Modulation of Proliferation and Differentiation of Human Bone Marrow Stromal Cells by Fibroblast Growth Factor 2: Implications for Tissue Engineering
Hankemeier, S. et al. 2005. Modulation of Proliferation and Differentiation of Human Bone Marrow Stromal Cells by Fibroblast Growth Factor 2: Potential Implications for Tissue Engineering. Tissue Engineering. 11(1/2): 41-49
Introduction:
A recent approach to ligament and tendon reconstruction in patients involves the use of human bone marrow stromal cells (BMSCs). BMSCs have the potential to differentiate into a number of mesenchymal cell lineages that are integral to ligament and tendon formation, such as fibroblasts and chondrocytes. In addition, the usage of BMSCs avoids the issue of transplant rejection and also avoids the ethical concerns associated with other possible stem cell therapies. In order for BMSCs to succeed as a tissue engineering material, they must be able to proliferate, properly differentiate, and have similar expression patterns to those of the desired cell type. In this paper, the effects of FGF-2, a protein of importance in tendon and ligament healing, on the properties of BMSCs were studied.
To study the effect of FGF-2 on BMSCs, the authors set up three cultures: 1) BMSCs with a low dose of FGF-2 (3 ng/mL), 2) BMSCs with a high dose of FGF-2 (30 ng/mL), and 3) BMSCs with no FGF-2. Proliferation of the cells in each culture was quantified using BrdU. Apoptosis rates were quantified using flow cytometry; apoptosis was determined by the presence of a specific fluorescent marker (annexin V). To study the gene expression of the cells in different cultures, RT-PCR was run with a number of different primers for various genes of interest integral to the formation of ligaments and tendons.
Results:
Each of the three cell cultures displayed different morphologies. The cell density in the low-dose FGF-2 culture was higher compared to the other two cultures, and the cell density in the high-dose FGF-2 culture stagnated after day 14 (Fig. 1). Compared to the control, the low-dose FGF-2 culture had a higher cell density after 28 days and it also showed a much higher degree of BrdU incorporation.
In contrast to the differences in cell proliferation, the apoptosis rates for cells in each culture were relatively similar and the difference from culture to culture was not statistically significant.
Each culture had a different pattern of gene expression (Fig. 4).
Though collagen I, collagen III, fibronectin, and alpha-SMA mRNAs were present in all cultures, each culture expressed them in different amounts. The low-dose FGF-2 culture expressed much more collagen III and vimentin than either the control or the high-dose culture. For all the genes tested, the low-dose FGF-2 culture expressed at least as much mRNA as any of the other cultures.
Discussion:
The initial proliferation in low-dose FGF-2 cells coupled with a steady increase in expression of several proteins such as collagen I and III suggests that FGF-2 acts on BMSCs in a biphasic manner: the first phase involves proliferation of cells, and the second phase involves an upregulation of certain genes that is associated with differentiation of these BMSCs into specific cell types. Furthermore, the low-dose FGF-2 cultures displayed a higher degree of cell proliferation and gene expression than the high-dose FGF-cultures, which suggests that the effects of FGF-2 on BMSCs act in a dose-dependent manner.
The authors conclude, therefore, that a low dose of FGF-2 has a positive effect on the proliferation and differentiation of BMSCs whereas a high dose of FGF-2 has an adverse effect on BMSCs. This dose-dependent behavior of FGF-2 on BMSC activity is a variable that should be taken into account either in culture on in the design of ligament/tendon reconstruction methods using BMSCs.
Comments:
Though the use of various concentrations of FGF-2 to examine its effect on BMSC makes sense, the authors never explicitly mention why they chose the concentrations they did. I would have liked to known why they chose the numbers they used, and, more importantly, I would liked to have known if the concentrations they used are physiologically relevant. The authors also do not mention how many samples they use to calculate the cell proliferation data, and it seems as if they only used a single sample for each culture. I think the data would be more reliable if the authors had calculated cell proliferation data for a number of samples.
The authors are also a bit flippant in describing the morphology of each cell culture. They write that “large flattened cells and star shaped cells were observed in smaller numbers”, but they make no effort to properly characterize these cells. They also do not explicitly compare the morphology of the two FGF-2 cultures with that of the control. Since a major part of their paper involves the differentiation of BMSCs as a result of FGF-2, I think it would be helpful if they described the morphology in more detail.
Finally, the paper presents a clear correlation between FGF-2 and proliferation and differentiation of BMSCs but makes no effort to elucidate the molecular basis of this interaction. Why, for example, does FGF-2 act in a dose-dependent manner? This paper only studies an input (different doses of FGF-2) and the corresponding products (cell proliferation, mRNA expression, etc), so further studies are necessary to examine the exact way in which BMSCs are affected by FGF-2.
4 comments:
One thing I’ve noticed about the RTPCR is that a lot of the control cells seem to also express the genes in very similar amounts as the experimental setups. It also seems like all the RT-PCRs were run on different gels, and thus need to be considered against the controls (0 dosage?) to have any significance whatsoever. The choices of genes here seem to be a little less than optimal, given the similarities of the gels. A q-RT-PCR, in this case, might be a little more informative because it would tell us exactly how the expression changed, potentially across different days.
I also find the dosage dependence very curious, and I don’t quite understand why there seems to be such a clear “optimum” dosage of FGF-2. Future work should definitely explore the pathways behind this.
I also noticed that the RT-PCR signals intensities seemed very similar from gene to gene. I feel like it is fair to compare the differences in signaling pattern (as in, was this gene expressed, yes or no). However, it does not seem correct for the authors to explicitly compare amounts of gene expression from culture to culture. Furthermore, I am not convinced that the authors chose the best gene set to study cell growth or proliferation. Why two types of Collagen and not some other gene that was more indicative of cell differentiation (or at least less redundant)? I agree that if the authors really wanted to make these comparisons and actually distinguish the differences in gene expression, qRT-PCR would be more appropriate.
I am still a little confused about the trend in cell morphology and growth observed in the three different cultures. If low FGF-2 enhanced cell morphology and growth and high FGF-2 decreased it, I would have expected that no FGF-2 would deliver the best results. It's interesting that such good cell development can occur at low doses of FGF-2 but not more. It would be interesting to see what the minimum or maximum threshold doses were for optimal cell growth. I also wonder if the cell density effect is linear or if there is some particular FGF-2 dosage that would cause a sudden exponential decrease in cell growth. I actually don't see a difference between the control and low dose FGF-2. It would be interesting to see comparisons of FGF-2 to other type growth factors. I don't understand clearly why they chose to study this one so extensively. It may have been more productive experimentally to first screen several growth factors, pin point huge differences in growth trend, and then study those select cultures more closer with PCR, etc.
@Charles I do agree with your critique of their RT-PCR, and the banding patterns they see do seem similar. A more specific, quantitative assay is needed to properly differentiate between the gene expression of the different cultures.
@aavestro Again, I agree that using their RT-PCR result for quantitative purposes is a bit suspect. As for your argument that the no FGF-2 culture should give the "best" results, I don't think that's necessarily true. It's reasonable to assume that FGF-2 acts in a biphasic manner, and I'm sure there are examples of many other molecules that have one effect at a certain concentration range and a different effect at other ranges.
I agree with the comments. It would be nice to have some justification for choosing 3ng and 30ng as the low and high concenrations. Since the 3ng produces better proliferation than 0ng or 30ng, it would be interesting to find the concentration where cell proliferation peaks.
Also, I wonder if cell migration was affected in anyway by the FGF-2 since it is also an important part of the healing process.
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