Congressionally 감독 의학 연구 기금 부여 (DAMD17 - 01-0264)에서 ML 커틀러와 ML 커틀러하는 방법 United States 군사 암 연구소에 기금이 작업을 지원합니다.

<ol><li> HC11 세포 10% 소태아 혈청, 5μg/ml 인슐린, 10 MM HEPES, 능력을 확립하기 위해 10ng/ml 표피 성장 인자 (EGF)와 보충 RPMI 1640 배지에서 6 일간 합류로 성장했다. </li><li> 세포는 PBS로 씻어 24 시간 동안 EGF없이 성장 미디어에 유지했다. </li><li> lactogenic 차별을 유발하는 세포 차별화 미디어, dexamethasone과 함께 즉 RPMI (10 -6 M), 인슐린 (5 μg / ML)과 같은 DIP라고 prolactin (5 μg / ML)에 incubated했다. </li><li> 명시된 시간 이후 DIP 추가에서 mammospheres이 찍은 디지털 카메라 올림푸스 현미경 IX71를 사용하여 위상 콘트라스트 현미경에 의해 열거. </li></ol> 대표 결과 : 정상 내유 상피 세포 monolayers 여러 mammospheres의 낮은 전력 확대뿐만 아니라 단일 mammosphere의 높은 전력 배율은 각각 그림 1에 표시됩니다. <img src="/files/ftp_upload/1265/1265fig1.jpg" alt="" /> 그림 1. Mammospheres가. () 일반 내유 상피 세포 monolayers가 합류 세포의 평면 레이어로 표시되어야합니다. (B) DIP 자극 3-5일 후 여러 mammospheres이 나타납니다. 사진은 5 일 DIP 또한 게시 촬영. DIP 또한 게시물 오일에 mammospheres를 포함하는 단일 필드 (C) 고등 확대.

<ol>
	<li>Blatchford, D. R., Hendry, K. A., Turner, M. D., Burgoyne, R. D., Wilde, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectorial+secretion+by+constitutive+and+regulated+secretory+pathways+in+mammary+epithelial+cells.">Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.</a> Epithelial Cell Biol. 4, 8-16 (1995).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid+induced+expression+of+connective+tissue+growth+factor+contributes+to+lactogenic+differentiation+of+mouse+mammary+epithelial+cells.">Glucocorticoid induced expression of connective tissue growth factor contributes to lactogenic differentiation of mouse mammary epithelial cells.</a> J Cell Physiol. 214, 38-46 (2008).</li></ol>

위해서는이 기술에 대한 성공과 최고의 품질의 상피 세포가 lactogenic 호르몬으로 유도되고 이전에 합류 유지되어야합니다. 부량 어려운 만드는 수많은 mammospheres가있다 상황이있을 수 있습니다. 따라서, 정량 목적은 mammospheres의 숫자가 계산되는 수있는 때 시점에서 mammospheres을 사진에 좋습니다. 내유 상피 세포의 분화 lactogenic에 노출된 정도를 확인하는 데 사용되는 다양한 ?...

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mouse mammary epithelial cells form mammospheres during lactogenic differentiation

일반적으로 내유 마우스 상피 세포 배양에 lactogenic 분화의 정도를 결정하는 데 사용 phenotypic 측정 mammospheres이라고 돔 모양의 세포 구조의 형성이다<sup&gt; 1</sup&gt;. HC11 세포 라인은 내유 lactogenic 분화 모두의 규제 연구를위한 모델 시스템으로 채용되었습니다<em&gt; 체외에서</em&gt;과<em&gt; 생체내에</em<sup&gt; 2</sup&gt;. HC11 세포 lactogenic 호르몬 치료에 대한 응답으로 우유 단백질을 차별하고 종합. 합류하는 HC11 마우스 상피 세포 내유의 성장에 따라, lactogenic 차별화는 dexamethasone, 인슐린 및 prolactin 포함한 lactogenic 호르몬 조합의 추가에 의해 유도된으로 DIP 언급했다. 차별화 유도 HC11 세포 1백20시간 게시물 분화의 유도 및 열거되었다 각 문화에 나타난 mammospheres의 수에 번에서 촬영했다. 개인 mammospheres의 크기는 분화의 정도와 연결합니다 그리고 이것은 차별 세포의 이미지에 그려져 있습니다.

Watch this Scientific Journal Video about Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation at JoVE.com

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

HC11 lactogenic 차별화는 mammospheres이라고 돔 구조의 형성에 의해 특징 수 있습니다. 구조 lactogenic 차별 quantifying에 투입 위상 콘트라스트 현미경에 의해 열거 수 있습니다.

마우스 내유 상피 세포는 Lactogenic 차별 동안 Mammospheres를 형성

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

mouse-mammary-epithelial-cells-form-mammospheres-during-lactogenic

Research

JoVE Journal

Biology

13.3K 조회수.  F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD. HC11 lactogenic 차별화는 mammospheres이라고 돔 구조의 형성에 의해 특징 수 있습니다. 구조 lactogenic 차별 quantifying에 투입 위상 콘트라스트 현미경에 의해 열거 수 있습니다.

비디오: Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

내유 상피 이식 절차

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

연구

생물학

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

세 가지 차원 혼합 셀 회전 타원체 공동 문화에서 Mammary 상피 세포의 분리

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

모터 뉴런에 마우스 배아 줄기 세포의 효율적인 분화

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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.

유도 만능의 줄기 세포에서 파생 마우스의 생성

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

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

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.

간엽 전환을 유도하고 상피의 분석

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

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

세균과 같은 세포로 신생아 마우스 피부 유래 줄기 세포의 분화<em&gt; 체외</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

상피 - 간엽 전환시 선택적 스 플라이 싱 (alternative splicing)의 검출

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

핵심 생물학 개념을 가르치는 마우스 유방 종양 세포를 사용 : 간단한 랩 모듈

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

마우스 유선의 냉동 섹션에 간접 면역 형광

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...