박사 Rountree 아이의 기적 네트워크, 건강의 국립 연구소, K08DK080928 및 R03DK088013, 그리고 미국 암 협회의 연구 학술 상, MGO - 11651에서 현재의 지원을 인정한다. 박사 Rountree는 소아 과학 개발 프로그램 (NICHD 부여 수상 K12 - HD00850)에 의해 재정 지원을하면서이 프로 시저가 처음 개발하고 세련된 것을 인정합니다.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+hepatic+tumor+progenitor+cells+in+Pten-null+mice+requires+liver+injury+and+is+reversed+by+loss+of+AKT2.">Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.</a> Gastroenterology. 139, 2170-2182 (2010).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+of+murine+liver+tumor+cells+promotes+invasion.">Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion.</a> Hepatology. 52, 945-953 (2010).</li></ol>

박사 Rountree은 바이엘의 조제학의 연구 지원을받습니다. Drs. 땡와 사기꾼과 부인 저런와 양 VanKirk는보고도 공개가 없습니다.

조혈 시스템과는 달리,있는 조혈 줄기 세포가 replacesnormal physiologic 설정 - 이상 leukocytes, 적혈구 및 혈소판, 간 줄기 세포, 또는 성인 간 전구 세포의 정상 간에 참여하지 않는 세포 차별화 시스템을 유지하는 책임이 있습니다 항상성은. 급성 간 손상 또는 일부 hepatectomy, hepatocytes, 후 5,6는 차별화된 간 상피로, 잃어버린 간 질량을 대체하는 확산 여러 순찰을 받고있다. 5,6만을 만성 부상 중에하는 것이 관찰 간 줄기 세포 아르 분아 따위에 의해 번식합니다. 1.5은 -11 이러한 성인, 장기 특정 줄기 세포는 1,5-8은 흥미롭게도, 간암의 대부분이 만성 부상의 배경에 개발하고 있습니다. hepatocytes과 cholangiocytes 모두에 차별화하는 제안이며, 따라서 간 줄기 세포도 될 가상 아르 간암의 하위 집합에 대한 엽 성의 전구 물질. 2-4,12-17 간장의 세포 활동을 막기위한 주요 과제 중 하나는 기능 분석을위한 세포의 희귀 인구의 격리입니다. 예를 들어, 간장의 세포의 대부분은 cholangiocytes 및 기타 작은 비 parenchymal 세포보다 훨씬 큰 hepatocytes입니다. 세포 구획 (- parenchymal 분수와 작은 세포 - 대형 hepatocytes 비 parenchymal 분율)으로 전체 간을 파괴함으로써, 더 나아가 CD45 음성 세포 (비 조혈 세포)에 대한 선택, 우리는에 의해 줄기 세포의 시작 인구를 풍성하게하실 수 있습니다 600 배. 통해, 우리는 CD133 +의 인구가 100 % 순수 줄기 세포 인구이지만, 분명히 다른 혈통과 repopulation 잠재력과 세포의 이종 인구를 대표하는 것을 나타내는 의미없이 노트 1,18-21 있습니다. 분야의 주요 한계 중 하나는 줄기 세포와 전구 세포의 정의입니다. 우리는이 작품에 더 널리 용어 &quot;줄기 세포&quot;를 사용하지만, 엄격한 정의에, CD133 + 비 parenchymal 세포는 이중 혈통 시조 인구를 나타냅니다. 간장에 현재의 줄기와 시조 연구의 상태 감안할 때,이 보고서는이 분야에 관심이있는 조사를위한 출발점을 제공한다. 새로운 마커 이러한 EpCAM, 22,23 또는 Sox9과 같은 전사 요소 24로, 등장으로 그들은 포함될 수 있습니다. 예를 들어, 우리는 EpCAM + 세포 및 CD133 + 세포 사이의 중복의 비교적 높은 비율을 발견했다. 이 보고서에서는, 우리는 세부 정상 murine 간뿐만 아니라 만성 간 손상 모델, 보여주 증가 간 줄기 세포 증식에​​서 줄기 세포 분리를위한 프로세스를. 라이브 세포를 사용하여 기능성 연구가 분리 후 수행할 수 있으므로이 방법은 냉동 또는 포르말린 고정 간 표준 immunohistochemistry를 통해 분명히 장점이 있습니다.이 과정 1,3,4 구체적인 목표는 수 간 줄기 세포의 비교적 순수한 인구를 분리하는 것입니다 clonally 체외로 확대됩니다. 이 절차에 대한 기본 제한은 고립된 세포의 대부분은 (문제 해결 섹션을 참조) 유동세포계측법 후 가능한되지 것입니다. 이것은 세포를 준비하는 데 필요한 시간의 결과이며, 사전에 격리하는 인구를 수정하는 데 필요한 다양한 절차. 간장은 즉각 FACS 분석을위한 단일 세포 현탁액에서 소화 경우, hepatocytes 및 기타 비 - parenchymal 세포 사이의 크기 차이가 효과 FACS 게이트 생성이 불가능하게됩니다. 간 이외의 parenchymal 세포는 조혈 세포의 제거없이 활용하는 경우, CD133 + 세포의 상당수가 소수를 오염시킬 수 있습니다 조혈 원산지 수있는 위험이 있습니다. 또한, Miltenyi 필터를 통해 세포를 처리하여, 단 하나의 세포와 세포의 아주 작은 클러스터는 회수됩니다. 이것은 예제에서는 FACS 흡기 바늘을 방해할되지 않습니다 보장합니다. 이 절차에 대한 대안은 CD49f, EpCAM 같은 다른 세포 표면 마커에 대한 수정, 또는 CD133 + EpCAM로 ​​마커의 조합을 포함 + 두 번째 출판 보고서가 아닌 parenchymal 분수에서 간 줄기 세포를 분리하기 위해 밀도 구배을 활용합니다. 이 절차는 울트라 원심 (8,000 XG)가 필요합니다, 절차에 상당한 시간을 추가하고, 우리의 경험에 크게 사전 FACS 세포 수율 및 사후 FACS 생존을 감소. 8 이후의 실험은 같은 HNF3, HNF4α 및 αfp.In 추가로 태아 간 유전자를 포함한 유전자 발현 분석의 광범위한 범위를 포함, 표현 단백질의 서양 얼룩 분석 및 immunocytochemistry는 문화에 세포의 RT - PCR 결과를 확인하기 위해 이용하실 수 있습니다. 참고 한 문제는 우리는 이제 정기적으로 (문제 해결 섹션을 참조) 별 모양의 세포 마커에 대한 우리의 샘플을 화면 25. 간성 별 모양의 세포에서 CD133 표현을 식별 최근 작업이고, ID가 없습니다우리 분수에 상당한 오염 entified. 이것은 간 소화하고 세포 분리의 다른 기술과 관련이있을 수 있습니다. 2,3,26은 세포의 대부분은 즉시 FACS 절연 후 가능한되지 않습니다 사실을 바탕으로, 우리는 동물에서 사용하기 전에, 대량 CD133 + 세포 단일 세포와 같은 하나의 세포가 도금하는 것이 좋습니다. 체외에서 5~7일 크게 종양의 분석 결과를 향상됩니다. 식민지가 대량 CD133 + 세포를 고립에서 확장 가능한 한 또한, 단일 세포 분리의 엄격 주어진이에만 실시해야합니다. 문화 간 줄기 및 전구 세포로 활용 될 수있는 다양한 문화 조건 및 비계 단백질의보다 철저한 논의가 잘 롤라 리드에 의해 특징되었습니다. 이 작품은 기본 절연 기술이 마스터되면 수사관들이 연구 프로그램에 통합할 수 있습니다 대안 조건 및 수정을 제공합니다. 27,28 박사는 리드의 작품 또한 혈통 생물학과 간장의 줄기 세포와 헌신 progenitors 사이에 성숙의 상세한 분석을 제공합니다. 생체내 종양의 분석 측면에서, 우리는 주로 1x10 6 세포를 사용하여 체외에서 clonally 확장 CD133 + 세포에서 갓 격리 세포와 세포를 사용하여 성공을 있었다. / - - 그리고 간 특정 Pten - / - 우리는 만성 간 손상의 ontwo 유전자 변종, MAT1a 초점이 마우스를, 우리는 누드 마우스 및 종양 성장을위한 숙주로 야생 형 생쥐를 모두 활용합니다. 그들이 사전 악성 아르 큰 간 손상 모델에서 격리하는 경우 우리의 경험에서 CD133 + 간 줄기 세포는 종양을 형성합니다. 참고 CD133에서 형성 종양 + 일반 종양에 대한 줄기 세포 또는 전구 세포의 기원을 제안, 간세포암 및 cholangiocarcinoma 기능을 모두 가지고 세포. 2-4,29 종양이 문서화 후 후속 작업은 종양 조직 (H &amp; E의 얼룩) 및 immunohistochemical 얼룩의 표준 pathologic 분석을 포함하고 있습니다. 또한, 종양은 FACS 분석하거나 다시 문화 다진 및 소화 수 있습니다. 2-4,30 결론적으로, 우리는 고립, 확장, 및 CD133 + 간 줄기 세포 및 CD133 + 암 줄기 세포의 기본 특성에 대한 절차를 자세히 있습니다. 문제 해결 : CD45 오염 : 3 단계에 대한 사전 FACS 분석 및 격리에 대한 CD45 - FITC AB를 추가하여 평가 수 있습니다 CD45 + 세포의 오염이있다면, CD45 microbead 항체가 만료되지 않도록 확인하고 여과액에만 동안 수집했다는 필터는 자석 홀더에. 필터가 자석 홀더가 CD45 + 세포를 포함하지 않을 동안 모든 여과물가 수집. 저가 휴대폰 번호 : 손상되지 않은 간, CD133의 총 숫자는 + 비 parenchymal 격리는 10,000보다 수 있습니다. 이러한 세포는 정지 간장에 드문 있습니다. 등 DDC 0.1 %의 음식으로 만성 부상 모델의 경우 숫자는 100,000 세포에 크게 증가합니다. 전체 휴대폰 번호가 크게 숫자보다 낮으면, 고려 하나의 문제는 FACS AB의 염색법이다. 가난한 얼룩이 가난한 항복의 결과 있으므로 CD133 AB는, 유효 기간이 만료되지 않도록 확인하십시오. 또한, 우리는 이전에 시도 FACS 절연에 상대적으로 인구를 결정하기 위해 간 비 parenchymal 세포 FACS 분석을 수행하는 것이 좋습니다. FACS 후 낮은 세포 생존 능력 : 생존의 문제 중 하나는 세포가 처리 그리고 시간이 지남에 따라하는 방법과 관련이있을 수 있습니다. 이상적으로, 전체 세포 격리 절차, 1-4 단계는 단계 사이에 지체없이 실시하고 같은 하루에 완료되어야합니다. 1-4 단계 사이에 상당한 지연이 크게 세포 생존을 줄일 수 있습니다. 세포 생존에 관한 두 번째 문제는 FACS 절연에 사용되는 외장 압력과 관련이있을 수 있습니다. 우리는 낮은 칼집 압력을 사용하는 것이 좋습니다. 정렬 후 얼음에 대한 상당한 저장 용량 또한 생존을 감소되므로 마지막으로, 세포가 고립되면, 그들은 즉시 도금한다. CD133 + 비 parenchymal 이질 : 최근 보고서는 간장 별 모양의 세포도 CD133 표현을 일부 소성을 할 수 있습니다 나타냅니다. 따라서 25, 알부민 및 Krt19와 BI - 힘의 유전자를 확인 이외에, 추가 검증 그런에 glial fibrillary 산성 단백질로 별 모양의 세포와 관련된 유전자를 포함시킬 수 있습니다 정지 별 모양의 세포와 활성 별 모양의 세포에있는 알파 평활근의 굴지 및 desmin가. 3,4,26은 또한, CD133 + 인구는 광범위한 시조 인구를 나타냅니다. 같은 EpCAM로 ​​두 번째 마커,의 또한 더욱 인구를 수정하는 데 도움이, 그리고 제한 이질 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> DMEM : F12 </td><td> Invitrogen </td><td> 10565-018 </td><td> 페놀 레드 </td></tr><tr><td> CD45 microbeads </td><td> Miltenyi </td><td> 130-052-301 </td><td></td></tr><tr><td> Hepatocyte 성장 인자 </td><td> 시그마 </td><td> H1404 </td><td></td></tr><tr><td> 표피 성장 인자 </td><td> 시그마 </td><td> E4127 </td><td></td></tr><tr><td> DNase </td><td> 시그마 </td><td> DN25 </td><td> 1g </td></tr><tr><td> Collagenase D </td><td> 호슈 </td><td> 1,088,874 </td><td></td></tr><tr><td> Pronase </td><td> 호슈 </td><td> 0,165,921 </td><td></td></tr><tr><td> 70 미크론 메쉬 스트레이너 </td><td> 어부 </td><td> 352350 </td><td></td></tr><tr><td> Omniscript RT </td><td> Quaigen </td><td> 205111 </td><td> 50 반응 </td></tr><tr><td> HotStarTaq </td><td> Quaigen </td><td> 203203 </td><td></td></tr><tr><td> Miltenyi LD 칼럼 </td><td> Miltenyi </td><td> 130-042-901 </td><td></td></tr><tr><td> CD133 - PE FACS AB </td><td> eBioscience </td><td> 12-1331-82 </td><td></td></tr><tr><td> Laminin 코팅 접시 </td><td> BD </td><td> 354410 </td><td> 잘 96 </td></tr><tr><td> 트립신 0.05 % EDTA (에틸렌 다이아 민 테트라 초산) </td><td> Invitrogen </td><td> 25300-354 </td><td> 100 ML </td></tr><tr><td> RNeasy 마이크로 키트 </td><td> Quaigen </td><td> 74,004 </td><td> 5x10 5 세포 이하 50 열 </td></tr><tr><td> 알약은 Lyse </td><td> BD </td><td> 555899 </td><td> 10X 농도 </td></tr></tbody></table>

isolation of cd133+ liver stem cells for clonal expansion

간 줄기 세포, 또는 타원형 세포, 만성 간 손상시 세포 분열 따위에 의해 번식하고, hepatocytes 및 cholangiocytes 모두에 차별화하는 제안입니다. 또한, 간 줄기 세포는 간암, 간세포암의 하위 집합에 대한 엽 성의 전구 물질로 가상하고 있습니다. 간 원하는 고체 장기의 세포 활동을 막기위한 기본 과제 중 하나는 자세한 분석을 위해 세포의 드문 인구의 격리입니다. 예를 들어, 간장의 세포의 대부분은 비 - parenchymal 세포보다 훨씬 큰 hepatocytes (parenchymal 소수)입니다. 간 (예 : parenchymal 및 비 - parenchymal 분수)의 특정 세포 구획을 풍요롭게하고, CD45 음성 세포에 대한 선택함으로써, 우리는을 위해 허용이 리포트에 proceduresdetailed 600 fold.The 이상에 의한 줄기 세포의 시작 인구를 풍성하게하실 수 있습니다 확실한 장기의 세포의 비교적 드문 인구는 효율적으로 정렬합니다. 이 프로세스는 증가 간 줄기 세포 증식을 보여주 정상 murine 간장에서 isolateliver 줄기 세포뿐만 아니라 만성 간 손상 모델에 활용할 수 있습니다. 라이브 세포를 사용하여 기능성 연구가 초기 공동 지역화 실험 이후에 수행할 수 있습니다대로이 방법은 냉동 또는 포르말린 고정 간 표준 immunohistochemistry를 통해 분명히 장점이 있습니다. FACS 절연의 세부 전문 계측 및 기본적인 흐름 cytometry 절차의 강력한 실무 지식에 크게 의존 본 보고서에 설명된 절차를 수행하려면 연구 기반 흐름 cytometry 코어와 함께 작동하는 관계는 강력하게 권장됩니다. 이 과정의 구체적인 목표는 clonally 체외에서 확장 가능한 간 줄기 세포의 인구를 분리하는 것입니다.

Watch this Scientific Journal Video about Isolation of CD133+ Liver Stem Cells for Clonal Expansion at JoVE.com

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

여기 우리는 전체 murine 간, 조직 소화, 세포 강화, 그리고 유동세포계측법 절연을 요구하는 과정에서 CD133 표현 간 줄기 세포와 암 줄기 세포의 절연을 설명합니다. 우리는 고급 단일 세포 분리 및 clonal 확장 메서드를 포함합니다.

Clonal 확장 CD133 + 간 줄기 세포의 분리

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

isolation-of-cd133-liver-stem-cells-for-clonal-expansion

Research

JoVE Journal

Biology

22.3K 조회수.  Pennsylvania State College of Medicine. 여기 우리는 전체 murine 간, 조직 소화, 세포 강화, 그리고 유동세포계측법 절연을 요구하는 과정에서 CD133 표현 간 줄기 세포와 암 줄기 세포의 절연을 설명합니다. 우리는 고급 단일 세포 분리 및 clonal 확장 메서드를 포함합니다.

비디오: Isolation of CD133+ Liver Stem Cells for Clonal Expansion

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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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 ...

Isolation of Mouse Salivary Gland Stem Cells

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

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

마우스 침샘에 줄기 세포의 분리

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

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

간엽 전환을 유도하고 상피의 분석

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

Isolation, Culture, and 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 ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

차 인간 간세포의 분리 및 비 실질 간 세포의 주요 집단 대응을위한 프로토콜

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...