우리는 그녀의 귀중한 도움 로라 클라크 감사합니다. 줄기 세포 네트워크, CIHR 및 NIH :이 작품은 제정신에 의해 지원됩니다.

1. 분석 해 봅시다 솔루션 및 효소 솔루션 준비 <ol><li> 인공 대뇌 척수 (ACSF)와 미리의 혈청이없는 미디어를 만들어 냉동. </li><li> Kynurenic 산성 (0.2 MG / ML)을 무게와 그것이 쉽게 용해되지 않으므로 미리 37 ° C waterbath에서 힐로 ACSF 10 ML에 디졸브. </li><li> 트립신 (1.33 MG / ML)과 Hyaluronidase (0.67 MG / ML) 한 15 ML 튜브의 위치를​​ 밖으로 무게 필요한 때까지 -20 ° C에서 보관하십시오. 이 튜브에 Kynurenic 산성 솔루션을 추가하고 사용하기 직전 22 μm의 주사기 필터를 사용하여 필터링합니다. </li><li> 트립신 억제제를 달다 (Ovamucoid : 1 MG / ML)과 따뜻한 세럼 - 무료 미디어 및 22μm 주사기 필터를 사용하여 필터에 디졸브. </li><li> FGF2 (10 NG / ML)과 헤파린 (2 μg / ML)이있는 세럼 - 무료 미디어 : 도금 미디어를 확인하십시오. </li></ol> 2. 마우스 아이의 망막 줄기 세포를 분리 <ol><li> 망막 줄기 세포의 분리는 기본 문화 실험 전용 특정 멸균 실에서 수행됩니다. 전에이 절차를 시작하기 위해 해부 현미경과 추위에 광원을 설정하고, 다음 멸균 해부 악기를 배치. 각 후드 단계 사이에 악기를 살균을 위해 뜨거운 구슬 살균기 있습니다. </li><li> 우리는 승인된 동물 윤리 프로토콜에 따라 희생 생쥐에서 그들의 제거 후 인공 대뇌 척수 (ACSF)를 포함하는 멸균 배양 접시에 바로 배치되었습니다 눈에서 망막 줄기 세포를 분리합니다. </li><li> 해부 현미경, 집게로 시선을 청소 : 머리와 각막 / scleral 국경에 첨부되어 결합 조직의 제거. 다음 ACSF로 새로운 접시에 눈을 전송합니다. </li><li> 부드럽게 톱니 모양의 집게로 고정 시선을 잡고 있지만, 안구 근육을 제거하는 직각 마이크로 해부 가위를 사용합니다. 그것이 아직도 눈에에 연결된 경우뿐만 아니라 시신경을 제거합니다. 이 과정에서 눈에 죽겠어하지보십시오. ACSF있는 새로운 요리에 눈을 전송. </li><li> 다음 사용 곡선 마이크로 해부 가위 절반에 눈을 잘라 : 시신경 시에 시작해서 각막의 중앙을 통해 컷, 컷이 시작되었다 시신경에 다시 회의. 이것은 다음 단계로 쉽게 때문에 안구의 두 가지 크기가 대략 동등한있다면 그것은 이상적입니다. </li><li> 두 눈 껍질 부드럽게 두 개의 집게를 사용하여 각막을 개최하는 것은 따로 반쪽. 제거하고 렌즈, 내장, 그리고 눈 껍질에서 신경 망막을 삭제. ACSF로 새로운 접시에 껍질을 전송합니다. </li><li> 이제 우리는 ciliary 상피를 분리 준비가되어 있습니다. 시작하려면, 오리엔트 눈 쉘은 때문에 각막이 오른쪽과 망막 색소 상피 (RPE)에있는 왼쪽에 있습니다. 부드럽게 RPE 측면에 바로 포셉와 눈 껍질을 핀. </li><li> 다음 부드럽게 메스에 아래로 밀고보다는 제재의 움직임을 사용하여 눈 ciliary 상피에서 떨어진 각막과 홍채를 잘라 메스를 사용합니다. </li><li> 그 후, 멀리 RPE에서 ciliary 상피를 잘라. ACSF을 포함하는 새로운 35 mm 요리에 ciliary 상피의 스트립을 전송하지 않는 톱니 모양의 집게를 사용하십시오. </li><li> 동일한 방식으로, 다른 눈 껍질에서 ciliary 상피를 분리. </li><li> 두 ciliary 상피 스트립이 수집되고 나면, Dispase 2 ML을 포함하는 35 mm 접시에 스트립을 전송하기만하면됩니다. 10 분 동안 37 ° C 배양기에서 스트립과 접시를 놓습니다. </li><li> 십분 후, 여과 트립신, Hyaluronidase, 그리고 Kynurenic 산성 2 ML을 포함하는 요리로 Dispase에서 스트립을 전송. 37 접시를 놓고 ° C를 10 분. </li><li> 이제 해부 현미경으로 돌아갑니다. 스트레이트 집게로 공막엔을 누른 상태에서 부드럽게 떨어져 공막엔에서 ciliary 상피를 긁어 곡선이 아닌 톱니 모양의 집게의 바닥을 사용합니다. </li><li> 접시에서 공막엔를 제거합니다. 이제 접시에 남아있을 것이 모든 관심과 효소 솔루션의 세포 수 있습니다. </li><li> 화재 - 세련된 솜 연결 피펫을 사용하여 14 ML 튜브에이 솔루션을 전송합니다. 부드럽게 피펫의에서 밖으로 솔루션을 강제하여 상피 세포를 분리 휴식이 솔루션을 30 번 씹다. </li><li> 1,500 RPM에서 5 분 동안 원심 튜브. 세포가이 단계에서 튜브의 바닥을오고하는 경향이 있기 때문에 원심 분리가 완료되면 조심스럽게 원심에서 튜브를 제거합니다. </li><li> 부드럽게 불타는 광택 피펫을 사용하여 표면에 뜨는의 대부분을 대기음 다음 세포로 세럼 - 무료 미디어 트립신 억제제 1 ML를 추가합니다. 그것이 단일 세포 현탁액 때까지 약 50 번 샘플을 씹다하는 작은 시추공 솜 연결 피펫을 사용합니다. </li><li> 1,500 RPM에서 5 분 동안 다시 튜브를 원심 분리기. </li><li> 뜨는을 제거하고 도금 매체 1 ML로 바꾸십시오. 세포를 resuspend에 불을 광택 유리 피펫을 사용하여 부드럽게 가루로 빻다. </li><li> 개수 후24 - 잘 판에서 원하는 농도에서 세포, 접시 그들을 주입. 우리는 일반적으로 판 10 세포 / 중간 각 잘 첫번째 입력하고 다음 미디어 500 μL의 최종 볼륨을 얻기 위해 세포 현탁액을 추가하여 μL. </li><li> 당신은 7 일 후 발생하는 분야를 계산하기 전까지 이동되지 않습니다 37 ° C CO 2 배양기에서 접시를 놓습니다. </li></ol> 3. 망막 줄기 세포를 분리 결과 <ol><li> 이 절차가 성공적으로 수행되면, 해부 ciliary 상피 세포는 저밀도 (그림 1)에서 dissociated 및 도금 후에 같이한다. </li><li> 문화 7 일 후에 발생하는 망막 줄기 세포 분야가 포함됩니다. 구형은 직경과 줄기 세포 파생된 영역으로 계산하는 자유 부동 75 μm의 이상되어야합니다. (그림 2). 그러나, 일부 세포는 크기의 기준을 충족하지 않으며 줄기 세포 파생된 분야로 계산되지 않습니다 제한된 증식 및 양식 spheroids을 것입니다, 이러한 회전 타원체의 예제는 (그림 2) 여기에 표시됩니다. </li></ol> 4. 대표 결과 <img src="/files/ftp_upload/2209/2209fig1.jpg" alt="그림 1" /> 그림 1. <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="그림 2" /> 그림 2.

<ol>
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관심 없음 충돌 선언하지 않습니다.

이 프로토콜은 마우스 눈 1-3의 ciliary 상피에서 망막 줄기 세포를 분리하는 방법에 대해 설명합니다. ciliary 상피의 스트립을 격리에 대한 방법론 그러나 단일 세포 현탁액을 달성을위한 효소와 방법론이 프로토콜에 최적화되었습니다, 다를 수 있습니다. 그것은 이러한 각각의 시선에서 격리 수있는 분야의 총 개수에 부정적인 영향을 미칠 것처럼부터 고립되어있다 ciliary 상피의 스트립이 RPE이나 각막의 다량을 포함하지 않는 것이 또한 중요합니다. 또한, 우리가 원래 두뇌 neurospheres 13 공식화 것을 확인하는 혈청이없는 미디어는 망막 줄기 세포 분야의 성장에 이상적입니다. 하나는 그들이 성장하는 분야가 함께 14 집계되지 않도록 도금 및 보육에 배치받은 후 세포가 방해되지 않도록해야합니다. 망막 줄기 세포가 clonal 분야를 형성되면, 그들은 눈 당 줄기 세포의 미래의 수치를 얻기 위해 계산하실 수 있습니다. 분야 다음 다음 단일 셀에 dissociated하고 확인할 자기 갱신 기능을 passaged 또는 다른 성장 요인의 조합 및 / 또는 단백질을 사용하여 신경 망막과 RPE의 서로 다른 세포 유형으로 차별화된 수 있습니다.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
</tr>
<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
</tr>
<tr>
<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
</tr>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
</tr>
<tr>
<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
<tr>
<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>VWR/BD </td>
<td>CACB354235</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
<td>&nbsp;</td>
<tr>
<td>Kynurenic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>K3375</td>
<td>1000 IU</td>
</tr>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
<td>&nbsp;</td>
<tr>
<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
<tr>
<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
<tr>
<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
</tr>
<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
</tr>
<tr>
<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
</tr>		</tbody>
		</table>
		</div>

isolation of retinal stem cells from the mouse eye

성인 마우스 망막 줄기 세포 (RSC)은 포유류의 눈 1,2,3의 ciliary 상피 (CE) 내에서 발견된 드문 정지 세포이다. 모두를 형성 CE가 아닌 색소 내부 및 색소 외부 세포 레이어로 구성되어 있으며, CE에서 단일 색소 세포에서 발생하는 clonal RSC 식민지는 차별화된 수있는 두 색소가 아닌 색소의 세포로 구성되어 있습니다 신경 망막과 RPE 세포의 종류. 분야 내의 모든 세포가 모두 적어도 몇 가지 색소 4가 포함되어 있는지 여부에 대한 논란이있다, 그러나 세포는 여전히 1-3 신경 망막 내에있는 다른 세포 유형을 형성 수 있습니다. 등 양서류와 물고기 종 (种)에서는, 그들의 눈을 그러나, 부상 5시 이후엔 재생 능력이있다, 포유류의 눈은 그런 재생 특성을 보여줍니다 않습니다. 우리는 심지어 부상 후뿐만 아니라 안구 손상의 수리 물리 또는 유전자 모델을 도와 세포의 잠재적인 소스로 그들을 사용하여 생체내에서 줄기 세포를 식별하고 6-8 포유류의 망막 줄기 세포가 정지 유지 메커니즘을 이해하기 위해 추구 9-12 통해 이식. 여기서 우리는 clonal 망막 줄기 세포 분야를 형성하기 위해 마우스 눈에서 ciliary 상피 세포를 분리과 문화에서 그들을 성장하는 방법에 대해 설명합니다. 생체내에있는 줄기 세포에 대한 알려진 마커가 없으므로,이 분야는 prospectively 눈 ciliary 상피 내에 줄기 세포의 인구를 식별하는 유일한 알려진 방법입니다.

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

이 비디오에서는, 우리는 마우스 눈 ciliary 상피에서 망막 줄기 세포를 분리하고 clonal 망막 분야를 형성하는 문화에서 그들을 성장하는 방법을 보여줍니다 것입니다. 절연되는 분야는 줄기 세포의 추기경 속성을 가지고 : 자기 갱신과 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-of-retinal-stem-cells-from-the-mouse-eye

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JoVE Journal

Biology

24.7K 조회수.  University of Toronto. 이 비디오에서는, 우리는 마우스 눈 ciliary 상피에서 망막 줄기 세포를 분리하고 clonal 망막 분야를 형성하는 문화에서 그들을 성장하는 방법을 보여줍니다 것입니다. 절연되는 분야는 줄기 세포의 추기경 속성을 가지고 : 자기 갱신과 multipotentiality.

비디오: Isolation of Retinal Stem Cells from the Mouse Eye

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

태반에서 조혈 줄기 세포의 분리 및 분석

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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생물학

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

마우스 침샘에 줄기 세포의 분리

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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 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.

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 ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

마우스 망막 안료 상피에서 1 차 셀 문화

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

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 ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

1차 마우스 망막 색소 상피 세포의 효율적인 해부 및 배양

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

돼지 망막 색소 상피 세포의 1 차 배양

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

일차 마우스 망막 색소 상피 세포의 분리

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...