proliferation of spermatogonial stem cells. At the moment the best culture system known still consists of a mixture of testicular cells with about 1.33% spermatogonial stem cells. Recently pure SV40 large T immortalized spermatogonial stem cell lines have been established. These c-kit-negative cell lines did not show any differentiation in vitro or in vivo. A telomerase immortalized c-kit-positive spermatogonial cell line has been established that was able to differentiate in vitro. Spermatocytes and even spermatids were formed. However, spermatogonial stem cell activity by means of the transplantation assay was not tested for this cell line. Both the primary long-term cultures and immortalized cell lines have represented a major step forward in investigating the regulation of spermatogonial self-renewal and differentiation, and will be useful for identifying specific molecular markers.     

Functional markers and the 'homogeneity' of human mesenchymal stem cells

Stem cells are characterized by the abilities to self-renew, generate large numbers of progeny and differentiate into at least one mature cell type. Bone marrow serves as a reservoir for several classes of adult stem cells. In addition to haematopoietic stem cells (HSCs), which can reconstitute the haematopoietic system of a myeloablated host, bone marrow contains a diverse population of marrow stromal cells ( Herzog et al. 2003 ). Included among these are mesenchymal stem cells (MSCs), which ex vivo can be isolated as a relatively homogeneous and undifferentiated cell population that produces multiple mature cell types including fat, bone and cartilage ( Pittenger et al. 1999 ). Under appropriate, but ill-defined conditions, human MSCs (hMSCs) can also differentiate into other cell types, including excitable cells with neuronal-, myogenic and cardiomyogenic-like phenotypes. Because MSCs are multipotent and readily expandable in vitro , these cells have already been employed in early clinical studies, including the treatment of human myocardial infarction. Transplantation of autologous or allogeneic MSCs therefore represents a novel form of cellular therapy, which shows substantial promise for the treatment of a number of human diseases.     

鸡胚胎干细胞分离方法和培养体系的优化

背景：胚胎干细胞是从动物早期胚胎的内细胞团或原始生殖细胞分离出来的具有发育全能性的一种未分化的无限增殖细胞系。而鸡胚胎干细胞则是从X期鸡胚的胚盘分离而来。 目的：优化鸡胚胎干细胞分离方法和离体培养体系。 方法：采用滤纸纸环-发环的方法从X期鸡胚分离胚盘细胞,并采用STO细胞作为饲养层和大鼠肝细胞(BRL)条件培养基(CM)+细胞因子作为离体培养体系对分离的胚盘细胞进行培养。 结果与结论：滤纸纸环-发环法获得的完整胚盘率为75%~85%,克隆形成率约为50%。BRL-CM+饲养层培养体系,鸡胚胎干细胞可传至7代,而BRL-CM+饲养层+细胞因子培养体系,鸡胚胎干细胞可传至25代。分离到的鸡胚胎干细胞,经碱性磷酸酶染色、SSEA-1染色鉴定,表明鸡胚胎干细胞处于未分化状态。提示,实验不仅优化了鸡胚胎的分离方法,获得完整且杂质少的胚盘,而且进一步优化了鸡胚胎干细胞体外培养体系。     

Susceptibility of chicken mesenchymal stem cells to infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the causative agent of one of the most important viral diseases affecting the poultry industry worldwide. The virus causes an acute, highly contagious and immunosuppressive disease in chickens. Previous studies have demonstrated that in addition to B cells, macrophages can support the replication of IBDV. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of hematopoietic precursors, the interaction between IBDV and mesenchymal stem cells was investigated. Mesenchymal stem cells were isolated from chicken bone marrow. The classical IM strain and the variant strain-E of IBDV, both adapted to grow in a chicken macrophage cell line, were used to infect mesenchymal stem cells. Primary chicken mesenchymal stem cells were highly susceptible to replication of IBDV. Both viruses induced cytopathic effects and replicated to high titers in mesenchymal stem cells. The finding that IBDV can replicate in mesenchymal stem cells provides new information on the susceptible target cell population within the host and contributes to the understanding of the pathogenic potential of the virus.     

Functional neural stem cells derived from adult bone marrow

Pluripotent hematopoietic cells from adult bone marrow may give rise not only to neurons, oligodendrocytes and astrocytes after transplantation into newborn brains, but also to neural stem cells (NSC). These NSC localize to both the ventricular epithelium and subventricular zone, persist in the transplanted brain, and may generate neurospheres 1 month after transplant, which after in vitro expansion differentiate into the different neural lineages. Furthermore, the bone marrow-derived NSC differentiate in vivo into functional oligodendrocytes and neurons following demyelinating lesions, thus, demonstrating the ability of adult bone marrow progenitors to generate self-renewing, functional neural stem cells, validating this approach as an alternative source of long-lasting neural stem cells with therapeutic implications in neurodegenerative diseases.     

Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells

Recent results have shown the ability of bone marrow cells to migrate in the brain and to acquire neuronal or glial characteristics. In vitro, bone marrow-derived MSCs can be induced by chemical compounds to express markers of these lineages. In an effort to set up a mouse model of such differentiation, we addressed the neuronal potentiality of mouse MSCs (mMSCs) that we recently purified. These cells expressed nestin, a specific marker of neural progenitors. Under differentiating conditions, mMSCs display a distinct neuronal shape and express neuronal markers NF-L (neurofilament-light, or neurofilament 70 kDa) and class III beta-tubulin. Moreover, differentiated mMSCs acquire neuron-like functions characterized by a cytosolic calcium rise in response to various specific neuronal activators. Finally, we further demonstrated for the first time that clonal mMSCs and their progeny are competent to differentiate along the neuronal pathway, demonstrating that these bone marrow-derived stem cells share characteristics of widely multipotent stem cells unrestricted to mesenchymal differentiation pathways.     

Functional and phenotypic characteristics of bovine natural cytotoxic cells

Bovine natural cytotoxic (NC) cell activity of peripheral blood leukocytes (PBL) was studied in a chromium release assay, using xenogeneic tumor cells (YAC-1, P815) and herpes virus-infected bovine fibroblasts. The activity pattern resembled that of murine natural killer cells, in that YAC-1 cells were readily lysed, whereas P815 cells were resistant. However, 10-16 h of incubation were usually required to give appreciable cell lysis. Low, but consistent NC-activity was expressed against virus-infected fibroblasts. Using various biophysical and biochemical cell-separation methods, it was found that the effector cell active against both the xenogeneic tumor cells and virus-infected fibroblast belonged to the mononuclear phagocyte system. The indications are that at least two subpopulations of the blood monocytes, differing perhaps in maturation or clonal derivation, express NC-activity.     

鸡精原干细胞分离培养的影响因素

目的:比较不同分离方法和培养体系对鸡精原干细胞(SSCs)生长的影响。方法:采用3种分离方法、6种培养体系对SSCs进行分离培养。结果:胶原酶 胰蛋白酶组合消化睾丸后获得的平均活细胞率显著地高于其余两种方法。在有和无饲养层细胞存在的条件下,SSCs在DMEM培养液中存活的时间分别为6.5 d和45.5 d,显著地长于在TCM199和RRMI1640中。在6种培养体系中,SSCs在有饲养层体系Ⅳ中存活时间和碱性磷酸酶(AKP)阳性克隆率分别为(45.5±3.2)d和31%±16%,显著地高于其他5种培养体系。SSCs在有饲养层体系Ⅳ中传代传至1、2和3代时,AKP阳性克隆率分别为31.6%、20%和18%。SSCs形成的克隆,经SSEA-1免疫染色,均为阳性。传代至第3代的SSCs,其染色体组型保持不变。结论:采用二酶组合法分离获取SSCs,而以DMEM为基础液形成的有饲养层体系Ⅳ较为适宜。     

小鼠诱导型多潜能干细胞定向分化为功能性肝细胞

目的:探讨在体外诱导型多潜能干细胞(IPS)能否遵循正常肝脏发育途径定向分化为具备良好生理功能的肝细胞。方法:根据正常肝脏体内发育的规律,序贯应用肝脏发育所需的生长因子,设计特色培养基,诱导IPS细胞遵循正常发育途径定向高效分化为肝细胞:首先将IPS细胞定向分化为前层确定性内胚层细胞(ADE),再进一步分化为肝细胞。结果:在本研究中IPS细胞在体外可循正常发育通路定向诱导为肝细胞:先分化为前层内胚层细胞,再继续分化为肝细胞。分化的各时段实时定量RT-PCR检测显示:在mRNA表达的水平,IPS细胞经历了肝细胞在体内从胚胎干细胞的发育过程,内胚层细胞特异性RNA如CXCR4、Sox17和FOXA2在分化的早期明显升高,而早期肝细胞特异性RNA如HNF4和AFP在分化的中期开始升高,肝细胞成熟特异性RNA如白蛋白在分化的晚期才达高峰,同时IPS细胞的未分化标记RNA如OCT4在早期就明显下调。同mRNA表达一致,在分化的第20d,大部分的肝脏细胞表达甲胎蛋白和白蛋白(阳性率80%)。更为重要的是IPS来源肝细胞在体外具有典型成熟肝细胞的功能,能够摄取和释放靛青绿(ICG)并具有药物代谢酶细胞色素P450酶的活性。结论:IPS细胞在体外能够遵循正常肝脏发育通路,序贯表达肝脏发育各阶段的特色基因和蛋白,能够高效分化为有功能的肝细胞,本研究可能为临床终末期肝病进行细胞治疗提供肝细胞。     

间充质干细胞的生物学特性及临床应用

背景：间充质干细胞的临床应用是干细胞移植研究的热点,但其安全性和有效性仍存在争议。 目的：回顾总结近年来对于间充质干细胞的生物学特性及临床应用前景的研究。 方法：计算机检索Pubmed(1990年1月至2013年11月)和中国知网数据库（2011年至2013年）,检索关键词为“间充质干细胞;分离培养;表面抗体;诱导分化;临床应用”。 结果与结论：间充质干细胞可表达多种表面抗原,但没有特异性,同时其具有自我更新和向骨细胞、脂肪细胞、软骨细胞、骨骼肌细胞和神经细胞等的多向分化能力。由于其多向分化潜力,且能分泌多种细胞因子,还具有免疫调节能力,已成为再生医学、血液学和免疫学方面细胞疗法的候选细胞。间充质干细胞临床试验已处于第三期,尽管数据还不多,已发现存在利于肿瘤生长和转移、增加侵袭性真菌感染等方面问题。即使如此,其在血液系统恶性肿瘤等疾病面方面仍存在良好的应用前景。所以,找出间充质干细胞在体内的作用原理及修复其产生的不良影响是目前干细胞移植研究的重点。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;组织工程全文链接：