Evaluation of Human Platelet Lysate as an Alternative to Fetal Bovine Serum for Potential Clinical Applications of Stem Cells from Human Exfoliated Deciduous Teeth

Abstract

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H₂O₂. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Keywords: DPSC; FBS alternative; SHED; cell therapy; clinical application of stem cells; hPL; human dental pulp stem cells; human platelet lysate; stem cells derived from human exfoliated deciduous teeth.

Figure 1

Comparison of cell morphology and proliferation effects between 10% FBS, 2% hPL, and 5% hPL. (A) Schematic representation of the experimental setup. Pulps isolated from two teeth obtained from one donor were cultured in media containing either 10% FBS or 5% hPL from the initial time point. Upon reaching passage 3, cells were frozen and subsequently cultured in media supplemented with 10% FBS, 2%, or 5% hPL for experimental purposes. (B) Cell morphologies in each group. F-actin and nucleus were stained with Phalloidin (green) and DAPI (blue), respectively. Cell roundness and area were analyzed using ImageJ software 1.54f with stained images. A total of 160 cells were analyzed, revealing a more spindle-shaped elongated morphology in the hPL groups, with no significant difference observed between the 2% and 5% hPL groups. (C) Proliferation effect. A total of 50,000 cells were seeded onto 60 mm culture plates. The number of cells was counted on days 1, 2, and 3, and the doubling time was calculated. The doubling time was found to decrease in the hPL groups, with shorter times observed at higher concentrations of hPL. (D) CFU (colony-forming units) assay for analysis of self-renewal capacity. A total of 500 cells were seeded onto 100 mm culture dishes, and the assay was conducted on day 10. Data are shown as mean ± SD (n = 3). * p  <  0.05, ** p  <  0.01, **** p  <  0.0001 using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 2

Effects of hPL and FBS on cell migration, and cell survival under oxidative stress. (A) Migration assay utilizing a transwell system. Quantities of 10% FBS-SHEDs or 5% Hpl-SHEDs were seeded with serum-free medium in the upper chamber, and medium containing 10% FBS or 5/10% hPL was added to the lower chamber. Both FBS-SHEDs and hPL-SHEDs exhibited migration toward the hPL-containing medium, while only minimal migration occurred toward FBS-containing media, indicating the cell recruitment capacity of hPL. (B) Protection against H₂O₂-induced reduction in cell viability. Cells were treated with H₂O₂ at concentrations of 0, 100, 200, and 400 μM for 24 h, and cell viability was assessed using Live and Dead staining (Green: Live cells; Red: Dead cells) and the CCK-8 assay. Cell viability decreased in FBS-supplemented medium in a concentration-dependent manner, significantly decreasing under 400 μM H₂O₂. Conversely, viability increased at 100 mM compared to the control (non-treated group) in both 2% and 5% hPL groups, reaching over 80–90% even at 400 mM. (C) Assessment of antioxidant ability following supplementation change from FBS to hPL or vice versa in the final culture condition, indicating that the antioxidative capability originates from hPL and is not retained under different culture conditions. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 3

Characteristics of hPL-SHEDs and FBS-SHEDs. (A) Analysis of stem cell markers (CD29, 44, and 73 for positive markers and CD14 for negative makers) by flow cytometry. (B) Chromosomal stability was assessed by karyotyping and chromosomal microarray (CMA). Any abnormalities in the number or structure of chromosomes were not detected in passage 10 samples.

Figure 4

Cell differentiation capacity of 2/5% hPL-SHEDs and 10% FBS-SHEDs. All differentiation media were used as commercial products not containing FBS or hPL for the same differentiation condition. (A) Osteogenic (RUNX2, ALP, ColA1, OCN, DMP1 and DSPP), (B) adipogenic (PPARg2, LPL, and FABP4), (C) chondrogenic (ACAN, Col II A, and SOX9), and (D) neurogenic (MASH-1, Neuron D1, Nestin, GFAP, and b-tubulin) gene expression levels were measured using qRT-PCR on days 3 and 7. (A) ALP and ARS Red S assays were performed to access ALP activity and mineralization on days 7 and 14, respectively. For the detection of neutral triglycerides, lipids, and polysaccharides, (B) Oil Red O and (C) Alcian blue staining were carried out on days 14 and 28, respectively. hPL-SHEDs exhibited significantly higher levels than FBS-SHEDs in most genes, although predominance varied between the 2% and 5% hPL groups. (D) The protein levels of GFAP and β-tubulin were assessed by ICC, revealing significantly higher expressions in the 2% hPL group. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 4

Cell differentiation capacity of 2/5% hPL-SHEDs and 10% FBS-SHEDs. All differentiation media were used as commercial products not containing FBS or hPL for the same differentiation condition. (A) Osteogenic (RUNX2, ALP, ColA1, OCN, DMP1 and DSPP), (B) adipogenic (PPARg2, LPL, and FABP4), (C) chondrogenic (ACAN, Col II A, and SOX9), and (D) neurogenic (MASH-1, Neuron D1, Nestin, GFAP, and b-tubulin) gene expression levels were measured using qRT-PCR on days 3 and 7. (A) ALP and ARS Red S assays were performed to access ALP activity and mineralization on days 7 and 14, respectively. For the detection of neutral triglycerides, lipids, and polysaccharides, (B) Oil Red O and (C) Alcian blue staining were carried out on days 14 and 28, respectively. hPL-SHEDs exhibited significantly higher levels than FBS-SHEDs in most genes, although predominance varied between the 2% and 5% hPL groups. (D) The protein levels of GFAP and β-tubulin were assessed by ICC, revealing significantly higher expressions in the 2% hPL group. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 5

In vivo evaluation of organ toxicity and tumor formation. Representative histological images illustrating the absence of tumor formation and structural alterations in organs (lung, spleen, kidney, heart, liver, brain, and skin) of nude mice transplanted with 10% FBS-SHEDs, 5% hPL-SHEDs, or PBS for 6 months. No evidence of tumor formation was detected, and organs exhibited normal histology without observable injuries. (n = 5).