Differentiation of Mouse Stem Cells into Neural Cells on PLGA Microspheres Scaffold

1.Introduction

      The term "tissue engineering" was coined at a committee meeting at the National Science Foundation during the fall of 1987 [1]. Tissue engineering is an interdiscipli-nary technology that applies the principles and methods of bioengineering and life science to assemble and synthesize biological substitutes that will restore, maintain and improve tissue functions following damage by either disease or thrauma. Basically it involves the combination of living cells with either natural or synthetic materials to establish a 3-dimonsional construct that is structurally functional and mechanically equal to, or better than the damaged organ or tissue to be replaced [2]. Research in this field rapidly expanded and resulted in development of different designs and materials used in the preparing of 3-dimensional scaffolds suitable for tissue engineering.

      A vast majority of biodegradable polymers belonging to the polyester family including polyglycolides and polylactides have been studied. Poly L-lactic-co-glycolic acid (PLGA), a biocompatible and biodegradable copolymer, has been used mostly for tissue engineering scaffold and drug delivery carriers because of good mechanical properties, non-toxicity, processibility and biodegradability [5, 6].

      Scaffolds can be prepared in various shapes such as film, fiber, foam and particle. Special structure properties of microspheres such as high surface area and capability to be injected resulted in expanding of cells, rapidly [3]. The key elements in nerve tissue engineering are supportive scaffolds, cells, and trophic factors. It was noted that a permanent material is more likely to provoke a chronic immunological rejection and has a higher risk of infection as well as the potential to compress the axons over time; thus, their scaffolds should be biodegradable but not bioactive, to ensure safety. In addition, they should be cell adhesive to facilitate subsequent proliferation and differentiation [1].

Figure 1. A scanning electron micrograph showing the surface morphology of the PLGA microspheres.

      Nerve tissue repair is a precious treatment concept in human health care as it directly impacts the quality of life. It is well known that the adult central nerve system can not be regenerated on its own after trauma or disease. Therefore, restoring its function is a challenge for neurobiologists and neurologists [4].

       Stem cells have the capacity of self-renewal and differentiation to all types of cells. Like other embryonal carcinoma and embryonic stem cells, P19 cells are develop-mentally pluripotent and appear to differentiate using the same mechanisms as normal embryonic cells [7]. P19 embryonic carcinoma cells have the potential to differentiate into cell types of all three germ layer and form all tissues in the body. Tera-tocarcinomas can develop in some mouse strains from early embryos transplanted from the uterus into ectopic sites. The differentia-tion of these cells can be controlled by nontoxic drugs. P19 cells treated with retinoic acid differentiate into a different spectrum of cell types that include neurons, astroglia and microglia cell types normally derived from the neuroectoderm [7, 8].

       Injuries to the nervous system are the result of mechanical, thermal, chemical or congenital pathologies and if function is not restored, they lead to loss of function, pain and impair sensation. Nerve injuries complicate successful rehabilitation because mature neurons like many other cells in the body do not replicate; that is, they don't undergo cell division [9]. Spinal cord injury interrupts the nerve connections between the brain and the rest of the body and results in paralysis and loss of sensation below the level of injury. Because in most cases of human spinal cord injury there is significant loss of spinal cord tissue, cavity formation is an important obstacle, impeding axonal regeneration [1]. Therefore, repair of the injured human spinal cord in many cases will require not only neural survival, axonal growth and remyelination, but also reconnection across the trauma cavity by means of bridging grafts [9]. There have been encouraging reports of deficit reduction and axonal regrowth by blocking inhibitory molecules and antagonizing secondary injury mechanism; myelin replacement by stem cell, schwann cell and olfactory ensheating cells; delivery of growth factor and small molecules; and implantation of fetal tissue and various types of scaffolds, such as fibers, films, microparticle and nanoparticle scaffolds, as yet there is no practical treatment for spinal cord injury [10].

Figure 2. Phase contrast of P19 cells cultured on the surface of PLGA microspheres (a) and histological examination with H & E stain (b) showing cell adhesion and attachment on the PLGA microspheres.

       The goal of this project was to investigate the use of biodegradable PLGA microspheres as microcarriers for pluripotent cells. In this study retinoic acid (RA) was used which is known as the vitamin an acid as inducer. It affects the differentiation of P19 cells into neuron, astrocytes and oligodendrocytes and cell types normally derived from the neuroectoderm.

2. Materials and methods

2.1. Preparation of PLGA microspheres

      PLGA polymer with uncapped end groups (type RG504H) was purchased from Boehringer-Ingelheim company and polyvinyl alchohol (PVA) (87-89% hydrolyzed, Mol. wt. 3l000-50000 g/mol) was purchased from Aldrich.

      The most commonly used method for preparing PLGA microspheres is the solvent evaporation technique based on the formation of a double emulsion (water-in-oil-in-water). Therefore, in this study, PLGA microspheres were prepared by solvent evaporation, water-in-oil-in-water technique [11, 12]. Briefly, 100 mg of PLGA were dissolved in 500 μl chloroform. This polymer solution was combined with 2 ml of a 2% w/v solution of PVA in distilled deionized water (ddH₂O) and homogenized for 2 min. This emulsion was then added to a 30 ml of stirring 2% w/v PVA/ddH₂O and continued to be stirred for 4 h. The microspheres were then collected via centrifugation using a centrifuge set at 11000 rpm, 20 °C, for 10 min. The PLGA microsphieres were then washed twice with ddH₂O and centrifuged under the previously stated conditions. The resulting microspheres were then freeze-dried for 2-3 days and stored at -20 °C before use.

2.2. Characterization of microspheres

      The diameters and surface morphology were determined using scanning electron microscopy (SEM) (Vega@ Tescan). Briefly, 5vmg of freeze-dried microspheres were resuspended in 500 μl of ddH₂O and air-dried onto aluminum plates. These samples were coated with gold-palladium at a thickness of 20 nm and were then viewed using a SEM.

2.3. Cell seeding

      P19 cell line was obtained from Pasteur Institute, Tehran, Iran. The cells were cultured in Dulbecos modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 μg/ml streptomycin. For loading these cells on strilled microsphere, P19 cells were then trypsinised, centrifuged and resuspended in DMEM containing FBS. For seeding on microspheres, 1.5×10⁵ cells/ml were loaded into each microsphere that previously were became wet with 70% ethanol and suspended in medium culture, containing DMEM and FBS by centrifuge at 1500 rpm for 5 min. After loading the cells, scaffold were suspended in bacterial grade Petri dishes for 6 days in the presence of RA, then the microspheres were plated onto tissue culture grade plastic Petri dishes. Aggregation, cell adhesion and expansion of P19 cells on the PLGA microspheres were examined by brightfield microscopy (Olympus IX51).

Figure 3. Scanning electron microscopy of attachment, expansion and cell differentiation on PLGA microspheres (a, b) and immunofluorescent staining for β-tubulinIII (c) and MapII (d) indicate neural differentiation and  axons formation.

2.4. Histology and immunofluorescent staining

      Microspheres were fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin. Five micrometer-thick transverse sections were placed on silanized slides for immunohistochemistry or staining with hematoxylin and eosin (H & E). For immunocytochemistry, these slides were deparafinized in xylene and dehydrated in 100%, 96% and 70% ethanol for 5 min. and rinsed in ddH₂O. Then, slides should be retrival transveres by neutral buffered trypsin and calcium chloride. The sections were incubated in PBS containing 0.2% TritonX-100 for 15 min. and then incubated in blocking buffer (10% normal goat serum) for lh in 37 °C, thereafter in primary antibodies (β-TubulinIII and MapII) for 12h at 4 °C diluted in blocking buffers. After 12h, the slides were incubated with secondary antibody which was IgG FITC for 1h in 25 °C. Then the slides were washed 3 times with PBS at 5 min./wash and were also stained with PI, a nuclear stain, by a 10 sec at room temperature and three washes in PBS, then analyzed using the fluorescence microscope (Olympus, BX51, Japan).

2.5. RT-PCR

      Total RNA was isolated by the NucleoSpin RNA II Kit (Macherey-Nagle, Duren, Germany). Prior to reverse transcription (RT), RNA samples were digested with DNase I (EN 0521; Fermentas) to remove contaminating genomic DNA. Extracted RNA was treated by DNase I (EN 0521; Fermentas) to avoid cross contamination of RNA by genomic DNA. Standard RT was performed using l μg total RNA, random hexamer primer and the RevertAid^TM H Minus First Strand cDNA synthesis Kit (Kl622; Fermentas) according to the manufacturer's instructions. The cDNA samples were subjected to polymerase chain reaction (PCR) amplification using mouse specific primers designed using different exons (Table1). Amplification conditions were as follows: Initial denaturation at 94 °C for 5 min. followed by 30 cycles of denaturation at 94 °C for 30s, annealing at 59-70 °C for 45s, extension for 45s at 72 °C and a final polymerization at 72 °C for 10 min. The PCR products were analyzed by gel elec-trophoresis on l.5% agarose and stained with ethidium bromide and visualized and photographed on a UV transilluminator (Uvidoc, UK).

Figure 4. RT-PCR confirmed the neural differentiation of P19 carcinoma stem cells on PLGA microspheres.

3. Results

3.1. Characterization of microspheres

      The particle size and surface morphology were determined by scanning electron microscopy (SEM). The average diameter of them was 13-100 μm and they had smooth outer surface (Figure 1).

3.2. Cell seeding

      P19 cells adhesion and growth on the surface of PLGA microspheres on days 1-6 in culture in bacterial grade Petri dishes was observed using phase contrast micrographs (Figure 2a). Microspheres were totally surrounded with P19 cells.

3.3. Histology

      H & E staining, on the slides, also demonstrated that P19 cells were placed on the surface of microspheres and surrounded them. The cells lined the outer surfaces of the scaffold, filling the area between microspheres, as demonstrated by histology and bright-field microscopy (Figure 2b).

3.4. Scanning electron microscopy (SEM)

      SEM imaging was established to observe attachment and expansion of P19 cells on PLGA microspheres and differentiation of them to neural cells. Scanning electromicro-graphs shows adhesion of loaded cells on the surface of microspheres. As seen in the Figures 3a and 3b, attached cells were proliferated and made them seem to be axon and dendrites.

3.5. Immunoflourescent analysis

      Immunoflourescent analysis using β-Tubulin III and Map II were performed on the PLGA microsphere that was loaded with P19 cells and PI used for staining of nuclei of cells. As is obsereved in Figures 3c and 3d, mature nervous structures (axons and dendrites) were stained by green dye of the β-Tubulin III and Map II.

Table 1. Primers and the reaction conditions for RT-PCR.

F: 5`- gag agg acc cat tat cca gat g -3`

R: 5`- gct gac tgt tca tgt gtg ttt g -3`