이 작품에 대한 부분적인 자금은 NSF - 경력 0,744,556 (라오)와 VCU - HHMI 과학 교육 및 연구 프로그램 (파스쿠알)을 통해 장학금에 의해 제공되었다.

1 부 : 서스펜션 Immunocytochemical 스테 이닝 <ol><li> 세포는 단일 세포 현탁액에 수확하고 있습니다. </li><li> 세포는 다음 1.5 2mL microcentrifuge 튜브로 옮겨 4 분 200g에 centrifuged 있습니다. </li><li> 세포 표면에 뜨는을 aspirating에 의해 씻겨 있으며, PBS의 1mL에 다시 일시 중지 - (MG이없는 + 및 CA 2 +) 4 분 200g에서 다시 centrifuged 다음합니다. 이것은 두 번 수행되어야합니다. </li><li> 세척 후, 세포를 그 다음 10 분 동안 상온에서 1mL 4 %의 paraformaldehyde (PFA)에 다시 보류 그들에 의해 해결됩니다. </li><li> 전지는 PBS의 1mL와 함께 두 번 다시 씻어 ​​있습니다 -. </li><li> 로 세척, 블록 전지 후 다시 정지 차단 솔루션 1mL (6 % serum/94 % PBS -)에 있습니다. 45 분 동안 실온에서 이들을 품어. </li><li> 후, 4 분 200g에 튜브를 원심 분리기 및 차단 솔루션을 대기음. 기본 항체 용액 (권장 희석에서 차단 솔루션으로 만들어진)의 1mL의 세포를 다시 일시 중지합니다. 세포 중 1 시간 또는 4 ° C 하룻밤 다음 단계를 진행하기 전에 실온에서 incubated 수 있습니다. </li><li> 전지는 다음 블록 솔루션 1mL로 두 번 씻어 있습니다. </li><li> 차 항체 용액 (권장 희석에서 차단 솔루션을 만든)과 1 시간 동안 실온에서 품어의 1mL에 다시 정지 세포, 후. </li><li> 일시간 후, 세포를 그 다음 블록 솔루션 1mL와 함께 두 번 다시 세탁해야합니다. </li><li> 차단 솔루션과 함께 세척 후, PBS의 1mL로 다시 세포를 씻어 -. </li><li> 다양한 세척 후의 1mL의 세포를 다시 중단 DAPI 핵 얼룩 (PBS에서 DAPI의 1:10,000 희석 -) 5 분 실온에서 알을 품다. </li><li> 5 분 후에, 4 분 200g에 튜브를 원심 분리기. - PBS의 1mL의 DAPI 핵 얼룩 솔루션과 다시 중단 세포를 대기음. </li></ol> 2 부 : cytospin 장치를 통해 생성된 슬라이드의 hPSCs의 설립 <ol><li> 플라스틱 슬라이드는 드릴 프레스와 그들의 구멍에서 원하는 금액을하여 준비가되어 있습니다. 직경 사용할 micropipette 팁 (들)의 넓은 직경보다 가장 큰 1mm에서해야합니다. </li><li> 필터 페이퍼는 가위로 플라스틱 슬라이드의 매개 변수로 절단됩니다. </li><li> 구멍은 다음 여과지에 만들어집니다. 구멍의 크기 (들) micropipette 팁 (S)는 그것을 통해 snuggly 맞는 충분히 커야합니다. </li><li> 후 준비 플라스틱 슬라이드 중 하나는 상단과 컷 필터 용지 (그림 1)의 하단에 위치에있는 유리 슬라이드에 배치됩니다. 레이어는 테이프로 안전하고 있습니다. </li><li> 파트 1에서 스테인드 세포 현탁액의 100μL의 최대 (원하는 monolayer 밀도에 따라 다름) 다음 micropipette 팁 (들)의 상단에 배치됩니다. </li><li> 필요한 경우, 셀 솔루션기구는 무게와 유사한 체중의 카운터 - 균형을 만들 수 있습니다. </li><li> cytospin 장치 및 카운터 - 균형은 다음 바탕 화면 원심 분리기에 배치 4 분 200g에 centrifuged 아르 수 있습니다. </li><li> 원심 분리 후, cytospin 장치는 신중하게 유리 슬라이드에서 세포를 이동시키다 나던 방법으로 분해됩니다. </li><li> 필요한 경우, 초과 주변 PBS - 유리쪽에에 밖으로 aspirated. </li><li> 세포는 다음 원하는 세포 monolayer가 달성되었는지 확인하기 위해 형광 현미경을 사용하여 볼 수 있습니다. </li></ol> 파트 3 : 슬라이드 마운트 <ol><li> 원하는 설치 미디어의 방울이 세포를 포함하는 각 지역의 중심에 직접 저장됩니다. </li><li> 후 커버 슬립이 부드럽게 가능한 경우 기포를 피하려고 슬라이드로 저하됩니다. </li><li> 초과 설치 미디어는 다음 슬라이드에서 제거됩니다. </li><li> 후, 커버 전표의 측면​​은 손톱 바니와 함께 봉인하고 있습니다. </li><li> 결과가 관찰하고 설명하는 때까지 슬라이드는 어두운 저장 영역에 보관하실 수 있습니다. </li></ol>

<ol>
	<li>Rao, R. R., Johnson, A. V., Stice, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+markers+in+human+embryonic+stem+cells.">Cell surface markers in human embryonic stem cells.</a> Methods in Mol. Bio. - Stem Cell Assays. 407, 51-61 (2007).</li><li>Sisino, G., Bellavia, D., Corallo, M., Geraci, F., Barbieri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homemade+cytospin+apparatus.">A homemade cytospin apparatus.</a> Anal. Biochem. 359, 283-284 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리 연구소의 목적을 위해, 마우스 배아 섬유아 세포의 (MEFs)에 성장 줄기 세포 문화 35mm 요리가 cytospin 장치와 함께 사용하기위한 immunocytochemical 얼룩 절차에 할당된 것입니다. 세포는 다음 단일 세포 현탁액에, 가능한 한 MEF 층의 제거, passaging 효소에 의해 수집됩니다. MEF 계층에서 더 효과적으로 줄기 세포 격리가 필요한 경우, 식민지를 수동으로 정지에 소화 다음 포착 수 있습니다. 피더 무료 ?...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lab Tek Permanox Chamber Slide</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>117437</td>
<td>Material for re-used plastic slides</td>
</tr>
<tr>
<td>Superfrost/Plus Microscope Slides Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>0.1-10&mu;L Filter Tips</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1121-3810</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>120542-B</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 280</td>
<td>&nbsp;</td>
<td>Richard-Allan Scientific</td>
<td>8311-4</td>
<td>Mounting Medium</td>
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<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Normal Goat Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10000C</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Anti-SSEA-4 Monoclonal Antibody</td>
<td>&nbsp;</td>
<td>Millipore/Chemicon</td>
<td>MAB4304</td>
<td>Primary Antibody for SSEA-4 staining</td>
</tr>
<tr>
<td>Rat Anti-SSEA-3 Monoclonal Antibody</td>
<td>&nbsp;</td>
<td>Millipore/Chemicon</td>
<td>MAB4303</td>
<td>Primary Antibody for SSEA-3 staining</td>
</tr>
<tr>
<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen/ Molecular Probes</td>
<td>A11029</td>
<td>Secondary Antibody for SSEA-4 staining</td>
</tr>
<tr>
<td>Alexa Fluor 488 Labeled Goat Anti-Rat IgG </td>
<td>&nbsp;</td>
<td>Invitrogen/ Molecular Probes</td>
<td>A11006</td>
<td>Secondary Antibody for SSEA-3 staining</td>
</tr>
<tr>
<td>DAPI, Dihydrochloride</td>
<td>&nbsp;</td>
<td>CalBioChem</td>
<td>268298</td>
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immunocytochemical analysis of human pluripotent stem cells using a self-made cytospin apparatus

인간 배아 줄기 세포 (hESCs)와 인간의 유도 pluripotent 줄기 세포 (hiPSCs)를 포함 인간 pluripotent 줄기 세포 (hPSCs)은 무한한 자기 갱신 기능 및 여러 세포 유형으로 구별하기 위해 잠재적으로 인해 흥미로운 셀 소스입니다. hPSCs의 pluripotent 상태는 일반적 qPCR, immunocytochemistry 같은 방법으로 평가, 그리고 기타로<em&gt; 체외에서</em&gt;과<em&gt; 생체내에</em여러 세포 유형으로&gt; 차별화 전략. 이러한 가운데, immunocytochemical 기법 후보 세포 및 세포 - 표면 생체 분석을 바탕으로 hPSCs의 undifferentiated 상태를 일상적인 특성화를 위해 개발되었습니다. 식민지 문제는 일상적인 기준으로 개별 세포 수준에서 이러한 마커의 표현을 quantifying의 발생으로 hPSCs 성장했다는 사실 감안할 때. 유동세포계측법 분석이 문제를 해결하지만, 필요 셀 번호 및 hPSC 문화의 일상적인 품질 관리 평가 정상적으로 도움이되는 없습니다 시약의 사용에 제공하고 있습니다. 따라서, 마커 평가에 대한 보존 무결성과 monolayer 세포 샘플을 만드는 실용적이고 재현할 방법의 개발은 줄기 세포 연구에 많은 장점이 있습니다. monolayer에서 개별 세포를 쉽게 관찰 및 특정 마커의 표현에 대한 계량 수 있기 때문에 이것은 크게 immunocytochemical 분석 혜택을 제공합니다. 이 목표는 자수 성 가한 cytospin 장치가 건설되었으며 immunocytochemical 얼룩와 함께 사용하기위한 최적화된. 두 세포 표면 마커 (SSEA3/SSEA4) 표현이 프로토콜의 목적을 위해 변형 BG01의 줄기 세포 라인에서 분석했다.

Watch this Scientific Journal Video about Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus at JoVE.com

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

세포 - 표면 마커에 대한 인간의 pluripotent 줄기 세포의 중지 immunocytochemical 얼룩 (hPSCs) (SSEA-3/SSEA-4)가 관측 및 부량에 대한 세포 monolayer을 생성하기 위해 스스로 만든 cytospin 장치의 사용을 기반으로 달성했다.

자수 성 가한 Cytospin 장치를 사용하여 인간 Pluripotent 줄기 세포의 Immunocytochemical 분석

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

immunocytochemical-analysis-human-pluripotent-stem-cells-using-self

Research

JoVE Journal

Biology

14.2K 조회수.  Virginia Commonwealth University. 세포 - 표면 마커에 대한 인간의 pluripotent 줄기 세포의 중지 immunocytochemical 얼룩 (hPSCs) (SSEA-3/SSEA-4)가 관측 및 부량에 대한 세포 monolayer을 생성하기 위해 스스로 만든 cytospin 장치의 사용을 기반으로 달성했다.

비디오: Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

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 ...

연구

생물학

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

Embryoid 기관의 Cryosections를 사용하여 Pluripotent 줄기 세포 분석

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

인간 유도 만능의 줄기 세포를 Transfecting 및 Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFP와 Retroviral 벡터를 사용하여 유도 Pluripotent 줄기 세포 (iPSCs)로 인간 체세포를 프로그래밍

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCA Lentiviral 벡터를 사용하여 말초 혈에서 인간 유도 만능 줄기 세포의 생성

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititre 판 형식으로 인간 만능의 줄기 세포에서 뉴런의 신속하고 효율적인 생성

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

인간의 Cardiomyocytes의 생성 : 피더없는 인간 유도 만능 줄기 세포에서 분화 프로토콜

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...