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.

Prelude

This blog will relate the importance of proteomics analysis with mesenchymal stem cells in human teeth. Before going into depth of the relation between stem cells and the human teeth, let us learn about some background in proteomics, methods of proteomics analysis, stem cells and the dental anatomy.

What is Proteomics?

According to News Medical, “proteomics is the large-scale study of proteins, particularly their structures and functions.” The term “proteomics” was used to make an analogy with “genomics” (the study of genomes and genes) since 1997.

http://www.news-medical.net/health/Proteomics-What-is-Proteomics.aspx

Why pay attention to proteomics?

In undergraduate biology courses, emphasis was made on protein structures influences their functions. Furthermore, mRNA is not always translated into protein. In other words, the amount of protein produced depends on the mRNA being transcribed from a particular gene due to the physiological state of the cell [News Medical]. Proteomics became an interesting area to study and research because it gives us better understanding an organism, some protein-related diseases. We can study the conditions in which the mRNA for the protein is transcribed and then translated. We can also study the mechanisms on the post-translational modifications in which affects the proteins’ functions. In diseases, the study of proteomics can help compare the different proteins expressed in healthy and diseased cells and basing on the proteins expressed and the specific functions of the proteins, a hypothesis of the rise of the diseased cells may be formulated which can lead to specific targeting on the proteins via various treatments.

Methods on Proteomics Analysis:

• Immunoassays: ELISA- uses antibodies and colors to identify a protein of interest in a sample

If you are interested, read more here: https://www4.vanderbilt.edu/vapr/elisa

• Two-dimensional Gel Electrophoresis (2D- DIGE) [Figures on the results of 2D-DIGE in the The Significance of Stem Cells: Proteomic Analysis of Normal vs Deep Carious Dental Pulp portion below] – separates molecules in 1D electrophoresis first then separate them by a second property in a direction of 90 degrees from the first. The proteins that have two distinct properties will be shown on the gel effectively.

What are Mesenchymal Stem Cells (MSCs)?

According to the National Institutes of Health website, stem cells “have the remarkable potential to develop into many different cell types in the body”. Stem cells are unspecialized cells that can renew themselves and under certain physiological/ environmental conditions, they can be induced to become specific/differentiated cells. In this blog, we are concerned about stem cells in dental pulp. This means that we can extract stem cells from dental pulp into other tissues for tissue regeneration.

Read more here: http://stemcells.nih.gov/info/basics/pages/basics1.aspx

The Basic Human Dental-Tooth Anatomy

In this blog, we are focusing on the stem cells in the dental pulp. We will be looking at the protein analysis in normal vs deep carious dental pulp. A human tooth has its pulp chamber in the center of the tooth where all the connective tissue- blood vessels and cells- odontoblasts lie. The pulp is like “the heart” of the tooth, injury to the dental pulp may lead to tooth-death where the cell activity and their signaling processes that help with repairing of the tooth is disrupted. A normal dental pulp is a healthy tooth without caries whereas a deep carious dental pulp is a tooth which has caries where the tooth enamel and dentin has been degraded by the bacterial acid byproduct during their food (sugar) consumption. Just a fun fact: dental plaque- a biofilm found on our teeth surface are in fact colonizing bacteria trying to attach themselves on our teeth. So often when a person just had a sugary meal, more plaque tend to form on the teeth. If plaque builds up on the sides of the crown between the gums, this will lead to bone recession and thus gum recession and eventually lead to gum disease and periodontal disease. So it is important to floss and get those dental plaque out!

The Significance of Stem Cells:
Proteomic Analysis of Normal vs Deep Carious Dental Pulp 

Ma et al. mentioned in their research paper that “human dental stem cells are generally applied in tissue and organ regeneration (1).” They carried out proteomic analysis of dental pulp stem cells (DPSCs) and carious dental pulp stem cells (CDPSCs) as they claimed that DPSCs are “ideal seed cells for tooth tissue regeneration, [and] can differentiate into functional odontoblasts in vivo when the tooth encounters external mild stimuli such as carious lesion, attition and abarsion…[but] the molecular mechanisms responsible for the biological differences between CDPSCs and DPSCs are still unclear (1).” In short, Ma et. al wanted to see what specific proteins play a role in stem cells in dental pulp that may help with dental tissue regeneration.

Before looking for the differences of proteins expressed in CDPSCs and DPScs, Ma et al. confirmed that CDPSCs has a higher proliferation rate than DPSCs by using the cell counting assay. The results were significant as P<0.01.

Fig. 1

In order to better understand the changes in molecular mechanisms of DPSCs when there are deep caries that lead to the biological differences between DPSCs and CDPSCs, Ma et. al then analyzed the proteins expressed in DPSCs and CDPSCs by using 2D-DIGE.

Fig 3 and Fig 4.

2D-DIGE of CDPSCs (3A), DPSCs (3B) and standard proteome map (3C).  Figure 4A and B shows the 18 different proteins expressed in CDPSCs and DPSCs. Ma et al. then selected proteins with functions of cell proliferation and differentiation: CCT2, stathmin and CLIC4.

CCT2: potential positive regulator of cell growth. Can also found in certain malignant tumors. The ability of CDPSCs to have a higher cell proliferation rate may be due to higher expression of CCT2.

Stathmin: involved in intracellular signaling pathways like cell proliferation, microtubule dynamics and activities (5). It was also suggested that stathmin promotes osteoblast differentiation. CDPSCs is more differentiated than DPSCs according to Ma et al. so the expression of this protein is expected to be higher in DPSCs.

CLIC4: found in transmembranes and intramembranes of cells. It is important for membrane trafficking, cell proliferation, differentiation and host defense. This can be evidence that there is an increased expression in CDPSCs due to bacterial infection.

Ma et al. isolated 3 proteins from the dental pulp stem cells that might be responsible for the biological differences between CDPSCs and DPSCs. Furthermore, these proteins are known to be involved with cell proliferation, differentiation, cell cytoskeleton and motility (8). So effort should be put into further studies on these proteins in order to help with dental tissue regeneration.

References

Ma. Dandan et. al. Proteomic Analysis of Mesenchymal Stem Cells from Normal and Deep Carious Dental Pulp. PLoS One, May 2014. Web. 6 Feb 2015.

Image: http://www.angelaharneydentistry.com/blog/post/tooth-anatomy.html