Novel Non-viral Nano Gene Delivery Systems For Cell Reprogramming

Since 2006 when the induced pluripotent stem cells (iPSCs) were first reported by Yamanaka and colleagues, somatic cell reprogramming has been a hotspot in stem cell research. Ectopic expression of a defined set of pluripotent factors (such as Oct4, Sox2, c-Myc, Klf4, etc) mediated by viral transduction can reprogram somatic cells to a pluripotent state and finally achieve phenotype and multiple differentiation potentials that are similar to those of embryonic stem cells (ESCs). With the ability of self-renewal and multiple differentiation potential into three-germ lineages, iPSCs has become an ideal alternative to ESCs in clinical application because they can be derived from patients’somatic cells so that to avoid ethic issues associated with ESCs. More importantly, iPSCs generated from patient somatic cells make it possible to provide individual-based treatment for patients. Therefore, iPSCs have attracted great attention worldwide. However, the clinical application of iPSCs has been impaired by safety issues caused by viral vectors and the involvement of transcription factors (like c-Myc) that are related to cancer. Meanwhile, the current non-viral methods for iPSCs generation suffered from low reprogramming efficiency. In order to meet the safety requirements and huge demand for iPSCs in clinical applications, it is urgent to develop safe and efficient reprogramming strategies. To this end, this study focuses on three aspects including construction of novel non-viral gene vectors, screening of new combinations of reprogramming factors, and conduction of somatic cell reprogramming in three-dimensional non-viral systems for the first time, to improve safety and enhance reprogramming efficiency of iPSCs generated from human umbilical cord mesenchymal stem cells (HUMSCs).Chapter 1 Review on research progress of cell reprogrammingThis section describes the concept of cell reprogramming systematically, provides a detailed review of the development status of somatic cell reprogramming and the great application potentials of iPSCs, and summarized the methods of cell reprogramming based on massive literatures. Meanwhile, the problems that iPSCs are facing have also been addressed, whereby the topic basis and design of this dissertation were presented. Altogether, this section offers a theoretical guidance for the development of this work.Chapter 2 Natural polysaccharides-based nano delivery of transcription factor OCT4This section lays the experimental basis for the development of novel non-viral gene vectors for somatic cell reprogramming. These non-viral vectors were derived from naturally occurred polysaccharides with appropriate cationic modification, followed by assessment of the ability to condense plasmid OCT4, examination of cytotoxicity and determination of transfection efficiency to select safe and efficient gene carriers for cell reprogramming. Specifically, the crude polysaccharides were isolated from four raw materials (represented by alphabets A, B, C and D, respectively) using water-extraction and alcohol-precipitation method and purified by column chromatography. Then, natural polysaccharides (A, B, C and D) with high purification and centralized molecular weight distribution were obtained. For cationic modification, three different methods, namely, spermine (Sp), ethylenediamine (Ed) and polyethyleneimine (PEI), were employed to chemically modify each polysaccharide. Agarose gel electrophoresis was conducted to screen out four cationized polysaccharides (A-Ed, B-PEI, C-Ed and D-Sp) that showed the optimal combination effect with plasmid OCT4 for the subsequent evaluations of transfection efficiencies. The findings of transmission electron microscopy, particle size measurement and Zeta potential determination demonstrated that these cationized polysaccharides could effectively condense and incorporate macromolecular plasmid OCT4 to obtain spherical and/or spheroidal cationized polysaccharides/OCT4 nanoparticles with positive surface charges. The MTT colorimetry proved that the cationized polysaccharides/OCT4 nanoparticles prepared in this study had excellent biocompatibility and extremely low toxicity. The enzyme linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and living cell imaging were employed to comprehensively assess the gene delivery effect of cationized polysaccharides/OCT4 nanoparticles from various aspects, including protein expression level, RNA transcriptional level as well as the real-time process of cellular uptake. These results fully confirmed that cationized polysaccharides hold huge potential to be developed into desirable non-viral gene vectors.Chapter 3 Screening of optimal combination of reprogramming factors for generating induced pluripotents stem cellsThis chapter is centralized on optimizing the combinations of reprogramming factors so as to broaden insights to improve the safety of iPSCs and reprogramming efficiency. In this section, the cationized polysaccharide D-Sp was selected as the representative gene carrier to co-deliver the negatively-charged plasmids, encoding OCT4, SOX2, and miR302-367, alone and in all possible double and triple combinations with each other (resulting in a total 7 different compositions). Quantitative RT-PCR (qRT-PCR) was carried out to examine the transfection efficiency and alkaline phosphatase staining was performed to screen out the positively stained clones, whereby the optimal combination was selected, that is, OCT4, SOX2 and miR302-367 (OS-miR). This combination was employed for subsequent, in-depth investigation for reprogramming human umbilical cord mesenchymal stem cells into iPSCs. The resulting D-Sp/OS-miR nanoparticles were characterized by a series of tests including agarose gel electrophoresis, transmission electron microscopy, particle size determination and Zela potential measurement. The findings showed that D-Sp could completely combine the plasmids and result in the smallest size (ranging from 70 to 150 nm) when the D-Sp/OS-miR weight ratio was 10:1. Furthermore, the D-Sp/OS-miR nanoparticles had regular spherical and/or spheroidal shapes, uniform distribution, and positive surface charge. QRT-PCR, immunofluorescence staining, western blot and hematoxylin-eosin (HE) staining were conducted to confirm that the iPSCs induced by D-Sp/OS-miR nanoparticles could positively express pluripotent markers and successfully differentiate into all three-germ layers both in vitro and in vivo.Chapter 4 Three-dimensional non-viral nano gene delivery systems for cell reprogrammingThis section represents an initial attempt to explore the possibility of somatic cell reprogramming in three-dimensional (3D) non-viral nano gene delivery systems, opening up a brand new avenue of generating iPSCs. The 3D collagen scaffold was prepared by direct freeze-drying of 2% collagen solution without addition of any cross-linking agent and characterized by morphology observation, porosity measurement and determination of water absorption. Then, polysaccharide-calcium phosphate hybrid nanoparticles were prepared via a reverse microemulsion method using cationized polysaccharide D-Sp, calcium phosphate, and the four Yamanaka plasmids as raw materials. Agarose gel electrophoresis demonstrated that the hybrid nanoparticles had excellent plasmid retention effect. Next, these hybrid nanoparticles were adsorbed on the inner surface of the scaffold to obtain the 3D gene-laden nanoparticle-collagen scaffold. The scanning electron microscopy showed that the resulting 3D gene-laden nanoparticle-collagen scaffold displayed a highly porous structure, with interconnected pores of the appropriate size, which effectively mimicked the 3D microenvironments of cells in vivo. After that, HUMSCs were seeded on the scaffolds for reprogramming. Over time, small iPSCs aggregates with compact spherical or ellipsoidal shapes, clear-cut and round edges could be observed via HE staining. The RT-PCR assay indicated that the 3D gene-laden nanoparticle-collagen scaffold had significantly higher reprogramming efficiency than the 2D systems. Furthermore, a variety of tests including immunohistochemistry, immunofluorescence staining, karyotype analysis and measurement of methylation of the promoter regions were conducted to confirm the pluripotency of the iPSCs aggregates from the aspects of protein expression level, integrity of chromatosomes, and epigenetic modification. More importantly, these induced iPSCs aggregates could be successfully expanded on the 2D feeder layers for more than 20 passages, indicating the establishment of stable iPSC cell lines. Finally, the in vivo teratoma assay proved that the induced iPSCs were able to differentiate into all three-germ lineages, indicating the successful reprogramming of HUMSCs in the 3D gene-laden nanoparticle-collagen scaffold.ConclusionsThis work successfully screened out non-viral gene vectors based on natural polysaccharides, which were then applied in the nano delivery of reprogramming factor OCT4. The characterization of these polysaccharide-based nanoparticles and the assessment of their gene transfection efficiency proved the huge potential that the cationized polusaccharides could be developed into non-viral gene vectors for reprogramming. By replacing the oncogenic factors C-MYC and KLF4 with the microRNA, we selected a new combination of factors, OCT4+SOX2+miR302-367, which was then used for successful induction of human iPS cells. Furthermore, a 3D tissue engineering system was first constructed for cell reprogramming, and the results showed that compared to the 2D system, the 3D system had facilitated reprogramming kinetics, higher reprogramming efficiency through long-lasting release of the plasmids from the scaffolds. Importantly, the iPS cell spheres generated in the 3D scaffolds could be expanded on the 2D feeder layers, providing experimental basis for clinical application iPS cells.