Contreras와 Watts Laboratories와 William Raphael 박사. 이 실험은 NHLBI F31 HL128035-01 (조직 분해 프로토콜 표준화), NHLBI 5R01HL117847-02 및 2P01HL070687-11A1 (동물) 및 NHLBI 5R01HL117847-02 (세포 분리 및 배양)에 의해 지원되었다.

이 문서에 설명 된 모든 절차는 Michigan State University의 기관 동물 관리 및 사용위원회 (IACUC)에서 제정 한 지침을 따릅니다. 모든 버퍼와 미디어는 빛으로부터 보호되어야합니다. 1. 버퍼, 미디어 및 기기 준비 <ol><li> Krebs Ringer Bicarbonate-Buffered 용액 (KRBB) 준비 : 135 mM 염화나트륨, 5 mM 염화칼륨, 1 mM 황산 마그네슘, 0.4 mM 인산이 수소 칼륨, 5.5 mM 포도당, 1 % 항생제 / 항균제 (10,000 units / mL 페니실린, 10,000 μg / mL 스트렙토 마이신, 25 μg / mL 암포 테리 신 B) 및 10 mM HEPES (pH = 7.4). 이 용액은 4 ℃에서 멸균 한 상태에서 3 주 동안 안정하다. </li><li> Collagenase Type 1 용액을 준비하십시오 : 4 % Bovine Serum Albumin (BSA)가 함유 된 KRBB 1mg / mL. 이 용액은 37 ° C에서 보관해야하며 4 시간 동안 안정합니다. </li><li> 적혈구 용해 용액 준비 : 154 mM ammonium chloride, 10 mM potassium bicarbonate, 및 0.1 mM EDTA. 4 ° C에서 최대 1 개월간 보관하십시오. </li><li> MCS 차단 버퍼 : DMEM / F12 미디어베이스, 10 % 태아 소 혈청 (FBS), 5 % 정상 당나라 혈청, 40 μL / ML F (ab) 단클론 항 쥐 IgG를 준비합니다. 4 ° C에서 최대 1 개월간 보관하십시오. </li><li> MCS 완충 용액 준비 : 인산염 완충 식염수 (PBS, pH = 7.5), 0.5 % BSA 및 2mM EDTA. 4 ° C에서 최대 1 개월간 보관하십시오. 유리 용기에 넣고 37 ° C로 가열 한 후 15 초 동안 진공을 가하여 De-gas 용액을 만든다. 사용하지 않은 상태로 봉인 된 상태로 두십시오. </li><li> FBS, FCS, 1 % 항생제 / 항균제 (10,000 units / mL 페니실린, 10,000 μg / mL 스트렙토 마이신, 25 μg / ml), Dalbecco &#39;s Modified Eagles Medium (DMEM) / ml amphotericin B), 44.05 mM 중탄산 나트륨, 100 μM 아스코르브 산, 33 μM 비오틴, 17 μM 판토텐산, 2 mM L- 글루타민 및 20 mM HEPES. 4 ° C에서 2 주 동안 무균 상태로 유지하십시오. </li><li> APC &amp;표피 성장 인자 10 ng / mL, 백혈병 억제 인자 (10 ng / mL), 혈소판 유도 성장 인자 BB (10 ng / mL) 및 염기성 섬유 모세포 성장 인자 5 ng / mL). 4 ° C에서 2 주 동안 무균 상태로 유지하십시오. </li><li> APC 유도 배지 : 10 % FBS, 2.5 μg / mL 인슐린, 0.5 mM 2-isobutyl-1-methylaxanthine (IBMX), 1 μM dexamethasone 및 200 pM T3 (triiodothyronine 갑상선 호르몬)가 포함 된 APC Media를 준비하십시오. 4 ° C에서 2 주 동안 무균 상태로 유지하십시오. </li></ol> 2. 지방 세포 전구 세포 분리 <ol><li> 기관 지침에 따라 쥐를 마취. 쥐를 등받이에 놓습니다. 발가락 핀치를 통한 마취 깊이와이 고통스러운 자극에 대한 반사 반응의 상실을 확인하십시오. 참고 :이 프로토콜은 10 주 된 Sprague Dawley 쥐와 70 mg / kg의 pentobarbital을 복강 내 주사를 통해 전달합니다. </li><li> 수직 정중 절개를 만들어라.흉골 부위의 흉골과 회음부의 가위. 복강에 접근하여 상 장간막 동맥, 작은 장간막 저항 혈관 (mPVAT) 및 흉부 대동맥 (aPVAT)을 노출시킵니다. <ol><li> 장간막과 대동맥에 모든 연결을 단절하고 동물에서 혈관을 제거하십시오. 해부 현미경과 KRBB로 가득 찬 페트리 접시를 사용하여 PVAT를 분리하고 혈관을 관찰하고 PVAT를 분리합니다. </li><li> 이 실험에서는 비 PVAT 지방 저장소를 나타내는 생식선 (Gonadal, GON)을 수집합니다. 10 밀리미터 HEPES (산도 = 7.4)와 KRBB에 얼음에 격리 지방 패드를 놓으십시오. </li><li> 바이오 안전성 (biosafety) 두드림에서 조직 약 50 mg을 콜라게나 제제 I 용액 1 mL가 들어있는 1.7 mL 튜브에 옮기고 조직 가위 (1 - 3 mm 조각)로 말하다. </li></ol></li><li> 1 시간 동안 rotisserie 인큐베이터 (또는 궤도 진탕기를 가진 인큐베이터)에서 37 ° C에서 배양하여 샘플을 소화합니다. biosafety 후드에서 100과 40을 통해 소화 된 물질을 순차적으로 걸러냅니다.<html> μm 세포 스트레이너를 50 mL 튜브에 넣었다. 생성 된 여과 액을 4 ℃에서 원심 분리하여 300 x g에서 10 분간 원심 분리한다. 참고 : 여기에서 프로토콜의 모든 단계는 세포를 살균 유지하기 위해 생물 안전성 보닛에서 수행되어야합니다. </li><li> SVX 세포가 들어있는 상등액과 resuspend 펠렛을 1X 적혈구 용적 완충 용액 1 mL에 넣고 1.7 mL 미세 튜브에 옮긴다. 빛으로부터 보호 된 RT에서 5 분간 세포를 인큐베이션하고 4 ℃에서 300 x g에서 5 분간 원심 분리한다. </li><li> SVF Basal Media에 상청액을 붓고 남아있는 세포 펠렛을 재용 해하십시오. Trypan 블루 솔루션으로 살아있는 세포를 계산하기 위해 20 μL 하위 샘플을 수집합니다. 참고 : 격리 할 수있는 SVF 셀의 수는 사이트마다 다릅니다. 조직 1mg 당 평균 SVF 수는 다음과 같습니다. aPVAT = 5.0 ± 2.0x10 3 , mPVAT = 1.04 ± 0.62x10 4 , GON = 2.4 ± 1.2x10. </li></ol> 3. 자기 활성화 셀 정렬 _content &quot;&gt; 참고 : 4 ℃에서 모든 단계를 수행하여 CD34 및 PDGFRα 세포 표면 마커를 기반으로 SVF에서 APC를 분리합니다. <ol><li> 300 x g에서 5 분간 세포를 회전시킨다. 뜨는을 붓고 1 X 10 6 세포 / ML에서 MCS 차단 버퍼에 세포 펠렛을 resuspend 20 분 동안 품어. 참고 : 1x10 6 - 2 x 10 8 세포 / mL의 세포 현탁액을 효과적으로 분리 할 수 ​​있습니다. </li><li> 4에서 30 분 동안 FITC - 결합 마우스 안티 CD34 (1 μg / 1 X 10 6 셀) 5 μL와 세포를 품어 ° C. 300 xg 및 4 ° C에서 5 분간 세포를 스핀. <ol><li> 항 -FITC 마이크로 비드 4 μL와 4 ° C에서 5 분간 MCS 완충액 96 μL (총 부피 100 μL)와 함께 항온 배양하여 CD34 + 및 CD34 - 세포를 분리한다. </li></ol></li><li> 자성 분리기를 스탠드에 부착하고 컬럼 날개가 앞쪽에있는 MultiSort (MS) 컬럼을 분리기에 넣으십시오. 5 mL포집 관을 상부 관 홀더의 MS 칼럼 아래에 놓는다. </li><li> MCS Buffer Solution으로 헹구어 MS Column을 준비하십시오. 컬럼 상단에 500 μL의 MCS Buffer를 넣고 완충액을 통과 시키십시오. 유출 물을 버리고 수거 튜브를 교체하십시오. </li><li> 준비된 MS 칼럼에 항체 표식 세포 현탁액을 적재하십시오. 레이블이없는 세포가 들어있는 플로우 스루를 수집하십시오. </li><li> 탈기 된 MCS 완충액 3x 500 μL로 MS 칼럼을 세척하십시오. 이전 단계에서 통과 흐름과 결합하여 전달 unlabeled 세포를 수집합니다. </li><li> 마그네틱 분리기에서 MS 컬럼을 제거하고 새로운 수집 튜브에 놓습니다. MS 칼럼에 MCS 완충액 1 mL 피펫. 즉시 자석으로 표시된 세포로 분획을 즉시 플러싱하지만, 칼럼과 함께 제공된 플런저를 천천히 적용하여 과잉 가스가 칼럼에 들어 가지 않도록하십시오. 참고 : 격리 된 셀 번호는 사이트마다 다릅니다. SVF 집단으로부터 분리 된 CD34 + APC의 평균 수조직의 mg은 aPVAT = 2.6 ± 0.43x10 2 , mPVAT = 9.6 ± 1.4x10 2 , GON = 1.3 ± 0.22x10 3 이다. </li><li> 300 × g 및 4 ℃에서 5 분간 세포를 스핀. 4시 30 분 1시 200 분 솔루션 / 1 X 10 6 세포 토끼 항 PDGFRα 10 μL에 수집 된 CD34 + 분만 품어 ° C. <ol><li> 300 XG 및 4 ° C에서 5 분 동안 세포를 다시 원심 분리하십시오. 분리 된 단계 3.3 ~ 3.7을 반복하여 안티 토끼 IgG microbeads 4 μL와 MCS 버퍼 96 μL로 표시된 세포를 품어. 참고 : 격리 된 셀 번호는 사이트마다 다릅니다. 조직 1mg 당 CD34 + 개체군으로부터 분리 된 PDGFRα + APC의 평균 수는 aPVAT = 2.4 ± 0.64, mPVAT = 8.4 ± 2.4, GON = 10.4 ± 1.9이며 이는 이전에 격리 된 개체군의 0.5-10 %이다. </li></ol></li></ol> 4. 세포 배양 및 Adipogenesis 유도 <ol><li> SVF 문화및 2 일마다 교체하는 기초 배지의 6-Well 조직 배양 판에서의 APC. 3 연속 통로 후, 8 웰, 24 웰, 48 웰 및 96 웰에서 평가되는 증식 검정을위한 1 x 10 2 세포 / 웰의 검정색 96 웰 웰 배양 플레이트 및 24 웰 플레이트 또는 50,000 세포 / 웰에서의 플레이트 질적 및 정량적 인 지방 생성 분석을위한 48-Well 조직 배양 플레이트에서 10,000 세포 / 웰. </li><li> 분화를 위해 표시된대로 뼈 형태 형성 단백질 4 (3.3 nmol / L)로 유도하기 전과 48 시간 후의 합류 후 APC 기초 배지 보충. APC 유도 미디어를 사용하여 세포를 부화시키기 위해 100 % confluency (0 일) 48 시간 후 세포를 유도하십시오. </li><li> 48 시간 후, 미디어를 변경하여 IBMX 및 dexamethasone이없는 APC 유도 매질에서 14 시간 동안 세포를 유지하고 매 48 시간마다 배지를 교환하십시오. </li><li> FACS에 의해 검증 된 MCS 격리. <ol><li> 자기 적으로 분리 된 50 μL (50,000 셀)의 하위 샘플을 가져옵니다.FACS 용액으로 세척 하였다. </li><li> 원심 분리기 세포와 PDGFRα + 세포를 라벨에 당나귀 안티 - 토끼 IgG Dylight 405의 1 : 1,000 솔루션 100 μL에 resuspend. 빛과 4 ℃에서 30 분 동안 인큐베이션하십시오. </li><li> Flow cytometer에서 488 nm (FITC) 및 405 nm (Dylight 405) 필터를 사용하여 분석 할 때까지 세포를 세척하고 2 % 포름 알데히드 용액 200 μL로 resuspend하십시오. 참고 : 배양 된 세포에 의한 지질 축적은 형광을 측정하는 마이크로 플레이트 판독기에서 지방 친화 지방 세포 형광 분석을 사용하여 정량적으로 평가되고 지질 축적을위한 교정자로 전 지방 세포를 사용합니다. 지질 축적은 또한 지질 염색 및 세포가 지질을 함유하지 않는 전체 세포의 비율을 관찰 할 수있는 역상 현미경에서 수행되는 영상화에 의해 정 성적으로 측정됩니다. 485 nm에서 여기와 572에서 방출로 형광을 측정 할 수있는 모든 플레이트 판독기nm는 디지털 이미지를 캡처 할 수있는 카메라가있는 모든 현미경뿐만 아니라 분석에도 적합합니다. </li></ol></li></ol>

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저자는 공개 할 것이 없습니다.

현재 실험의 중심은 PVAT 저장소에서 APC를 분리, 확장 및 지방 생성 유도하는 것입니다. 여기서 우리는 표면 마커 CD34 및 PDGFRα를 발현하는 세포의 동정에 기초한 APC의 분리 프로토콜을 제시한다. 이러한 표면 단백질은 이전 APC에서 높은 증식 속도와 다양한 지방 저장소 14 , 15 에서 흰색 또는 갈색 지방 세포로 분화 할 잠재력으로 확인되었습니다. 이러한...

혈관과의 근접성 때문에 혈관 주위 지방 조직 (Perivascular Adipose Tissue, PVAT)은 혈관 기능 1 에서 중요한 파라 크린 신호 전달 요소입니다. 이 지방 조직의 팽창은 지방 세포 전구 세포 (Adipocyte Progenitor Cells, APC)의 표현형에 따라 다릅니다 2 , 3 . 지방 세포로부터의 분리는 주요 지방 세포가 약하고 부력이 있고 크기가 다양하기 때문에 어렵습니다. 특정 분리 기술은 또한 염증 단백질 합성을 증가시키고 지방 생성 유전자 발현을 감소시킴으로써 세포 표현형 및 형태를 변화시킬 수 있으며, 세포의 완전성을 유지하는 프로토콜의 중요성을 강조한다. 1 차 세포 ​​및 특이적인 전 지방 세포 모집단의 배양 은 생체 내 성장 에 대한 환원 주의적 접근법을 제공하고, 동등한 세포 유전 학적 구성을 유지한다.이 세포들을 가지고있는 노화는 노화 또는 노화로 인한 악화로 인해 제한됩니다 6 . 피하 및 망막 저장소를 비롯한 다양한 지방 저장소의 전 지방 세포는 또한 특정 해부학 적 부위에서 세포를 수집하는 중요성을 강조하는 증식의 차이를 보여준다. 비 PVAT 백색 지방 저장소의 선구 세포는 이전 연구 7,8,9에서 특징 지어졌지만 PVAT APC 표현형에 대해서는 알려진 바가 적다. 여기에 설명 된 기술은 형태와 생존력, 증식 및 분화 가능성에 미치는 영향을 최소화하면서 특정 APC 집단과 정의 된 APC 집단을 분석 할 수 있습니다. MCS (Magnetic-Activated Cell Sorting)는 배양액과 같이 다운 스트림 응용 분야에 적합합니다. 구슬이 세포를 변경하지 않고 용해되기 때문입니다. MCS는 또한 경제적이며 일단 항체가 손상되면농도가 표준화 되었기 때문에 유동 세포 계측법의 필요성은 미미합니다. PVAT 선구 물질에 대한 시험 관내 연구는 또한 이러한 일차 세포가 가질 수있는 잠재력을 엿볼 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="757">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:325px;">Tissue Dissection</td>
			<td style="width:168px;"></td>
			<td style="width:108px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Dishes</td>
			<td>Handmade with Silicone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture Petri Dish</td>
			<td>Pyrex</td>
			<td></td>
			<td>7740 Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 170</td>
			<td>Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Braided Silk Suture</td>
			<td>Harvard Apparatus</td>
			<td>51-7615</td>
			<td>SP104</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope MZ6</td>
			<td>Leica</td>
			<td></td>
			<td>10447254</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomaster Microscope Fiber-Optic Light Source</td>
			<td>Fisher Scientific</td>
			<td>12562-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splinter Forceps</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>105-400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KRBB Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Phosphate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>7758-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic/Antimicotic</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type 1</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Thermo-Fisher Scientific</td>
			<td>2876</td>
			<td>Reciprocal Shaking Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Cabinet</td>
			<td>Thermo-Fisher Scientific</td>
			<td>1385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotisserie Incubator</td>
			<td>Daigger</td>
			<td>EF4894C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-549</td>
			<td>Yellow</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Blue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OctoMACS Kit</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(DMEM):F12 Medium</td>
			<td>Corning</td>
			<td>90-090</td>
			<td>Medium Base</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35016CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Donkey Serum</td>
			<td>AbCam</td>
			<td>AB7475</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD34</td>
			<td>Santa Cruz</td>
			<td>SC-7324</td>
			<td>FITC conjugated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PDGFR&#945;</td>
			<td>Thermo-Fisher Scientific</td>
			<td>PA5-17623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-007-003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS 10X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 Cell Incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>51030285</td>
			<td>Heracell 160i&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48- Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Plates, Black Wall</td>
			<td>Corning</td>
			<td>353376</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>144-55-8</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Calf Serum</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>58-85-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>137-08-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Corning</td>
			<td>61-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone Morphogenic Protein 4</td>
			<td>Prospec Bio</td>
			<td>CYT-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal Growth Factor</td>
			<td>PeproTech</td>
			<td>400-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia Inhibitory Factor</td>
			<td>PeproTech</td>
			<td>250-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived Growth Factor BB</td>
			<td>Prospec Bio</td>
			<td>CYT-740</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic Fibroblast Growth Factor</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Corning</td>
			<td>25-800-CR</td>
			<td>ITS Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>28822-58-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>50-02-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T3 (Triiodothyronine)</td>
			<td>Sigma-Aldrich</td>
			<td>6893-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyQUANT Proliferation Assay</td>
			<td>Thermo-Fisher Scientific</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AdipoRed Fluorescence Assay Reagent</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O Lipid Dye Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>O1391</td>
			<td>In Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M1000 Microplate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse Inverted Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Sight DS-Qil Camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

Paracrine 신호를 통한 혈관 기능의 주요 조절자인 Perivascular Adipose Tissue (PVAT)의 확장은 비만 동안 고혈압 발생과 직접적으로 관련이 있습니다. 비대 및 증식의 정도는 저장소 위치, 성별 및 존재하는 지방 세포 전구 세포 (Adipocyte Progenitor Cell, APC) 표현형의 유형에 달려 있습니다. 지난 10 년 동안 APC와 전 지방 세포 분리에 사용 된 기술은 특정 세포 표면 마커를 기반으로 개별 세포를 식별 할 수있는 정확성을 획기적으로 향상 시켰습니다. 그러나 APC와 지방 세포의 분리는 세포의 취약성으로 인해 어려움이 될 수 있습니다. 특히 손상되지 않은 세포가 세포 배양에 사용되어야하는 경우 특히 그렇습니다. MCS (Magnetic-activated Cell Sorting )는 지방 조직의 무게 단위 당 더 많은 수의 APC를 분리하는 방법을 제공합니다. MCS에 의해 수확 된 APC 는 시험 관내 프로토콜에 사용되어 prea세포에 의해 유지되는 다작 및 지방 형성 잠재력의 분석을 허용하는 성장 인자 칵테일의 사용을 통해 지방 세포로 이들을 분화시킨다. 이 실험은 대동맥 및 장간막 PVAT 저장소에 초점을 두었습니다.이 저장소는 확장 중에 심혈관 질환 발병에서 중요한 역할을합니다. 이 프로토콜은 정의 된 APC 인구를 분리, 확장 및 차별화하는 방법을 설명합니다. 이 MCS 프로토콜을 사용하면 최소한의 장비 또는 교육으로 세포 정렬이 필요한 모든 실험에서 격리를 사용할 수 있습니다. 이러한 기술은 세포 표면 마커의 존재를 기반으로 특정 세포 인구의 기능을 결정하기 위해 추가 실험을 도울 수 있습니다.

전 지방 세포의 증식 능력과 지방 세포 전구체의 지방 형성 잠재력은 시험 관내에서 유지 되는 특성이다 11 . 정량적 DNA 분석을 사용하여 도금 후 8, 24, 48 및 96 시간에 수컷 랫트의 aPVAT, mPVAT 및 GON으로부터 분리 된 SVF 및 APC의 시험 관내 증식을 평가 하였다. 같은 장소의 SVF 세포와 비교하여 96 시간까지 증식이 적은 aPVAT의 APC를 제외하?...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

여기에서는 MCS (Magnetic-activated Cell Sorting)를 사용하여 Perivascular Adipose Tissue (PVAT)에서 지방 세포 전구 세포 (Adipocyte Progenitor Cell, APC) 개체군을 분리하는 방법을보고합니다. 이 방법은 FACS (Fluorescence-Activated Cell Sorting)와 비교할 때 지방 조직 1g 당 APC의 증가 된 분리를 허용합니다.

자기 활성화 세포 정렬에 의해 분리 된 혈관 주위 지방 조직으로부터의 지방 전구 세포 전구 세포의 확장 및 지방 생성 유도

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

expansion-adipogenesis-induction-adipocyte-progenitors-from

Research

JoVE Journal

Developmental Biology

9.4K 조회수.  Michigan State University.  여기에서는 MCS (Magnetic-activated Cell Sorting)를 사용하여 Perivascular Adipose Tissue (PVAT)에서 지방 세포 전구 세포 (Adipocyte Progenitor Cell, APC) 개체군을 분리하는 방법을보고합니다. 이 방법은 FACS (Fluorescence-Activated Cell Sorting)와 비교할 때 지방 조직 1g 당 APC의 증가 된 분리를 허용합니다. 

비디오: Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

인두 아치 전의 VIVO 문화는 심장과 근육 전구 세포와 그 틈새를 연구

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

연구

발생학

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

인간 태반 조직에서 추출 분리 및 중간 엽 줄기의 확장 / 기질 세포

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

인간의 심장 조직에서 혈관 주위 능성 전구체 세포 집단의 분리

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin 주입에 의한 급성 골격 근육 재생 유도

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

원심 중력에 의해 지방이 유래 줄기 세포의 연골 분화 유도

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Flow Cytometry에 의한 마우스 태아의 적혈구 전구 세포 (Erythromyeloid Progenitors)와 그 자손의 동정

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

부고환에서 분리 된 1 차 상피 세포의 전체 세포 패치 - 클램프 기록

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

비 계, 3 차원 인슐린 표현 Pancreatoids 마우스 췌 장 창시자에 체 외에서 의 세대

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.