The video demonstrates a procedure for immunophenotyping murine adipose-derived stem cells. It details the preparation and incubation of cell suspensions with specific antibodies, followed by washing and centrifugation. Finally, it explains how to analyze the cell population using dot plots, revealing high CD105 positivity and low CD31 expression.

flow cytometric detection of adipose-derived stem cell expression markers

지방 조직 유래 줄기세포 분리 및 특성 분석

Mesenchymal stem cells (MSCs) have strongly impacted regenerative medicine due to their high capacity for self-renewal, proliferation, migration, and differentiation into different cell lineages1,2. Currently, a great deal of research is focusing on their potential for the treatment and diagnosis of various diseases.
There are different sources of mesenchymal cells: bone marrow, skeletal muscle, amniotic fluid, hair follicles, placenta, and adipose tissue, among others. They are obtained from different species, including humans, mice, rats, dogs, and horses3. Bone marrow-derived MSCs (BMSCs) have been used for many years as a major source of stem cells in regenerative medicine and as an alternative to the use of embryonic stem cells4. However, adipose-derived MSCs, or adipose-derived stem cells (ASCs), are an important alternative with great advantages due to their ease of collection and isolation, as well as the yield of cells obtained per gram of adipose tissue5,6. It has been reported that the harvest rate of ASCs is generally higher than that of BMSCs7. It was initially proposed that the reparative/regenerative capacity of ASCs was due to their ability to differentiate into other cell lineages8. However, research in recent years has reinforced the primary role of paracrine factors released by ASCs in their reparative potential9,10.
Adipose tissue (AT), in addition to being an energy reserve, interacts with the endocrine, nervous, and cardiovascular systems. It is also involved in postnatal growth and development, the maintenance of tissue homeostasis, tissue repair, and regeneration. The AT is composed of adipocytes, vascular smooth muscle cells, endothelial cells, fibroblasts, monocytes, macrophages, lymphocytes, preadipocytes, and ASCs. The latter possess an important role in regenerative medicine due to their low immunogenicity11,12. ASCs can be obtained by enzymatic digestion and mechanical processing or by adipose tissue explants. Primary cultures of ASCs are easy to maintain, grow, and expand. Phenotypic characterization of ASCs is essential to verify the identity of the cells by assessing the expression of specific membrane markers using methods such as immunofluorescence and flow cytometry13. The International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) have defined that ASCs express CD73, CD90, and CD105, while lacking the expression of CD11b, CD14, CD19, CD45, and HLA-DR14. These markers, both positive and negative, are therefore considered reliable for the characterization of ASCs.
This project was focused on describing a procedure for the isolation and identification of adult mesenchymal cells extracted from rats' AT, as this source of cells does not present ethical challenges, unlike embryonic stem cells. This solidifies the procedure as a viable option because of the ease of access and minimally invasive method compared to bone marrow-derived stem cells. 
Mesenchymal cells from this tissue source have an important role in regenerative medicine because of their immunomodulatory capabilities and low immune rejection. Therefore, the present study is a fundamental part of future research into their secretome and their application as regenerative therapy in different diseases, including metabolic diseases such as diabetes.

저자 스포트라이트: Sprague Dawley Rats의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별  

Sprague Dawley 쥐의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별

Mesenchymal stem cells have a really broad role in cellular communication due to their paracrine function. This protocol obtains adipose-derived mesenchymal stem cells with morphological, immunophenotypical, and adequate functionality for use in studying therapies for pancreatic regeneration and diabetes. There has been increased use of cell therapies in the treatment of diabetes.These therapies include allogenic transplants of pancreatic cells, autologous stem cell-derived products of insulin-producing cells, and 3D-printed pancreatic tissue using biological inks. Most research primarily studied the secretome components and the role in the regulation of different cellular processes. New techniques like microarrays and next-generation sequencing can investigate the function of miRNAs.A challenge in this area is to achieve greater stimulation of mesenchymal cells, reaching precise molecular composition in the secretome, and directing it to the treatment of a specific conditions. We will be evaluating how an miRNAs contribute to pancreatic tissue degeneration exposed to oxidative stress. The study will involve usage of stem cells from adipose tissue.

Watch this Scientific Journal Video about Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers at JoVE.com

Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers  

지방유래 줄기세포 발현 마커의 유세포 검출

5% 소 태아 혈청과 함께 PBS를 사용하여 해동된 쥐 지방 유래 줄기 세포 현탁액의 농도를 조정하는 것으로 시작합니다. 100, 000 세포를 포함하는 현탁액을 100 마이크로리터 분취하여 1.5 밀리리터 원심분리 튜브에 넣는다. 제조업체의 지침에 따라 적절한 양의 항-CD105 AF594 및 항-CD31 AF680을 추가합니다.섭씨 4도의 어둠 속에서 20분 동안 배양합니다. PBS/FBS로 세척하고 250 x g에서 5분 동안 원심분리하여 과도한 얼룩을 제거합니다. 300 마이크로 리터의 1 % 포르말린으로 세포를 재현 탁하십시오.다각형 기호를 클릭하여 점도표 그래프를 만들고 관심 있는 부분모집단을 선택하고 FSC-A 대 SSC-A 게이팅을 사용하여 파편 영역을 제외합니다. 그런 다음 다각형 기호를 클릭하여 새 도트 플롯을 생성하고 SSC-H 대 SSC-A, FSC-H 및 FSC-A를 사용하여 이중 및 다중 셀을 제외합니다. AF594에는 Y610-mCHERRY를, AF680 형광단에는 R660 APC-A 검출기를 각각 사용합니다.사분면 기호를 클릭하고 자가형광 세포에 대한 각 검출기의 임계값을 설정합니다. CD105 및 양성 모집단으로 표지된 샘플을 Q1LR에 표시하십시오. 다음으로, Q1LR에 표시할 CD31 및 양성 모집단으로 표지된 샘플을 선택합니다.면역 표현형 분석은 세포 집단의 98.7%가 CD105에 대해 양성인 반면 5.88%만이 CD31 마커를 발현하는 것으로 나타났습니다.

flow-cytometric-detection-adipose-derived-stem-cell-expression

Research

JoVE Journal

Biology

Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers

107 조회수.  Universidad Autónoma Metropolitana. 5% 소 태아 혈청과 함께 PBS를 사용하여 해동된 쥐 지방 유래 줄기 세포 현탁액의 농도를 조정하는 것으로 시작합니다. 100, 000 세포를 포함하는 현탁액을 100 마이크로리터 분취하여 1.5 밀리리터 원심분리 튜브에 넣는다. 제조업체의 지침에 따라 적절한 양의 항-CD105 AF594 및 항-CD31 AF680을 추가합니다.섭씨 4도의 어둠 속에서 20분 동안 배양합니다. PBS/FBS로 세척하고 250 x g에?...

비디오: Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers  

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

연구

생물학

Murine Adipose Tissue Excision

Shave the abdominal and inguinal regions of two sedated adult male Sprague Dawley rats. Disinfect the shaved regions with an iodine solution. Make a medial longitudinal incision from the sternum to the testicular region, Using a pair of sterile forceps, remove the adipose tissue from the epididymidis and the kidneys.Place the tissue in conical tubes containing 20 milliliters of cold, sterile PBS-AA solution.

쥐 지방 조직 절제

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization

Inside a biosafety cabinet, remove the murine adipose tissue with sterile forceps. Once excess PBS is removed, transfer the tissue to another petri dish and wash twice for five minutes each with 10 milliliters of PBS AA solution. Add 20 milliliters of prepared collagenase solution into a crystal beaker containing one square centimeter sized fragmented tissue and a magnetic bar.Incubate the sealed beaker at 37 degrees Celsius with continuous agitation. Filter the homogenate through a 40-mesh 0.38-millimeter stainless steel aperture Filter on a petri dish and collect the cell suspension in a sterile 50-milliliter conical tube. Carefully suspend the stromal vascular fraction in 20 milliliters of warm supplemented DMEM and centrifuge the suspension.After discarding the supernatant, gently wash the cells twice with 40 milliliters of non-supplemented DMEM medium. Suspend the palate in five milliliters of supplemented DMEM. Transfer the suspension to a 25-square centimeter T bottle and incubate.Wash cells three times with five milliliters of warm PBS AA and twice with non-supplemented DMEM. To remove any cell debris and non-murine cells add five milliliters of fresh, warm supplemented DMEM and change the medium every three to four days. Once the cells reach 90 to 100%confluency, add one milliliter of warm trypsin EDTA solution to the DMEM washed cells.Incubate for five to seven minutes at 37 degrees Celsius. Add four milliliters of supplemented DMEM to the detached cell monolayer and gently disaggregate the suspension. Suspend the cells in five milliliters of warm supplemented DMEM.After centrifuging and discarding the supernatant, gently mix the pellet in one milliliter of supplemented DMEM. Stain the cells with trypan blue and count them in a Neubauer chamber. Seed cells in T-75 flasks.To ensure colony formation and high proliferation, observe the morphology of the hematoxylin and eosin-stained cells under an optical microscope. hematoxylin and eosin staining reveal extensive cytoplasm with elongated prolongations and abundant intracellular microfilaments.

쥐 지방 유래 중간엽 세포 분리, 유지 및 특성 분석

Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers

Begin by washing murine adipose-derived stem cells in a six-well plate with two milliliters of wash buffer. Add one milliliter of blocking reagent after incubating and washing the cells thrice. Incubate the cells with slow agitation for 20 minutes.Add 200 microliters of the primary antibodies to each well and incubate overnight at four degrees Celsius. After washing the plates thrice as demonstrated previously, incubate with 200 microliters of the secondary antibodies. Counterstain the cell nuclei with 100 microliters of DRAQ7 for 20 minutes after washing the plates thrice.Repeat washing and mount slides. Visualize the mounted slides under epifluorescence confocal microscopy. Adipose-derived stem cells were 100%positive for CD9 surface markers in all subpassages tested.All cells expressed CD63 markers in the subpassages one, three and nine, while 49.3%of the cells expressed it in passage seven. CD34 markers were absent in passages one, seven, and nine, but visible in passage three.

지방유래 줄기세포 발현 마커의 면역형광 검출

Begin by adjusting the concentration of thawed murine adipose-derived stem cell suspension using PBS with 5%fetal bovine serum. Aliquot 100 microliters of the suspension containing 100, 000 cells into a 1.5 milliliter centrifuge tube. Add appropriate amounts of anti-CD105 AF594 and anti-CD31 AF680 as per the manufacturer's instructions.Incubate for 20 minutes in the dark at four degrees Celsius. Remove excess stain by washing with PBS/FBS and centrifuge at 250 x g for five minutes. Resuspend the cells in 300 microliters of 1%formalin.Create a dot plot graph by clicking on the polygon symbol to select the subpopulation of interest and exclude the debris areas using FSC-A versus SSC-A gating. Next, click on the polygon symbol to generate a new dot plot and exclude the duplexes and multiples cells using SSC-H versus SSC-A followed by FSC-H and FSC-A. Use Y610-mCHERRY for the AF594 and the R660 APC-A detectors for the AF680 fluorophores, respectively.Click on the quadrant symbol and set each detector's threshold for autofluorescence cells. Select samples labeled with CD105 and positive population to be shown on Q1LR. Next, choose samples labeled with CD31 and positive population to be shown on Q1LR.Immunophenotyping showed that 98.7%of the cell population was positive for CD105, while only 5.88%expressed the CD31 marker.

Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage

Begin by seeding a six well plate with 10, 000 adipose-derived stem cells per square centimeter. Once cells become 60 to 80%confluent add the differentiation medium. On day 12, wash the cells trice with PBS and incubate them with 500 microliters of 4%formalin for one hour at room temperature.Once the cells are washed with distilled water, wash them with 60%isopropanol for five minutes and let them dry. Add 500 microliters of 0.5%oil red O staining dye diluted in 60%alcohol. After incubation at room temperature wash the wells twice with one milliliter of distilled water and observe the plate under an inverted microscope.Adipose-derived stem cells differentiated towards the adipogenic lineage showing reduced cell size and rounded shape within 48 hours. After seven days, lipid vesicles were visible, which were positive for oil red staining 12 days after differentiation.