Magnetofection Mediated Transient NANOG Overexpression

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Magnetofection mediated transient Nanog overexpression enhances proliferation and myogenic differentiation of human hair follicle derived mesenchymal stem cells Seoyoung Son, Mao-shih Liang, Pedro Lei, Xiaozheng Xue, Edward P. Furlani, and Stelios T Andreadis Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/bc5005203 • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Bioconjugate Chemistry

Magnetofection mediated transient Nanog overexpression enhances proliferation and myogenic differentiation of human hair follicle derived mesenchymal stem cells

Running Title: Magnetofection can effectively over express Nanog in hHF-MSCs

Seoyoung Son1#, Mao-Shih Liang1#, Pedro Lei1, Xiaozheng Xue1, Edward P. Furlani1,3, and Stelios T. Andreadis1,2,4, §

1

Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, NY 14260-4200 2 Department of Biomedical Engineering, 3 Department of Electrical Engineering University at Buffalo, State University of New York, Amherst, NY 14260-4200 4 Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203

#: Contributed to this work equally

§

Address for all Correspondence:

Stelios Andreadis, Ph.D. Professor Bioengineering Laboratory, 908 Furnas Hall Department of Chemical and Biological Engineering, Department of Biomedical Engineering, and Center of Excellence in Bioinformatics and Life Sciences University at Buffalo, The State University of New York Amherst, NY 14260-4200, USA Tel: (716) 645-1202 Fax: (716) 645-3822 email: [email protected]

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Abstract We used magnetofection (MF) to achieve high transfection efficiency into human mesenchymal stem cells (MSCs). A custom-made magnet array was generated and characterized. Theoretical predictions of magnetic force distribution within each well demonstrated that there was no magnetic field interference among magnets in adjacent wells. An optimized protocol for efficient gene delivery to human hair follicle derived MSCs (hHF-MSCs) was established using an EGFPencoding plasmid, reaching approximately ~50% transfection efficiency without significant cytotoxicity. Then we applied the optimized MF protocol to express the pluripotency-associated transcription factor Nanog, which was previously shown to reverse the effects of organismal aging on MSC proliferation and myogenic differentiation capacity. Indeed, MF-mediated Nanog delivery increased proliferation and enhanced the differentiation of hHF-MSCs into smooth muscle cells (SMCs). Collectively, our results show that MF can achieve high levels of gene delivery to MSCs and therefore, may be employed to moderate or reverse the effects of cellular senescence or reprogram cells to the pluripotent state without permanent genetic modification.

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Introduction Mesenchymal Stem Cells (MSCs) have the potential to differentiate into multiple lineages including osteocytes, chondrocytes, adipocytes and myocytes. They can be isolated from various autologous sources such as bone marrow 1, adipose tissue 2 or hair follicles 3-6. In addition, the immune-privilege and paracrine effects of MSCs are great advantages for many regenerative medicine applications 7-9. However, donor aging and culture senescence reduce the proliferation and differentiation potential of MSC significantly, limiting their culture time to about 8-10 passages and preventing their expandability to the large cell numbers required for cellular therapies 10-14. This is a major concern, as the patients mostly in need for cellular therapies are elderly. Nanog is a divergent homeodomain transcription factor that is necessary to maintain embryonic stem cell (ESCs) pluripotency and self-renewal in synergy with Oct4 and Sox2 15, 16. Whereas ectopic expression of Nanog enhanced proliferation of NIH-3T3 17, 18 or bone marrow derived (BM)-MSCs 19-21, the effects of Nanog on differentiation are unclear and contextdependent. Terminal differentiation of myogenic progenitors into muscle was not affected by Nanog expression but transdifferentiation into osteocytes was impaired 22. Interestingly, coexpressing Nanog and Oct4 lowered the efficacy of myoblast progenitor terminal differentiation 23

. On the other hand, in human BM-MSC, ectopic expression of Nanog enhanced

chondrogenesis and osteogenesis but inhibited adipogenesis 19, 20. Our group previously demonstrated that ectopic expression of Nanog in adult MSCs using lentivirus enhanced MSC proliferation and completely restored the diminished myogenic differentiation potential, as evidenced by expression of SMC marker proteins and contractile function 21. These data suggest 1



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that ectopic expression of Nanog may be employed as a strategy to overcome the effects of cellular senescence, either due to aging or extensive in vitro culturing, thereby increasing the potential of MSC for use in regenerative medicine. Despite these promising results, using lentivirus has some drawbacks including permanent integration of lentiviral vector into the target cell genome, which increases the likelihood of activating oncogenes or inactivating tumor suppressor genes 24, thereby hampering clinical applications. This prompted us to seek alternative strategies to overexpress Nanog in MSCs. Although non-viral delivery of plasmid DNA into cells is considered safer 25, the efficiency of gene transfer into difficult-to-transfect cells such as MSCs is very low 26. Several strategies have been proposed to enhance the transfection efficiency in primary cells including MSC such as using cationic liposome based methods e.g. Lipofectamine 2000 27, Fugene6 or PEI 28

but some of them suffer from cytotoxic effects and their efficiency remains cell type

dependent. On the other hand, physical methods may be used to enhance gene transfer efficiency. In particular, magnetofection (MF) uses nano-magnetic particles (MPs) to form complexes with DNA, and then shuffle the DNA toward the cells in the presence of magnetic force, thereby significantly increasing the transfection efficiency. This simple method has been shown to yield higher transfection efficiency both in vitro and in vivo 29, 30. Different MP surface modifications including cell penetrating peptide (CPPs) 31 or endosomal escaping reagents (e.g. PEI) have also been proposed for further enhancement of MF efficiency 32. Although MF demonstrated dosedependent toxicity on cells, using suitable MP:DNA ratios and proper magnetic field intensity/exposure time can decrease the detrimental impacts of MF on cells 33, resulting in 2


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successful transfection to stem cells and other difficult-to-transfect cells such as human endothelial cells 29, 34, 35, neural stem cells 36, 37, neurons 38, 39 and other primary cell types 40. In this study, we hypothesized that MF may be employed for efficient gene transfer to MSC, enabling ectopic expression of Nanog to levels necessary to promote proliferation and enhance the differentiation potential into smooth muscle cells (SMC). To this end, an effective MF protocol was established using an EGFP-encoding plasmid for gene transfer to human hair follicle derived mesenchymal stem cells (hHF-MSCs), a relatively easily accessible source of stem cells. Unlike epidermal stem cells that originated from the epidermal compartment of the hair follicle, these cells were derived from the dermal papilla or dermal sheath and have been shown to be able to differentiate into bone, cartilage, fat as well as smooth muscle cells under proper differentiation conditions 3-6, 41-43. The optimization parameters included the MP:DNA ratio, the duration of MP:DNA complex incubation with cells, and the number of MF applications. The optimal MF protocol was then employed to deliver Nanog-encoding plasmid and to investigate its effects on proliferation and myogenic differentiation of hHF-MSCs.

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Results Magnetic field and force analysis To perform magnetofection (MF), cells were seeded in a 24-well tissue culture plate overnight, followed by addition of the transfection complex. Subsequently, the plate was aligned with a custom-designed magnetic plate containing an array of 24 cylindrical rare-earth magnets, which matched well-to-well to a 24-well plate (Fig. S1). Each magnet produces a non-uniform magnetic field exerting an attractive force on magnetic nanoparticles within each respective cell culture well. Based on the dimensions and properties of the magnets (see Materials and Methods), the magnetic field produced by these structures was characterized (Fig. 1A). It is noteworthy that when using our theoretical model to back-calculate the intensity of the magnetic field, Br

44-46

, it

resulted in a value of 1.26 T, which was very close to the maximum remnant magnetization value of 1.28 T. This indicated that the magnets were essentially magnetized to saturation. The measured field components Bx, By and Bz on different planes above the magnet are also shown (Fig. 1A). The theoretical field predictions are in excellent agreement with the measured data as demonstrated in Fig. 1B, which shows both sets of data for Bz over a 24 mm x 24 mm square area at distance z = 1 mm above the magnet. Next, the force experienced by the magnetic particles was determined by using equations 1-3 (see Materials and Methods). The radial and axial force components Fmr and Fmz on the particle were plotted along a line that spans the diameter of the magnet. It should be noted that these forces are axisymmetric due to the cylindrical symmetry of the magnet and hence Fmr and

Fmz (Fig. 2A) were displayed here in a cross-sectional view as a function of normalized distance x/Rm from the center of the magnet. Note that the magnetic force on the particle is on the order 4


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of femto-Newtons (fN). It is instructive to compare field-directed particle transport with Brownian motion. To this end, we compare the magnetic energy expended in moving a particle a distance equal to its diameter (Dp) i.e. Emag = Fmag ⋅ Dp, with the thermal energy, kT. For this analysis we use the average magnetic force 1 mm above the magnet (Fig. 2A) and find that near the magnet, Emag is on the same order as kT. Thus particles close to the magnet will be captured and the concentration gradient that results will accelerate the downward diffusion of more distant particles, which will ultimately be captured as well. Moreover, the particles can aggregate into clusters during transport due to attractive dipole-dipole interactions. This would result in accelerated capture due to a stronger effective magnetic force on the particle cluster. Finally, a surface plot of Fmz at z=1 mm above the entire array of 24 magnets is shown in Fig. 2B. This analysis shows that there is negligible overlap in the forces of neighboring magnets, i.e. the magnetic field of a given magnet does not impact particle motion in the neighboring wells.

MF293T significantly improved gene delivery efficiency in 293T cells but had detrimental effects on MSCs First we used 293T cells to develop a MF protocol for efficient gene transfer to target cells. After a series of optimization steps, we derived a protocol that resulted in almost 100% transfected cells and significant enhancement in transgene copies delivered to cells, as evidenced by increased green fluorescence intensity (GFI) (Fig. S2). Briefly, 0.5:2 (µg of Magnetic particles (MPs): µg of DNA) were first mixed in serum free DMEM for 20 min to allow MP:DNA complex formation before applying on top of each well of 293T cells that were cultured in DMEM supplemented with 10% FBS. Subsequently, the magnetic field was applied under the 5



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cells for 20 min followed by 20 hours of incubation at 37°C before replenishing with fresh medium. One day later, the cells were ready for analysis (Fig. 3A). Our data demonstrated that the optimized MF293T protocol significantly enhanced the percentage of transfected cells as well as the number of gene copies per cell as compared to the conventional calcium phosphate precipitation method (CP) (Fig. 3B). The fraction of EGFP+ cells increased significantly from 83.42±4.66% with CP to 99.60±0.64% with MF293T (p

Magnetofection Mediated Transient NANOG Overexpression

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