作者感谢王秀萍的初步帮助与DESC文化。提交人由来自美国国立卫生研究院（K99-DE022059到AHJ，K12-GM081266到MGC，K08-DE022377-02 OH和R01-DE021420到ODK）奖学金和助学金资助了一部分。

1，解剖下半侧下颌骨从成年小鼠<ol><li>在此之前执行此协议，获得必要的机构批准，并一定要遵守所有的动物护理指南。使用标准IACUC批准程序安乐死的动物。对这项工作的CO 2窒息用于颈椎脱位。 <ol><li>可选：使用剃刀刀片的身体的其余部分分离头。 </li></ol></li><li>从下唇除去皮肤的领口。 </li><li>使用手术刀，使两个下门牙，它们松散地连接之间的切口。当施加压力时，下颌骨将被分成两部分。 </li><li>楔颌骨髁突和颞下颌关节和削减沿着下颚肌肉之间的手术刀分离的下巴。 </li><li>卸下所有肌肉，肌腱，韧带和直到只剩骨头仍然存在。 </li></ol> 2，隔离切牙<ol><li>使用＃15手术刀仔细刮掉骨以45°角从前​​端向膜区域的近端。使用手术刀的尖端挑走任何剩余的骨头，包括骨头碎片的边缘。慢慢开始随到随走在颌骨舌侧骨皮质，开始只是下面的第三磨牙。 </li><li>继续下去，直到切牙是干净的，精心赶制含CL的区域。 </li><li>卸下下牙槽神经束。 </li><li>做一个干净的切割，以先移除骨组织近端到CL。 </li><li>做第二切口远端用手术刀，大致低于第二磨牙。近端门齿区域，然后解剖出通过插入骨和近侧门牙的内侧表面之间的手术刀。这必须谨慎进行，以免撕裂组织。切口运动是近 - 远。 可供选择的步骤： 删除尽可能多的骨尽可能沿着我上方的区域ncisor。一旦区域被清除，使用5号镊子处理釉质部最靠近发光区域小心地取出整牙。 </li><li>放置切取的组织（无论是分离的发光如在图3E中 ，或整个门齿作为替代步骤2.5）中在1X PBS 2％胶原酶为3-4小时，在4℃下在具有低粘附板。为1-10门齿，用每孔100μl的12孔板中。对于大于10门齿，用每孔500μl的在6 - 孔低粘附板。 </li></ol> 3，Microdissect的唇颈环<ol><li>从胶原酶和地方在寒冷的DMEM/F12培养基取出发光区域。 </li><li>无论使用5号镊子或胰岛素注射器（1毫升，28 G½）在CL下端轻轻一拉;间质会土崩瓦解，而上皮细胞保持完整。 CL和相邻上皮细胞的横向为“翼状”结构延伸将是可见的。 </li><li>清除从相邻的上皮通过使两侧的V形切口，并立即在冷的DMEM/F12放置在冰上的顶芽。 </li><li>重复所有CLS，收集1.5 ml离心管中。 </li><li>降速离心管样品，取出介质，替换为100微升细胞脱落的解决方案。 </li><li>在孵育30分钟，细胞脱离组织液在37°C </li><li>离解的组织通过使用1,000微升低粘性枪头轻轻地上下吹打来产生单细胞悬浮液。细胞也可以通过无菌细胞过滤器放置来实现解离。 </li><li>计数细胞，血球，板对组织培养塑料或其他所需材料。 </li></ol> DESCs 4。原代培养<ol><li>板的细胞在6孔板中以1-6×10 4个细胞/ ml的降序媒体，它由DMEM/F12补充有小鼠表皮生长因子的重组蛋白在20 n的浓度克/毫升，在浓度为25 ng / ml和1×B27添加剂和1％抗生素溶液（青霉素，100 U / ml的链霉素，50微克/毫升）的FGF重组蛋白质。 </li><li>在5％CO 2的1毫升降序媒体的生长细胞接种在6孔板中，在37℃。电镀，使细胞粘附和集落形成后请勿打扰初步培养5天。 </li><li>对于第一媒体的变化，随着新媒体的一半体积（500微升）取代一半旧媒体的体积（500微升）的。检查菌落显微镜下变化的媒体的时候。 </li><li>第一媒体的变化后，继续改变介质（1-2毫升），每2天。检查菌落显微镜下变化的媒体的时候。 </li></ol> 5。通道DESCs <ol><li>吸媒体和洗涤细胞3倍预热的PBS中。 </li><li>添加预热的0.25％胰蛋白酶-EDTA在盐溶液中，并在37℃下进行10-15分钟的DESCs分离。 </li><li>添加DESC媒体中和胰蛋白酶，轻轻地沿着组织培养板中吸取收集细胞，并将其放置在一个15毫升的锥形管。 </li><li>降速细胞在800×g离心2分钟。 </li></ol>

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该作者有没有利益冲突披露。

上皮细胞首次成功培养了40年前21-24，以及最近的上皮干细胞25-27成功分离了我们先进的上皮生物学知识。我们报告一个协议，用于分离成年小鼠切牙的DESCs，干细胞的相对充分研究人口，有产生了重要的见解牙科生物学和牙釉质形成的可能性。该协议最初是基于对上皮干细胞的分离从毛囊28以前的报告。而许多协议使用饲养层和血清维持上皮干细胞，我们的方法是一项联接...

之一的脊椎动物的独特特征是牙齿的演化。牙齿已成为许多研究领域一个重要的模型系统，作为分子途径和相关与这个器官形态特化进行了研究从几个方面，包括发展和进化生物学家5。最近，再生医学领域已经开始使用牙作为一种模式来获得有价值的见解。尤其，牙科上皮干细胞的发现是一个重要的进步6-13。 所有啮齿类动物具有不断增长的门牙，其增长是得益于干细胞，使这些牙齿可访问的模型系统来研究成体干细胞的调控。标记实验在20世纪70年代10,11，接着遗传谱系追踪实验8,9,12,14，已经证明，DESCs驻留在门牙的近侧区域。干细胞的后代在唇侧移动了公认的小生，被称为唇颈环（CL）的上皮车厢，并有助于细胞称为运输扩增（TA）细胞（ 图1）的人口。具体来说，DESCs驻留在外部釉上皮（OEE）和星状网（SR）8,9,14，和内釉上皮细胞（IEE）产生了对TA的细胞通过细胞周期的数量有限的进步，然后移动远侧沿着门齿的长度（ 图1）。在小鼠中切牙成釉细胞分化继续向远侧移动沿切牙以约350微米15,16一天一个惊人的速度。因为他们移动，细胞分化为成熟的成釉细胞和中间层（SI）的细胞。沉积釉质基质的整个厚度后，许多成釉细胞发生凋亡，而其余的细胞收缩的尺寸和调节搪瓷成熟&lt;燮&gt; 17。在唇侧发光，如SR，其他类型的细胞谱系是不太清楚，并就干细胞在间充质18和舌CL数据才刚刚开始出现。 使用鼠标切牙模型，一些团体一直在努力阐明遗传途径和参与自然干细胞为基础的器官重建细胞生物学过程。但是，阴唇CL包含相对少量的细胞，估计为约5000每只小鼠切牙，这使得与原代细胞具有挑战性的工作。因此，已作出努力，以文化，以打开新的大门，以实验方法是无法达到在体内扩大这些细胞在体外 6,16,19,20。最近的研究表明，这些细胞可以既自我更新和分化成釉原蛋白表达细胞的培养时，13。在这里，我们描述和展示为次的方法E可靠和一致的从鼠标唇发光细胞的培养。

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isolation and culture of dental epithelial stem cells from the adult mouse incisor

了解背后牙齿再生和更新的细胞和分子机制已成为极大的兴趣1-4个主题，鼠标切牙提供了一个模型，这些过程。这一显着的器官在整个动物的生命持续增长，并产生所有从容纳在阴唇（朝向唇）和舌（朝向舌）颈环（CL）的区域的成体干细胞的活性池所需的细胞类型。只有从阴唇CL中的牙齿干细胞生成搪瓷，牙齿的外壳，在唇面釉细胞产生。这种不对称的釉质形成允许磨损在切牙尖，和祖细胞和干细胞在近端门齿确保牙齿组织正在不断地补充。隔离和生长这些祖细胞或干细胞在体外的能力允许其扩张和打开大门，大量的实验不能达到体内 ，如H潜在的干细胞调控因子的IGH吞吐量测试。在这里，我们描述并演示从小鼠切牙的唇CL可靠和一致的方法来培养细胞。

鼠标半侧下颌骨包含一个不断增长的门牙三根臼齿（ 图1A）。所有的牙齿由牙本质和牙釉质，牙冠的两个矿化成分（ 图1A和1A&#39;）的。门齿装有两个干细胞小生境称为CL唇和舌CL和搪瓷是在唇侧（ 图1A&#39;）完全形成。 DESCs负责门齿釉质形成与被容纳在阴唇CL，特别是OEE和SR（ 图1A&#39;）8,9。唇CL也包含独立外部评价，TA细胞，?...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

不断增长的鼠标切牙提供了一个模型，以研究牙源性上皮干细胞（DESCs）牙齿组织的重建。一个强大的系统，从切牙持续，可靠地获得这些细胞，并扩大它们在体外这里报道。

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

Research

JoVE Journal

Biology

15.0K 次观看.  University of California, San Francisco. 不断增长的鼠标切牙提供了一个模型，以研究牙源性上皮干细胞（DESCs）牙齿组织的重建。一个强大的系统，从切牙持续，可靠地获得这些细胞，并扩大它们在体外这里报道。 

视频: Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...