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

Aislamiento y caracterización de células madre derivadas del tejido adiposo

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.

Autor destacado: Aislamiento e identificación de células madre mesenquimales derivadas del tejido adiposo de ratas Sprague Dawley  

Aislamiento e identificación de células madre mesenquimales derivadas del tejido adiposo de ratas Sprague Dawley

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  

Detección inmunofluorescente de marcadores de expresión de células madre derivadas de tejido adiposo

Comience lavando las células madre murinas derivadas de tejido adiposo en una placa de seis pocillos con dos mililitros de tampón de lavado. Agregue un mililitro de reactivo bloqueador después de incubar y lavar las células tres veces. Incubar las células con agitación lenta durante 20 minutos.Agregue 200 microlitros de los anticuerpos primarios a cada pocillo e incube durante la noche a cuatro grados centígrados. Después de lavar las placas tres veces como se demostró anteriormente, incubar con 200 microlitros de los anticuerpos secundarios. Contrateñir los núcleos celulares con 100 microlitros de DRAQ7 durante 20 minutos después de lavar las placas tres veces.Repita el lavado y monte las diapositivas. Visualice los portaobjetos montados bajo microscopía confocal de epifluorescencia. Las células madre derivadas de tejido adiposo fueron 100% positivas para los marcadores de superficie CD9 en todos los subpasajes probados.Todas las células expresaron marcadores CD63 en los subpasajes uno, tres y nueve, mientras que el 49,3% de las células lo expresaron en el pasaje siete. Los marcadores CD34 estaban ausentes en los pasajes uno, siete y nueve, pero visibles en el pasaje tres.

immunofluorescent-detection-adipose-derived-stem-cell-expression

Research

JoVE Journal

Biology

Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers

146 vistas. Comience lavando las células madre murinas derivadas de tejido adiposo en una placa de seis pocillos con dos mililitros de tampón de lavado. Agregue un mililitro de reactivo bloqueador después de incubar y lavar las células tres veces. Incubar las células con agitación lenta durante 20 minutos.Agregue 200 microlitros de los anticuerpos primarios a cada pocillo e incube durante la noche a cuatro grados centígrados. Después de lavar las placas tres veces como se demostró anterio...

Video: Immunofluorescent 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 ...