우리는 Myf5 + / nLacZ 마우스를 제공하기 위해 박사 Shahragim Tajbakhsh 감사합니다. 우리는 또한이 원고의 중요한 읽기 알렉산더 Hron 마이클 Baumrucker 감사합니다. 이 작품은 근육질 영양 장애 협회 (MDA)와 그레고리 Marzolf 주니어 MD 센터 대상에서 교부금에 의해 지원되었다.

동물은 SPF 환경에서 보관하고, 연구 동물 자원 미네소타 대학의 (RAR)에 의해 감시했다. . 동물 (에버 틴 (250 ㎎ / ㎏)의 IP 주입으로 마취 된 후 CO 2 흡입 또는 KCl을 주입 적절한 방법에 의해 안락사 된 모든 프로토콜은 기관 동물 케어 및 사용위원회 (IACUC, 코드 번호에 의해 승인되었습니다 : 1304-30492 ) 미네소타 대학의. 마우스 골격근에서 단핵 세포의 1. 격리 <ol><li> 제대로 1 또는 2 젊은 성인 마우스 (3-8 주)가 희생. <ol><li> 핀치와 날카로운 가위로 복부의 피부를 슬릿. 완전히 (반대 방향으로 피부를 당겨) 삼두근과 뒷다리의 근육을 보여주기 위해 피부를 벗겨. </li><li> 가위로 뼈를 따라 모든 다리 골격 근육 (전 경골근, 비복근, 그리고 대퇴사 두근)과 삼두근을 제거합니다. 그런 다음 10cm 접시에 얼음처럼 차가운, 멸균 PBS에 근육을 전송. </li></ol></li><li> 새로운 멸균 6cm 판에 PBS에있는 근육 및 전송 근육의 혈액을 씻어 : 1 판을 1-2 마우스의 경우. </li><li> 해부 현미경으로 결합 조직, 혈관, 신경 번들 및 지방 형성 조직을 제거합니다. </li><li> (그림 1A 및 1B) 잘라 부드러운 펄프로 조직을 말하다, 안과 용 가위를 사용. 그들은 효소 용액에 의해 쉽게 분해되지 않으므로, 큰 조각을 남기지 않도록하십시오. </li><li> 팔콘 50 ML 튜브에 다진 근육을 전송하고, 콜라게나 제 용액 (0.2 % 콜라게나 제 유형 DMEM에있는 10 %의 FBS) 5 ㎖를 추가합니다. 60 분 동안 37 ° C에서 알을 품다. </li><li> 씹다 (상하 18 G 바늘) 혼합물 (그림 1C)을 균일화 할 수 있습니다. 그런 다음 추가로 15 분 동안 37 ° C에서 혼합물을 품어. </li><li> 단일 세포 현탁액으로 해리 된 혼합물을 균질화 다시 씹다. 최대 단일 세포 현탁액에 50 ml의 DMEM에서 2 % FBS를 추가하고 혼합잘. </li><li> 팔콘 50 ML 튜브 (그림 1D)에 셀 스트레이너 (70 μm의)를 배치합니다. 셀 스트레이너 상에 해리 된 세포를 포함하는 상등액을 전송하며, 그것을 통해 통과 할 때까지 상하 필터에 세포 현탁액을 피펫. </li><li> 혈구에 의해 세포 수를 계산합니다. 5 분 동안 4 ° C에서 2,000 rpm으로 원심 분리기 튜브; 대기음 상층 액을 버린다. </li><li> DMEM에 10 ㎖의 2 %의 FBS로 재현 탁. 5 분 동안 4 ° C에서 2,000 rpm으로 원심 분리기 튜브; 대기음 상층 액을 버린다. </li><li> 200 DMEM에서 2 % FBS ㎕의 1.5 ML의 microcentrifuge 튜브에 세포 현탁액을 전송로를 Resuspend. 일반적으로, 약 2 × 10 6 세포는 1 쥐의 근육에서 채취해야한다. 세포는 DMEM에서 2 % FBS 100 ㎕의 1 X 10 6 세포의 농도로 희석한다. </li></ol> 2. 항체 염색법과 맥 분리 다음 홍보 중ocedures, 멸균 버퍼를 사용하여 무균 조건을 유지한다. 항체의 각각의 양을 첨가하고, 세포 현탁액 매질 한 쥐의 근육 전체에서의 셀들에 대해 계산된다. 세포는 2 이상의 마우스에서 수확하는 경우, 시약의 양을 최적화해야한다. <ol><li> 1 ㎕의 CD31-PE, CD45-PE, 무서-1-PE 및 세포 현탁액 200 μL에 인테 α7 항체를 각각 추가합니다. 30 분 동안 얼음에 품어. </li><li> 세포를 세척 : 부화 후, 1.5 ML 튜브에 세포 현탁액에 DMEM에서 1 ㎖ 2 %의 FBS를 추가하고, 3 분 동안 4 ° C에서 2,000 rpm으로 원심 분리기. 두 번이 단계를 반복합니다. </li><li> 기음과 상층 액을 버린다. </li><li> 200 DMEM에서 2 % FBS의 μL 및 안티-PE 자석 구슬의 10 μl를 추가로 세포를 Resuspend. 30 분 동안 얼음에 품어. </li><li> 세포를 세척 : 부화 후, 1.5 ML의 microcentrifuge 튜브에 세포 현탁액에 MACS 버퍼 1 ㎖를 추가 한 다음 3 분 동안 4 ° C에서 2,000 rpm으로 원심 분리기. 이 단계의 TW를 반복얼음. 세포는 자기 칼럼에 의해 분리되기 전에 MACS 버퍼로 세척해야합니다. </li><li> 기음과 상층 액을 버린다. MACS 완충액 1.0 ㎖로 세포를 재현 탁. </li><li> 자석 보드에 LD 열을 설정하고 MACS 버퍼 (그림 1E)의 2.0 ㎖로 컬럼을 씻어. </li><li> LD 칼럼 상에 세포 현탁액을 전송하며, 1.5 ㎖의 튜브에 관류 분획을 수집한다. 이 부분은 PE-음성 세포가 포함되어 있습니다. </li><li> 3 분, 대기음 상층 액을 버린다 4 ° C에서 2,000 rpm으로 원심 분리기. </li><li> 200 DMEM에서 2 % FBS의 μL 및 안티 - 마우스 IgG 자석 구슬의 10 μl를 추가로 세포를 Resuspend. 30 분 동안 얼음에 품어. </li><li> 세포를 세척 : 부화 후, 1.5 ML 튜브에 세포 현탁액에 MACS 버퍼 1 ㎖를 추가 한 다음 3 분 동안 4 ° C에서 2,000 rpm으로 원심 분리기. 기음과 상층 액을 버린다. 두 번이 단계를 반복합니다. MACS 버퍼 500 μL와 세포를 Resuspend. </li><li> 자석 보드에 MS의 열을 설정하고 MACS 버퍼 (그림 1 층) 500 μL에 열을 씻어. </li><li> 전송 MS 칼럼 상 세포 용액을 현탁하고, 플로우 스루 분획 (인테그린 α7-음성 세포)를 버린다. </li><li> MACS 버퍼의 1 ㎖로 씻어, 두 번이 단계를 반복합니다. </li><li> 세척 한 후, 분리의 자기장에서 열을 제거합니다. 칼럼 상 MACS 완충액 1.0 ㎖를 적용하고, 칼럼의 상부로부터 주사기 플런저를 밀어 1.5 ml의 마이크로 원심 튜브에 자기 적으로 표지 된 세포 (인테그린 α7-양성 세포)를 용출한다. 1.5 ML 튜브에 흐름을 통해 수집합니다. MACS 버퍼의 1.5 ㎖로 용출을 반복하고 흐름을 통해 수집합니다. </li><li> 3 분, 대기음 상층 액을 버린다 4 ° C에서 2,000 rpm으로 원심 분리기. </li><li> 근원 세포 배지 1 ㎖로 정제 된 세포를 재현 탁 (20 % FBS가 bFGF를 함께 햄 F-10을 포함) 및 마트 리겔 - 코팅 10cm 접시에 세포근원 세포 중간의 8 ㎖ (6cm 판에 5 ㎖) (그림 1G)와 함께 접시. 1-2 × 10 5 세포는 잠재적으로 1 그대로 마우스 근육에서 분리 될 수 있습니다. </li></ol> 3. 유지 보수 <ol><li> 근원 세포 배지로 매일 세포에게 공급. 근육 아세포 성장의 외형은 핵에서 MyoD (도 2)와 Pax7 (데이터는 도시하지 않음)을 발현하는 작고 둥근 형상이다. </li><li> 세포 융합을 시작할 때 근육 아세포가 50 % 합류하기 전에 계대 또는해야한다. PBS로 한번 세척 한 후, CO 2 배양기에서 3 분 동안 37 ° C에서 0.25 % 트립신 용액으로 세포를 배양하고 근원 세포 배지로 해리 된 세포를 수집합니다. 원심 분리기 세포 (5 분 1,000 RPM) 한 후, 근원 세포 매체를 일시 중단하고 새로운 리겔 코팅 된 플레이트에 세포를 replate. 콜라겐 코팅 된 플레이트는 통로 (3) 후 사용할 수 있습니다. 한 판은 보통 3-5 플레이트로 분할 할 수 있습니다. </li></ol> 4. Differentiation <ol><li> 매일 분화 배지로 재 공급. </li><li> 차별화 중간에 하루 1으로 근육 아세포는 세포주기를 종료하고 마이 오신 중쇄 (MHC) 양성 근육 세포로 분화를 받고있다. 이 myoctyes는 다핵 myotubes를 생성하기 위해 서로 세포 융합을 시작한다. 일반적으로 대부분의 근원 세포는 MHC-긍정적 인 차별화 단핵 근육 세포 또는 myotubes 차별화 중간에 하루 3-5로 (그림 2)이된다. </li></ol> 골격 근육의 재생을위한 마우스에 5. 근 모세포 이식 <ol><li> 복강 내 (IP) 주입에 의해 에버 틴 (250 ㎎ / ㎏)와 마우스를 마취시키다. </li><li> 전 경골근 (TA) 근육의 피부 머리 주위를 면도. 주야의 근 모세포 이식하기 전에, 10 μM CTX (50 μL)은 근육 면도 피부 (그림을 통해 31 G 인슐린 주사기를 통해 근육의 재생을 유도하는 끄덕임 / SCID 마우스 TA 근육에 주입60, 3). </li><li> 근육 아세포 증식은 0.25 % 트립신 용액으로 분해하고, 5 분 동안 1000 rpm에서 원심 분리된다. 기음과 상층 액을 버린다. DMEM에서 2 % FBS의 50 μL로 재현 탁 1 X 10 6 세포. 31 G 인슐린 주사기로 일시 중단 된 세포를 전송합니다. </li><li> 받는 마우스는 IP 주입하여 에버 틴 (250 ㎎ / kg)로 마취되고, 1 × 106 아세포 (도 2)을 근육 내 TA 근육 재생 주입된다. </li><li> 조직 학적 분석을위한 세포 주입 (그림 3) 후 1~4주으로 수확 TA 근육. </li></ol>

<ol>
	<li>Fukada, S., 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=Purification+and+cell-surface+marker+characterization+of+quiescent+satellite+cells+from+murine+skeletal+muscle+by+a+novel+monoclonal+antibody.">Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody.</a> Exp. Cell Res. 296, 245-255 (2004).</li><li>Hirai, H., Verma, M., Watanabe, S. C. T., Asakura, Y., Asakura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD+regulates+apoptosis+of+myoblasts+through+microRNA-mediated+down-regulation+of+Pax3.">MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3.</a> J. Cell Biol. (191), 347-365 (2010).</li><li>Asakura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+adult+skeletal+muscle.">Stem cells in adult skeletal muscle.</a> Trends Cardiovasc. Med. 13, 123-128 (2003).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+that+participate+in+regeneration+of+skeletal+muscle.">Cells that participate in regeneration of skeletal muscle.</a> Gene Ther. 9, 752-753 (2002).</li><li>Collins, C. A., 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=Stem+cell+function,+self-renewal,+and+behavioral+heterogeneity+of+cells+from+the+adult+muscle+satellite+cell+niche.">Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.</a> Cell. 122, 289-301 (2005).</li><li>Montarras, D., 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=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. 456, 502-506 (2008).</li><li>Conboy, M. J., Cerletti, M., Wagers, A. J., Conboy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-analysis+and+FACS+sorting+of+adult+muscle+fiber-associated+stem/precursor+cells.">Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells.</a> Methods Mol. Biol. 621, 165-173 (2010).</li><li>Yokoyama, T., Huard, J., Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+therapy+for+stress+urinary+incontinence+and+bladder+dysfunction.">Myoblast therapy for stress urinary incontinence and bladder dysfunction.</a> World J. Urol. 18, 56-61 (2000).</li><li>Menasche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+satellite+cell+transplantation.">Skeletal muscle satellite cell transplantation.</a> Cardiovasc. Res. 58, 351-357 (2000).</li><li>Huard, J., 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=Myoblast+transplantation+produced+dystrophin-positive+muscle+fibres+in+a+16-year-old+patient+with+Duchenne+muscular+dystrophy.">Myoblast transplantation produced dystrophin-positive muscle fibres in a 16-year-old patient with Duchenne muscular dystrophy.</a> Clin. Sci. 81, 287-288 (1991).</li><li>Tremblay, J. P., 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=Results+of+a+triple+blind+clinical+study+of+myoblast+transplantations+without+immunosuppressive+treatment+in+young+boys+with+Duchenne+muscular+dystrophy.">Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy.</a> Cell Transplant. 2, 99-112 (1993).</li><li>Gussoni, E., Blau, H. M., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+individual+myoblasts+after+transplantation+into+muscles+of+DMD+patients.">The fate of individual myoblasts after transplantation into muscles of DMD patients.</a> Nat. Med. 3, 970-977 (1997).</li><li>Palmieri, B., Tremblay, J. P., Daniele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past,+present+and+future+of+myoblast+transplantation+in+the+treatment+of+Duchenne+muscular+dystrophy.">Past, present and future of myoblast transplantation in the treatment of Duchenne muscular dystrophy.</a> Pediatr. Transplant. 14, 813-819 (2010).</li><li>Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hydrodynamic+forces+and+apoptosis:+a+complex+question.">Acute hydrodynamic forces and apoptosis: a complex question.</a> Biotechnol. Bioeng. 98, 772-788 (2007).</li><li>Asakura, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+cells+from+diverse+adult+tissues+are+capable+of+in+vitro+hematopoietic+differentiation.">Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation.</a> Exp. Hematol. 30, 1339-1345 (2002).</li><li>Asakura, A., 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=Increased+survival+of+muscle+stem+cells+lacking+the+MyoD+gene+after+transplantation+into+regenerating+skeletal+muscle.">Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 16552-16557 (2007).</li><li>Gerard, X., 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=Real-time+monitoring+of+cell+transplantation+in+mouse+dystrophic+muscles+by+a+secreted+alkaline+phosphatase+reporter+gene.">Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.</a> Gene Ther. 16, 815-819 .</li><li>Tajbakhsh, S., Rocancourt, D., Cossu, G., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+genetic+hierarchies+controlling+skeletal+myogenesis+Pax-3+and+Myf-5+act+upstream+of+MyoD.">Redefining the genetic hierarchies controlling skeletal myogenesis Pax-3 and Myf-5 act upstream of MyoD.</a> Cell. 89, 127-138 (1997).</li><li>Beauchamp, J. R., 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=Expression+of+CD34+and+Myf5+defines+the+majority+of+quiescent+adult+skeletal+muscle+satellite+cells.">Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.</a> J. Cell Biol. 151, 1221-1134 (2000).</li><li>Sabourin, L. A., Girgis-Gabardo, A., Seale, P., Asakura, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+differentiation+potential+of+primary+MyoD-/-+myogenic+cells+derived+from+adult+skeletal+muscle.">Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle.</a> J. Cell Biol. 144, 631-643 (1999).</li><li>Bischoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+single+skeletal+muscle+fibers+in+vitro.">Regeneration of single skeletal muscle fibers in vitro.</a> Anat. Rec. 182, 215-235 (1975).</li><li>Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J. Cell Biol. 159, 123-134 (2002).</li><li>Kuang, S., Kuroda, K., Le Grand, F., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+self-renewal+and+commitment+of+satellite+stem+cells+in+muscle.">Asymmetric self-renewal and commitment of satellite stem cells in muscle.</a> Cell. 129, 999-1010 (2007).</li><li>Cerletti, M., 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=Highly+efficient,+functional+engraftment+of+skeletal+muscle+stem+cells+in+dystrophic+muscles.">Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.</a> Cell. 134, 37-47 (2008).</li><li>Farina, N. H., 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=A+role+for+RNA+post-transcriptional+regulation+in+satellite+cell+activation.">A role for RNA post-transcriptional regulation in satellite cell activation.</a> Skelet. Muscle. 2, 21-21 (2012).</li><li>Tanaka, K. K., Hall, J. K., Troy, A. A., Cornelison, D. D., Majka, S. M., Olwin, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndecan-4-expressing+muscle+progenitor+cells+in+the+SP+engraft+as+satellite+cells+during+muscle+regeneration.">Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration.</a> Cell Stem Cell. 4, 217-225 (2009).</li><li>Pallafacchina, G., 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=An+adult+tissue-specific+stem+cell+in+its+niche:+a+gene+profiling+analysis+of+in+vivo+quiescent+and+activated+muscle+satellite+cells.">An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells.</a> Stem Cell Res. 4, 77-91 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜에서, 정지 위성 세포 간단 콜라게나 제 소화 표면 항체 - 매개 MACS 분리에 의해 생쥐의 성인 골격근으로부터 정제 될 수있다. 이 방법은 약 6 시간 소요하고 FACS 기계와 같은 고가의 장비를 필요로하지 않는다. 또한,이 방법은 표면 항체 - 매개 FACS 분리에 비해 상대적으로 저렴합니다. FACS 레이저 노출은 분리 중에 15 세포 죽음을 유도하는 경향이 이후 정지 위성 세포의 높은 수?...

근육 위성 세포가 골격근 섬유의 기저판 아래에 위치한 근육 조직 줄기 세포의 작은 집단이다. 그들은 Pax7, PAX3, C-메트, M-cadherin의, CD34, Syndecan-3의 발현을 특징으로하고, 칼시토닌 1된다 - 3. 위성 세포는 근육 줄기 세포로 근육의 재생을위한 책임을 입증했다. 성인 근육 위성 세포는 4-8 일반적으로 mitotically 정지합니다. 손상 후, 위성 세포가 MyoD 발현을 개시하고, 그 자손을 확장 세포주기를 입력, 활성화, 근육 조직 전구체 세포 또는 근육 아세포 3 칭했다. 세포 분열 몇 차례 한 후, 근육 아세포는 성숙한 근육 섬유 이어 멀티 핵 myotubes로 차별화를 받아야하기 위해 각각의 다른 세포주기 및 퓨즈를 종료합니다. 성인 근육에서 분리 된 근육 아세포는 쉽게 생체 확장 할 수 있습니다. 근육 재생과에 근육 섬유가 될 수 아세포의 용량nonmuscle 조직의 형태 자궁외 근육 섬유의 근 모세포 이식, Duchenne 근육 퇴행 위축 (DMD) 4에 대한 잠재적 인 치료 방법, 비뇨기 장애 (9), 심장 마비 (10)에 의해 이용된다. 사실, 근육 아세포는 성공적으로 모두 MDX (DMD 모델) 마우스와 DMD 환자의 11 ~ 14의 근육에 이식되었다. 주입 된 정상 근원 세포는 병에 걸린 근육의 조직 학적 및 기능 향상을 위해 호스트 근육 섬유와 융합. 이전 작업은 근육 아세포의 모집단이 더 세포와 같은 줄기 근육 재생 5시 근육에 이상 미분화 상태로 유지하는 것을 보여 주었다. 최근 작품은 성인 근육 갓 고립 된 위성 세포는 근육 5-8 재생에보다 효율적으로 생착과 자기 갱신의 활동을 나타내는 줄기 세포와 같은 인구를 포함하는 것으로 나타났습니다. 따라서 성인 골격 MU로부터 정지 위성 세포의 순수 인구 정화용scle는 위성 세포, 근육 아세포와 근육 재생의 생물학을 이해하고, 세포 기반 치료법​​의 개발을 위해 필수적이다. 그러나, 정지 위성 세포의 현재 장래 정제 방법은 고가의 형광 - 활성 세포 선별 (FACS) 기계 1,2,6-8의 사용을 필요로한다. 또, FACS 레이저 노광 정지 위성 세포 (15)의 낮은 수율로 인해 분리시 세포 사멸을 유도하는 경향이있다. 여기, 우리는 성인 마우스 골격 근육에서 정지 위성 세포의 급속한 경제적이고 신뢰할 수있는 정제를위한 새로운 방법을 제시한다. 이 방법은 (MACS)를 정렬 자기 활성화 된 셀 다음에 효소 분해를 이용한다. 순수한 정지 위성 세포의 분리 후,이 세포는 여러 통로 후에 근육 아세포 다수 구하는 배양 될 수있다. 우리는 또한 보여 이들 갓 고립 된 정지 위성 세포 또는 전 VI의 근육 내 주사할머니 확장 아세포가 cardiotoxin에 이식 할 수있다 (CTX) 근육 섬유뿐만 아니라 자기 갱신 활동의 시험 위성 세포 구획에 재생에 기증자 유래 세포의 기여를 검사하는 마우스 골격 근육을 재생 유도.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Collagenase Type 2</td>
			<td>Worthington</td>
			<td>CLS-2</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Marigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen</td>
			<td>10569010</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>100 mg (3-4 mg/ml)</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>bFGF, human, Recombinant</td>
			<td>Gibco-Invitrogen</td>
			<td>PHG0263</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A5611-1G</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F10 Medium</td>
			<td>Gibco-Invitrogen</td>
			<td>11550-043</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>3600511</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco-Invitrogen</td>
			<td>26050088</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycin</td>
			<td>Gibco-Invitrogen</td>
			<td>15640055</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco-Invitrogen</td>
			<td>14190144</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco-Invitrogen</td>
			<td>25200072</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>18G needle with 12cc Syringe</td>
			<td>Fisher Scientific</td>
			<td>22-256-563</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#956;m)</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 ml tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10 ml disposable pipet</td>
			<td>BD Biosciences</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody-PE</td>
			<td>eBiosciences</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45 antibody-PE</td>
			<td>eBiosciences</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sca1 antibody-PE</td>
			<td>eBiosciences</td>
			<td>Dec-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;7 antibody</td>
			<td>MBL International</td>
			<td>ABIN487462</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini &#38; MidiMACS Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>LD Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma Aldrich</td>
			<td>C9759-1MG</td>
			<td>Stock 10 &#956;M in PBS</td>
		</tr>
		<tr>
			<td>31G Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>328438</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge (Microfuge 22R)</td>
			<td>Beckman Coulter</td>
			<td>368826</td>
			<td></td>
		</tr>
		<tr>
			<td>S241.5 Swinging Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>368882</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (Allegra X-22R)</td>
			<td>Beckman Coulter</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr>
			<td>Nod/Scid immunodeficient mice</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code 394</td>
			<td>Use 2 months old mice</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Name of the reagent</td>
			<td></td>
			<td></td>
			<td>Recipie</td>
		</tr>
		<tr>
			<td>10% and 2% FBS DMEM</td>
			<td></td>
			<td></td>
			<td>DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>0.2% Collagenase solution</td>
			<td></td>
			<td></td>
			<td>Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM.</td>
		</tr>
		<tr>
			<td>10% Matrigel solution</td>
			<td></td>
			<td></td>
			<td>Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20&#176;C until use.</td>
		</tr>
		<tr>
			<td>Matrigel-coated plate</td>
			<td></td>
			<td></td>
			<td>Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37&#176;C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20&#176;C for reusing.</td>
		</tr>
		<tr>
			<td>0.01% Collagen solution</td>
			<td></td>
			<td></td>
			<td>Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O.</td>
		</tr>
		<tr>
			<td>Collagen-coated plate</td>
			<td></td>
			<td></td>
			<td>Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months.</td>
		</tr>
		<tr>
			<td>bFGF stock solution</td>
			<td></td>
			<td></td>
			<td>bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 &#956;l in 500 &#956;l microcentrifuge tubes and kept in -80&#176;C.</td>
		</tr>
		<tr>
			<td>Myoblast medium</td>
			<td></td>
			<td></td>
			<td>500 mL HAM&#8217;S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 &#956;g of bFGF (20 &#956;l of bFGF stock).</td>
		</tr>
		<tr>
			<td>Differentiation medium</td>
			<td></td>
			<td></td>
			<td>500 mL DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>10 &#956;M Cardiotoxin stock</td>
			<td></td>
			<td></td>
			<td>1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS.</td>
		</tr>
	</tbody>
</table>

isolation, culture, and transplantation of muscle satellite cells

근육 위성 세포는 근육 섬유에 sublaminal 핵 2 ~ 5 %를 차지, 출생 후의 골격 근육 발달 및 재생에 필요한 줄기 세포 집단이다. 성인 근육 위성 세포는 일반적으로 mitotically 정지합니다. 손상 후, 그러나, 위성 세포는 근육의 재생을 중재하는 아세포, 그들의 자손을 생산하는 세포 증식을 시작. 위성 세포 유래 근육 모세포 이식 널리 근육 영양 장애, 심장 마비, 및 비뇨 장애 등 여러 가지 재생 질환에 대한 가능한 치료로 연구되고있다. 이영 골격 근육, 심장 경색 및 dysfunctioning 요도 관에 근 모세포 이식 접목 근원 세포가 호스트 조직에서 근육 섬유로 분화 및 이러한 질병에 부분적인 기능 개선을 표시 할 수있는 것으로 나타났습니다. 따라서 골격 muscl에서 정지 위성 세포의 효율적인 정제 방법의 개발전자뿐만 아니라 위성 세포 유래 근원 세포 배양 및 근육 아세포를위한 이식 방법의 확립으로, 위성 세포의자가 재생, 활성화, 차별화 뒤에 분자 메커니즘을 이해하는 데 필수적이다. 또한, 근육의 영양 장애 및 기타 재생 질환에 대한 세포 기반 치료법​​의 개발은 이러한 요인에 따라 달라집니다. 그러나, 정지 위성 세포의 현재 장래 정제 방법은 고가의 형광 - 활성 세포 선별 (FACS) 기계의 사용을 필요로한다. 여기, 우리는 (MACS)를 정렬 자기 활성화 된 셀 다음에 효소 분해에 의해 성인 마우스 골격 근육에서 정지 위성 세포의 급속한 경제적이고 신뢰할 수있는 정제를위한 새로운 방법을 제시한다. 순수한 정지 위성 세포의 분리 후,이 세포는 여러 통로 후에 근육 아세포 다수 구하는 배양 될 수있다. 이 갓 고립정지 위성 세포 또는 생체 확장 아세포는 cardiotoxin에 이식 할 수있다 (CTX) 근육 섬유 재생에 기증자 유래 세포의 기여를 검사하는 마우스 골격 근육을 재생 유도뿐만 아니라 자기 갱신의 시험 위성 세포 구획에 활동.

갓 고립 된 정지 위성 세포는 정지 위성 세포에 대한 최종 마커로 작고 둥근 모양 (그림 1G), 간편 Pax7를 표시합니다. 갓 고립 된 세포의 90 % 이상이 Pax7 (그림 1H와 1I)를 표현한다. 대부분의 오염 된 세포가 효율적으로 근원 세포 배양 조건 다음 체외에서 성장하지 않습니다 혈액 세포에서합니다. 따라서, 위성 세포 유래 근육 아세포는 문화에 지배. 선택적으로, 우리?...

Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Isolation, Culture, and Transplantation of Muscle Satellite Cells

정지 위성 세포의 순수 인구의 분리 및 배양은 근육 줄기 세포 집단은 근육 줄기 세포 생물학 및 재생뿐만 아니라, 근육 퇴행 위축 및 기타 퇴행성 질환의 치료를위한 줄기 세포 이식의 이해에 중요하다.

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

isolation-culture-and-transplantation-of-muscle-satellite-cells

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Fibroblast Transformation and Muscle Stem Cells

SCIENTISTS IN ACTION

41.2K 조회수.  University of Minnesota Medical School.  정지 위성 세포의 순수 인구의 분리 및 배양은 근육 줄기 세포 집단은 근육 줄기 세포 생물학 및 재생뿐만 아니라, 근육 퇴행 위축 및 기타 퇴행성 질환의 치료를위한 줄기 세포 이식의 이해에 중요하다. 

비디오: Isolation, Culture, and Transplantation of Muscle Satellite Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

분리 및 조혈 줄기 세포의 이식 (HSCs)

연구

생물학

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

인간의 탯줄 아티 평활근 세포 절연 (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

조류 배아 판막병 전구 세포의 분리 및 문화

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

마우스 기본 췌장 포상 세포의 분리 및 문화

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

신생아 쥐의 폐 동맥 평활근 세포의 분리

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...