成年绒山羊皮肤干细胞克隆定向诱导分化为成骨细胞

成年绒山羊皮肤经0.02%中性蛋白酶4℃过夜消化,分离表皮,37℃消化30min,经100μg/mlIV型胶原处理的培养皿黏附10min筛选干细胞,培养到第二代时采用有限稀释法进行单细胞克隆纯化,角蛋白15、角蛋白19免疫荧光染色部分细胞强阳性,添加成骨细胞诱导液定向诱导分化,经碱性磷酸酶、茜素红染色鉴定为阳性,表明分离的细胞是皮肤干细胞且有能力诱导分化为成骨细胞。     

造血干细胞的研究进展

干细胞研究是一门新兴的学科。经过50多年的努力,造血干细胞的研究已经成为当今生物医学领域中发展最快的领域。介绍了造血干细胞的来源、分离纯化和检测方法以及“可塑性”等方面的研究情况,并详细说明了一些主要的造血干细胞表面标志以及造血干细胞在干细胞移植、细胞治疗和基因治疗等方面的临床应用和前景。     

恒河猴皮肤干细胞的体外培养纯化与鉴定

探索恒河猴皮肤干细胞的体外培养及纯化条件,为进一步的研究奠定基础. 通过组织块培养法和消化培养法 在体外培养恒河猴表皮细胞,然后用Ⅳ型胶原吸附法吸附20 min,获得快吸附细胞. 对快吸附细胞进行克隆培养,并进行免疫细胞化学双标染色、RT PCR鉴定 β1 整合素和角蛋白15的表达,用流式细胞仪鉴定纯化前后的细胞中 β1 整合素和角蛋白15的阳性细胞比例,并通过透射电镜观察细胞的超微结构. 组织块培养法和消化培养法均可获得表皮细胞,Ⅳ型胶原纯化后的细胞胞体较小,饱满,核/浆比例大,细胞镶嵌状排列. 细胞克隆分析显示,细胞全克隆生长率高. 细胞免疫荧光显示,分选后的细胞显示 β1 整合素和角蛋白15阳性. RT PCR检查呈现 β1 整合素和角蛋白15的特异性片段. 流式细胞仪检查显示,纯化前的细胞中角蛋白15阳性细胞占总细胞中的比例为8％, β1 整合素阳性细胞的比例为10.7％;纯化后,角蛋白15阳性细胞的比例为89.4％, β1 整合素阳性细胞的比例为88.5％. 通过组织块培养法和消化培养法均可培养获得活性良好的表皮细胞,Ⅳ型胶原吸附法是一种简便、有效的皮肤干细胞分离方法,可以为进一步的眼表上皮替代重建眼表提供足量的高纯度的干细胞建立可靠的物质基础.     

骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。     

骨髓间充质干细胞在组织工程研究中的应用新进展

骨髓间充质干细胞又称为骨髓源性间充质干细胞,是指存在于骨髓基质细胞系统中的一类干细胞,具有高度稳定的体外扩增能力和多向分化潜能等特点。骨髓间充质干细胞因其取材方便,易于分离和培养,以及在适当条件下可诱导分化为皮肤、骨骼、内脏、血液、神经等多种组织细胞的独特优势,目前被广泛应用于药物开发、免疫调节、组织修复、器官重建等多个研究领域。近年来,骨髓间充质干细胞作为种子细胞在组织工程领域有着非常诱人的潜在应用前景。本文就骨髓间充质干细胞在组织工程学研究中应用的最新进展作一综述。     

调节表皮干细胞增殖和分化的信号通路

表皮干细胞能够维持正常表皮的新陈代谢、毛囊周期循环以及参与创伤情况下创面的修复,皮肤肿瘤的发生也与其密切相关。表皮干细胞的增殖和分化受到严格的调控,了解表皮干细胞增殖与分化的调控机制将有助于治疗脱发、创伤以及皮肤肿瘤等疾病。文章着重概述了Wnt和Bmp信号对于控制干细胞命运的重要作用。     

表皮干细胞研究进展

哺乳动物表皮中包含有多种不同类型的表皮干细胞, 它们共同维持了表皮组织结构的稳态并在皮肤创伤的修复中起重要作用。表皮干细胞具备干细胞两大基本特征: 自我更新和分化, 两者间平衡的破坏通常是皮肤肿瘤和其他皮肤疾病的根源。文章着重叙述了表皮干细胞存在的证据、两大基本特征、分裂模式、调节表皮干细胞的信号通路以及维持其稳态的微观和宏观环境。     

水牛精原干细胞研究进展

精原干细胞(SSCs)是一类能够不断地进行自我更新和分化,并能保证遗传物质在亲子代之间有效传递的成体干细胞。伴随着各种移植技术、细胞体外培养及冻存等技术的发展,精原干细胞的应用成为可能,其在生产转基因动物及保护珍稀物种等方面有着广阔的应用前景。本文对水牛SSCs的生成、分离、纯化、培养、鉴定以及冻存的研究进行了系统的阐述。     

表皮干细胞的生物学特性及其潜在应用

表皮是维持生命及保障各个组织器官发挥功能的重要屏障,而表皮干细胞能够分化为皮肤各层组织,从而使表皮始终处于持续不断的增殖、分化、凋亡的状态,在维持表皮平衡,表皮损伤后修复的过程中发挥重要的作用。随着细胞生物学的飞速的发展,人们越发认识到表皮干细胞的重要性,使其成为人们目前的研究热点之一。针对表皮干细胞研究进展,着重叙述了表皮干细胞的分类、定位、特征、功能以及应用前景,旨为相关研究者对表皮干细胞生物学特性及应用有更全面的认识。     

表皮干细胞中CK15和CD49f阳性率与年龄关系的初步探究

目的:探究人体包皮组织中分离的表皮干细胞CK15和CD49f阳性表达率与年龄的关系。方法:利用双酶法从1、11、28、31岁4个年龄组皮肤组织中分离表皮干细胞,用CK15和CD49分别进行标记,用流式细胞仪检测阳性率。结果:在研究的4个年龄组中,11岁年龄组皮肤中的细胞CK15和CD49标记物阳性率最高,分别为70.19%和25.27%;流式细胞仪分选出的CK15阳性和CD49f阳性表皮干细胞在体外可呈现克隆生长。结论:11岁年龄组的表皮干细胞活性较强,更适宜组织工程种子细胞的选用。     

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β₁ integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.     

Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.     

Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin

In this review we focus on epidermal stem cells in the normal regeneration of the skin as well as in wounded and psoriatic skin. Furthermore, we discuss current data supporting the idea of cancer stem cells in the pathogenesis of skin carcinoma and malignant melanoma. Epidermal stem cells present in the basal layer of the interfollicular epidermis and in the bulge region of the hair follicle play a critical role for normal tissue maintenance. In wound healing, multipotent epidermal stem cells contribute to re-epithelization. It is possible that defects in growth control of either epidermal stem cells or transit amplifying cells constitute a primary pathogenetic factor in the epidermal hyperproliferation seen in psoriasis. In cutaneous malignancies mounting evidence supports a stem cell origin in skin carcinoma and malignant melanoma and a possible existence of cancer stem cells.     

Embryonic stem cells and skin: from cellular model to therapeutic potential

Embryonic stem (ES) cells are pluripotent cells able to differentiate into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported their efficient ability to recapitulate ectodermal and epidermal fates and form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. Thus, ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, characteristic of embryonic skin formation. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation. We further demonstrated that p63 stimulates ectodermal cell proliferation and is necessary for epidermal commitment. We provided further evidence that this unique cellular model provides a powerful tool to identify the molecular mechanisms controlling normal skin development and to investigate human ectodermal dysplasia congenital pathologies linked to p63 (in p63-ectodermal dysplasia human congenital pathologies). Epidermal stem cell activity has been used for years to repair skin injuries, but ex vivo keratinocyte amplification has limitations and grafted skin homeostasis is not totally satisfactory. Human ES cells raise hopes that the understanding of developmental steps leading to the generation of epidermal stem cells will once be translated into therapeutic benefit. We recently demonstrated that human embryonic stem cells can give rise to a stable somatic ectodermal cell population. Its finite population doubling, normal cell cycle kinetics and the absence of teratoma formation strongly suggest that, although derived from human embryonic stem cells, these ectodermal cells represent a clinically safe somatic cell population. They could thus be particularly useful as a source for committed, homogeneous, non-tumorigenic cell populations to be employed in clinical trials for epithelial stem cell loss.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

Glycome profiling by lectin microarray reveals dynamic glycan alterations during epidermal stem cell aging

Aging in the epidermis is marked by a gradual decline in barrier function, impaired wound healing, hair loss, and an increased risk of cancer. This could be due to age‐related changes in the properties of epidermal stem cells and defective interactions with their microenvironment. Currently, no biochemical tools are available to detect and evaluate the aging of epidermal stem cells. The cellular glycosylation is involved in cell–cell communications and cell–matrix adhesions in various physiological and pathological conditions. Here, we explored the changes of glycans in epidermal stem cells as a potential biomarker of aging. Using lectin microarray, we performed a comprehensive glycan profiling of freshly isolated epidermal stem cells from young and old mouse skin. Epidermal stem cells exhibited a significant difference in glycan profiles between young and old mice. In particular, the binding of a mannose‐binder rHeltuba was decreased in old epidermal stem cells, whereas that of an α2‐3Sia‐binder rGal8N increased. These glycan changes were accompanied by upregulation of sialyltransferase, St3gal2 and St6gal1 and mannosidase Man1a genes in old epidermal stem cells. The modification of cell surface glycans by overexpressing these glycogenes leads to a defect in the regenerative ability of epidermal stem cells in culture. Hence, our study suggests the age‐related global alterations in cellular glycosylation patterns and its potential contribution to the stem cell function. These glycan modifications detected by lectins may serve as molecular markers for aging, and further functional studies will lead us to a better understanding of the process of skin aging.     

Epidermal stem cells: targets for carcinogenic chemicals

Epidermal stem cells long have been considered a target for carcinogenic chemicals, but these stem cells have never been identified or isolated. Toward this goal, this report examines two-stage carcinogenesis in light of the stem cell model for cellular replacement in the epidermis and considers characteristics that may be useful in the identification and the isolation of epidermal stem cells. Firstly, the carcinogenesis experiments in mice have indicated that the population of target cells normally remains in the epidermis for the life of the animal despite the continual cellular turnover. Hence, the slowly cycling (label-retaining) keratinocytes from the epidermis and hair follicles are potential targets. Secondly, the results of carcinogenesis experiments have also indicated that the target cells are necessarily ones with a high potential for proliferation relative the most of the proliferative population. The keratinocyte colony forming units (kCFU) from the epidermis of normal and treated adult mice are consequently a quantifiable indicator of proliferative potential and another possible target. Further application of the stem cell concepts of quiescence and of self-renewal is expected to yield additional tools for the identification and isolation of the epidermal targets for chemical carcinogens.     

Enrichment of epidermal stem cells of rats by Vario magnetic activated cell sorting system

Epidermal stem cells (ESCs) play an important role in skin homeostasis, wound repair, and tumorigensis which have great potential in scientific research and clinical application. So, the efficient isolation of these infrequent stem cells is very important for researchers to solve the problem of low purity and insufficient quantity of stem cells in vitro. The aim of this study was to investigate a method for the enrichment of ESCs by magnetic activated cell sorting system. The isolation strategy was CD71 depletion followed by α6-integrin positive selection. The percentage of α6^briCD71^dim cells in isolated cells was 94.59%. Transmission electron microscopy results revealed that α6^bri CD71^dim cells exhibited some typical characteristics like progenitor cells, such as big nucleus, obvious nucleolus, large nuclear–cytoplasm ratio, and few organelles in cytoplasm. When cultured in vitro, the α6^briCD71^dim cells had greater proliferating potential and higher colony-forming ability, and high levels of epidermal stem cell markers were expressed in our positive cells. ESCs have been successfully isolated from neonatal epidermis using Vario MACS and cultured in vitro. This isolation method is simple, fast, and inexpensive, providing an important tool for tissue engineering and cell transplantation studies.     

Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/β-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active β-catenin, two key members of the Wnt/β-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/β-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.