이 연구는 중국의 자연 과학 재단 (http://www.nsfc.gov.cn/ Grant No. 30971665)의 Li Ka Shing Foundation, 광동 고 수준 대학 프로젝트 &quot;해양 산업을위한 녹색 기술&quot; 81172894, 81370925) 및 광동 교육부 (http://www.gdhed.edu.cn/ 보조금 번호 cxzd1123).

8-12 주된 BALB / c 마우스는 Shanghai Experimental Animal Center (Shanghai, China)에서 구입하여 특정 병원균이없고 공기가없는 동물 시설에서 자랐습니다. 동물 관리 및 실험 절차는 산 터우 대학 (Shantou University)에 의해 설립 된 실험용 동물의 사용 및 관리에 대한 지침을 준수했습니다. 1. 장치의 조립 <ol><li> 무균 상태를 유지하려면 사용하기 전에 30 분 동안 조직 배양 후드의 자외선을 켭니다. </li><li> 일회용 25 mm 필터 유닛의 나사를 풀고 캡과 장치의 바닥을 75 % 에탄올이 채워진 200 mL 유리 컵에 30 분간 담근다. 의료용 폴리 프로필렌으로 만들어졌습니다. </li><li> 포셉을 사용하여 장치의 뚜껑과 바닥을 잡고 남은 에탄올을 손으로 털어 내고 부품을 후드에서 10 분 동안 공기 건조시킵니다. </li><li> PBS에 19mm 폴리 카보네이트 멤브레인을 적신 다음 조심스럽게 보토의지지 매트릭스 위에 올려 놓습니다m입니다. 고분자 필름은 매끄럽고 평평한 표면을 가지고 있으며 3 μm의 공극을 트랙 에칭했습니다. </li><li> 캡을 바닥에 단단히 조여서 장치를 다시 조립하십시오. 멤브레인이 중앙에서 빠지지 않았는지 확인하십시오. </li><li> 1 ML 인슐린 주사기의 바늘을 제거하고 PBS 1 ML을 그립니다. 주사기를 필터 장치에 연결 한 다음 PBS를 장치에 밀어 넣어 어셈블리를 적시고 누출이 있는지 테스트하십시오. </li></ol> 2. 플라즈마 막 소포 (PMVs)의 생성 <ol><li> Nemeth et al. 에 의해보고 된대로 BMSC 문화를 확립 하십시오. 15 , 15 % FBS 및 1 % 페니실린 / 스트렙토 마이신을 함유하는 DMEM 배양 배지에서 배양 하였다. 37 ° C에서 5 % CO 2 가 포함 된 humidified 인큐베이터에서 6 잘 접시에있는 세포를 육성. </li><li> 세포를 증식시키기 위해 배양 배지를 제거하고 칼슘이없는 PBS 0.5 mL로 잘 헹구십시오. </li><li> 0.25 % 트립신 - EDTA 0.5 ML을 추가하고 37에서 세포를 품어 ° C3 분 동안. 손바닥에 대해 접시를 몇 번 두드려 세포 분리를 촉진하십시오. 배양액 1 mL를 넣어 소화를 멈춘다. </li><li> 벤치 상단 원심 분리기에서 2 분 200 XG 15 ML 원뿔 튜브와 회전에서 세포를 수집합니다. 배지 4 ML에 세포를 Resuspend하고 6 - 웰 플레이트의 두 우물에 세포를 나누어지는. 참고 : 세포는 대개 24 시간 내에 합류합니다. </li><li> PMVs를 생성하기 위해 트립신 소화에 의해 6-well plate의 한 well (약 5 x 10 5 cell)에서 세포를 수확하고 0.5 mL의 배양 배지에서 monodisperse시킨다. </li><li> 인슐린 주사기에 세포를 넣고 필터 장치에 연결 한 다음 플런저를 빠르게 밀어 필터를 통과하는 세포를 쥐어 짜십시오. 내부에 몇 개의 PMV 만 있어야하기 때문에 압출 된 매체를 버립니다. </li><li> 주사기에 추가로 0.5 mL의 배지를 넣고 신속하게 필터를 통과시킵니다. 포함 된 두 번째 압출 매체 수집다양한 크기의 PMV. </li><li> 수집 된 매체 150 μL를 35 mm 유리 바닥 접시에 넣고 PMV를 약 10 분 동안 바닥에 고정시킵니다. 20X 대물 렌즈를 사용하여 CCD 카메라가 장착 된 역상 위상차 현미경으로 PMV를 검사합니다. </li></ol> 3. 공 촛점 현미경을 이용한 PMV 내의 내용물 검사 <ol><li> BMSC에서 트랜 스펙 션을 수행하려면 50 μL의 무 혈청 DMEM에 2 μg의 supercoiled plasmid DNA와 6 μL의 양이온 transfection reagent ( 예 : PolyJet)를 희석하십시오. 소용돌이가 잘 섞이고 튜브의 측면에서 모든 액체를 수집하기 위해 잠시 회전합니다. </li><li> 희석 된 DNA 용액에 희석 된 용액 (단계 3.1에서)을 적가하고, 1 분 동안 강하게 와동시키고, 짧게 회전시키고, 실온에서 10 분 동안 인큐베이션한다. </li><li> 하룻밤 동안 약 30 %의 농도로 배양 된 BMSC의 새 배지를 2 mL의 새로운 배양 배지로 교체하고 DN형질 전환 혼합물을 적가합니다. </li><li> 형질 감염 12 시간 후 신선한 매체 2 ML로 바꿉니다. </li><li> 세포질로 현지화 된 단백질을 추적하려면 위에 설명한대로 EGFP 발현 카세트 (pEGFP-N1)를 포함하는 플라스미드로 세포를 형질 감염시킵니다. 위에서 설명한대로 PMV 생성에 대한 transfection 48 시간 후 트립신 소화에 의해 세포를 수확. </li><li> 미토콘드리아를 추적하려면 37 ° C에서 30 분 동안 신선한 배지에서 미토콘드리아 염료 (1 μM, MitoTracker)로 세포를 염색하십시오. </li><li> 미토콘드리아의 막 전위를 검출하기 위해, 시아닌 염료 JC-1 (5,5 &#39;, 6,6&#39;- 테트라 클로로 -1,1&#39;, 3,3&#39;- 테트라 에틸 벤즈 미미 - 다 졸릴 카르 보시 아닌 요오드화물) (10 μg / ML) 37 ° C에서 30 분 신선한 배지에서. <ol><li> 위에서 설명한대로 PBS 0.5 ML의 세 번 세포를 씻어 후 PMV 생성에 대한 트립신 소화에 의해 세포를 수확. </li><li> 150 μL의 수집 된 med로드ium을 35mm 유리 바닥 접시에 넣고 PMV를 약 10 분 동안 바닥에 내려 놓습니다. 100X 오일 객관적인 렌즈를 사용하여 공 촛점 현미경하에 PMVs를 검사하십시오. </li></ol></li><li> EGFP 또는 미토콘드리아 염료를 검출하려면 488 nm 레이저를 켜고 505-530 nm에서 신호를 수집하십시오. JC-1을 감지하려면 488nm 및 543nm 레이저를 켜고 505-530nm 및 560-615nm에서 신호를 수집합니다. 핀홀 값을 약 1.4로 설정하십시오. </li></ol> 4. 원심 분리에 의한 농축 된 PMV의 제조 <ol><li> 트립신 소화에 의해 세포를 수확하고 위에서 설명한대로 PMVs를 0.5 ML 문화 매체에 생성합니다. </li><li> 상온에서 5 분 동안 벤치 탑 원심 분리기에서 1,200 xg로 PMV를 돌립니다. 조심스럽게 상등액을 기음과 부드러운 소용돌이와 문화 매체의 100 μL에 펠렛을 resuspend. </li><li> 35mm 유리 바닥 접시에 50 μL의 PMV를로드하고 약 10 분 동안 실온에서 안정되게하십시오. PMV 검사40X 오일 대물 렌즈를 사용하는 공 촛점 현미경 하에서. </li><li> 1 시간 동안 BMSC 배양 물에 일련의 희석 된 폴리에틸렌 이민 (PEI) 용액을 첨가하고 세포 독성의 징후를 확인하십시오. 참고 : BMSC에서 독성의 징후가 발견되지 않았으므로 여기서 2 μg / mL의 PEI를 선택했습니다. </li><li> 트립신 소화하여 세포를 수확하고 응집을 방지하는 수단으로 세포막을 충전 1 H 2 μg / ML에 PEI를 추가합니다. 위에서 설명한대로 0.5 ML 문화 매체에서 PMVs를 생성합니다. 위에서 설명한대로 PMV를 집중시키고 공 촛점 현미경으로 검사하십시오. </li></ol> 5. 비복근 근육에 PMV 주사 <ol><li> 막을 염색하기 위해 30 분 동안 BMSCs에 신선한 문화 매체 1 ML에 CM - DiI (chloromethyl dialkyl - indocarbocyanine) (10 μm의) 염료를 추가합니다. </li><li> 트립신 소화에 의해 세포 (약 5 x 10 5 )를 수확하고 배양 배지 0.5 mL에 그들을 재현 탁한다. PEI (2 μg / mL)를 1h. 위에 설명한대로 PMV를 생성하고 100 μL의 배지에 집중시킵니다. </li><li> 12 시간의 금식 후 sodium barbital (10 mg / kg)을 주사하여 BALB / c 마우스를 마취시킨다. </li><li> 75 % 에탄올로 위의 근육의 외부를 청소하십시오. 천천히 30 G 인슐린 주사기를 사용하여 비복근 근육에 PMV (100 μL)를 주입합니다. </li><li> 마취를 시행 한 후, 실험 기간 동안 눈 위에 젖은 거즈를 놓고 마우스가 깨어날 때까지 백열등으로 따뜻하게합니다. 개별 환기 케이지에 마우스를 홀로 두십시오. </li><li> 수술 12 시간 후 gastrocnemius 근육을 수확하려면 경추 탈구로 마우스를 죽이십시오. </li><li> gastrocnemius 근육 위에 75 % 에탄올을 뿌리고, 가위로 피부를 열고 양끝에서 전체 gastrocnemius 근육을 잘라내십시오. </li><li> PBS로 근육을 헹구고 20X 객관적인 렌즈를 사용하여 형광 현미경으로 검사하십시오. </li><li> 근육을 손질해라.날카로운 면도날로 형광이없는 부분을 제거합니다. 약 9mm 3의 입방체로 강한 붉은 색 형광으로 부품을 자릅니다. </li><li> 최적의 절삭 온도 매체 (OCT)에 각 큐브를 삽입하고 5 분 동안 액체 질소에 담그고 -20 ° C의 냉동고에 보관하십시오. </li><li> 냉동 섹션을 저온 유지 장치를 사용하여 20 μm 두께로 자르고 각 섹션을 유리 슬라이드에 부착하십시오. PBS로 절편을 한 번 헹굽니다. </li><li> 얼룩 원형 펜으로 섹션 주위에 원을 그려 DAPI (1 μg / ML) 100 μL를 추가합니다. 어둠 속에서 실온에서 10 분 잠복 후 DAPI 솔루션을 대기음과 PBS로 세 번 씻어. </li><li> 시험편 위에 오일을 떨어 뜨리고, 유리 슬립으로 덮고 매니큐어로 슬라이드를 밀봉하십시오. 100X 오일 객관적인 렌즈를 사용하여 공 촛점 현미경하에 섹션을 검사하십시오. </li><li> CM-DiI를 검출하기 위해서는 543 nm 레이저를 켜고 560-615 nm에서 신호를 수집하십시오. DAPI를 감지하려면 405를 켭니다.420-480 nm에서 신호를 수집합니다. 핀홀 값을 약 1.4로 설정하십시오. </li></ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+for+extracellular+vesicles+in+tissue+engineering+and+regenerative+medicine.">Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine.</a> Tissue Eng B. 21 (1), 45-54 (2015).</li><li>Baumgart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+fluid/fluid+phase+separation+of+proteins+and+lipids+in+giant+plasma+membrane+vesicles.">Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.</a> Proc Natl Acad Sci. 104 (9), 3165-3170 (2007).</li><li>Sezgin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+membrane+structure+and+protein+behavior+using+giant+plasma+membrane+vesicles.">Elucidating membrane structure and protein behavior using giant plasma membrane vesicles.</a> Nat Protoc. 7 (6), 1042-1051 (2012).</li><li>Lingwood, D., Ries, J., Schwille, P., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membranes+are+poised+for+activation+of+raft+phase+coalescence+at+physiological+temperature.">Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.</a> Proc Natl Acad Sci. 105 (29), 10005-10010 (2008).</li><li>Pandey, A. P., Sawant, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine:+A+versatile,+multifunctional+non-viral+vector+for+nucleic+acid+delivery.">Polyethylenimine: A versatile, multifunctional non-viral vector for nucleic acid delivery.</a> Mater Sci Eng C Mater Biol Appl. 68, 904-918 (2016).</li></ol>

저자는 공개 할 것이 없습니다.

Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor...

<html> 유전자, 효소 및 세포 대체 요법에 대한 접근법을 수립하는데 엄청난 노력이 기울여왔다. 이것은 획기적인 발전을 가져 왔고 심지어 임상 적용 1 , 2 , 3을 가져 왔습니다. 최근 핵 전달 기술에 기반을 둔 논란이되고있는 미토콘드리아 보충 요법이 ​​노년기 여성 또는 치명적인 mitochondrial DNA 돌연변이를 가지고있는 체외 수정에 적용되었습니다. 아테롬성 동맥 경화증, 2 형 당뇨병, 알츠하이머 병 및 암을 포함한 연령 관련 질환에서 발견되는 결함은 대개 다각적입니다. 지질 방울의 축적이 문서화되어있다. 아밀로이드 단백질의 침전; 소포체에서 전개되지 않은 단백질의 유지; autophagosome, mitochondria가 이들 질병의 발달 또는 악화에 기여한다.&quot;xref&quot;&gt; 5 , 6 , 7 , 8 , 9 , 10 , 11 . 현재, 노화 및 노화 표현형을 일으키는 세포질 및 세포 기관에서의 오작동의 직접적인 개선을 목표로하는 이용 가능한 메커니즘이 없다. 우리는 이전에 포유 동물 세포의 기계적 압출을 통해 플라즈마 막 소포 (PMVs)의 생성에 대해보고했습니다 12 . 핵을 제외하고는 미토콘드리아와 같은 세포 소기관뿐만 아니라 단백질과 RNA를 비롯한 막 또는 세포질의 구성 요소가 PMV에서 발견되었습니다. 본질적으로, PMV는 소형 핵 세포로 간주 될 수 있습니다. 더 중요한 것은, 미토콘드리아 결핍 Rho0 세포와 PMVs의 융합은 정상적인 문화 조건 하에서 mitotic 활동을 복원했다. 이것은 첫 번째 보고서입니다.세포질 대체 요법을위한 잠재적으로 효율적인 접근 방법을 제시하고있다. 골수 간엽 줄기 세포 (bone marrow mesenchymal stem cells, BMSCs)는 골수에서 일상적으로 생성되는 다 분화 선조 세포 (multipotent progenitor cell)이며 배양 물에서 쉽게 확장됩니다. 배아 줄기 세포 마커 Oct4, Nanog 및 SOX2는 MSCs 13 에서 낮은 수준으로 검출되었다. 텔로 머라 아제 활성도 측정 가능합니다. 또한 MSC에서의 낮은 HLA Class I 발현뿐만 아니라 공동 자극 분자 및 인간 백혈구 항원 (HLA) 클래스 II 분자의 부재는 동종 이성체 또는 &quot;기성품 인&quot;사용에 이상적인 세포로 작용합니다. 재생 의학 및 면역 조절 응용 14 . 여기, 우리는 3 μm의 공극을 가진 폴리 카보 네이트 멤브레인을 통해 압출을 통해 마우스 BMSC에서 PMV를 준비하는 방법, 미토콘드리아의 존재를 결정하는 방법 및 PMV에서 멤브레인 잠재력의 유지를 검사하는 방법을 설명합니다.현미경 검사법으로 원심 분리에 의해 응축 된 PMV가 아닌 농축 된 PMV를 준비하고 PMV를 생쥐의 비복근 내로 주입합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IsoporeTM membranes</td>
			<td>Millipore</td>
			<td>TSTP04700</td>
			<td>3 mm pore</td>
		</tr>
		<tr>
			<td>Disposable filter unit</td>
			<td>Xinya, Shanghai, China</td>
			<td>25 mm</td>
			<td>Medical grade polypropylene</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328446</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>pN1-EGFP</td>
			<td>Clontech</td>
			<td>&#160;6085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
			<td>Green FM, 1 &#956;M</td>
		</tr>
		<tr>
			<td>JC-1</td>
			<td>Beyotime, Haimen, China</td>
			<td>C2006</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Beyotime, Haimen, China</td>
			<td>C1036</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td>Mn = 75000</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE 2000</td>
			<td>With CCD camera</td>
		</tr>
		<tr>
			<td>Confocol Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 510 Meta</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet</td>
			<td>SigaGen</td>
			<td>SL100688</td>
			<td>For cell transfection</td>
		</tr>
	</tbody>
</table>

preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy

우리는 이전에 포유류 세포의 기계적 압출을 통한 원형질막 소포 (PMVs)의 생성에 대해보고했다. 미토콘드리아 결핍 Rho0 세포와 PMVs의 융합은 정상적인 배양 조건 하에서 유사 분열 활성을 회복시켰다. 죽상 동맥 경화증, 제 2 형 당뇨병, 알츠하이머 병 및 암은 다양한 세포 유형의 세포질 및 세포 소기관에서 여러 가지 기계적 및 기능적 결함과 관련이 있다고보고 된 연령 관련 질환입니다. 골수 간엽 줄기 세포 (bone marrow mesenchymal stem cells, BMSCs)는 다 기능성을 유지하면서자가 재생 능력을 보유한 골수에서 유래 된 독특한 세포 집단을 대표합니다. PMVs의 융합을 통한자가 BMSCs로부터의 어린 세포질을 가진 노화 세포의 보충은 노화와 연관된 표현형을 개선하거나 심지어 역전시키는 유망한 접근법을 제공한다. 이 프로토콜은 압출을 통해 BMSC에서 PMV를 준비하는 방법을 설명합니다.1, 공극, 미토콘드리아의 존재를 결정하고 공 촛점 현미경을 사용하여 PMV 내의 막 전위의 유지를 검사하고, 원심 분리에 의해 PMV를 농축하고 생쥐의 비복근 내로 PMV의 생체 내 주입을 수행한다.

PMV의 성공적인 제조의 열쇠는 1 mL의 PBS를 멤브레인을 통과시켜 시험 할 수있는 필터 유닛 ( 그림 1 )의 정확한 조립에 크게 의존합니다. 누출이 발생하면 필터 장치를 다시 조립하고 다시 테스트하십시오. 그러나 누설은 세포가 멤브레인을 통과 할 때만 신뢰성있게 테스트 할 수 있습니다. 단지 10X 대물 렌즈를 사용하는 일반적인 현미경으로 PMV가 몇 개 발견되...

Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

연령 관련 질환은 세포질 구성 요소의 여러 결함과 관련이 있습니다. 여기, 우리는 골수 간엽 줄기 세포에서 플라즈마 막 소포를 준비하는 프로토콜을 제시합니다. 이 기법은 연령 관련 phenotypes을 개선하거나 심지어 심지어 세포질 대체 요법의 수단으로 잠재적으로 사용할 수 있습니다.

잠재적 인 세포질 대체 요법을위한 골수 간엽 줄기 세포로부터의 혈장 막 소포의 제조

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

preparation-plasma-membrane-vesicles-from-bone-marrow-mesenchymal

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Developmental Biology

7.5K 조회수.  Shantou University.  연령 관련 질환은 세포질 구성 요소의 여러 결함과 관련이 있습니다. 여기, 우리는 골수 간엽 줄기 세포에서 플라즈마 막 소포를 준비하는 프로토콜을 제시합니다. 이 기법은 연령 관련 phenotypes을 개선하거나 심지어 심지어 세포질 대체 요법의 수단으로 잠재적으로 사용할 수 있습니다. 

비디오: Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

인간 중간 엽 줄기 세포의 분리 및 다공성 뼈 매트릭스에 자신의 재배

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

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An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

효소 방법은 응고 골수 샘플에서 중간 엽 줄기 세포를 구출

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

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 ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

사람 골수에서 분리 Mesangiogenic 전구 세포 (MPC와)

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

마우스의 인간 골수 미세 환경의 생체 공학 및 실시간 영상화에 의한 시각화

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

수평 한 전체적인 거치 : 피부의 두껍고, 3 차원 조직 단면도를위한 비발 한 가공 그리고 화상 진찰 의정서

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

절연, 문화, 및 골 수 기질 세포와 생쥐에서 Osteoclast 창시자의 차별화

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...