CADD522

Effects of labeling human mesenchymal stem cells with superparamagnetic zinc–nickel ferrite nanoparticles on cellular characteristics and adipogenesis/osteogenesis differentiation

Solaleh Ghanbarei . Naghmeh Sattarahmady . Farzaneh Zarghampoor .Negar Azarpira . Mahdokht Hossein-Aghdaie

Abstract

Objective An attractive cell source for stem cellbased therapy are WJ-MSCs. Hence, tracking WJMSCs using non-invasive imaging procedures (such as MRI) and contrast agents (Zn0.5Ni0.5Fe2O4, NFNPs) are required to evaluate cell distribution, migration, and differentiation.
Results Results showed that the bare and dextrincoated NFNPs were internalized inside the WJ-MSCs and had no effect on the cell viability, proliferation, apoptosis, karyotyping, and morphology of WJ-MSCs up to 125 lg/mL. Besides, treated WJ-MSCs were differentiated into osteo/adipocyte-like cells. The expression of RUNX 2, SPP 1 (P\0.05), and OCN (P[0.05) genes in the WJ-MSCs treated with dextrin-coated NFNPs was higher than the untreated WJ-MSCs; and the expression of CFD, LPL, and PPAR-c genes was reduced in WJ-MSCs treated with both NFNPs in comparison with the untreated WJMSCs (P[0.05).
Conclusion Overall, results showed that dextrincoated NFNPs had no adverse effect on the cellular characteristics, proliferation, and differentiation of WJ-MSCs, and suggesting their potential clinical efficacy.

Keywords Wharton’s jelly mesenchymal stem cells Differentiation Osteogenesis Adipogenesis

Introduction

Stem cells such as mesenchymal stem cells (MSCs) are of great interest for Cellular therapies because of their high regenerative potential. MSCs can be isolated from various tissues and organs such as bone marrow, skin, brain, umbilical cord (UC), fat tissue, and bone. MSCs have the potential to differentiate into bone, fat tissue, chondrocytes, and myocytes (Malgieri et al. 2010). Besides, several studies have shown that an injection of MSCs can promote the regeneration of injured/ischemic tissue and neuroprotection. Consequently, one of the main aspects of using MSCs regenerative therapies is the ability to survive, track, and integrate within the host tissue and undertake the desired cellular differentiation (Weiss et al. 2006; Jomura et al. 2007; Yang et al. 2008). However, an effective method for detecting infused-MSCs in vivo is required. Cell tracking in vivo is the main procedure in the improvement of stem cell therapies. Currently, noninvasive imaging procedures are used to assess the migration and function of MSCs. Several pre-clinical imaging techniques are available for cell tracking including computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), single-photon emission computer tomography (SPECT), optical imaging, and ultrasound imaging (Taboada et al. 2007; Wang et al. 2011; Riegler et al. 2013).
MRI is one of the non-invasive and informative techniques for imaging transplanted stem cells for long periods of time (Guzman et al. 2008; Ren et al. 2011). MRI has several advantages than other methods (such as PET, SPECT, fluorescence and bioluminescence imaging) including high spatial resolution, widespread availability, and that it does not expose the patient to ionizing radiation. This method can provide anatomical and pathological information on the surrounding tissue such as edema and inflammation surrounding the transplantation site. Therefore, MRI can provide additional information for clinicians, and it can help to understand all aspects of certain cellular therapy (Anderson et al. 2005; Iwanami et al. 2005). To achieve this, the transplanted stem cells need to be treated with a contrast agent before transplantation (Deddens et al. 2012). Several magnetic nanoparticles can be used for visualization and tracking of stem cells or MSCs that lead to cell detection as positive or negative contrast on MR images (Ferreira et al. 2008; Cromer Berman et al. 2011; Guenoun et al. 2012; Astolfo et al. 2013; Bao et al. 2014). However, the variance between the contrast of the images of normal and abnormal tissues is not always adequate. Thus, using an effective contrast agent can significantly increase the contrast of MR images (Ito et al. 2005; Ma et al. 2008; Liu et al. 2011).
Magnetic nanoparticles (MNPs) are of great interest for scientists in biomedicine applications because of their properties such as non-toxicity, biocompatibility, injectability, and high-level of accumulation in the target tissue or organ (Elsherbini et al. 2011; Heli et al. 2016). MNPs cause negative contrast in MRI and distinguishes transplanted stem cells from endogenous tissue by altering one or more of the time constants (T1 and T2 relaxation times) (Ito et al. 2005; Hai et al. 2008). MNPs can be classified into several groups including superparamagnetic, paramagnetic, ferromagnetic, and ferrimagnetic nanoparticles (Weissleder et al. 1990; Bulte and Kraitchman 2004; Shapiro et al. 2004, 2006). Ferrite nanoparticles (such as Zn0.5Ni0.5Fe2O4 nanoparticles, NFNPs) are a class of superparamagnetic iron oxide nanoparticle (SPIONs) contrast agents. Studies have shown that the Superparamagnetic behavior of SPIONs is decreased as soon as it accumulates (Raynal et al. 2004), thus coating of NFNPs by polymeric shells inhibits the accumulation and increases the biocompatibility for in vivo applications (Portet et al. 2001; Niemirowicz et al. 2012). Up to now, different polymers have been used for coating contrast agents, among which starch-based materials, such as dextrin, due to their biodegradable property, are a good choice for coating contrast agents (Wong and Mooney 1997; Kim et al. 2001; Marques et al. 2002; Wei et al. 2011). In a previous study, the Zn0.5Ni0.5Fe2O4 nanoparticles were coated with starch-based materials (dextrin) to enhance membrane permeability and inhibit NFNPs accumulation (Sattarahmady et al. 2016).
In the current study, we initially evaluated the uptake of bare and dextrin-coated NFNPs by human Wharton’s jelly mesenchymal stem cells (WJ-MSCs) and then the effect of both NFNPs on the proliferation, viability, apoptosis, karyotyping, and morphology of WJ-MSCs was explored. Furthermore, we induced adipo/osteogenesis differentiation in the treated and untreated WJ-MSCs and then assessed the level of expression of adipogenesis and osteogenesis genes.

Materials and methods

Synthesis of dextrin-coated NFNPs

All chemicals for the synthesis of the bare and dextrincoated NFNPs were purchased from Merck (Germany) or Scharlau (Spain). The Zn0.5Ni0.5Fe2O4 nanoparticles were synthesized by an aqueous precipitation technique and then coated with dextrin to increase solubility and biocompatibility. The details of construction and Characterization (morphology, size, structure, and magnetic properties) were previously described (Sattarahmady et al. 2016). The size distribution of dextrin-coated NFNPs was a mean diameter of about 20.5 ± 3.2 nm. Furthermore, the results showed that these nanoparticles have superparamagnetic properties and can be used as an effective negative contrast agent in MRI.

WJ-MSC isolation and expansion

This research was approved by the council and ethics committee of Shiraz University of medical SciencesIran with the ethical gridlines of the Helsinki Declaration of 1975 (the ethics code IR.SUMS.REC. 1397.752). The umbilical cords were collected from volunteer donors and immediately transferred to the laboratory in serum-free Dulbecco’s Modified Eagle Medium F12 (DMEM/F-12; Gibco, USA). After washing, samples were cut into 2–3 cm pieces and the umbilical vessels removed. The Wharton’s jelly was then minced into pieces and plated in tissue culture flasks containing DMEM/F-12 medium supplemented with 10% FBS (Gibco, USA) and incubated at 37 C and 5% CO2 (WJ-MSCs media). After 48 h, non-adherent MSCs were removed by washing. The WJ-MSCs were sub-cultured at 70% confluence following the treatment with 0.25% Trypsin–EDTA (Sigma-Aldrich, USA).

Iron uptake test

To detect the cellular iron uptake, the WJ-MSCs were seeded in the 6-well plates (15 9 104 cells/well), cultured in WJ-MSC media, and then treated with bare and dextrin-coated NFNPs. After the cell washing with Xylene and deionized water, a prepared solution containing 10% of potassium ferrocyanide, HCL, and potassium ferrocyanide added and the mix incubated for 20 min. Subsequently, the cells were washed and stained by eosin for 1 min. Finally, the cell analysis was performed using an invert microscope.

Cell viability assay

To assess the effect of bare and dextrin-coated NFNPs on cell viability, MTT assay was performed. For this purpose, WJ-MSCs were seeded in 96-well plates (2 9 104 cell/well) a day prior to the experiment and then treated with different concentrations of 5, 25, 125, and 725 lg/mL of bare and dextrin-coated NFNPs. The untreated WJ-MSCs was considered as a control group. The viability was evaluated 24, 48, 72, and 96 h post-treatment. The WJ-MSCs media was removed and MTT (Sigma, USA) made up in the medium to a final concentration of 0.5 mg/mL was added. After 3 h of incubation at 37 C, the plate was centrifuged for 5 min in 21009 g and then MTT solution removed and 100 ll/well DMSO added. The plate was incubated at 37 C for 30 min and the absorbance (570/630 nm) measured using a microplate reader (BioTek, USA). The results indicated that the bare and dextrin-coated NFNPs up to 125 lg/mL had no adverse effect on the cell viability and consequently, other tests were performed with 125 lg/mL concentration.

WJ-MSC growth curve

The trypan-blue staining and cell counting were performed to estimate the effect of bare and dextrincoated NFNPs on the proliferation. For this purpose, the WJ-MSCs were seeded in the 24-well plates (5 9 103 cells/well) and then treated with two groups of NFNPs (125 lg/mL). After five different times of incubation (2, 4, 6, 8, and 10 days), the cells were collected and stained with trypan blue dye and counted. Finally, the results were demonstrated as the percentage of live cells.

Cell apoptosis assay

The effect of bare and dextrin-coated NFNPs on cell apoptosis was evaluated using the AnnexinV-FITC staining and flow cytometry method (BD FACS Calibur, USA). After culturing, both types of NFNPs (125 lg/mL) were added and then cell apoptosis assay was performed using the Phosphatidyl Serine Detection kit (IQ products, Netherlands) according to the manufacturer’s instructions. Untreated cells were examined as a control group, as well.

Genotoxicity evaluation

For evaluation of genotoxicity of bare and dextrincoated NFNPs on the WJ-MSCs, the karyotype was assessed in treated and untreated cells. For this purpose, the WJ-MSCS were seeded in the 6-well plates (15 9 104 cells/well) and treated with two types of NFNPs (125 lg/mL). Afterwards, 0.1 lg/mL colcemid (KaryoMAX- Gibco, USA) was added to the WJ-MSCs media and incubated for 2 h. The trypsinized cells were centrifuged at 5289g for 5 min. Next, the cells were treated in a hypotonic solution (KCl 0.56%) for 45 min at 37 C and fixed in a freshly prepared 3:1 mixture of methanol and acetic acid (Merck, Germany). After seeding the WJ-MSCs on glass slides, were dried and soaked in diluted 1.5% trypsin with PBS solution. The slides were washed with PBS three times and soaked in Giemsa (Merck, Germany) for 4 min. Finally, the analysis of chromosome structural aberrations was performed by GenAysis software.

Cell phenotypes analysis

To evaluate the effect of the bare and dextrin-coated NFNPs on cell phenotypes, the CD90 marker was examined by flow cytometric analysis after being treated with two groups of NFNPs. For this purpose, WJ-MSCs were seeded at 15 9 104 cells/well in 6-well plates. Next, cells were treated with bare and dextrin-coated NFNPs. After a day, the untreated and treated cells were trypsinized and suspended with PBS. Subsequently, the cells were incubated with 10 lL of FITC label anti-Human CD90 antibody (Biolegend-USA # 328108) for 20 min at 4 C. Finally, the cells were washed twice with PBS, then suspended in PBS and analyzed by flow cytometry method (BD FACS Calibur, USA). FlowJo software (version 10.6) was used for analyzing flow cytometry data.

WJ-MSCs differentiation

To evaluate the effect of the bare and dextrin-coated NFNPs on the osteo/adipogenesis differentiation, the WJ-MSCs were seeded at 15 9 104 cells/well in 6-well plates (Sigma-Aldrich, UK). Upon reaching 60–70% confluence, the WJ-MSCs media was replaced with differentiation medium. To induce osteogenesis differentiation, StemProTM Osteogenesis Differentiation Kit (Gibco, USA) and 125 lg/mL of the bare and dextrin-coated NFNPs were used. The osteogenesis differentiation medium was replaced every 3 days for 21 days. For adipogenesis assays, the WJ-MSCs media was replaced with adipogenesis media (StemProTM Adipogenesis Differentiation Kit; Gibco, USA) and 125 lg/mL of the bare and dextrincoated NFNPs. The adipogenesis differentiation medium was replaced every 3 days for 14 days. The untreated WJ-MSCs were cultured in WJ-MSCs media as a control group.

Alizarin Red S staining

To confirm the osteogenesis differentiation, Alizarin Red S (Sigma, USA) staining was performed, as follows: the osteogenesis differentiation media from each well was carefully aspirated and then washed with 2 mL PBS. Subsequently, the cells were fixed with 4% formaldehyde and incubated at room temperature (RT) for 30 min. The fixative solution was carefully removed and rinsed three times with an excess of distilled water. Then, 2% Alizarin Red Stain Solution was added and incubated at RT for 3–4 min. Finally, the excess dye was aspirated and the cells washed four times with deionized water.

Oil Red O staining

Oil Red O (Merck, Germany) Staining was used to confirm the adipogenesis differentiation. The staining steps were as follows: the adipogenesis differentiation media from each well was carefully removed and then washed with 2 mL PBS. After, the cells were fixed with 10% formaldehyde and incubated at RT for 30–60 min. The fixative solution was carefully removed and rinsed three times with an excess of distilled water. Subsequently, isopropanol (60%) was added and incubated at RT for 5 min. The isopropanol was then removed and 2 mL Oil Red O dye added and incubated at RT for 5 min. Finally, the excess dye was removed and the cells washed four times with deionized water.

Real-time PCR assay

The effect of bare and dextrin-coated NFNPs on the expression of osteo/adipogenesis cell markers (osteogenesis markers: OCN, SPP 1, and RUNX 2; and adipogenesis markers: LPL, CFD, and PPAR-c) were examined by quantitative real-time PCR analysis. After induction osteo/adipogenesis differentiation in the WJ-MSCs, the cells trypsinized and total RNA was extracted using TRIzol reagent (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Next, the RNA was reversely transcribed by a cDNA synthesis kit (Takara, Japan). The quantitative real-time PCR analysis was performed using the ABI step one plus Real-time PCR System (Applied Biosystem, USA) and SybrGreen PCR Master Mix (Takara, Japan). The RNA of adipogenesis induced cells was extracted on day 14 and osteogenesis RNA was extracted on days 3, 6, and 8 after induction. The GeneRunner and AlleleID softwares were used to design the primers of interesting genes. Subsequently, the primer specificity was confirmed by Primer-BLAST (www.ncbi.nlm.nih.gov/ tools/primer-blast) and In-Silico PCR (https:// genome.ucsc.edu/cgi-bin/hgPcr). The sequence of primers was used in the current study are listed in Table 1. The GAPDH gene (Glyceraldehyde-3-phosphate dehydrogenase) was used as the housekeeping gene, as it is known to have only minor fluctuations versus the comparatively higher fluctuations of other genes.

Statistical analysis

All experiments were repeated at least three times. The SPSS software was used for data analysis. One-way analysis of variance (ANOVA) and Tukey test were used to determine whether there is any statistically significant difference between the experiments. The p\0.05 was considered statistically significant. Data were presented as mean ± SEM.

Results

WJ-MSC isolation and expansion

Wharton‘s jelly-derived mesenchymal stem cells were grown from the edge of Wharton’s jelly explants about 5 days post culture. After 7 to 11 days, the cells reached 70% confluency and were subcultured (Fig. 1).

Iron uptake test

The cellular uptake of bare or dextrin-coated NFNPs was explored by the potassium ferrocyanide staining. As shown in Fig. 2, compared to untreated WJ-MSCs, the WJ-MSCs treated with the bare or dextrin-coated NFNPs were positive for staining with potassium ferrocyanide.

Cell viability assay

The effect of bare and dextrin-coated NFNPs (5, 25, 125, and 725 lg/mL) on the cell viability were evaluated using MTT assay at four different times (24, 48, 72, and 96 h). As shown in Fig. 3, there were no statistically significant differences between the viability of treated and untreated cells after 24, 48, and 72 h on any group (5, 25, and 125 lg/mL). However, the cell viability of WJ-MSCs treated with bare and dextrin-coated NFNPs (725 lg/mL) at 72 and 96 h was significantly reduced (P-value\0/05). Therefore, other tests were performed at 125 lg/mL of the bare and dextrin-coated NFNPs. After determining the appropriate concentration of bare and dextrin-coated NFNPs (125 lg/mL), cell viability was examined (with 125 lg/mL) in a long time and no adverse effect was observed (p[0/05).

WJ-MSC growth curve

The effect of the bare and dextrin-coated NFNPs (125 lg/mL) on the proliferation was evaluated by trypan blue staining and cell counting (on days 0, 2, 4, 6, 8, and 10). As shown in Fig. 4, the proliferation of WJ-MSCs treated with bare and dextrin-coated NFNPs was similar to the proliferation of untreated WJ-MSCs until day 10.

Cell apoptosis assay

To determine whether bare and dextrin-coated NFNPs treatment (125 lg/mL) induce WJ-MSC apoptosis, we were examined the treated WJ-MSCs using AnnexinV-FITC staining. As shown in Fig. 5, we observed no increases in apoptosis of WJ-MSCs treated with bare and dextrin-coated NFNPs.

Genotoxicity evaluation

For evaluation of genotoxicity of bare and dextrincoated NFNPs on the WJ-MSCs, the karyotype was assessed in treated and untreated WJ-MSCs. The distribution of the chromosomes in treated and untreated WJ-MSCs at metaphase shown in Fig. 6. The results of classical G-banding showed normal diploid karyotype in each group.

Cell phenotypes analysis

To evaluate the effect of bare and dextrin-coated NFNPs on cell phenotypes, the CD90 marker was examined by flow cytometric analysis on the untreated and untreated WJ-MSCs. As shown in Fig. 7, both NFNPs treated WJ-MSCs were as positive for CD90 marker as untreated WJ-MSCs. These results further confirm that there are no obvious influences of the bare and dextrin-coated NFNPs treating the properties of WJ-MSCs.

WJ-MSCs differentiation

To confirm the osteo/adipogenesis differentiation, Alizarin Red S and Oil Red O staining were performed. The results of Alizarin Red S showed calcium deposition in the cells, which confirmed the establishment of an osteocyte-like phenotype. In addition, cultured WJ-MSCs in adipogenesis differentiation medium displayed the development of fat vacuole in the cells that confirmed the establishment of an adipocyte-like phenotype. However, we observed no difference in the control samples of any groups (Fig. 8).

Real-time PCR assay

To assess the effect of the bare and dextrin-coated NFNPs on the expression of osteo/adipogenesis marker genes, a real-time PCR assay was performed. For evaluation osteogenesis marker, expression of RUNX 2, OCN, and SPP 1 genes on day 3, 6, and 8 after osteogenesis induction was assessed. As shown in Fig. 9, the expression of RUNX 2, SPP 1 (P\0.05), and OCN (P[0.05) genes in the WJ-MSCs treated with dextrin-coated NFNPs was higher than the untreated WJ-MSCs. The expression of OCN and RUNX 2 genes was reduced in the WJ-MSCs treated with bare NFNPs in comparison with the untreated WJ-MSCs. In addition, the expression of the SPP 1 gene in the WJ-MSCs treated with bare NFNPs was higher than untreated WJ-MSCs. On the other hand, dextrin-coated NFNPs the expression of RUNX 2, OCN, and SPP 1 genes in the WJ-MSCs treated with dextrin-coated NFNPs compare to WJ-MSCs treated with bare NFNPs was significantly increased (P\0.05).
The expression of adipogenesis gene markers was evaluated on day 14 after adipogenesis induction. As shown in Fig. 10, the expression of CFD, LPL, and PPAR-c genes in the WJ-MSCs treated with the dextrin-coated NFNPs compare to untreated WJMSCs was reduced (P[0.05). In addition, the expression of the CFD, PPAR-c (P[0.05), and LPL (P\0.05) genes in the WJ-MSCs treated with bare NFNPs was lower than untreated WJ-MSCs. However, the level of gene expression (CFD, LPL, and PPAR-c) in WJ-MSCs treated with bare and dextrincoated NFNPs was not significantly different from each other.

Discussion

Stem cells, particularly WJ-MSCs, are an attractive cell source due to their multipotent, immunomodulatory properties, accessibility, and reparative abilities (Rosenberg et al. 2018; Volarevic et al. 2018; Kabat et al. 2020), which can be used for stem cell-based therapy in treating ischemic stroke and many other ischemia-associated, neurodegenerative maladies, and other cell death-related diseases. Hence, tracking WJMSCs using non-invasive imaging procedures to determine the fate and survival of WJ-MSCs are required to evaluate cell distribution, migration, and differentiation (Ren et al. 2011). Molecular imaging exploits multimodal imaging to achieve important information at the cellular and molecular levels in the in vivo characterization and measurement of biological processes (Forte et al. 2020). There are several pre-clinical imaging procedures are available for cell tracking, of which MRI is widely used for tracking transplanted stem cells for highly sensitive, noninvasive, high spatial resolution, and non-radiation detection protocols (Wang et al. 2011). Besides, the MRI technique can be serially used for long-term tracking of cells after transplantation (Yahyapour et al. 2018; Stroh et al. 2019). Although PET (positron emission tomography) and SPECT (single-photon emission computerized tomography) approaches are highly sensitive for cell tracking, they have low spatial resolution and radionuclides used in these methods have a short half-life and emit ionizing radiation. Moreover, fluorescence and bioluminescence imaging have limited penetration depth and poor spatial resolution (Jasmin et al. 2017). Therefore, MRI possesses favorable benefits for non-invasively tracking cell transplants, which make it a viable alternative to traditional histological analysis.
For the purposes of labeling cells, a range of molecules including SPIONs, fluorescent dyes, or radionuclides can be used (Hong et al. 2010; Youn and Hong 2012). One of the favorable contrast agents for tracking stem cells by MRI is the SPIONs (such as Zn0.5Ni0.5Fe2O4 nanoparticles, NFNPs) (Fan et al. 2011; Li et al. 2013). Since uncoated SPIONs do not efficiently label stem cells, commercially available SPIONs include particles that are coated with polyamines, polysaccharides, or high negative charges polymer (Li et al. 2013). The labeling efficiency of WJ-MSCs is extremely dependent on interactions between NFNPs and cell membranes. These interactions and labeling can be the influence of various target factor including antibodies, cellular receptors, nanoparticles coating properties (such as charge and hydrophobicity/hydrophilicity), physical and chemical properties of nanoparticles (such as particle size, surface charge, roughness, and surface curvature) and a negative charge on the cell membrane (Decuzzi and Ferrari 2007; Mahmoudi et al. 2010). In a previous study, the NFNPs was coated with starch-based materials (dextrin) to enhance membrane permeability and inhibit NFNPs accumulation. The result of MR imaging using phantom agar (1% (w/v) agarose gel in 96-well ELISA plates) showed that dextrin-coated NFNPs could be used as a good negative contrast agent. In addition, the ultra-small size of dextrincoated NFNPs (approximately 20 nm) was very appropriate for clinical application (Sattarahmady et al. 2016).
There are various endocytosis pathways in mammalians cells such as WJ-MSCs including clathrin, caveolin, flotillin 1, GRAF1 kinases, small G proteins, actin, and dynamin (Gillingham and Munro 2007; Girao et al. 2008; Doherty and McMahon 2009; Howes et al. 2010; Patil et al. 2018). During cell labeling, deformation of the plasma membrane of cells occurs, and this mechanism of endocytosis requires the organized action of proteins (Mahmoudi et al. 2010). Hence, the clathrin-mediated and caveolinmediated pathways may be the possible mechanism to take up NPs into cells (Ahn et al. 2019).
As mentioned, one of the physical properties of nanoparticles that influences on their cellular uptake is the size of NPs. Studies have shown that SPIONs with a diameter of 20 nm or particles larger than 900 nm are less taken up by the cells than Resovist with a diameter of 62 nm (Lee et al. 2010). Another study also indicated that Resovist was more effectively taken up into human MSCs than Feridex (a dextrancoated SPIONs) (Maila¨nder et al. 2008). Consequently, it is suggested that the difference in biopolymers could influence the efficiency of take up particles into cells. In the present study, bare and dextrin-coated NFNPs was also successfully incorporated into the WJ-MSCs. The bare and dextrin-coated NFNPs is a SPIONs contrast agent designed for cell labeling (Sattarahmady et al. 2016) and have a mean diameter of about 20.5 ± 3.2 nm.
After confirmed the uptake of bare and dextrincoated NFNPs by WJ-MSCs, we evaluated the effect of both types of NFNPs on the WJ-MSCs properties. We found that the bare and dextrin-coated NFNPs had no effect on the cell viability of WJ-MSCs up to 125 lg/mL. In addition, our results showed that bare and dextrin-coated NFNPs (125 lg/mL) had no effect on the proliferation of WJ-MSCs. Taken together, the bare and dextrin-coated NFNPs (125 lg/mL) do not affect the proliferative capacity of cells, karyotype, and morphology of cells. The previous studies demonstrated that iron oxide nanoparticles (ION) such as MIRB with concentrations above 30 lg/mL for 20 h (Molday ION Rhodamine B) reduced cell viability due to generate reactive oxygen species (ROS) in the cells and leading to oxidative stress and cellular toxicity (Addicott et al. 2011; Luo et al. 2015).
The ability of multipotency of WJ-MSCs to produce various mesenchymal cell lineages such as osteocyte- and adipocyte-like is crucial for cells to be defined as MSCs. We found that WJ-MSCs treated with bare and dextrin-coated NFNPs (125 lg/mL) differentiated into osteo/adipocyte-like cells and no cytotoxicity on labeling of WJ-MSCs was observed, which was consistent with the previous results of hMSC labeling with SPIONs (Bulte et al. 2001; Bulte and Kraitchman 2004; Bulte 2005; Hsiao et al. 2007). To corroborate the osteo/adipogenesis differentiation at the molecular level, the expression of gene markers after differentiation induction was assessed. The results showed that the expression of RUNX 2, OCN, and SPP 1 genes in the WJ-MSCs treated with dextrincoated NFNPs was higher than the untreated WJMSCs and treated with bare NFNPs. Although the expression of adipogenesis gene markers was reduced in WJ-MSCs treated with bare and dextrin-coated NFNPs in comparison with the untreated WJ-MSCs, this difference was not statistically significant. In the previous study, the effect of SPIONs on 22 and 29 risk genes for obesity and Type 2 diabetes in human adipocytes. The results showed that the expression of some genes severely induced, while the expression of other genes such as LPL was down-regulated (Sharifi et al. 2013). Other studies have also reported that hMSCs labeling with SPIONs did not have an inhibition effect on the osteogenic and adipogenic differentiation of MSCs, which is compatible with our results (Wang et al. 2015; Liao et al. 2016; Silva et al. 2016). A study by Chen et al. showed that SPIONs (300 lg/mL) downregulated the osteogenesis gene marker such as alkaline phosphatase and inhibited the osteogenic differentiation (Chen et al. 2010). They have also indicated inhibition of osteogenesis differentiation was dose-dependent. The SPIONs with high concentration induce cell migration and activate the signaling molecules including b-catenin, cancer/testis antigen, SSX, and matrix metalloproteinase 2 (MMP2) and finally inhibit the differentiation pathway. Therefore, these conflicting results in various studies likely to be related to the differences in concentration and incubation time. Although the present study shows that NFNPs will not have too much effect on WJMSCs’ behavior, we suggest the effect of NFNPs evaluate in the animal model by in vivo imaging.

Conclusion

In summary, we demonstrated that dextrin-coated NFNPs were effectively uptake by WJ-MSCs and did not exert toxic effects on the cell viability and proliferation, apoptosis, as well as the surface stem cell marker expression and the multi-differentiation potentials. Consequently, dextrin-coated NFNPs could be used as a biocompatible agent for WJ-MSCs tracking. Overall, our findings provide new information and introduce a noninvasive technique for tracking transplanted cells into injured sites.

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