This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.

참고 : 장골에서 인간의 골수 흡인는 루체른의 광저우의 윤리위원회의 승인을 기증자를 동의에서 수집되었다. 장골에서 송곳니 골수 흡 인물은 (약. 20 ㎖) 개 오너 consent.Human으로 수집 하였다 송곳니 (약. 10 ㎖) 골수 흡인했다 항 응고 즉시 철수 후 3.8 %의 구연산 나트륨 15ml를 첨가하여 조작 극장에서. 샘플로서 인출 당일 처리 실험실 환경에 옮겼다. 유로키나제 1. 준비 (이전에 1 차 사용에) <ol><li> 제조업체의 지시에 따라 멸균 인산 완충 식염수 (PBS)를 사용하여 재구성 유로키나제 : 10 ㎖ 주사기를 사용하여 격막-입구 플라스크에 10 ㎖의 PBS를 추가한다. 1 mL 당 50,000 사출 유닛 (U)을 얻었다 소용돌이에 의해 건조 물질을 녹이고. </li><li> 의에 500 ㎕를 분주 (25,000 U 각)를 준비terile 튜브. 스토어 씩 -20 ° C 및 6 개월 이상 필요할 때 사용. </li></ol> 2. 제조 단계 (이전에 모든 골수 치료에) <ol><li> 유로키나제 중 하나 나누어지는 응고 골수 흡인 당 (25,000 U)를 (25 ml의에 흡인 볼륨이 성공적으로 25,000 U의 단일 나누어지는을 사용하여 처리 된) 해동. </li><li> 37 ° C의 물 목욕이나 진탕 배양기를 예열한다. </li></ol> 응고 3. 효소 다이제스트 <ol><li> 처리 중 골수 샘플의 미생물 오염을 방지하기 위해 층류 바이오 안전성 캐비닛의 모든 작업을 수행합니다. 감염 가능성이 인간의 물질로 작업 할 때, 보호 장갑뿐만 아니라 취급에주의의 사용을 권장합니다. </li><li> 멸균 50 ML 튜브의 상단에 100 μm의 셀 스트레이너를 놓습니다. 조심스럽게 스트레이너를 기울 또는 응고 물질 주위를 이동, 셀 스트레이너를 통해 골수 흡인 액을 붓는다. 대한 멸균 피펫 팁을 사용하여필터 망을 통해 더 나은 흐름. 참고 : PBS와 혈전을 세척하여, 예를 들면, 혈전의 희석을 피하십시오. 유로 키나아제는 간접적으로 작용하고 혈청의 구성 요소 (예 :., 플라스 미노 겐) 효과 다이제스트에 대한 생검이 필요합니다. 응고 물질과 절차를 계속하면서, 여액을 단계 3.6에 더 사용할 때까지 실온에서 유지 될 수있다. </li><li> 무균 핀셋을 사용하여 빈 세포 배양 접시에 골수 응고 재료를 전송합니다. 약 2mm 3 멸균 메스를 사용하여 작은 조각으로 파편을 잘라. </li><li> 무균 핀셋을 사용하여 50 ㎖ 반응 관에 혈병의 작은 조각을 전송. 다진 응고 젖은 모습을 가지고 있는지 확인하십시오. 이 마른 것 같으면, 축축 3.1 단계에서 여과 액의 일부를 사용합니다. </li><li> 로 pipetting 아래 5 ml의 마이크로 타이 피펫을 사용하여 5 회에 의해 혈전을 씹다. 샘플 유로키나제의 1 나누어지는을 추가합니다. 37 ° C에서 어느 수욕 또는 30 분 동안 인큐베이션진탕 (부드럽게) 인큐베이터. </li><li> 로 pipetting 아래 5 ML의 피펫을 사용하여 5 회에 의해 씹다. 바이오 안전성 캐비닛이 단계를 수행합니다. 37 ° C에서 또 다른 30 ​​분 동안 품어. 배양 후, 5 ㎖ 피펫을 사용하여 다시 5 회 연화. 단계 3.2에서 여과와 풀에 신선한 100 μm의 셀 스트레이너를 통해 전달합니다. </li><li> 원심 분리기 (500)의 X g에서 10 분 동안 주위 온도에서 세포 현탁액. 상층 액을 제거한다. 설명 또는 MSC 확장 문화에 대한 실험실의 표준 절차에 따라 같은 매체에서 세포 펠렛을 다시 일시 중지합니다. </li></ol> 확장 세포 배양 및 CFU 플레이트 4. 시드 <ol><li> 알파 MEM, 10 % 소 태아 혈청 (FBS), 100 U / ㎖ 페니실린, 100 ㎎ / ㎖ 스트렙토 마이신 및 2.5 ㎎ / ㎖의 암포 테리 신 B로 보충 : 기초 배지 50 ㎖ 중의 (적혈구 및 단핵 세포로 구성됨) 세포를 재현 탁 . 세포에게 노이 바 우어 chambe를 사용하여 트리 판 블루 용액 희석 1:10 카운트R. </li><li> 5 × 107 세포 / ㎠의 밀도로 세포 배양 플라스크에서 세포를 플레이트 및 37 ° C에서 가습 배양기에서 배양한다. 가능하면, 저산소 (5 % 산소) 조건을 사용합니다. CFU 분석 제어시 10cm 직경의 세포 배양 접시에 세포를 10 9 접시. </li><li> 다른 세포 현탁액에 남아있는 동안 배양 3 일 후, 중간 엽 줄기 세포는 세포 배양 접시에 부착된다. 5 NG / ㎖ 염기성 섬유 아세포 성장 인자 (bFGF)이 보충 된 기본 배지로 미디어를 변경한다. CFU 분석 제어를 위해, 마찬가지로 세포 배양 접시를 치료. </li><li> 미디어 일주일에 세 번 교환, 이주의 총 세포 배양을 계속합니다. 세포가 80 % 컨 플루 언시를 초과하면, 트립신 처리에 의해 분리. CFU 분석 제어를 위해, 마찬가지로 미디어를 교환하지만, 2 주 코스의 세포를 분할하지 않는다. </li></ol> CFU 분석 5. 김사 얼룩 <ol><li> 접시 당 김사-용액 10 ml의 준비 : DIL을UTE 원액 (글리세롤 7.6 g / L 김사 : 메탄올) 1시 10분 멸균 물 (항상 신선한 작업 희석을 준비). </li><li> 세포 배양 접시에서 미디어를 제거하고 PBS로 세포를 씻어. 페트리 접시에 액체를 적용 할 때 매우 precautious하고 세포를 세척을하지 마십시오. </li><li> RT에서 5 분 동안 순수한 메탄올에 세포를 고정. 메탄올을 폐기하십시오. 김사-솔루션을 추가하고 37 ° C에서 가습 인큐베이터에서 60 분 동안 품어. </li><li> PBS로 2 회 세척 할 것. 공기는 종이 타월에 먼저 판 머리를 건조. 수동으로 식민지를 계산합니다. 이것은 최상의 플레이트의 후방에 마커 펜을 사용하여 달성된다. </li></ol> 6. MSC 차별화 <ol><li> 개 중간 엽 줄기 세포의 분화 참고 : 개 중간 엽 줄기 세포는 4 주 동안 해당 매체를 자극하여 연골 세포, 골 세포 및 지방 세포 계통으로 분화되었다. </li><li> 용 지방 세포 분화, 4 × 10 5 CE에서 단일 층의 문화 엽 줄기 세포다음 LLS / cm 2 개의 상이한 배양 조건 교번; <ol><li> DMEM + 루타, 3 % FBS, 100 유닛 / ㎖ 페니실린, 100 ㎎ / ㎖ 스트렙토 마이신, 250 ㎍ / ml의 암포 테리 신 B, 170 mM의 인슐린을 함유하는 지방 형성 유지 배지에서 배양. </li><li> 3 % FBS, 5 % 토끼 혈청, 1 μM 덱사메타손으로 보충 유지 배지 지방 형성 유도 배지에서 배양, 500 μM 3- 이소 부틸 -1- 메틸 크 33 μM 바이오틴, 5 μM 로시글리타존 16 17 μM 판토텐산. </li><li> 오일 레드 O로 염색하여 지질 방울을 르벨. 간단히, 10 % 포름 알데히드와 세포를 해결 PBS로 세척 및 이소프로판올 0.35 % 오일 레드 O로 염색. </li></ol></li><li> 7 × 103 세포 / cm (2)와 다음의 자극 단층에서의 조골 세포 분화 중간 엽 줄기 세포 배양의 경우 : <ol><li> 고급 DMEM (GIBCO) 루타 + 5 % FBS, 100 유닛 / ㎖ 페니실린, 100 ㎎ / ㎖ 스트렙토 마이신, 250 ㎍ / ml의 암포 테리 신 B 501; M L 아스코르브 산 -2- 포스페이트, 10mM의 글리세의 ß-10​​0 nm의 덱사메타손. </li><li> 폰 코사 염색 (5 % AGNO 3)에 의한 광물 예금을 확인합니다. 간단히, 10 % 포름 알데히드와 세포를 고정 5 % 증류수에 AGNO 3 PBS와 얼룩 씻는다. </li></ol></li><li> 연골 세포의 분화 <ol><li> 스폰지 형 의료 장치로부터 절단 조각 (면당 3mm)는, 동결 건조 된 콜라겐 형 I로부터 구성되고 셀 (17)을지지하는 지지체 재료로서 사용된다. </li><li> 4 × 106 세포 / ml (~ 70,000 세포 / 큐브)의 농도로 큐브의 위에 시드 엽 줄기. </li><li> 매체의 첨가에 앞서, 세포를 30 분 동안 큐브에 부착 할 수있다. </li><li> DMEM / F12 + 루타, 2.5 % FBS, 100 단위 / ml의 페니실린, 100 ㎎ / ㎖ 스트렙토 마이신, 2.5 ㎍ / ml의 암포 테리 신 B, 40 NG / ㎖ 덱사메타손 (dexamethasone), 50 ㎍ / ㎖의 아스 코르 빈산으로 구성된 연골 미디어 MSC-콜라겐 구조를 유지 산 -2- 포스페이트,50 ㎍ / ㎖의 L 프롤린, 1X 인슐린 - 트랜스페린 - 셀렌 X 및 성장 인자 β1 변형 10 ng / ml이되게 준비 하였다. </li><li> 구조 섹션에 프로테오글리칸의 축적을 시각화하는 알 시안 블루 염색을 사용합니다. 간단히, 0.01 %의 H 2 SO 4 및 0.5M 구아니딘 하이드로 클로라이드에 용해하고 0.4 % 알 시안 블루 섹션 O / N 얼룩. 다음, 세척 섹션은 40 % DMSO와 0.05 M의 MgCl 2에서 30 분 동안 세척 하였다. 세포 핵 빠른 빨간색으로 대조되었다. </li></ol></li></ol>

<ol>
	<li>Jiang, Y., 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=Pluripotency+of+mesenchymal+stem+cells+derived+from+adult+marrow.">Pluripotency of mesenchymal stem cells derived from adult marrow.</a> Nature. 418 (6893), 41-49 (2002).</li><li>Uccelli, A., Moretta, L., Pistoia, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+health+and+disease.">Mesenchymal stem cells in health and disease.</a> Nat Rev Immunol. 8 (9), 726-736 (2008).</li><li>Bartholomew, 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=Mesenchymal+stem+cells+suppress+lymphocyte+proliferation+in+vitro+and+prolong+skin+graft+survival+in+vivo.">Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.</a> Exp Hematol. 30 (1), 42-48 (2002).</li><li>Wang, S., Qu, X., Zhao, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+mesenchymal+stem+cells.">Clinical applications of mesenchymal stem cells.</a> J Hematol Oncol. 5, 19 (2012).</li><li>Baksh, D., Song, L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells:+characterization,+differentiation,+and+application+in+cell+and+gene+therapy.">Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.</a> J Cell Mol Med. 8 (3), 301-316 (2004).</li><li>Malempati, S., Joshi, S., Lai, S., Braner, D. A., Tegtmeyer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Videos+in+clinical+medicine.+Bone+marrow+aspiration+and+biopsy.">Videos in clinical medicine. Bone marrow aspiration and biopsy.</a> N Engl J Med. 361 (15), e28 (2009).</li><li>Cuthbert, 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=Single-platform+quality+control+assay+to+quantify+multipotential+stromal+cells+in+bone+marrow+aspirates+prior+to+bulk+manufacture+or+direct+therapeutic+use.">Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use.</a> Cytotherapy. 14 (4), 431-440 (2012).</li><li>Veyrat-Masson, 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=Mesenchymal+content+of+fresh+bone+marrow:+a+proposed+quality+control+method+for+cell+therapy.">Mesenchymal content of fresh bone marrow: a proposed quality control method for cell therapy.</a> Br J Haematol. 139 (2), 312-320 (2007).</li><li>Lazarus, H. M., Haynesworth, S. E., Gerson, S. L., Rosenthal, N. S., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+and+subsequent+infusion+of+human+bone+marrow-derived+stromal+progenitor+cells+(mesenchymal+progenitor+cells):+implications+for+therapeutic+use.">Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use.</a> Bone Marrow Transplant. 16 (4), 557-564 (1995).</li><li>Bertolo, 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=An+in+vitro+expansion+score+for+tissue-engineering+applications+with+human+bone+marrow-derived+mesenchymal+stem+cells.">An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.</a> J Tissue Eng Regen Med. , (2013).</li><li>Casiraghi, F., Remuzzi, G., Abbate, M., Perico, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+mesenchymal+stromal+cell+therapy+and+risk+of+malignancies.">Multipotent mesenchymal stromal cell therapy and risk of malignancies.</a> Stem Cell Rev. 9 (1), 65-79 (2013).</li><li>Bonab, M. 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=Aging+of+mesenchymal+stem+cell+in+vitro.">Aging of mesenchymal stem cell in vitro.</a> BMC Cell Biol. 7, 14 (2006).</li><li>Niku, S. D., Hoon, D. S., Cochran, A. J., Morton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+lymphocytes+from+clotted+blood.">Isolation of lymphocytes from clotted blood.</a> J Immunol Methods. 105 (1), 9-14 (1987).</li><li>De Vis, J., Renmans, W., Segers, E., Jochmans, K., De Waele, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+immunophenotyping+of+leukemia+cells+in+clotted+blood+and+bone+marrow.">Flow cytometric immunophenotyping of leukemia cells in clotted blood and bone marrow.</a> J Immunol Methods. 137 (2), 193-197 (1991).</li><li>St Antoine, 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=Application+of+thrombolytic+drugs+on+clotted+blood+and+bone+marrow+specimens+to+generate+usable+cells+for+cytogenetic+analyses.">Application of thrombolytic drugs on clotted blood and bone marrow specimens to generate usable cells for cytogenetic analyses.</a> Arch Pathol Lab Med. 135 (7), 915-919 (2011).</li><li>Kisiel, A. 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=Isolation,+characterization,+and+in+vitro+proliferation+of+canine+mesenchymal+stem+cells+derived+from+bone+marrow,+adipose+tissue,+muscle,+and+periosteum.">Isolation, characterization, and in vitro proliferation of canine mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum.</a> Am J Vet Res. 73 (8), 1305-1317 (2012).</li><li>Bertolo, 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=Influence+of+different+commercial+scaffolds+on+the+in+vitro+differentiation+of+human+mesenchymal+stem+cells+to+nucleus+pulposus-like+cells.">Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells.</a> Eur Spine J. 21, S826-S838 (2012).</li><li>Makridakis, M., Roubelakis, M. G., Vlahou, 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:+insights+into+the+secretome.">Stem cells: insights into the secretome.</a> Biochim Biophys Acta. 1834 (11), 2380-2384 (2013).</li><li>Benvenuti, 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=Rosiglitazone+stimulates+adipogenesis+and+decreases+osteoblastogenesis+in+human+mesenchymal+stem+cells.">Rosiglitazone stimulates adipogenesis and decreases osteoblastogenesis in human mesenchymal stem cells.</a> J Endocrinol Invest. 30 (9), RC26-RC30 (2007).</li><li>Jaiswal, N., Haynesworth, S. E., Caplan, A. I., Bruder, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+purified,+culture-expanded+human+mesenchymal+stem+cells+in+vitro.">Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.</a> J Cell Biochem. 64 (2), 295-312 (1997).</li><li>Patel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+banking+for+research--bench+to+bedside+and+back--myth,+reality+or+fast+fading+reality+at+the+dawn+of+a+personalised+healthcare+era.">Tissue banking for research--bench to bedside and back--myth, reality or fast fading reality at the dawn of a personalised healthcare era.</a> Cell Tissue Bank. 12 (1), 19-21 (2011).</li><li>Smith, H. W., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell+signalling+by+uPAR.">Regulation of cell signalling by uPAR.</a> Nat Rev Mol Cell Biol. 11 (1), 23-36 (2010).</li></ol>

The authors have nothing to disclose.

환자가 약간의 추가 작업 만이 동작 극장 요원에 의해 수행되어야하는 이점, (우리의 경우 주로 척추 수술) 수술을하는 동안 일상적으로 우리는 골수 샘플. 샘플은 즉시 인출 후 구연산 나트륨과 혼합 되더라도 그들이 처리 실험실에 도착했을 때, 많은 샘플이 부분적으로 응고했다. 이 단계에서, 응고 표본을 대체 할 리샘플링은 별도의 추가 작업이 다시 로컬 또는 전신 마취 6 필요로 할 ?...

중간 엽 줄기 세포 (중간 엽 줄기 세포)는 재생 의학 및 조직 공학에 중요한 역할을합니다. 그들은 그들에게자가 치료 2,3를위한 이상적인 후보자를 표현하는 다양한 세포 유형 1로 분화와 접목, 마이그레이션 할 수 있습니다. 최근, 뼈와 연골 수리를 위해 중간 엽 줄기 세포를 이용하여 임상 시험은, 호스트 질환이나 심장 질환 이식편 대 4를 출시했다. 이러한 중간 엽 줄기 세포는 제대혈이나 지방 조직에서 수확 할 수 있지만 가장 유망한 결과는 골수 유래 줄기 세포 5 얻었다. 장골 골수 상당한 양의를 수집 할 수 있으므로 흡인 6의 메인 사이트의 역할을한다. 그러나 대기음의 품질 인출 골수 부피가 증가함에 따라 감소한다. 골수 흡 인물의 제 5 ㎖ 고품질의 중간 엽 줄기 세포를 포함하는 반면, 많은 양의 취하 말초 혈액 F와 흡 인물의 희석에 이르게높은 혈관 뼈 7 롬. 항응고제를 사용하지 않고서 본 거핵 혈소판, 골수 흡 인물은 응고되기 쉽다. 그러나 심지어 항응고제, 혈전이 발생할 수 있습니다. 골수에서 중간 엽 줄기 세포는 전체 풀 (8)의 단지 작은 부분을 나타내고, 대부분의 조직 공학 또는 치료 응용 4 배양에서 확장해야한다. 이러한 문화의 품질은 크게 초기 셀 풀, 즉에 따라 달라집니다., 다양성과 높은 기동 번호 9. 인출에서 중간 엽 줄기 세포의 낮은 숫자는 부분적으로 기증자의 변화에​​ 의해 설명 될 수있다. 한편, 낮은 품질의 샘플들로부터 중간 엽 줄기 세포의 수를 달성하기 위해 더 긴 시간 배양 및 계대 확장을 필요로한다. 어느 경우에도, 연장 계대는 세포 노화의 소스이고 10 분화 잠재력의 손실을 초래할 수있다. 따라서, 셀 (Y)을 극대화 할 수있는 프로토콜을 최적화과의 ield 11,12 악영향이 개발 될 필요가 방지. 우리가 개 중간 엽 줄기 세포와 함께 작업을 시작했을 때 다행히 응고 인간의 샘플 (열에서 하나) 덜 자주있는 동안, 우리는 3 ~ 4 개에서 골수 샘플 혈전 포함 된 것을보고 깜짝 놀랐다. 반면에 우리가 응고 샘플에서 중간 엽 줄기 세포의 훨씬 낮은 수익률을 관찰하는 것이, 놀랍지 않습니다. 응고 된 샘플의 반복 문제를 해결하기 위해, 우리는 리샘플링 대신 혈전 용해 약물 유로 키나아제를 사용하는 프로토콜을 개발했다. 혈전 용해 요법은 심장 마비, 뇌졸중 또는 때문에 원치 않는 응고의 색전증을 일으키는 혈관의 폐색과 같은 생명을 위협하는 상황에 대처 할 수 있습니다. 그들은 플라스 민과 효소 플라스 미노 겐 활성제에 의한 섬유소의 효소 분해를 통해 혈전의 분해에 의해 작동합니다. 환자의 치료에 널리 사용에도 불구하고, 매우 소수의 서적은 활용도가 혈전​​ 활동하는 것이 존재실험실 응용 프로그램이 응고 샘플을 구출하기 위해, 대부분의 림프구에 초점을 맞추고. 1987 년, 니쿠 등. (13)와 4 년 뒤 기능 림프구의 결과로 혈전을 용해 스트렙토의 사용을 설명, 드 비스 등. 유세포 애플리케이션 (14)에 대한 혈액 및 골수에서 백혈병 세포를 분리 스트렙토의 사용을 확장. 최근 간행물은 암 진단 15 Alteplase의 사용을 제안한다. 동일한 효소 적 방법을 사용하는 동안, 우리는 프로토콜 줄기 세포 연구 분야를위한 툴을 제공하는 MSC의 분화능 양식 골수의 분리에 초점을 맞춘다.

<table border="0" cellpadding="0" cellspacing="0" width="674">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Basal Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PenStrep 100X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human FGF-basic</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MEM Alpha w/ Nucleoside, w/ stable Glutamine</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-23S50-I</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">FBS Heat Inactivated</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">2-01F36-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Amphotericin B</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1907</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Adipogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DMEM-HAM F12 + GlutaMAX</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-26F09-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rabbit serum&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">16120099</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Dexamethasone</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">D4902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5879</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biotin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">B4639</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rosiglitazone&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">R2408</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pantothenate&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5155</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oil Red-O&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">O0625</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Osteogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-ascorbic acid 2-phosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A8960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#223;-glycerophosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">G9422</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:328px;">Silver nitrate (AgNO3)</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">S6506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chondrogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biopad - sponge shaped medical device&#160;</td>
			<td style="width:227px;">Euroresearch</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-proline&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5607</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin-Transferrin-Selenium X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">51500056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human transforming growth factor-&#946;1&#160;</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Alcian Blue 8GX</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A3157</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Nuclear fast red</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">N8002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Generic</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tri-Sodium citrate dihydrate</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3901</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PBS</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">964.9100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Urokinase</td>
			<td style="width:227px;">Medac</td>
			<td style="width:119px;">1976826</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">0.5% Trypsin-EDTA</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15400054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Giemsa stain</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0885</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0877</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid (H2SO4)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0655</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1584</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidine hydrochloride</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1499</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Consumables</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">50 mL reaction tube</td>
			<td style="width:227px;">Axygen</td>
			<td style="width:119px;">SCT-50ML-25-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10 mL syringe</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4606108V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Sterican needle (22G)</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4657624</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">1.7 mL Microtubes</td>
			<td style="width:227px;">Brunschwig</td>
			<td style="width:119px;">MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">100 &#956;m cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">6.05935</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile forceps</td>
			<td style="width:227px;">Bastos Viegas, SA</td>
			<td style="width:119px;">489-001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile scalpel</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">5518059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Primaria cell cuture dish</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">353803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">C-Chip Neubauer Improved</td>
			<td style="width:227px;">Bioswisstech</td>
			<td style="width:119px;">505050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">cell culture flask - Flask T300</td>
			<td style="width:227px;">TPP</td>
			<td style="width:119px;">90301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Microbiological biosafety cabinet class II</td>
			<td style="width:227px;">Skan</td>
			<td style="width:119px;">82011500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">water bath</td>
			<td style="width:227px;">Memmert</td>
			<td style="width:119px;">1305.0377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Stripettes Serological Pipette 5ml</td>
			<td style="width:227px;">Corning</td>
			<td>4487-200ea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">microscope</td>
			<td style="width:227px;">Olympus</td>
			<td style="width:119px;">CKX41</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">humidified incubator Heracells 240</td>
			<td style="width:227px;">Thermo scientific</td>
			<td>51026331</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Heraeus Multifuge 1S-R</td>
			<td style="width:227px;">Thermo scientific</td>
			<td style="width:119px;">75004331</td>
		</tr>
	</tbody>
</table>

an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples

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

함께 그들은 우리의 실험실 (그림 1A)에 도착했을 때 개 골수 샘플의 74 % (N = 54) 혈전을 포함한다는 사실은 우리가 중간 엽 줄기 세포의 상당수가 혈전 내에 갇혀 있다고 생각했고, 이러한 샘플에서 MSC 수율을 감소 . 실제로, 단면 응고 재의 간단한 DAPI 얼룩은 고밀도 (도 1B)에 유핵 세포의 존재를 확인한다. 이것은 궁극적 유로키나제를 사용하여 프로토콜을 개발하기 위해 ?...

Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

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

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

효소 방법은 응고 골수 샘플에서 중간 엽 줄기 세포를 구출

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

an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

Research

JoVE Journal

Developmental Biology

9.1K 조회수.  Swiss Paraplegic Research. Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

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

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

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

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

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

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

연구

발생학

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

성인 피부 섬유 아 세포에서 인슐린 분비 세포를 얻을 수있는 안전하고 간단한 방법으로 성적인 변환

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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

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

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

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

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

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

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

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

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

사람 골수에서 분리 Mesangiogenic 전구 세포 (MPC와)

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

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

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

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

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

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

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

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

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

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

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

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

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

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

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

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

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

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

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

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

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

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

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

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

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

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

절연 및 Endomyocardial Bioptic 샘플의 Arrhythmogenic 심장 근육 병 증 환자에서 심장 중간 엽 기질 세포의 특성

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...