배아 줄기 세포의 분화 <ol><li> ESC 트립신과 치료를 통해 배아 줄기 세포 (ESC)의 근처에 합류 플라스크를 수집합니다. </li><li> 차별화 미디어 5 ML에서 세포를 Resuspend가 아닌 젤라틴 코팅 T25로 전송할 수 있습니다. 37 품어 ° C를 45 분 정도 사용합니다. 뜨는 회수하여 원심 분리를 통해 세포를 수집합니다. 참고 : ESC 문화에서 마우스 배아 섬유아 세포 (MEFs)가 아닌 젤라틴 코팅 T25에 쉽게 부착하고 있으므로이 도금 단계에서 문화 고갈됩니다. 당신이 MEFs에 culturing 귀하의 ESC를하지 않은 경우, 당신은 사전 플레이트를 건너뛸 수 있습니다. </li><li> 333,333 cells/50 ML에서 차별화 미디어에 Resuspend MEF - 고갈 ESC. 이 농도 100 ESC/15 ML 결과. </li><li> 를 사용하여 다중 채널 pipettor, 15cm이 배양 접시에 100 cells/15 ML 드롭에 플레이트 ESC. 8 채널 pipettor으로, 당신은 접시마다 방울의 18-22 행 (접시 당 5ml에 대한)에 대한 맞게 수 있어야합니다. 참고 :이 프로토콜의 경우, 방울 2-5 15cm이 요리 충분보다되며, 2-5 X 10 6 6 번째 EB - 파생 세포를 얻을 것입니다. </li><li> 부드럽게 방울을 반전하는 접시를 플립. ° C 5 % CO 2 48 시간 37 알을 품다. </li><li> 풀 부드럽게 15 ML 원뿔 관에 접시와 전송을 소용돌이로 방울. PBS의 4-6 ML과 접시를 씻어와 같은 15 ML 원뿔 추가할 수 있습니다. 참고 : 다섯 매달려 드롭 판까지 풀 수 있습니다. 그들이 차별을 계속하고 너무 집중되어있다면 그들은 큰 대단히 짧은 시간을 형성하는 경향으로 EBS는 자랄거야. </li><li> 풀링 EBS 중력 (약 10 분)에 의해 정착하도록 허용합니다. EBS 방해없이 뜨는을 대기음. 부드럽게 10 ML 차별 미디어와 10cm 비 점착 성의 배양 플레이트에 전송에 resuspend. </li><li> 37 50 RPM과 부화에 플레이트 흔드는 일에 배양 접시 ° C 5 % CO 2. </li><li> 24 시간 후에 (분화의 일 사), 소용돌이 모양의 접시는 페트리 접시의 중앙에 EBS를 집중. 조심스럽게 교환 5 ML 신선한 차별화 미디어와 미디어의 50 % (5 ML). 이틀 더 배양기에 플레이트를 반환합니다. </li></ol> MSCV - HoxB4 - IRES - GFP와 EB 해리와 감염 <ol><li> 분화의 6 번째에서 15 ML 원뿔 관에 EBS를 전송할 수 있습니다. 중력에 의해 해결하도록 허용합니다. </li><li> 10 ML PBS에서 미디어 및 resuspend을 대기음. EBS는 중력에 의해 다시 정착하도록 허용합니다. 대기음 PBS. </li><li> 250 ML 분리 효소 믹스 1 ML PBS를 추가합니다. 가끔 효소와 EBS를 섞어 관을 소용돌이, 20 분 동안 37 ° C의 물을 욕조에 품어. </li><li> 8 ML 효소 무료 분리 버퍼를 추가합니다. </li><li> EBS 완전히 dissociated 때까지 혼합물은 (; 과도한 pipetting는 EB - 유도 세포 준비 내에 사망 증가 10 번) 5 ML의 피펫을 사용하여 가루로 빻다. <ol><li> 혼합물은 EBS가 교제를 끊다로 세포 흐리고 될 것입니다. </li><li> 분쇄하는 동안 원뿔 튜브의 바닥에 대한 피펫 팁을 게재하면 크게 분리를 촉진하는 전단 힘을 만들어집니다. </li></ol></li><li> 원심 분리를 통해 세포를 수집합니다. </li><li> Resuspend 10 % IMDM 5 ML에서 세포를 EB - 파생와 trypan 파랑 배제를 사용하여 계산 참고 : 분화의 일 넷이서 6 번째 사이에, EBS cavitate있는가, 일 6 시에, apoptosis와 세포 death.Thus 많은 양의 결과 이상 EB - 파생 세포의 30 % trypan과 청색의 착색 수 있습니다. </li><li> Resuspend EB - 파생 100,000 세포에서 세포 / 2 ML 10% IMDM + 바이러스 뜨는 같은 것을 뫄 50-10의가 8 MG / ML의 최종 농도 프로타민 황산을 achieved.Add입니다. 참고 : 판 사 6 - 잘 접시 (물론이 ML / 가정) 정도 EB / 바이러스 뜨는 믹스를 준비합니다. </li><li> 플레이트 2 ML EB / 바이러스 뜨는 믹스 당 우물의 OP9 기질 세포 (아래 참조)로 사전 도금 사 육 잘 접시. </li><li> 90 분간 상온에서 2,500 rpm으로 번호판을 원심 분리기. 37 인큐베이터에 번호판을 전송 ° C 5% CO 2. </li><li> 4-6시간 후, 접시에서 뜨는 수확하고 원심 분리를 통해 정지에 남아있는 잠재적인 세포를 수집합니다. </li><li> 48 ML 10 % IMDM에 Resuspend 펠렛 (작은 수) + 크린 시토킨. 2 ML 하는걸 / 잘 감염이 수행되었으며 뜨는 수집어진 오리지널 사 6 잘 접시에 resuspension니다. 참고 : 항상 사용 시간에 신선한 10% IMDM + 크린 시토킨를 준비합니다. </li><li> ° C 5 % CO 2 이레 동안 37 번호판을 품어. 식민지 하루 푸르 포스트 감염 및 대형 및 7 일째에 의해 잘 형성에 의해 명확해야합니다. 강력한 확장 / 잘 일 7 40-60 식민지를 얻을 것입니다. </li><li> 7 일째 이후의 감염에 트립신과 치료의 모든 우물의 모든 세포를 (OP9 기질 포함) 수집 및 수영장. 참고 : 뜨는 또는 트립신 treatment.At 이때 문화 중에 고용 PBS 세척을 삭제하지 마십시오, 일부 식민지는 느슨하게 자기편 수 많은 세포가 floa있다suspension.Collect과 수영장 모든 세척 및 뜨는에서 팅은 잠재적으로 가치있는 세포가 폐기되지 않도록합니다. </li><li> 8 ML 신선한 10 % IMDM에 Resuspend 세포 + 크린 시토킨. 네 T75 flasks 2 개의 MLS / 플라스크를 배포합니다. 각 플라스크에 추가 13 MLS 10 % IMDM의 이상 크린 시토킨를 추가합니다. 37 품어 ° C 5% 사흘 동안 CO 2 (10 일 게시물 감염 총). </li></ol> 문화 OP9 기질의 도금 <ol><li> 20% - 멤의 표준 프로토콜에 따라 OP9 기질을 유지합니다. confluency로 성장하면 OP9 전지는 속성을 변경합니다. 항상 더 이상 1시 3분 이상의 분할에 confluency 80 % 분할. </li><li> 24 이전에 / 잘 20% - 멤 네 개의 6 - 잘 접시의 MSCV - HoxB4 - IRES - GFP, 플레이트 25,000 OP9 세포와 6 번째 EB - 파생 세포의 감염 시간. </li></ol> 수집, 분류 및 이식 <ol><li> 트립신과 치료를 통해 열흘 동안 OP9 기질에 대한 확장된 세포를 수집합니다. 세포를 세어보세요. 참고 : 뜨는 또는 PBS를 폐기하지은 트립신 처리하는 동안 고용 씻는다. 문화의이 시점에서, 일부 식민지는 느슨하게 자기편 수 있으며 suspension.Collect과 수영장에 잠재적으로 가치있는 세포가 폐기되지 않도록 모든 세척 및 뜨는을 부동 많은 세포가있다. 참고 : 좋은 확장 160-200 X 10 6 세포의 총, 40-50 X 10 6 세포 / 감염 원래 6 자 플레이트 사이에 양보해야합니다. </li><li> 세포는 프로토콜이 시점에서 표준 기술에 따라 자기 구슬 선택이나 외과 통해 fractionated 수 있습니다. </li><li> 이식의 경우, 방사선 조사 2.5 시간의 9.25 GY의 분할 선량의 대상이 Rag-2/gc 결함 수신자 (15~22g 사이 무게)로 역전사 궤도 또는 꼬리 정맥 주입을 통해 세포를 제공합니다. 분리. 참고 : 동물의 무게는 방사선 조사의 시간에 15~22그램 사이에 떨어질 것이 중요 좋습니다. 그들이 22g보다 큰 경우, 9.25 GY 해당 절제를 보장하고 세포가 치명적인 조사 및 / 또는 합치다 조혈 구획의 구조를 중재하지 않을 수 있습니다 방사선의 충분한 복용량되지 않습니다. 큰 동물을 이식하는 경우, 적절한 방사선 선량은 실험적으로 결정되어야합니다. </li><li> 2-5 X 10 6 세포 / 동물의 최소 복용은 치명적인 조사에서 구출을 보장하기 위해 필요합니다. 세포를 fractionating 경우, 2-5 X 10 6 세포에 해당하는 금액 (예 : 관심의 인구가 세포의 unfractionated 수영장의 10 %를 대표하는 경우, 2-5 X 10 5 세포 / 동물을 주사) 주사. 참고 :&gt; 2 주입하면 X 10 6 세포는 항상 세포의 높은 번호가 복고풍 - orbitally 전송하는 경우 발생할 수 있습니다 궤도에서 기형 종의 형성을 피하기 위해 꼬리 정맥을 통해 주입. </li><li> 유지 래그 - 2 - / - / GC - / - autoclaved 새장에 부족한받는 사람. 동물 시설의 &quot;청결&quot;에 따라 사후 이식 동물 치명적인 방사선 다음 5 주 동안 산성의 물 또는 Trimethiprim - Sulfasoxazole (Septra)의 관리를 요구할 수 있습니다. </li><li> 삼주 후 이식에서 말초혈에서&gt; 90 % GFP + ES - 파생 leukocytes 것으로 기대하고 있습니다. lymphocytes이 retroviral HoxB4 - IRES - GFP를 침묵하는 것으로 알려져 있지만 Engraftment는 멀티 혈통해야합니다. </li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

줄기 세포가 수있는 능력을 가진 세포로 정의 모두 자기 갱신 여러 차별 자손을 생성합니다. 배아 줄기 세포 (ESC)은 초기 배아의 blastocyst에서 파생된 및 differentiative 능력 pluripotent입니다. 그들의 광대한 differentiative 가능성들을 임상적으로 유용한 세포 유형을 생성하기 위해 그들을 동축 케이블하는 방법 deducing을 중심으로 많은 연구의 초점이되었다. 마우스 ESC에서 조혈 줄기 세포 (HSC)의 성공적인 유도가 최근 완성되었으며이 비디오 프로토콜의 시각하실 수 있습니다. HSC, 거의 틀림없이 가장 임상적으로 악용 세포 인구는 조혈 malignancies 및 장애의 무수한를 치료하는 데 사용됩니다. 그러나, HSC 치료 혜택을 누릴 수 많은 환자가 적합한 기증자에 대한 액세스를 부족합니다. ESC는 이러한 환자 HSC의 대체 소스를 제공할 수. 다음 프로토콜은 ESC - HSC이 공부하고 인간 ESC에서 HSC를 격리하는 노력을 알려 될 수있는 기준을 설정합니다. 이 프로토콜에서는 ESC가 상용 혈청 사전 검사를 최적의 조혈 차별화 6 일 동안 embryoid 기관 (EBS)로 구별됩니다. EBS 다음 dissociated과 retroviral HoxB4에 감염됩니다. 마우스, 그리고 열흘 동안 크린 시토킨를 추진하고 조혈의 존재 공동 교양 - 감염된 EB - 파생 전지 OP9 기질, M - CSF - /의 calvaria에서 파생된 골수 stromal 세포 라인에 도금입니다. 이 공동의 문화 중에 감염된 세포는 세대 세포의 수백만의 100s의 이기종 수영장 결과 크게 확장합니다. 이러한 전지는 다음 구조와 reconstitute lethally 조사 마우스로 사용할 수 있습니다.

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

이 프로토콜 세부 마우스 배아의 줄기 세포 (ESC)와 lethally 조사받는 마우스에 그들의 이후의 주입에서 transplantable 조혈 줄기 세포의 유도. 간단히, ESC는 다음 retroviral HoxB4 및 OP9 stromal 세포와 조혈 크린 시토킨 공동 양식에 감염된 embryoid 단체로 구별됩니다.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

derivation-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

13.2K 조회수.  Harvard Medical School. 이 프로토콜 세부 마우스 배아의 줄기 세포 (ESC)와 lethally 조사받는 마우스에 그들의 이후의 주입에서 transplantable 조혈 줄기 세포의 유도. 간단히, ESC는 다음 retroviral HoxB4 및 OP9 stromal 세포와 조혈 크린 시토킨 공동 양식에 감염된 embryoid 단체로 구별됩니다.

비디오: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

인간의 배아 줄기 (ES) 세포의 전파

연구

생물학

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

분리 및 조혈 줄기 세포의 이식 (HSCs)

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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. ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Murine 난황과 AGM에서 조기 조혈 줄기 세포의 분리

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Blastocysts에서 마우스 Trophoblast의 줄기 세포의 유도

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Murine 조혈 줄기 세포와 리니지 - 헌신 Progenitors의 Phenotypic 분석 및 절연

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...