Name: isolating mesangiogenic progenitor cells (mpcs) from human bone marrow
Uploaded: 2016-07-15T15:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

저자는 특히 골수 샘플과 인간의 골관절염 조상에서 자신의 전문 지식을 제공하기 위해, 박사 파올로 Parchi, 외과의 부서, 의료 및 분자 병리학 및 중환자 의학, 피사의 대학을 감사드립니다

참고 : 서면 동의 후, HBM의 샘플 고관절 교체 정형 외과 수술 중 얻었다. 즉시 헤파린의 500 UI를 포함하는 20 ML의 주사기를 리밍 대퇴 경부 절골술 후 대퇴 전에, 새로운 BM을 흡인하는 데 사용되었다. 프로토콜은 BM 소스 널리 적용을 고려하여야한다. 사람 골수 단핵 세포의 1. 분리 (HBM-다국적 기업) <ol><li> 둘 베코 인산염 완충 식염수 (D-PBS)을인가 해, 50 ml의 신선한 BM 10 ㎖와 혼합하여 반전 - 5 희석. 마찬가지로, 두 개의 새로운 50 ML 원뿔 튜브에 25 ml의 배포 각각의 튜브에 D-PBS 25 ml에 추가하고 반전으로 섞는다. </li><li> 튜브를 용액으로부터 광물 뼈 조​​각의 분리 및 지방 들어, 실온에서 10 분간 방치한다. </li><li> 조심스럽게 미네랄 뼈 조각 펠렛을 방해하지 않고 70 μm의 필터를 통해 멸균 파스퇴르 피펫 및 필터 세포 현탁액에 떠있는 지방을 제거합니다. </li> &lt;리&gt; 불연속 밀도 구배 원심 분리 배지 15 ㎖ (1.077 g / ml)로 4 개의 50 ㎖ 튜브를 설정한다. 이 매체는 실온에 있는지 확인합니다. </li><li> 밀도 구배 배지 위에서 희석 BM 25 ㎖의 - 부드럽게 20 누워. 혼합 층을 방지하기 위해 튜브 벽에 세포 현탁액의 물방울셔서,주의 깊게이 작업을 수행합니다. </li><li> 비활성화 브레이크 실온에서 30 분 동안 400 XG에서 밀도 구배 원심 분리를 수행한다. </li><li> 멸균 파스퇴르 피펫을 사용하여 두 단계 사이에 위치하는 셀의 백탁 링 모아서 신선한 50 ㎖ 튜브에 옮긴다. </li><li> 신선한 배지로 세포를 세척 : 페놀 레드가없는 저 글루코스 (1,000 ㎎ / ℓ) 둘 베코 변형 이글 중간 (DMEM), 10 % (v / v)로 풀링 된 인간 AB 형 혈청 (PhABS), 2 mM L- 글루타민 및 항생제 (DMEM / 10 % PhABS). 5 분 동안 400 XG에 원심 분리기. </li><li> 신선한 DMEM 10 ㎖ / 10 % PhABS - 기음 뜨는 및 5 펠렛을 다시 일시 중지합니다. </li><li> 세포 수를 진행합니다.트리 판 1 희석 (1)에 의한 백혈구의 수를 결정한다. 혈구이 적용하고, 위상차 현미경 하에서 관찰한다. 셀 수에서 작은 완벽하게 둥근 적혈구와 블루 스테인드 죽은 세포를 제외합니다. 주 : 매우 MPC 복구 성능을 위해 화면 PhABS 배치하는 것이 좋습니다. 다른 출처의 대부분의 혈청은 MSC와 같은 세포의 높은 비율과 MPC 문화 초래하면서 미국의 소스에서 PhABS 최상의 결과를 제공하고있다. </li></ol> HBM-다국적 기업에서 MPC와의 2. 분리 <ol><li> 신선한 DMEM / 10 % PhABS 15 ml의 소수성 T-75 플라스크를 설정하고 pH 및 온도는 30 분 동안, 5 % CO 2에서 37 ℃에서 미리 인큐베이션 평형하자. </li><li> 시드 4 - 플라스크 당 6 × 10 7 HBM-다국적 기업과 48 시간 동안 5 % CO 2에서 37 ° C에서 품어. </li><li> 기음과 플라스크에서 매체 및 비 부착 세포를 폐기합니다. 신선한 DMEM / 10 % PhABS의 15 ML을 추가하고 37 ° C에서 부화5 %의 CO 2. 매체마다 48 시간을 변경, 팔일 - 6 문화를 유지한다. OPTIONAL : MPC 수율을 높이기 위해 2.3에서 비 부착 성 세포는 새로운 배양 플라스크에 다시 도금 일차 배양 기술 한 바와 같이 유지 될 수있다. </li><li> 대기음하고, 플라스크에서 배지를 버리고 신선한 DMEM으로 씻어 솔루션을 분리 동물 무료 프로테아제 2 ㎖를 추가합니다. 15 분 (장기간 배양을 방지) - (5) 37 ℃에서 인큐베이션. </li><li> 5 분 동안 400 XG에 세포 현탁액을 원심 분리하고, 신선한 DMEM / 10 % PhABS 10ml의 추가 대기음 </li><li> 기음과 뜨는 버리고 1 펠렛을 다시 중단 - 신선한 DMEM 2 ㎖ / 10 % PhABS. 단계 1.10에 설명 된대로 세포 수를 진행합니다. 참고 : 시약을 분리으로 트립신 / EDTA를 사용하지 마십시오. MPC와 내성 트립신된다. 배양의 형태 학적 검사는, 세포를 수확하기 전에 높은 방추형 MSC 유사 세포의 존재를 평가하기 위해 추천된다. MSC와 같은 세포의 경우에는 상당한 양의 D에있다etected는 선택적으로 오염 된 세포를 제거함으로써 세포 생성물의 순도를 증가시킨다. 이를 위해 트립신 소화 2 분 동안 트립신 2 ㎖ / EDTA 0.05 %를 첨가하여, MPC 수확하기 전에 수행 될 수있다. 워시 문화를 두 번 DMEM / 10 % PhABS 5 ㎖로, 다음과 같은 이상 치료를 프로테아제로 진행합니다. </li></ol> 3. 세포의 특성 분석 <ol><li> 유동 세포 계측법 <ol><li> 세척 용액 105 새롭게 분리 된 세포 시료의 중복 설정 : D-PBS 0.5 % (v / v)의 소 혈청 알부민 (BSA)과 0.02 % (w / v)의 아 지드 화 나트륨으로 보충. 5 분 동안 400 XG에 원심 분리기. 주의 : 나트륨 아 지드 독성이다. </li><li> 세척 용액을 200 μL의 펠릿을 다시 중지 및 항 CD90, 항 CD45, 항 CD73과 형광 염료 ( &quot;TEST&quot;)에 접합 된 항 CD31 항체를 추가; 병렬 아이소 타입 컨트롤 ( &quot;CTRL&quot;)을 설정합니다. 참고 : 항체 염색의 금액이 적정 또는 코르에 의해 결정되어야한다제조업체의 지침에 땡. </li><li> 30 분 동안 4 ° C에서 샘플을 품어. </li><li> 5 분 동안 400 XG에 원심 분리기. 세정액 500 ㎕의 세포를 다시 일시 7 9 10 사이토 다색 흐름을 적어도 5 × 104의 이벤트를 취득. </li><li> 도트 플롯으로 결과를 분석하고 사분면을 설정하는 &quot;CTRL&quot;기록 된 이벤트를 사용합니다. 참고 : MPC 문화와 문화를 정의하기 위해 CD73 음수 CD90의 음수의 CD45 + CD31 + 세포의 비율이 95 % 이상이어야한다. 98 % - 일부 특정 애플리케이션의 경우, 즉, 유전자 발현 분석, 복구 차단 97로 증가되어야한다. </li></ol></li><li> 네 스틴 탐지 및 F - 굴지 조직 분석 <ol><li> 문화 챔버 슬라이드 플레이트 갓 격리 된 MPC (20,000 / cm 2). 세포가 5 % CO 2, 37 ℃에서 하룻밤 배양하여 부착 할 수 있습니다. </li><li> 세척 용액 Fi를에 세포를 씻으4 %의 X 15 분 동안 실온에서 (w / v)의 파라 포름 알데히드. 정착이, 세정액을 추가 2 분 동안 품어과 부어 제거합니다. </li><li> 두 번 세척을 반복합니다. </li><li> D-PBS에서 Permeabilize 하시려면 세포는 실온에서 15 분 동안 0.05 % (v / v) 트리톤 X-100을 보충. </li><li> 단백질이없는 신호 인핸서 (RT에서 30 분) 또는 표준 블로킹 용액, 반응 정지 (D-PBS 3 %로 보충 (W / v)의 BSA). </li><li> 솔루션을 차단 / 신호 증강을 제거합니다. </li><li> 7 μg의 / ㎖를 추가 항 - 인간 스틴 차 항체 (V / w)와 가습 실에서 밤새 4 ° C에서 품어. 평행이 아닌 특정 형광 신호를 평가하는 제어 이소 타입 항체를 사용한다. </li><li> D-PBS를 추가하여 슬라이드를 씻으, 2 분 동안 떠나 붓는다. 두 번 반복합니다. </li><li> 2 μg의 / ㎖를 추가 형광 염료 복합 이차 항체 (V / w)와 어둠 속에서 1 시간 동안 4 ° C에서 품어. </li><li> 워시는 위의 슬라이드. </li><li> 형광 Phalloidin의 (5 UI / ㎖)를 추가, 30m RT에서 떠나그리고 어둠 속에서하는 D-PBS로 3 회 세척한다. </li><li> 챔버 벽을 제거하고 핵을 검출 antifade 시약 4 &#39;, 6-diamidino -2- 페닐 인돌 (DAPI)로 보충 수성 장착 매체에 슬라이드 마운트. 7 9 10 이미징로 이동합니다. </li></ol></li></ol> MPC와의 4 Mesengenic 차별화 <ol><li> 플레이트 (2) × 104 / ㎠ TC 처리 T75 배양 플라스크에서 갓 격리 된 MPC 세포는 5 % CO 2에서 37 ° C에 DMEM / 10 % PhABS 밤새 부착하자. </li><li> 중간 엽 기질 세포 확장 (MSC-RS 매체) 용으로 설계된 표준 감소 혈청 배지, 약 200 μL / cm 2로 DMEM / 10 % PhABS를 교체합니다. 유도에서 7 ~​​ 10 일에서 일반적으로 (P1-중간 엽 줄기 세포)를 합류하는 세포를 성장. 2 일마다 배지 새로 고칩니다. </li><li> 대기음하고, 플라스크에서 배지를 버리고 신선한 MSC-RS 배지로 세척 및 솔루션을 분리 동물 무료 프로테아제 2 ㎖를 추가합니다. 37 품어76; 5 C - 15 분 (연장 보육을 방지). </li><li> 5 분 동안 400 XG에 세포 현탁액을 원심 분리하고, 신선한 MSC-RS 배지 10 ㎖를 추가 대기음 </li><li> 신선한 MSC-RS 배지 2 ㎖ - 대기음 뜨는 및 1 펠렛을 다시 일시 중지합니다. 단계 1.10에 기재된 바와 같이 세포 수로 진행 </li><li> 5 × 103 세포 / cm 2-3를 파종하여 그 하위 문화로 넘어갑니다. (P2-중간 엽 줄기 세포)를 합류하는 세포를 성장. </li><li> 4.5 단계 및 3 장에 설명 된대로 특성을 진행 단계 4.3에서 설명 된 바와 같이 단백질 분해 효소 소화에 의해 수확 세포. 주 : CD73 + CD90 + CD45 CD31 NEG의 NEG 세포의 비율 95 % 이상 만 편미분을 나타내며, 또 배양 통로를 요구한다. </li><li> 플레이트 P2-중간 엽 줄기 세포 2 × 여섯 잘 TC-처리 판 10 4 / cm 2 및 성장에는 MSC-RS 매체에 합류합니다. </li><li> 마크 &quot;아니오의 Diff&quot;로 두 우물과는 MSC-RS 매체를 새로 고칩니다. </li><li> 마크이 아니라&quot;Osteo&quot;특히 MSC 분화 용으로 설계된 200 μL / 표준 골 형성 매체의 cm 2와 매체를 교체 등의. </li><li> 마크 두 개의 &quot;Adipo&quot;로 우물 특히 MSC 분화 용으로 설계된 200 μL / 표준 지방 세포 배지 cm 2와 매체를 교체하십시오. </li><li> 전체 미디어 각 48 시간을 변경하여 5 % CO 2 37 ° C에서 문화를 유지한다. 참고 : 높은 시험 재현성 표준 상업적으로 이용 가능한 차별화 된 미디어를 사용하는 것이 좋습니다. 세포 내 지질 방울의 축적이 지방 세포에 의한 세포에서 분명하다 동안 차별화 된 조건에서 2/3 주 후, 칼슘 예금, 골 형성 유도 문화에 나타납니다. </li><li> 기음 문화 미디어 다음 D-PBS로 씻어 버리고. </li><li> 실온에서 15 분 동안 파라 포름 알데히드 (/ V W) 4 % 1 ㎖를 첨가하여 배양을 수정. </li><li> 정착이, D-PBS를 추가 2 분 동안 품어과 부어 제거합니다. 반복 세탁두번. </li><li> 두 &quot;Adipo는&quot;200 nM의 나일 레드 솔루션에 우물을 표시와 함께 두 개의 &quot;Osteo는&quot;함께 하이드 록시 아파타이트 특정 형광 용액 일 &quot;아니오의 Diff&quot;에 우물을 표시와 함께 한 &quot;아니오의 Diff&quot;를 얼룩. 실온 (11, 12)에서 30 분 동안 인큐베이션. </li><li> 솔루션을 염색 제거를 두 번 D-PBS로 씻는다. </li><li> 추가, D-PBS를 제거 D-PBS는 50 % (v / v)의 글리세린 보충 및 이미징 (7) (9) (10)를 진행합니다. </li></ol> 5. MPC 회전 타원체 돋 분석 <ol><li> 3D 회전 타원체를 생산하기 위해 페트리 접시 뚜껑의 내부 표면에 갓 격리 MPC 현탁액 (1.5 × 104 세포 / 드롭)의 20 μl의 방울을 배치합니다. 참고 : 취급 상품들이 파열로 이어질 수있는 바와 같이, 그것은 초과를 배치하는 것이 매우 추천입니다. </li><li> 조심스럽게 D-PBS가 걸려 증발을 삭제 방지하기 위해 포함 된 배양 접시를 정리해하기 위해 뚜껑을 사용합니다. CO 2 5 %에서 37 ° C에서 품어하룻밤 세포를 3 차원 회전 타원체에 통합 할 수 있습니다. </li><li> 미리 냉각 24- 웰 배양 플레이트에 표준 ECM 단백질을 300 ㎕의 분취 량을 첨가하여 쥐의 세포 외 기질 (ECM) 단백질의 두꺼운 겔을 설정하고, 30 분 동안 37 ℃에서 배양한다. </li><li> 조심스럽게 페트리 접시의 뚜껑을 뒤집어 부드럽게 멸균 파스퇴르 피펫을 사용하여 회전 타원체를 선택합니다. </li><li> 상기 ECM 단백질 겔상에서 구 상체를 마련 표준 VEGF 풍부한 내피 세포 성장 배지 700 ㎕의 분취 액을 추가 5 % CO 2에서 37 ℃에서 배양한다. </li><li> 24 시간 후와 문화 7 일에서 4 배 확대 배율에서 3D 문화의 사진을 찍을. 마지막 침입 셀 및 구형 에지 사이의 반경 방향 거리를 측정하여 화상 해석 소프트웨어를 적용 타원체 돋아 평가한다. 적어도 20 서로 다른 방향을 따라 반복 측정합니다. 평균 거리는 경우 50 μm의 이상 긍정적으로 간주된다. </li></ol>

<ol>
	<li>Stoltz, J. 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The authors have no competing financial interests or other conflicts of interest.

In the last decades, MSCs have been extensively researched and pre-clinically evaluated for possible application in the treatment of various bone/articular, immunological, neurological, cardiovascular, gastrointestinal and hematological disorders14,15. The easy and inexpensive isolation of multipotent MSCs, from many different tissues, together with their lack of significant immunogenicity16, contribute to make these cells one of the most interesting cell population to be applied in cell based therapies. Nonetheless, the very low frequency in the tissue of origin represents a great limitation to the MSCs application in clinics, forcing the expansion of these cells, in vitro, before the infusion or transplantation. 
Expanded MSC cultures have revealed high grades of heterogeneity and variability17-19 making it difficult to reach a consensus about MSC production and characterization protocols. Moreover, recent investigations suggested the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to culture heterogeneity10,20. In fact, it has been proposed that particular culture conditions possibly select or simply promote specific sub-populations of MSCs progenitors present, in various percentages, in "crude" and unprocessed samples like bone marrow (hBM-MNCs) or adipose tissues (stromal vascular fraction)2. Thus, the variability in MSC-initiating cell populations together with the great number of different enrichment/isolation and culture protocols applied, represent a great obstacle to the definition of feasible MSC-based therapies.
A crucial factor affecting heterogeneity of MSC cultures is serum supplementation21. In our hands replacement of FBS with PhABS in primary cultures from hBM-MNCs, combined with high density seeding on hydrophobic plastics, led to the isolation of a novel highly adherent cell population with distinct biological features named MPCs5,6. We observed that the addition of small percentages of PhABS to FBS primary cultures also allowed MPC isolation, suggesting the presence of MPC inducing agents in the human serum6. At the moment, the MPC isolation/characterization protocol is a unique method available to obtain almost pure MPCs. The protocol has been carefully adjusted and it is highly reproducible for quality screening of MPC preparations before further applications.
MPCs could be used as a source for MSC production, thus limiting the variability introduced by use of unfractionated starting material. The precise definition of the multiple steps characterizing MPC mesengenic differentiation reported9 would allow synchronized mesenchymal cell expansion. Nonetheless, this latest condition could be realized exclusively applying highly purified MPC population, as a consequence the characterization of the cell products obtained by the protocol described here, results of crucial importance. This isolating method has been reported allowing MPC recovery with purity generally around 95%. However, donor/patient variability together with the variability related to the different batches of human pooled serum applied, could lead to a significant percentage of MSC-like cells co-isolated together with MPCs, under selective conditions. 
It is not clear if these "contaminating" MSC-like cells could arise from the other different in vivo progenitors described in bone marrow22 or from uncontrolled and spontaneous MPC differentiation. In any case, a consistent percentage of MSC-like cells in the MPC products nullify the possibility to applying these cells as homogeneous starting material for the MSC expansion. Thus, here it has been suggested a simple and inexpensive method, based on the MPC resistance to trypsin digestion, increasing the purity of the MPC products. Similar or even better results in purifying MPC cultures could be achieved by fluorescent or magnetic cell sorting performing CD73 and/or CD90 depletion, but significantly prolonging the process time and increasing the costs.
Moreover, MPCs showed expression of pluripotency-associated markers and Nestin, all rapidly lost during mesengenic differentiation7. Sprouting assay revealed MPC ability to invade murine ECM protein gel. Taken together these results indicate that MPCs have to be considered a more immature progenitor, retaining angiogenic potential. Nonetheless, the initial enthusiasm about mesodermal differentiation potential of MPCs is actually waning. In fact, after more than 7 years of studies on MPCs, mesengenic and angiogenic potential have been extensively described5-9, but differentiation toward any other cells of mesodermal origin is still lacking. Thus, here we propose a new, and more rigorous, definition of these cells as "Mesangiogenic Progenitor Cells", maintaining the acronym MPCs.
We also believe that most controversies about MSC angiogenic potential could be related to the heterogeneous composition of expanded cultures consisting of sub-populations of MPCs and MSCs in variable percentages23.
Finally, MPCs could also play a crucial role for the implementation of CBMPs applicable for tissue reconstruction, as these cells could also support the neo-vascularization. In fact, future studies on regeneration should take in consideration that the newly formed tissue growth should be supported by concomitant neo-angiogenesis. The co-existence of mesengenic and angiogenic potential in MPCs could significantly improve the regeneration potential of new therapeutic approaches that involve these interesting cells.

간엽 기질 세포 (MSC들)은 그들의 다중 계통 분화 능력과 성장 인자 / 사이토 카인을 분비 할뿐만 아니라 면역 조절 하나의 역할을하는 조혈을지지하는 그들의 능력에 대한 중요한 임상 적 가치가있다. MSC 기반 치료법 셀 생산 및 적용의 정의에서 의약품 (CBMP) 치료 3 계 전지의 안전성과 효능에 대해 특정 국제 규정에 특별한주의와 광범위한 임상 및 전임상 연구 (2)의 대상이되어왔다. 인간 중간 엽 줄기 세포는 태아 소 혈청 (FBS) 및 소 트립신으로 보충과 동물 기원의 시약을 포함하는 배지에서 광범위하게 배양. 따라서, 세포 조작과 관련된 감염 위험이 환자는 프리온에게 노출뿐만 아니라 단백질, 펩타이드 또는 동물 유래의 다른 생체 분자에 연결 면역 학적 위험에 직면 함께와 세포 수확 및 transplan 후 계속 수있는테이션 4. 이 문제를 회피하기 위해, 우리는 풀링 된 인간 AB 형 혈청 (PhABS)를 FBS를 대체​​ 인간 골수 (HBM) 동물 배지에서 유래 중간 엽 줄기 세포를 배양 하였다. 이러한 조건에서 중간 엽 줄기 세포 성장과 함께, 우리는 새로운 세포 집단을 확인 하였다. 이러한 세포 형태학 및 중간 엽 줄기 세포에서 표현형 달랐다 독특한 유전자 발현 프로파일과 특성 성장 / 접착 성을 나타내었다. 그들은 유지 모두 mesengenic 및 잠재적 인 혈관 신생 따라서 선정됐다 중배엽 전구 세포 (MPC와) 5. 다음으로, 우리는 순도 6 높은 등급의 MPC를 생성하도록 선택적 및 재현성 배양 조건을 정의 할 수 있었다. 우리는 더 MPC와의 형태 학적 및 생물학적 특성을 조사 하였다. 된 MPC는 자전거에 긍정적 - 네 스틴 느린 것으로 보여, 기-67 음성, 긴 텔로미어 5 특징 염색체와. 그들은 plur 표현ipotency 관련 전사는 요인 10 월 4, Nanog를하기보다는 MSC 마스터 조절기 Runx2와 Sox9 7. 중간 엽 마커 CD73, CD90, CD166이 부족한 상태에서 표현형, MPC와는 중간 엽 줄기 세포보다 낮은 수준에서 endoglin (CD105)를 표명했다. MPC와는 구체적으로 podosome 같은 구조 팔을 지탱 접착 PECAM (CD31)의 일관된 표현을 특징으로 분자 인테그린 αL (CD11a에), αM (CD11b를) αX (중 CD11c)과 인테그린 β2 (CD18)의 독특한 패턴을 보여 주었다 . 표준 MSC 확장 미디어에서 MPC와 신속 9 신호 Wnt5 / 칼 모듈 린 세포의 활성화를 갖춘 중간 단계를 통해 중간 엽 줄기 세포로 분화. 쥐의 세포 외 기질 (ECM) 단백질의 3 차원 문화 회전 타원체에서 새싹 할 수있는 능력에 의해 입증으로하여 MPC는 또한, 혈관 신생 속성을 유지했다. 혈관 신생 가능성은 빠르게 mesengenic 혈통을 따라 MPC 분화 후 끊어졌습니다. 여기에서 우리는 프로 제시tocols은 분리 및 HBM 혈액 샘플로부터 고도로 정제 된 MPC의 특성을 최적화. MPC의 mesengenic 및 혈관 분화 재현 프로토콜도 설명되어 있습니다.

<table><tbody><tr><td>Matrigel Basement Membrane Matrix</td><td>BD Bioscience (San Jose, CA-USA)</td><td>354230</td><td>Murine ECM proteins&lt;br/&gt; Stock Concentration: 100% (9 - 12 mg/ml)&lt;br/&gt; Final Concentration: 100%</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (D-PBS)</td><td>Sigma (St. Louis, MO, USA)</td><td>D8537</td><td></td></tr><tr><td>70 &amp;mu;m Filters</td><td>Miltenyi Biotec (BergischGladbach, Germany)</td><td>130-095-823</td><td></td></tr><tr><td>Ficoll-Paque PREMIUM&amp;nbsp;</td><td>GE Healthcare (Uppsala, Sweden)</td><td>17-5442-03</td><td>medium for discontinuos density gradient centrifugation</td></tr><tr><td>Pooled human AB type serum (PhABS)</td><td>LONZA (Walkersville MD-USA)</td><td>14-490E</td><td>Final Concentration: 10%</td></tr><tr><td>Glutamax-I</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>35050-038</td><td>Stabilized L-Glutamine&lt;br/&gt; Stock Concentration: 100x&lt;br/&gt; Final Concentration: 2 mM</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma (St. Louis, MO, USA)</td><td>A8412</td><td>Stock Concentration: 7.5%&lt;br/&gt; Final Concentration: 0.5%</td></tr><tr><td>Sodium Azide</td><td>Sigma (St. Louis, MO, USA)</td><td>S8032</td><td>Final Concentration: 0.02%</td></tr><tr><td>Penicillin/Streptomycin (Pen Strep)</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td>Antibiotics&lt;br/&gt; Stock Concentration: 5,000 UI/ml penicillin, 5,000 &amp;#956;g/ml Streptomycin&lt;br/&gt; Final Concentration: 50 UI/ml penicillin, 50 &amp;#956;g/ml Streptomycin</td></tr><tr><td>T-75 culture flask for suspension cultures</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658 190</td><td></td></tr><tr><td>T-75 culture flask TC treated</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658170</td><td></td></tr><tr><td>TrypLE Select</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12563-011</td><td>Animal-free proteases detaching solution&lt;br/&gt; Stock Concentration: 1x&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Trypsin/EDTA</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>15400-054</td><td>Phenol red free&lt;br/&gt; Stock Concentration: 0.5%&lt;br/&gt; Final Concentration: 0.25%</td></tr><tr><td>anti-CD90 APC antibody&amp;nbsp; (CD90)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-402</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD45 APC-Vio770 antibody (CD45)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-609</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD73 PE antibody (CD73)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-182</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD31 PE Vio-770 antibody (CD31)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-105-260</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 APC antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-846</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG2a APC Vio770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-637</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-845</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE Vio-770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-563</td><td>Final Concentration: 1:40</td></tr><tr><td>Low Glucose Dulbecco&#039;s Modified Eagle Medium&amp;nbsp; (DMEM)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>13-1331-82</td><td>Phenol red-free minimal essential medium&lt;br/&gt; Stock Concentration: 1,000 mg/L glucose</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp; (FBS)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>10500</td><td>Stock Concentration:0.2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Prolong Gold antifade reagent with 4&amp;rsquo;,6-diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>P-36931</td><td>Aqueous mounting medium + DAPI&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Paraformaldehyde</td><td>Sigma (St. Louis, MO, USA)</td><td>P6148</td><td>Fixative&lt;br/&gt; Final Concentration: 4%</td></tr><tr><td>LAB-TEK two-well chamber slides</td><td>Sigma (St. Louis, MO, USA)</td><td>C6682</td><td></td></tr><tr><td>Anti-Nestin antibody [clone 10C2]</td><td>Abcam (Cambridge, UK)</td><td>ab2035</td><td>Stock Concentration: 1 mg/ml&lt;br/&gt; Final Concentration: 7 &amp;mu;g/ml</td></tr><tr><td>Alexa Fluor 555 Phalloidin</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A34055</td><td>Stock Concentration: 200 UI/ml&lt;br/&gt; Final Concentration: 5 UI/ml</td></tr><tr><td>Triton X-100</td><td>Euroclone (Milan, Italy)</td><td>EMR237500</td><td>Final Concentration: 0.05%</td></tr><tr><td>MesenPRO RS Medium&amp;nbsp; (MSC-RS medium)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12746-012</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse SFX kit</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A31619</td><td>Goat anti-mouse secondary antibody + Signal enhancer&lt;br/&gt; Stock Concentration: 2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Pasteur Pipette</td><td>Kartell Labware&amp;nbsp; (Noviglio (MI), ITALY )</td><td>329</td><td></td></tr><tr><td>StemMACS AdipoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-679</td><td></td></tr><tr><td>StemMACS OsteoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-678</td><td></td></tr><tr><td>Osteoimage Bone mineralization Assay</td><td>LONZA (Walkersville MD-USA)</td><td>PA-1503</td><td>Hydroxyapatite specific fluorescent staining solution</td></tr><tr><td>50 ml Polystyrene conical tube</td><td>Greiner bio-one (Kremsm&amp;uuml;nster&lt;br/&gt; Austria)</td><td>227261</td><td></td></tr><tr><td>Nile Red</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>N1142</td><td>Fluorescent staining solution for lipids&lt;br/&gt; Stock Concentration: 100 mM&lt;br/&gt; Final Concentration: 200 Nm</td></tr><tr><td>Glycerin</td><td>Sigma (St. Louis, MO, USA)</td><td>G2289</td><td>Final Concentration: 50%</td></tr><tr><td>Polistirene Petri dishes</td><td>Sigma (St. Louis, MO, USA)</td><td>P5606</td><td></td></tr><tr><td>24-well plates TC-treated</td><td>Greiner Bio-one GmbH (Frickenhausen, Germany)</td><td>662160</td><td></td></tr><tr><td>Endothelial Growth Medium, EGM-2 BulletKit&amp;nbsp; (EGM-2)</td><td>LONZA (Walkersville MD-USA)</td><td>CC-3162</td><td>VEGF-rich endothelial cell growth medium</td></tr><tr><td>Leica Qwin Image Analisys Software</td><td>Leica (Wetzlar, Germany)</td><td></td><td>Image analysis software</td></tr></tbody></table>

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".

여기에서 설명한 선택적 배양 조건은 HBM-다국적 1.0 %의 신규 점착 거의 단형 세포군의 분리를 허용 할 (0.5-2.0 × 105 내지 6 HBM-다국적 - 신선한 BM 시료 10 mL) 중 5,6- . MPC와 5로, 대기, 기-67 음성 세포를 반올림, - 우리는이 큰 (60 μm의 직경 40)를 확인했다. 형태 학적으로, 그들은 (그림 1 A.1에 흰색 ​​화살표)을 높은 배율 전력에서 filopodia을 많이 보여주는 평면 얇은 주변에 둘러싸여 두꺼운 핵심 영역과 특유의 기름에 튀긴 된 계란 모양을 특징으로한다. 외부 셀 경계 폴라 신도들은 (도 1에 검은 색 화살표 A.1)를 관찰한다. 이러한 형태는 표준 MSC 문화에보고 된 전형적인 방추형 중간 엽 기질 세포 모양에서 분명히 다르다. 유동 세포 계측법 m 동안 CD31과 CD45를 표현하는 갓 격리 된 MPC의 95 % 이상을 보여 주었다esenchymal 관련 마커 CD90 및 CD73 (13)는 탐지 (그림 1 A.2)이었다. 우리는 MPC와 네 항원이 제한 집합으로 나타내는 생각. MPC와의 또 다른 구별 특징은 세포에서 감지되지 않습니다 (그림 1 A.3 빨간색)을 podosome 같은 구조의 수를 공개 점 F-굴지의 유통 및 네 스틴의 강렬한 식 (그림 1 A.3 녹색)입니다 이소 타입 대조군 항체로 염색 (데이터는 보이지 않음). 표준 RS에서 배양 된 MPC는 매체 기하 급수적으로 증가 MSC와 같은 세포 (그림 1 B.1)에 급속한 분화 MSC 확장 결과를 디자인했다. 이 구절 후 세포는 마지막으로 CD73 + CD90 + CD45 음수 CD31의 NEG (그림 1 B.2)에 CD73 음수 CD90의 음수 CD45 + CD31 +에서 자신의 표현형을 전환합니다. 이 과정에서 MPC와 재 ORGA네 스틴 표현은 몇 가지 희귀 세포 (그림 1 B.3)에 국한된다 동안 nize 스트레스 섬유로 F는 액틴. 명확한 MSC와 같은 표현형에 MPC mesengenic 분화는 다른 세포의 형태학에 의해 밝혀 두 가지 단계를 통해 발생합니다. MSC RS 매체 일주 MPC 유사 세포의 잔류 인구 후에도 (도 2의 P1-MSC들) 평평한 다각형의 다 분지 셀 합류 층 내에서 검출된다. 추가의 통로 (도 2 (A)에 P2-MSC들) 방추형 MSC 유사 세포의 거의 단형 배양을 얻을 필요가있다. 따라서 자신의 MSC 자연을 확인, 최소 2 주 동안 선택적 미디어에 전송 할 때이 기하 급수적으로 증가 세포는 쉽게 조골 세포 나 지방 세포로 분화 할 수 있습니다. 골 형성 유도 배양 물에 칼슘 침착은 어느 비색 알리자린-S 염색 또는 특정 형광 염료 (도 2의 B 녹색)에 의해 검출 될 수있다. 지방 세포 유도 세포 후(그림 2 B 빨간색) 중 하나 비색 오일 레드 또는 형광 나일 레드 얼룩에 의해 계시 된 지질 축적을 보여줍니다. MPC 입력은 혈관 신생 분석을 돋에 의해 확인되었다. MPC와는 24 시간 VEGF-자극 (그림 2 C) 후 3 차원 회전 타원체에서 쥐 ECM 단백질 젤 침공 (μm의 50 이상) 할 수있는 능력을 보여 주었다. 600 μm의 거리 - 일주 침입 세포 (300)에서 검출 한 후. 반대로, 침략 용량은 mesengenic 분화 (그림 2 D) 후 P2-중간 엽 줄기 세포에서 분실되었다. <img alt="그림 1" src="/files/ftp_upload/54225/54225fig1.jpg" /> 그림 1. 갓 고립 된 MPC는 독특한 특징을 가지고있다. DMEM / 10 % PhABS에서 배양 HBM-다국적 기업을 이레 동안은 중간 엽 줄기 세포와 쉽게 구별 정지 된 MPC (A)의 인구 (B)을 발생시킨다 </strong&gt; 형태의 점 (A.1, B.1, 스케일 바 = 100 μm의), 표현형 (A.2, B.2), F - 굴지 분포 (A.3 빨간색, B.3), 및 네 스틴 식 (A.3, B.3 녹색, 스케일 바는 20 μm의 =). <a href="https://www.jove.com/files/ftp_upload/54225/54225fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54225/54225fig2.jpg" /> 2. MPC와 중간 MSC 확장을 위해 설계 상업적으로 이용 가능한 RS와 DMEM / PhABS의 체외. 교체의 표준 중간 엽 줄기 세포 및 표시 돋 혈관으로 분화 그림 MPC와의 mesengenic 유도를 트리거합니다. 문화 일주 후 몇 잔류 된 MPC는 여전히 MSC-RS 매체에 추가 통로 합류 MSC와 같은 세포 집단 (P2-중간 엽 줄기 세포, 스케일 바 = 100 μm의)로 연결하는 동안 검출 (P1-중간 엽 줄기 세포)입니다. 피칼슘 침착 (B 녹색) 및 지질 축적 (B 빨간색, 스케일 바 = 200 μm의)에 의해 계시 된 2 중간 엽 줄기 세포는 말기 각각 적절한 자극에서 골 세포 나 지방 세포로 분화. MPC와는 P2-중간 엽 줄기 세포 (D, 스케일 바 = 1.0 mm)과의 차이에 쥐 ECM 단백질 겔 (C)의 회전 타원체에서 일관된 발아을 보여줍니다. <a href="https://www.jove.com/files/ftp_upload/54225/54225fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

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 ...

isolating-mesangiogenic-progenitor-cells-mpcs-from-human-bone-marrow

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JoVE Journal

Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

8.0K 조회수.  University of Pisa. 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.

비디오: Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

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

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

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

효소 방법은 응고 골수 샘플에서 중간 엽 줄기 세포를 구출

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

연구

발생학

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

인간의 심장 조직에서 혈관 주위 능성 전구체 세포 집단의 분리

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

인간 탯 줄 혈액에서 내 피 조상 세포의 고립

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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 Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

생물학

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

식별 및 Oligopotent 및 계보 커밋된 골수성 창시자 마우스 골 수에서 격리

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

면역학 및 감염병학

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

생체공학

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

인간의 골격 근육에서 혈관 유래 다 능성 전구체의 분리

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

의학

Rat Jaw Bone Marrow 중간엽 줄기 세포의 틈새 기반 분리

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

저자 스포트라이트: 조직 공학에서의 고순도 턱뼈 골수 중간엽 줄기 세포의 응용

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells(쥐 턱뼈 골수 중간엽 줄기세포의 분리 및 배양 기법)

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...