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.     

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.     

绵羊羊膜上皮细胞治疗兔桡骨缺损模型的研究

为了确定绵羊羊膜上皮细胞在体内向骨组织的分化能力,实验在分离培养绵羊羊膜上皮细胞并对其进行干细胞特性的鉴定的基础上,制作新西兰大白兔桡骨13mm骨缺损模型,随机分组对其进行注射绵羊羊膜上皮细胞实验。高剂量组:移植细胞5×107个;低剂量组:移植细胞5×106个;对照组:生理盐水。细胞移植后2、4、8周拍摄X光片观察骨缺损部位的缺损修复情况;相应时段取骨缺损部位新生骨进行组织学观察:分析骨小梁生成数量和骨的改建时期。实验结果显示,高剂量实验组在移入细胞第8周,骨缺损完全修复,且同期高剂量组新骨生成的数量和质量明显高于低剂量组,低剂量组优于对照组。由此可见,绵羊羊膜上皮细胞不仅可以在不同种动物间进行移植,而且对骨缺损有良好的修复能力。     

胚胎干细胞源性表皮干细胞在肾被囊微环境中分化潜能的研究

目的 探讨胚胎干细胞源性表皮干细胞在肾被囊微环境中的分化情况,为研究其在不同微环境中的分化潜能和稳定性及寻找新的皮肤工程种子细胞奠定基础。方法129小鼠E14胚胎干细胞与人羊膜共培养4d,定向诱导其分化为呈β1整合素、CK15和CK19阳性的表皮样干细胞克隆,无菌手术下移植入129小鼠双侧肾被囊内,术后4周、6周和8周取材。对移植后细胞的分化情况进行形态学和CEA和CK18免疫组织化学观察。结果小鼠胚胎干细胞源性表皮干细胞在129小鼠肾被囊内4周,分化为由单层或复层上皮样细胞构成的管状和泡状结构,种植6周和8周,除上述结构外,可见角化复层扁平上皮、汗腺样、皮脂腺样及毛囊样等结构。免疫组化结果汗腺样结构呈CEA和CK18阳性。结论研究结果表明小鼠胚胎干细胞源性表皮干细胞在肾被囊微环境下,可具有分化为角化复层上皮、汗腺样、皮脂腺样及毛囊样结构的潜能。     

羊膜及羊膜匀浆液在眼科学中应用的研究进展

羊膜应用于医学已有一个多世纪,应用于眼科也有70余年的历史。因种种原因,在很长一段时间里,对羊膜的研究停滞不前,但随着生物医学技术和其它相关技术的发展,羊膜的解剖结构及生物学特性研究得越来越深入。而随着羊膜的解剖和生物学特性日益明确,羊膜在医学和组织工程学中的应用也越来越广泛,并且应用前景广阔。近年来,由于羊膜的诸多解剖和生物学特性符合眼科学发展的需要,羊膜在眼科中的应用也日益广泛,并取得了良好的临床效果。羊膜的研磨提取液-羊膜匀浆液保留了羊膜中的有效生物成分,在治疗眼部疾病方面也取得了一定的进展。国内外关于羊膜及羊膜匀浆的研究文献有很多,为了更深入了解羊膜的特性及其在眼科疾病治疗方面的优势,该文将对羊膜的应用历史、羊膜及羊膜匀浆液的制备及保存、生物学特性及其在眼科学中的应用作一综述。     

Late extraembryonic morphogenesis and its zen-induced failure in the milkweed bug Oncopeltus fasciatus

Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen^RNAi eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen^RNAi defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic ‘bouncing’ activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen^RNAi defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen^RNAi eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event – rupture of the ‘serosal window’ structure – as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis.     

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.     

Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy

Cell-based regenerative medicine is of growing interest in biomedical research. The role of stem cells in this context is under intense scrutiny and may help to define principles of organ regeneration and develop innovative therapeutics for organ failure. Utilizing stem and progenitor cells for organ replacement has been conducted for many years when performing hematopoietic stem cell transplantation. Since the first successful transplantation of umbilical cord blood to treat hematological malignancies, non-hematopoietic stem and progenitor cell populations have recently been identified within umbilical cord blood and other perinatal and fetal tissues. A cell population entitled mesenchymal stromal cells (MSCs) emerged as one of the most intensely studied as it subsumes a variety of capacities: MSCs can differentiate into various subtypes of the mesodermal lineage, they secrete a large array of trophic factors suitable of recruiting endogenous repair processes and they are immunomodulatory.Focusing on perinatal tissues to isolate MSCs, we will discuss some of the challenges associated with these cell types concentrating on concepts of isolation and expansion, the comparison with cells derived from other tissue sources, regarding phenotype and differentiation capacity and finally their therapeutic potential.     

Ultrastructure of human amnion and amniotic plaques of normal pregnancy

Summary The fine structure of amniotic and amniotic-plaque epithelia has been studied from normal term pregnancies. The columnar/cuboidal amniotic epithelial cells usually have apical or central nuclei, some free ribosomes, patches of granular endoplasmic reticulum, juxtanuclear Golgi complexes, rod-shaped mitochondria, lipid droplets and some glycogen granules. They have short, blunt microvilli which frequently branch and bathe in the amniotic fluid. The lateral plasma membranes enclose tortuous intercellular spaces which are always interrupted by variously folded processes and desmosomes. The epithelial cells rest on a basal lamina and exhibit highly folded basal processes. The amniotic epithelial cells are neither distinctly Golgi and fibrillar types nor light and dark in appearance.Amnion from near the umbilical cord contains many microscopic and several large plaques. Similar structures are not found on the reflected amnion. The microscopic plaques are whitish and translucent, whereas the large ones are opaque. The large plaques vary between 1–3 mm in diameter, and are over 15 cell layers thick. Each large plaque has a main central region and edges continuous with either the microscopic plaque or the simple amniotic epithelium. The main region shows four zones, namely, stratum basale, stratum spinosum, stratum granulosum and stratum corneum. Such zones are not distinct at the edges. The fine structure of basal cells compares with the amniotic epithelial cells, but the cells of spinosum and granulosum layers possess variable amounts of tonofibrils, keratohyalin granules, free ribosomes and other cytoplasmic organelles and inclusions. The corneum cells are keratinized and are frequently separated by intercellular spaces. They slough into the amniotic cavity singly or as a sheet, and contribute towards the composition of the amniotic fluid. The plaques are of amniotic origin, and are not formed by adhesion of either squamous cells or fetal skin cells (masses of keratinized squames). The present observations suggest that the occurrence of amniotic plaques is normal. The presence of plaques may not be necessarily associated with fetal abnormality. However, increase in numbers of plaques may be caused by conditions of fluid imbalance. The homology and significance of plaques in eutherian mammals have been discussed.This research was supported by USPHS Grant AM-11376 and NIH Grant 69-2136.     

羊膜移植联合低浓度丝裂霉素治疗原发性青光眼的临床疗效

目的:探究羊膜移植联合低浓度丝裂霉素在治疗原发性青光眼的临床疗效。方法:选取2013年6月~2014年1月我院收治的原发性青光眼患者58例(80眼),按照随机数字表法分为观察组(30例,40眼)和对照组(28例,40眼),观察组行小梁切除、羊膜移植联合低浓度丝裂霉素治疗,对照组行小梁切除治疗,比较两组的手术效果及术后并发症情况。结果:观察组术后优良率为97%(38/40),显著优于对照组的75%(28/40),差异有统计学意义(x2=9.764,P=0.008);两组术后1、6个月眼压较术前均明显降低(P0.05),且观察组较对照组更低(P0.05);观察组功能性滤泡明显多于对照组(x2=4.943,P=0.026),术后并发症明显低于对照组(x2=4.501,P=0.034),复发情况明显低于对照组(x2=3.914,P=0.047)。结论:羊膜移植联合低浓度丝裂霉素治疗原发性青光眼能够有效控制眼压,减缓患者视力的丧失,术后并发症及复发率均较低,值得临床推广。