Genome Editing of MSCs as a Platform for Cell Therapy.

The rapid growing of genome editing technology leads to an optimistic expectation for the treatment of many incurable diseases. The core of genome editing relies on DNA-repairing processes which occur independently in each cell, thus generating a mix of genetic variation cells. Here, we describe the protocol of using mesenchymal stem cells as a platform to generate high purity of ZFN-edited patients' cell clones which may be useful in conjunction with therapeutic cell conversion and reprograming.

Doxorubicin-conjugated dexamethasone induced MCF-7 apoptosis without entering the nucleus and able to overcome MDR-1-induced resistance.

BACKGROUND: Doxorubicin (DOX) is the most widely used chemotherapeutic agent that has multimodal cytotoxicity. The main cytotoxic actions of DOX occur in the nucleus. The emergence of drug-resistant cancer cells that have the ability to actively efflux DOX out of the nucleus, and the cytoplasm has led to the search for a more effective derivative of this drug. MATERIALS AND METHODS: We created a new derivative of DOX that was derived via simple conjugation of the 3' amino group of DOX to the dexamethasone molecule. RESULTS: Despite having a lower cytotoxic activity in MCF-7 cells, the conjugated product, DexDOX, exerted its actions in a manner that was different to that of DOX. DexDOX rapidly induced MCF-7 cell apoptosis without entering the nucleus. Further analysis showed that Dex-DOX increased cytosolic oxidative stress and did not interfere with the cell cycle. In addition, the conjugated product retained its cytotoxicity in multidrug resistance-1-overexpressing MCF-7 cells that had an approximately 16-fold higher resistance to DOX. CONCLUSION: We have synthesized a new derivative of DOX, which has the ability to overcome multidrug resistance-1-induced resistance. This molecule may have potential as a future chemotherapeutic agent.

Efficient ZFN-Mediated Stop Codon Integration into the CCR5 Locus in Hematopoietic Stem Cells: A Possible Source for Intrabone Marrow Cell Transplantation.

We reported a simple genome editing approach that can generate human immunodeficiency virus-1 (HIV) coreceptor defective cells, which may be useful for latent viral eradication treatment. Samples of bone marrow leftover after diagnostic procedures and crude bone marrow from aviremic HIV patients were subjected to zinc finger nuclease-mediated stop codon insertion into chemokine receptor 5 (CCR5) loci. Locked nucleic acid-based polymerase chain reaction was used to estimate the amount of insertion in the expandable CD34+ cells. The results showed that about 0.5% of CD34+ cells carried stop codon insertions in CCR5 loci. Cells edited using this simple protocol have the potential to be infused back into the bone marrow.

Cytoprotective effect of glutaraldehyde erythropoietin on HEK293 kidney cells after silver nanoparticle exposure.

The toxic effects from exposure to silver nanoparticles (AgNPs), which are broadly present in many consumer products, have long raised concerns. Many studies have focused on the mechanisms of nanosilver, which cause toxicity in human cells, but little is known about prevention of this type of injury. This study investigated the in vitro effects of glutaraldehyde erythropoietin (GEPO), a cytoprotective compound derived from erythropoietin, in terms of cell protection against AgNP-induced injury. HEK293 cells were pretreated with or without GEPO before administration of AgNPs. The protective effects of GEPO in this cell line were assessed by the percentage of viable cells, alterations of cell morphology, and the proliferative capability of the cells. In addition, we assessed the role of GEPO in lowering cellular oxidative stress and regulating expression of the anti-apoptotic protein Bcl2. The results showed rescue effects on the percentage of viable and proliferative cells among GEPO pretreated cells. Pretreatment with GEPO maintained the normal cell shape and ultrastructural morphology. Moreover, GEPO reduced the generation of reactive oxygen species in cells and activated expression of Bcl2, which are the major mechanisms in protection against cellular toxicity induced by AgNPs. In conclusion, our study showed that the cytotoxic effects from exposure to AgNPs can be prevented by GEPO.

Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones.

BACKGROUNDS: Homozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest. RESULTS: Taking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual's cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells. CONCLUSION: We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.