This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

기관 심사위원회가 승인 한 환자 동의서는 인간 세포의 사용을 취득해야합니다. 사람 말초 혈액 단핵 세포의 1. 밀도 구배 분리 (PBMC를) <ol><li> 25 mL를 피펫으로 50 ㎖ 튜브에 전체 혈액을 헤파린 25 ML을 추가합니다. <ol><li> 인산 완충 식염수 (PBS) 25 ㎖로 희석하여 세포. </li></ol></li><li> 새로운 50 ㎖ 튜브에 피콜 - 페이크 밀도 구배의 15 mL를 넣고 튜브 틸팅 동안 전혈의 믹싱으로 존재하지 않도록 매우 조심스럽게 천천히는 밀도 구배 위에 희석 세포 현탁액 25 ㎖에 추가 밀도 구배, 즉., 혈액 - 밀도 구배 인터페이스의 방해없이. </li><li> 원심 분리기 20 ° C에서 브레이크없이 30 온도 조절 스윙 버킷 로터에서 분 40 400 × g에서 1.2 단계에서 정지. 참고 : 세 가지 층은 원심 분리 후 명백하다 : 일즉 상층 존재 플라즈마; 바닥 클리어 층은 피콜 - 페이크 밀도 구배되며; 및 얇은 층은 중간 셀룰러 PBMC를 되 </li><li> 기음과는 단핵 세포 (즉, 림프구, 단핵구, 혈소판)의 계면 층을 방해하지 않도록주의로, 파스퇴르 피펫으로 흡입하여 상층을 버린다. </li><li> 조심스럽게 새로운 50 ㎖ 원심 분리 튜브에 10 ㎖ 피펫이 단핵 세포층을 수집한다. </li><li> , PBS의 20 ㎖와 튜브를 채우 잘 혼합하고, 20 ℃에서 10 분 동안 300 × g에서 원심 분리기. 원심 분리 후 완전히 뜨는을 제거합니다. </li><li> 20 ℃에서 10 ~ 15 분 동안 200 × g에서 세포 PBS 20ml에 펠렛을 재현 탁하고 원심 분리기. 원심 분리 후 완전히 뜨는을 제거합니다. 참고 :이 단계는 혈소판 - 원치 않는-내에서 PBMC를하다을 제거; 이 단계는 혈소판의 완전한 제거를 보장하기 위해 반복 될 수있다. </li><li> 특정 집단의 선택 범위가 없으면세포 수 (16)를 수행 한 후, 배양을 위해 바람직한, 백혈구 완전 배지에 재현 탁 된 세포 펠릿 (10 % 소 태아 혈청 (FBS), 1 % L- 글루타민, 1 % 페니실린 / RPMI-1640 배지에서 스트렙토 마이신)을 1 ml의 중단을 다음 단계로 진행하기 전에 10 7 PBMC를 당 매체. </li></ol> 백혈구 인구 2. 자기 라벨링 (특정 집단의 어떤 선택이 필요없는 경우 4 단계로 건너 뜁니다) <ol><li> 완전히 10 분, 흡인 상층 액 300 × g에서 원심 분리기 PBMC 서스펜션. </li><li> 10 7 총 세포 당 PBS의 80 μL에 재현 탁 세포 펠렛. </li><li> 10 7 총 세포 당 (T 림프구의 분리를위한 단핵 세포, CD4 자석 구슬의 격리를 위해 즉, CD14 자석 구슬) 지정된 자석 구슬의 20 μl를 추가합니다. </li><li> 잘 섞어 냉장고에 8 ° C에 2에서 15 분 동안 품어. </li><li> 씻어 10 7 세포 당 PBS의 1-2 ML을 추가하고, 원심 분리기10 분 동안 300 × g에서. </li><li> 대기음 상층 액을 완전히 PBS 500 μL에 10 8 셀을 재현 탁. </li></ol> 백혈구의 부분 집단 3. 자기 선택 참고 : 자기 비드 구분과 열의 다양한 제조 업체의 수와 세포 표면 마커에 의한 백혈구의 특정 인구의 분리에 사용할 수 있습니다. 격리 하나 또는 몇 가지 긍정적 인 마커 또는 원치 않는 인구의 고갈 후 음의 선택에 기반한 선택에 의해 할 수있다. (즉, 단핵구에 대한 CD4 T 헬퍼 림프구에 대한, 또는 CD14) 한 마커를 사용하여 긍정적 인 선택을 수행하는 일반적인 방법은 아래에 설명되어 있습니다. <ol><li> 준비 및 주요 마그네틱 분리기는 제조업체의 지침에 따라 분리 컬럼을 포함. </li><li> 표시 PBMC를의 샘플이 포함 튜브를 적용합니다. 긍정적으로 표지 및 unlabel의 수집이 15 ml의 원심 분리기 튜브를 제공합니다제조업체의 지침에 따라 에드 셀 분수,. </li><li> 107 세포 당 1 ml의 배지를 이용하여 세포 수를 카운트하고 15 백혈구 완전 배지에서 긍정적으로 선택된 백혈구 (예, CD4 + T 림프구 또는 CD14 + 세포)를 재현 탁. </li></ol> 대량 살상 무기 확산의 평가를위한 백혈구 4. CFSE 얼룩 주 : CFSE 널리 생체 내 및 시험 관내 연구에서 모두, 면역 학적 조사에 사용되었다. 우리의 프로토콜은 MSC / PBMC (또는 다른 백혈구)의 상호 작용의 체외 연구를 위해 최적화되었습니다. CFSE 체외 라벨을 수행에 관련된 일반적인 단계는 유사하지만, 프로토콜 (14)의 특정 용량과 타이밍의 차이가있을 수 있습니다. 이 때문에 다수의 인자, 즉, 사용 된 특정 세포 유형, 세포 수로 사용할 수 있고-있는 가능성 CFSE 형광 신호의 강도에 영향을 미칠 수있다. <ol&gt; <li> 10 μm의 (CFSE - 작업 솔루션)의 원하는 작업 농도로 PBS에서 CFSE 주식 솔루션 (10 mm)를 희석. CFSE-작동 용액으로 라벨링 10 7 세포 (즉, PBMC를 또는 T 세포)를 준비한다. </li><li> 10 분 동안 300 × g에서 원심 분리기는 세포 펠렛을 얻고 뜨는을 대기음합니다. </li><li> 부드럽게 예열 (37 ° C) CFSE-작동 용액 1 ㎖로 세포를 재현 탁하고, 37 ℃에서 10 분 동안 세포를 배양한다. </li><li> 과량 CFSE을 씻어, 예비 냉각 (4 ℃) RPMI 배지를 10 % FBS를 함유하는 (부피 기준)으로 10 배의 세포 현탁액을 희석. 5 분 300 XG에서 원심 분리하여 세포 침전물과 상층 액을 버린다. 두 번 더 이런 방식으로 세포 펠렛을 씻으십시오. </li><li> 원심 분리하여 세포를 다시 펠렛 세포 수 (15)를 계산. 1 ㎖의 백혈구 데워진 신선한 완전 배지에서 10 7 세포 (PBMC를 또는 T 세포를) 재현 탁. </li></ol> M 5. 공동 문화백혈구 및 백혈구의 활성화와 희주 <ol><li> 이하에서 30 분 동안 37 ° C까지 미리 따뜻한 MSC 완전 배지 (최적 MSC 성장을위한 테스트 된 10 % FBS (), 1 % L- 글루타민, 1 % 페니실린 / DMEM 낮은 포도당 배지에서 스트렙토 마이신). </li><li> 24 웰 플레이트에서 MSC 완전 배지 1 ㎖에 50,000 세포 (MSC 밀도 : 25,000 세포 / ㎠)에서 시드 엽 줄기 37 ° C의 배양기에 부착 O / N에 대한, 줄기 세포 수는 80 % 합류에 도달한다. </li><li> 시드 MSC 번호를 기반으로 대기음 매체와는 CFSE 표지 PBMC를 추가 (이전에 표시된 제발 위의 프로토콜 4 단계 참조)에 백혈구 완전 배지 1 ㎖에 (24 웰 플레이트에 전날 시드) 배양 된 중간 엽 줄기 세포에 1시 10분 (셀 비율)로 PBMC를 엽 줄기 세포의 공동 배양 비율. </li><li> 1 ㎖의 백혈구 완전 배지의 총 부피에서 10 μg의 / ml의 최종 농도로, 미토 겐의 피토 헤 마글 루티 닌 (PHA), 비특이적 백혈구 활성화 제를 추가 웰당. <ol><li> Alternativ엘리는, 구체적으로 T 림프구의 활성화를 자극하기 위해 : α-CD3 / 28 마이크로 비드를 사용하여 (1)의 비드 간 T 림프구의 비율을 얻기 위해 두 세포 공동 배양에 추가 1. </li></ol></li><li> 50 PBMC를 / 웰 음성 대조군, 플레이트 (; 세포 밀도 또는 특정 이펙터 백혈구 인구 : 25 만 백혈구 / ㎠)의 경우 만 1 ㎖의 백혈구 완전 배지와 24 웰 플레이트에; 양성 대조군을 위해, 추가로, PBMC를 웰 / 동일한 수의 도금을 10 μg / ML의 최종 농도로 PHA를 추가한다. </li><li> 3와 공동 배양 실험 5 일에, 형광에 적합한 488 nm의 여기 및 방출 필터와 유세포 분석 (17)에 의해 CFSE 표지 백혈구의 증식 (둥근 바닥 튜브에 배치)을 평가한다. 주 : 유세포 분석을 위해 세포 내 사이토 카인 염색은 또한 MO 같은 백혈구 사이토 카인 발현 양상의 변화를 평가하기 위해이 시점에서 이러한 CFSE로 표지 백혈구를 위해 수행 될 수있다중간 엽 줄기 세포에 의해 dulated. CFSE이 형광 용 필터에 적절한 평가되기 때문에, 항체는 각종 사이토 카인이 형광 또는 유사한 스펙트럼 이외의 형광 색소에 접합 될 필요가 평가하도록 선택 (즉, 피코 에리 트린, 엽록소 단백질 복합체 (를 PerCP) peridinin). </li></ol> 6. 변화 : 이펙터 억제 분석 마그네틱 활성화 CFSE 표지 이펙터 CD4 + T 세포에 면역 백혈구를 MSC 유도, 비드 선택 <ol><li> 6 웰 플레이트에 MSC 전체 매체의 3 ㎖에 25 만 세포 (MSC 밀도 : 25,000 세포 / ㎠)에서 종자 중간 엽 줄기 세포 37 ° C 배양기에 부착 O / N를 들어, 줄기 세포 수는 80 %의 합류에 도달합니다. 적어도 3 개의 6- 웰 플레이트 모집단 선택 충분한 MSC-PBMC를 공 배양을 보장하기 위해 필요하다. </li><li> 대기음 매체는, 및 1 단계에 따라하지만 / 웰 (세포 밀도 2.5 × 10 6 세포에서 6 웰 플레이트에서 분리 PBMC를 추가 : 250,000 르를백혈구 전체 매체의 3 ㎖와 ukocytes / ㎠). 37 ° C 배양기에서 48 ~ 72 시간 동안 공동 문화. </li><li> 자기 섹션 2-3에 따라 MSC에 의한 면역 백혈구 (즉, CD14 + 세포)의 특정 인구 비드-선택합니다. 주 : 유세포 분석을 위해 세포 내 사이토 카인 염색은 백혈구 사이토 카인의 발현 프로필, 즉, 발현의 변화를 평가하기 위해이 시점에서 수행 될 수있다 인터루킨 10와 같은 MSC들에 의해 변조. </li><li> 24 웰 플레이트 섹션 2-4에 따라 생성 된 동종 CD4 + T 세포를 CFSE 표지 된 추가 한 백혈구 완전 배지 (T 세포 밀도 250,000 세포 / ㎠) ml의 다양한 비율로 비드 선택된 MSC 유도 백혈구에 즉, 1:10, 1 : 5, 1 : 2, 1 : 1 비율 (셀로 셀). </li><li> CD4 + T 세포를 자극하기 위해, (1)의 비드 대 세포 비율을 구하는 α-CD3은 / 28 공액 마이크로 비드를 추가 1. </li><li> 음성 대조군은 5 판00,000 CD4 + T 세포 / 웰로 24- 웰 플레이트 (T 세포 밀도 250,000 세포 / ㎠)에 단 1 ㎖의 백혈구 완전 배지와; 양성 대조군을 위해, 추가로 (1)의 비드 대 세포 비율을 구하는 α-CD3은 / 공액 마이크로 비드 (28)를 추가 / 웰의 CD4 + T 세포의 수를 동일한 도금 : 1이다. </li><li> 공동 배양 3 일째에, 플루오 레세 적합한 488 nm의 여기 및 방사 필터를 사용하여 유세포 분석 (16)에 의해 (둥근 바닥 튜브에 배치) CFSE 표지 된 CD4 + T 세포의 증식을 평가. </li></ol>

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C., Lawrence, D. A., Reed, D. J., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNIT+18.8+Immune+cell+phenotyping+using+flow+cytometry.">UNIT 18.8 Immune cell phenotyping using flow cytometry.</a> Curr Protoc Toxicol. , 18.8.1-18.8.24 (2005).</li><li>Phelan, M. C., Lawler, G., Robinson, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APPENDIX+3A+Cell+counting.">APPENDIX 3A Cell counting.</a> Curr Protoc Cytom. , A.3A.1-A.3A.4 (2001).</li><li>Gebler, A., Zabel, O., Seliger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunomodulatory+capacity+of+mesenchymal+stem+cells.">The immunomodulatory capacity of mesenchymal stem cells.</a> Trends Mol Med. 18, 128-134 (2012).</li><li>Le Blanc, 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=Mesenchymal+stem+cells+for+treatment+of+steroid-resistant,+severe,+acute+graft-versus-host+disease:+a+phase+II+study.">Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.</a> Lancet. 371, 1579-1586 (2008).</li><li>Tan, 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+therapy+with+autologous+mesenchymal+stem+cells+in+living-related+kidney+transplants:+a+randomized+controlled+trial.">Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial.</a> JAMA. 307, 1169-1177 (2012).</li><li>Le Blanc, 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=Mesenchymal+stem+cells+inhibit+and+stimulate+mixed+lymphocyte+cultures+and+mitogenic+responses+independently+of+the+major+histocompatibility+complex.">Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.</a> Scand J Immunol. 57, 11-20 (2003).</li><li>Li, X. 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=Long-term+culture+in+vitro+impairs+the+immunosuppressive+activity+of+mesenchymal+stem+cells+on+T+cells.">Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells.</a> Mol Med Rep. 6, 1183-1189 (2012).</li><li>Moll, 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=Do+cryopreserved+mesenchymal+stromal+cells+display+impaired+immunomodulatory+and+therapeutic+properties?.">Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?.</a> Stem Cells. 32, 2430-2442 (2012).</li><li>Ho, P. J., Yen, M. L., Tang, B. C., Chen, C. T., Yen, B. 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We have nothing to disclose.

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

인간 중간 엽 줄기 세포 (중간 엽 줄기 세포는) 몇 extramesodermal 계통 5뿐만 아니라, 뼈, 연골 및 지방 조직 1-4의 근축 중배엽 계통으로 분화 할 수 체세포 조상이다. 먼저 성인의 골수에서 분리,이 multilineage 조상은 지금 임상 응용 프로그램 9-12에 매우 의무 표시 강한 면역 특성을 보여, 예기치 않게, 많은 조직 6-8에서 발견되었다. 면역 효과에 관련된 자세한 메커니즘은 적극적으로 특정 질병의 실체에 효과적인 적용을 위해 연구되고있다. 면역을 평가하는 가장 간단한 방법 중 하나는 이펙터 백혈구 증식 (13)의 억제에 대해 평가하는 것이다. 자극 또는 활성화되면 이러한 T 림프구 및 단핵구와 같은 대부분의 이펙터 백혈구는 비범 증식. 면역 기능이 평가 될 수있는 경우의 진압확산은 입증된다. 전통적으로, 이펙터 백혈구 증식 DNA에 [3 H] 티미 딘의 결합 검출에 의해 평가되었다. 그러나,이 방법은 복사 현상 및 후 처리의 이용에 상당한 관심 단점뿐만 아니라, 필요한 복잡한 장비를 갖는다. 세포 증식을 평가하기위한 비 방사능 분석이 있지만, 카르복시 숙신 이미 딜 에스터 (CFSE) 분석은 여러 유형의 세포를 포함하는 공동 배양 실험에 특히 유용하다 특정 세포 개체수의 식별을 허용하는 다른 이점을 갖는다. CFSE은 유세포 분석에 의해 평가 될 수 셀룰러 형광 염료이다. 세포 분열,이 휴대 라벨의 강도는 비례 적으로 감소된다; 이뿐만 아니라 전반적인 세​​포 증식의 판정을 가능하게 할뿐만 아니라 형광 DET 곤란해진다 전에 8 분할 최대 세포 분열 횟수에 평가를 허용요법 배경 신호에 대하여. 또한, 형광 CFSE의 안정성은 많은 세포가 14 개월까지 가시화 될 수있는 표지 된 세포를 생체 내 추적을 허용한다. 이 분석은 또한 이펙터 백혈구 또는 특정 집단의 면역 기능의 특정 유형을 평가하기 위해 변화 될 수의 자성 비드 표면 마커의 선택을 행함으로써 면역 백혈구 - 인터루킨 10 (IL-10)의 제조 CD14 + 단핵구 15를 MSC를 유발 이전에 또는 적절한 공동 배양 한 후 관심의 세포 집단. 우리 프로토콜 (흐름도가 도시는 (플로우 차트는도 1에 도시) 이펙터 백혈구에서 중간 엽 줄기 세포의 면역 조절 효과를 평가의 기본적인 분석 및 동종 CD4 + 이펙터 T 림프구에 MSC 유도 백혈구 면역 평가를위한 기본적인 분석의 변형을 설명 그림 4).

<table><tbody><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>71-7167-00 AG</td><td>Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td></tr><tr><td>Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td><td>Life Technologies</td><td>V12883</td><td>Cellular label for detection of cell division</td></tr><tr><td>Phytoagglutinin (PHA)</td><td>Sigma</td><td>L8902</td><td>Activation of human PBMCs</td></tr><tr><td>Dynabeads Human T-Activator CD3/28</td><td>Life Technologies</td><td>111.32D</td><td>Activation of human T lymphocytes, &lt;em&gt;e.g.&lt;/em&gt; CD4&lt;sup&gt;+&lt;/sup&gt; T cells, CD8&lt;sup&gt;+&lt;/sup&gt; T cells, &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>autoMACS&amp;trade; Separator</td><td>Miltenyi Biotec</td><td>autoMACS&amp;trade; Separator</td><td>Magnetic based cell separator</td></tr><tr><td>autoMACS&amp;reg; Columns</td><td>Miltenyi Biotec</td><td>130-021-101</td><td>separation columns</td></tr><tr><td>CD14 microbeads, human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-050-201</td><td>For positive selection of CD14&lt;sup&gt;+&lt;/sup&gt; human monocytes and macrophages from PBMCs</td></tr><tr><td>CD4 microbeads, human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-045-101</td><td>For positive selection of CD4&lt;sup&gt;+&lt;/sup&gt; human T lymphocytes from PBMCs</td></tr><tr><td>RPMI 1640 Medium</td><td>Life Technologies</td><td>11875</td><td>Human PBMC/leukocyte culture medium</td></tr><tr><td>DMEM, Low glucose, pyruvate</td><td>Life Technologies</td><td>11885</td><td>Human mesenchymal stem cell (MSC) culture medium</td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>25030-081</td><td>Supplementation for MSC complete medium</td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td>Supplementation for PBMC/leukocyte and MSC complete medium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>1) Hyclone, for MSC culture&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td><td>1) SH30070.03M 2) 10091-148</td><td>Pre-test lots for support of MSC &lt;em&gt;in vitro&lt;/em&gt; culture</td></tr><tr><td>24-well cell culture plate</td><td>Corning</td><td>COR3524</td><td>Co-culture plate</td></tr><tr><td>50 ml centrifuge tube</td><td>Corning</td><td>430291</td><td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td></tr><tr><td>15 ml centrifuge tube&amp;nbsp;</td><td>Corning</td><td>430766</td><td>Collection of the labeled and unlabeled cell fractions</td></tr><tr><td>Round-bottom tubes</td><td>BD Falcon&amp;nbsp;</td><td>352008</td><td>Collection of cells for flow cytometric analysis</td></tr></tbody></table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

도 1은 실험의 전반적인 스키마를 나타내고,도 2는 위상차 도립 현미경으로 가시화로서 다양한 세포 배양 조건의 모양을 보여준다. PBMC를하고 백혈구가 작은 원형 비 부착 세포 반면 중간 엽 줄기 세포는 섬유 모세포, 스핀들 모양의 형태와 부착 세포이다. 이 두 형태 학적으로 상이한 세포 유형은 명확 공존 배양에서 볼 수있다. 시험의 끝에, PBMC를 (또는 leuckotyes)를 유세포위한 흡인 될 ?...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

인간 중간 엽 줄기 세포 (MSC)의 면역 특성은 임상 응용 프로그램에 대해 점점 더 관련이 나타납니다. 형광 염료 카르복시 숙신 이미 딜 에스터 (CFSE)로 미리 염색 엽 줄기 세포 및 말초 혈 백혈구의 공존 배양 시스템을 사용하여, 우리는 이펙터 백혈구 증식 및 특정 부분 집단에 MSC의 면역의 시험 관내 평가를 설명한다.

인간 중간 엽 줄기 세포의 면역 속성 (MSCS)의 평가

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

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Immunology and Infection

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

12.1K 조회수.  National Health Research Institutes (NHRI).  인간 중간 엽 줄기 세포 (MSC)의 면역 특성은 임상 응용 프로그램에 대해 점점 더 관련이 나타납니다. 형광 염료 카르복시 숙신 이미 딜 에스터 (CFSE)로 미리 염색 엽 줄기 세포 및 말초 혈 백혈구의 공존 배양 시스템을 사용하여, 우리는 이펙터 백혈구 증식 및 특정 부분 집단에 MSC의 면역의 시험 관내 평가를 설명한다. 

비디오: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

제노없는 조건에서 3-D 문화의 생산과 치료 중간 엽 줄기의 관리 / 기질 세포 (MSC) 타원체 뇌관

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

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발생학

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 ...

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 ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

분리 및 랫 Mesenchymal 줄기 세포의 농축 (MSCS)와 MSCS를 유래 단일 식민지의 분리

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

생물학

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

초음파 유도 Intracardiac 주입 실험 선 동적인 장 질병의 Murine 모델에 사용 하기 위해 내장을 추적 증가 인간 중간 엽 줄기 세포의

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

의학

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

대식세포식세포의 중간엽 줄기세포 조절; 수량 및 이미징

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

면역학 및 감염병학

<meta charset="utf8"></head><body>BAMBI높은MFGE8높은 UC-MSC 하위 모집단의 기능 분석

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

<meta charset="utf8"></head><body>Human BAMBIhighMFGE8high Umbilical Cord Derived Mesenchymal Stromal Cells의 분리

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

Human BAMBIhighMFGE8high Umbilical Cord Derived Mesenchymal Stromal Cells의 분리

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

hiPSC-Driving MSC의 표면 항원 및 증식 능력 특성화

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

저자 스포트라이트: iPSC 및 유전 질환 연구의 발전 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

인간 유도만능줄기세포(Human Induced Pluripotent Stem Cells)를 중간엽 기질세포(Mesenchymal Stromal Cell)로 분화하기 위한 두 가지 대표적인 방법의 비교

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

인간 중간 엽 줄기 세포의 면역 속성 (MSCS)의 평가

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제노없는 조건에서 3-D 문화의 생산과 치료 중간 엽 줄기의 관리 / 기질 세포 (MSC) 타원체 뇌관

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

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

분리 및 랫 Mesenchymal 줄기 세포의 농축 (MSCS)와 MSCS를 유래 단일 식민지의 분리

초음파 유도 Intracardiac 주입 실험 선 동적인 장 질병의 Murine 모델에 사용 하기 위해 내장을 추적 증가 인간 중간 엽 줄기 세포의

대식세포식세포의 중간엽 줄기세포 조절; 수량 및 이미징

Human BAMBI^highMFGE8^high Umbilical Cord Derived Mesenchymal Stromal Cells의 분리

Human BAMBI^highMFGE8^high Umbilical Cord Derived Mesenchymal Stromal Cells의 분리

Human BAMBI^highMFGE8^high Umbilical Cord Derived Mesenchymal Stromal Cells의 분리

인간 유도만능줄기세포(Human Induced Pluripotent Stem Cells)를 중간엽 기질세포(Mesenchymal Stromal Cell)로 분화하기 위한 두 가지 대표적인 방법의 비교