Direct Conversion of Human Dermal Fibroblasts into Cardiomyocyte-Like Cells Using CiCMC Nanogels Coupled with Cardiac Transcription Factors and a Nucleoside Drug.

Using direct conversion technology, normal adult somatic cells can be routinely switched from their original cell type into specific differentiated cell types by inducing the expression of differentiation-related transcription factors. In this study, normal human dermal fibroblasts (NHDFs) are directly converted into cardiomyocyte-like cells by drug and gene delivery using carboxymethylcellulose (CMC) nanoparticles (CiCMC-NPs). CMC-based multifunctional nanogels containing specific cardiomyocyte-related genes are designed and fabricated, including GATA4, MEF2C, and TBX5 (GMT). However, GMT alone is insufficient, at least in vitro, in human fibroblasts. Hence, to inhibit proliferation and to induce differentiation, 5-azacytidine (5-AZA) is conjugated to the hydroxyl group of CMC in CiCMC-NPs containing GMT; in addition, the CMC is coated with polyethylenimine. It is confirmed that the CiCMC-NPs have nanogel properties, and that they exhibit the characteristic effects of 5-AZA and GMT. When CiCMC-NPs-containing 5-AZA and GMT are introduced into NHDFs, cardiomyocyte differentiation is initiated. In the reprogrammed cells, the mature cardiac-specific markers cardiac troponin I and α-actinin are expressed at twofold to threefold higher levels than in NHDFs. Engineered cells transplanted into live hearts exhibit active pumping ability within 1 day. Histology and immunohistology of heart tissue confirm the presence of transplanted engineered NHDF cells at injection sites.

Author Correction: Sequential transfection of RUNX2/SP7 and ATF4 coated onto dexamethasone-loaded nanospheres enhances osteogenesis.

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Sequential transfection of RUNX2/SP7 and ATF4 coated onto dexamethasone-loaded nanospheresenhances osteogenesis.

The timing of gene transfection greatly influences stem cell differentiation. Sequential transfection is crucial for regulation of cell behavior. When transfected several days after differentiation initiation, genes expressed at the late stage of differentiation can regulate cell behaviors and functions. To determine the optimal timing of key gene delivery, we sequentially transfected human mesenchymal stem cells (hMSCs). This method can easily control osteogenesis of stem cells. hMSCs were first transfected with RUNX2 and SP7 using poly(lactic-co-glycolic acid) nanoparticles to induce osteogenesis, and then with ATF4 after 5, 7, and 14 days. Prior to transfecting hMSCs with all three genes, each gene was individually transfected and its expression was monitored. Transfection of these genes was confirmed by RT-PCR, Western blotting, and confocal microscopy. The pDNAs entered the nuclei of hMSCs, and RUNX2 and SP7 proteins were translated and triggered osteogenesis. Second, the ATF4 gene was delivered when cells were at the pre-osteoblasts stage. To induce the osteogenesis of hMSCs, the optimal timing of ATF4 gene delivery was 14 days after RUNX2/SP7 transfection. Experiments in 2- and 3-dimensional culture systems confirmed that transfection of ATF4 at 14 days after RUNX2/SP7 promoted osteogenic differentiation of hMSCs.

Transfection of gene regulation nanoparticles complexed with pDNA and shRNA controls multilineage differentiation of hMSCs.

Overexpression and knockdown of specific proteins can control stem cell differentiation for therapeutic purposes. In this study, we fabricated RUNX2, SOX9, and C/EBPα plasmid DNAs (pDNAs) and ATF4-targeting shRNA (shATF4) to induce osteogenesis, chondrogenesis, and adipogenesis of human mesenchymal stem cells (hMSCs). The pDNAs and shATF4 were complexed with TRITC-gene regulation nanoparticles (GRN). Osteogenesis-related gene expression was reduced at early (12 h) and late (36 h) time points after co-delivery of shATF4 and SOX9 or C/EBPα pDNA, respectively, and osteogenesis was inhibited in these hMSCs. By contrast, osteogenesis-related genes were highly expressed upon co-delivery of RUNX2 and ATF4 pDNAs. DEX in GRN enhanced chondrogenic differentiation. Expression of osteogenesis-, chondrogenesis-, and adipogenesis-related genes was higher in hMSCs transfected with NPs complexed with RUNX2 and ATF4 pDNAs, shATF4 and SOX9 pDNA, and shATF4 and C/EBPα pDNA for 72 h than in control hMSCs, respectively. Moreover, delivery of these NPs also increased expression of osteogenesis-, chondrogenesis-, and adipogenesis-related proteins. These alterations in expression led to morphological changes, indicating that hMSCs differentiated into osteoblasts, chondrocytes, and adipose cells.

Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs).

Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become increasingly important in the context of stem cell therapy. Methods: Functional nanoparticles (Sunflower typed nanoparticles; SF-NPs) were fabricated by coating heparin pluronic F127 gels with quantum dot nanoparticles (QDs), and then bound the SOX9 gene to the QD nanogels. The resultant nanoparticles were transferred into stem cells, and the effect on genetic stability was monitored. To determinate gene delivery efficacy and long-term genomic stability of cells transfected with QD nanogels, hMSCs were transfected with nanogels at passage 4 (T1; Transfected cells 1) and then sub-cultured to passage of (T4). Following transplantation of transfected T1-T4 cells, the cells were monitored by in vivo imaging. The genetic stability of cells treated with nanoparticles was confirmed by chromosomal analysis, copy number variation (CNV) analysis, and mRNA profiling. Results: After 21 days of pellet culture after sub-culture from T1 to T4, hMSCs treated with QD nanogels complexed with SOX9 plasmid DNA (pDNA) significantly increased expression of specific extracellular matrix (ECM) polysaccharides and glycoproteins, as determined by Safranin O and Alcian blue staining. Moreover, the T4 hMSCs expressed higher levels of specific proteins, including collagen type II (COLII) and SOX9, than P4 hMSCs, with no evidence of DNA damage or genomic malfunction. Microarray analysis confirmed expression of genes specific to matured chondrocytes. Stem cells that internalized nanoparticles at the early stage retained genetic stability, even after passage. In in vivo studies in rats, neuronal cartilage formation was observed in damaged lesions 6 weeks after transplantation of T1 and T4 cells. The degree of differentiation into chondrocytes in the cartilage defect area, as determined by mRNA and protein expression of COLII and SOX9, was higher in rats treated with SF-NPs. Conclusion: The QD nanogels used in this study, did not affect genome integrity during long-term subculture, and are thus suitable for multiple theranostic applications.

Gene expression profiling of chondrogenic differentiation by dexamethasone-conjugated polyethyleneimine with SOX trio genes in stem cells.

BACKGROUND: During differentiation of stem cells, it is recognized that molecular mechanisms of transcription factors manage stem cells towards the intended lineage. In this study, using microarray-based technology, gene expression profiling was examined during the process of chondrogenic differentiation of human mesenchymal stem cells (hMSCs). To induce chondrogenic differentiation of hMSCs, the cationic polymer polyethyleneimine (PEI) was coupled with the synthetic glucocorticoid dexamethasone (DEX). DEX/PEI could be polyplexed with anionic plasmid DNAs (pDNAs) harboring the chondrogenesis-inducing factors SOX5, SOX6, and SOX9. These are named differentiation-inducing nanoparticles (DI-NPs). METHODS: A DI-NP system for inducing chondrogenic differentiation was designed and characterized by dynamic light scattering and scanning electron microscopy (SEM). Chondrogenic induction of hMSCs was evaluated using various tools such as reverse-transcription polymerase chain reaction (RT-PCR), Western blotting, confocal fluorescent microscopy, and immunohistochemistry analysis. The gene expression profiling of DI-NP-treated hMSCs was performed by microarray analysis. RESULTS: The hMSCs were more efficiently transfected with pDNAs using DI-NPs than using PEI. Moreover, microarray analysis demonstrated the gene expression profiling of hMSCs transfected with DI-NPs. Chondrogenic factors including SOX9, collagen type II (COLII), Aggrecan, and cartilage oligometric matrix protein (COMP) were upregulated while osteogenic factors including collagen type I (COLI) was downregulated. Chondrogenesis-induced hMSCs were better differentiated as assessed by RT-PCR, Western blotting analyses, and immunohistochemistry. CONCLUSION: DI-NPs are good gene delivery carriers and induce chondrogenic differentiation of hMSCs. Additionally, comprehensive examination of the gene expression was attempted to identify specific genes related to differentiation by microarray analysis.