이 작품은 재생 의료의 캘리포니아 연구소 (RN2 - 00921-1) 및 NIH 투자 국가 연구 상 (F32-HL104924)에서 새로운 교수 상 II가 추진하는 사업

1. 마우스 배아 공급기의 작성 <ol><li> hESCs의 공동 문화에 사용되는 MEFs는 이전에 다음 부서를 겪고에서 세포를 억제하기 위해 조사 또는 미토 마이신-C에 의해 처리되어야합니다. </li><li> MEF를 심는 이전 두 시간, 코트는 각 60mm 플라즈마는 0.05 % 젤리 2 ML과 문화 요리를 취급. </li><li> 처리 MEFs의 약병과 ~ 20,000 세포 / cm 2에 판을 해동. </li><li> 세포가 밤새 준수 할 수 있도록 허용합니다. </li></ol> 2. MEFs로 공동 심는 hESCS <ol><li> 새로운 식민지는 다음과 같이 새로운 MEF-코팅 요리에 cryopreserved 문화 나 passaging 현재 문화에서 시작 될 수 있습니다. </li><li> 예열 1X PBS의 3 ML로 1 회를 passaged 할 큰 hESC의 식민지가 들어있는 판을 씻으십시오. </li><li> Collagenase IV / PBS 용액 (1 MG / ML) 3 ML을 추가하고 37 5-10 분 동안 품다 ° C. (참고 : 세포가 뚜렷한이나 공 등의 트립신으로 본이되지 않습니다.) </li><li> 세포는 아직있는 동안collagenase, 5 ML의 pipet 팁의 가장자리를 사용하여, 작은 세포 clumps으로을 깨고, 식민지에 그리드 패턴을 만들 수 있습니다. </li><li> 표면의 모든 셀을 dislodging, 문화 판의 바닥을 긁어 pipet 팁 (문화 판에 수직 방식에서 개최)의 끝을 사용합니다. </li><li> 5 분을위한 (1000 RPM)에서 매 관과 스핀으로 세포를 수집합니다. </li><li> 원심 분리 후, 매 튜브에 세포 펠렛을두고 매체를 대기음. </li><li> 전에 MEFs 도금 날부터 MEF 매체를 대기음. </li><li> 재 판 원본 판에서 1시 3분 비율 셀 및 hESC 매체의 3 ML과 피드 </li></ol> 3. 공동 문화에서 MEFs를 없애고 참고 : 인간 ESC 문화는 일반적으로 문화 10 14 일 사이에 고밀도에 있어야합니다. <ol><li> 접시의 가장자리에서 aspirating pipet의 팁을 놓고 흡입은 부드럽게 MEF 시트의 가장자리를 당겨 할 수 있습니다. </li><li> R요리에서 미디어를 emove, 그리고 끝이 세포 시트를 잡아 천천히 접시 위의 arced 방식으로 끝을 이동 할 수 있습니다. 참고 : 시트 pipet의 끝에서 방해 할 수 있지만 수동으로 문화 판의 표면에서 시트를 당기는 사용자를 방지하지 말아야으로이 문제가되지 않습니다. 세포 흡인기의 끝에 붙어있는 경우에는 전체 열망에 대한 세포 시트를 깨 배양 접시의 상단을 사용할 수 있습니다. </li><li> 즉시 hESC 매체를 교체하고, 보육에 다시 판을 넣어. </li></ol> 4. 대표 결과 MEF 제거 프로세스의 최종 결과는 작은 방해받지 hESC의 pluripotency 업체 SSEA-4의 표현을 유지하면서 매우 큰 식민지 (그림 2)에 상당한 확장을 받아야 할 수 식민지 (그림 1D), 10 월 ¾, 그리고 Tra 1-81 (그림을 생산 3). 1 그림. ESC의 콜로니의 형태학 MEF 제거 이전과 이후. 문화 요리로 심는 후 A) 일 1 B) 2 일에서의 인간의 배아 줄기 세포 식민지. C) 고밀도 인간 배아 줄기 세포의 식민지 (H7)은 MEFs로 둘러싸여. 두 hESC의 식민지가 빨간색 대시에 설명되어 있습니다. D)이 설명 된 흡인 기술을 사용하여 MEF 제거 후, 우리는 그대로 식민지를 둘러싼 거의 MEFs과 격리 된 hESC의 식민지로 남아 있습니다. <img alt="그림 2" src="/files/ftp_upload/3951/3951fig2.jpg" /> 그림 2. 10 일 MEF 제거 후 ESC의 콜로니 확장. 바로 MEF 고갈 후 원래 hESC의 식민지가 (왼쪽) 매우 큰 식민지 (오른쪽) appro으로 확장 할 수 있습니다10X ximately 800 μm. 원래 식민지가 복합 식민지와 같은 배율 (10 배)에 이미지로되어 있습니다. <img alt="그림 3" src="/files/ftp_upload/3951/3951fig3.jpg" /> 그림 3. MEF-소모의 hESC의 식민지 Immunostaining. 식민지의 Immunofluorescent 식별가 MEF 고갈부터 10 일까지하는 것은 식민지가 10월 ¾, SSEA-4에 의해 분명 같은 pluripotency 마커의 표현, 그리고 Tra-1-81 착색을 유지 것으로 나타났습니다. 또한, 이러한 hESC의 식민지가 mesodermal 운명으로 차별화를 전시하지 Flk-1의 부재로 표시. A, E, I, 그리고 M) immunoflouscently 스테인드 식민지, 20, 10, 10, 2 배의 전송 빛 이미지이며, .. 각각 B, F, J, 그리고 N) 핵 표시 - 각각 immunofluorescently 스테인드 식민지, 20, 10, 10, 배, CD)의 DAPI 스테인드 셀의 표현을 보여인간의 줄기 세포 마커 만 SSEA-4, 및 복합 이미지, 20x. GH)은 인간의 줄기 세포 마커 10월의 표현을 보여 ¾ 만하고 복합 이미지, 10 배. KL)은 인간의 줄기 세포 마커의 표현 Tra 만 1-81 표시 , 그리고 복합 이미지, 10 배. MP) 마지막 행은 이미지가 확대, 배,이 식민지에서 볼 전형적인 부드러운 테두리를 설명해 식민지을 묘사 아닌 경우 명시 적 O) Flk-1, 중배엽 차별화의 초기 마커 않으며, 그랬지 DDR2 착색의 부재에 의해 표시된대로 그들은 P)은 섬유 아세포가 포함되어 있습니다. 보충 비디오. 시트 흡인에 의해 섬유 아세포를 삭제를. <a href="http://www.jove.com/files/ftp_upload/3951/3951movie1.mp4" target="_blank">동영상을 보려면 여기를 클릭하십시오</a> .

<ol>
	<li>Bongso, A., Fong, C. Y., Ng, S. C., Ratnam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+inner+cell+mass+cells+from+human+blastocysts.">Isolation and culture of inner cell mass cells from human blastocysts.</a> Hum. Reprod. 9, 2110-2117 (1994).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat. Biotechnol. 18, 399-404 (2000).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+scalable+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Automated, scalable culture of human embryonic stem cells in feeder-free conditions.</a> Biotechnol Bioeng. 102, 1636-1644 (2009).</li><li>Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+surfaces+for+human+embryonic+stem+cell+culture.">Synthetic surfaces for human embryonic stem cell culture.</a> J. Biotechnol. 146, 143-146 (2010).</li><li>Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum. Reprod. 18, 1404-1409 (2003).</li><li>McKay, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cell+line+for+derivation+and+culture+of+hESc/hiPSc.">Human feeder cell line for derivation and culture of hESc/hiPSc.</a> Stem Cell Res. 7, 154-162 (2011).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Stein, G. S., Shi, M. J., Stencel, K., Borowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thawing, Seeding, and Changing Medium of Human Embryonic Stem Cells. , (2010).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol. Biol. 767, 107-123 .</li></ol>

관심 없음 충돌이 선언 없습니다.

이 논문에 제시된 방법은 인간 배아 세포 문화에서 섬유 아세포 피더 세포를 제거에 신속하고 적은 비용이 많이 드는 대안을 제공합니다. 섬유 아세포의 성공적인 제거 더 이상 줄기 세포 문화에 개발이 세포의 단단히 합류 monolayer의 존재에 따라 달라집니다. 7~10일 후, 성장 hESC의 식민지는 식민지 사이에 점점 더 조밀 한 섬유 아세포 monolayer를 생성, 바깥 방향으로 피더 섬유 아세포를 밀어 것입?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> DDR2 </td><td> 산타 크루즈 (Santa Cruz) </td><td> 7555 </td><td> 섬유 아세포 마커 </td></tr><tr><td> Dapi </td><td> Calbiochem </td><td> 268298 </td><td> 세포 핵 마커 </td></tr><tr><td> SSEA-4 </td><td> Millipore </td><td> MAB4304 </td><td> 인간 ESC 마커 </td></tr><tr><td> Oct 4분의 3 </td><td> 산타 크루즈 (Santa Cruz) </td><td> 9081 </td><td> 인간 ESC 마커 </td></tr><tr><td> Flk-1 </td><td> BD 제약 </td><td> 555307 </td><td> 조기 차별화 마커 </td></tr><tr><td> Tra-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> 인간 ESC 마커 </td></tr></tbody></table> 표 1. 특정 정말이에요 표신사는 hESC를 immunostaining에 사용됩니다.

rapid fibroblast removal from high density human embryonic stem cell cultures

마우스 배아 섬유 아세포의 (MEFs)는 blastocyst 절연 1 일 이후 인간 배아 줄기 세포 (hESCs) 문화를 확립하는 데 사용되었습니다. 이 피더 시스템은 셀 확장하는 동안 자연스럽게 차별화를 겪고에서 hESCs을 유지합니다. 그러나이 공동 문화에있어서, 집중적 인 노동이 고도로 숙련 된 인력을 필요로하고, 수율 낮은 hESC의 순도 4. 많은 실험실 hESC 문화의 피더 세포의 수를 (즉, 매트릭스 코팅 요리 또는 기타 피더 세포 유형 5-8을 포함) 최소화하기 위해 시도했습니다. 이러한 수정 문화 시스템은 몇 가지 약속을 표시했지만, 미토 마이신 C-처리 마우스 embyronic의 섬유 아세포로 배양의 hESCs hESC 문화의 원치 않는 자연 차별화를 병신하기 위해에 대한 표준 방법을 supplanted 없습니다. 따라서, hESC 확장에 사용되는 피더 세포는 분화 실험을하는 동안 제거해야합니다. 여러 기술 hESC의 공동 정화에 사용할 수 있지만피더에서 lonies은 (FACS, 맥, 또는 약물 내성 벡터의 사용), 이러한 기술은 노동 집약적 인 비용 및 / 또는 hESC에 파괴됩니다. 이 프로젝트의 목적은 hESCs의 순수한 인구의 수확을 가능하게 정화하는 방법을 발명하는 것이 었습니다. 우리는 합류 hESC 문화에서, MEF 인구는 MEF 시트의 간단하고 빠른 흡인을 사용하여 제거 할 수 관찰했다. 이 제거 &quot;측면 휴대 전화의 스티렌 배양 접시에 낮은 바인딩 친화력을 가지고 MEFs의 결합, 그리고 자신의 세대 동안 바깥쪽으로 섬유 아세포를 누르는 줄기 세포 콜로니의 능력 등 여러 가지 요인에 따라 달라집니다 틈새 &quot;. hESC 그런 다음 pluripotency의 유지 보수를 보장하기 위해 MEF 제거 후 10 일 SSEA-4, Oct3 / 4, Tra 1-81 표현에 조사되었다. 또한, hESC의 식민지는 hESC 확장의 추가 레벨을 제공 MEF 제거 후 큰 형태로의 성장을 계속 할 수있었습니다.

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

전환 문화에 피더 - 무료 조건에 지속적인 노력에도 불구하고, 인간 배아 줄기 세포 (hESC)의 유도와 문화 마우스 배아 피더 (MEFs)와 공동 문화에 달려 남아 있습니다. 여기, 우리는 신속하게 이전 실험에 hESC 문화에서 피더를 제거 소설 방법을 보여줍니다.

고밀도의 인간 배아 줄기 세포 문화의 급속한 섬유 아세포 제거

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

rapid-fibroblast-removal-from-high-density-human-embryonic-stem-cell

Research

JoVE Journal

Biology

11.7K 조회수.  University of California, Merced. 전환 문화에 피더 - 무료 조건에 지속적인 노력에도 불구하고, 인간 배아 줄기 세포 (hESC)의 유도와 문화 마우스 배아 피더 (MEFs)와 공동 문화에 달려 남아 있습니다. 여기, 우리는 신속하게 이전 실험에 hESC 문화에서 피더를 제거 소설 방법을 보여줍니다.

비디오: Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

트립신과 Passaging 색채 인간의 배아 줄기 세포 - 라인

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

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


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

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

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

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

마우스 표피 신경 크레스트 줄기 세포 (에피 NCSC) 문화

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

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

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

동물 세럼 - 무료 인간 줄기 세포 배양을위한 효율적인 보충으로 풀링 인간 혈소판 Lysate (pHPL)의 준비

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

설치류에서 기본 성인 Fibroblast 문화를 구축

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.