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      Mesenchymal Stromal Cell Proliferation, Gene Expression and Protein Production in Human Platelet-Rich Plasma-Supplemented Media

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          Abstract

          Background

          Platelet-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.

          Methods

          Human 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.

          Results

          10% 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.

          Conclusions

          Mesenchymal 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.

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          Most cited references19

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          Protein synthesis and secretion in human mesenchymal cells derived from bone marrow, adipose tissue and Wharton’s jelly

          Introduction Different 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. Methods We 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. Results Cell 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. Conclusions Mesenchymal 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.
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            Clinical applications of mesenchymal stem cells

            Mesenchymal stem cells (MSC) have generated a great amount of enthusiasm over the past decade as a novel therapeutic paradigm for a variety of diseases. Currently, MSC based clinical trials have been conducted for at least 12 kinds of pathological conditions, with many completed trials demonstrating the safety and efficacy. This review provides an overview of the recent clinical findings related to MSC therapeutic effects. Roles of MSCs in clinical trials conducted to treat graft-versus-host-disease (GVHD) and cardiovascular diseases are highlighted. Clinical application of MSC are mainly attributed to their important four biological properties- the ability to home to sites of inflammation following tissue injury when injected intravenously; to differentiate into various cell types; to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation and to perform immunomodulatory functions. Here, we will discuss these four properties. Moreover, the issues surrounding clinical grade MSCs and principles for MSC therapeutic approaches are also addressed on the transition of MSCs therapy from bench side to bedside.
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              Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy.

              Adult stem cells from human bone marrow stroma, referred to as mesenchymal stem cells or marrow stromal cells (hMSCs), are attractive candidates for clinical use. The optimal conditions for hMSC expansion require medium supplemented with fetal calf serum (FCS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FCS proteins. By a sensitive fluorescence-based assay we determined that 7 to 30 mg of FCS proteins are associated with a standard preparation of 100 million hMSCs, a dosage that probably will be needed for clinical therapies. Here we present ex vivo growth conditions for hMSCs that reduce the FCS proteins to less than 100 ng per 100 million hMSCs, approximately a 100,000-fold reduction. The cells maintain their proliferative capacity and sustain their ability for multilineage differentiation. Experiments in rats demonstrate that rat MSCs grown in 20% FCS induce a substantial humoral response after repeated administrations, whereas cells grown under the conditions described in this study reduce the immunogenicity in terms of IgG response over 1000-fold to barely detectable levels. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs and perhaps other cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                12 August 2014
                : 9
                : 8
                : e104662
                Affiliations
                [1 ]Excellion Biomedical Services S.A., Petrópolis, Rio de Janeiro, Brazil
                [2 ]National Institute of Metrology, Quality and Technology, Xerém, Rio de Janeiro, Brazil
                National Institutes of Health, United States of America
                Author notes

                Competing Interests: PRA, MVVT, RBVC and RB were employees at Excellion Biomedical Services S.A. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Results reported here have no connections or influence on company 's products. The authors declare that no competing financial and/or private interests exist.

                Conceived and designed the experiments: PRA MVTT RBVC JMG RB. Performed the experiments: PRA MVTT RBVC. Analyzed the data: PRA MVTT RB. Contributed reagents/materials/analysis tools: JMG RB. Contributed to the writing of the manuscript: PRA MVTT RBVC JMG RB. Manipulated human samples: RBVC.

                Article
                PONE-D-14-19495
                10.1371/journal.pone.0104662
                4130592
                25115920
                413d4693-fbd3-4832-aa5a-9825d61b94ef
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 5 May 2014
                : 14 July 2014
                Page count
                Pages: 9
                Funding
                This work was funded by Excellion Serviços Biomédicos S.A. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biotechnology
                Biomaterials
                Cell Biology
                Cell Processes
                Cell Proliferation
                Cellular Types
                Animal Cells
                Stem Cells
                Cell Physiology
                Molecular Cell Biology
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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