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Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci.

ABSTRACT: Induced pluripotent stem cells (iPSCs) are a promising source of mesenchymal stem cells (MSCs) for clinical applications. In this study, we transformed human iPSCs using a non-viral vector carrying the IL24 transgene pHrn-IL24. PCR and southern blotting confirmed IL24 integration into the rDNA loci in four of 68 iPSC clones. We then differentiated a high expressing IL24-iPSC clone into MSCs (IL24-iMSCs) that showed higher expression of IL24 in culture supernatants and in cell lysates than control iMSCs. IL24-iMSCs efficiently differentiated into osteoblasts, chondrocytes and adipocytes. Functionally, IL24-iMSCs induced in vitro apoptosis in B16-F10 melanoma cells more efficiently than control iMSCs when co-cultured in Transwell assays. In vivo tumor xenograft studies in mice demonstrated that IL24-iMSCs inhibited melanoma growth more than control iMSCs did. Immunofluorescence and histochemical analysis showed larger necrotic areas and cell nuclear aggregation in tumors with IL24-iMSCs than control iMSCs, indicating that IL24-iMSCs inhibited tumor growth by inducing apoptosis. These findings demonstrate efficient transformation of iPSCs through gene targeting with non-viral vectors into a rDNA locus. The ability of these genetically modified MSCs to inhibit in vivo melanoma growth is suggestive of the clinical potential of autologous cell therapy in cancer.

SUBMITTER: Liu B

PROVIDER: S-EPMC5522332 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci.

Liu Bo B Chen Fei F Wu Yong Y Wang Xiaolin X Feng Mai M Li Zhuo Z Zhou Miaojin M Wang Yanchi Y Wu Lingqian L Liu Xionghao X Liang Desheng D

Oncotarget 20170601 25

Induced pluripotent stem cells (iPSCs) are a promising source of mesenchymal stem cells (MSCs) for clinical applications. In this study, we transformed human iPSCs using a non-viral vector carrying the IL24 transgene pHrn-IL24. PCR and southern blotting confirmed IL24 integration into the rDNA loci in four of 68 iPSC clones. We then differentiated a high expressing IL24-iPSC clone into MSCs (IL24-iMSCs) that showed higher expression of IL24 in culture supernatants and in cell lysates than contro ...[more]

PMID: 28388559

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Project description:The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone has been unpredictable. We hypothesized that given the multi-stage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time-point of healing. Utilizing a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome-proliferator-activated-receptor-γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were co-administered with ECM, they remained at the injury well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized defects in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be employed at optimal times to substantially and reproducibly improve bone repair. To gain insight into the superior healing potential of GW-hMSCs and also what might be accounting for their extended engraftment, microarray analyses on the RNA extracted from the calvarial tissue recovered after days 5 and 14 were performed. Equal amounts of total RNA from 4 animals per group and time point were pooled and animals receiving control (DMSO) or peroxisome proliferator-activated receptor-gamma inhibitor GW9662-treated hMSCs were compared with the assumption that murine cross-hybridization would be constant throughout the samples and thus be subtracted from the analysis.

Project description:BackgroundCellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations.MethodsIn the present study, the bone marrow samples of patients with ONFH (n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation.ResultsThe morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head.ConclusionReprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.

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Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci.

Publications

Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci.

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