Name: Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers
Uploaded: 2023-04-07T14:30:00
Description: Watch this Scientific Journal Video about Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers at JoVE.com

The video outlines a protocol for washing and staining murine adipose-derived stem cells using primary and secondary antibodies, followed by counterstaining and visualization under confocal microscopy. It details the expression of surface markers (CD9, CD63, and CD34) across various cell passages, indicating their positivity and variability.

immunofluorescent detection of adipose-derived stem cell expression markers

Adipose Tissue-Derived Stem Cell Isolation and Characterization

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.

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

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

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 Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers at JoVE.com

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.

Research

JoVE Journal

Biology

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