Why is it Hard to Make Use of Mesenchymal Stem Cells (MSCs)?

According to Mimeault et al., their research paper Stem Cells: A Revolution in Therapeutics- Recent Advances in Stem Cell Biology and Their Therapeutic Applications in Regenerative Medicine and Cancer Therapies, “stem cells can be used in biomedical research, drug discovery, and toxicity testing, as a model in understanding diseases and for therapeutic purposes in regenerative medicine (252).” However, Akpinar et al. pointed out in their research paper Phenotypic and Proteomic Characteristics of Human Dental Pulp Derived Mesenchymal Stem Cells from a Natal, an Exfoliated Deciduous, and an Impacted Third Molar Tooth that in previous researches, in order to use stem cells successfully, homogeneous populations of stem cells have to be isolated. The level of heterogeneity of MSC lines is very high which leads to lack of consistency in research studies over MSCs. Factors that contribute to the heterogeneity problem include genetic background, physiological/ physical conditions of the donor, cell cycle and environmental conditions that lead to different gene expressions etc.

Phenotypic Profiling of Stem Cells- Yes! Proteomic Approach!

According to Akpinar et al., gene expression profiles are preferred. However this method of profiling for stem cells “vary greatly with the organisms’ state and environment in ways that cannot be easily interpreted (2).” So they proposed that “the signature obtained from analysis of the total cell proteome or cell surface proteome (“protein barcodes”) is promising and proteomic approaches can be powerful in characterizing the entire protein profile of stem cell phenotype from different niches (2).”

Purpose of the Research Paper

The purpose of the research was to “understand the level of heterogeneity among MSCs (2).” In order to do so, Akpinar et al. isolated MSCs from dental pulps of a natal (Fig. A: uncommon teeth present at birth, not well formed: has little root structure), an exfoliated deciduous (erupted baby tooth), and an impacted third molar tooth (Fig. B) of three different donors (which is their research paper title). Stem cells from the three types of teeth were isolated and cultured under the same growth conditions. They then compared the basis of cellular morphology and expression of MSC specific markers and transcription factors, telomerase activity and protein expression profiles.

Fig. A: Natal teeth
http://www.forp.usp.br/bdj/t1062.html

Fig. B Impacted third molar
http://www.bangsardental.com/dental-treatment/surgical-removal.html

Results

First the researches showed the results from the differences and similarities of the 3 tooth samples:

Fig. 1a: Morphological analysis of stem cells in 3 different tooth types- they all have large, flattened, or fibroblast-like shape.
Fig. 1b: Growth curves (proliferation rates) for the 3 different tooth types over 25 days. DPSCs showed a lower growth rate than the other two.

Table 1: Highlighted MSC markers are expressed by all three stem cell lines

Fig. 2: Cell cycle analysis showed that the majority of stem cells are in G1 phase. In this phase, cells are growing in size and synthesizing mRNA and proteins.

Fig. 3: Telomerase shortening is an indicator of stem cell aging. This figure shows that as the donor age increases, the telomerase activity decreases. There is a 30% decrease in SHED compared to SHED and DPSCs has the lowest telomerase activity.

Then they analyzed the proteomics of 3 different tooth types:

Fig. 5: (a) shows the proteins isolation for each tooth type via SDS-PAGE. (b) shows that there are 183 protein spots that are matched by all 3 tooth types via 2DIGE. (c) shows the amount of proteins conserved (green), upregulated(blue) and downregulated (red) within the matching spots.

The researches identified the conserved proteins and analyzed them:

Fig.6 The 61 identified protein classification based on their molecular function and involvement in biological pathways.

Figure 6 shows that the majority of the isolated proteins play a role in cell architecture (11 proteins) and protein folding/ inducible chaperons expressed under stress conditions. All the other functions showed in the pie chart help explain the cell ability of self-renewal and proliferation.

Discussion- The proteins

Structure:

– Actin Related Protein Complex regulates tight junctions and function in establishment of branched actin networks. However the existence of this protein has not been described in MSCS (6).

– Calponin regulates smooth muscle contraction and was found in human hair follicle derived stem cells and bone marrow derived stem cells in previous studies (6).

– Caldeson indicates slow growth rate. This may be indication of the defined medium of the experiment.

Metabolism/ detoxification:

– glutathion S-transferase-P: detoxification in cells from xenobiotics and decreases susceptibility to cancer. Previous studies showed that expression of this protein indicates that cells have multipotent MSC properites.

– SOD (Superoxide dismutase): reactive oxygen species found in the 3 types of tooth stem cells. Previous studies showed that these proteins protect cells from antioxidants and may have proliferative effects by eliminating peroxides (14).

– DJ-1 is a chaperone protein that protects cells against oxidative stress and cell death (15). Role in Parkinson’s is well known but the significance in stem cells are not well studied.

Calcium binding Metabolism

– S100 helps with cell proliferation, survival and differentiation of human osteosarcoma cells (15).

Transcription/Nucleotide Metabolism:

– PSPC1’s function is still not well known. Previous studies reported that this type of protein only occur upon differentiation (embryonic stem cells). However, Akpinar et al. showed that this protein is expressed in human mesenchymal stem cells and the expression is not required by a differentiation process (15).

Protein-folding:

– PIN1 accelerates folding of proteins. Previous studies showed that this protein promotes survival, enhance repair, improve differentiation, and antagonize senescence (15). Akpinar et al. suggested that this protein may have significant implications in regenerative medicine.

Conclusion

With the isolated proteins, the researchers have listed out the heterogeneity and characteristics/ functions of these proteins among the 3 tooth types. In which the isolated proteins are conserved throughout the life cycle of humans. In other words, the features related to morphology, proliferation rates, expression of various cell surface markers, and differentiation potentials are similar in these conserved proteins (17). As listed in the discussion part, there are proteins that are not well studied such as DJ-1 that plays a role in Parkinson’s disease. By listing out these proteins and analyzing their characteristics and functions, it served an important base-work for future MSC studies to further understand these proteins and how they can help with regenerative medicine.

References

Akpinar, Gurler et al. Phenotypic and Proteomic Characteristics of Human Dental Pulp Derived Mesenchymal Stem Cells from a Natal, an Exfoliated Deciduous, and an Impacted Third Molar Tooth. Stem Cells International, Oct. 2014. Web. Feb. 28, 2015.