Dataset Information

Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression.

ABSTRACT:

Background

Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity.

Methods

In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid.

Results

TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24-36% GFP-expressing cells with a viability of 85-96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system.

Discussion

We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.

SUBMITTER: Cheung WY

PROVIDER: S-EPMC6323439 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression.

Cheung Winston Y WY Hovey Owen O Gobin Jonathan M JM Muradia Gauri G Mehic Jelica J Westwood Carole C Lavoie Jessie R JR

Stem cells international 20181224

<h4>Background</h4>Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity.<h4>Methods</h4>In order to find a transfection method that would address the c ...[more]

PMID: 30675166

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Project description:The focus of both clinical and basic studies on stem cells is increasing due to their potentials in regenerative medicine and cell-based therapies. Recently stem cells have been genetically modified to enhance an existing character in or to bring a new property to them. However, accomplishment of declared goals requires detailed knowledge about their molecular characteristics which could be achieved by genetic modifications mostly through nonviral transfection strategies. Capable of differentiating into multiple cells, human unrestricted somatic stem cells (hUSSCs) and human mesenchymal stem cells (hMSCs) seem to be suitable candidates for transfection approaches. Involvement of microRNAs (miRNAs) in many biological processes makes their transfection evaluation valuable. Herein we investigated the efficacy and toxicity of four typically used transfection reagents (Arrest-In, Lipofectamine 2000, Oligofectamine and HiPerfect) systematically to deliver fluorescent labeled-miRNA and Green Fluorescent Protein (GFP) expressing plasmid into hUSSCs and hMSCs. The authenticity of stem cells was verified by differentiation experiments along with flow cytometry of surface markers. Our study revealed that stemness properties of these stem cells were not affected by transient transfection. Moreover the ratios of cell viability and transfection efficiency in both analyzed stem cells were reversed. Considering cell viability, the highest fraction of GFP-expressing cells was obtained using Oligofectamine (~50%) while the highest transfection rate of miRNA was achieved by Lipofectamine 2000 (~90%). Moreover dependency of hMSCs to size of transfected nucleic acid and time-dependency of Oligofectamine and their affection on the yield of transfection were observed. Cytotoxicity assessments also showed that hUSSCs are sensitive to HiPerFect. In addition cells treated by Lipofectamine showed morphological changes. Representing the efficient nucleic acid transfection, our research facilitates comprehensive genetic modification of stem cells and demonstrates powerful approaches to understand stem cell molecular regulation mechanisms, which eventually improves nonviral cell-mediated gene therapy.

Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

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Dataset Information

Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression.

Background

Methods

Results

Discussion

Publications

Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression.

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