Qingtong Yu

MicroRNA Replacing Oncogenic Klf4 and c-Myc for Generating iPS Cells via Cationized Pleurotus eryngii Polysaccharide-based Nanotransfection

Induced pluripotent stem cells (iPSCs), resulting from the forced expression of cocktails out of transcription factors, such as Oct4, Sox2, Klf4, and c-Myc (OSKM), has shown tremendous potential in regenerative medicine. Although rapid progress has been made recently in the generation of iPSCs, the safety and efficiency remain key issues for further application. In this work, microRNA 302-367 was employed to substitute the oncogenic Klf4 and c-Myc in the OSKM combination as a safer strategy for successful iPSCs generation. The negatively charged plasmid mixture (encoding Oct4, Sox2, miR302-367) and the positively charged cationized Pleurotus eryngii polysaccharide (CPEPS) self-assembled into nanosized particles, named as CPEPS-OS-miR nanoparticles, which were applied to human umbilical cord mesenchymal stem cells for iPSCs generation after characterization of the physicochemical properties. The CPEPS-OS-miR nanoparticles possessed spherical shape, ultrasmall particle size, and positive surface charge. Importantly, the combination of plasmids Oct4, Sox2, and miR302-367 could not only minimize genetic modification but also show a more than 50 times higher reprogramming efficiency (0.044%) than any other single or possible double combinations of these factors (Oct4, Sox2, miR302-367). Altogether, the current study offers a simple, safe, and effective self-assembly approach for generating clinically applicable iPSCs.

Cationic carbon quantum dots derived from alginate for gene delivery: One-step synthesis and cellular uptake

UNLABELLED Carbon quantum dots (CQDs), unlike semiconductor quantum dots, possess fine biocompatibility, excellent upconversion properties, high photostability and low toxicity. Here, we report multifunctional CQDs which were developed using alginate, 3% hydrogen peroxide and double distilled water through a facile, eco-friendly and inexpensive one-step hydrothermal carbonization route. In this reaction, the alginate served as both the carbon source and the cationization agent. The resulting CQDs exhibited strong and stable fluorescence with water-dispersible and positively-charged properties which could serve as an excellent DNA condensation. As non-viral gene vector being used for the first time, the CQDs showed considerably high transfection efficiency (comparable to Lipofectamine2000 and significantly higher than PEI, p<0.05) and negligible toxicity. The photoluminescence properties of CQDs also permitted easy tracking of the cellular-uptake. The findings showed that both caveolae- and clathrin-mediated endocytosis pathways were involved in the internalization process of CQDs/pDNA complexes. Taken together, the alginate-derived photoluminescent CQDs hold great potential in biomedical applications due to their dual role as efficient non-viral gene vectors and bioimaging probes. STATEMENT OF SIGNIFICANCE This manuscript describes a facile and simple one-step hydrothermal carbonization route for preparing optically tunable photoluminescent carbon quantum dots (CQDs) from a novel raw material, alginate. These CQDs enjoy low cytotoxicity, positive zeta potential, excellent ability to condense macromolecular DNA, and most importantly, notably high transfection efficiency. The interesting finding is that the negatively-charged alginate can convert into positively charged CQDs without adding any cationic reagents. The significance of this study is that the cationic carbon quantum dots play dual roles as both non-viral gene vectors and bioimaging probes at the same time, which are most desirable in many fields of applications such as gene therapy, drug delivery, and bioimaging.

Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles

This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS) with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a) into human umbilical cord mesenchymal stem cells (HUMSCs). After modification with branched low-molecular-weight (1,200 Da) polyethylenimine, the cationized PPS (CPPS) was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles). Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa). These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1) had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system.

Pleurotus eryngii Polysaccharide Promotes Pluripotent Reprogramming via Facilitating Epigenetic Modification.

Pleurotus eryngii is a medicinal/edible mushroom with great nutritional value and bioactivity. Its polysaccharide has recently been developed into an effective gene vector via cationic modification. In the present study, cationized P. eryngii polysaccharide (CPS), hybridized with calcium phosphate (CP), was used to codeliver plasmids (Oct4, Sox2, Klf4, c-Myc) for generating induced pluripotent stem cells (iPSCs). The results revealed that the hybrid nanoparticles could significantly enhance the process and efficiency of reprogramming (1.6-fold increase) compared with the CP nanoparticles. The hybrid CPS also facilitated epigenetic modification during the reprogramming. Moreover, these hybrid nanoparticles exhibited multiple pathways (both caveolae- and clathrin-mediated endocytosis) in their cellular internalization, which accounted for the improved iPSCs generation. These findings therefore present a novel application of P. eryngii polysaccharide in pluripotent reprogramming via active epigenetic modification.

A novel formulation of [6]-gingerol: Proliposomes with enhanced oral bioavailability and antitumor effect

[6]-Gingerol, one of the components of the rhizome of Ginger, has a variety of biological activities such as anticoagulant, antioxidative, antitumor, anti-inflammatory, antihypertensive, and so forth. However, as one of the homologous phenolic ketones, [6]-gingerol is insoluble in water which limits its applications. Herein, we prepared [6]-gingerol proliposomes through modified thin-film dispersion method, which was spherical or oval, and physicochemically stable with narrow size distribution. Surprisingly, in vitro release of [6]-gingerol loaded proliposome compared with the free [6]-gingerol was significantly higher and its oral bioavailability increased 5-fold in vivo. Intriguingly, its antitumor effect was enhanced in the liposome formulation. Thus, our prepared [6]-gingerol proliposome proved to be a novel formulation for [6]-gingerol, which significantly improved its antitumor effect.

Photoluminescent Cationic Carbon Dots as efficient Non-Viral Delivery of Plasmid SOX9 and Chondrogenesis of Fibroblasts

With the increasing demand for higher gene carrier performance, a multifunctional vector could immensely simplify gene delivery for disease treatment; nevertheless, the current non- viral vectors lack self-tracking ability. Here, a type of novel, dual-functional cationic carbon dots (CDs), produced through one-step, microwave-assisted pyrolysis of arginine and glucose, have been utilized as both a self-imaging agent and a non-viral gene vector for chondrogenesis from fibroblasts. The cationic CDs could condense the model gene plasmid SOX9 (pSOX9) to form ultra-small (10–30 nm) nanoparticles which possessed several favorable properties, including high solubility, tunable fluorescence, high yield, low cytotoxicity and outstanding biocompatibility. The MTT assay indicated that CDs/pSOX9 nanoparticles had little cytotoxicity against mouse embryonic fibroblasts (MEFs) compared to Lipofectamine2000 and PEI (25 kDa). Importantly, the CDs/pSOX9 nanoparticles with tunable fluorescence not only enabled the intracellular tracking of the nanoparticles, but also could successfully deliver the pSOX9 into MEFs with significantly high efficiency. Furthermore, the CDs/pSOX9 nanoparticles-mediated transfection of MEFs showed obvious chondrogenic differentiation. Altogether, these findings demonstrated that the CDs prepared in this study could serve as a paradigmatic example of the dual-functional reagent for both self-imaging and effective non-viral gene delivery.

Neural differentiation of fibroblasts induced by intracellular co-delivery of Ascl1, Brn2 and FoxA1 via a non-viral vector of cationic polysaccharide

Direct reprogramming of other somatic cells into neurons is an alternative strategy for the recovery of an injured nervous system. In this work, we developed a new non-viral gene carrier based on Porphyra yezoensis polysaccharide (PYP). After modification with ethylenediamine, the cationized PYP (Ed-PYP) was combined with plasmids encoding Ascl1, Brn2 and FoxA1 to form spherical nanoscale particles (Ed-PYP-pABF nanoparticles). Cytotoxicity assays proved that Ed-PYP-pABF nanoparticles had a better safety profile than Lipofectamine 2000 and polyetherimide. Characterization tests illustrated that the Ed-PYP-pABF nanoparticles at an Ed-PYP:pABF weight ratio of 40:1 is a potential candidate for gene delivery, which was further supported by Western blot and plasmid encoding enhanced green fluorescence protein transfection. Based on this transfection strategy, we co-delivered pABF to 3T6 cells using Ed-PYP. ELISA indicated that the levels of brain-derived neurotrophic factor, nerve growth factors and sonic hedgehog reached a maximum at 14 days after the last transfection. Immunofluorescence and Western blot further exhibited positive expression of neurofilament 200, Nestin, glial fibrillary acidic protein, growth associated protein-43, β-3tubulin, and microtubule associated protein 2, proving the successful conversion of 3T6 cells into neurons. Taken together, these results illustrated that a natural polysaccharide-based gene co-delivery system is a promising strategy for neural reprogramming.

Prolonged Three-Dimensional Co-Delivery of Yamanaka Factors for Cell Reprogramming.

Reprogramming somatic cells into a pluripotent state has been widely investigated in two-dimensional (2D) systems but not described in the more biologically faithful three-dimensional (3D) scaffolds. Here, we devise a 3D porous tissue engineering scaffold that could achieve successful and efficient induction of pluripotency. To construct this 3D scaffold, nonviral hybrid nanoparticles were fabricated beforehand by employing calcium phosphate and cationized Pleurotus eryngii polysaccharide to codeliver plasmids OCT4, SOX2, KLF4 ,and C-MYC (pOSKM). These hybrid nanoparticles were then loaded into a 3D porous collagen scaffold to obtain the so-called pOSKM-activated 3D scaffold. This 3D scaffold could reprogram human umbilical cord mesenchymal stem cells (HUMSCs) into a pluripotent state, generating 3D cell spheres which showed positive expression of pluripotency markers in the 3D scaffolds and tightly packed colonies when transferred to 2D feeder layers. Besides sharing similar morphology, epigenetic modification, and expression of pluripotency genes with the embryonic stem cells, the 3D system-generated colonies could also be expanded on feeder layers for more than 20 passages, indicating the successful establishment of stable induced pluripotent stem cell (iPSC) lines. Our findings represent a first employment of porous 3D scaffolds to achieve successful reprogramming via a one-time transfection, offering a safe, simple, and effective alternative strategy for iPSC generation.

Human chorionic plate-derived mesenchymal stem cells transplantation restores ovarian function in a chemotherapy-induced mouse model of premature ovarian failure

BackgroundPrevious studies have reported that transplantation of mesenchymal stem cells (MSCs) from many human tissues could ameliorate ovarian dysfunction. However, no study has revealed the therapeutic efficiency of MSCs derived from the chorionic plate (CP-MSCs) for premature ovarian failure (POF).MethodsWe investigated the restorative effects of CP-MSCs on cyclophosphamide (CTX)-induced POF. The POF mouse models were established via intraperitoneal injection of 50 mg/kg CTX into female mice for 15 consecutive days. After that, CP-MSCs were intravenously transplanted into the mice once a week for 4 weeks. The serum estradiol (E2) and follicle-stimulating hormone (FSH) levels in the mouse models were detected using enzyme-linked immunosorbent assay (ELISA) before and after treatment. Ovarian function was evaluated through counting the follicles, estrous cycles, and oocytes.ResultsCP-MSC transplantation restored the serum hormone level and ovarian function of the mice in the mouse model of POF induced by CTX. The levels of serum E2 and FSH in the POF model group was 232.33 ± 17.16 pg/mL and 4.48 ± 0.29 mIU/mL, respectively, after 6 weeks of treatment, which were similar to the values in the wild-type (WT) group. The superovulation demonstrated that ovarian function was significantly improved compared with nontreated POF model mice. The CP-MSC transplantation could restore CTX-induced ovarian dysfunction.ConclusionsOur results offer a potential application for human CP-MSCs in POF treatment.