Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.

1. 미디어 준비 참고 : 1 비율 : 일반적으로 1, B27 보충제로 보충 수정 N2 보충과로 Neurobasal 보충 DMEM / F12 : 프로토콜은 두 개의 별도의 미디어 믹스의 사용에 의존한다. <ol><li> 15 ㎖의 튜브에 성분을 혼합함으로써 변성 N2 보충제를 준비한다. 소용돌이 나이를 필터링하지 마십시오; 용액이 깨끗해질 때까지 튜브를 반전시켜 혼합한다. <ol><li> 다음 (0.01 M 염산에서 만든) 25 ㎎ / ㎖ 인슐린 1 ㎖를 추가하고 튜브를 반전하여 잘 혼합, DMEM / F12의 7.2 ml의 피펫 팅으로 시작합니다. 이 솔루션은 분명하다 때까지 몇 분 정도 걸립니다. </li><li> (물에 만든) 100 mg을 1 ㎖ / ml의 아포 트랜스페린, ml의 progesternone (에탄올에 만든) / 0.6 mg을 33 μL, 160 ㎎ / ㎖ (1 M) 퓨 트레 신 (putrescine)의 100 μl를 추가 (물에서 만든 ), 물 (10)에 만들어진 3 mM의 나트륨 셀레 나이트 () 및 7.5 % 소 혈청 알부민의 666 μL의 μL 잘 튜브를 혼합한다. -20 °에서 2.5 ml의로 나누어지는 및 저장최대 2 개월 동안 C. </li></ol></li><li> 미디어를 준비합니다. <ol><li> DMEM / F12 250 ml의 N2의 2.5 ml를 추가합니다. 잘 섞는다하지만 흔들거나 필터가 없습니다. </li><li> 로 Neurobasal 250 ml의 미디어는 B27 보충 5 mL를 추가 할 수 있습니다. 잘 섞는다하지만 흔들거나 필터가 없습니다. </li><li> : 1의 비율로 1 단계 1.2.1과 1.2.2에서 미디어를 섞는다. 1 일부의 경우 : 3 혼합이 요구 될 수있다 (단계 : 1.2.4 1 혼합 1로 보충). </li><li> 혼합 L- 글루타민 (최종 농도 0.2 mM의) 및 2- 머 캅토 에탄올 (최종 농도 0.1 mM)을 3.5 μL의 0.5 ml에 추가하고 흔들림없이 잘 섞는다. 최대 3 주 동안 4 ℃에서 미디어를 저장하고 빛에 노출시키지 마십시오. 이 마지막 믹스 N2B​​27이라고합니다. </li></ol></li><li> 젤라틴 용액을 준비합니다. <ol><li> 15 분 동안 121 ° C, 15 psi에서 초순수 및 오토 클레이브 1 % 젤라틴 용액을 준비한다. 그것은이 사용 세제 병 살균제 완전히 깨끗 것이 중요하므로 새로운 병 것이 좋다이 사용하고 오직 용도 사이 초순수 세정. 50 ㎖ 씩에 1 % 용액을 나누어지는 달 4 ℃에서 보관하십시오. </li><li> 용해 될 때까지 37 ° C의 수조에 1 % 젤라틴 분취 따뜻한 따뜻한 인산염 완충 식염수 (PBS) 500㎖의 추가. 잘 섞는다 잘 또는 플레이트 각의 바닥을 커버하기에 충분 피펫. 적어도 30 분 동안 코트에 젤라틴을 표면을 허용합니다. </li></ol></li></ol> 2. 도금 세포 참고 :이 프로토콜은 백혈병 억제 인자 (LIF), LIF 뼈 형태 형성 단백질 4 (BMP4) 또는 MEK와 GSK3 억제제 (2I 미디어)와 LIF 또는 혈청이없는 매체와 혈청 교체와 10 %의 혈청에서 재배 마우스 ESC에 동일하게 적용 또는 LIF없이. 그러나, 분화의 타이밍 및 효율 미디어 세포 (설명을 참조)에 따라 달라질 수있다. 여기에 표시된 실험을 위해 우리는이 EGFP 기자는 O에 노크가 (46C 마우스 ESC 라인을 사용10 % 혈청 및 LIF로 GMEM 재배 Sox1 내인성 대립 유전자의 NE). 최적의 결과는 세포가 해리와 N2B27 미디어에 플레이 팅하는 것이 중요하다; N2B27 항상 세포 replating에 비해 감소 된 분화 효율 결과를 단순히 GMEM / 혈청 / LIF의 용지 변경. <ol><li> 10 % 혈청, 1 mM 나트륨 피루 베이트, 1X 비 필수 아미노산 GMEM에서 세포를 성장 0.1 M 2- 머 캅토 에탄올, 100 유닛 / ㎖ LIF (13). 마우스 ESC, 판의 subconfluent 문화를 얻으려면 2 걸릴 것 T25 문화 플라스크에 넣고 1 × 10 6 세포 - 3 일. </li><li> 밝은 필드 현미경으로 세포를 관찰한다. 세포가 통로 subconfluent과 준비가되면, PBS로 두 번을 씻어. 세포 분리 시약 1 ㎖를 추가하고 2 인큐베이터로 돌아 - 5 분​​. 트립신 및 다른 효소는 세포의 분리를 위해 사용될 수 있지만, 도금 밀도가 될 필요가 있으므로, 그들은 adju 세포 부착에 부정적인 영향을 미칠 수있다다네. </li><li> 세포는 플레이트로부터 리프트 시작되면, 단일 세포 현탁액으로 그들을 이동시키다 용기를 탭. 15 mL의 원심 분리 튜브에 용지 및 세포 분리 및 시약 피펫 10 ㎖의 전체에 세포를 수집. </li><li> RT에서 3 분 동안 300 XG에 세포를 스핀. </li><li> 조심스럽게 뜨는을 대기음 아래 3로 pipetting에 의해 미리 예열 N2B27 10ml에 펠렛을 재현 탁 - 5 회. 아래 현탁액을 피펫 팅 할 때, 튜브의 측면에 대한 피펫은 거품을 만들지 않도록합니다. 자동 세포 계수기 또는 혈구와 세포를 세어 농도를 기록한다. </li><li> 단위 부피 당 세포의 수를 포함하는 세포 현탁액은 미리 예열 N2B27에 웰 당 도금 될 준비한다. 6 잘 접시에 cm 2 당 10,500 세포의 밀도는 (즉, 6 잘 요리 잘 당 1 × 10 5 세포)에 최적입니다. CM 2 괞 찮아 당 세포의 제안 수는 표 1을 참조하십시오세포 배양 용기의 다양한 팅 매체 볼륨. </li><li> 배양 용기로부터 젤라틴 대기음 표 1에 제시 및 부피 밀도에 따라 세포 현탁액을 접시.이 셀 중앙에 집중되므로 플레이트 소용돌이 마십시오. 37 ℃, 5 % CO 2에 습기가 인큐베이터에서 문화를 놓습니다. </li><li> 신선한 N2B27 모든 미디어를 대체하여 2 일 - 매 1 미디어를 변경합니다. 부드럽게 피펫 밀도에 영향을 다음 날에 분화 효율을 줄일 수있는 세포를 플러싱 등. 도금 후 초기 생존이 나쁜 경우, 하나의 셀을 판 : N2와 B27-보충 미디어의 3 믹스 처음 두 일 후에 N2B27로 변경. 세포 분화하기 시작하고 4 내에서 (여기에 사용 된 46C 세포주에서 기자의 녹색 형광으로 표시) Sox1 식 표시해야합니다 - 6 일. </li></ol> 3. 면역 형광 염색법 아니TE는 : 어떤 프로토콜 타입 용기에 적용될 수있다. 각 시약의 부피는 6 웰 플레이트 잘마다 설명하고 표면 영역을 확장 할 수 있습니다. Replating 나중에 낮은 가능성으로 인해 사용하지 않는 것이 좋습니다. <ol><li> 차별화 매체를 제거하고 4 %의 500 μL (v / v)의 파라 포름 알데히드를 첨가하여 세포를 고정, 15 분 동안 둡니다. </li><li> 워시 ((v / v)의 트윈 20 0.5 %와 인산염 완충 식염수)을 두 번 PBS-T의 2 ml의 세포를 Permeabilize 하시려면. 세포를 4 ℃에서 최대 2 개월 동안 PBS-T에서 유지 될 수있다. </li><li> 10 % (이차 항체와 같은 종) (v / v)의 혈청과 PBS-T의 1 ml의 PBS-T를 교체합니다. 비 특이 적 결합을 차단하는 RT에서 로커 플레이트에 1 시간 부화. </li><li> 1 시간 후, PBS-T 2 ㎖로 두 번 세포를 씻어 βIII - tubulin에 대한 500 μL 마우스 IgG의에서 부화 (10 % 혈청 PBS-T에서 1000 1 희석). 판 로커에 넣고 4 ℃에서 O / N 두십시오. </li><li> 이 단계에서, 항상 용기를 보호빛에서. 다음, (10 % 혈청 PBS-T에서 2 μg의 / ㎖로 희석) 형광 표지 항 마우스 IgG 항체 500 ㎕를 추가, T-PBS로 두 번 세포를 씻어. 실온에서 1 시간 동안 판 로커에 넣어. </li><li> 다음 DAPI의 500 μl를 추가, PBS-T로 두 번 세포를 씻어 (PBS-T에서 0.5 ㎍ / ml의 희석)과 5 분 동안 둡니다. </li><li> 다시 세포를 세척하고, PBS-T에 보관하십시오. 세포는 촬영 준비가 4 ° C에서 최대 2 주 동안 유지 될 수있다. 그것은 고정 생균 또는 다른 시간에 고정 된 세포의 GFP 강도를 비교하는 부적절한 그래서 GFP 신호가 시간이 지남에 따라 페이드 참고. </li></ol>

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The authors have nothing to disclose.

단층 신경 분화 프로토콜은 십 년 6 이상 사용되어왔다. 프로토콜 한정 배지 구성되며, 전임상위한 시스템이 더 적용될 수 단층 시스템에서 수행, 고도로 효율적인 (예, 약물 스크리닝)을 사용한다. 그러나 분화 효율을 결정하는 몇 가지 중요한 요소가있다. 이 문서에서는 이러한 요인과 각 장애물에 대한 솔루션을 지적한다. 분화 조건 도금 후 세포 밀도는 아?...

배아 줄기 세포는 동계 또는 면역 호스트로 (키메라를 형성하여) 적절한 단계 배아 내로 재 도입 다음 모든 성인 세포 유형으로 분화 할 수있는 능력, 주입을 유지하면서 시험 관내에서 무기한 증식 용량 초기 배아 유래의 다 능성 세포이다 (기형 종 형성에 의해) 또는 해당 큐 1 체외 주제에. 신경 계통에 마우스 배아 줄기 세포의 체외 분화는 1995 년에 처음 설명과 morphogen 레티노 산 2-4 보충 혈청 함유 미디어에서 다세포 서스펜션 집합체의 형성 (배아 체, EBS을 (를)) 참여했다. 이후 다양한 프로토콜 신경 분화 5를 허용하도록 개발되어왔다. 여전히 응집을 이용하는 많은 다른 세포 유형으로 유도 배양 공동 여러 혈청이없는 배지의 사용을 포함한다. 모든 프로토콜은 장점을 가지고차 단점과 신경의 정확한 특성 또는 신경 세포는 또한 사용되는 프로토콜에 따라 달라집니다 생산. 이상적인 프로토콜, 강력한 확장 성이 완벽하게 정의 된 미디어와 기판을 활용, 분화 과정의 비 침습적 모니터링 의무가하고 수 신경 전구 세포의 순수한 인구의 발생을 초래할 외부 단서에 의해 구별하는 패턴 할 것 상대적으로 짧은 시간에 높은 효율과 수율 모든 신경 세포와 교세포 하위 유형으로. 지난 12 년에 우리는 저밀도, 무 혈청 접착 단층 배양 6-10 ESC 마우스에서 신경 전구 세포 및 신경 세포를 생성하는 방법을 사용하고있다. 이 프로토콜은 위에 명시된 기준 중 많은 충족 : 우리 손에 분화의 효율이 상당히 오랜 세월에 걸쳐 일관되고 다양한 세포주왔다가 상하 또는 축소 될 수있다 (우리는 성공적으로 96 웰 플레이트에서 용기를 사용 15cm 직경 디급히 드릴 말씀) 사용 된 미디어는 잘 정의되어 있습니다. 프로세스 (도파민 뉴런 6 예, 쉬와 Fgf8) 신경 가지 유형의 서브 타입의 생성을 유도하기 위해 첨가 될 수 분화의 모니터링 및 패터닝 단서의 다양한 timelapse에 현미경 의무가있다. 그럼에도 불구하고,이 프로토콜의 성공적인 응용 프로그램에 몇 가지 문제가있다. 주요 측면 중 하나는 미디어의주의 준비입니다. 우리는 항상 상업 대안의 가능성에도 불구하고 사내 매체를 준비합니다. 사용되는 보조제 중 하나 (N2, 프로토콜 참조)는 표준 상업적으로 이용 가능한 버전에 비해 수정이 있습니다. 마지막으로,이 방법의 성공적인 적용을위한 가장 중요한 단계 중 하나는 도금에 최적의 셀 밀도이다. 유도 신호 중 하나 (Fgf4 11)의 분비 자연이 필요로하는 동안 때문에 충분한 세포가 최적의 viabil을 허용 할 수 있는지 주로너무 높은 밀도의 분화에 성만과 차별화, (인해 LIF (12)의 분비 생산 가능한 부분) 손상된다. 그것은 두 매체 준비와 세포 도금 신중하게 수행하고 지속적으로 최적의 결과를 보장하는 것이 중요하다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Reconstitute with sterile 0.01M HCl</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Reconstitute with ethanol</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V</td>
			<td>Life Technologies</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Life Technologies</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukaemia inhibitory factor</td>
			<td colspan="3">prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Mouse anti betaIII tubulin IgG antibody</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence-labelled anti-Mouse IgG antibody</td>
			<td>Life Technologies</td>
			<td>A31571</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>25cm2 tissue culture flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
	</tbody>
</table>

neural differentiation of mouse embryonic stem cells in serum-free monolayer culture

신경 전구 세포에 마우스 배아 줄기 세포가 분화하는 능력 (ESC)는 신경 사양뿐만 아니라, 더 깊은 연구 성숙한 신경 세포 유형의 발생을 제어하는​​ 메커니즘의 연구를 허용한다. 이 프로토콜에서 우리는 무 혈청, 단층 문화를 사용하여 신경 전구 세포에 ESC의 분화하는 방법을 설명합니다. 이 방법은 효율성, 확장 성 및 4 내 70 %의 신경 전구 세포 ~의 생산 결과 - 6 일. 이것은 다양한 조건 하에서 성장 다양한 균주에서 ESC에인가 될 수있다. 신경 전구 세포는 현미경으로 기능적 뉴런 및 신경교 또는 분석으로 더 분화 세포 계측법 또는 분자 기법을 흐르게 할 수있다. 분화 과정은 시간 경과 현미경에 순종하며 신경 특정 처리를 모니터링하는 리포터 라인의 사용과 결합 될 수있다. 우리는 프로세스가 ㄱ 할 수 있도록 미디어 준비 및 세포 밀도 최적화에 대한 자세한 지침을 제공E는 가장 ESC 라인 및 세포 배양 용기의 다양한 적용.

이 실험에서, 우리는 신경 분화를 추적하기 위해, 내인성 Sox1-GFP 리포터와 마우스 배아 줄기 세포를 46C 세포주 (14)를 사용했다. Sox1, 신경 전구 세포에 대한 마커의 세포주, 식을 사용하여 녹색 형광으로 검출 할 수있다. 밀도 도금하여 신경 분화를 달성하는 중요한 요인이다. 마우스 배아 줄기 세포 / cm 2 10,500 88,500 개의 세포로부터 다양한 다른 밀도로 6 웰 플레이트에 플레이 팅 <st...

Watch this Scientific Journal Video about Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture at JoVE.com

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

혈청이없는 단층 문화 마우스 배아 줄기 세포의 신경 분화

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

neural-differentiation-mouse-embryonic-stem-cells-serum-free

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Developmental Biology

14.0K 조회수.  University of Dundee. This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

비디오: Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

기본 및 전임상 연구 밀폐 세포 배양 시스템에서 인간 다 능성 및 신경 줄기 세포를 배양

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

연구

발생학

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

배아 줄기 세포와 조직의 심장 차별화의 성적인 규정

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

약 발견에 적합 EMT 조사를위한 효소 - 및 혈청이없는 신경 줄기 세포 배양 모델

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

기본 및 확실 한 조 혈 창시자 인간 만능 줄기 세포에서의 감독

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

인간 다 능성의 체외 분화에서 융모 세포로 줄기 세포

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

두와 입체 배아 체에서 마우스 배아 줄기 세포의 레티노 산에 의한 신경 분화 분석

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

인간의 배아 또는 유도 만능 줄기 세포에서 망막 안료 상피 세포의 급속 한, 감독 차별화

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

인간 다능성 줄기 세포를 간 세포로 효율적으로 분화

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.