Extensive study of human stem cells and the cellular processes underlying differentiation of these cells towards specialized cell types opens up the possibility for the targeted and specific treatment of diseases by cell-based therapies, also known as regenerative medicine.
Human mesenchymal stem cells (hMSC) are multipotent adult stem cells that possess a high capacity for self-renewal evolving from perivascular progenitor cells . With the use of appropriate differentiation media, MSC can be differentiated in vitro towards chondrocytes, adipocytes, neurons, and osteoblasts .
This article describes the results of an investigation aimed at detecting phenotypic changes during MSC differentiation through automated, real-time analysis using the Spark® multimode reader and a number of proven and reliable cell-based assays to assess and quantify cell cytotoxicity, viability, and apoptosis developed by PromoCell.
Custom-made solutions to suit any life science research application are offered by Tecan’s Spark® multimode microplate reader platform. The platform possesses various features and functions for cell-based analyses such as an automated cell imaging module, and environmental control features such as temperature, humidity, and CO2 control within the reader.
Material and methods
Stem cell culture and differentiation
Human MSC from adipose tissue (hMSC-AT, PromoCell C-12977) were propagated in MSC Growth Medium DXF (PromoCell, C-28019) on cell culture vessels. The culture vessels were pre-coated with 10 μg/ml of human fibronectin (PromoCell, C-43060) in HEPES Buffered Saline Solution (PromoCell, C-40020) at 37 °C for 20 minutes.
For differentiation, MSC were resuspended at 2-5 x 104 cells/ml in DXF medium, followed by plating 100 μl per well on black 96-well plates with μClear bottom (Greiner Bio-One, 655 087). A Spark® multimode plate reader equipped with a standard fluorescence module was employed, integrated with humidity cassette, injector system and cell chip adapter, for counting, vessel coating, seeding, and growth of MSC.
DXF medium was substituted by 200 μl/well of ready-to-use differentiation media after MSC adhesion. The following differentiation media were used:
- adipogenic (PromoCell C-28016)
- neurogenic (PromoCell C-28015)
- chondrogenic (PromoCell C-28012)
- osteogenic (PromoCell C-28013)
MSC differentiation was monitored for up to 60 hours. Cells were seeded into the microplate and allowed to adhere overnight. Then, the plate was placed into the Humidity Cassette; the fluid reservoirs of the plate filled with deionized water.
The outer wells of the plate were filled only with culture medium without cells in order to eliminate the edge effects due to evaporation. The Spark® reader was set to 5% CO2 and 37 °C. Cell differentiation was observed by automated measurement of cell confluence, absorbance, fluorescence, or luminescence at defined intervals.
Reagents and measurement
For viability assessment, the Fluorometric Cell Viability Kit I (Resazurin; PromoCell, PK-CA707-30025-0) was used. This assay is based on the conversion of resazurin to its fluorescent product resorufin by a chemical reduction in living cells. The resazurin solution was directly added to the culture medium at a dilution of 1:20. Blank wells containing culture medium without cells, but with resazurin, were added.
The LDH Cytotoxicity Kit I (PromoCell, PK-CA577-K314) was used for the analysis of cytotoxicity to detect lactate dehydrogenase (LDH) enzyme activity in cell culture supernatant as an indicator for necrotic cell death. The Bioluminescent Cell Viability Kit I (ATP, PromoCell, PK-CA577-K254-200) was used to quantify adenosine triphosphate (ATP) levels. The manufacturer’s instructions were followed for the usage of all kits.
Differentiation of human MSC in vitro needs to be investigated or monitored, spread over several days to weeks so as to observe tissue-specific morphological changes and expression of cell surface markers .
However, early upon the onset of differentiation, slight changes in the cells’ phenotype, for example, metabolic activity, may take place. Thus, it had to be decided whether those changes could be revealed by real-time measurement, hours after subjecting the MSC to the various differentiation media.
For this purpose, MSC were exposed to osteogenic, adipogenic, neurogenic, or chondrogenic differentiation, and a non-toxic, cell-permeable non-fluorescent blue dye, such as resazurin, was directly added to the culture media.
Resazurin is changed to the red-fluorescent product resorufin because of the reducing power of living cells, thus, providing a measurement for cell metabolism or cell viability. The experiment was conducted by propagating MSC within the Spark® multimode plate reader for the entire length of the experiment, so that online measurement is enabled.
In comparison to all other conditions applied, MSC in adipogenic differentiation medium revealed a substantial increase in the conversion of resazurin, three hours post induction. This constant increase lasted for more than 24 hours before reaching a plateau, as shown in Figure 1.
With respect to MSC which had been left in non-differentiating DXF medium, chondro-, osteo-, or neurogenic differentiation conditions demonstrated a slight decrease in the conversion of resazurin, the plateau-phase being reached by around 48 hours. This result illustrates that, after induction of differentiation, the change in the phenotype of MSC becomes apparent within few hours.
Figure 1. Automated analysis of metabolic activity during differentiation of stem cells.
The presence of LDH activity in MSC culture supernatant was examined as a measure for ruptured (necrotic) cells in order to determine if the difference in resazurin conversion reflects changes in cell metabolism or cell viability.
Interestingly, cytotoxicity during MSC differentiation is observed; the most toxic was the osteogenic differentiation, as shown in Figure 2. However, the cells in adipogenic differentiation medium with the highest rate of resazurin conversion (as seen in Figure 1) did not show lowest cytotoxicity.
Figure 2. Cytotoxicity measured during stem cell differentiation.
Therefore, difference in resazurin conversion was not caused by differences in cell viability. Therefore, it was investigated whether changes in metabolic activity during differentiation can be caused by differences in resazurin conversion.
For this purpose, the ATP levels at the time of MSC differentiation were determined. The measurements revealed that the adipogenic differentiation condition had the highest ATP content, followed by non-differentiated MSC, as shown in Figure 3.
This result correlated well with the measurement of resazurin shown in Figure 1, demonstrating that changes in metabolic activity occur early during stem cell differentiation.
Figure 3. ATP levels during differentiation of stem cells.
Based on the above summarized findings, it could be shown that after induction of differentiation towards adipocytes, human MSC show an early and significant elevation of cell metabolism, indicated by the production of ATP and cells’ reducing power.
On the other hand, neurogenic, chondrogenic, and osteogenic differentiation substantially reduces the MSC’s metabolic activity. It may be noted that in vitro differentiation of MSC with ready-to-use cell culture media is robust and reliable.
It was reliable and convenient to perform cell counting, plating, and culture of stem cells for several days within the Spark® multimode reader, thus providing a good basis for the automated multiparameter measurement of cell characteristics. Moreover, adding resazurin to the culture medium to a maximum of 60 hours proved to be nontoxic, yet sensitive to stem cells.
Produced from materials originally authored by Associate Professor Dr. Rüdiger Arnold1, Dr. Britt Lemke2 and Dr. Katrin Flatscher3 from:
1 University of Heidelberg
2 PromoCell Academy
3 Tecan Austria.
The authors wish to thank Dr. Hagen Wieland (PromoCell) for advice on mesenchymal stem cells and Dr. Jürgen Becker (PromoCell) for his help in selecting appropriate assays.
- Crisan M, Yap S, Casteilla L, et al., Cell Stem Cell 2008; (3):301–13.
- da Silva Meirelles L, Caplan AI, Nardi NB., Stem Cells 2008; 26(9):2287–99.
- Caplan AI., Cell Stem Cell 2008; 3(3):229–30.
- ATP adenosine triphosphate
- LDH lactate dehydrogenase
- MSC mesenchymal stem cells
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