Name: Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage
Uploaded: 2023-04-07T14:30:00
Description: Watch this Scientific Journal Video about Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage at JoVE.com

The video outlines a protocol for differentiating adipose-derived stem cells into adipocytes. It details the seeding process, the addition of differentiation medium, fixation with formalin, staining with oil red O, and observing morphological changes and lipid vesicle formation under an inverted microscope over a 12-day period.

adipose-derived stem cell differentiation to the adipogenic lineage

Isolierung und Charakterisierung von Stammzellen aus Fettgewebe

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 im Rampenlicht: Isolierung und Identifizierung mesenchymaler Stammzellen aus Fettgewebe von Sprague-Dawley-Ratten  

Isolierung und Identifizierung von mesenchymalen Stammzellen aus dem Fettgewebe von Sprague-Dawley-Ratten

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 Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage at JoVE.com

Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage  

Differenzierung von Stammzellen aus Fettgewebe zur adipogenen Linie

Beginnen Sie mit der Aussaat einer Sechs-Well-Platte mit 10.000 Stammzellen aus Fettgewebe pro Quadratzentimeter. Sobald die Zellen zu 60 bis 80 % zusammenfließen, fügen Sie das Differenzierungsmedium hinzu. Waschen Sie die Zellen am 12. Tag mit PBS und inkubieren Sie sie mit 500 Mikrolitern 4%Formalin für eine Stunde bei Raumtemperatur.Sobald die Zellen mit destilliertem Wasser gewaschen sind, waschen Sie sie fünf Minuten lang mit 60%igem Isopropanol und lassen Sie sie trocknen. Fügen Sie 500 Mikroliter 0,5%igen ölroten O-Färbefarbstoff hinzu, der mit 60%Alkohol verdünnt ist. Nach der Inkubation bei Raumtemperatur werden die Vertiefungen zweimal mit einem Milliliter destilliertem Wasser gewaschen und die Platte unter einem inversen Mikroskop betrachtet.Aus Fettgewebe gewonnene Stammzellen differenzierten sich innerhalb von 48 Stunden in Richtung der adipogenen Linie und zeigten eine reduzierte Zellgröße und eine abgerundete Form. Nach sieben Tagen waren Lipidvesikel sichtbar, die 12 Tage nach der Differenzierung positiv für die Ölrotfärbung waren.

Research

JoVE Journal

Biology

Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage

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 ...

Forschung

Biologie

Murine Adipose Tissue Excision

Exzision von murinem Fettgewebe

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization

Isolierung, Aufrechterhaltung und Charakterisierung von mesenchymalen Zellen aus murinem Fettgewebe

Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers

Immunfluoreszenzdetektion von Stammzellexpressionsmarkern aus Fettgewebe

Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers

Durchflusszytometrische Detektion von aus Fettgewebe gewonnenen Stammzellexpressionsmarkern