1. 배아 제거 및 placental 해부 <ol><li> 시작하려면, 배아 먼저 임신 댐에서 격리해야합니다. </li><li> 동물이 승인 절차에 따라 euthanized 있습니다 - 우리 사건에서 우리는 마취 isoflurane의 치사량을 사용하는거야 </li><li> 첫째, 에탄올과 복부를 스프레이하고 가위로 작은 절개를합니다. </li><li> 복부를 발견하고 복막을 열고 잘라 양손으로 피부를 떠날. </li><li> 포셉로 자궁 뿔을 선택하고 각 말초 끝에 가위를 사용하여 수집합니다. PBS 얼음에 장소로 가득 배양 접시에있는 자궁 뿔를 놓습니다. 또한 태반을 분리하기 전에 PBS로 여러 번 씻어 기억 해요. 우리는 이제 태반을 분리하실 수 있습니다. </li><li> 이 집게로 조심스럽게 배아 conceptuses를 둘러싼 자궁 내막 조직을 물러나고 시작합니다. 하나는 안 찔린 또는이 이후 절개 단계를 복잡하므로 별도의 배아에 구조적 피해를 입힐 신중해야합니다. </li><li> 얼음 PBS와 함께 새로운 배양 접시에있는 고립된 conceptuses를 배치하고 모든 conceptuses이 endometrium에서 해방 때까지 계속합니다. </li><li> 현미경 거리에 태반에서 decidua 껍질이 집게와 배치 PBS와 함께 새로운 배양 접시 하나 conceptus를 전송합니다. decidua 세포는 단일 세포 현탁액 및 유동세포계측법 방해하므로 가능한 decidua의 많은를 제거합니다. 부드러운 필링 지속적인 운동은 최상의 결과를 달성하는 것이 좋습니다. </li><li> 두 기관 사이의 교차점에있는 태반에서 멀리 난황을 잘라 부드럽게 태반에서 떨어져 난황 및 vitelline 혈관을 가져옵니다. 관리는 특히 초기 태아와 태반의 chorionic 번호판을 방해하지 않도록해야합니다. </li><li> 조심스럽게 한 쌍의 집게의 다른과 탯줄과 태반의 chorionic 접시를 들고, 멀리 태반에서 탯줄과 연결된 태아를 가져옵니다. </li><li> 단일 세포 현탁액의 준비 기간 동안 clumping 휴대폰을 최소화하기 위해 태반의 가장자리에 여분의 거대 세포 조직을 제거합니다. </li><li> PBS 5% FCS와 얼음 등 15 ML 팔콘 튜브로 적당한 용기에 태반을 수집합니다. 해부 모든 placentas에 대해이 작업을 수행합니다. </li><li> 이 시점에서, 여러분은 이러한 체외 문화 또는 이식처럼 유동세포계측법, 기능 assays를위한 단일 세포 현탁액의 준비와 진행하거나 조직 고정 및 최종 immunohistochemistry에 대한 삽입 고정 냉동 블록 전체가 태반을 준비합니다. </li></ol> 2. 태반 조직의 단일 세포 현탁액의 준비 <ol><li> 외과 분석을위한 태반에서 단일 세포 현탁액을 생성하는 방법을 우리는 지금이 표시됩니다. </li><li> 단세포 태반에서 서스펜션, 기계 및 효소 분해를 준비하는 것이 필요합니다. 먼저 10% FCS, 1 % 페니실린 / 스트렙토 마이신 솔루션으로 PBS에 0.​​1 % Collagenase 솔루션을 준비합니다. </li><li> 당신은 얼마나 많은에 따라 placentas에 collagenase 솔루션의 적절한 볼륨을 추가합니다. 두 번 태반 볼륨 2 - 5ml 사이에 볼륨을 권장합니다. </li><li> 기계식 바늘을 3 회를 통해 collagenase 솔루션과 태반을 passaging하여 조직을 방해하는 5 ML 주사기에 장착 16 G 바늘을 사용합니다. 18 G 바늘로 반복합니다. </li><li> 45 분 동안 37 ° C 5 % CO 2 배양기에서 샘플을 놓으십시오. </li><li> collagenase 20 G 바늘을 통해 통과 셀 솔루션, 그리고 37, 추가 45 분 동안 품어 ° C.에 45 분 부화 후 </li><li> 각각의 바늘로 22 - G 25 - G의 바늘을 통해 collagenase, 통로 세포 솔루션에서 1.5 시간 배양 후 총 3 회. </li><li> 새로운 15 ML 팔콘 튜브에 연결된 50 μm의 필터를 통해 샘플을 필터, 4 PBS 5% FCS와 원심 분리기로 씻어 ° C를 5 분간 300 × G. </li><li> 이 시점에서, 우리는 외과의 분석 또는 기능 assays에 적합한 단일 세포 현탁액 있습니다. </li></ol> 3. 냉동 섹션에 대한 조직의 고정 <ol><li> 즉시 각 배아에서 태반을 분리 후, 그들은 차가운 1X PBS에 배치됩니다. 보통 96 - 웰 플레이트 젊은 placentas (E8.5 - 11.5)에 가장 잘 작동하지만, 오래된 placentas과 E12.5 세 이상은 24 잘 플레이트 더 편리합니다. </li><li> 각각의 태반은 갓 해동 4% paraformaldahyde 또는 PFA 잘 채워진 새로 전송됩니다. E12.5 placentas 경우, 조직은 손으로 뉴 웰로 이동하지만, E10.5 위해서는 피펫과 함께 신중하게 PBS를 제거하고 PFA를 추가할 수 있습니다. 그것은 태반이 완전히 조직의 나이와 크기에 따라, 4 ° C에서 2~4시간 위해 잠겨 것이 중요합니다. </li><li> 30 % 자당에 조직을 전송하거나, 조직은 매우 작고 연약 경우, PFA를 대기음 직접 자당을 추가합니다. 이 단계에서는 placentas는 솔루션에 떠있는 것입니다. 조직은 4 박하자 ° C.</li><li> 30 % 자당의 야간 단계 후, 조직은 궁극적으로 적절한 cryopreservation 나타내는 우물의 바닥에 침몰한다. 자당 솔루션의 절반을 제거하고 OCT로 볼륨을 교체하고 ° C 1-2시간 4에서 다시 조직을 놓으십시오. 이것은 OCT 침투를 용이하게합니다. </li><li> 그것이 임베디드 수있는 후 1 시간 100 % OCT 수있는 조직을 전송합니다. </li></ol> 4. 고정 조직을 포함 <ol><li> 적절한 조직 식별 (쇼 예를 들어 곰팡이)와 레이블 플라스틱 금형. </li><li> OCT 솔루션의 조직을 당겨 아래로 향하게 탯줄 사이드와 디스크 모양의 태반을 orienting 조심스럽게 깨끗한 면도날이되도록 압력을 방출하기 전에 앞뒤로 블레이드를 이동해야하는과 깔끔히하여 절반으로 태반을 잘라 심지어 컷. 태반은 나중에 placental HSCs을 시각화하는 가장 좋은 prientation을 달성하기 반으로 있습니다. </li><li> 금형의 하단 표면에 접촉 컷 가장자리와 금형의 하단에 놓으 태반의 절반. 다른 반쪽에 대한 새로운 금형과 반복합니다. E10.5 placentas 들어, 두 반쪽은 동일한 기술을 사용하여 동일한 금형에 게재될 수 있습니다. </li><li> 금형에 OCT를 따르고 때, 그것은 적절한 방향 placentas을 유지하기 어려울 수 있습니다. 포셉와 조직 절반을 누른 위치에 태반을 확보. 천천히 금형에 OCT 솔루션을 부어하고 거품을 생산하지 마십시오. E10.5 placentas 들어, 포셉 한 조직 절반을 보유하고 포셉의 외부에 대한 다른 조직 절반을 나머지. 천천히 같은 E12.5 placentas에 대한 설명 OCT 솔루션을 부어. </li><li> 곰팡이가 OCT 가득되면, 신속하게 드라이 아이스로 금형을 배치, OCT는 조직을 냉동 흰 플래시를 설정합니다 </li><li> Drierite을 포함하는 작은 비닐 봉지에 곰팡이를 놓고 즉시 -80 ° C의 냉동고에 저장합니다. </li></ol>

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

이 프로토콜에서 설명하는 실험 절차는 placental 조직 및 조혈 줄기 및 전구 세포의 분리 및 시각화를위한 수 있습니다. 태아 발달에 걸쳐 태반 및 기타 태아 조혈 장기에 HSCs의 예상 세포 수율과 전화 번호에 대한 포괄적인 요약을 위해 우리는 Gekas, 외를 참조하십시오. 2005. 태반의 개발 HSCs 및 기타 조혈 세포의 지방화에 대한 우리 로즈, 외를 참조하십시오. 2008.

자료 목록 : 100mM 포도당 10mM NaN3 15 ML 원뿔 튜브 (BD) 16 G, 18 G, 20 G, 22 - G 25 - G의 바늘 (BD) 30 % 자당 솔루션 35mm 배양 접시 (BD) 4 % Paraformaldehyde 50 - m 전지 필터 (Celltrics) 5 ML의 주사기 (BD) 알카라인 인산 가수 분해 효소 기판 키트 I (벡터 빨강, 벡터) 알카라인 인산 가수 분해 효소 기판 키트 III (벡터 블루, 벡터) 항생제 (페니실린 / 스트렙토 마이신, P / S) 솔루션 Collagenase 유형 I (시그마) Cryomold 일회용 비닐 스피스먼 금형 (10mm X 10mm X 5mm, 조직 테크) Cytokeratin 차 항체 (DakoCytomation) 해부의 포셉, 스테인레스 스틸이나 티타늄, 숫자 5 55 Drierite 태아 소 혈청 (FBS) Fisherbrand 플라스틱 현미경 슬라이드 메일러 (피셔) 포도당 산화 효소의 1000u/ml Levamisole (벡터) 마운트 미디어 수성 기반 장착 중간 (VectamountTM AQ) 일반 호스 세럼 (벡터) 최적의 절삭 온도 (OCT, 조직 테크) 퍼옥시데이즈 기판 키트 DAB (벡터) 인산은 칼슘 / 마그네슘 (W Ca2 + / Mg2 +)와 살린 (PBS)를 버퍼 인산은 칼슘 / 마그네슘 (W / O Ca2 + / Mg2 +)없이 살린 (PBS)를 버퍼 플라스틱 슬라이드 박스 (100 슬라이드를 보유) 플라스틱 관 (Nalgene 180 PVC 무독성 Autoclavable) 폴리 프로필렌 라운드 - 히프 테스트 튜브, 5 ML (12x75 mm, BD 팰컨) Proteinase K 20mg/ml 솔루션 (Amresco) 연구 제품 국제 코프 슈퍼 아빠 펜 HT * 슬라이드 마커 (2,5 mm, 피셔) 조직 문화 U 형 (원형 - 하단) 처리 96 자 또는 24 - 잘 접시 (BD 팰컨) Tween20, SigmaUltra (시그마) Tyramide 증폭 키트 (Invitrogen) Vectastain ABC 알카라인 인산 가수 분해 효소 표준 키트 (벡터) Vectastain ABC 표준 엘리트 키트 (벡터)

isolation and analysis of hematopoietic stem cells from the placenta

조혈 줄기 세포 (HSCs)는 자기 갱신과 개인의 일생 동안 혈액 lineages 모든 세포 유형을 생성하는 기능이 있습니다. 모든 HSCs는 수영장 크기가 셀프 갱신 세포 분열에 의해 유지되는 후, 배아 발달 동안 등장. 많은 해부 학적 사이트가 태아의 조혈 동안 참여로 HSC 발전의 과정을 규제 HSCs하고 중요한 발달 사건의 해부 출처를 확인하는 것은 복잡하고 있습니다. 최근, 우리는 HSCs가 (Gekas, 외 2005, 로즈, 외 2008) 생성과 독특한 microenvironmental 틈새에 확장하는 중요한 조혈 기관으로 태반을 확인. 따라서 태반들이 출현 및 초기 확장 중에 HSCs의 중요한 원천입니다. 이 문서에서는, 우리는 E10.5 각각 HSCs의 개시와 태반에서 HSC 수영장의 크기에 최대의 발달 단계에 해당 E12.5 태아, 태반에서 murine의 격리에 대한 해부 기술을 보여줍니다. 또한, 우리는 유동세포계측법 또는 기능 assays에 사용하기 위해 단일 ​​세포 현탁액에 placental 조직의 효소 및 기계적 분리에 대한 최적화된 프로토콜을 제시한다. 우리는 태반의 단일 세포에 대한 사용 중지 collagenase의 사용 HSCs의 충분한 수율을 제공 것으로 나타났습니다. 태반에서 HSC 수율에 영향을 미치는 중요한 요소는 사전에 기계적 분리의 정도, 그리고 효소 치료의 기간입니다. 우리는 또한 정확한 세포 틈새에 immunohistochemistry으로 HSCs을 개발의 시각화를위한 고정 냉동 placental 조직 섹션의 준비를 위해 프로토콜을 제공합니다. 조혈 특정 항원이 파라핀 임베디드 섹션의 준비 기간 동안 보존되지 않습니다, 우리는 정기적으로 placental HSCs과 progenitors을 번역을위한 고정 냉동 섹션을 사용합니다.

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

우리는 개발하는 동안 중요한 조혈 기관으로 태반을 확인했습니다. 우리는 조혈 줄기 세포 (HSCs)이 생성되는 독특한 microenvironmental 틈새의 태반에서 확장 둘 발견했습니다. 여기, 우리는 마우스 태반에 HSCs의 격리 및 시각화에 필요한 실험 기법을 설명합니다.

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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20.3K 조회수.  University of California, Los Angeles. 우리는 개발하는 동안 중요한 조혈 기관으로 태반을 확인했습니다. 우리는 조혈 줄기 세포 (HSCs)이 생성되는 독특한 microenvironmental 틈새의 태반에서 확장 둘 발견했습니다. 여기, 우리는 마우스 태반에 HSCs의 격리 및 시각화에 필요한 실험 기법을 설명합니다.

비디오: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

분리 및 조혈 줄기 세포의 이식 (HSCs)

연구

생물학

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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 derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

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 ...

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 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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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 ...

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 ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

쥐과 배아의 레트로 바이러스 감염은 조혈 분화 분석을위한 세포 유래 배아 바디 줄기 세포

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNA 시퀀싱 분석하여 EML 조혈 전구체 세포의자가 재생 (self-renewal)과 분화를 조절하는 중요한 요소의 식별

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

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 ...