An increasing interest in mesenchymal stem cells and their role in regenerative and reparative medicine has brought many concerns and limitations that should be taken into consideration before their future broader use, especially in human medicine. One of the limitations is fetal bovine serum, the widely used standard medium supplement and source of growth factors for cell culture. Regenerative cells occur in low doses in origin tissues. Therefore, they must be amplified after their isolation to obtain a suitable dose for clinical application. hNDP-SCs are not an exception. Even though the problems associated with using FBS are well known (risks of xenoimmunization against bovine antigens, the transmission of pathogens, ethical issues associated with FBS collection) (9–11, 37), FBS remains the standard growth factor supplement in most laboratory cultivation protocols. Therefore, it is rationale to find suitable human alternatives for in vitro cell propagation. This has become more important with the rapidly growing field of advanced cell therapy, where the use of FBS should be avoided due to international guidelines.
Over the past two decades, various human alternatives have been tested for their ability to sustain the proliferation and differentiation of cells in culture. The use of human serum might seem the most straightforward solution. Unfortunately, studies have indicated that this method is not reliable, and MSC proliferation is slow – cells reach the desired confluence only with difficulty (38). Platelet-rich plasma has been shown to enhance MSC proliferation (12, 39–41), but the debris present in PRP might disturb cell culture. Furthermore, it is necessary to activate thrombocytes to release growth factors. The use of human platelet lysate was first described in 2005 (16). So far, hPL enriched in growth factors (such as platelet-derived growth factor) is used predominately for human MSCs, endothelial, and fibroblast culture. Whereas FBS is readily available as a by-product of slaughterhouse procedures, hPL is generated by the freeze-thaw process (in which waste products form after the expiration of platelet units). Variations exist between individual hPL, but that issue can be resolved through pooling. One major drawback of hPL, however, is that it is rarely distributed commercially (16, 26). Furthermore, all human blood-derived substituents pose a risk of transmitting human diseases through viruses such as human immunodeficiency virus and human T-lymphotropic virus. Nevertheless, these threats could be decreased by strict adherence to blood bank quality standards.
Our study aimed at verifying the effect of hPL-supplemented culture medium on hNDP-SCs by studying the proliferation capacity, viability, expression of specific markers, and relative telomere length. We sought to determine the consequences of hPL on hNDP-SC multipotency. The study included four lineages of hNDP-SCs isolated from two newborns (one male, one female).
The hPL used in the study was generated from the blood of five healthy donors to eliminate variations. The amount of growth factors in the hPL suggests a possible mechanism of action for cell proliferation. The inconsistent data caused by different preparation protocols, different blood sources, and the different concentrations of platelets or growth factors make it difficult to establish an optimal hPL concentration. However, many recent studies have agreed that increasing the concentration of hPL negatively affects the MSC proliferation rate (42, 43). Chen et al. concluded that, when dental pulp stem cells were cultivated in 10% hPL, significant inhibition of cell proliferation was observed; 1% and 5% hPL enhanced the cell growth, but 5% was the most effective concentration for the proliferation and mineralization of DPSCs (43). We expanded four lineages of hNDP-SCs in α-MEM culture medium supplemented with either 2% FBS or 2% hPL. In one of our previous studies, 2% concentration of human blood components was established as the most effective in dental pulp-related stem cell cultivation (44).
hPL in the culture medium accelerated the proliferation rate of hNDP-SCs at the beginning of the cultivation (2nd passage – 5th passage). hPL-cultivated hNDP-SCs showed approximately two times shorter PDT (22.65 ± 0.10 hours) compared with the FBS-cultivated group (44.69 ± 1.36 hours), while the PDs per passage was approximately the same (3.97 ± 0.35 vs. 3.98 ± 0.38). Our results are comparable with other studies (26, 39, 42, 43, 45, 46). At the end of cell growth, we observed the prolongation of population doubling time in the hPL-treated group. For potential clinical application, the total cell count would need to be amplified in the initial stages following isolation; this, however, should pose no issue given that hPL-treated cells revealed extensive proliferation capabilities particularly in the beginning of the cultivation process. The initial viability measured using trypan blue exclusion methods was also not statistically significantly higher. In contrast, we observed higher percentages of viable cells cultivated with FBS in one of the later passages (11th passage).
Interestingly, hPL showed a high expression of all tested markers (< 90%). These results are different from other studies where no effect of the medium supplement was observed (46–48). There was no statistical difference in mesenchymal stem cells markers (CD29, CD44, CD73, CD90) and stromal associated markers (CD13 and CD166). These markers were also highly positively expressed on hNDP-SCs cultivated in FBS. However, markers CD10, CD34, CD45, CD105, CD146 varied significantly between groups. Since our results are different from other studies (49), we can only hypothesize the reasons for significant variances in the expression of tested CD markers. On the other side, phenotype changes were seen in recent study focusing on hPL in the medium culture for mesenchymal progenitors derived from human-induced pluripotent stem cells (50).
A recent study determined that higher CD10 expression identifies high proliferation in perivascular progenitor cells (51). The CD34 marker is taken as a hematopoietic stem cell marker; however, certain studies have called this into question (52, 53), and further inquiry is needed. CD105, also known as endoglin, is a type I membrane glycoprotein that functions as an accessory receptor for TGF-beta superfamily ligands. Higher expression of CD105 might be explained by the fact that hPL is rich in several growth factors, including insulin-like growth factor 1 (IGF‑1) and transforming growth factor-beta (TGF‑β1, TGF‑β2). Ma et al. concluded that the expression level of CD146 showed a positive correlation with proliferation, differentiation, and immunomodulation, suggesting that CD146 can serve as a surface molecule to evaluate the potency of human dental pulp stem cells cultivated in the serum-free medium (54). To summarize all the above, it seems that hNDP-SCs are more affected by changes in serum-free growth medium than other mesenchymal stem cells, and that hPL keeps hNDP-SCs less differentiated and prepared for wider differentiation into mature cells lines. Nevertheless, since the disadvantages of using flow cytometry as a tool for immunophenotyping have already been published (55), and using two methods for phenotype analysis is recommended, further investigation is needed before we would be able to reach such a conclusion.
Undifferentiated hNDP-SCs kept their ability to express specific markers (Beta3-tubulin, Nestin, neurofilaments, and Nanog) independently of the nutrient supplement used in the cultivation medium, suggesting these cells display some of the characteristics for pluripotency.
We studied the effect of hPL in the cultivation medium on relative telomere length. We evaluated cells in the 3rd and 14th passages using qPCR. We observed shorter relative telomere lengths in hNDP-SCs grown in hPL in the 3rd passage than in hNDP-SCs grown in FBS. In our previous study, we observed that the compensatory mechanism of telomerase activity might be time-dependent. The necessary and excessive in vitro cultivation leads to telomere attrition. This idea is supported in the current study as well; we observed significant telomere attrition in both groups of cells when comparing figures between the 3rd and 14th passages. However, hNDP-SCs cultivated in hPL proliferated faster in initial passages, and the compensatory effect of telomerase was not efficient due to lack of time. The proliferation rate slowed down at the end of the cultivation and, therefore, the compensatory effect was sufficient in comparison with hNDP-SCs cultivated in the standard cultivation medium.
We also triggered osteogenesis, chondrogenesis, and adipogenesis in both groups of cells. We determined the successful differentiation using histological staining and immunocytochemistry. We did not observe any variances. All hNDP-SCs were able to keep their multipotency and differentiate into mature cell lines independent of the nutrient supplement used in the growth medium.