본 연구는 건강 연구 보조금 R01-DE021683-01 및 건강 연구 보조금 R01 - DE019434의 국립 연구소 MTL에 국립 연구소에 의해 지원되었다; MTCDCW 하워드 휴즈 의학 연구소의 연구 활동은 ACS 프랭클린 마틴 교수 연구 활동, 소아 재생 의학 Hagey 연구소와 스탠포드 대학 아동 건강 연구소 학부 학술 수상에 의해 지원되었다.

참고 : 모든 환자 샘플 동의 얻었다, 실험 프로토콜을 검토하고 스탠포드 대학의 임상 시험 심사위원회 (프로토콜 # 2188과 # 9999)에 의해 승인되었다. 1. 세포 분리 및 문화 : <ol><li> 일반 / 국소 마취하에 복부, 옆구리, 및 / 또는 허벅지 영역의 선택 과목 lipoaspiration를 받고 건강한 여성 환자에서 인간의 피하 지방 조직을 얻습니다. 임상 시험 심사위원회 (IRB) 승인 인체 조직에서의 ASC를 분리의 프로토콜 얻어되었는지 확인하고, 그러한 물질들로 작업하는 동안 제도적 안전주의 사항을 따르십시오. </li><li> lipoaspirated 지방 조직에서 SVF를 얻으려면, 먼저 1X 멸균 인산 완충 생리 식염수를 같은 부피 (PBS)와 리포 애스 퍼 레이트 세 번 씻는다. 조심스럽게 기음과 바닥 수성 층을 버린다. </li><li> 내가 행크의에서 콜라 0.075 % 유형 : 콜라겐 소화 버퍼를 준비균형 소금 솔루션 (HBSS). FACS 버퍼를 준비 : 2 % FBS, 1 % P188 및 PBS에 1 % 펜 연쇄상 구균을. 시판 0.22 ㎛의 공극 크기 빠른 유동 폴리 필터를 사용하여 두 용액을 필터. </li><li> 세척 된 지방 조직을 소화, 37 ° C (약 180 흔들기 / 분)에서 60 분 동안 진탕 수욕 안전하게 분해 용기를 콜라게나 제 소화 버퍼의 동일 부피를 추가 배치. 참고 :이 떨고 동안 최대한의 소화 수 있습니다대로, 요구되는 것보다 더 큰 볼륨 분해 용기를 사용하는 것이 가장 좋은 방법입니다 (즉, 콜라겐 소화 버퍼 250 ml의, 1L 멸균 플라스크에 씻어 지방 조직의 250 ㎖). </li><li> FACS 완충액의 동량을 첨가하고 5 분 동안 RT에 앉아 있도록함으로써 효소 활성을 중화. 다음, 4 ℃에서 20 분 동안 233 XG에 원심 분리기. </li><li> 조심스럽게 기음과 고밀도 SVF 펠렛을 방해하지 돌보는, 상층 액을 제거한다. </li><li> RT 레드 5-10 ml의 펠렛을 재현 탁세포 펠렛의 크기에 기초하여, 용해 완충액. 실온에서 5 분 동안 233 XG에서 5 분 원심 분리기에 대한 RT에 앉아 솔루션을 남겨주세요. </li><li> 기음 상등액과 펠릿 크기에 따라 기존의 성장 배지 5-10 ml를 첨가하여 펠릿을 재현 탁 (둘 베코 변형 이글 중간 [DMEM] / 10 % FBS / 1 % 페니실린 - 스트렙토 마이신 용액 [펜 연쇄상]). </li><li> 세포 파편을 제거하기 위해 100 ㎛ 나일론 세포 여과기를 통해 현탁액을 여과. </li><li> 4 ° C에서 5 분 233 XG에 원심 분리기 및 펠렛을 방해하지 않고 상층 액을 버린다. </li><li> 펠릿의 크기에 기초하여 기존의 성장 배지 5~10 ㎖,에 펠렛을 재현 탁. </li><li> 15cm 표준 배양 접시에 2 × 10 6 세포를 놓고 37 ° C / 21 % O 2, 5 % CO 2에서 차 문화 O / N을 설정합니다. 37 ° C / 21 % O 2, 성장 배지에서 5 % CO 2에서 자발적인 분화를 방지하기 위해 서브 합류 수준에서 유지한다. 참고 : 완전히의 셀 밀도합류 15cm 플레이트는 약 4-6 × 106 세포이다. </li></ol> 2. 염색 <ol><li> 염색하기 전에 36 시간 동안 37 ° C / 21 % O 2, 5 % CO 2, 전통적인 성장 배지에서 배양의 ASC는 / 정렬. </li><li> (제조사의 프로토콜에 따라) Accutase를 사용하여 배양 접시에서 리프트의 ASC. </li><li> 한 번 (2 % FBS와 1 % 펜 연쇄상 구균과 PBS) FACS 완충액으로 세포를 씻으십시오. </li><li> 4 ℃에서 5 분 동안 233 XG에 원심 분리기. </li><li> 펠렛을 방해하지 않고 상층 액을 버린다. FACS 완충액 0.5 ml의 세포를 재현 탁 (세포 수 및 항체의 원하는 양에 따라). 주 : 세포 현탁액의 부피는, FACS 항체 제조자의 권고에 따라, 항체 농도를 결정한다. 그러나, 항체의 농도가 적정, 하나의 출발 농도를 사용 : 그것은 항체를 사용하는 최초 시간 인 경우 (100), 일반적으로 권장된다. </li><li> 총 수를 계산혈구를 사용하여 세포. </li><li> 1.5 ml의 원심 분리기 튜브에서 예약 100 UL 분취 흠 샘플 (사용하는 형광 항체의 수에 따라) 단일 색상 조절을 위해 사용된다. </li><li> (제조업체의 지침에 따라 소정의 농도에서) 적절한 형광 표지 된 단일 클론 항체를 추가하고 빛으로부터 보호 얼음에 20 ~ 30 분 동안 품어. <ol><li> 골 형성 모집단 레이블을 지정하려면 : 1시 50분의 농도로 FACS 버퍼에 안티 CD90 (APC 안티 - 인간 CD90 (Thy1)) 또는 안티 CD105 (FITC 안티 - 인간의 CD105 (endoglin))를 희석. </li><li> 다른 세포 집단 (예를 들어, 조혈 및 내피 세포)에 레이블을 지정하려면 : 안티 CD45 (안티 - 인간 CD45 태평양 블루), 항 CD34 (안티 - 인간 CD34 APC), 및 / 또는 항 CD31 (안티 - 인간 CD31 PE를 희석 ) FACS 1:50의 농도로 버퍼. </li><li> 직접 접합 된 항체를 사용하지 않는 경우, 적절한 스트렙 타비 딘 - 접합 제와 비오틴 차 항체를 사용하여하나의 희석 등 스트렙 타비 딘 PE-Cy7로 진 항체, 100. </li></ol></li><li> 상층 액을 흡입, 4 ° C에서 5 분 동안 233 XG에 FACS 버퍼와 원심 분리기와 튜브를 채우기 : 부화 후, 두 번 세포를 씻으십시오. </li><li> 세포 덩어리를 제거하기 위해 70 μm의 나일론 셀 스트레이너를 통해 400 ~ 500 μL FACS 버퍼 및 필터 샘플에서 세포를 재현 탁. </li><li> 필터 상단으로 FACS 튜브에 표시된 세포를 전송하고 분석 기간 동안 얼음에 샘플을 유지. </li></ol> 3. 형광 활성화 셀 정렬 주 : 다음 단계는 이전 형광 활성화 된 세포에서 지식 정렬 (FACS) 또는 숙련 된 기술자의 도움을 의무화. <ol><li> 포워드 산란 (FSC), 측면 산란 (SSC)를 조사하기 위해 설계 분석 플롯을 적절한 FACS 소프트웨어를 이용하여, 피코 에리 트린 (PE) - 텍사스 레드, 퍼시픽 블루, 형광 염료 (FITC), 알로 피코시 아닌 (APC)와 다른 형광 물질을 사용D는 세포 레이블을. </li><li> 셀 소터, 잠시 소용돌이 세포를 재현 탁 각 샘플에로드하기 전에. </li><li> 세포 파편을 제외하고,베이스 라인 형광도를 결정하기 위해, 전체 세포 집단을위한 게이트를 설정하기 위해 흠 견본의 분석을 시작. </li><li> 흠 견본에 요오드화 프로피 듐 (PI)를 첨가하고, 전체 세포 집단 내에서 살아있는 세포 집단을 시각화하기 위해 그것을 분석. 이 인구 주위에 게이트를 구축합니다. </li><li> 형광 보정을 설정하기 위해 각각의 형광 색소에 대한 단색 대조군 샘플을 분석한다. </li><li> 정렬 게이트의 위치를​​ 설정하는 스테인드 샘플을 분석 할 수 있습니다. </li><li> 죽은 세포를 염색하기 위해 염색 된 샘플에 PI를 추가합니다. </li><li> 표지 된 세포 집단을 정렬합니다. 정렬 1.5 ml를 원심 분리기 튜브 또는 배양 배지를 함유하는 원뿔형 튜브 (15 mL) 중 하나에. </li><li> 수동 셀들의 수량이 필요한 구하면 정렬 중단. </li><li> 우리FACS를 보내고, 극히 일부를 분석 (예., 500 세포) 취득 된 세포 집단의 순도에 의해 검사를 수행한다. 참고 :이 단계는 정렬 된 세포 집단의 순도를 확인합니다. 이상적으로, 순도 90-95 % 이상이어야한다. </li><li> 39 °에서 5 분 동안 원심 분리기 (233) XG 세포를 즉시 10 % FBS를 함유하는 새로운 배지 1 ㎖에 정렬 완료하고 다음을 재현 탁시켰다. 주 : 사용 된 용지의 양이 세포 펠렛의 크기에 따라 증가 될 수있다. </li><li> 젤라틴 코팅 플레이트에 플레이트는 세포 생존 능력을 향상시킬 수 있습니다. 최종 세포 수율, 판 6 웰 플레이트에 웰 당 30 만 셀, 12 웰 플레이트에 웰 당 10 만 셀에 따라. </li></ol>

<ol>
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T., 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=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li></ol>

저자 중에이 원고에서 언급 된 제품, 장치 또는 약물의에 재무 적 이해 관계가 없습니다. 저자 중에보고 어떤 경쟁 금융 관심이 없다.

현재, 인간 지방 조직의 SVF에서의 ASC의 동종 개체군의 분리는 바람직한 목표 그래도 도전 남아있다. 이러한 세포는 골격 조직의 형성 및 항상성을 연구하는데 이용 될 수있는 친 조골 ASC의 개체군의 분리는, 특히 바람직하다. 그러나, 지방 조직의 SVF는. 셀 용량과 분화 가능성을 막기위한 관련이 이질성에 대한 분자 기초는 세포의 풀링 된 인구에서 이해 될 수없는 11 이질성을 품고, 대신 ?...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

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			<td style="width:152px;">BD Pharmingen</td>
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			<td height="42" style="height:42px;width:275px;">Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&#160;</td>
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			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">12-0319-41</td>
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			<td height="21" style="height:21px;width:275px;">Streptavidin PE-Cyanine7</td>
			<td style="width:152px;">eBioscience</td>
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			<td height="21" style="height:21px;width:275px;">BD FACS Aria II instrument</td>
			<td style="width:152px;">BD Biosciences</td>
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			<td style="width:152px;">BD Biosciences</td>
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isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

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

인간의 ASC (그림 1A, 1B)의 고농축 인구의 분리 향상된 골 형성의 결과 세포 마커로서 CD90 사용. 태평양의 ASC는 블루 - 공액 항 - 인간 CD45, FITC 접합 된 항 - 인간 CD105 및 APC 공액 항 - 인간 CD90으로 염색 하였다. 정렬 후 분석으로 정량으로 정렬 한 후 순도의 수준은 98 % 이상이었다. 두 개의 새로운 개체군의 미래의 격리를 위해 허용 전사 프로필을 기반으로 세포의 그...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

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

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

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

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

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

Research

JoVE Journal

Developmental Biology

11.4K 조회수.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

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

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

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

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

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

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

연구

발생학

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

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

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

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

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

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

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

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

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

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

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

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

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

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

원심 중력에 의해 지방이 유래 줄기 세포의 연골 분화 유도

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

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

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

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

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

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

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

마우스 자궁 내막 상피와 기질 세포의 분리에 대한<em&gt; 체외</em&gt; 탈락

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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 ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

분리 및 소설 표면 마커 조합에 의해 확인 된 간 전구 부분 집합의 농축

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

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 ...