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Silver nanoparticle modified surfaces induce differentiation of mouse kidney-derived stem cells

In this paper, we interrogate the influence of silver nanoparticle (AgNPs)-based model surfaces on mouse kidney-derived stem cells (mKSCs) differentiation. The widespread use of silver in biomedical and consumer products requires understanding of this element's effect on kidney cells. Moreover, the potential for using stem cells in drug discovery require methods to direct their differentiation to specialized cells. Hence, we generated coated model substrates containing different concentrations of surface immobilized AgNPs, and used them to evaluate properties and functions of mKSCs. Initially, mKSCs exhibited reduced viability on higher silver containing surfaces. However, longer culture periods assisted mKSCs to recover. Greater degree of cell spreading and arborization led by AgNPs, suggest podocyte differentiation. Proximal tubule cell marker's expression revealed differentiation to the specific lineage. Although the exact mechanism underpinning these findings require significant future efforts, this study demonstrate silver's capacity to stimulate mKSC differentiation, which may provide opportunities for drug screenings.


Introduction
Silver and silver nanoparticles (AgNPs) have been utilized in a number of consumer products because of their capacity to kill bacteria. [1][2][3][4][5][6][7] In medicine, silver coated catheters, wound dressings and a range of other devices are currently used on a daily basis. 3,8 With the potential benets brought by the antibacterial properties of silver come concerns regarding toxicity to mammalian cells, tissues and overall human health. Although mammalian cells can generally tolerate greater amounts of silver than bacteria, the element can also be toxic to tissues if used in high concentrations. [9][10][11] The inammatory response to silver is still an open question with the area being rather controversial. Many studies claim reduced inammation or no alteration of inammatory response while others report adverse responses. Another area that needs further attention is related to the effect of silver on stem cell fate. We have recently looked at the effect of silver on the differentiation of mesenchymal stem/stromal cell (MSC) and found that oxidative stress caused by the silver led to preferential differentiation to adipocytes rather than osteoblasts. 12 This report demonstrated the need for greater attention to how silver may affect stem cells.
Chronic kidney disease (CKD) is recognized as a serious global health problem. 13 End stage renal disease (ESRD) is the nal stage of CKD and is associated with high rates of morbidity and mortality, as well being nancially costly, with the United States alone spending >US$ 50,000 for each patient suffering from ESRD. Although current therapeutic interventions slow down the progression of CKD to ESRD, the current state of treatment does not generate satisfactory outcomes in all patients. 14 Therefore, it is extremely important to develop new treatment strategies where stem cells can be directed to differentiate to specialized renal cell types that could be useful to treat CKD in order to prevent ESRD progression.
The kidney is a highly complex organ with several distinct cell types, 15 but two cell types in particular, podocytes and proximal tubule cells (PTCs), play crucial roles in maintaining normal kidney function. For instance, podocytes are required to maintain the glomerular ltration barrier, and PTCs are necessary for secreting metabolites into the tubular lumen, and for the absorption of various molecules from the glomerular ltrate. PTCs also play a role in drug metabolism, and consequently, drug-induced nephrotoxicity is a major cause of kidney disease. [16][17][18] Podocytes and PTCs are difficult cells to culture because the former are post-mitotic and the latter rapidly lose their phenotype following isolation from the kidney. There has thus been great interest in generating these cell types from stem cells in order to understand their physiology, investigate how this becomes altered in kidney disease, and to use them in drug screening programs.
We have previously isolated stem cells from neonatal mouse kidneys that are able to generate podocytes and proximal tubule cells (PTCs) in vitro, and ex vivo when transplanted into mouse embryo kidneys. 19,20 Recent reports have also demonstrated that the chemistry and nanotopography of cell culture surfaces regulate the proliferation and differentiation of various stem cell lines. 14,21,22 MacGregor-Ramiasa et al. have investigated the effect of concentration gradient of -NH 2 functional groups on the differentiation of a clonal kidney stem cell (mKSCs) line derived from neonatal mouse kidney. Differentiation of proximal-tubule-like cells was observed on the surfaces with low amine content by the expression of specic markers. 23 With the increased promise of cell therapies and the wide application of silver containing products in medicine and everyday life, it becomes important to evaluate the effect of silver based antibacterial coatings on mKSC fate. In this work, we used established platforms for generation of antibacterial surfaces combining plasma polymer coatings and silver nanoparticles. 6 These types of surfaces were selected since they were demonstrated in our previous work to have excellent antibacterial properties but no toxicity to primary human broblasts or MSCs. The method of preparation also allowed us to generate two different silver nanoparticle surface concentrations which facilitated examination of dose dependent effects. 6,12 The effect of the silver nanoparticle modied surface on the attachment, proliferation, viability, spreading and differentiation of mKSCs was evaluated via microscopic and immunostaining techniques.

Plasma deposition of 2-methyl-2-oxazoline
A custom built capacitively coupled bell-chamber reactor with a 13.56 MHz plasma generator and a matching network (Advanced Energy, USA) described elsewhere 24 was used for plasma deposition. 13 mm glass coverslips and silicon wafers were cleaned with ethanol, dried with nitrogen stream and stored in airtight containers until further use. The substrates were cleaned using air plasma (50 W, 5 minutes, 2 Â 10 À1 mbar) prior to any plasma treatment. Deposition of 2-methyl-2oxazoline was carried out at 50 W for 2 minutes at a precursor pressure of 2 Â 10 À1 mbar. To stabilize the plasma coating, the substrates were kept overnight at room temperature in sealed containers prior to any further modication.

Synthesis of AgNPs@MSA
Silver nanoparticles were synthesized by sodium borohydride reduction of silver nitrate. 6 Briey, 12 ml of 2 mM AgNO 3 was mixed with 5 ml of 2 mM MSA under ice cold condition. 0.5 ml of 0.5 M of NaBH 4 was added to the solution under vigorous stirring. The color of the solution mixture turned to reddish brown aer the addition of NaBH 4 indicated the formation of AgNPs. The AgNPs were stored in light proof container until further use. The solutions remain stable for several months without showing any visible aggregation of particles.

Immobilization of AgNPs@MSA on pPOX substrates
The plasma modied substrates were incubated at room temperature in AgNPs solution for time intervals of 6 and 24 h. Depending on the AgNPs@MSA immobilization time the samples will be represented as S L and S H (6 and 24 h AgNPs@MSA immobilization time respectively) in this paper. Aer incubation the AgNPs@MSA solution was discarded and the samples were rinsed 3 times with MilliQ water to remove any loosely bound AgNPs. Then the samples were dried with nitrogen stream and stored in airtight container until further use.
Glass coverslips were used for atomic force microscope (AFM) and cell culture studies, while silicon wafers were used for ellipsometry and X-ray photoelectron spectroscopy (XPS).

Surface characterization
Ellipsometry. A Variable Angle Spectroscopic Ellipsometer (VASE) (J. A. Woollam Co. Inc.) equipped with a HS-190 high speed monochromator and a VB-400 control module, was used to determine the thickness of plasma polymer lms. All measurements were carried out at different angles from 65 to 75 at an interval of 5 and, over a wavelength range of 250 to 1100 nm with 10 nm increment. WVASE32 soware was used for running the setup, calibration, data collection and analysis. Inbuilt Cauchy algorithm was used to t the data. For each plasma deposition condition, thickness measurements were conducted on three technical replicates of the coating.
Atomic force microscopy (AFM). The topographical analysis of the prepared samples was carried out by an NT-MDT NTEGRA SPM (Moscow, Russia) AFM in non-contact mode. Silicon nitride non-contact cantilevers with gold coating on the reective side (NT-MDT, NSG03) and resonance frequencies between 50 and 150 kHz were used. The tips spring constant is between 0.35 and 6.06 N m À1 , as measured by the Sader method. 25 The amplitude of oscillation was 10 nm, and the scan rate for 5 mm Â 5 mm images was 1.0 Hz. The scanner used had a maximum range of 100 mm and was calibrated in x, y and z directions using 1.5 mm standard grids with a height of 22 nm.
Scanning electron microscopy (SEM). A scanning electron microscope (Carl Zeiss Microscopy Merlin with GEMINI II column) was used to investigate the silicon wafers substrates aer the immobilization of AgNPs@MSA. The microscope was operated at 2 kV with high-resolution column mode (probe current: 100 pA). The secondary electron images were recorded by on-axis in-lens secondary electron detector.
X-ray photo electron spectroscopy (XPS). XPS was used to analyze the chemical composition of the surfaces before and aer the plasma polymerization and AgNPs@MSA immobilization. All surfaces were characterized using a Spec SAGE XPS equipped with a monochromatic Mg radiation source operating at 10 kV and 20 mA. For the quantication of the atomic concentrations of all samples, survey spectra were recorded over the binding energy range of 0-1000 eV at a pass energy of 100 eV and with 0.5 eV resolution. All the binding energies were referenced to the C1s neutral carbon peak at 285 eV, to compensate for the effect of surface charging. Casa XPS soware was used for the processing and curve tting of all resulting spectra.

Cell culture
All solutions and buffers used during cell culture were prewarmed to 37 C prior to use. The stem cell line was derived from neonatal mouse kidney by Fuente Mora et al. 19 Cells were cultured in DMEM in standard tissue culture plates (TCPs). The medium was changed every 2-3 days and cells were incubated with 5% CO 2 at 37 C. 13 mm coated and uncoated glass coverslips were placed in 24 well TCPs and mKSCs were seeded at a density of 10 000 cells per well and incubated for a period of 48 and 96 h. Care was taken to ensure that the cell suspension was evenly distributed during plating.
Cell viability. To determine cell viability, aer each incubation period, DMEM was aspirated and cells were washed 3 times with PBS. 150 mL of 10% CCK-8 (diluted in DMEM 1 : 10) was added to each well and cells were incubated for 1 h. Then 100 mL of the reaction solution was transferred into a 96 well plate. The absorbance of the solution was measured by an Anthos Labtec LP400 spectrophotometer at wavelength of 450 nm for detection (620 nm reference lter). The background of the culture medium was measured and subtracted from all values.
Cell proliferation, population doubling time (PDT) and extent of cell spreading. Cells were xed using 4% (w/v) paraformaldehyde (PFA) for 15 minutes and then incubated in phalloidin (diluted in PBS 5 : 200) labelled with Alexa Fluor® 488 (Invitrogen) for 30 minutes at room temperature in the dark and then nuclei were stained with 4 0 ,6-diamidino-2phenylindole (DAPI). Samples were imaged under epiuorescence illumination using a Leica DM2500 microscope tted with a Leica DFC420C camera. An average of 10 images per coverslip was taken and cell number quantied by counting stained nuclei from the images obtained. All images are representative for the entire surface from the corresponding sample. The extent of cell spreading (phalloidin-stained cells) was measured using ImageJ.
From the total cell numbers at different time points, the mKSC population doubling times (PDTs) were calculated with the help of non-linear regression analysis using the following equation: Cytologymethylene blue (MB) staining. For morphological evaluation, cells were xed with 4% PFA for 15 minutes, washed with PBS and stained with a solution of 200 mL 0.5% (v/v) methylene blue in dH 2 O for 10 minutes at room temperature and washed with PBS. Samples were analyzed using a light microscope.
Podocyte quantication. Phalloidin-labelled cells were imaged aer 96 h and podocyte-like cells were identied based on their morphology including arborized appearance, high cytoplasmic-to-nuclear ratio and binucleation. Podocytes were quantied microscopically following established procedures. 19,20,23,26 10 images per coverslip were acquired and analyzed. Quantication of podocyte-like cells was carried out relative to the cells that did not show typical podocyte morphology. All images are representative for the entire surface from one sample.
Immunouorescence. For immunouorescence, cells were xed at room temperature with 4% (w/v) PFA for 5 minutes, washed 3Â with PBS and then incubated for 1 h at room temperature in blocking solution (10% (v/v) normal goat serum). The serum was removed aer the rst incubation and a second incubation was carried out overnight at 4 C with primary antibody solutions that contained 1% (v/v) goat serum, 0.1% (w/v) Triton-X 100 in PBS and the primary antibodies (Table 1) at the required concentrations. The following day, cells were washed 3Â with PBS, and incubated for 2 h at room temperature with the required concentration of secondary antibody (Table 1) in 1% (v/v) goat serum and 0.1% (w/v) Triton-X 100 in PBS. Cells were then washed 3Â with PBS and counterstained with DAPI.
For quantication of images, an average of 10 images were taken from each sample and number of positive cells was quantied. Images shown represent the entire sample surface as well as an average of all experiments.
Alkaline phosphatase activity assay. On day 4 aer seeding, cells were washed three times with PBS and xed in 4% (w/v) PFA for 5 minutes. Then the cells were equilibrated in Tris-HCl (pH 9.2) for 5 minutes at room temperature, followed by an incubation in a solution containing 2 mg 0.02% (w/v) Naphthol AS-MX phosphate and 10 mg 0.1% (w/v) Fast Red TR in 10 ml Tris-HCl (pH 9.2) for 15 minutes at room temperature in the dark. The reaction solution was then removed and cells were washed once with Tris-HCl pH 9.2 and twice with PBS. Cells were co-stained with DAPI. All experiments were performed with three biological and technical replicates. Methyl-oxazoline plasma polymerized surfaces and uncoated glass coverslips were used as positive (PC) and negative controls (NC) respectively for all cell culture experiments.

Substrate preparation and characterization
To generate model substrates which facilitate understanding of the effect of silver-based coating on the fate of mKSCs, we used silver nanoparticles (AgNPs@MSA) that we reported earlier. 6 We knew that coatings generated using these nanoparticles have excellent antibacterial properties and are non-cytotoxic to primary human broblasts and mesenchymal stem cells. 6,12 Our early approach to immobilize these nanoparticles to the surface was via electrostatic coupling to allylamine plasma polymer lms which are known to retain signicant number of amine groups. 27,28 In this work, we used covalent attachment of the nanoparticle to the substrate since it presents a more stable platform compared to electrostatic binding. 29 To achieve this, we used relatively recent plasma polymer coatings developed by our group which are prepared from oxazoline (pPOX) based precursors. The resultant lms have been demonstrated to retain a population of intact oxazoline functionalities which can be used to covalently bind nanoparticles and ligands containing carboxylic acid groups. [30][31][32] The conditions selected for the plasma deposition resulted in a lm thickness of 25.01 AE 0.03 nm as measured by ellipsometry. AgNPs@MSA were immobilized by immersion of the plasma polymer modied surface in a solution of silver nanoparticles for predetermined time (Fig. 1A).
Aer AgNPs@MSA immobilization, the samples showed a homogenous light brownish colour which is due to the plasmon resonance adsorption of the silver nanoparticles (Fig. 1B,  II). The AgNPs@MSA were spherical with an average size of 12.27 nm similar to those in ref. 6. The SEM analysis showed uniform particle distribution on the plasma modied surfaces and absence of aggregations (Fig. 1C). AFM imaging before the immobilization of the AgNPs@MSA (Fig. SI 1A †) demonstrated that the coatings are continuous, pin hole free with a surface roughness of 0.569 nm (Fig. SI 1C †). The surface roughness increased to 5.511 nm aer the deposition of AgNPs@MSA (Fig. SI 1D †). XPS survey spectra of the unmodied glass coverslips (NC), pPOX coated substrates (PC) and pPOX surfaces with the immobilized AgNPs@MSA is shown in Fig. 1D. As expected, only three elements are present on the bare glass surfacecarbon, oxygen and silicone. Substrates with pPOX plasma polymer lm showed the presence of nitrogen, in addition to carbon and oxygen, which is consistent with the chemical composition of the precursor. Along with the carbon, nitrogen and oxygen a distinct Ag3d peak was detected when AgNPs@MSA were immobilized on the plasma polymer modi-ed samples. The atomic concentration of chemical elements on the surface of the samples is presented in Table 2. S H and S L denote different time of nanoparticle immobilization for 24 h and 6 h, respectively. As anticipated the atomic concentration of silver increased with the time of immobilization from 8.61 AE 0.3% (6 h, samples S L ) to 13.95 AE 0.1% (24 h, samples S H ). Longer immobilization time resulted in greater silver concentration corresponding to greater number of nanoparticles binding to the surface. This time dependent immobilization process allowed us to generate surfaces with different and controlled concentration of silver nanoparticles. Both silver surface concentrations are sufficient to completely eliminate bacterial growth as we demonstrated in ref. 6, however, this allows us to test the inuence of different silver surface concentration on the fate of mKSCs.

Effect of silver nanoparticle concentration on mKSC viability, proliferation and morphology
The viability of mKSCs cultured on silver nanoparticle modied surfaces and controls was determined using the CCK-8 kit assay (Fig. 2A). The results indicate strong reduction in cell viability  aer 48 h in culture, particularly for the higher silver nanoparticle concentration. However, the cells recovered aer 96 h, especially at the lower nanoparticle concentration where cell viability was only 15-20% lower compared to than on control substrates without silver. The reduced initial viability and attachment of the cell on the silver containing samples could be due to the substantial amount of released silver ions from the surfaces. 6,12 The silver ions are the active species that make silver such a potent antibacterial agent but the element could be toxic to mammalian cells too. In a pioneering study we demonstrated the mammalian cells can tolerate greater amount of silver than bacteria. Our nding was also conrmed by others and our follow up studies 3,6,12 which showed that amounts of silver lethal to bacteria did not signicantly affect primary human broblasts and mesenchymal stem cells (MSCs). However, mammalian cells may have different tolerance to silver and our current study demonstrate that, although capable of recovery, the viability of mKSCs was reduced in the early stages of growth. Overall, these data suggest both time-and concentration-dependent effects of silver nanoparticles on mKSC viability. We also assessed the mKSC population doubling time (Fig. 2B). It was greater on the silver nanoparticle modied surface i.e. 45 h and >52 h for S L and S H , respectively. In comparison, the PDT was <40 h on control surfaces.

Podocyte differentiation
mKSCs have been shown to differentiate on surfaces to podocytes and PTCs. 19,20,23 To investigate if the propensity of cells to undergo podocyte differentiation varied between different substrates, we rst measured the extent of cell spreading, because podocyte-like cells tend to have a large cytoplasm : nuclear ratio. F-Actin staining was used to facilitate evaluation of cell spreading area (Fig. 3A). The average spreading areas of cells seeded on silver nanoparticle modied surfaces was nearly twice larger compared to glass coverslip control with (PC) or without (NC) a methyl oxazoline plasma polymer coating. The signicant increase of cell spreading area on samples containing nanoparticles strongly suggests that the silver nanoparticles play a role in driving the observed phenomenon.
In some cells, F-actin staining showed a pattern typical of that in primary podocytes. These cells tended to be highly arborized and were oen binucleate, all of which are typical characteristics of podocytes. The percentage of podocyte like cells on silver treated samples particularly on the S H samples was signicantly higher than the controls (Fig. 3B). Upon differentiation, on both samples containing AgNPs@MSA, mKSCs increased in cell size, developed binucleate cells with arborized morphology (Fig. 3A) aer 96 h. Immunostaining analysis showed the expression of podocyte specic marker podocalyxin by mKSCs cultured on the silver containing substrates. Representative images are provided in the ESI (Fig. SI 2 †). The data also suggests that the greater silver concentration facilitate differentiation of mKSCs into podocytelike cells.
Actin cytoskeleton have key roles in physiological function of podocytes and maintenance of glomerular ltration barrier. [33][34][35] Several studies have reported that mutation or changes in the actin cytoskeleton lead to dysfunctional podocytes and subsequent proteinuria. 33,[36][37][38] No disruption in the actin cytoskeleton organization was observed on the silver treated substrates (Fig. 3A). Our results therefore conrm that the silver did not induce any derangement of the actin cytoskeleton.

Proximal tubule cell differentiation
To examine the differentiation of mKSCs to PTCs we have carried out staining for megalin. The data showed a greater number of megalin expressing cells on silver nanoparticles modied surfaces and particularly on those with lower silver concentration (S L ). The plasma polymer only coating did not show any notable difference to that of glass coverslip control (Fig. 4A), and these cells are signicantly greater in number on the S L surfaces (Fig. 4B).
To further conrm the differentiation to PTCs we carried out staining for alkaline phosphatase, an enzyme that is expressed by PTCs. 39 The images presented in Fig. 4C show signicantly stronger alkaline phosphatase activity of mKSCs grown on S L . This is in good agreement with the data obtained from the megalin assays and further strengthen the nding that silver may be stimulating mKSCs to differentiate into PTCs.
The silver nanoparticle modied surfaces appeared to trigger differentiation of mKSCs to podocyte and PTCs lineage. There are at least two possible explanations for the observed phenomena. Silver nanoparticles have been shown to cause oxidative stress in MSCs and in this way drive the differentiation of these cells. 12 It may be possible that this is the reason behind the current ndings pointing to increased differentiation of the mKSCs when silver was present. On another hand, the silver nanoparticles create surface roughness (Fig. S1 †). We have demonstrated recently that tailored surface roughness, including roughness comparable with this inherent for the model samples used in this study, was capable of guiding the differentiation of mKSCs to podocytes. 23 This nding was not surprising since it is now well accepted that surface nanotopography has signicant inuence on the fate of various types of cells, including immune and stem cells. 27,40,41 Conclusions Novel clinical approaches that involve the use of nanoengineered biomaterials to support and/or direct the differentiation and/or renewal of podocytes and PTCs, are urgently required to combat ESRD. Furthermore, the increasing use of silver in biomedical and consumer products require better understanding of the effect of this element on the fate of kidney stem cells. The results of this study showed that the AgNPs@MSA containing substrates (S L and S H ) affect the viability, spreading, proliferation, population doubling time and differentiation of mKSCs to podocytes and PTCs as evaluated from the expression of markers. Bioreactors, cell culture substrates, cell therapies and tissue engineering constructs may be able to benet from the antibacterial effect of silver nanoparticles and their capacity to stimulate mKSCs differentiation. However, before the later becomes a real opportunity much further work is required to fully and mechanistically understand how and in what direction silver nanoparticles affect the fate of kidney cells. Whether, the observed increased cell differentiation is due to surface nanoroughness or due to generation of reactive oxygen species, the combined effect of both, or due to other factors coming into play will be the subject of future studies.

Conflicts of interest
There are no conicts to declare.