Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

참고 : 환자 및 / 또는 보호자는 연구를 승인 심사위원회 (Institutional Review Board)에 참여하는 동의를 제공해야합니다. 1. 버퍼 및 미디어 준비 <ol><li> 세척 버퍼 : 세척 버퍼 100 ㎖를 준비 인산염 완충 식염수 (PBS)의 99 ml의 100 배 펜 / 연쇄상 구균 솔루션 (스트렙토 마이신의 100 U / ml의 페니실린이 + 100 μg의 / ㎖) 1 ㎖를 추가합니다. </li><li> 소변 전구 세포 (UPC) 중간 : UPC 미디어를 준비 케 라티노 세럼 무료 (KSF) 중간 + 전구 세포 매체의 동일한 볼륨을 혼합합니다. <ol><li> KSF 중간 : 표피 성장 인자의 5 ng를 / ㎖ (EGF), 소 뇌하수체 추출물의 50 ng를 / ㎖ (BPE), 콜레라 독소의 30 ng를 / ㎖, 펜 / 연쇄상 구균 용액 (100 U를 추가, 각질 세포 미디어를 준비 / ml의 페니실린 500 ml의 KSF 매체, 100 ㎍ / ml의 스트렙토 마이신). </li><li> 전구 세포 매체 : FBS, 0.4 ㎍ / ㎖의 하이드로 코티손, 0.1 햄 F12 미디어의 1/4과 DMEM의 4 분의 3에 추가하고 10 % 보완nM의 콜레라 독소, 5 NG / ml의 인슐린, 1.8 × 10 -4 M 아데닌, / ㎖ 트랜스페린, 2 × 10 -9 M의 triiodo의 로닌, 10 NG / ml의 EGF, 펜 / 패 혈성 솔루션 5 μg의. </li></ol></li><li> 배아 줄기 중간 : 20 % KO-세럼 교체 (K-SR), 1X MEM 비 필수 아미노산, 2 mM L- 글루타민, 100 μM의 β 머 캅토 에탄올, 20 NG / ㎖의 bFGF 및 펜 / 패 혈성 추가 400 ㎖의 DMEM / F12 배지. </li></ol> 2. 소변 샘플 컬렉션 <ol><li> 소변 적절한 양의 (~ 30 ~ 40 ml)을 수집 할 수 있도록 소변 컬렉션에 30 분 전에 음료수를 마시지하는 환자를 지시합니다. </li><li> 환자 및 / 또는 보호자 다음 항목이 포함 된 소변 수집 키트를 제공한다 : (1) 멸균 또는 클린 캐치 소변 샘플을 구하는 방법에 대한 작성 지침, (2) 습기 항균 toilettes, (3) 100 ml의 멸균 표본 수집 컵. 샘플을 수집하는 환자를 지시합니다. </li><li> 즉시 얼음에 소변 샘플을 배치하고 LABORA로 전송쿨러 토리. 즉시 세포 분리를위한 소변을 처리합니다. 지연이 불가피한 경우, 세포 생존 능력의 손실을 최소화하면서 최대 4 시간 동안 얼음에 표본을 저장합니다. </li></ol> 3. 분리 및 소변 세포의 확장 참고 : BSL2 생물 안전 캐비닛에서 무균 상태에서 다음 단계를 수행하십시오. <ol><li> 멸균 피펫을 사용하여, 전송 소변 샘플은 50 ml의 원심 분리 튜브 멸균한다. RT에서 10 분 동안 400 × g에서 원심 분리한다. </li><li> 튜브에 1 ㎖를 떠나 뜨는을 기음; 세포 펠렛을 방해하지 않도록주의하십시오. </li><li> 재현 탁은 세척 완충액 7 ㎖ 중에 다중 튜브의 펠렛을 결합하고 RT에서 10 분 동안 400 × g에서 원심 분리를 반복하고. </li><li> 조심스럽게 세포 펠렛 및 상등액 ~ 0.2 ~ 0.5 ml를 떠나, 뜨는을 대기음. 소변 전구 세포 (UPC) 매체의 2-3 용액에 재현 탁과 유엔의 4-6 우물에 정지를 전송코팅 된 24 웰 플레이트. </li><li> 72 시간 후, 잘 당 신선한 UPC 매체의 0.5 ml의 문화를 보완하고 2 ~ 3 일 후에 매체 변경합니다. 판에 부착하지 않는 적혈구 및 편평 상피 세포 매체 변화에 제거됩니다. </li><li> 4-6일 후 문화를 모니터링합니다. 참고 : 작은 2-4 세포 식민지가 나타 시작합니다. 세포는 둥근 또는 유형 I 또는 신장 상피 (RE) 세포를 각각 3 종류 II를 대표하는 연장됩니다. </li><li> 세포가 표시되면, 그들은 빠른 확장 단계를 거쳐야하기 때문에 2-3 일마다 매체를 변경합니다. </li><li> 세포가 80-90 %, 도금 후 약 9-15일에 도달하면, 추가 확장 24 웰 플레이트의 2-4 우물에 세포 (15,000-18,000 세포 / cm 2) 해리와 통로. 세포 통로 1 (P1)로이를 표시합니다. </li><li> 유세포 분석을 통해 혈통 사양 소변 세포를 특성화, immunohistochem적인 염색 또는 역전사 효소 - 중합 효소 연쇄 반응을 통한 (RT-PCR). </li><li> 장기 cryostorage에 대한 (DMEM 10 % 혈청과 10 % DMSO로 보충) 절연 소변 세포 (섹션 4)를 다시 프로그램 또는 중간 동결에 그들을 동결. </li></ol> 센다이 바이러스를 사용하여 4. 소변 세포 재 프로그래밍 참고 : 트랜 에이전트를 조작하는 동안 적절한 취급과 PPE (개인 보호 장비)를 사용하는 것이 좋습니다. BSL2 생물 안전 캐비닛에서 무균 상태에서 다음 단계를 수행하십시오. 에이전트 및 / 또는 형질 감염된 세포를 형질의 적절한 폐기는 환경과 건강 위험의 위험을 방지하는 것이 좋습니다. 아래에 설명 된대로 소변 세포 재 프로그래밍은 제조업체의 공급에 의존하는 프로토콜의 수정 센다이 리 프로그래밍 키트를 사용합니다. <ol><li> 센다이 바이러스를 사용하여 고효율의 리 프로그래밍을 보장하기 위해 사용되어 신속한 뇨 세포 1-5 통로해동 분할. 세포가 잘 복제하거나 노화가되지 않는 경우가 효율적으로 재 프로그래밍되지 않으므로 취소하십시오. </li><li> 시드 6 웰 플레이트의 두 우물에 잘 60,000 세포. 마크 일 -2이. 셀 시드 농도를 조정 (5 × 10월 4일에서 9일까지 × 10 4 세포 아니라 당) 일 0으로 80-90 %에 도달하기 위해 필요에 따라. </li><li> 0 일째 (셀 도금 후 48 시간)에서, 네 OSKM 요소 각각을 함유 센다이 리 프로그래밍 벡터 (SEV)을 제조. 미리 예열 UPC 매체의 1 ml의 벡터의 각을 추가합니다. 프로그램을 다시 소변 세포에 충분한 1.5의 MOI (5-7.5 × 10 5 CIU)를 사용합니다. 매체를 대기음 천천히 소변 세포의 잘 하나에 1 ml의 UPC + SEV를 추가합니다. 전달 음성 대조군으로 두 번째 잘 사용합니다. </li><li> 1 일에, 신선한 UPC 매체와 UPC + SEV 매체를 교체합니다. 일부 세포의 죽음으로 인해 바이러스의 세포 독성에 24 시간 후에 볼 수 있습니다. </li><li> 재 프로그램 클론의 효율성에 따라전자 형성하고 컴팩트 한 형태는 하루 6 또는 7까지 매일 매체를 계속 변경. </li><li> 이전에 형질 세포 (5 일 또는 6), 미토 마이신 C (3 시간 10 ㎍ / ㎖)을 0.1에 5 × 104 세포 / cm 2의 밀도에서 처리-MEFs에 도금하여 MEF 피더 플레이트를 준비 해리에 어느 날 % 젤라틴은 100mm 2 배양 접시를 코팅. </li><li> 다음 날 (6 또는 7 일), 0.25 % 트립신으로 세포를 해리 UPC 매체 플레이트 5 × 104 세포에 재현 탁 - 2 × 105 세포 / 100mm이 MEF 피더 플레이트. </li><li> 다음 날, 배아 줄기 매체로 전환 한 매체 매일을 변경합니다. 형질 전환 된 세포를 모니터링 현미경으로 세포를 관찰한다. 참고 : 세포 특성 조약돌 형태와 세포질 비율이 더 높은 핵을 가진 (12-18일 게시물 전달)와 클론 집계를 형성한다. </li><li> 2~3주 내에서 전달 후, 식민지를 선택하고 클론에 대한 새로운 접시로 전송알 확장. </li><li> 37 ℃에서 1 시간 동안 TRA-1-81 항체로 세포를 배양하여 IPSC 클론을 선택하기위한 생균 염색을 수행 ° C에서 CO는 CO 2 인큐베이터에서 37 ° C에서 1 시간 동안 특정 형광 염료와 접합 된 이차 항체 염색에 의해 대향 인큐베이터 하였다. </li><li> 에 26 G 1.5 인치 바늘을 사용하여 작은 동일한 크기의 조각으로 큰 TRA-1-81 긍정적 인 IPSC 클론 (복제 당 4-6 개) 크로스 - 부화. </li><li> 선택 및 전송 매트 리겔의 각 웰 (10 ㎍ / cm 2)으로 여러 클론에서 10 ~ 20 크로스 해치 조각 배아 줄기 매체 24 웰 플레이트를 코팅 처리하는 멸균 피펫 팁을 사용합니다. 참고 : 하나의 우물에 개별적으로 도금 단일 클론에서 조각 확장 또는 능성을 유지하지 않습니다. </li><li> 재 프로그래밍 클론 마트 리젤의 표면에 부착 한 후 IPSC 매체에 배아 줄기에서 24 ~ 48 시간을 변경합니다. </li><li> 마트 리겔 코팅 플레이트, 수동 scra 유지 보수시PE-오프 완전히 재 프로그래밍과 만능 이영 IPSC (MD-IPSC) 클론 풍부하게 피펫 팁을 사용하는 차별화 된 세포 또는 오염 MEF 피더 세포를. </li><li> 개별 클론 ~ 100 ㎛의 크기에 도달 한 후, 3 분 동안 0.48 mM의 EDTA (에틸렌 디아민 테트라 아세트산) 용액으로 해리 및 IPSC 배지에 현탁 MD-iPSCs 작은 세포 집합체를 도금함으로써 클론 확장 (5 μM Y27632, RHO 키나제 보충 억제제 ) 신선한 마트 리겔 (10 ㎍ / cm 2) 12 웰 플레이트를 코팅. 매일 IPSC 매체를 변경합니다. </li><li> 각 클론의 완전한 리 프로그래밍은 OSKM 유전자 및 다 능성 마커 (OCT3 / 4, TRA-1-81)의 면역 조직 화학 염색을 모두 유전자 발현 분석에 의해 결정될 수있다. 만능 전위의 엄격한 평가가 완전히 재 프로그램 IPSC 라인 세 배엽으로 분화 할 수 확인하기 위해 채용되어야한다. 이 배아 체 (EB) 형성과 분화 분석 또는 운에 의해 중 수행 할 수 있습니다atoma 형성 분석법. </li></ol>

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The authors declare that they have no competing financial interests.

iPSCs를 사용하여 심혈관 질환을 모델링하는 것은 유전 적 기여 4-6를 이해하는 일반적인 방법이되고있다. 환자 세포로부터 샘플을 얻는 몇몇 예상치 못한 문제, 특히 어린 아이들, 이러한 소변 수집 같이 비 침습적 접근하는 옵션을 제공함으로써 회피 할 수있다. 이 젊은 환자 집단에서, 재 프로그래밍을위한 충분한 세포 소변을 수득하기에 충분한 양의 소변을 수집하는 것이 곤란하다. 유?...

심근증은 뒤 시엔 느와 베커 근이영양증 (MD) 환자에서 두 번째로 높은 사망 원인이다. X-연결 디스트로핀 유전자의 돌연변이가 하나 발생하더라도 3,500 남성 출산, 약간은 진보적 인 심장 근육 손상을 유도하는 분자 및 세포 이벤트에 대한 알려져있다. 근이영양증 환자 유래의 인간 유도 다 능성 줄기 세포는 기저 질환 메커니즘을 연구 약물 1,2- 스크리닝에 사용하는 신규 한 도구로 나왔다. 피부 조직 검사 또는 혈액 샘플의 예상되는 불편 함은 연구 참여에 대한 동의를하는 젊은 환자 및 / 또는 보호자를 설득 할 수 있습니다. 소변 샘플은 리 프로그래밍을 수정할 수있는 방법이다 체세포 비 침습적 원천이다. 우리는 최근 근이영양증 환자로부터 수집 된 소변 세포를 배양 할 수 있다는 것을 도시하고 효율적 야마나카 인자를 사용하여 레트로 바이러스 형질 도입 iPSCs (내로 재 프로그램 한OCT3 / 4, SOX2, Klf4 및 c-Myc와; OSKM) 1. 레트로 바이러스 유전자 전달의 단점은 숙주 염색체로 유전자의 재 프로그래밍 랜덤 통합이다. 이러한 한계를 극복하기 위해, 우리는 소변 세포 리 프로그래밍에 대한 비 통합 센다이 바이러스를 사용 하였다. 이 프로토콜은 추가 연구를위한 심근 세포 또는 다른 종류의 세포로 분화 할 수 근이영양증 환자의 격리 소변 세포의 센다이 바이러스의 재 프로그래밍에 대해 자세히 설명합니다. 이 프로토콜은 또한 특정 환자 다른 질환을 위해 더 적응 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1144px;">List of Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 Oz. Specimen Cup with Lid</td>
			<td>STL Medical Supply</td>
			<td>M9AMSAS340</td>
			<td>For Urine Sample Collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune-iPS Sendai Reprogramming Kit</td>
			<td>Life Technologies</td>
			<td>A1378-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen-Strep w/o Glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/o Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>0050129SA</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 (1:1)</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene, 1:5000&#160;</td>
			<td>Life Technologies</td>
			<td>15040066</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bFGF (10 &#181;g)</td>
			<td>Life Technologies</td>
			<td>13256-029</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counter Cartridges</td>
			<td>Life Technologies</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum (500 mL Bottle)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recovery Cell Culture Freezing Medium</td>
			<td>Life Technologies</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, High Glucose, Glutamax</td>
			<td>Life Technologies</td>
			<td>10566-016</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ham&#39;s F-12 Nutrient Mix&#160;</td>
			<td>Life Technologies</td>
			<td>11765-054</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGF Recombinant Human Protein, Liquid Form</td>
			<td>Life Technologies</td>
			<td>PHG0311L</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human Recombinant, Zinc Soln</td>
			<td>Life Technologies</td>
			<td>12585-014</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TeSR-E8</td>
			<td>Stem Cell Technologies</td>
			<td>5940</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ROCK Inhibitor (Y-27632)</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>hESC Qualified Matrix, LVED Free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>170-8890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo-Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS-Qualified</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADENINE BIOREAGENT</td>
			<td>Sigma Aldrich</td>
			<td>A2786-5G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHOLERA TOXIN FROM VIBRIO CHOLERAE</td>
			<td>Sigma Aldrich</td>
			<td>C8052-.5MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HYDROCORTISONE</td>
			<td>Sigma Aldrich</td>
			<td>H0888-1G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HOLO-TRANSFERRIN FROM HUMAN</td>
			<td>Sigma Aldrich</td>
			<td>T0665-50MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#39;,5-TRIIODO-L-THYRONINE SODIUM SALT&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6397-100MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-3598</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoromount Aquous mounting medium</td>
			<td>Sigma Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% Paraformaldehyde</td>
			<td>Alfa-Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">List of Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD44 - labelled with FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>560977</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD146 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD73 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-&#945;-smooth muscle actin</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-Vimentin</td>
			<td>Abcam</td>
			<td>ab8978-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Anti - TRA-1-81</td>
			<td>Life Technologies</td>
			<td>411100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Oct 3/4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:415px;">Mouse Anti Dystrophin</td>
			<td>Leica Biosystems</td>
			<td>NCL-DYSB</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells

이영 심근 병증은 근육 이영양증 제대로 이해 결과이다. 근이영양증 환자에서 유도 다 능성 줄기 세포 (iPSCs)를 생성하면 체외 질병 모델 시스템을위한 귀중한 셀룰러 소스 및 약물 검사 연구에 사용할 수 있습니다. 환자 뇨 - 유래 세포는 1 이영양증 심근증을 모델링하기 위해 유도 된 다 능성 줄기 세포로의 성공적인 리 프로그래밍에 사용되어왔다. 벡터 시스템을 통합의 안전성 문제를 해결하는, 우리는 야마나카의 전달은 근이영양증 환자로부터 수집 된 소변 세포에 인자 비 통합 센다이 바이러스 벡터를 이용하여 프로토콜을 제시한다. 이 프로토콜은 완전히 2-3주 내에서 클론을 재 프로그램 생성합니다. 만능 세포 통로 (13)에 의해 벡터 무료입니다. 이 이영 iPSCs는 심근 세포로 분화 및 질병 메커니즘 또는 약물 검사에 대한 공부를 사용할 수있다.

(각각 97.37 %, 97.09 %, 97.3 %; 그림 1A 및 1B) 인간의 소변에서 분리 된 대부분의 전구 세포는 CD44, CD73, 및 CD146 등 uroepithelial 전구 및 혈관 주위 세포 마커에 대한 긍정적이다. 이 세포는 또한 알파 - 평활근 액틴과 멘틴 (그림 1B)와 같은 다른 중간 엽 마커를 표명했다. RT-PCR 분석 아세포-7의 약한 표현이 점에서 문화 세포의 혼합 인구의 증거를 제공합니다 (CK-7) 및 Urop...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

근육 영양 장애 환자에서 유도 만능 줄기 세포의 생성 : 소변 유래 세포의 효율적인 통합이없는 프로그램을 다시

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

generation-induced-pluripotent-stem-cells-from-muscular-dystrophy

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11.1K 조회수.  Medical College of Wisconsin. This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. 

비디오: Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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의학

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

전립선 암 환자의 발생은 종양 세포를 순환에서 이종 이식 모델을 파생

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

C2C12 근육 모세포의 세포에 근긴장 성 이영양증 1 모델링

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

에서 재조합 효소 중재 카세트 교환에 의해 재조합 인간 다 능성 줄기 세포의 신속하고 효율적인 생성<em&gt; AAVS1</em&gt; 로커스

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

평가 의해 유도 된 Exon 포함의 결합 스위칭 센스 Oligonucleotides SMA 환자의 섬유 아 세포에서

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

인간 탯 줄 혈액 파생 CD34에서 조 혈 줄기 세포의 생체 내 확장 전+ 세포 Valproic 산 사용 하 여

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

초기 산후 발달에 있는 에머리 드레이푸스 근 이영양증의 Murine 모형에서 단 근섬유 격리 및 문화

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...