这项工作是由NIH / NCI资助R01CA - 118916

1。从人体皮肤中提取的上皮干细胞<ol><li>开始之前隔离上皮干细胞的过程，需要准备各自的媒体和试剂（见表1）。 </li><li>收集新鲜成人换装程序或打孔活检人类头皮，然后孵化的DMEM / 10％FBS / Dispase（4毫克/毫升）一夜之间在4 ° C。潜伏期为2-4小时，在37 ° C也是有效的。皮肤件应为1厘米，最大宽度允许酶渗透。 </li><li>消毒的培养皿转移到皮肤，从皮肤拉断每个头发抓住附近的皮肤表面发干，拉动坚决和顺利。选择基于他们在解剖显微镜下的形态休止阶段的卵泡，切出的凸起区域（图1A）转移卵泡到15毫升的消毒管。生长期毛囊也可以用来切上部1 / 3的卵泡，如果获得毛囊“隆起”的地区（图1B）。 </li><li>孵育15-20分钟，在室温下晃动定期在0.05％胰酶- EDTA（GIBCO）和Versene（0.53毫米EDTA 4Na，GIBCO公司）（1:1）混合的孤立的卵泡片段，添加4毫升的DMEM + 10 ％胎牛血清停止反应，5分钟800转的旋转。此外，毛囊组织片段植可以放在没有胰蛋白酶消化，从单一的毛囊细胞培养文化。 </li><li>弃上清小心（〜0.2-0.5毫升保存，以避免失去细胞），并重新暂停在1毫升KCM公司（角质形成细胞的培养基），无表皮生长因子。 </li></ol> 2。文化小学的上皮干细胞<ol><li>前培养的上皮干细胞，丝裂霉素C - 3T3 - 6300320细胞（饲养层细胞），应准备如下处理。 3T3 - 6300320细胞培养的DMEM与10％FBS治疗前。删除培养媒体，用PBS洗细胞两次，治疗与15μg/ mL的丝裂霉素C的DMEM无血清在2小时37 ° C，5％的CO 2的细胞。删除丝裂霉素C的DMEM，用PBS洗两次，细胞已经准备好使用。 （丝裂霉素C处理的3T3 - 6300320细胞还可以准备以前保存于-80 ° C.解冻和种子细胞培养初级上皮干细胞前一天，于37孵育℃，5％的CO 2。 150,000-200,000 6孔板，每孔细胞3T3 - 6300320建议。 </li><li>步骤1.5种子孤立毛囊干丝裂霉素细胞的C - KCM公司3T3 - 6300320细胞没有表皮生长因子在37℃过夜治疗℃，5％CO 2，含有表皮生长因子- KCM公司改为第二天。细胞是生长在37 ° C在潮湿的大气中含有5％的CO 2 。所有的角质形成细胞是美联储含有表皮生长因子与KCM公司每2天，并成长为14-20天。每天检查细胞在显微镜下。毛囊干细胞会形成菌落（图2A）。毛囊植后，将形成10-14天（图2B）的副产物。 </li></ol> 3。通道上皮干细胞<ol><li>用PBS洗涤细胞一次。卸下Versene（预热至RT）治疗饲养层细胞3T3 - 6300320。孵育文化Versene抛出5分钟，然后轻轻摇动和吸饲养层细胞。 </li><li>毛囊干细胞Versene或PBS一次清洗。 </li><li>加入预热（至37 ° C，临界）胰蛋白酶- EDTA（2X），并在37 ° C时7分钟或更长的时间，必要时对毛囊干细胞的分离。最长不超过15分钟是最好的。 </li><li>添加KCM公司W / O型表皮生长因子，以阻止胰蛋白酶，轻轻吸管分散细胞，在200克旋转下来，5分钟。 </li><li>与KCM公司暂停细胞无表皮生长因子，计数和丝裂霉素C处理的3T3 - 6300320细胞层上的重制版。 </li></ol> 4。不朽上皮干细胞原代细胞，甚至成人干细胞，达到串行通道和文化个月后的衰老。基层干细胞永生化是一种有效的的方式来缓解这个问题。 <ol><li>板小学在饲养层上的1通过上皮干细胞（3T3 - 6300320）（〜35万细胞/ 6孔板）和2天的文化。 </li><li>文化PA317 LXSN 16E6E7细胞株有一天播种后用DMEM含10％胎牛血清的主要上皮干细胞。 （此生产线生产amphotropic的逆转录病毒LXSN16E6E7编码的HPV16型E6和E7开放读码框架，可用于稳定感染和不朽多种细胞类型）。 </li><li>治疗2小时，用丝裂霉素C（3T3 - 6300320细胞相同的协议）LXSN 16E6E7 PA317细胞系。加丝裂霉素C治疗PA317 LXSN 16E6E7细胞株主上皮干细胞，共培养6天与表皮生长因子KCM公司。与表皮生长因子媒体续订新鲜KCM公司每2天。 </li><li>删除3T3 - 6300320 16E6E7 LXSN PA317细胞与Versene。添加丝裂霉素C处理3T3 - 6300320 NHP细胞（新霉素，潮霉素，嘌呤霉素抗性〜20细胞，每孔）的上皮干细胞。细胞被选中下添加0.2毫克/毫升的G418（GIBCO）itional 6天。续订与EGF媒体每隔2天的新鲜G418包含KCM公司。 尚存的上皮干细胞将是永生的干细胞。不PA317 LXSN 16E6E7细胞共同培养的主上皮干细胞的井应设置控制G418筛选，在这口井的所有细胞被杀死后经G418选择6天。 </li></ol> 5。代表性的成果通过皮肤上皮干细胞和永生化上皮干细胞的早期形式组成严密的殖民地饲养层细胞包围的小角质形成细胞（图2A）。如果在无饲养层，无血清媒体定义的补充的培养，干细胞会分散，不能形成紧殖民地（他们成长为单个细胞和小群）。永生上皮干细胞的维持稳定的表型为12个月的连续传代，但往往比主细胞形成更紧密的殖民地。他们没有形成锚地独立的殖民地，在软琼脂实验表明，这些永生细胞所不具备的特点癌细胞。小学和永生化上皮干细胞表达毛囊干细胞标记的细胞角蛋白15（图3A），并能分化成表皮，毛囊和皮脂腺的谱系（图3B - D）。 <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="图1" /> 图1。弹拨的毛发。 dispase治疗皮肤采摘后，毛囊会出现一个细胞周围的头发底部球无论是俱乐部的休止期头发（一）或（B）与上皮细胞周围的头发长度的袖子生长期毛发。休止期隆起地区形成了一个独特的微解剖形态学地区，而生长期隆起明显。生长期灯泡包含矩阵角质形成细胞，占过境放大，也可以分离和培养的细胞。 <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="图2" /> 图2干细胞培养。 （一）上皮干细胞，从皮肤形成紧上皮殖民地（通过电子邮件指定）时，KCM公司培养的饲养层细胞（由F指定）。 （二）毛囊植，引起上皮的副产物。休止期隆起地区（指定由T）连接到的菜和上皮细胞集落一起饲养层细胞包围。 <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="图3" /> 图3。干细胞的分化。 （一）干细胞群，维持上皮标志物如细胞角蛋白15的表达。 （二）皮肤上皮干细胞可分化K6Hf表达确定的沿毛囊血统。 （三）被培养的细胞可以去epidermalized真皮或其他矩阵在气 - 液界面上，将形成一个与分层表皮角化层，颗粒层，棘层。 （四）干细胞可诱导沿油红积极球皮脂腺血统。

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细胞的提取和培养方法是惊人的轻便和可重复性。我们产生了几十个人在广泛的年龄范围，包括遗传性皮肤缺损18患者的上皮干细胞培养。最好是组织收获的一天开始的过程，但是细胞会继续生存在冰上媒体好几天了，如果需要，促进通宵航运。弃置换装皮肤产生数百个可行的毛囊细胞提取单细胞悬液。对于更有限的组织，如冲床从头皮活检，，外植体的文化可能会更有效，因为细胞胰酶...

角质形成细胞的媒体（KCM） [DMEM和火腿小号F12（GIBCO公司，3:1），腺嘌呤（Sigma公司，180毫米），10％胎牛血清（GIBCO公司），霍乱毒素（ICN，0.1纳米），青霉素/链霉素（GIBCO公司，为100 U / ml的100毫克/毫升，分别），氢化可的松（Sigma公司，0.4毫克/毫升，1.1毫米），T/T3（转GIBCO公司;，5微克/毫升，649纳米和三碘- L -甲状腺素，西格玛2纳米）胰岛素（Sigma公司，5毫克/毫升，862纳米），EGF（Sigma公司，10毫微克/毫升，1.6海里），pH值7.2]

isolation and culture of adult epithelial stem cells from human skin

所有组织自我更新的动态平衡，是依赖于成人干细胞。由于未分化的干细胞进行不对称分裂，它们所产生的子细胞，保留干细胞表型和过境扩增细胞（助教细胞），移植的干细胞小生境，进行快速增殖和晚期分化重新填充组织。 在表皮，毛囊和肠上皮干细胞已被确定为细胞在体外增殖潜能高，慢骑自行车的标签保留细胞在 体内 1-3 。成年后，组织特异性干细胞再生的组织在其居住，在正常的生理营业额，以及在压力4-5倍。此外，干细胞一般认为是多有力，拥有的能力上升到多种类型的细胞内组织6。例如，啮齿类动物毛囊干细胞可以生成表皮，皮脂腺和毛囊7-9。我们已经表明，从人类毛囊隆起地区干细胞表现出多潜能 10 。 干细胞已经成为在生物医学研究的宝贵工具，由于其作为体外研究发育生物学，分化，肿瘤的发生和其可能的治疗用途的系统实用工具。成人上皮干细胞很可能将在如外胚层发育不良，monilethrix，瑟顿综合征，Menkes病，遗传性大疱性表皮松解症和alopecias 11-13等疾病的治疗非常有用。此外，如烧伤，慢性伤口和溃疡等皮肤问题，将有利于干细胞相关治疗 14,15 。鉴于潜在的成年细胞重新编程为国家多能干（iPS细胞）16,17，可以提供方便和可扩展的成人干细胞在人体皮肤细胞诱导和下游治疗的疾病范围广泛，包括一个非常宝贵的资源糖尿病和帕金森氏病。

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

隔离可行的成人上皮干细胞的人体皮肤快速，可靠的方法是描述。该方法利用酶消化，皮肤胶原质基质，毛囊和隔离的单细胞悬液或细胞培养的组织碎片采摘。

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 ...

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Research

JoVE Journal

Biology

28.2K 次观看.  University of Massachusetts Medical School. 隔离可行的成人上皮干细胞的人体皮肤快速，可靠的方法是描述。该方法利用酶消化，皮肤胶原质基质，毛囊和隔离的单细胞悬液或细胞培养的组织碎片采摘。

视频: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

科研

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

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 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 ...