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Gene Therapy for Bone Repair Using Human Cells: Superior Osteogenic Potential of Bone Morphogenetic Protein 2-Transduced Mesenchymal Stem Cells Derived from Adipose Tissue Compared to Bone Marrow.

ABSTRACT: Ex vivo regional gene therapy strategies using animal mesenchymal stem cells genetically modified to overexpress osteoinductive growth factors have been successfully used in a variety of animal models to induce both heterotopic and orthotopic bone formation. However, in order to adapt regional gene therapy for clinical applications, it is essential to assess the osteogenic capacity of transduced human cells and choose the cell type that demonstrates the best clinical potential. Bone-marrow stem cells (BMSC) and adipose-derived stem cells (ASC) were selected in this study for in vitro evaluation, before and after transduction with a lentiviral two-step transcriptional amplification system (TSTA) overexpressing bone morphogenetic protein 2 (BMP-2; LV-TSTA-BMP-2) or green fluorescent protein (GFP; LV-TSTA-GFP). Cell growth, transduction efficiency, BMP-2 production, and osteogenic capacity were assessed. The study demonstrated that BMSC were characterized by a slower cell growth compared to ASC. Fluorescence-activated cell sorting analysis of GFP-transduced cells confirmed successful transduction with the vector and revealed an overall higher but not statistically significant transduction efficiency in ASC versus BMSC (90.2 ± 4.06% vs. 80.4 ± 8.51%, respectively; p = 0.146). Enzyme-linked immunosorbent assay confirmed abundant BMP-2 production by both cell types transduced with LV-TSTA-BMP-2, with BMP-2 production being significantly higher in ASC versus BMSC (239.5 ± 116.55 ng vs. 70.86 ± 24.7 ng; p = 0.001). Quantitative analysis of extracellular deposition of calcium (Alizarin red) and alkaline phosphatase activity showed that BMP-2-transduced cells had a higher osteogenic differentiation capacity compared to non-transduced cells. When comparing the two cell types, ASC/LV-TSTA-BMP-2 demonstrated a significantly higher mineralization potential compared to BMSC/LV-TSTA-BMP-2 7 days post transduction (p = 0.014). In conclusion, this study demonstrates that transduction with LV-TSTA-BMP-2 can significantly enhance the osteogenic potential of both human BMSC and ASC. BMP-2-treated ASC exhibited higher BMP-2 production and greater osteogenic differentiation capacity compared to BMP-2-treated BMSC. These results, along with the fact that liposuction is an easy procedure with lower donor-site morbidity compared to BM aspiration, indicate that adipose tissue might be a preferable source of MSCs to develop a regional gene therapy approach to treat difficult bone-repair scenarios.

SUBMITTER: Bougioukli S

PROVIDER: S-EPMC5909082 | biostudies-other | 2018 Apr

REPOSITORIES: biostudies-other

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Gene Therapy for Bone Repair Using Human Cells: Superior Osteogenic Potential of Bone Morphogenetic Protein 2-Transduced Mesenchymal Stem Cells Derived from Adipose Tissue Compared to Bone Marrow.

Bougioukli Sofia S Sugiyama Osamu O Pannell William W Ortega Brandon B Tan Matthew H MH Tang Amy H AH Yoho Robert R Oakes Daniel A DA Lieberman Jay R JR

Human gene therapy 20180314 4

Ex vivo regional gene therapy strategies using animal mesenchymal stem cells genetically modified to overexpress osteoinductive growth factors have been successfully used in a variety of animal models to induce both heterotopic and orthotopic bone formation. However, in order to adapt regional gene therapy for clinical applications, it is essential to assess the osteogenic capacity of transduced human cells and choose the cell type that demonstrates the best clinical potential. Bone-marrow stem ...[more]

PMID: 29212377

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Project description:Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ?0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application.

Project description:Bone morphogenetic protein-2 (BMP-2) is a growth factor used to stimulate bone regeneration in clinical applications. However, there are contradicting reports on the functionality of BMP-2 in human adipose stem cells (hASCs), which are frequently used in tissue engineering. In this study, we analyzed the effects of BMP-2 on SMAD1/5 signaling, proliferation, and differentiation in hASCs. Our results indicated that BMP-2 induced dose-dependent (25-100 ng/ml) activation of SMAD signaling. Furthermore, the cell proliferation analysis revealed that BMP-2 (100 ng/ml) consistently decreased the proliferation in all the cell lines studied. However, the analysis of the differentiation potential revealed that BMP-2 (100 ng/ml) exhibited a donor-dependent dual role, inducing both osteogenic and adipogenic differentiation in hASCs. The quantitative alkaline phosphatase (qALP) activity and mineralization levels were clearly enhanced in particular donor cell lines by BMP-2 stimulus. On the contrary, in other cell lines, qALP and mineralization levels were diminished and the lipid formation was enhanced. The current study also suggests that hASCs have accelerated biochemical responsiveness to BMP-2 stimulus in human serum-supplemented culture medium compared with fetal bovine serum. The production origin of the BMP-2 growth factor is also important for its response: BMP-2 produced in mammalian cells enhanced signaling and differentiation responses compared with BMP-2 produced in Escherichia coli. These results explain the existing contradiction in the reported BMP-2 studies and indicate the variability in the functional end mechanism of BMP-2-stimulated hASCs.This study examined how bone morphogenetic protein-2 (BMP-2) modulates the SMAD signaling mechanism and the proliferation and differentiation outcome of human adipose stem cells (hASCs) derived from several donors. The results indicate that BMP-2 triggers molecular SMAD signaling mechanisms in hASCs and regulates differentiation processes in human serum-culture conditions. Importantly, BMP-2 has dual activity, inducing osteogenic and adipogenic differentiation, subject to hASC donor line studied. These findings explain contradictory previous results and highlight the importance of further studies to understand how signaling pathways guide mesenchymal stem cell functions at the molecular level.

Project description:BackgroundMesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model.MethodsBM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice.ResultsBM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model.ConclusionsWe showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Dataset Information

Gene Therapy for Bone Repair Using Human Cells: Superior Osteogenic Potential of Bone Morphogenetic Protein 2-Transduced Mesenchymal Stem Cells Derived from Adipose Tissue Compared to Bone Marrow.

Publications

Gene Therapy for Bone Repair Using Human Cells: Superior Osteogenic Potential of Bone Morphogenetic Protein 2-Transduced Mesenchymal Stem Cells Derived from Adipose Tissue Compared to Bone Marrow.

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