Effect of Human Platelet Lysate as Cultivation Nutrient Supplement on Human Natal Dental Pulp Stem Cell In Vitro Expansion

Despite several scientific or ethical issues, fetal bovine serum (FBS) remains the standard nutrient supplement in the mesenchymal stem cell cultivation medium. Cell amplification plays an important role in human stem cell therapies. Increasing interest in this field has supported attempts to find suitable human alternatives to FBS for in vitro cell propagation. Human platelet lysate (hPL) has recently been determined as one of them. Our study aimed to evaluate the influence of 2% hPL in the growth medium for in vitro expansion of human natal dental pulp stem cells (hNDP-SCs). The effect was determined on proliferation rate, viability, phenotype profile, expression of several markers, relative telomere length change, and differentiation potential of four lineages of hNDP-SCs. As a control, hNDP-SCs were simultaneously cultivated in 2% FBS. hNDP-SCs cultivated in hPL showed a statistically significantly higher proliferation rate in initial passages. We did not observe a statistically significant effect on mesenchymal stem cell marker (CD29, CD44, CD73, CD90) or stromal-associated marker (CD13, CD166) expression. The cell viability, relative telomere length, or multipotency remained unaffected in hNDP-SCs cultivated in hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.


Introduction
The occurrence of natal and neonatal teeth in humans is an uncommon anomaly of premature tooth eruption. Natal teeth are teeth present at birth, and neonatal teeth are teeth that erupt within the first month of life [1]. They might further accompany various difficulties, such as pain on sucking and refusal to feed, faced by the mother or child [1,2]. Although the eruption of the lower deciduous incisors is normal at birth in many mammals, natal and neonatal teeth are rare in humans. The incidence of natal teeth ranges from 1:2000 to 1:3500 live birth [1][2][3][4][5]. Only 1-10% of natal and neonatal teeth are supernumerary, and more than 90% of natal teeth and neonatal teeth are prematurely erupted primary teeth [6]. Natal teeth are three times more common than neonatal teeth. They most frequently occur in the mandibular central incisor region.
The pulp of natal teeth is a source of a unique type of dental pulp-related stem cells that display some of the characteristics of pluripotency [7,8]. In comparison with dental pulp stem cells isolated from the permanent (DPSCs) of deciduous teeth (SHED), human natal dental pulp stem cells (hNDP-SCs) have a higher proliferation rate and express similar surface markers CD13, CD29, CD44, CD73, CD90, CD146, and CD166. Interestingly, hNDP-SCs express detectable levels of factors, such as Nanog, octamer-binding transcription

Materials and Methods
Study guidelines were approved by the Ethics Committee of the University Hospital Hradec Kralove (201812 S07P, approved 8 November 2018). Legal representatives of donors signed informed written consents before natal tooth collection.

Culture Media
Cells seeded in adherent surface culture dishes were cultivated under standard conditions in a humidified atmosphere containing 5% CO2 at 37 • C. Every three days, we removed the detritus and non-adherent elements by washing dishes with a phosphate-buffered saline (PBS, (Sigma-Aldrich) and exchanged each culture medium. Each lineage was detached from the adherent surface using 0.05% Trypsin-EDTA (Gibco, Thermo Fisher Scientific) and passaged when hNDP-SCs reached approximately 70% confluence. Subsequently, it was reseeded in a final concentration of 5000 cells/cm 2 . All cell lines were terminated in the 14th passage (14p).
The hPL was obtained from the Transfusion department, University Hospital Hradec Kralove, Czech Republic. Briefly, the preparation protocol started with platelet-rich plasma (PRP) units derived from buffy coats. After a sterility check, PRP units were frozen to at least −20 • C in the original storage bags. When the bacterial test was negative, hPL units were thawed at 37 • C (water bath) until the ice clots disappeared. To decrease individual platelet variation, one hPL unit was pooled from five randomly chosen units donated by healthy individuals, subject to guidelines for the selection of blood donors. To increase the rate of platelet fragmentation and the number of released growth factors, hPL units were further re-frozen and re-thawed. Subsequently, the hPL units were centrifuged to discard the platelet pellets and keep the supernatant rich in factors.

Effect on hNDP-SC Proliferation and Viability
We count a total cell count in each passage using a Z2-Counter (Beckman Coulter, Miami, FL, USA). For each measurement, we analyzed 100 µL/1 mL cell suspension (approximately 1.5 × 10 6 cells/1 mL). Proliferation capacity was evaluated as population doublings (PDs) and population doubling time in hours (Equation (1)). Proliferation activity was compared from the 1st passage when hNDP-SCs were seeded into two different growth media.
Equation (1). N x is the total passage cell count calculated using the Z2-Counter (Beckman Coulter), and N 1 is the initial cell count seeded into the culture dish (5000 cells/cm 2 ).
To determine the number of viable hNDP-SCs cells from each sample in the 3rd and 11th passage, we used the trypan dye exclusion method by Vi-Cell analyzer (Beckman Coulter). This method is based on the principle that viable cells do not take up the trypan blue dye, whereas non-viable cells do due to disturbed cell membrane. For each analysis, we utilized 250 µL of cell suspension (approximately 1.5 × 10 6 cells/1 mL) and 250 µL PBS.

Effect on Relative Telomere Length
To assess the effect of cultivation with hPL on the relative telomere length measured using real-time polymerase chain reaction (qPCR) in the 3rd and 14th passages (Equation (2)). The analysis protocol was used in previous studies [27][28][29]. After the DNA isolation using a DNeasy Tissue Kit (Qiagen, Hilden, Germany), we calculated its concentration in each sample using a spectrophotometer Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA).
We performed the qPCR in 96-well plates and analyzed each sample in triplicates at the same well position on an ABI 7500 HT detection system (Applied Biosystems, Foster City, CA, USA). Each 20 µL re-action consisted of 20 ng DNA, 1 × SYBR Green master mix (Applied Biosystems), 200 nM forward telomere primer (CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT), and 200 nM reverse telomere primer (GGC TG TCT CCT TCT CCT TCT CCT TCT CCT TCT CCT). We used the following primer pairs for the housekeeping gene analysis: 36B4u, CAG CAA GTG GGA AGG TGT AAT CC; 36B4d, CCC 135 ATT CTA TCA TCA ACG GGT ACA A. The cycling of each qPCR analysis (for both telomere and housekeeping gene) started with a ten-minute cycle at 95 • C, followed by 15-s cycles at 95 • C, ending with a one-minute cycle at 60 • C.

Effect on hNDP-SC Multipotency
For osteogenic, chondrogenic, or adipogenic induction, hNDP-SCs from the 4th passage were seeded into separated culture dishes and grown to 80-100% confluence. Subsequently, they were incubated in differentiation media.

Osteogenic Differentiation In Vitro
After hNDP-SCs reached confluence, we induced osteogenic differentiation with the FBS-free differentiation medium containing α-MEM (Sigma-Aldrich), 0.5 mM ascorbic acid (Bieffe Medital), 10 mM of β-glycerophosphate (Sigma-Aldrich), 0.1 µM of dexamethasone (Bieffe Medital), and 10% hPL (Transfusion Department, University Hospital Hradec Kralove, Czech Republic). As a standard osteogenic differentiation medium, we used the Human Mesenchymal Stem Cell Osteogenic Differentiation Medium BulletKit TM (Lonza, Basel, Switzerland). Both media were exchanged after the washing step using PBS twice a week. hNDP-SCs were cultivated under differentiation conditions for 3 weeks. At the end of the third week, the pellets were fixed using 10% formalin, dehydrated in ascending concentrations of ethanol, embedded in paraffin, and cut into 7 µm thick sections. Osteogenic differentiation was assessed via staining with Alizarin Red S and von Kossa staining to locate calcium deposits in the extracellular matrix. Osteocalcin was detected using immunocytochemistry. After deparaffination, samples were exposed to a primary mouse IgG antibody (1:50, Millipore, Burlington, MA, USA) and donkey anti-mouse secondary IgG antibody (1:250, Jackson ImmunoResearch Labs, West Grove, PA, USA).

Chondrogenic Differentiation In Vitro
In hNDP-SCs cultivated in FBS free cultivation medium, we induced chondrogenic differentiation using a medium containing α-MEM (Sigma-Aldrich), 0.5 mM ascorbic acid (Bieffe Medital), 10 mM of β-glycerophosphate (Sigma-Aldrich), 0.1 µM of dexamethasone (Bieffe Medital), and 50 ng/mL TGF-β1 (Stem Cell Technologies, Vancouver, BC, Canada). Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium BulletKit TM (Lonza) was used as a control medium supplemented with FBS. We changed both media twice a week and cultivated cells for 3 weeks. After three weeks, we prepared 7 µm thick paraffin sections from fixed differentiated cell pellets. Afterward, we detected collagen and procollagen in the extracellular matrix using blue Masson's trichrome stain and specified type II collagen with immunochemical detection. Slices were incubated with a primary mouse IgM antibody (1:500, Sigma-Aldrich) and Cy3TM-conjugated goat anti-mouse secondary IgM antibody. Cell nuclei were counterstained with 4 -6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Furthermore, we also stained acid mucopolysaccharides in the chondrogenic matrix using Alcian blue histological staining.

Adipogenic Differentiation In Vitro
To induce pro-adipogenic conditions, HNDP-SCs grown without FBS were cultivated using the Mesenchymal Stem Cell Adipogenic Differentiation Medium kit (Cyagen Biosciences, Santa Clara, CA, USA), differentiation Basal Medium A and B containing 10% hPL instead of FBS. hMSC Adipogenic medium kit (Lonza), induction medium, and maintenance medium induced adipogenesis in cells cultivated in a medium with FBS. Cells were exposed to pro-adipogenic conditions for four weeks. Two different media from each kit were subsequently used and switched every three days for three weeks. Last week, hNDP-SCs were cultivated only in the hMSC Adipogenic Maintenance medium or Differentiation Basal Medium B. Cultures were then fixed with 10% formalin and rinsed with 50% ethanol. The presence of intracellular lipid droplets, which indicates that adipogenic differentiation occurred, was confirmed by Oil Red O staining.

Statistical Analysis
The data are presented as the mean ± SD. All statistical analyses were performed using the statistical software GraphPad Prism 9.3.0 (San Diego, CA, USA). The Shapiro-Wilk or Kolmogorov-Smirnov tests were used for normal distribution evaluations. The statistical significances (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) were calculated using either the paired t-test for continuous variables or the Wilcoxon matched-pairs test for nonparametric variables.

Results
Independently on the culture medium, hNDP-SCs initially had a rounded spindle-like shape with elongated processes reaching the surrounding cells ( Figure 1). letKit (Lonza) was used as a control medium supplemented with FBS. We changed both media twice a week and cultivated cells for 3 weeks. After three weeks, we prepared 7 μm thick paraffin sections from fixed differentiated cell pellets. Afterward, we detected collagen and procollagen in the extracellular matrix using blue Masson's trichrome stain and specified type II collagen with immunochemical detection. Slices were incubated with a primary mouse IgM antibody (1:500, Sigma-Aldrich) and Cy3TM-conjugated goat antimouse secondary IgM antibody. Cell nuclei were counterstained with 4′-6-diamidino-2phenylindole (DAPI, Sigma-Aldrich). Furthermore, we also stained acid mucopolysaccharides in the chondrogenic matrix using Alcian blue histological staining.

Adipogenic Differentiation In Vitro
To induce pro-adipogenic conditions, HNDP-SCs grown without FBS were cultivated using the Mesenchymal Stem Cell Adipogenic Differentiation Medium kit (Cyagen Biosciences, Santa Clara, CA, USA), differentiation Basal Medium A and B containing 10%hPL instead of FBS. hMSC Adipogenic medium kit (Lonza), induction medium, and maintenance medium induced adipogenesis in cells cultivated in a medium with FBS. Cells were exposed to pro-adipogenic conditions for four weeks. Two different media from each kit were subsequently used and switched every three days for three weeks. Last week, hNDP-SCs were cultivated only in the hMSC Adipogenic Maintenance medium or Differentiation Basal Medium B. Cultures were then fixed with 10% formalin and rinsed with 50% ethanol. The presence of intracellular lipid droplets, which indicates that adipogenic differentiation occurred, was confirmed by Oil Red O staining.

Statistical Analysis
The data are presented as the mean ± SD. All statistical analyses were performed using the statistical software GraphPad Prism 9.3.0 (San Diego, CA, USA). The Shapiro-Wilk or Kolmogorov-Smirnov tests were used for normal distribution evaluations. The statistical significances (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) were calculated using either the paired t-test for continuous variables or the Wilcoxon matched-pairs test for nonparametric variables.

Results
Independently on the culture medium, hNDP-SCs initially had a rounded spindlelike shape with elongated processes reaching the surrounding cells ( Figure 1).
The same trend, but less significant, was seen till the 8th passage when the proliferation rate of both groups became nearly the same. The proliferation rate of hNDP-SCs cultured in the hPL-culture medium was faster again between the 10th and 12th passage, but with a lower figure for PD in the 12th passage (cumulative PD in the 12th passage was 53.94 ± 0.89) compared with the hNDP-SCs cultured in the FBS-culture medium (55.87 ± 0.95). At the end of cultivation, the proliferation rate for hPL-cultivated hNDP-SCs c slowed down. Figure 2a,b illustrates the proliferation capacity of both groups displaying cumulative PDs and PDT in hours.
After several passages, they became prolonged and more spindled. Interestingly, during initial passages (mainly 2p-5p), hNDP-SCs grown in the hPL-culture medium proliferated faster than their counterparts. The average PDT (2p-5p) per passage for hNDP-SCs grown in the hPL-culture medium was 22.65 ± 0.10 h (average PD per passage (2p-5p) was 3.97 ± 0.35). The figure for hNDP-SCs grown in the FBS-culture medium was 44.69 ± 1.36 h (PD = 3.98 ± 0.38). This difference was statistically significant (p < 0.0001). The same trend, but less significant, was seen till the 8th passage when the proliferation rate of both groups became nearly the same. The proliferation rate of hNDP-SCs cultured in the hPLculture medium was faster again between the 10th and 12th passage, but with a lower figure for PD in the 12th passage (cumulative PD in the 12th passage was 53.94 ± 0.89) compared with the hNDP-SCs cultured in the FBS-culture medium (55.87 ± 0.95). At the end of cultivation, the proliferation rate for hPL-cultivated hNDP-SCs c slowed down. Figure 2a,b illustrates the proliferation capacity of both groups displaying cumulative PDs and PDT in hours. The percentages of viable cells were established using the trypan blue exclusion method in the 3rd and 11th passages. The figures for hPL-cultivated and FBS-cultivated hNDP-SCs were over 90% for the entire cultivation time (Figure 3). The percentages of viable cells were established using the trypan blue exclusion method in the 3rd and 11th passages. The figures for hPL-cultivated and FBS-cultivated hNDP-SCs were over 90% for the entire cultivation time (Figure 3).
After several passages, they became prolonged and more spindled. Interestingly, during initial passages (mainly 2p-5p), hNDP-SCs grown in the hPL-culture medium proliferated faster than their counterparts. The average PDT (2p-5p) per passage for hNDP-SCs grown in the hPL-culture medium was 22.65 ± 0.10 h (average PD per passage (2p-5p) was 3.97 ± 0.35). The figure for hNDP-SCs grown in the FBS-culture medium was 44.69 ± 1.36 h (PD = 3.98 ± 0.38). This difference was statistically significant (p < 0.0001). The same trend, but less significant, was seen till the 8th passage when the proliferation rate of both groups became nearly the same. The proliferation rate of hNDP-SCs cultured in the hPLculture medium was faster again between the 10th and 12th passage, but with a lower figure for PD in the 12th passage (cumulative PD in the 12th passage was 53.94 ± 0.89) compared with the hNDP-SCs cultured in the FBS-culture medium (55.87 ± 0.95). At the end of cultivation, the proliferation rate for hPL-cultivated hNDP-SCs c slowed down. Figure 2a,b illustrates the proliferation capacity of both groups displaying cumulative PDs and PDT in hours. The percentages of viable cells were established using the trypan blue exclusion method in the 3rd and 11th passages. The figures for hPL-cultivated and FBS-cultivated hNDP-SCs were over 90% for the entire cultivation time (Figure 3). passages. The data are presented as the mean ± SD. The Shapiro-Wilk test or Kolmogorov-Smirnov test were used for normal distribution evaluations. The statistical analysis was calculated using the paired t-test. The difference was not statistically significant.

Effect on hNDP-SC Phenotype Profile and Specific Factor Expressions
Defined markers exist that specifically and uniquely identify mesenchymal stem cells. The flow cytometry analysis of all common mesenchymal, hematopoietic stem cell markers indicated that hNDP-SCs grown in the hPL-culture medium were highly positive (>95%) to most of the tested markers. On the other hand, the same trend was not observed in FBS-cultivated hNDP-SCs. We did not observe statistically significant variances in CD markers defined as mesenchymal stem cell markers CD29, CD44, CD73, CD90, or stromal-associated markers CD13, CD166. Both groups showed high average expression of these markers (<90%). The protein tyrosine phosphatase (CD45) was highly expressed in hPL-cultivated hNDP-SCs, but lowly expressed in FBS-cultivated hNDP-SCs (>10%). The markers CD34, CD105, and CD146, differed statistically significantly in comparison between groups (Figure 4).   Remarkably, hNDP-SCs express detectable levels of the embryonic stem cell (ESC) markers or neuronal markers. We performed immunocytochemistry to determine whether this spontaneous ability is independent of culture medium composition. Undifferentiated hNDP-SCs were stained with primary antibodies against Beta3-tubulin (an early neuronal marker [30]), Nestin (neural progenitor marker [30]), neurofilaments (a late neuronal marker [30]), and Nanog (a marker known for its functions in ESC pluripotency, maintenance, and self-renewal [31]). The following pictures showed that the markers mentioned above were positively expressed in most of the cells independently whether we used FBSor hPL-supplemented medium for their cultivation (Figures 5-8).
Remarkably, hNDP-SCs express detectable levels of the embryonic stem cell (ESC) markers or neuronal markers. We performed immunocytochemistry to determine whether this spontaneous ability is independent of culture medium composition. Undifferentiated hNDP-SCs were stained with primary antibodies against Beta3-tubulin (an early neuronal marker [30]), Nestin (neural progenitor marker [30]), neurofilaments (a late neuronal marker [30]), and Nanog (a marker known for its functions in ESC pluripotency, maintenance, and self-renewal [31]). The following pictures showed that the markers mentioned above were positively expressed in most of the cells independently whether we used FBS-or hPL-supplemented medium for their cultivation (Figures 5-8).  used for normal distribution evaluations. The statistical analyses were calculated using the paired t-test (* p < 0.05, *** p < 0.001, **** p < 0.0001).
Remarkably, hNDP-SCs express detectable levels of the embryonic stem cell (ESC) markers or neuronal markers. We performed immunocytochemistry to determine whether this spontaneous ability is independent of culture medium composition. Undifferentiated hNDP-SCs were stained with primary antibodies against Beta3-tubulin (an early neuronal marker [30]), Nestin (neural progenitor marker [30]), neurofilaments (a late neuronal marker [30]), and Nanog (a marker known for its functions in ESC pluripotency, maintenance, and self-renewal [31]). The following pictures showed that the markers mentioned above were positively expressed in most of the cells independently whether we used FBS-or hPL-supplemented medium for their cultivation (Figures 5-8).

Effect on Relative Telomere Length
Many studies have reported that the telomere length shortening is a hallmark of cell senescence, and the maintenance of their length is essential for cell self-renewal ability and differentiation potential [32,33]. Therefore, we performed qPCR to explore whether there are variances in relative telomere length between hNDP-SCs cultivated in two different media. We studied the differences in relative telomere length changes between the 3rd and 14th passages. Independently of the culture medium, we observed the statistically significantly shorter relative telomere length in the 14th passage than in the 3rd passage. The shortening was more noticeable in the case of hNDP-SCs cultivated in the standard FBS-culture medium (Figure 9).

Effect on Relative Telomere Length
Many studies have reported that the telomere length shortening is a hallmark of cell senescence, and the maintenance of their length is essential for cell self-renewal ability and differentiation potential [32,33]. Therefore, we performed qPCR to explore whether there are variances in relative telomere length between hNDP-SCs cultivated in two different media. We studied the differences in relative telomere length changes between the 3rd and 14th passages. Independently of the culture medium, we observed the statistically significantly shorter relative telomere length in the 14th passage than in the 3rd passage. The shortening was more noticeable in the case of hNDP-SCs cultivated in the standard FBS-culture medium (Figure 9).

Effect on Relative Telomere Length
Many studies have reported that the telomere length shortening is a hallmark of cell senescence, and the maintenance of their length is essential for cell self-renewal ability and differentiation potential [32,33]. Therefore, we performed qPCR to explore whether there are variances in relative telomere length between hNDP-SCs cultivated in two different media. We studied the differences in relative telomere length changes between the 3rd and 14th passages. Independently of the culture medium, we observed the statistically significantly shorter relative telomere length in the 14th passage than in the 3rd passage. The shortening was more noticeable in the case of hNDP-SCs cultivated in the standard FBS-culture medium (Figure 9). Biomolecules 2022, 12, x FOR PEER REVIEW 12 of 21 Figure 9. Average relative telomere length measured between 3rd and 14th passages using qPCR. Both groups of hNDP-SCs experienced the shortening of relative telomere length in the 14th passage (* p < 0.05), but it was more noticeable in the case of hNDP-SCs cultivated in the medium with 2% FBS. The data are presented as the mean ± SD. The Shapiro-Wilk test or Kolmogorov-Smirnov test were used for normal distribution evaluations. The statistical analyses were calculated using the paired t-test.

Effect on hNDP-SC Multipotency
The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human mesenchymal stem cells, where hNDP-SCs belong. One criterion is that they must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. We proved that hNDP-SCs cultivated in an hPLor FBS-culture medium kept the ability to differentiate in osteogenic, chondrogenic, and adipogenic cell lines. To confirm our results, we used histologic staining and immunocytochemistry. The following Figures 10-14 show our results.
(a) (b) (c) Figure 9. Average relative telomere length measured between 3rd and 14th passages using qPCR. Both groups of hNDP-SCs experienced the shortening of relative telomere length in the 14th passage (* p < 0.05), but it was more noticeable in the case of hNDP-SCs cultivated in the medium with 2% FBS. The data are presented as the mean ± SD. The Shapiro-Wilk test or Kolmogorov-Smirnov test were used for normal distribution evaluations. The statistical analyses were calculated using the paired t-test.

Effect on hNDP-SC Multipotency
The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human mesenchymal stem cells, where hNDP-SCs belong. One criterion is that they must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. We proved that hNDP-SCs cultivated in an hPL-or FBS-culture medium kept the ability to differentiate in osteogenic, chondrogenic, and adipogenic cell lines. To confirm our results, we used histologic staining and immunocytochemistry. The following Figures 10-14 show our results.
Biomolecules 2022, 12, x FOR PEER REVIEW 12 of 21 Figure 9. Average relative telomere length measured between 3rd and 14th passages using qPCR. Both groups of hNDP-SCs experienced the shortening of relative telomere length in the 14th passage (* p < 0.05), but it was more noticeable in the case of hNDP-SCs cultivated in the medium with 2% FBS. The data are presented as the mean ± SD. The Shapiro-Wilk test or Kolmogorov-Smirnov test were used for normal distribution evaluations. The statistical analyses were calculated using the paired t-test.

Effect on hNDP-SC Multipotency
The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human mesenchymal stem cells, where hNDP-SCs belong. One criterion is that they must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. We proved that hNDP-SCs cultivated in an hPLor FBS-culture medium kept the ability to differentiate in osteogenic, chondrogenic, and adipogenic cell lines. To confirm our results, we used histologic staining and immunocytochemistry. The following Figures 10-14 show our results.

Discussion
An increasing interest in mesenchymal stem cells and their role in regenerative and reparative medicine has brought many concerns and limitations that should be considered before their future broader use, especially in human medicine. One of the limitations is the widely used standard medium supplement and source of growth factors for cell culture, fetal bovine serum. Regenerative cells occur in low doses in origin tissues. Therefore, they must be amplified after isolation to obtain a suitable dose for their clinical applica-

Discussion
An increasing interest in mesenchymal stem cells and their role in regenerative and reparative medicine has brought many concerns and limitations that should be considered before their future broader use, especially in human medicine. One of the limitations is the widely used standard medium supplement and source of growth factors for cell culture, fetal bovine serum. Regenerative cells occur in low doses in origin tissues. Therefore, they must be amplified after isolation to obtain a suitable dose for their clinical application. hNDP-SCs are not an exception. Even though the risks of xenoimmunization against bovine antigens, the transmission of pathogens, and ethical issues associated with FBS collection are well known [9][10][11]34], FBS remains the standard growth factor supplement in most laboratory cultivation protocols. Therefore, it is a rational and multiple basic scientific research supported 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 regarding the 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. Using human serum (HS) might seem the most straightforward solution. Unfortunately, it has been published that this method is unreliable, MSC proliferation is slow, and cells have difficulty reaching the desired confluence [35]. The platelet-rich plasma has been shown to enhance MSC proliferation [12,[36][37][38], but the present debris in PRP might disturb cell culture. We concluded the same results in our previous study [39]. Although we came up with sufficient results in the hNDP-SC cultivation in 2% PRP, we had to deal with handling difficulties thanks to debris during the entire cultivation. Furthermore, it is necessary to activate thrombocytes to release growth factors. The use of human platelet lysate was first described in 2005 [16]. Thus far, hPL enriched in growth factors (such as platelet-derived growth factor) is particularly used for human MSCs, endothelial, and fibroblast cultures. Variations exist between individual hPL, but that can be solved by pooling. It is rarely distributed commercially [16,26]. Furthermore, all human blood-derived substituents remain a threat to the transmission of human diseases by known or unknown 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 to verify the effect of hPL supplemented culture medium on hNDP-SCs. The effect determination was based on studying the proliferation capacity, viability, expression of specific markers, and relative telomere length. The study aimed to determine the consequences of hPL on hNDP-SC multipotency. The study included four lineages of hNDP-SCs isolated from two newborns (male and female).
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, blood sources, and the different concentrations of platelets or growth factors make establishing optimal hPL concentration difficult. However, most recent studies have agreed that increasing the concentration of hPL negatively affects the MSC proliferation rate [40,41]. Chen et al. concluded that when dental pulp stem cells were cultivated in 10% hPL, cell proliferation was significantly inhibited. The 1% and 5% hPL enhanced the cell growth, but 5% was the most effective concentration for the proliferation and mineralization of DPSCs [40]. We expanded four lineages of hNDP-SCs in α-MEM culture medium supplemented with either 2% FBS or 2% hPL. Our previous studies established the 2% concentration of human blood components as the most effective in dental pulp-related stem cell cultivation [42]. Therefore, we established 2% of FBS as a standard.
hPL in 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 ( [26,36,39,40,43,44]. At the end of cell growth, we observed the prolongation of population doubling time in the hPL-treated group. For potential clinical application, it would be necessary to amplify the total cell count initially, and hPL-treated cells revealed extensive proliferation, especially in the beginning. The initial viability measured using trypan blue exclusion methods was also non statistically significantly higher. Oppositely, we observed higher percentages of viable cells cultivated with FBS in the later passage (11th passage).
Interestingly, hPL showed high expression of all tested markers (<90%). These results differ from other studies where no effect of the medium supplement was observed [44][45][46]. There was no statistical difference in mesenchymal stem cell 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, and CD146 varied significantly between groups. Since our results are different from other studies, we can only hypothesize the reasons for significant variances in the expression of tested CD markers. A recent study determined that higher CD10 expression identifies high proliferation in perivascular progenitor cells [47]. CD34 marker is taken as a hematopoietic stem cell marker. However, it has been published that it is not always true [48,49], and it should be re-evaluated. The CD45 marker is considered to be expressed in cells of the hematopoietic system. Still, it has also been published that CD45+ mesenchymal stem cell morphology is similar to CD45− and the multilineage differentiation potential of CD45+ MSCs is well preserved [50]. The stem cell marker 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 insulinlike growth factor 1 (IGF-1), 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 [51]. To summarize all the above, hNDP-SCs seem more affected by changes in serum-free growth medium than other mesenchymal stem cells, and 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 [52], and using two methods at least for phenotype analysis is recommended, further investigation is needed before we can be able to conclude such results. Independently on the nutrient supplement used in the cultivation medium, undifferentiated hNDP-SCs kept their ability to express specific markers (Beta3-tubulin, Nestin, neurofilaments, and Nanog), suggesting that these cells display some of the characteristics of 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 length in hNDP-SCs grown in hPL in the 3rd passage than in hNDP-SCs grown in FBS, where the trend was opposite in the 14th passage. In our previous study, we observed that the compensatory mechanism of telomerase activity might be time-dependent. The necessary excessive in vitro cultivation leads to telomere attrition. This idea is supported in the current study because we also observed significant telomere attrition in both groups of cells when we compared figures between the 3rd and 14th passages. However, hNDP-SCs cultivated in hPL proliferated faster in initial passages. Therefore, the compensatory effect of telomerase was inefficient due to lack of time, but at the end of cultivation, the proliferation rate slowed down. Therefore, the compensatory effect was sufficient compared to hNDP-SCs cultivated in the standard cultivation medium [29].
We also triggered osteogenesis, chondrogenesis, and adipogenesis in both groups of cells. We determined the successful differentiation using histological staining or immunocytochemistry. We did not observe any variances. Independently on the nutrient supplement used in the growth medium, all hNDP-SCs were able to keep their multipotency and differentiate in mature cell lines.
Since the hNDP-SCs seem to be able to stay in the less differentiated state due to their embryonic origin from the neural crest and neurotropic character, it would be interesting to study their therapeutic potential through paracrine action of extracellularly released components. The therapeutic applicability of mesenchymal dental stem cells and exosomes in dental practice, maxillofacial defects, neurodegenerative diseases, and many other difficultly treatable diseases has been recently published [53], while the importance of evolutionally young stem cells for future regenerative therapies was stressed. The extracellular vesicles have also been discussed as an important diagnostic marker and indicator of targeted cancer therapies. Natal teeth are the source of evolutionally young stem cells; therefore, they might be promising for future regenerative therapies.

Conclusions
Our study aimed to evaluate the effect of hPL as a nutrient supplement during in vitro expansion of hNDP-SCs in compassion with standard FBS. Both supplements were in the total concentration of 2% in the cultivation medium. hNDP-SCs cultivated in hPL showed a significantly higher proliferation rate in initial passages. We did not observe the statistically significant effect on mesenchymal stem cells marker (CD29, CD44, CD73, CD90) or stromalassociated marker (CD13, CD166) expression. Cell viability, relative telomere length, or multipotency of HNDP-SCs remained unaffected during cultivation in 2% hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.  Informed Consent Statement: Informed consent was obtained from all subject legal representatives involved in the study. Data Availability Statement: All data generated or analyzed during this study are included in this published article.