저자는 DESC 문화와 초기 도움을 수수 핑 왕 감사합니다. 저자는 (OH에 MGC, K08-DE022377-02 AHJ, K12-GM081266에 K99-DE022059 및 만듭니다에 R01-DE021420) 건강의 국립 연구소에서 장학금과 보조금에 의해 부분적으로 투자됩니다.

성인 마우스 1. 해부 낮은 헤미 - 턱 <ol><li> 이전에이 프로토콜을 수행하기에 필요한 기관의 승인을 받아야하고 모든 동물 보호 지침을 준수해야합니다. 표준 IACUC이 절차를 승인하여 동물을 안락사. 이 작품에 대한 CO 2 질식 자궁 전위 다음에 사용되었다. <ol><li> OPTIONAL : 면도날을 사용하여 신체의 나머지 부분으로부터 분리 된 헤드. </li></ol></li><li> 목 둘레에 아랫 입술로부터 피부를 제거합니다. </li><li> 메스를 사용하여, 느슨하게 연결되어있는 두 개의 낮은 앞니 사이에 절개를합니다. 압력을 적용하면, 하악은 두 부분으로 분할됩니다. </li><li> 턱을 분리 턱의 관절 돌기 및 시간 하악 관절과 턱 근육과 함께 컷 사이의 메스 웨지. </li><li> 단지 뼈가 남을 때까지 모든 근육, 힘줄 및 인대를 제거합니다. </li></ol> 2. 앞니를 분리 <ol><li> # 15 메스를 사용하여조심스럽게 막 지역의 기단에 말단부에서 45 ° 각도로 뼈를 깎아. 가장자리에 뼈 조각 등의 남은 뼈를 멀리 선택하는 메스의 팁을 사용합니다. 천천히 단지 3 차 몰 이하로 시작, 하악의 설측 피질골 판에서 떨어져 선택하기 시작합니다. </li><li> 앞니가 CL을 포함하는 지역을 향해 조심스럽게 작업, 깨끗해질 때까지 계속합니다. </li><li> 하치 신경 다발을 제거합니다. </li><li> 먼저 CL에 인접한 뼈와 조직을 제거하는 깨끗한 상처를 확인합니다. </li><li> 대략 2 차 몰 아래의 메스와 원심 두 번째 컷을 확인합니다. 근위 앞니 영역은 다음 뼈와 인접 앞니의 안쪽 표면 사이의 메스를 삽입에 의해 해부된다. 조직을 찢어하지 않도록이 신중하게 수행해야합니다. 절개 운동은 근위-말단이다. 대안 STEP : 나는 위의 지역에 따라 가능한 한 많은 뼈를 제거ncisor. 지역이 해제되면, 조심스럽게 에나멜 부분에게 CL 지역에 가장 가까운을 처리하기 위해 크기에게 5 개의 집게를 사용하여 전체 앞니를 제거합니다. </li><li> 낮은 부착 판에 4 ° C에서 3 ~ 4 시간 동안 1X PBS의 2 % 콜라게나 제의 해부 조직 (대체 단계 2.5에서와 같이 그림 3E와 같이 고립 된 CL, 또는 전체 앞니 중 하나)을 놓습니다. 1-10 앞니를 들어, 12 - 웰 플레이트에 웰 당 100 μl를 사용합니다. 보다 큰 10 앞니의 경우, 6 자 낮은 부착 플레이트에 웰 당 500 μl를 사용합니다. </li></ol> 3. Microdissect 순음 자궁 루프 <ol><li> 차가운 DMEM/F12 미디어의 콜라게나과 장소에서 CL 영역을 제거합니다. </li><li> 크기 5 겸자 또는 인슐린 주사기 (1 CC, 28 G ½) 중 하나를 사용 CL의 하단부에 부드럽게 당겨; 상피 세포는 그대로 유지하면서 중간 엽 떨어져 떨어질 것이다. &quot;날개 같은&quot;구조로 옆으로 확장 CL과 인접한 상피 볼 수 있습니다. </li><li> 제거양쪽에 V 모양의 절개를하고, 즉시 얼음에 차가운 DMEM/F12에 배치하여 인접한 상피 세포의 꼭대기의 꽃 봉오리. </li><li> 1.5 ML의 미세 원심 분리 튜브에 수집, 모든 CL에서 반복합니다. </li><li> , 검체의 미세 튜브를 스핀 다운 매체를 제거, 100 ㎕의 세포 분리 솔루션으로 교체합니다. </li><li> 37 ℃에서 30 분 동안 세포 분리 솔루션의 조직을 품어 </li><li> 부드럽게 피펫 팅 상하 1000 μL 저 밀착성 피펫 팁을 사용하여 단일 세포 현탁액을 생성하는 조직을 해리. 세포는 또한 분리를 달성하기 위해 멸균 셀 스트레이너를 통해 배치 될 수있다. </li><li> 조직 배양 플라스틱 또는 기타 원하는 재료에 혈구, 접시에 세포를 계산합니다. </li></ol> DESCs 4. 차 문화 <ol><li> 6 - 웰 플레이트에 플레이트 세포 1-6에서 X 10 ~ 20 N의 농도로 마우스 EGF 재조합 단백질로 보충 된 DMEM/F12 구성 DESC 미디어에서 4 세포 / ㎖,㎍ / ㎖, 25 겨 / ㎖의 농도, 1X B27 보충, 1 % 항생제 용액 (페니실린, 100 U / ㎖, 스트렙토 마이신, 50 ㎍ / ㎖)에서 FGF 재조합 단백질. </li><li> 5 % CO 2 DESC 미디어 1 ㎖에 37 ° C에서 6 잘 접시에 도금 세포를 성장. 세포 부착 및 콜로니 형성 할 수 있도록 도금 후 5 일의 초기 문화를 방해하지 않습니다. </li><li> 첫 번째 미디어의 변화를 위해, 새로운 미디어의 절반 볼륨 (500 μL) 오래 된 용지의 양 (500 μL)의 절반을 교체합니다. 미디어를 변경할 때 현미경으로 식민지를 확인합니다. </li><li> 첫 번째 미디어를 변경 한 후 2 일마다 미디어 (1-2 ㎖) 변화하고 있습니다. 미디어를 변경할 때 현미경으로 식민지를 확인합니다. </li></ol> 5. 통로 DESCs <ol><li> 기음 미디어와 세포를 세척은 미리 예열 PBS로 3 배. </li><li> 생리 식염수에 미리 예열 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)을 추가 파견 할 DESCs 10 ~ 15 분 동안 37 ° C에서 알을 품다. </li><li> 트립신을 중화하는 DESC 미디어를 추가, 부드럽게 15 ML 원뿔 튜브에 세포 및 장소를 수집하는 조직 문화 판을 따라 피펫. </li><li> 2 분 800 XG에서 세포를 스핀 다운. </li></ol>

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+FGF+signaling+loop+sustains+the+generation+of+differentiated+progeny+from+stem+cells+in+mouse+incisors.">An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors.</a> Development. 135, 377-385 (2008).</li></ol>

저자는 공개 할 관심의 충돌이 없습니다.

상피 세포 40 년 전 21 ~ 24에 걸쳐 성공적으로 첫 번째 배양, 그리고 더 최근의, 상피 줄기 세포 25 ~ 27의 성공적인 분리 상피 생물학에 대한 우리의 지식을 전진했다. 우리는 성인 마우스 전치 DESCs 치과 생물학 및 에나멜 형성에 중요한 통찰력을 얻을 수있는 잠재력을 가지고 줄기 세포의 상대적 understudied 아르 집단을 분리하기위한 프로토콜을보고한다. 이 프로토콜은 처음에 모낭 <...

척추 동물의 독특한 특징 중 하나는 치아의 진화이다. 분자 경로와이 기관과 관련된 형태의 전문 분야가 발달하고 진화 생물 학자 (5)에 의해를 포함하여, 여러 가지 관점에서 조사 된 바와 같이 치아는, 연구의 많은 분야에 대한 중요한 모델 시스템이되고있다. 최근 재생 의학의 필드는 모델로서 치아를 사용하여 귀중한 통찰력을 얻기 위해 시작했다. 특히, 치과 상피 줄기 세포의 발견은 중요 미리 6-13왔다. 모든 설치류는 성체 줄기 세포의 조절을 연구하기 위해 이러한 치아에게 접근 모델 시스템을 만들고, 그 성장 줄기 세포에 의해 연료가 공급되어 지속적으로 성장 앞니를 가지고있다. 유전 적 혈통 추적 실험 8,9,12,14 다음에 1970 년대, 11의 레이블 실험, DESCs은 앞니의 인접 지역에 거주하는 것을 증명하고있다. 줄기세포 입술 자궁 루프 (CL)로 알려진 추정 틈새 시장 중 입술 측면 이동 상피 구획의 자손, 그리고 대중 교통 증폭 (TA) 세포 (그림 1)라는 세포의 인구에 기여한다. 특히, DESCs는 외부 에나멜 상피 (OEE) 및 성상 세망 (SR) 8,9,14에 상주하고, 내부 에나멜 상피 (IEE)의 다음 움직임을 세포 횟수에 제한을 통해 진행 TA 세포에 상승을 제공하고 원심 앞니의 길이 (그림 1)을 따라. 마우스 앞니의 차별화 ameloblasts는 하루 (15, 16)에 약 350 ㎛의 놀라운 속도로 앞니를 따라 원심 이동을 계속합니다. 그들이 움직일 때, 세포는 성숙한 ameloblasts 및 지층 매개물 (SI) 세포로 분화. 에나멜 매트릭스의 전체 두께를 증착 한 후, ameloblasts 많은 아폽토시스를 받아야하고, 나머지 셀의 크기가 축소 및 에나멜 성숙 조절 &lt;&gt; 17 일 저녁을 먹다. 이러한 SR 등 순측 CL,에 다른 세포 유형의 계보는 덜 선명하고 간엽 18 및 설측 CL의 줄기 세포에 관한 데이터 만 등장하기 시작하고있다. 절치 마우스 모델을 사용하여, 그룹의 개수는 유전 경로 및 천연 줄기 세포 기반 장기 내구성에 관련된 세포 생물학적 과정을 명료하게하기 위해 노력했다. 그러나, 입술 CL은 약 5,000 일차 전지는 도전 작업을하게 마우스 앞니, 당 것으로 추정 세포의 상대적으로 적은 수를 포함합니다. 따라서, 노력은 문화에 만들어 생체 내에서 달성 할 수없는 실험적인 접근 방식에 새로운 문을 열기 위해 체외 6,16,19,20에서 이러한 세포를 확장하고 있습니다. 가장 최근의 연구는이 세포가 자기 갱신 때 배양 13 세포를 발현하는 유전자 성별 감식로 분화 할 수있는 것을 모두 보여 주었다. 여기, 우리는 일하기위한 방법을 설명하고 보여마우스 입술 CL에서 세포의 전자 신뢰할 수 있고 일관성있는 문화.

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

치아 재생 및 갱신 기초가되는 세포 및 분자 메커니즘을 이해하는 것은 큰 관심을 1-4의 화제가되고, 마우스의 앞니는 이러한 프로세스에 대한 모델을 제공하고있다. 이 놀라운 기관은 동물의 삶 전반에 걸쳐 지속적으로 성장하고 (혀 쪽) (입술 쪽)으로 아랫 입술과 언어 자궁 루프 (CL) 지역에 자리 성체 줄기 세포의 활성 풀에서 필요한 모든 종류의 세포를 생성합니다. 순측 CL로부터 남은 치과 줄기 세포 순측 표면에 에나멜, 치아의 외피를 생성 ameloblasts로 야기. 이 비대칭 에나멜 형성 절치 팁에서 마모를 허용하고, 근위 앞니의 원종 줄기 세포는 치아 조직이 지속적으로 보충되도록. 체외에서이 전구 또는 줄기 세포를 분리하고 성장하는 능력은 자신의 확장을 허용하고 시간과 같은 생체 내에서 달성 할 수없는 수많은 실험에 문을 엽니 다잠재적 인 줄기 세포 규제 요인 고등학교의 처리량 테스트. 여기, 우리는 설명하고 마우스 앞니의 입술 CL에서 문화 세포에 안정적이고 일관성있는 방법을 보여줍니다.

마우스 헤미 - 하악 하나에게 연속적 성장 앞니와 삼 뿌리 어금니 (도 1a)를 포함한다. 모든 치아는 상아질과 에나멜, 치아 크라운의 두 광물 구성 요소 (그림 1A 및 1A &#39;)로 구성되어 있습니다. 앞니의 집은 두 개의 줄기 세포 틈새는 입술 CL 및 언어를 구사하는 CL이라고 에나멜은 입술 쪽 (그림 1A &#39;)에 독점적으로 형성되어있다. DESCs는 앞니의 ?...

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

지속적으로 성장 마우스 앞니는 치아 상피 줄기 세포 (DESCs)에서 치아 조직의 재생을 연구하기위한 모델을 제공합니다. 일관되고 안정적으로 앞니에서 이러한 세포를 획득하고 체외에서 그 확장을위한 강력한 시스템은 여기에보고됩니다.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

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

15.1K 조회수.  University of California, San Francisco.  지속적으로 성장 마우스 앞니는 치아 상피 줄기 세포 (DESCs)에서 치아 조직의 재생을 연구하기위한 모델을 제공합니다. 일관되고 안정적으로 앞니에서 이러한 세포를 획득하고 체외에서 그 확장을위한 강력한 시스템은 여기에보고됩니다. 

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

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.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

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

연구

생물학

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.

마우스 아이의 망막 줄기 세포의 분리

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 ...

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.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

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.

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 ...

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.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

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&#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.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

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.

간엽 전환을 유도하고 상피의 분석

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

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

격리, 문화 및 성인 마우스 심근 세포의 형질 도입

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

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.

절연, 특성, 및 성인 마우스 심장에서 심장 줄기 세포의 분화

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 ...