우리는 귀중한 조언을 Nobuyuki Onai, 히토시 타키자와 마르쿠스 Manz 감사드립니다. 이 작품은 스위스 국립 과학 재단, 스위스 암 리그와 Fondazione Ticinese마다 라 RIcerca ㅡ Cancro에 의해 재정 지원되었다.

1. 골수의 세포 현탁액의 준비 <ol><li> 마우스를 안락사와 복부 뒤로 향해 스테인리스 냄비와 스프레이 70 % 에탄올에 동물을 배치하십시오. 대퇴골과 경골 뒷다리에서, 그리고 척추를 수집합니다. </li><li> 정확하게 날카로운 팁 가위로와 거즈로 다리 뼈에서 척추에서 모든 연조직 잔류물을 제거합니다. 50 μm의 β-메르 캅 토 에탄올, 100 μm의 제품 MEM 비 필수 아미노산과 보충 10% 열 inactivated 태아 소 혈청 (FBS),, 1 밀리미터 MEM 나트륨 Pyruvate, 2를 포함하는 RPMI 매체 30 ML과 50 ML 팔콘 튜브의 상점 뼈 MM L-글루타민, 100 μg / ML의 스트렙토 마이신 및 100 U / ML 페니실린. </li><li> 무균 필터와 이전에 소독을 절구의 바닥을 커버. </li><li> 절구에 뼈를 전송 및 마찰 표면을 조립하기 위해 필터 계층으로 그들을 포괄합니다. </li><li> 팔콘 필터와 함께 제공되는 50 ML 팔콘 튜브를 준비합니다. </li><li> 뼈를 부술균일한 세포 현탁액을 얻기까지 유봉의 도움으로. </li><li> 튜브로 정학을 전송합니다. </li><li> 남아있는 세포를 씻고 기증 신선한 RPMI 전체 매체의 박격포 5 ML에 추가합니다. </li><li> 균질 뼈를 확실히 나타날 때까지 마지막 두 포인트를 반복합니다. </li><li> 나머지 조직 파편을 제거하기 위해 세포 현탁액을 전송하고 새 튜브로 필터링합니다. </li><li> 5 분을위한 1,500 rpm으로 튜브를 원심 조심스럽게 흡인에 의해 뜨는을 제거합니다. 세포 펠렛을 방해하지 않도록하십시오. </li></ol> 2. Phenotypic 분석 <ol><li> Resuspend 및 PBS와 2% FBS의 15 ML에서 개별 생쥐의 세포를 씻는다. </li><li> 5 분을위한 1,500 rpm으로 튜브를 원심 조심스럽게 흡인에 의해 뜨는을 제거합니다. 펠렛을 방해하지 않도록하십시오. </li><li> 5 ML PBS와 2퍼센트 FBS 및 카운트 세포에 펠렛을 일시 중지합니다. </li><li> 이전 1 × 10 6 세포 /도 96 라운드 우물 밑바닥에서판. </li><li> 5 분을위한 1,500 rpm으로 번호판을 원심 분리기. </li><li> 다음과 같은 표면 마커 향한 mAbs의 혼합을 준비합니다. 그들을 희석은 PBS에 표시된 및 2퍼센트 FBS가 세포를 얼룩. <ul type="disc"><li> CD4, CD8, 인 경우에는 3 번 CD, CD45R, CD19, Gr1, Ter119, NK1.1 PE-Cy5 1:200으로 복합 </li><li> APC 1:100으로 C-키트 복합 </li><li> 비오틴 1시 50분에 복합 Sca1 </li><li> FITC 1시 50분에 복합 CD34 </li><li> PE 1:100으로 복합 FcγR </li><li> PE-Cy7 1:100으로 IL-7R 복합 </li></ul></li><li> 뜨는을 무시하고 각 잘으로 mAbs 믹스의 50 μL를 추가합니다. pipetting하여 단일 세포로 분열시키다. </li><li> 4 ° C.에 어둠 속에서 20 분 동안 세포를 품어 </li><li> PBS 150 μL 및 2% FBS로 두 번 세포를 씻으십시오. </li><li> 5 분을위한 1,500 rpm으로 접시를 원심 분리기와 반전 판에 의해 뜨는 버리고. </li><li> PBS에 streptavidin을 희석하고 2 % FBS는 세포를 얼룩이 지적했다. <ul type="disc"><li> Streptavidin conjuAPC-Cy7 1:300으로 문이 </li></ul></li><li> 각도에 대한 해결책 50 μL를 추가합니다. pipetting하여 단일 세포 현탁액으로 교제를 끊다. </li><li> 4 ° C.에 어둠 속에서 10 분 대한 세포를 품어 </li><li> PBS 150 μL 및 2% FBS로 두 번 세포를 씻으십시오. </li><li> 5 분을위한 1,500 rpm으로 접시를 원심 분리기와 뜨는 버리고. </li><li> PBS와 2% FBS 100 μL의 세포를 Resuspend. </li><li> FACS에서 샘플을 습득. </li></ol> 3. C-키트 + 세포의 자성 농축 <ol><li> PBS, 산도 7.2, (BSA) 0.5 % 소 혈청 알부민, 2 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산)를 포함하는 완충 용액을 준비한다. </li><li> APC에 복합 C-키트 mAb를 포함하는 완충 용액의 1 ML에서 최소 10 생쥐에서 추출한 뼈 marrows의 세포 펠렛을 Resuspend은 1시 50분을 희석. </li><li> 4 ° C.에 어둠 속에서 20 분 동안 세포를 품어 </li><li> 완충 용액의 40 ML로 두 번 세포를 씻으십시오. </li><li> 5 분을위한 1,500 rpm으로 튜브를 원심 조심스럽게 T를 제거그는 흡인에 의해 뜨는. 펠렛을 방해하지 않도록하십시오. </li><li> Resuspend 세포 펠렛은하고 펠릿 직접 각 euthanized 마우스에 대한 안티 - APC의 MicroBeads 50 μl를 추가합니다. </li><li> 잘 섞어서 4 ° C.에 어둠 속에서 20 분 동안 품어 </li><li> 5 분을위한 1,500 rpm으로 완충 용액 및 원심 분리기 40 ML을 추가하여 세포를 씻으십시오. 완전히 뜨는 기음. </li><li> 사흘에 마우스 euthanized 완충 용액 4 ML의 여러 관련 Resuspend 세포 펠릿. </li><li> 적당한 맥에서 구분 기호의 자기장의 장소는 맥에서 LS 열을. 사흘에 마우스 euthanized 한 컬럼을 사용합니다. </li><li> 완충 용액 4 ML과 rinsing하여 컬럼을 준비합니다. </li><li> 컬럼에 세포 현탁액을 적용합니다. </li><li> 통과 및 완충 용액 4 ML로 칼럼을 씻고 레이블이 지정되지 않은 세포를 수집합니다. 완충 용액 4 ML 세 번 추가하여 세척 단계를 수행합니다. 열 저수지가납니다 새로운 버퍼를 추가합니다. </li><li> 구분 및 plac에서 열 제거전자 15 ML 팔콘 튜브 위에 각각. </li><li> 각 열에 진입 완충 용액의 피펫 4 ML. 즉시 열에 플런저를 밀어 자기 (磁 气)라고 표시된 전지를 살아가기. </li><li> 5 분을위한 1,500 rpm으로 튜브를 원심 조심스럽게 흡인에 의해 뜨는을 제거합니다. </li><li> 한 15 ML 팔콘 튜브의 완충 용액 및 그룹 알약 1 ML 각 펠렛을 Resuspend. </li></ol> 4. 조혈 줄기 세포와 리니지 헌신 Progenitors으로 정렬 <ol><li> 다음과 같은 표면 마커 향한 mAbs의 혼합을 준비합니다. 그들을 희석은 세포를 얼룩이 500 μL PBS와 2% FBS에 표시된. <ul type="disc"><li> CD4, CD8, 인 경우에는 3 번 CD, CD45R, CD19, Gr1, Ter119, NK1.1 PE-Cy5 1:200으로 복합 </li><li> APC 1:100으로 C-키트 복합 </li><li> 비오틴 1시 50분로 SCA-1 복합 </li><li> FITC 1시 50분에 복합 CD34 </li><li> PE 1:100으로 복합 FcγR </li></ul></li><li> previou에서 튜브를 원심 분리기님의 5 분을위한 1,500 rpm으로 섹션을 조심스럽게 흡인에 의해 뜨는을 제거합니다. </li><li> 펠렛에 mAbs 믹스를 추가합니다. pipetting하여 단일 세포 현탁액으로 교제를 끊다. </li><li> 4 ° C.에 어둠 속에서 20 분 동안 세포를 품어 </li><li> PBS의 14mL 및 2% FBS로 두 번 세포를 씻으십시오. </li><li> 5 분을위한 1,500 rpm으로 튜브를 원심 분리기와 뜨는 버리고. </li><li> streptavidin을 희석은 500 μL PBS에 표시된와 2 %의 FBS 세포를 얼룩. <ul type="disc"><li> APC-Cy7 1:300으로 복합 Streptavidin </li></ul></li><li> 튜브에 솔루션을 추가합니다. pipetting하여 단일 세포 현탁액으로 교제를 끊다. </li><li> 4 ° C.에 어둠 속에서 10 분 대한 세포를 품어 </li><li> PBS의 14 ML 및 2% FBS로 두 번 세포를 씻으십시오. </li><li> 5 분을위한 1,500 rpm으로 튜브를 원심 분리기와 뜨는 버리고. </li><li> PBS와 2% FBS 500 μL의 세포를 Resuspend. </li><li> 엘리를 위해 새로운 5 ML 관의 세포 현탁액을 전송 및 필터링분류 절차를 방해할 수 세포 집계를 minate. </li><li> 정렬 인구에서 7-AAD + 죽은 세포를 제외하기 위해 세포 현탁액에 7-AAD 5 μL를 (이것은 1:100 희석 사용) 추가합니다. </li><li> 다음 phenotypes에 따라 BD FACSAria로 정렬 세포 : <ul type="disc"><li> LKS 세포 : 린 - C-키트 + 무서-1 + </li><li> LT-HSCs : LKS CD34 - </li><li> ST-HSCs : LKS CD34 + </li><li> GMPs : 린 - C-키트 + 무서-1 낮은 FcγR 높은 CD34 + </li><li> MEPs : 린 - C-키트 + 무서-1 낮은 FcγR 낮은 CD34 - </li><li> CMPs : 린 - CD34 C-키트 + 무서-1 낮은 FcγR 낮음 + </li></ul></li></ol> 5. 대표 결과 한 쇼 및 전자 게이팅 절차의 예제와 그림린과 같은 점수를 일반적인 림프 progenitors (CLPs) - / C - 키트 싸다 / 인터루킨 (IL)-7R + 세포, 린 - / C-키트 + / 무서-1 + (LKS) 및 린 - / C-키트 + / 무서 -1 이오 (C-키트 +) 세포, C-키트 + 게이트에서, 일반적인 골수양 progenitors (CMPs)는 FcγR 싸다, CD34 + 세포, FcγR 하이 CD34 + 세포 및 / megakaryocyte 같은 과립 / 단핵세포 progenitors (GMPs)로 식별됩니다 FcγR 싸다, CD34와 같은 적혈구 progenitors (MEPs) - 세포, LKS 게이트, LT-HSC와 ST-HSC에서는 CD34으로 식별됩니다 - 그리고 CD34 + 세포, 각각. 그러면 위에서 설명한 표현형에 따라 CD34 표현뿐만 아니라 다른 progenitors에 따라 정렬하실 수 있습니다 LT-HSC와 ST-HSC,이 골수 세포 현탁액은 자기 구슬 (그림 2)와 C-키트 + 세포에 대해 풍부하게 할 수 있습니다. HSCs 아나 수<html> 세포가 염기 결합 화합물 quinacrine 11뿐만 아니라 핵 빨간색 (DRAQ5) 물들일되었습니다 - CD34는 그림 4와 같이 라이브 이미징 공촛점 현미경으로 lyzed. HSCs에 있지만 GMPs 아닌 quinacrine - 긍정 vesicles는 세포질 12 볼 수 있습니다. + LT-HSC에 노출시 purinergic P2 수용체의 활성화에 의한 cytosolic 칼슘 2의 증가를 보여주는 그림 5에서 보여준대로 더욱이, 정렬 HSCs는 기능적 실험에 사용할 수 아데노신-triphosphate (ATP)와 ionomycin 12. <img alt="그림 1" src="/files/ftp_upload/3736/3736fig1.jpg" /> 그림 1. 린 - BM 전지의 대표 점 플롯 분석. 프로토콜 텍스트에 설명된대로 골수 세포 현탁액에서 조혈 progenitors과 HSCs를 파악하고 채점하는 흐름 cytometer에서 전자 게이팅 절차 묻다. pload/3736/3736fig2.jpg &quot;/&gt; 그림 2. 골수에서 C-키트 + 세포의 선택적 농축을위한 제도. <img alt="그림 3" src="/files/ftp_upload/3736/3736fig3.jpg" /> 그림 3 전자 문이 LKS CD34의 세포주기 분석 -. 건강한 컨트롤 및 KI-67 항체와 DAPI 물들일 IBD와 생쥐의 HSCs. 세포가 수정 및 / Lyse 고정 및 1 μg / ML에서 FITC KI-67 복합 및 DAPI로 더럽히는 것 다음에 버퍼를 파마로 permeabilized되었다. 사이클링 세포들은 거의 LKS CD34의 모든 감지의 경우 - 건강한 컨트롤 12 HSCs 구획. <img alt="그림 4" src="/files/ftp_upload/3736/3736fig4.jpg" /> . 뉴클레오 티드 결합 화합물 quinacrine 및 핵 빨간색 (DRAQ5) 물들일 HSCs과 GMPs - 그림 4는 CD34을 정렬 FACS의 이미징 공촛점 현미경을 살고 있습니다. 작은 quadrants는 quinacrine POS를 보여itive vesicles는 HSCs의 세포질에서 발견되지만 GMPs 없습니다. <img alt="그림 5" src="/files/ftp_upload/3736/3736fig5.jpg" /> 그림 5. Cytosolic 칼슘 2 + Fura-2로로드되고 ATP와 ionomycin으로 자극 정렬 HSCs의 고도. 단일 세포의 성분이 표시됩니다. 모든 세포는 + ATP가 추가 후 cytosolic 칼슘 2의 현저한 증가를 보여주는 세포외 ATP에 반응했다.

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Commun. 331, 1429-1438 (2005).</li><li>Casati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+regulation+of+hematopoietic+stem+cell+cycling+activity+by+ATP.">Cell-autonomous regulation of hematopoietic stem cell cycling activity by ATP.</a> Cell Death Differ. 18, 396-404 (2011).</li><li>Bouma, G., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunological+and+genetic+basis+of+inflammatory+bowel+disease.">The immunological and genetic basis of inflammatory bowel disease.</a> Nat. Rev. Immunol. 3, 521-533 (2003).</li><li>Takizawa, H., Regoes, R. R., Boddupalli, C. S., Bonhoeffer, S., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+variation+in+cycling+of+hematopoietic+stem+cells+in+steady+state+and+inflammation.">Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.</a> J. Exp. Med. 208, 273-284 (2011).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

여기서 설명한 방법은 개별 생쥐 (그림 1) 조혈의 신속하고 정확한 분석을 가능하게합니다. murine 염증의 모델, autoimmunity, 면역 결핍, 퇴행성 질환, 대사 질환 및 암 등 다양한 실험적 설정,이 분석은 조혈에 대한 병리 학적 조건의 영향을 해결하실 수 있습니다. 그림 3은 전자 문이 LKS의 세포 사이클링 활동의 분석을 보여줍니다 CD34 - KI-67 mab 및 DAPI로 더럽?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> RPMI 1640 </td><td> Gibco </td><td> 42,401 </td></tr><tr><td> MEM NEAA 100X </td><td> Gibco </td><td> 11,140 </td></tr><tr><td> 나트륨 Pyruvate </td><td> Gibco </td><td> 11,360 </td></tr><tr><td> PenStrep </td><td> Gibco </td><td> 15,070 </td></tr><tr><td> PBS </td><td> Gibco </td><td> 20,012 </td></tr><tr><td> FBS </td><td> Gibco </td><td> 16,000 </td></tr><tr><td> 셀 스트레이너 40 μm의 </td><td> BD 팔콘 </td><td> 352,340 </td></tr><tr><td> 7-AAD의 염색법 솔루션 </td><td> BD Pharmingen </td><td> 559,925 </td></tr><tr><td> 버퍼를 Lyse / 수정 </td><td> BD Pharmingen </td><td> 558,049 </td></tr><tr><td> 버퍼 III를 파마 </td><td> BD Pharmingen </td><td> 558,050 </td> &lt;/ TR&gt; <tr><td> KI-67 </td><td> BD Pharmingen </td><td> 556,026 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> CD4 (GK1.5) </td><td> eBioscience </td><td> 150,041 </td></tr><tr><td> CD8 (53-6.7) </td><td> eBioscience </td><td> 150,081 </td></tr><tr><td> 인 경우에는 3 번 CD (145-2C11) </td><td> eBioscience </td><td> 150,031 </td></tr><tr><td> CD45R (RA3-6B2) </td><td> eBioscience </td><td> 150,452 </td></tr><tr><td> CD19 (6D5) </td><td> eBioscience </td><td> 150,193 </td></tr><tr><td> Gr1 (RB6-8C5) </td><td> eBioscience </td><td> 155,931 </td></tr><tr><td> Tre119 (TER-119) </td><td> eBioscience </td><td> 155,921 </td></tr><tr><td> NK-1.1 (PK136) </td><td> eBioscience </td><td> 455,941 </td></tr><tr><td> C-키트 (2B8) </td><td> eBioscience </td><td> 171,171 </td></tr><tr><td> SCA-1 (D7) </td><td> eBiosci줬어 </td><td> 135,981 </td></tr><tr><td> CD34 (RAM34) </td><td> eBioscience </td><td> 110,341 </td></tr><tr><td> FcγR (2.4G2) </td><td> eBioscience </td><td> 553,145 </td></tr><tr><td> 안티 APC의 MicroBeads </td><td> Miltenyi Biotec </td><td> 130-090-855 </td></tr><tr><td> LS 열 </td><td> Miltenyi Biotec </td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

골수는 HSCs보다 성숙한 혈액 세포 가계 progenitors가 거주하고 성인 유기체에서 차별 교장 사이트입니다. HSCs는 삶의 시간을 1 모든 혈액 세포 lineages을 생성할 수있는 pluripotent 세포 분 세포 인구를 구성합니다. 골수에서 HSCs 항상성의 분자 해부는 조혈, 종양학 및 재생 의료에서​​ 중요한 의미를 가지고 있습니다. 우리는 형광 항체 및 개별 생쥐의 조혈 전구 하위 집합 및 HSCs 분포 (그림 1) 득점하기 유동세포계측법의 전자 게이팅 절차 라벨 프로토콜을 설명합니다. 또한 광범위하게 조혈 progenitors뿐 아니라 장기 (LT) 및 C-Kit를 표현하는 세포의 자성 농축하여 풀링된 골수 세포 현탁액에서 HSCs를 reconstituting 단기 (ST)를 풍부하게하는 방법을 설명합니다. 그 결과 세포 준비는 체외에 대해 선택한 하위 집합을 정렬하는 데 사용할 수 있습니다생체내 기능 연구에 대하여 (그림 2). 두 trabecular osteoblasts 2,3 및 sinusoidal 내피의 4 골수에서 HSCs을 지원하는 기능성 틈새를 구성합니다. N-cadherin + osteoblasts 3의 하위 집합과 그 리간드 angiopoietin-1 5 HSCs로 표현 수용체 티로신 키나제의 Tie2의 상호 작용을 포함 osteoblastic 틈새에서 몇몇 메커니즘은 HSCs의 정지를 결정하는데 동의합니다. 골수에서 &quot;최대 절전 모드&quot;는 과도한 사이클링 활동 6시 복제 및 최종 피로로부터 HSCs을 보호하기 위해 중요합니다. 이러한 수신자 같은 수용체 리간드 7, 인터페론-α 6 등 타고난 면역 계통의 세포에 따르고 외인성 자극에도 혈통이 커밋된 progenitors으로 확산 및 HSCs의 분화를 유도하실 수 있습니다. 최근 린 내에서 휴면 마우스 HSCs의 인구 - C-키트 + 무서-1 + CD150 + CD48 - CD34 - 인구가 8을 설명했습니다. 세포 정렬 여기에 설명된대로 조혈 progenitors 강화 세포 현탁액에서 CD34 표현에 따르면 허용 정지 자체 갱신 LT-HSCs과 ST-HSCs 9 모두의 고립. 혈통 긍정적인 세포의 고갈과 CD48와 Flk2 항체와 LT-HSC의 분류를 기반으로 유사한 절차는 이전에 10을 설명했습니다. 현재 보고서에서는 phenotypic 특성화 및 다른 생리 및 병리 학적 조건에서 조혈을 모니터하는 데 유용할 수 있습니다 조혈 progenitors의 전직의 생체내 세포주기 분석을 위해 프로토콜을 제공합니다. 더욱이, 우리는 요인 및 세포 생물학 및 신호 전달 장치 assays에서뿐만 아니라 이식에 대한 그들의 자기 갱신, 확장 및 분화를 조절 메커니즘을 정의하는 데 사용할 수 HSCs를위한 절차를 정렬 FACS에 대해 설명합니다.

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

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

유동세포계측법의 골수 조혈 progenitors의 유통뿐만 아니라 효율적으로 분리 고도 정화 조혈 줄기 세포 (HSCs)를 분석하는 방법을 설명합니다. 분리 절차는 본질적으로 세포 및 분자 연구를위한 HSCs를 정화 정렬 C-키트 + 세포와 세포의 자성 농축에 근거한다.

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 ...

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

Research

JoVE Journal

Biology

18.5K 조회수.  Bellinzona (Switzerland). 유동세포계측법의 골수 조혈 progenitors의 유통뿐만 아니라 효율적으로 분리 고도 정화 조혈 줄기 세포 (HSCs)를 분석하는 방법을 설명합니다. 분리 절차는 본질적으로 세포 및 분자 연구를위한 HSCs를 정화 정렬 C-키트 + 세포와 세포의 자성 농축에 근거한다.

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

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 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. ...

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 ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

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

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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 ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

유세포측정에 의한 조혈줄기세포 및 T세포의 미토콘드리아 질량 및 막 전위 측정

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...