; 사우스 이스턴 위스콘신의 바이오 메디컬 기술 제휴, Milwauke, WI, 미국, 브라이언 Riesch 마비 재단, 밀워키, WI, 미국, 이글스의 성 주문, 중서부 장 USPHS 부여 NS38500, 신경계 장애의 국립 연구소와 stroke, NIH, 미국 : 지원 미국, 국립 척수 부상 협회, 밀워키 장, 미국, 노스 이스트 잉글랜드 줄기 세포 연구소, 뉴캐슬 대학, 영국.

성인 마우스 위스커의 모공에서 벌지의 해부 <ol><li> 우리는 10 주 6 개월 생쥐를 사용합니다. 젊은 생쥐는 종종 더 많은 세포를 얻을 수 있습니다. betadine (요오드 용액)와 약 3 분 과산화수소 (약국에서 구할 수)​​의 1시 1분 혼합으로 마우스와 잠수함 전체 동물을 안락사. </li><li> 물총 전체 마우스, 특히 얼굴의 75 % 에탄올과 지역, 그리고 층류 흐름 후드의 해부 현미경으로 마우스를 수행합니다. </li><li> 조심스럽게 머리 전구로 절단하고, HBSS에 수영장을 피하기 위해, 수염 패드를 해부하다. </li><li> 상온에서 HBSS에 수염 머리카락과 수영장을 해부하다. 포셉로 머리 뿌리 옆에있는 피부를 개최하고, 다음 바로 가위로 뿌리 주위를 절단하여이 작업을 수행합니다. 머리 전구를 손상하지 않도록 깊이 잘라. </li><li> 수염 패드의 수염 뿌리를 허용하고 신선한 HBSS와 새 접시에 넣어. </li><li> 수염의 모공이 청소까지, 버퍼의 squirts을 느슨하게 adipous과 피부 조직을 플러시. 필요한 경우, 수염 뿌리에 신경을 절단하고 힘든하고 HBSS의 반복 플러시하여 자기편 조직을 제거합니다. </li><li> 뿌리 옆에있는 피부 부분에 집중을위한 microforceps를 사용하여 날카롭게 텅스텐 바늘을 사용하여 Sylgard - 코팅 유리 페트리 접시에 깨끗한 머리 follilce 핀. </li><li> 컷 수염은 microblade와 longitudically 대과. 이것은 팽창을 다치게하므로, 너무 깊은 절단하지 마십시오. 사라 때까지 HBSS의 반복 squirts와 혈흔을 제거합니다. 혈액의 외관자를 충분히 깊이 것을 좋은 표시이다. longitudial 상처가 충분히 오래되지 않은 경우, 그것은 벤트 microscissors로 길어 수 있습니다. </li><li> 동굴 부비동의 수준 이상의 가로 잘라 고리 부비동 내에 수준에서 후 두 번째 가로 절단을 만들고, 피부에 닫습니다. </li><li> 이제 캡슐 내부의 팽창을 볼 수 있습니다. </li><li> 포셉과 콜라겐 캡슐에의 끝을 잡고 구부러진 텅스텐 바늘로 팽창을 롤. 이제 빈 캡슐과 격리 팽창을 볼 수 있습니다. </li><li> 수영장 실온에서 HBSS에 별도의 문화 접시에 bulges을 분리. 다른 조직은 풀링된 bulges을 훼손하지 있는지 확인하십시오. </li></ol> 팽창의 Explants의 문화 <ol><li> 50 μl 콜라겐과 서로 다음 10 μl 살균 6퍼센트 NaCl을 배치 코트 콜라겐과 35mm 문화 번호판. 이중 벤트 Pasteyr의 pipet과 잘 혼합하고 문화 판의 가장자리쪽으로 밀어. 깨끗한 dessicator에 야간 부화하지만, 건조하게하지 않습니다. 사용하기 전에 생리와 접시를 씻어. </li><li> 문화 매체로 약 3 시간에 대한 사전 품어 콜라겐 - 코팅 및 씻어서 문화 번호판입니다. 문화 매체 85% 알파 - 수정 멤 10 % 태아 소 혈청 및 5 % 일 11 여자의 배아 추출물로 구성되어 있습니다. </li><li> 다시 부화 후 접시에서 배지를 제거하고 가능한 한 작은 중소 여러 bulges를 추가합니다. 초과 문화 매체를 제거합니다. </li><li> 10 %의 산소와 5% CO 2 humidified 분위기에서 세포 배양기에 1 시간을 위해 품어,하지만 더 이상. </li><li> 1 시간 후, 부드럽게 문화 매체의 1.5 ML를 추가합니다. 벌지의 explants는 콜라겐 하층을 준수해야합니다. 일일 문화 매체의 50 %를 교체하십시오. </li><li> 3 이내 - 4 일, 높은 철새 세포는 팽창의 explants에서 이주 것입니다. 그들의 별 모양의 형태, 운동성, 그리고 세포 세포 접촉의 주된 부재를 적어 둡니다. 앞으로 몇 일 동안, 더 많은 세포가 이주하고, 세포가 빠르게 세포 분열 따위에 의해 번식합니다 이민 것입니다. </li><li> 날카롭게 텅스텐 바늘로 에피 NCSC의 이주의 발병 후 3 일 - 팽창 2 제거합니다. bulges가 더 이상 남아있다면, 그들은 평평하고 불가능한 순수 에피 NCSC 문화를 얻을 수 있도록하는 경향이있다. </li><li> 중요 : 에피 NCSC보다 몇 일 후에 가끔은 겉보기되며, 평평 형태와 희귀 전지 에피 NCSC되지 않습니다 덜 운동성이있는 있지만, putative 표피 줄기 세포 / progenitors. 에피 NCSC와 putative 표피 줄기 세포 / progenitors를 포함하는 이러한 세포로 구성된 문화, 또는 혼합 문화는 폐기해야합니다. </li><li> 힌트 : 그들이 putatively paracrine / autocrine 성장 인자의 신호로 인해, 높은 세포 밀도에서 급속하게 차별하는 경향으로, 기본 explant 매체에서 너무 오랫동안 전지를 보관하지 마십시오. </li></ol>

<ol>
	<li>Sieber-Blum, M., Grim, M., YF, H. u., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Sieber-Blum, M., Grim, M., Tuan, R., Grim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+hair+follicle+-+cradle+for+pluripotent+neural+crest+stem+cells.">The adult hair follicle - cradle for pluripotent neural crest stem cells.</a> Birth Defects Research (Part C). 72, 162-172 (2004).</li><li>Sieber-Blum, M., Schnell, L., Grim, M., Schneider, R., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+epidermal+neural+crest+stem+cell+(EPI-NCSC)+grafts+in+the+lesioned+spinal+cord.">Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.</a> Pub. 32, 67-81 (2006).</li><li>Hu, Y. F., Zhang, Z. -. J., Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidermal+neural+crest+stem+cell+(EPI-NCSC)+molecular+signature.">An epidermal neural crest stem cell (EPI-NCSC) molecular signature.</a> Stem Cells. 24, 2692-2702 (2006).</li></ol>

자신의 철새 능력을 가진, 에피 NCSC는 체외에서 확장 가능한 줄기 세포의 높은 순수 인구로 격리 수 있습니다. 성인 위치에서 배아 잔존물로서, 에피 NCSC는 향후 세포 대체 요법, 생명 의학 공학 및 / 또는 재생 의료에 대한 잠재적으로 매력적인 후보입니다. 그들이 바람직한 특성을 보여 우리는 척수 손상의 마우스 모델에서 에피 NCSC을 테스트했습니다. LongSAGE (www.ncbi.nlm.nih.gov / 지리적, 시리즈 번호 GSE4680)에 의해 ...

mouse epidermal neural crest stem cell (epi-ncsc) cultures

에피 NCSC는 성인 위치, 모낭의 팽창에 배아 신경 문장의 잔해입니다. 그들은 뉴런, 신경 세포 지원, 평활근 세포, 뼈 / 연골 세포와 melanocytes를 포함한 차별화된 세포 유형의 다양한 배열을 생성하는 생리 특성을 보유 multipotent 줄기 세포입니다. 에피 NCSC는 털이 피부에 쉽게 접근할 수 있으며 줄기 세포의 높은 순수 인구로 격리 수 있습니다. 이 동영상은 수염의 모공에서 마우스 에피 NCSC의 문화를 준비 상세한 프로토콜을 제공합니다. 성인 마우스의 수염 패드는 제거하고, 수염 머리카락이 해부하는 것입니다. 모공은 다음 팽창 지역 위 아래 길이 방향과 이후 transversely 절단됩니다. 팽창은 콜라겐 캡슐에서 제거하고 문화 접시에 배치됩니다. 에피 NCSC 3 ~ 4 일후 팽창의 explants에서 이민을 시작합니다.

Watch this Scientific Journal Video about Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures at JoVE.com

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

여기 마우스 표피 신경 능선의 줄기 세포를 (에피 NCSC) 분리하기 위해 방법을 보여줍니다. 기술과 관련된 마이크로 해부, 수염 모공 볼록한 분리하고 조직 문화에 그것을 placeing. 사일 - 에피 NCSC 3 이내 하층에 팽창의 explants에서 이민을 시작합니다.

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 ...

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Biology

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

11.5K 조회수.  Newcastle University. 여기 마우스 표피 신경 능선의 줄기 세포를 (에피 NCSC) 분리하기 위해 방법을 보여줍니다. 기술과 관련된 마이크로 해부, 수염 모공 볼록한 분리하고 조직 문화에 그것을 placeing. 사일 - 에피 NCSC 3 이내 하층에 팽창의 explants에서 이민을 시작합니다.

비디오: Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES 세포 - 파생 Neuroepithelial 셀 문화

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

연구

생물학

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

마우스 신경 줄기 세포 엽 성의 전구 물질의 문화

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC 마우스 태아의 해부

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

인간 신경 줄기 세포를 계산

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

아가로 오스 겔 기반 Microfluidic 장치를 사용 신경 줄기 세포의 운동성 평가

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

마우스 신경 조직에서 단일 셀 Suspensions의 생성

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

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 ...

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 ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

세포 운동성 및 Zebrafish 태아의 개별 뉴런과 신경 크레스트 세포의 굴지의 Cytoskeleton의 라이브 영상

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

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 ...