이 작품은 찰스 후드 재단의 의학 및 아동 보건 연구 상을 예일 학원에 의해 재정 지원되었다.

1. 프로그래밍 요소를 표현 레트로 바이러스에 의해 프로그래밍 <ol><li> 인간 섬유아 세포는 fibroblast 매체 (10 % 펜 / Strep과 DMEM에 FBS)에서 배양해 있습니다. </li><li> 6 - 잘 접시들이 한에 감염 전에 언젠가, 플레이트 1x10 5 인간 섬유아 세포. </li><li> 죽은 세포를 제거하고 신선한 fibroblast 매체 2 ML을 추가하는 매체를 기음. 5 μg / ML의 최종 농도에서 프로타민 황산염을 추가합니다. </li><li> 조심스럽게 감염의 다수 (방어) 5 5에 해당하는 각 GFP-표현 바이러스의 적절한 금액을 추가합니다. </li><li> 두 ML의 fibroblast 매체를 추가 후, 어느 날 감염 후, 바이러스 표면에 뜨는을 제거 2 ML PBS로 세 번 씻는다. </li><li> 사흘 감염 후, GFP의 형광을 확인하고 두 ML의 fibroblast 매체와 우물을 보충. </li><li> 향해 fibroblast 매체에서 4 일 감염 후 접시 1x10은 조사 마우스 배아 fibroblast 4 / ㎝ 2 (MEFs) 피더 세포10 cm 배양 접시는 0.1 % 젤라틴으로 코팅. 37 ° C 하룻밤 사이에 알을 품다. </li><li> 닷새 감염 후, 1 ML 0.05 % typsin / EDTA (에틸렌 다이아 민 테트라 초산)을 15 분 37 ° C 및 200g에서 5 분간 원심 분리기에 감염된 인간의 섬유아 세포를 분리. 매체를 기음과 fibroblast 매체 10ml로 세포를 resuspend. 미리 코팅된 10-cm 판으로 세포를 전송합니다. </li><li> 24 시간 이내에, hESC 배양 매체 (20 % 노크 아웃 혈청 교체, DMEM/F12, 0.1 ML 비 필수 아미노산, 4 NG / ML bFGF, 펜 / Strep / 글루 탐 산염, 베타 merceptoethanol)로 매체를 교체하십시오. 매일 매체를 변경합니다. ESC와 같은 식민지 감염 후 20-27 일에 의해 나타나기 시작합니다. </li></ol> 2. iPSCs의 분리 및 확장 <ol><li> 형광 현미경에서 hESCs에 유사한 형태를 보여줍니다 식민지에서 GFP 형광의 부재를 확인한다. </li><li> 10 μl 피펫을 사용하여 개별 iPSC 식민지를 선택하고 젤라틴 및 MEF-COA 잘 하나에 넣어테드 12 잘 플레이트와 hESC 매체와 보충. 매일 매체를 변경합니다. </li><li> passaging 들어, DMEM/F12 1 ML과 접시를 씻으 다음, collagenase 0.5 ML을 추가, 37시 10 분 품어 ° C를 </li><li> DMEM/F12으로 두 세포를 씻으십시오. </li><li> 신선한 hESC 매체 2 ML을 추가합니다. 셀 리프터를 사용하여 작은 조각으로 식민지를 헤어지게하고 접시에서 남아있는 세포를 분리. </li><li> 젤라틴 및 MEF-코팅 6 - 잘 접시의 잘 하나에 콜로니 resuspended 조각을 전송합니다. </li></ol> 3. Pluripotent 마커 Immunofluorescence 분석 <ol><li> PBS로 세포가 세 번 씻어하고 실온에서 20 분에 대한 4 % paraformaldehyde로 고정시킨다. </li><li> 부드럽게으로 0.2 % Triton은 30 분 대한 PBS의 X-100. PBS로 세포가 세 번 씻고 permeabilize </li><li> 두 시간 동안 PBS에서 3 % BSA와 세포를 잠복기가 아닌 특정 바인딩을 차단합니다. </li><li> 4에서 하룻밤 일차 항체와 세포를 품어 ° C. </li> &lt;리&gt; PBS로 세포가 세 번 씻고 빛으로부터 차폐, 실온에서 한 시간 만이라도 특정한 이차 항체와 세포를 품어. </li><li> 5 분간 실온에서 배양 다음 마지막으로 세척하는 동안 PBS와 추가 DAPI로 세포 세 번 씻는다. </li><li> 형광 현미경으로 염색법을 검색. </li></ol> 4. Pluripotent 마커에 대한 정량 실시간 PCR 분석 <ol><li> Qiagen의 RNeasy 키트를 사용하여 인간의 섬유아 세포에서 파생된 인간 iPSCs에서 총 RNA를 분리. </li><li> 윗첨자 II 역방향 Transcriptase를 사용하여 첫 번째 가닥 cDNA를 합성. </li><li> 이전에 보고된 primers를 사용하여 pluripotency 유전자를 검색할 qPCR을 수행 6. </li></ol> 5. 대표 결과 <ol><li> 프로그래밍 중에 형태학의 변화 우리는 OCT4, SOX2, KLF4 및 MYC 들고 retroviruses의 칵테일과 함께 인간의 섬유아 세포에게 BJ1와 디트로이트 551 감염,하고 (그림 1) 프로그래밍을하는 동안 형태학의 변화를 감지할 수 있었다. 스물 일일 감염 후, 우리는 그들의 hESC 같은 형태로 작은 iPSC 식민지 인식하고 있습니다. 더욱이, 우리는 GFP의 형광에 의해 iPSCs을 인식하고 있습니다. 이러한 ESCs 및 iPSCs 같은 Pluripotent 줄기 세포는 proviral 유전자 발현에게 7-9을 주체할하는 분자 기계를 표현. 우리 고유의 retroviral 벡터는 retroviral LTR에 의해 유전자 프로그래밍을 함께 GFP를 표현합니다. 따라서 지속적으로 GFP를 표현하는 세포 proviral 유전자 입을없이 transgenes을 표현하는 것으로 간주됩니다. pluripotency 분자 네트워크를 취득 충실하게 다시 프로그램 iPSC 식민지는 GFP 표현식 (그림 2) 10의 부재를 보여줍니다. </li><li> 인간 iPSCs의 pluripotency의 특성화 우리는 Tra-1-81, Tra-1-60, SSEA-4, SSEA-3, OCT4 및 NANOG의 항체 (과 immunohistochemistry를 통해 디트로이트-551 섬유아 세포에서 파생된 식민지를 분석 <stroNG&gt; 표 2). 성공적으로 다시 프로그램 iPSCs 이러한 마커 (그림 3A)을 모두 표현. 우리는 또한 정량 RT-PCR 분석을 통해 유전자 발현을 분석했다. 우리는 OCT4의 표현은, SOX2, KLF4, MYC와 NANOG 크게 부모 fibroblast 세포에 비해 증가했지만 H9 hESCs (그림 3B)의와 상응하는 것을 관찰했다. </li></ol><img alt="그림 1" src="/files/ftp_upload/3804/3804fig1.jpg" /> 1 그림. 레트로 바이러스에 감염된 인간 섬유아 세포의 형태학의 변경됩니다. (AD) 프로그래밍 요소에 감염된 디트로이트 - 551 섬유아 세포에서 식민지의 프로 그레시브 형태학의 변화. 5 일 (A), 10 일째 (B) 일 14 (C) 일 21 (D). 셀은 21 일 후에 hESC 같은 형태를 보여줍니다. <img alt="그림 2" src="/files/ftp_upload/3804/3804fig2.jpg" /> 그림 2. 언어학 재활 치료를 받고 셀에 대표 GFP 형광 표현. </strong&gt; BJ와 디트로이트 551 섬유아 세포가 레트로 바이러스 네 개의 프로그래밍 요소에게 4 표현에 감염, 그리고 4 주 동안 hESC 매체에 incubated했다. BJ 섬유아 세포 (A, B)와 디트로이트 551 (C, D)는 형태학의 유사한 표시됩니다. 일 21 일부터, GFP 부정적인 식민지는 타고난 iPSCs 10을 대표하는 형성되기 시작합니다. (E, F)의 적절한 언어학 재활 치료를받은하지 않은 디트로이트 551 세포를 변형 보여줍니다. 위상 콘트라스트보기 미만 (A, C, E) 식민지. (B, D) 제대로 GFP의 입을 보여주 세포를 다시 프로그램. 변환 콜로니 (F) 밝은 GFP 발현. <img alt="그림 3" src="/files/ftp_upload/3804/3804fig3.jpg" /> 그림 3. 인간 유도된 pluripotent 줄기 세포의 특성화. () 휴먼 551-IPS-K1 세포 식민지는 pluripotent 세포에 공통 마커를 표현. DAPI의 염색법, 분야마다 전체 셀 내용을 나타냅니다. 부모 F의 (B)를 정량 실제 OCT4 표현을위한 시간을 PCR (RT-qPCR), SOX2, KLF4, MYCibroblast, 551-IPS-K1의 iPSCs하고, H9 인간 배아 줄기 세포 (hESCs). 데이터는 β-actin의 가사 유전자에 대한 정규화와 부모 fibroblast 세포 4의 표현 수준에 상대적으로 꾸몄다되었다.

<ol>
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<p class="jove_content"&gt; 우리는 공개 할게 없다.</p

iPSCs 내지 네 전사 요인 reprograms 인간 섬유아 세포의 표현. 많은 시도가 임상적으로 안전 iPSCs을 생성하는 비 통합 또는 비 유전자 접근 방법을 사용하여 인간 iPSCs를 생성하는 데 있었다. 지금까지 이러한 방법은 매우 낮은 효율을 보여 주며 재현성 11-14 향상을 위해 더욱 최적화가 필요합니다. 레트로 또는 lentiviral 방식은 쉽게 도출 및 바이러스성 통합으로 인한 안전 문제에 덜 의존 체?...

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">hESC medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>80%</td>
			<td>Invitrogen</td>
			<td>11330057</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>20%</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>2 mM</td>
			<td>Invitrogen</td>
			<td>25030081</td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids (10 mM)</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>11140050</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol (14.3 M) or MTG</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>M-6250</td>
		</tr>
		<tr>
			<td>bFGF-2 10 &mu;g/ml</td>
			<td>4 ng/ml</td>
			<td>GIBCO/BRL</td>
			<td>GF003AF</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td colspan="4">Fiboblasts Medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
			<td>Invitrogen</td>
			<td>11965118</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
			<td>Invitrogen</td>
			<td>10407028</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
	</tbody>
</table>
Table 1. Culture Medium
<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab19857</td>
		</tr>
		<tr>
			<td>SSEA3</td>
			<td>1:100</td>
			<td>Milipore</td>
			<td>MAB4303</td>
		</tr>
		<tr>
			<td>SSEA4</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560218</td>
		</tr>
		<tr>
			<td>Tra-1-81</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560173</td>
		</tr>
		<tr>
			<td>Tra-1-60</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560174</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab21624</td>
		</tr>
		<tr>
			<td>Alexa-Flur 488</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa-Flur 555</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:5000</td>
			<td>Invitrogen</td>
			<td>D1306</td>
		</tr>
		<tr>
			<td colspan="4">Plasmids</td>
		</tr>
		<tr>
			<td>pMIG-OCT4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17225</td>
		</tr>
		<tr>
			<td>pMIG-SOX2</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17226</td>
		</tr>
		<tr>
			<td>pMIG-KLF4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17227</td>
		</tr>
		<tr>
			<td>pMIG-MYC</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>18119</td>
		</tr>
		<tr>
			<td colspan="4">Other Materials</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>1mg/ml</td>
			<td>Invitrogen</td>
			<td>17104019</td>
		</tr>
		<tr>
			<td>Gelatin, Porcine</td>
			<td>0.1%</td>
			<td>Sigma</td>
			<td>G 1890</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>0.2%</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>4%</td>
			<td>Sigma</td>
			<td>47608</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>3%</td>
			<td>American Bioanalytical</td>
			<td>AB01800</td>
		</tr>
		<tr>
			<td>MEF feeder cells</td>
			<td>&nbsp;</td>
			<td>Millipore</td>
			<td>PMEF-N</td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>&nbsp;</td>
			<td>Corning</td>
			<td>3008</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td colspan="4">Fluorescent microscopy: inverted microscope with GFP filter</td>
		</tr>
	</tbody>
</table>
Table 2. Reagents and equipment.

reprogramming human somatic cells into induced pluripotent stem cells (ipscs) using retroviral vector with gfp

인간 배아 줄기 세포는 (hESCs) pluripotent 및 체외 질병 모델링과 의학 1 재생에 대한 소중한 휴대 소스입니다. 그것은 이전에 인간의 체세포은 4 전사 요인 자궁외 표현 (Oct4, Sox2, Klf4과 Myc)에 의해 pluripotency로 다시 프로그램 및 유도된 pluripotent 줄기 세포 (iPSCs에게) 2-4이 될 수있는 것으로 나타났습니다. hESCs 마찬가지로, 인간 iPSCs는 pluripotent과 autologous 세포에 대한 잠재적인 소스입니다. 여기 GFP 함유 retroviral 등뼈 네에 복제된 네 개의 프로그래밍 요소와 인간 fibroblast 세포를 재설 정할 프로토콜을 설명합니다. 다음과 같은 프로토콜을 사용하여, 우리는 인간 ESC의 배양 조건 하에서 3~4주 인간 iPSCs를 생성합니다. 인간 iPSC의 식민지가 밀접한 형태의 hESCs 닮은 및 retroviral transgene의 입을의 결과로 GFP 형광의 손실을 표시합니다. iPSC의 식민지는 형광 microsco하에 기계적으로 분리PE는 hESCs과 비슷한 방식으로 동작합니다. 이러한 세포에서는 여러 pluripotency 유전자 및 표면 마커의 표현을 감지.

Watch this Scientific Journal Video about Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP at JoVE.com

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

OCT4의 레트로 바이러스 - 매개 자궁외 표현을 통해 인간의 유도된 pluripotent 줄기 세포 (iPSCs) 생성하는 방법은, SOX2, KLF4 및 MYC가 설명되어 있습니다. GFP 발현을 바탕으로 인간 iPSC 식민지를 식별하기위한 실용적인 방법도 설명되어 있습니다.

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 ...

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

20.4K 조회수.  Yale School of Medicine. OCT4의 레트로 바이러스 - 매개 자궁외 표현을 통해 인간의 유도된 pluripotent 줄기 세포 (iPSCs) 생성하는 방법은, SOX2, KLF4 및 MYC가 설명되어 있습니다. GFP 발현을 바탕으로 인간 iPSC 식민지를 식별하기위한 실용적인 방법도 설명되어 있습니다.

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

4 전사 인자와 마우스 배아 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성, Doxycycline는 Lentiviral 전달 시스템 Inducible

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

연구

생물학

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Stemgent 인간 TF의 Lentivirus와 인간의 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성 세트

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

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 ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

인간 유도 Pluripotent 줄기 세포의 선택 및 분리 식민지는 성인 섬유아 세포에서 다시 프로그램

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

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 ...