Mesenchymal stem cells (MSCs) have the capacity for multi-lineage differentiation, giving rise to a variety of mesenchymal phenotypes such as osteoblasts (bone), adipocytes (fat), and chondrocytes (cartilage). Stem cell therapy holds immense promise of delivering the next generation of future medical breakthroughs. In this respect, multipotent progenitor cells, such as hMSCs, have attracted high clinical interest because of their ability to differentiate into various cell types and their immunoregulatory properties. Together, these features enable the allogeneic use of hMSCs and thus make them an attractive target for commercial therapeutic development. Below you will find step-by-step protocols used to isolate, expand and differentiate MSC’s properly in stem cell cultures.
Mesenchymal stem cells have been isolated from a variety of tissues including human bone marrow, adipose tissue, umbilical cord and dental pulp. Below is a simple general protocol that can be used to derive MSCs from a variety of tissue sources.
Note: MSC populations will vary from donor to donor and might require further optimization.
Tissue culture plastic- or glassware plates should be coated with 0.1% gelatin as follows:
Figure 1.Phase contrast images of Human Mesenchymal Stem Cells one (A) and two (B) days after thawing. Note spindle shaped, fibroblast-like morphology. Right before passaging, cells should be ~80% confluent.
Figure 2. ICC staining of cultured human bone marrow-derived mesenchymal stem cells with antibodies provided in the Human Mesenchymal Stem Cell Characterization Kit. Human Mesenchymal Stem Cell express H-CAM (CD44) (A, CBL154; 1/500 dilution), THY-1 (CD90) (B, CBL415: 1/500 dilution) and STRO-1 (C, MAB4315: 1/500 dilution). Nuclei of the cells were visualized with DAPI (blue). Expression of hematopoietic stem cell markers, CD19 (MAB1794) and CD14 (MAB1219) and endothelial marker, CD146 (MAB16985) were not observed in human mesenchymal stem cells (data not shown).
Using a human fibronectin, 20 µg/mL (FC010) or a 0.1% gelatin (ES-006-B) coated 48 well tissue culture plate seed 20K cells/well in 0.5 mL normal MSC growth media (SCM015 or SCM045). After an overnight incubation replace the culture media with OsteoMAX-XF™ Differentiation Media (SCM121). Replace media every 2-3 days for a total of 14-21 days. Alizarin-Red (TMS-008) stained osteocytes will start forming around day 7 with maximum expression around day 14.
Using 24 well tissue culture plate seed 60K cells/well in 1 mL normal MSC growth media (SCM015 or SCM045). After an overnight incubation replace the culture media with AdipoMAX™ Differentiation Media (SCM122). Replace media every 2-3 days for a total of 14-21 days. Oil-Red-O (O1391) stained adipocytes will start forming around day 14 with maximum expression around day 21.
Mesenchymal stem cells can be induced to differentiate into chondrocytes using three-dimensional micromass cultures of the cells in aggregates.
Figure 3.Staining of differentiated human mesenchymal stem cell cultures with Oil-Red-O (Adipocytes), Alizarin-Red (Osteocytes), Safranin-O (Chondrocytes) and Alcian-Blue (Chondrocytes).
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