Enhanced Proliferation and Accelerated Adipogenic Differentiation of Mesenchymal Stem Cells via Fe 3 O 4 -Insulin Conjugates Stimulation

The culture of human Umbilical Cord derived Mesenchymal Stem Cells (UC-MSCs), as well as the control of its differentiation toward different tissue lineages, is a very important part of regenerative medicine. In the present work, a systematic study of Fe 3 O 4 nanoparticles and its insulin conjugates for growth and differentiation of UC-MSCs were carried out for the first time. The cell viability assay was performed using a number of analytical methods including MTT assay and cumulative population doubling time assay. Fe 3 O 4 -insulin conjugates were prepared and used for UC-MSC adipogenic differentiation. Results showed that the strong ability of Fe 3 O 4 -insulin conjugates allow it to act as a preconcentration platform for adipogenic induction, which accelerates MSC growing on it toward the adipogenic lineage. It was also found that the Fe 3 O 4 nanoparticles had no effect on UC-MSC cellular phenotype. This work demonstrates that the biological activity of insulin is not affected when conjugated to Fe 3 O 4 nanoparticles, which would help to enhance the growth and adipogenic differentiation of mesenchymal stem cells.


Introduction
Mesenchymal Stem Cells (MSCs) comprise a population of multipotent progenitors which can be originally isolated from bone marrow, adipose tissue, muscle, placenta, umbilical cord and umbilical cord blood [1,2]. Under appropriate microenvironments, MSCs can differentiate into multiple cell types, including adipocytes, chondrocytes, and cardiomyocytes, also into non-mesodermal-derived cells, including hepatocytes and neurons [3][4][5][6]. Recently, MSCs are being evaluated for their use in tissue engineering and cell-based therapies because of their no ethical restriction and low immunogenicity [7,8].
Obesity has been a common health problem worldwide and it is considered a major risk for a variety of metabolic disorders, such as diabetes mellitus, cardiovascular diseases, cholelithiasis, fatty liver disease and so on. Studies have shown that adipose differentiation and regulation disorders have been associated with the pathological processes involved in diabetes mellitus, fatty liver, hyperlipidemia and certain cancers [9]. Therefore, it's essential to study the mechanism and regulation of adipogenic differentiation for the prevention and treatment of these diseases and related drug screening. Furthermore, adipose tissue regeneration technology, which is a hot field of regenerative medicine, is required by the regeneration and repair of adipose tissue loss and damage due to NorCal Open Access Publications . 01 . NORCAL aging or pathological factors. And also, adipocytes are the source of seeding cells and the driving force of adipose tissue regeneration. Both of the above applications need effective and stable adipogenic differentiation method to obtain large quantity and high-quality adipocytes. However, the current differentiation strategy, which employs growth factor inducers, is of low efficiency and often needs weeks of culture for maturation of the specific lineage.
Recently, there are accumulating clues indicating that nanomaterials with particular properties and biocompatibility can modulate stem cell function, such as proliferation and differentiation [5,10]. For instance, nano-scaled materials such as polymeric, liposomal and silica nanoparticles act as potential carriers for delivery of genes, drugs or bioactive factor for stem cell differentiation [11].
With clear formation of neurites on layer-by-layer assembled single-walled carbon nanotube, mouse embryonic neural stem cells from the cortex were successfully differentiated into neurons, astrocytes and oligodendrocytes [12,13]. Dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer was designed to enhance the osteogenesis for rat bone marrow stromal cells in vitro [14,15]. Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through a stress mechanism relied on p38/MAPK signaling pathway [16].
Fe 3 O 4 nanoparticles, a member of Superparamagnetic Iron Oxide (SPIO) nanoparticles that are emerging as an ideal MRI probe for in vivo cell tracking and cellular imaging, have recently drawn attention as a means to increase cell growth, which can accelerate cell cycle progression by the free Iron (Fe) released from lysosomal degradation [17][18][19]. The research conducted by Song and Schafe respectively, showed that the cartilage and osteogenic differentiation of mesenchymal stem cells were not affected by SPIO [20,21]. However, whether Fe 3 O 4 nanoparticles have effect on MSC adipogenic differentiation remains unclear. Herein, we reported a systematic study on the effects of the Fe 3 O 4 nanoparticles and Fe 3 O 4 -insulin conjugates on the adipogenic differentiation of UC-MSCs. Adipogenic assays revealed that pure Fe 3 O 4 nanoparticles had no effect on cellular ultrastructure and adipogenic differentiation. Under the optimized Fe 3 O 4 nanoparticle concentration that has little cytotoxicity on UC-MSCs and no effects on the UC-MSC phenotype, Fe 3 O 4 -insulin conjugates were prepared and used for UC-MSC adipogenic differentiation. Results showed that the biological activity of insulin was not affected when conjugated to Fe 3 O 4 nanoparticles, which enhanced the growth and adipogenic differentiation of UC-MSCs.

Materials and Methods
All experiments were performed in compliance with the relevant laws and institutional guidelines and Guangzhou Institutes of Biomedicine and Health Ethical Committee have approved the experiments. All volunteer mothers who donated umbilical cord samples have provided their written informed consent and the Ethics Committees have approved this consent procedure.

Preparation of Fe 3 O 4 nanoparticles and Fe 3 O 4 -insulin conjugates:
Fe 3 O 4 nanoparticles were prepared by chemical precipitation method [22]. Briefly, dissolving FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O iron salts separately in distilled water at 80℃ to prepare 1M Fe 3+ and 0.5M Fe 2+ solution. Mixing the two solutions continuously and 25% ammonia water solution was added, following oleic acid addition 1 minute later. The reaction proceeded for 1h at 80℃. After then, the Fe 3 O 4 nanoparticles were washed twice with ethanol and many more times with water until they reached a neutral pH level. Adding a water solution of KMnO 4 into the Fe 3 O 4 nanoparticles and sonicated for 8h and washed with water. Finally, the Fe 3 O 4 nanoparticles were separated through magnetic enrichment and the resulted Fe 3 O 4 nanoparticles were carboxyl modified, which made them showed negatively charged.
The Fe 3 O 4 nanoparticles were then incubated with insulin for 16h at 4℃ and insulin was conjugated to the Fe 3 O 4 nanoparticles through electrostatic adherence. In this case, the release of insulin is facile and faster in the biological system as reported previously [23]. Following repetitive washing of Fe 3 O 4 -insulin conjugates and removal of unconjugated insulin by magnetic enrichment, the Fe 3 O 4 -insulin conjugates were resuspended in PBS for future usage. The unconjugated insulin presented in the supernatant was determined using the Bradford method. The amount of conjugated insulin was calculated according to the formula below [24]: q=(W ti -W si) /W Fe 3 O 4 where q was the amount of insulin conjugated onto a unit mass of Fe 3 O 4 (mg/mg). W ti , W si , and W Fe 3 O 4 represented the total amount of insulin added, the amount of insulin in supernatant after conjugation, and the mass of Fe 3 O 4 , respectively.
Isolation and culture of UC-MSCs: Umbilical Cords (UCs) were aseptically collected from full-term cesarean-section patients with their consent at Guangzhou Red Cross Hospital. UC-MSC isolation was performed as described previously with some modifications. Briefly, Wharton's jelly was cut into 2 to 3 mm 3 pieces, and then cultured in a 37℃ incubator in a 175 culture flask with growth medium containing DMEM-LG supplemented with 10%(v/v) FBS, 2mM glutamine, 5 ng/mL basic fibroblast growth factor, 100 U/ mL penicillin, 100 mg/mL streptomycin, and 1 mg/mL amphoterin B. After 10-14 days of culture, UC-MSCs migrated from tissues. When cells reached 90-95% confluence, cells were harvested with 0.25% trypsin. The medium was changed every 3 days.  morphology tests were carried out using a method published previously [25]. Briefly, UC-MSCs were seeded in a 96-well tissue culture plate at the density of 4×10 4 cells per well and incubated for 3 days. After the addition of Fe 3 O 4 nanoparticles at different concentrations (50, 100, 200, 300 and 400 nM), the cells were further incubated for 24h, 48h and 72h. Then, the adherent cells were harvested for MTT assay. Briefly, 20 μl of MTT (5.0 mg/ ml in PBS) was added and incubated for another 4 h at 37℃. The supernatant was removed and DMSO was added and incubated for 10 min. After all crystals were dissolved, the optical density of each well was measured at 570 nm using a microplate reader. Cells without treatment were used as control.

Cellular assays for UC-MSCs cultured with and without
Cells were cultured for 24h, 48h, and 72h, and were harvested with trypsin and counted after staining with trypan blue. The mean value of cell counts was calculated and mean Population Doubling time (PD) was obtained for each passage according to the formula: PD=(lgN t -lgN 0 )/lg2, where N 0 is the initial cell number and N t is the harvested cell number.
In addition, the effects of pure Fe 3 O 4 nanoparticles on the morphology of UC-MSCs were also investigated. Briefly, cells were cultured in a monolayer for 24 h and fed with pure Fe 3 O 4 nanoparticles at different concentrations (50, 100, 200, 300 and 400 nM) for another 72 h. Then, the media was removed, and the cells were washed with PBS and observed under the microscope. Cells without treatment were used as control.
Adipogenic differentiation: Cells were induced to differentiate into adipocytes as described previously with some modifications [26]. Briefly, cells were cultured in a 24-well plate at a density of 1×10 4 cells/cm 2 . After cells reached 70% confluence, the growth medium was changed to traditional adipogenic differentiation medium consisting of DMEM-LG supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 60 mM indomethacin, 1 mM dexamethasone, 0.5 mM IBMX, and 5 mg/mL insulin solution or adipogenic differentiation medium consisting of DMEM-LG supplemented with Fe 3 O 4 -insulin conjugates instead of insulin alone and other compounds same to the traditional adipogenic differentiation medium. Cells induced with traditional method and our new method which means the induction medium was supplemented with Fe 3 O 4 -insulin conjugates instead of insulin alone were incubated for indicated days, and the medium was changed every 3 days. Generation of neutral lipid vacuoles was visualized by staining with Oil Red O.

Real-Time Polymerase Chain Reaction (Real Time RT-PCR):
Total RNA was isolated with RN easy mini kit following manufacturer's instructions and treated with DNase I. Complementary DNA synthesis was performed with 1 μg of total RNA using Prime Script RTase. Real-time PCR was conducted as follows: 95℃, 15 seconds; 60℃, 15 seconds; 72℃, 30 seconds for 40 cycles using ABI 7300 Real-Time PCR System. Expression level of b-actin gene was used as the internal control.
The PCR primers and probes were as follows: hPPAR-γ2 sense: 5'-GCGAGGATAGTTCTGGAA-3' hPPAR-γ2 antisense: 5'-GGATAAGTCACCGAGGAG-3' Image analysis: Image analysis was carried out following a previously published method [25]. Briefly, at least three random fields of each of triplicate wells of Oil Red O assay were photographed and the software Image J downloaded from National Institutes of Health was used for measurement of adipocyte percentage. Briefly, color images were converted to eight-bit gray-scale and binarized or thresholded to gain good contrast and brightness against the background. Adipocyte percentage was calculated according to the number of pixels in the thresholded images.
Statistical analysis: Data were expressed as mean±standard error of the mean. Statistical comparisons were performed using Student's t-test. * means p-value < 0.05 and were considered statistically significant, ** means 0.01 < p-value < 0.05 and *** means 0.001 < p-value < 0.01.

Effects of Fe 3 O 4 nanoparticles on UC-MSC proliferation
The principle of MSC induction was shown in figure 1. Fe 3 O 4 -insulin conjugates were synthesized according to the electrostatic adsorption. When added to the cultured cells, they entered the cells through endocytosis, then Fe 2+ and Fe 3+ produced by Fe 3 O 4 nanoparticles in the cells can promote cell proliferation.  After cells migrated out from small pieces of umbilical cord Wharton's jelly during primary culture, they were cultured with and without Fe 3 O 4 nanoparticles. As shown in figure 2A, both UC-MSCs cultured with and without Fe 3 O 4 nanoparticles showed similar fibroblast-like morphology.
On the basis of the observed cellular effects and previous reports, we hypothesized that Fe 3 O 4 nanoparticles may interact with cells by membrane adsorption and subsequent internalization through endocytosis, possibly serving as mechanical stimuli on UC-MSCs to activate mechanosensitive signaling pathway in the cells and thus induce adipogenic differentiation. Previous study showed that all cells require iron for the proper uptake of oxygen from their environment and mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Figures 2B and C shows that Fe stimulates growth-related enzyme activity in UC-MSCs. . 04 .

Effects of Fe 3 O 4 -insulin conjugates on adipogenic differentiation of UC-MSCs
As previously reported, insulin is one of the important supplements of adipogenic induction and adipogenic maintenance media [27][28][29]. In the present study, the effects of  O staining, percentage area of the staining was increase following the increase of insulin concentration until 20mg/ml ( Figure 3D). And when it comes to 40mg/mL, the efficiency is lower than that of 20mg/mL. The results indicated that 20mg/mL of Fe 3 O 4 -insulin concentration produces the highest efficiency in MSC adipogenic differentiation induction.

Expression analysis of adipogenic-specific marker by real-time RT-PCR
The messenger RNA expression of the adipogenic-specific marker hPPAR-γ2 was analyzed by real-time RT-PCR. The marker hPPAR-γ2 gene was not expressed before MSCs were incubated in the adipogenic differentiation medium. Expression was detected after the cells were incubated in the adipogenic differentiation medium for different periods of time, and the expression of this marker was several times higher in Fe 3 O 4 -insulin conjugates containing medium than in the traditional differentiation medium ( Figure 4).

Discussion
When given suitable stimulations, the human UC-MSCs in both . 06 . several times higher in Fe 3 O 4 -insulin conjugates induction method than in the traditional differentiation method demonstrating that Fe 3 O 4 -insulin conjugates can dramatically accelerate adipogenic differentiation of UC-MSCs. According to the previous report [23], we hypothesized that cells uptake Fe 3 O 4 -insulin conjugates through endocytosis and the efficiency is higher than that of insulin alone, and the release of insulin from Fe 3 O 4 -insulin conjugates in cells are easy for the conjugates are formed through electrostatic adherence. In conclusion, mesenchymal stem cells can get enough insulin at a short time to promote adipogenic differentiation when incubated with Fe 3 O 4 -insulin conjugates, which result in higher adipogenic differentiation efficiency. The short induction period can satisfy the scientific utility and can get to the wanted adipogenic differentiation state within a short time, which will promote the adipogenic-related study. So the method is time saving and may also promote the clinical study of some disease which need adipose tissue transplantation.

Conclusion
In our work, a systematic study of Fe 3 O 4 nanoparticles and its insulin conjugates for growth and differentiation of UC-MSCs were carried out for the first time. And we verified that Fe 3 O 4 -insulin conjugates promote adipocyte differentiation of UC-MSCs better than that of traditional induction medium. The underlying mechanism maybe that cells are more easily to uptake Fe 3 O 4 -insulin conjugates through endocytosis and the insulin can release from the conjugates to play roles in cell differentiation in the cells [23].