IntroductionPlatelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed.MethodsPRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure.ResultsOur method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF.ConclusionsOur study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.

IntroductionDifferent mesenchymal stromal cells (MSC) have been successfully isolated and expanded in vitro and nowadays they are tested in clinical trials for a wide variety of diseases. Whether all MSC express the same cell surface markers or have a similar secretion profile is still controversial, making it difficult to decide which stromal cell may be better for a particular application.MethodsWe isolated human mesenchymal stromal cells from bone marrow (BM), adipose tissue (AT) and Wharton’s jelly (WJ) and cultured them in fetal bovine serum supplemented media. We evaluated proliferation, in vitro differentiation (osteogenic, adipogenic and chondrogenic potential), expression of cell surface markers and protein secretion using Luminex and ELISA assays.ResultsCell proliferation was higher for WJ-MSC, followed by AT-MSC. Differences in surface expression markers were observed only for CD54 and CD146. WJ-MSC secreted higher concentrations of chemokines, pro-inflammatory proteins and growth factors. AT-MSC showed a better pro-angiogenic profile and secreted higher amounts of extracellular matrix components and metalloproteinases.ConclusionsMesenchymal stromal cells purified from different tissues have different angiogenic, inflammatory and matrix remodeling potential properties. These abilities should be further characterized in order to choose the best protocols for their therapeutic use.

BackgroundPlatelet-rich plasma (PRP) is increasingly used as a cell culture supplement, in order to reduce the contact of human cells with animal-derived products during in vitro expansion. The effect of supplementation changes on cell growth and protein production is not fully characterized.MethodsHuman mesenchymal stromal cells from bone marrow, adipose tissue and Wharton's Jelly were isolated and cultured in PRP-supplemented media. Proliferation, in vitro differentiation, expression of cell surface markers, mRNA expression of key genes and protein secretion were quantified.Results10% PRP sustained five to tenfold increased cell proliferation as compared to 10% fetal bovine serum. Regarding cell differentiation, PRP reduced adipogenic differentiation and increased calcium deposits in bone marrow and adipose tissue-mesenchymal stromal cells. Wharton's Jelly derived mesenchymal stromal cells secreted higher concentrations of chemokines and growth factors than other mesenchymal stromal cells when cultured in PRP-supplemented media. Bone marrow derived mesenchymal stromal cells secreted higher concentrations of pro-inflammatory and pro-angiogenic proteins. Mesenchymal stromal cells isolated from adipose tissue secreted higher amounts of extracellular matrix components.ConclusionsMesenchymal stromal cells purified from different tissues have distinct properties regarding differentiation, angiogenic, inflammatory and matrix remodeling potential when cultured in PRP supplemented media. These abilities should be further characterized in order to choose the best protocols for their therapeutic use.

BackgroundQuantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately.ResultsIn the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Whartońs Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions.ConclusionMost stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively.

BackgroundMesenchymal stromal cells (MSC) can be obtained from potentially any tissue from the human body, but cells purified from different sources are undoubtedly different, and for each medical application, the MSC with the best regenerative potential should be chosen.ResultsBone marrow-derived mesenchymal stromal cells (BM-MSC), adipose tissue-derived mesenchymal stromal cells (AT-MSC) and Wharton’s Jelly-derived mesenchymal stromal cells (WJ-MSC) were isolated from human tissues and were cultured under differentiation media supplemented with fetal bovine serum. We quantified the expression of stem cell and adipocyte genetic markers using quantitative real time PCR, as well as the secretion of cytokines, extracellular matrix components and growth factors using Luminex and ELISA. All three MSC differentiated into adipogenic cells. AT-MSC showed the highest shift in ADIPOQ, CEBPA and PPARG mRNA expression. BM-MSC kept high expression levels of stem-cell markers SOX2 and POU5F1. WJ-MSC showed the lowest increase in mRNA expression when cells were induced to differentiate into adipocytes. Regarding protein secretion, adipocyte-like cells generated from WJ-MSC secreted the highest chemokine levels. AT-MSC-derived adipocyte-like cells secreted the lowest cytokine amounts and the highest quantity of collagen types I and III. Adipocyte-like cells obtained from BM-MSC secreted high amounts of most angiogenic factors, growth factors TGF-β1 and TGF-β2, collagens type II and IV, heparan sulfate, laminin and aggrecan.ConclusionMesenchymal stromal cells purified from different tissues have a different behavior when induced to differentiate into adipocyte-like cells.