현재 연구는 상하이 혁신 자금 (1402 H 294300) 과학 및 기술 위원회의 상하이 시 (CN) 박사 원 왕 후원에 의해 지원 되었다. 우리는 그의 기술 지원 및이 원고에 대 한 과학적 조언에 대 한 광 저 우 박사 (조직 공학 센터의 중국 국가)를 감사 하 고 싶습니다. 우리 또한 싶습니다 씨 Huitang 쌰 (상해 9 사람들의 병원) 감사 하 고 동물 복지에 그의 도움에 대 한.

동물 주제와 관련 된 절차 동물 고통 최소화 하는 노력으로 현지 기관 동물 관리 및 윤리 위원회에 의해 승인 했다. 다음 프로토콜 기관 동물 관리 및 사용 위원회 (IACUC) 상해 9 사람들에 의해 승인 되었다 &#8217; 병원 프로토콜 상하이 교통 대학의과 대학에 가입 하는 s 번호 [2017] 063.
 1. Surgically-Induced 쥐 무릎 관절염 모델의 설립 <li>이 수술, 250, 300 g. 사이 몸 무게와 함께 사용 8-12 주 오래 된 남성 Sprague Dawley (SD) 쥐에 대 한</li> <ol>
	<li>40 mg/kg tiletamine와 근육 주사를 통해 40 mg/kg zolazepam 동물 anesthetize.</li>
	<li>열띤된 무대에서 왼쪽된 측면 위치에 동물을 배치합니다. 면도기와 함께 오른쪽 무릎 철저 하 게 면도. 외과 영역 10 %providone-요오드 솔루션 뒤에 70% 에탄올으로 소독, 두 번 더 반복 하 고 다음 10 %providone-요오드 솔루션 그리고 외과 패드 비 외과 영역을 커버.</li>
	<li>는 jointcapsules를 노출 하는 슬 개 골 힘 줄을 따라 옆으로 2cm 절 개 하 게 수술 메스를 사용 하 여.</li>
	<li>수술 메스를 사용 하 여 transect 중간 부수적인 인 대. 대 퇴 골 쪽으로 초승달 모양 반영.</li>
	<li>연골 밖으로 건조 하지 않도록 하려면 작업 중에 관절 연골의 표면 살 균 0.9% 염화 나트륨 솔루션을 똑.</li>
	<li>중간 초승달 모양 집게로 잡아와 메스 ( 그림 1A)와 tibial 첨부 파일에서 그것의 가장 좁은 지점에는 초승달을 극복. 연골 부상 방지.</li>
	<li>공동 캡슐 4-0 흡수 봉합을 닫습니다.</li>
	<li>그들은 식이 되 찾을 때까지 외과 쥐 모니터링.</li>
	<li>주사 감염 및 수술 후 통증을 완화 하는 근육 경로 통해 0.05 mg/kg buprenorphine를 방지 하기 위해 20 mg/kg gentamicin.</li>
	<li>발병 KOA 3-8 주 동안 12 h 명암 주기 아래 온도 제어 동물 시설에 쥐를 유지합니다. 동물 계속 진통제, 통증 증상이 계속 되 면 Buprenorphine (0.05 mg/kg, 인스턴트 메신저) 및 Gentamicin (20 mg/kg, 인스턴트 메신저), 하 되이 국한 되지 않음 등 수신.</li>
</ol> 2. 조직학 평가의 쥐 KOA 모델 <ol><li>희생 과도 한 CO 2 흡입 (추가 챔버에 기존 공기에 이산화탄소와 분당 챔버 볼륨의 약 30%의 채우기 비율이 동물 의식 있도록와 쥐 호흡 후 1 분의 2-3 분 최소 유지 CO2 흐름 내에서 정지 한다.) 에 나타난 시간 점 (대표 결과 KOA 유도 3에서 8 주까지 8 주에 표시 됩니다) 수술.</li> <li>피부와 무릎 관절 주위 근육을 해 부하 고 메스와 무릎 관절을 잘라.</li>
	<li>실 온에서 3 일 동안 4 %paraformaldehyde (PFA)에서 무릎 관절을 수정합니다. 이후에, 4 ° C에서 약 4 주 동안 20 %ethylenediaminetetraacetic 산 (EDTA)에서 샘플을 알려줍니다 하 고 EDTA 3 일 마다 변경. 
	주의: Paraformaldehyde 독성, 적절 한 개인 보호 장비 (PPE), 장갑, 안경 등을 착용.</li>
	<li>탈수 자동 조직 프로세서를 사용 하 여 샘플. 플러시 주기를 다음과 같이 시작 메뉴 설정: 1 h;에 대 한 70% 에탄올 다음 80% 에탄올과 1 시간에 대 한 95% 에탄올 각각; 뒤에 1 h;에 대 한 100% 에탄올의 2 주기 그 후, 크 실 렌 1 시간에 두 번 각 시간과 각 시간 1 h 58 ° C에서 3 번 파라핀. 
	주의: 크 실 렌은 독성, 그래서 적절 한 보호구, 장갑, 안경 등을 착용. 
	참고: 필요에 따라 사용 Coplin 항아리 자동 조직 프로세서로 동일한 설정 수동 탈수.</li>
	<li>그대로 공동 얼굴의 중간 측면 내려 놓고 가운데에 포함 하는 조직 파라핀 블록에 포함. 
	참고: 필요에 따라 사용 하 여 집게 파라핀 블록으로 샘플을 수동으로 포함.</li>
	<li>로타리 톰 30 µ m 간격 2 직렬 섹션 5 µ m 화살 섹션 중간 마진에서 공동의 인하 시작.</li>
	<li>유리 슬라이드에 섹션을 탑재 하 고 크 실 렌에 슬라이드를 deparaffinize. 2 직렬 섹션 사이 얼룩 되며 한 슬라이드 &#38; 오신 (H &#38; E) 얼룩: 0.1% 되며 15 분, 1 %10 s 및 0.5% 아세트산 해결책 오신 10 분. 그리고 다른 슬라이드 Safranin O/빠른 그린 얼룩과 얼룩: 0.1% 되며 15 분, 0.02% 빠른 그린 3 분, 1% 초 산 솔루션을 10 s와 0.1% Safranin O 3 분 
	주의: 크 실 렌은 독성, 장갑, 안경 등 적절 한 보호구를 착용.</li>
	<li>쥐 6 관절염 연구 학회 인터내셔널 (OARSI) Histopathology 평가 시스템을 사용 하 여 스테인드 슬라이드 점수.</li>
</ol> 3. 라벨의 인간 중간 엽 줄기 세포와 형광 염료 않았다 <ol><li>1 m m의 농도에서 100% 에탄올에 셀 라벨 솔루션 준비 했 어. 빛에서 셀 라벨링 솔루션을 보호 하 고 6 개월까지 실내 온도에 저장.</li>
	<li>성장 Dulbecco에서 표준 세포 문화 절차와 haMSCs &#39; s 수정이 글 &#39; s 매체 (DMEM) 1% 페니실린 및 스와 10% 태아 종 아리 혈 청 (FCS) 10 cm 배양 접시에서 보충.</li>
	<li>1 mM EDTA 합류 이전에 약 80%와 haMSCs 2 mL 0.25 %trypsin 분리.</li>
	<li>4 mL 문화 매체와 트립 신을 중화 하 고 실내 온도에 3 분 동안 800 x g에서 셀 회전.</li>
	<li>1 x 10 6 셀/mL 5 mL 혈 청 무료 문화 매체에서의 조밀도에 셀 중단.</li>
	<li>50를 추가 하 여 셀 라벨 µ L 일 분 동안 37 ° C에 5 mL 혈 청 무료 문화 매체에서 셀 라벨링 솔루션</li>
	<li>3 분에 대 한 800 x g에서 레이블이 지정 된 셀을 회전</li>
	<li>5 mL 인산 염 버퍼 식 염 수 (PBS)에서 3 분 동안 800 x g 2 셀 번 더 세척.</li>
	<li>이라는 haMSCs Cy5 필터 세트 형광 현미경을 사용 하 여 확인 하십시오.</li>
	<li>Trypan 푸른 얼룩과 셀 고 주사에 대 한 100 µ L PBS에 10 6 가능한 셀 x 2.5.</li>
</ol> 4. Vivo에서 형광 트랙 haMSCs 이미징 <ol><li>(1 단계), 수술 후 8 주 25 mg/kg tiletamine 및 25 mg/kg zolazepam의 근육 주사 하 여 KOA 쥐 anesthetize.</li>
	<li>공동 영역을 노출 하는 면도기와 함께 오른쪽 무릎을 철저 하 게 면도. 70% 에탄올과 공동 지역 소독.</li>
	<li>2.5 x 10을 주사 26 G 주사기 바늘을 사용 하 여 슬 개 골 인 대, 내측 대 퇴 관절 돌기, 그리고 중간 tibial 관절 돌기 (의 중간 측에 의해 형성 된 6 삼각형 지역의 센터에서 100 µ L PBS에 셀 표시 그림 1B).</li>
	<li>이미징 소프트웨어를 열고 초기화는 vivo에서 생물 발광 이미징 시스템.</li>
	<li>뒤로 눕은 위치 쥐 이미징 시스템의 중앙 무대에 놓고 챔버의 문을 닫습니다.</li>
	<li>체크는 &#34; 형광 &#34; 확인란의 선택 형광 필터 세트 여기, 및 680 nm 방출 ( 그림 2A)에 대 한.</li>
	<li>선택 필드 볼 디 설정 픽셀 binning 매체 (8), F를 / 2, 노출 중지는 자동 시간. 최적의 유지 전체 연구 ( 그림 2A)에서 유사한 결과 얻기 위해 일관 된 노출 시간.</li>
	<li>무대 고 마 취에서 회복에 대 한 모니터에서 동물 제거. 
	참고: 장소 동물 난방 패드에 덮여 toweling의 2-3 층.</li>
	<li>형광 ( 그림 2B)의 측정에 대 한 복사 효율의 단위를 선택합니다. (ROI) 분석에 대 한 관심의 영역을 선택 하 고 각 이미지 ( 그림 2C) 형광 신호를 계량.</li>
	<li>Vivo에서 화상 진 찰 haMSCs 관절에 종 보존 연구를 순차적으로 각 쥐의 절차를 반복. 
	참고: 우리는 동물을 이미징 제안 &#39; 주입 후 첫 주에 대 한 일에 한 번 그리고 한 번 매주 마다 신호를 감지할 수 없을 때까지 vivo에서 이미징 시스템에 의해 형광 신호.</li>
</ol>

<ol>
	<li>Loeser, R. F., Goldring, S. R., Scanzello, C. R., Goldring, 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=Osteoarthritis:+a+disease+of+the+joint+as+an+organ.">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis Rheum. 64 (6), 1697-1707 (2012).</li><li>Lane, N. E., Shidara, K., Wise, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+year+in+review+2016:+clinical.">Osteoarthritis year in review 2016: clinical.</a> Osteoarthritis Cartilage. 25 (2), 209-215 (2017).</li><li>Wang, W., Cao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+osteoarthritis+with+mesenchymal+stem+cells.">Treatment of osteoarthritis with mesenchymal stem cells.</a> Sci China Life Sci. 57 (6), 586-595 (2014).</li><li>Burke, 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=Therapeutic+potential+of+mesenchymal+stem+cell+based+therapy+for+osteoarthritis.">Therapeutic potential of mesenchymal stem cell based therapy for osteoarthritis.</a> Clin Transl Med. 5 (1), 27 (2016).</li><li>Bendele, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+osteoarthritis.">Animal models of osteoarthritis.</a> J Musculoskelet Neuronal Interact. 1 (4), 363-376 (2001).</li><li>Gerwin, N., Bendele, A. M., Glasson, S., Carlson, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OARSI+histopathology+initiative+-+recommendations+for+histological+assessments+of+osteoarthritis+in+the+rat.">The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rat.</a> Osteoarthritis Cartilage. 18, S24-S34 (2010).</li><li>Janusz, M. 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=Induction+of+osteoarthritis+in+the+rat+by+surgical+tear+of+the+meniscus:+Inhibition+of+joint+damage+by+a+matrix+metalloproteinase+inhibitor.">Induction of osteoarthritis in the rat by surgical tear of the meniscus: Inhibition of joint damage by a matrix metalloproteinase inhibitor.</a> Osteoarthritis Cartilage. 10 (10), 785-791 (2002).</li><li>Li, 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=In+vivo+human+adipose-derived+mesenchymal+stem+cell+tracking+after+intra-articular+delivery+in+a+rat+osteoarthritis+model.">In vivo human adipose-derived mesenchymal stem cell tracking after intra-articular delivery in a rat osteoarthritis model.</a> Stem Cell Res Ther. 7 (1), 160 (2016).</li><li>Zhou, B., 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=Administering+human+adipose-derived+mesenchymal+stem+cells+to+prevent+and+treat+experimental+arthritis.">Administering human adipose-derived mesenchymal stem cells to prevent and treat experimental arthritis.</a> Clin Immunol. 141 (3), 328-337 (2011).</li><li>Desando, 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=Intra-articular+delivery+of+adipose+derived+stromal+cells+attenuates+osteoarthritis+progression+in+an+experimental+rabbit+model.">Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experimental rabbit model.</a> Arthritis Res Ther. 15 (1), 22 (2013).</li><li>Riester, S. 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=Safety+Studies+for+Use+of+Adipose+Tissue-Derived+Mesenchymal+Stromal/Stem+Cells+in+a+Rabbit+Model+for+Osteoarthritis+to+Support+a+Phase+I+Clinical+Trial.">Safety Studies for Use of Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells in a Rabbit Model for Osteoarthritis to Support a Phase I Clinical Trial.</a> Stem Cells Transl Med. 6 (3), 910-922 (2017).</li><li>Bai, 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=Tracking+long-term+survival+of+intramyocardially+delivered+human+adipose+tissue-derived+stem+cells+using+bioluminescence+imaging.">Tracking long-term survival of intramyocardially delivered human adipose tissue-derived stem cells using bioluminescence imaging.</a> Mol Imaging Biol. 13 (4), 633-645 (2011).</li><li>Wolbank, 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=Labelling+of+human+adipose-derived+stem+cells+for+non-invasive+in+vivo+cell+tracking.">Labelling of human adipose-derived stem cells for non-invasive in vivo cell tracking.</a> Cell Tissue Bank. 8 (3), 163-177 (2007).</li><li>Heymer, 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=Iron+oxide+labelling+of+human+mesenchymal+stem+cells+in+collagen+hydrogels+for+articular+cartilage+repair.">Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair.</a> Biomaterials. 29 (10), 1473-1483 (2008).</li><li>Hemmrich, K., Meersch, M., von Heimburg, D., Pallua, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applicability+of+the+dyes+CFSE,+CM-DiI+and+PKH26+for+tracking+of+human+preadipocytes+to+evaluate+adipose+tissue+engineering.">Applicability of the dyes CFSE, CM-DiI and PKH26 for tracking of human preadipocytes to evaluate adipose tissue engineering.</a> Cells Tissues Organs. 184 (3-4), 117-127 (2006).</li><li>Shim, 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=Pharmacokinetics+and+in+vivo+fate+of+intra-articularly+transplanted+human+bone+marrow-derived+clonal+mesenchymal+stem+cells.">Pharmacokinetics and in vivo fate of intra-articularly transplanted human bone marrow-derived clonal mesenchymal stem cells.</a> Stem Cells Dev. 24 (9), 1124-1132 (2015).</li><li>Chen, B. K., 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+safety+study+on+intrathecal+delivery+of+autologous+mesenchymal+stromal+cells+in+rabbits+directly+supporting+Phase+I+human+trials.">A safety study on intrathecal delivery of autologous mesenchymal stromal cells in rabbits directly supporting Phase I human trials.</a> Transfusion. 55 (5), 1013-1020 (2015).</li><li>Chan, M. M., Gray, B. D., Pak, K. Y., Fong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+in+vivo+imaging+of+arthritis+in+a+collagen-induced+murine+model+with+phosphatidylserine-binding+near-infrared+(NIR)+dye.">Non-invasive in vivo imaging of arthritis in a collagen-induced murine model with phosphatidylserine-binding near-infrared (NIR) dye.</a> Arthritis Res Ther. 17, 50 (2015).</li><li>Texier, I., 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=Cyanine-loaded+lipid+nanoparticles+for+improved+in+vivo+fluorescence+imaging.">Cyanine-loaded lipid nanoparticles for improved in vivo fluorescence imaging.</a> J Biomed Opt. 14 (5), 054005 (2009).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carbocyanine+dyes+allow+living+neurons+of+identified+origin+to+be+studied+in+long-term+cultures.">Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.</a> J Cell Biol. 103 (1), 171-187 (1986).</li><li>Rahmati, M., Mobasheri, A., Mozafari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mediators+in+osteoarthritis:+A+critical+review+of+the+state-of-the-art,+current+prospects,+and+future+challenges.">Inflammatory mediators in osteoarthritis: A critical review of the state-of-the-art, current prospects, and future challenges.</a> Bone. 85, 81-90 (2016).</li><li>Detante, O., 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=Intravenous+administration+of+99mTc-HMPAO-labeled+human+mesenchymal+stem+cells+after+stroke:+in+vivo+imaging+and+biodistribution.">Intravenous administration of 99mTc-HMPAO-labeled human mesenchymal stem cells after stroke: in vivo imaging and biodistribution.</a> Cell Transplant. 18 (12), 1369-1379 (2009).</li><li>Hu, S. L., 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=In+vivo+magnetic+resonance+imaging+tracking+of+SPIO-labeled+human+umbilical+cord+mesenchymal+stem+cells.">In vivo magnetic resonance imaging tracking of SPIO-labeled human umbilical cord mesenchymal stem cells.</a> J Cell Biochem. 113 (3), 1005-1012 (2012).</li><li>Xia, Q., 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=Intra-articular+transplantation+of+atsttrin-transduced+mesenchymal+stem+cells+ameliorate+osteoarthritis+development.">Intra-articular transplantation of atsttrin-transduced mesenchymal stem cells ameliorate osteoarthritis development.</a> Stem Cells Transl Med. 4 (5), 523-531 (2015).</li><li>Jasmin, , 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=Optimized+labeling+of+bone+marrow+mesenchymal+cells+with+superparamagnetic+iron+oxide+nanoparticles+and+in+vivo+visualization+by+magnetic+resonance+imaging.">Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and in vivo visualization by magnetic resonance imaging.</a> J Nanobiotechnology. 9, 4 (2011).</li><li>Lehmann, T. 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=Coculture+of+human+nucleus+pulposus+cells+with+multipotent+mesenchymal+stromal+cells+from+human+bone+marrow+reveals+formation+of+tunnelling+nanotubes.">Coculture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes.</a> Mol Med Rep. 9 (2), 574-582 (2014).</li><li>Wang, W., 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=Human+adipose-derived+mesenchymal+progenitor+cells+engraft+into+rabbit+articular+cartilage.">Human adipose-derived mesenchymal progenitor cells engraft into rabbit articular cartilage.</a> Int J Mol Sci. 16 (6), 12076-12091 (2015).</li></ol>

멩 리 밍 커, 원 왕은 현재 직원 및 셀룰러 의학 그룹의 스톡 옵션 보유자 (나스닥: CBMG). 다른 저자는 충돌의 관심을 선언합니다.

우리는 머리 맡에 벤치에서 코 아에 대 한 재생 줄기 세포 치료를 가져올 수 있는 전에 안전 표준 및 줄기 세포 치료의 biodistribution 연구 절실히 필요는 합니다. 그러나, 질병의 병 적인 환경 지 속성에 biodistribution 이식된 haMSCs10의 중요 한 역할을 재생합니다. 최근, 우리의 그룹 보다 정상적인 조건을8에서 주사 병 적인 KOA 환경 haMSCs의 내부 관절 주사 이상 유지는...

무릎 관절염 (KOA) 관절 연골 손실 및 진보적인 염증, 되 고 있다는 세계1주위 노인에서 주요 만성 질환 인 퇴행 성 질환 이다. 그러나, 항 염증 제 약물, 물리 보충, 및 수술 절차를 사용 하 여 현재 치료만 증상 통증2에 대 한 임시 구호를 제공할 수 있습니다.
무릎 관절염, 연골 재생, immunomodulatory 속성3, 잠재적인 multipotent 그들의 차별화 때문에 유망한 재생 치료 되 고 인간 지방 유래 중간 엽 줄기 세포 (haMSCs) 4. 약리학 노선 vivo에서행동의 메커니즘을 조사와 비교 하면, 현재 유익에 대 한 근거 및 임상 응용 프로그램 이전 haMSC 치료의 타당성을 확립 하는 작은 KOA 동물 모델에서 라이브 haMSCs를 추적. 전 임상 시험에 대 한 중간 meniscectomy (MM) destabilizes 일관 재현성5상대적으로 가능한 모델을 제공 합니다 쥐, KOA를 유도 하 관절의 기계적 부하. KOA m M에 의해 유도 된의 발병은 전방 십자 인 대 transection 혼자 또는 결합 부분 중간 meniscectomy6보다 이전. 따라서, KOA의 병 적인 microenvironment로 주입 된 haMSCs 간의 장기 상호 작용 종종 m M7,8에 의해 유도 된 쥐에 평가 됩니다.
비록 haMSCs의 치료 효능에 광범위 하 게 보고, 관련 지식이 하고있다 내 관절 (IA) 주입을 통해 이식된 haMSCs의 비보에 지 속성 부족9,10입니다. 따라서, 다양 한 세포 표시 방법은 개발 되었다, immunohistology11, luciferase12, 녹색 형광 단백질13 transfection, 자기 공명 영상 (MRI)14에 대 한 라벨 산화 철을 포함 하 여 , 그리고 수많은 형광 셀 염료8,,1516. 노동 집약적인 조직학 분석 비교, vivo에서 비 침략 적 영상 실시간 유통 및 형광 신호10,17으로 표시 하는 셀의 역학을 감지 하는 광학 장치를 사용 합니다. 기능 라이브 셀 이미징에 대 한 cytocompatible 형광 라벨 줄기 세포 이식18후 세포 활동을 공개 하는 정교한 무료 방사성 추적 기술입니다. 또한, 다 색 형광 닥터지 염료 친수성 아미노산 반응성 염료 나 형광 단백질, 그들의 향상 된 세포 침투성 등 향상 된 형광 양자 수율19이점을 있습니다.
따라서, 여기에 포함 된 프로토콜 활용 빨간색 레이저 질 성 carbocyanines로 표시 하는 세포를 자극 했다 (DilC18(5)), 빨강 형광 Dil (dialkylcarbocyanines) 아날로그20입니다. 빨간색 이동 여기 및 방출 스펙트럼 autofluorescent 간섭 방지 않았다 하 고 깊은 조직 동물8시간의 긴 기간 동안 이미징 있습니다. 추적의이 방법은 세포 vivo에서 표시 한은 이해 하 고 현재 줄기 세포 재생 치료 개선에 필수적인 동물 모델에서 haMSCs 같은 이식된 줄기 세포 모니터링에 적합.

<table>
	<tbody>
		<tr>
			<td>Matrx VMR animal anesthesia system</td>
			<td>Midmark</td>
			<td>VIP3000</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 suture</td>
			<td>Shanghai Jinhuan</td>
			<td>KC439</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Pritech</td>
			<td>LD-9987</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Zhejiang Jindakang Animal Health Product Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium chloride solution</td>
			<td>Hunan Kelun Pharmaceutical Co., Ltd.</td>
			<td>H43020455</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Shanghai Kangfu chemical pharmaceutical Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>16005</td>
			<td>Dilute to final concentration of 10% in PBS</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td>Dilute to final concentration of 20% in PBS</td>
		</tr>
		<tr>
			<td>0.1% Hematoxylin Solution, Mayer&#8217;s</td>
			<td>Sigma-Aldrich</td>
			<td>MHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Eosin Y solution, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma-Aldrich</td>
			<td>S8884</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Excelsior ESTM Tissue Processor</td>
			<td>Thermo Fisher</td>
			<td>A78400006</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Histocentre&#8482; 3 Tissue Embedding Center</td>
			<td>Thermo Fisher</td>
			<td>B64100010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully Automated Rotary Microtome</td>
			<td>Leica</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>Molecular Probes, Life 
			Technologies</td>
			<td>V-22887</td>
			<td></td>
		</tr>
		<tr>
			<td>D-MEM High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO, Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200-114</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>Corning</td>
			<td>V118877</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>Optima MAX-TL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr>
			<td>Titetamme</td>
			<td>Virbac (Zoletil 50)</td>
			<td>1000000188</td>
			<td></td>
		</tr>
		<tr>
			<td>Zolazepam</td>
			<td>Virbac (Zoletil 50)</td>
			<td>1000000188</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile hyposermic syringe for single use 26G</td>
			<td>Shanghai Misawa Medical Industry</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging 4.0 software</td>
			<td>PerkinElmer</td>
			<td>None</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo tracking of human adipose-derived mesenchymal stem cells in a rat knee osteoarthritis model with fluorescent lipophilic membrane dye

무릎 관절염 (KOA)에 대 한 인간 지방 유래 간 엽 줄기 세포 (haMSC) 치료의 임상 적용을 지원 하기 위해 셀 지의 효능과 동물 모델에서 haMSCs의 biodistribution을 검사 합니다. 우리는 질 성 형광 염료와 haMSCs의 세포 막에 레이블을 하는 방법을 설명 했다. 그 후, 수술로 유도 KOA와 쥐에 있는 레이블된 셀의 내부 관절 주사는 vivo에서 이미징 시스템에서 동적으로 모니터링 했다. 우리는 질 성 고용 carbocyanines 않았다 (DilC18 (5)), 조직 주변에서 자연 녹색 autofluorescence의 자극을 피하기 위해 빨간색 레이저를 활용 하는 빨강 형광 Dil (dialkylcarbocyanines) 아날로그. 또한, 레드 이동 방출 스펙트럼의 살아있는 동물에서 깊은 조직의 이미징 및 레이블 프로시저 발생 세포 독성 효과 또는 haMSCs 기능 손상을 허용 했다. 이 접근은 쥐 KOA 모델에서 haMSCs에 대 한 효율적인 추적 방법으로 보였다. 이 방법의 응용 프로그램 또한 최적의 관리 경로 및 전 임상 연구에서 다른 소스에서 MSCs의 복용량을 결정 하기 위해 사용 될 수 있습니다.

KOA 모델을 유도 하기 위하여 m M SD 쥐 (그림 3)의 오른쪽 무릎 관절에 수행 되었다. 수술 후 8 주 쥐 희생 했다 고 무릎 관절의 직렬 섹션 두 H로 평가 했다 &#38;와 Safranin O/빠른 그린 얼룩 (그림 4). H에 대 한 &#38; 전자 얼룩, 관절 연골의 표면 전시 보다 수술을 하지 않고 정상적인 관절 수술 무릎에 거칠어 테두리. Safranin O/빠른 그린 ...

Watch this Scientific Journal Video about In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye at JoVE.com

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Vivo에서 인간 지방 유래 중간 엽 줄기 세포 형광 질 성 막 염료와 쥐 무릎 관절염 모델에서의 추적

이 프로토콜에는 빨강 형광 라벨 내부 관절 (IA) 주입을 통해 쥐 무릎 관절염 (KOA) 모델에 의해 셀 속성과 biodistribution 인간 지방 유래 중간 엽 줄기 세포 (haMSCs)의 모니터링 하는 효율적인 방법을 설명 합니다.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

in-vivo-tracking-human-adipose-derived-mesenchymal-stem-cells-rat

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JoVE Journal

Developmental Biology

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

8.9K 조회수.  Cellular Biomedicine Group, China. 이 프로토콜에는 빨강 형광 라벨 내부 관절 (IA) 주입을 통해 쥐 무릎 관절염 (KOA) 모델에 의해 셀 속성과 biodistribution 인간 지방 유래 중간 엽 줄기 세포 (haMSCs)의 모니터링 하는 효율적인 방법을 설명 합니다.

비디오: In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

분리 및 인간의 농축 향상된 골 연장술에 대한 기질 세포를 지방이 유래

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

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발생학

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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

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

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

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

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

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

지방 창고 별 SCA1의 면역 자기 분리<sup&gt; 높은</sup&gt; 지방이 유래 줄기 세포 (의 ASC)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

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 ...

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

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

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

원심 중력에 의해 지방이 유래 줄기 세포의 연골 분화 유도

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

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +의 신속한 격리 두개골 결함 치유 모델에서 사용을위한 기질 세포를 지방이는 유래

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

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

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

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

기본 및 확실 한 조 혈 창시자 인간 만능 줄기 세포에서의 감독

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

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

생체 외에서 기능 Cardiomyocyte-같은 세포로 인간 중간 엽 줄기 세포의 분화

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

세 인간 Bruch 막의 Ex Vivo 모델에서 망막 안료 상피 세포의 배양

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...