Effect Of Human Platelet Lysate Research Articles

Assessment of the Dose-Dependent Effect of Human Platelet Lysate on Wharton's Jelly-Derived Mesenchymal Stem/Stromal Cells Culture for Manufacturing Protocols.

Mesenchymal stem/stromal cells (MSCs)-based products have unique characteristics compared to other drugs because of their inherently variable effects depending on culture conditions and microenvironment. In some cases, cells can be produced individually, one batch at a time, for personalized therapy. Therefore, it is very important to optimize both culture conditions and medium composition under Good Manufacturing Practice (GMP) standards. MSCs properties have been exploited as potential cell therapies in regenerative medicine. The main mechanism of their protective and regenerative effect is based on their secretory activity. Simultaneously, their secretome is highly variable and sensitive to any change in environmental conditions. Depending on the type of damage and the target application, it is desirable to enhance the secretion of therapeutic factors. Changes in the modulation of environmental conditions can affect survival, migration ability, and both proliferative and clonogenic potentials. This study cultured Wharton's jelly-derived MSCs (WJ-MSCs) in media with varying concentrations of human platelet lysate (hPL). Two groups were created: one with low hPL concentration and another with a high hPL concentration. The effects of these different hPL concentrations were analyzed by assessing mesenchymal phenotype retention, secretory activity, clonogenic potential, proliferation, and migration capabilities. Additionally, the secretion levels of key therapeutic factors, such as Hepatocyte Growth Factor (HGF), Brain-Derived Neurotrophic Factor (BDNF), and Chemokine Ligand 2 (CCL-2), were measured. WJ-MSCs maintained their mesenchymal phenotype regardless of hPL concentration. However, a higher concentration of hPL promoted cell clonogenic potential, proliferation, migration, and increased secretion of therapeutic factors. Adjusting the hPL concentration in the culture medium modulates the response of WJ MSCs and enhances their therapeutic potential. Higher hPL concentration promotes increased secretory activity and improves the regenerative capacity of WJ-MSCs, suggesting a promising strategy to optimize MSC-based therapies.

Cytoprotective Effects of Human Platelet Lysate during the Xeno-Free Culture of Human Donor Corneas.

We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm2) compared to 2%FBS (2113 ± 331 cells/mm2; p = 0.03), and endothelial cell loss was lower (ECL HPL = -0.7% vs. FBS = -3.8%; p = 0.01). There were no significant differences in cell morphology between TP1 and 2, or between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial cells and 217 genes in stromal cells. It was found that 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2), and the downregulation of pro-inflammatory/apoptotic genes (e.g., CXCL14, SIK1B, PLK5, PPP2R3B, FABP5, MAL, GATA3). 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and producing potentially beneficial alterations in gene expression.

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.

Improved biological performance of human cartilage-derived progenitors in platelet lysate xenofree media in comparison to fetal bovine serum media

Primary articular cartilage-derived cells are among the preferred contenders for cell-based therapy approaches for cartilage repair. Limited access to primary human cartilage tissue necessitates the process of in vitro cell expansion to obtain sufficient cells for therapeutic purposes. Therapeutic outcomes of such cell-based approaches become highly dependent on the quality of the in vitro culture-expanded cells. The objective of this study was to determine the differential biological effects of human platelet lysate (hPL) xeno-free defined media vs FBS containing traditional media on primary human cartilage-derived cells. Our goal in pursuing this work was to identify a preferred xenofree media alternative, that can be used as a platform for expansion of cells intended for clinical applications. Primary cartilage-derived cells obtained from five patients were simultaneously cultured in two expansion media's: (1) traditional (DMEM+10%FBS+1%P/S) and (2) defined xenofree (Nutristem® complete media+0.5%hPL). Connective tissue progenitors (CTPs) were assayed by standard colony forming unit assay, morphology, proliferation in early and late passages, expression of MSC associated cell-surface markers (CD73, CD90 and CD105) and trilineage differentiation (adipogenesis, osteogenesis and chondrogenesis) were considered for comparison of biological performance. Early biological performance of primary cartilage-derived cells was significantly improved in Nutristem® expansion media in comparison to traditional expansion media with respect to (1) Colony forming efficiency tended to be higher (p = 0.058) and (2) CTPs formed larger colonies with respect to total cells per colony and colony area (p < 0.01). In the culture expanded cell population, Nutristem® expansion media was superior to traditional expansion media with respect to: (1) overall proliferation rate through passages 1–4 (p = 0.027), (2) total cells harvested at end of passage 4 (p = 0.028) and (3) total positive stain area of CD73 (p = 0.006), CD90 (p = 0.001) and CD105 (p = 0.049). Nutristem®-hPL expanded cells when differentiated in respective xenofree serum-free defined MSCgo™ differentiated media's, also showed significant improvement in adipogenic, osteogenic and chondrogenic marker expression. Overall, we convincingly demonstrated that a low concentration of hPL in combination with defined xenofree media is an effective and economic growth supplement to culture expand primary cartilage-derived cells. It can be manufactured under cGMP conditions to improve clinical-grade cell products’ quality for therapeutic applications.

Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor.

PurposeCorneal endothelial cell (CEC) therapy can be used as a promising therapeutic option for patients with various corneal endothelial dysfunctions. In this study, we compared the proliferative effect of human platelet lysate (HPL), as a xeno-free medium supplement, with Y-27632 Rho/rho-associated protein kinase (ROCK) inhibitor, as a well-known proliferative and adhesive agent for CECs, and fetal bovine serum (FBS) as the control, in the culture medium of human corneal endothelial cells (HCECs).MethodsWe isolated HCECs from human donors and treated the cells as three different treatment groups including 20% HPL only, 10 μM Y-27632 ROCK inhibitor, combination of 20% HPL and 10 μM Y-27632 ROCK inhibitor, and 20% FBS as the control group. ELISA cell proliferation assay and cell counting was performed on the treated cells. Finally, HCECs were characterized by morphology and immunocytochemistry (ICC).Results There was no significant proliferative effect of HPL on cell proliferation compared with the cells treated with Y-27632 ROCK inhibitor or the combination of HPL and Y-27632 ROCK inhibitor, but all the respected treatments had significant inducible effect on cell proliferation as compared with FBS-treated cells. The cells grown in all three treatment groups exhibited CEC morphology. Also, there was a higher expression of Na+/K+-ATPase and ZO-1, as CEC characteristic markers, in the culture of HCECs treated with HPL as compared with FBS.ConclusionHPL offers a xenofree and affordable medium supplement for CEC expansion that can be used in clinical applications.

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets.

Adipose-derived stem cell (ASC) is a valuable source of cell therapy. By stimulating extracellular matrix (ECM) secretion, ASC sheets can be fabricated with enhanced regenerative capabilities. In recent years, human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for the ex vivo expansion of ASCs for clinical use. However, the effect of HPL on ASC sheet formation has not been previously determined. In this study, we compared ECM composition and cellular characteristics of ASC sheets cultured in growth medium supplemented with either FBS or HPL. HPL supplement significantly enhanced ASC proliferation without obvious change in the expression pattern of cell surface markers. We found that culturing ASCs with HPL rendered thicker cell sheets with significantly more ECM deposition, including collagen and fibronectin. Proteomic analysis of the FBS or HPL-cultured cell sheets showed diversity in ECM composition. HPL-cultured ASC sheets exhibited up-regulation of interleukin-6 and an anti-inflammatory cytokine, C1q/tumor necrosis factor-related protein-3. Conditioned medium of HPL-cultured ASC sheets significantly enhanced fibroblast migration and tube formation of endothelial cells in vitro, while it inhibited the migration of macrophages toward stimulated macrophages in vitro. TGF-β1-stimulated fibroblasts cultured in ASC sheet-conditioned medium showed down-regulation of α-SMA and TGF-β1. By adding an anti-hepatocyte growth factor (HGF) neutralizing antibody in conditioned medium, we indicated that an anti-fibrosis effect of HPL-cultured ASC sheets is partially mediated through the increased secretion of HGF. Moreover, chick embryo chorioallantoic membrane (CAM) assay showed comparable capillary density after applying either FBS or HPL-cultured ASC sheets, both of which were significantly higher than the control. In conclusion, robust ECM formation with altered ECM composition was noted in ASC sheets cultured in HPL-supplemented medium. Their immunomodulatory and pro-angiogenesis capabilities were largely maintained. Our findings paved the way to elucidate the potential of HPL-cultured ASC sheets for clinical application in tissue regeneration.

The effects of human platelet lysate versus commercial endothelial growth medium on the endothelial differentiation potential of human amniotic fluid mesenchymal stem cells

To differentiate stem cells into endothelial cells, vascular endothelia growth factors (VEGF) serve as the major signal for stimulating the cells. However, there are other cytokines or growth factors associated with endothelial cell development and differentiation. Human platelet lysate (hPL) has been a promising reagent in cell-based therapy since it is considered as a source of bioactive molecules and growth factors. The aim of this study was to investigate the in vitro differentiation of human amniotic fluid mesenchymal stem cells (hAF-MSCs) into endothelial-like cells under hPL together with VEGF or endothelial cell growth medium 2 (EGM-2), a commercially induced medium. In this study, hAF-MSCs were isolated from human amniotic fluid cells (hAFCs) using the direct adherence method. The cells expressed CD44, CD73, CD90, and HLA-ABC at high levels and expressed Oct-4 (octamer-binding transcription factor 4) at low levels. The cells were negative for CD31, CD34, CD45, CD105 and HLA-DR. This study found that hAF-MSCs induced with hPL and VEGF had the ability to differentiate into endothelial-like cells by presenting endothelial specific markers (vWF, VEGFR2 and eNOS), forming a network-like structure on Matrigel, and producing nitric oxide (NO). This outcome was similar to those of experiments involving EGM-2 induced cells. The present findings indicate that hPL + VEGF can induce hAF-MSCs to express endothelial cell characteristics. Our findings represent an important step forward in the development of a clinically compliant process for the production of endothelial cell-derived hAF-MSCs, and their subsequent testing in future clinical trials.

Platelet lysate promotes the healing of long-standing diabetic foot ulcers: A report of two cases and in vitro study

Long-standing foot ulcers present a great challenge in diabetes care. Platelet products have been suggested as a possible therapeutic option. However, nor the effect of an injectable form of platelet lysate on the healing of ulcers nor that on primary cells of the epidermis have been studied. In the current study, we present two cases of an ongoing clinical trial showing the positive effect of autologous platelet lysate injected perilesional. Both clinical cases treated with injections of hPL showed complete healing of previously un-healed within 8 weeks of treatment. Further, we describe the in vitro effect of human platelet lysate (hPL) on primary human epidermal keratinocytes (HEK) in terms of chemotaxis, migration and proliferation. In vitro, HEK showed enhanced chemotaxis towards the hPL compared to keratinocyte-defined media (p < 0.0001). Their migration was also stimulated especially at hPL concentration of 10%V/V (p < 0.0001). In contrast, hPL significantly inhibited HEK proliferation measured through MTT assay (p < 0.0001). In conclusion, the findings presented here provide preliminary evidence of an explanatory mechanism for the effect of hPL on primary keratinocytes and therefore of their potential use in a clinical setting. hPL promotes keratinocyte migration and therefore closure of foot ulcers.

Evaluation of the effect of human platelet lysate versus fetal bovine serum on human osteoarthritic cartilage derived chondroprogenitors

Purpose: Articular chondroprogenitors are a sub-population of cells exhibiting clonal growth, propensity for enhanced chondrogenesis and reduced hypertrophy. Since these cells have been classified as mesenchymal stem cells (MSCs) as per International Society for Cellular Therapy criteria, they may be a suitable contender for cell-based therapy in cartilage repair following disease and degeneration, as seen in osteo-arthritis. Fetal bovine serum (FBS) is a widely used culture additive for expansion and differentiation of chondroprogenitors (CPs). However due to concerns related to potential transmission of zoonoses and possibility of transplant rejections following cell implantation, there is a need for a human derived alternative. Human platelet lysate (HPL) has been extensively used for expanding various cell lines due to existent pool of abundant growth factors, but data related to its use with chondroprogenitors has not been reported. We therefore aimed to culture human chondroprogenitors in HPL and compare their growth, senescence, phenotypic expression and differentiation capacity to that of FBS. Methods: CPs were isolated from three human osteo-arthritic knee joints (Age: 53.3 ± 10.21 years) following differential fibronectin adhesion assay. Passage 0 cells grown in a)10% FBS and b)10% HPL were considered for assessment of trilineage differentiation, growth kinetics (cell cycle analysis and galactosidase assay), surface marker expression (CD105, CD73, CD90, CD49e, CD29, CD146, CD166, CD34, CD45 and HLA-DR) and RT-PCR (markers of chondrogenesis: Collagen II, SOX9, Aggrecan; hypertrophic chondrocyte marker: Collagen I and that of hypertrophy: Collagen X, MMP13, RUNX2). Latent TGFß1 levels were also measured for each culture medium used. Data was compared using Student t-test and a P value of < 0.05 was considered significant. Results: Results showed that cellular proliferation was significantly higher in cells grown with HPL (P<0.05). Surface marker expression was comparable except in CD 146 where HPL group had significantly lower values (P<0.05). Comparison of mRNA expression revealed notably low values of Aggrecan, Collagen II, Collagen I and Collagen X (P<0.05). Trilineage differentiation was seen in both groups with higher uptake in the HPL group noted for alizarin red. There was also significantly higher level of latent TGFß1 in the medium containing HPL as compared to FBS (P<0.001). Conclusions: This is the first in-vitro xeno free study to affirm that HPL can serve as an optimal growth supplement for expansion of articular chondroprogenitors, although an in-depth assessment of resident growth factors and evaluation of different dilutions of HPL is required to assess suitability for use in translational research.

The effect of human platelet lysate on corneal nerve regeneration

AimThis study aimed to test whether human platelet lysate (HPL) has neurotrophic ability for corneal nerve regeneration.MethodsWe measured the neurotrophic factors in human peripheral serum (HPS) and two commercially available...

Effect of Human Platelet Lysate in Differentiation of Wharton's Jelly Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are highly preferred in clinical therapy for repair and regeneration of diseased tissues for their multipotent properties. Conventionally, MSCs have been cultured in media supplemented with animal derived serum, however, it is ideal to expand MSCs in media containing supplements of human origin for clinical therapy. Currently, a number of human derived products are being studied as an alternative to animal sources. Amongst these, platelet lysate (PL) has gained interest in the culture of MSCs without affecting their phenotypic property. In this study, we used various concentration of PL (2.5, 5, 7.5 & 10%) in the growth medium of MSCs to identify the least concentration of PL that could be an effective alternative to animal products. MSCs were isolated from Wharton's Jelly by using explant method and expanded in various concentration of PL supplemented medium against the standard FBS containing medium. WJ-MSCs were characterised as per the minimal criteria proposed by International Society for Cell therapy (ISCT), Proliferation study by BrdU assay, gene expression study by qRT-PCR, sterility test for bacteria, Mycoplasma by PCR and endotoxin detection by LAL assay. Whartons jelly derived MSCs (WJ-MSCs) cultured using standard medium supplemented with various concentration of PL exhibited enhanced proliferation and differentiation potential, unaltered immunophenotypic property and genetic stability when compared with the commercial medium containing 10% FBS. The least concentration of PL for an ideal expansion of MSCs was found to be 2.5% and was comparable to FBS.

The effect of human platelet lysate on the differentiation ability of human adipose-derived stem cells cultured on ECM-coated surfaces

Human mesenchymal stem cells (hMSCs), such as human adipose-derived stem cells (hADSCs), present heterogeneous characteristics, including varying differentiation abilities and genotypes. hADSCs isolated under different conditions exhibit differences in stemness. We isolated hADSCs from human fat tissues via culture on different cell culture biomaterials including tissue culture polystyrene (TCPS) dishes and extracellular matrix protein (ECM)-coated dishes in medium supplemented with 5% or 10% serum-converted human platelet lysate (hPL) or 10% fetal bovine serum (FBS) as a control. Currently, it is not clear whether xeno-free hPL in the cell culture medium promotes the ability of hMSCs such as hADSCs to differentiate into several cell lineages compared to the xenomaterial FBS. We investigated whether a synchronized effect of ECM (Matrigel, fibronectin, and recombinant vitronectin) coatings on TCPS dishes for efficient hADSC differentiation could be observed when hADSCs were cultured in hPL medium. We found that Matrigel-coated dishes promoted hADSC differentiation into osteoblasts and suppressed differentiation into chondrocytes in 10% hPL medium. Recombinant vitronectin- and fibronectin-coated dishes greatly promoted hADSC differentiation into osteoblasts and chondrocytes in 5% and 10% hPL media. hPL promoted hADSC differentiation into osteoblasts and chondrocytes compared to FBS on the fibronectin-coated surface and recombinant vitronectin-coated surface.

220 - Effect of human platelet lysate supplemented medium on human mesenchymal stem cell identity and immunomodulatory capacity

220 - Effect of human platelet lysate supplemented medium on human mesenchymal stem cell identity and immunomodulatory capacity

Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia

Background aims. Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. Methods. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. Results. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Conclusion. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions.

Scalable efficient expansion of mesenchymal stem cells in xeno free media using commercially available reagents.

BackgroundThe rapid clinical translation of mesenchymal stem cells (MSC) has resulted in the development of cell-based strategies for multiple indications. Unfortunately one major barrier to widespread implementation of MSC-based therapies is the limited supply of fetal calf serum (FCS) used to expand cells to therapeutic numbers. Additionally, the xenogeneic element of fetal calf serum has been previously demonstrated to stimulate antibody mediated reactions and in some cases sensitization leading to anaphylaxis.MethodXcytePLUS™ media, a human platelet lysate based product, was used to supplement the culture medium at 5, 7.5 and 10% and compared to fetal calf serum at 10%, for human umbilical cord MSC expansion. Properties of the expanded cells were investigated.ResultsThis study demonstrated equivalent or superior effects of human platelet lysate compared to standard FCS supplemented media, based on doubling rate, without loss of identity or function, as demonstrated with flow cytometry characterization. Differentiation into osteocytes, adipocytes and chondrocytes was comparable from cells expanded in either media supplement.ConclusionsThese data support the implementation of human platelet lysate supplemented media as an alternative to xenogeneic containing preparations which may lead to safer MSC products with therapeutic uses.

The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars

Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) in the large-scale expansion of dental pulp stem cells (DPSCs). However, the biological effects and the optimal concentrations of PL for the proliferation and differentiation of human DPSCs remain unexplored. We isolated and expanded stem cells from the dental pulp of extracted third molars and evaluated the effects of PL on the cells’ proliferative capacity and differentiation potential in vitro and in vivo. Before testing, immunocytochemical staining and flow cytometry-based cell sorting showed that the cells derived from human dental pulp contained mesenchymal stem cell populations. Cells were grown on tissue culture plastic or on hydroxyapatite-tricalcium phosphate (HA/TCP) biomaterials and were incubated with either normal or odontogenic/osteogenic media in the presence or absence of various concentrations of human PL for further investigation. The proliferation of DPSCs was significantly increased when the cells were cultured in 5% PL under all testing conditions (P < 0.05). However, this enhancement was inconsistent when the cells were cultured in 1% PL or in 10% PL; 10% PL significantly inhibited cell proliferation and was therefore excluded from further differentiation testing. Culture medium containing 5% PL also significantly promoted the mineralized differentiation of DPSCs, as indicated by the measurement of alkaline phosphatase activity and calcium deposition under mineral-conditioned media (P < 0.05). Scanning electron microscopy and modified Ponceau trichrome staining showed that the cells treated with 5% PL and mineralizing media were highly capable of integrating with the HA/TCP biomaterials and had fully covered the surface of the scaffold with an extensive sheet-like structure 14 d after seeding. In addition, 5% PL showed significantly positive effects on tissue regeneration in two in vivo transplantation models. We conclude that the appropriate concentration of PL enhances the proliferation and mineralized differentiation of human DPSCs both in vitro and in vivo, which supports the use of PL as an alternative to FBS or a nonzoonotic adjuvant for cell culture in future clinical trials. However, the elucidation of the molecular complexity of PL products and the identification of both the essential growth factors that determine the fate of a specific stem cell and the criteria to establish dosing require further investigation.

Effect of Platelet Lysate on the Functional and Molecular Characteristics of Mesenchymal Stem Cells Isolated from Adipose Tissue

Mesenchymal stem cells (MSCs) are non-hematopoietic, adult, fibroblast-like, multipotent cells that are plastic adherent in standard culture conditions. They can be isolated from several tissues, but it is always necessary to expand them for clinical practice. We investigated the effect of human platelet lysate (hPL) on the expansion of human MSCs isolated from adipose tissue (AT), comparing it with fetal bovine serum (FBS) and human platelet-poor plasma (hPPP). Human AT-MSCs, hPL and hPPP were obtained from 7 healthy subjects. AT-MSCs were seeded at 1500 cells/cm(2) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 10% hPPP or 10% hPL. Cells were harvested, counted and analyzed by flow cytometry every 7 days for 5 passages (P). The differentiation assays, RNA isolation and co-culture with allogeneic lymphocytes were performed at the end of P2. AT-MSCs achieved a better proliferation rate when cultured with hPL than with hPPP or FBS (20 ±2 versus 8 ±3 and 6 ±3, respectively, at the end of P5 [p<0.01]). hPL preserved the differentiation capacity and typical expression of surface antigens, avoiding the risks associated with the use of animal derivatives. AT-MSCs demonstrated a stronger inhibitory effect on lymphocyte proliferation with hPL than with other culture conditions, even at a AT-MSCs:T cells ratio of 1:10. The transcriptional level of matrix metalloproteinase 2, used to evaluate stemness, was very high in all conditions tested. hPL represents an effective and safe supplement for MSC expansion to be used in the clinical setting.

Effects of platelet lysates on select bone cell functions.

Although platelet-rich plasma and platelet concentrates have been used to promote bone healing in orthopaedic and maxillofacial surgery, the underlying cellular-level mechanisms remain poorly understood. The present in vitro study investigated the effects of human platelet lysate (PL) on selected functions of cultured bone cells. Cells from 18-day-old fetal rat calvaria were isolated by a collagenase digestion procedure. PL was added at different concentrations on pre- or post-confluent cell stage. After 1 day, bone cell proliferation was maximal and half-maximal in the presence of PL from 3 x 10(8) and 0.5 x 10(8) platelets/ml, respectively. During 17 h, the number of bone cells traversing the scrape border of a scrape wound model increased by 16-fold in the presence of PL from 3 x 10(8) platelets/ml. The presence of PL from 3 x 10(8) platelets/ml in pre-confluent bone cell cultures for 48 h resulted in a threefold decrease of alkaline phosphatase (ALP) specific activity. In the case of confluent bone cells, the presence of PL (from 1 x 10(6) to 3 x 10(8) platelets/ml) for 11 days, the ALP specific activity and total calcium content decreased in a PL dose-dependent manner and reached a minimum in the presence of PL from 3 x 10(8) platelets/ml. In summary, short-term PL exposure (up to 24 h) promotes the proliferative and chemotactic bone cell functions while long-term PL exposure results in a decrease of both ALP activity and mineral formation. These data show that the soluble components contained in PL may affect the bone healing process by modulating differently bone cell functions.