Keywords

Keratinocyte; Cytokeratin 18; Cellulose Film; Mesenchymal Stem Cell.

Introduction

Our body surface is covered by epithelial tissue which provides a protective barrier. Damaging to this tissue must be healed by cell operation. One of the possible origins of the cells involved in tissue repair after damaging is stem-like progenitor cells, which are the most important but scarce components of the proliferative compartment of an epithelial tissue [1]. Primary efforts in order to accelerate wound healing and promoting the quality of healing in chronic wounds or burn injuries, included the use of biological or artificial coverings. Although these materials were effective but did not cause any permanent health effects because eventually for access the full recovery, we need transplantation and this due to damaging to the part of the donor’s body is undesirable [2]. Developing tissue engineered constructions made major changes in treatment of an ulcer [3-5]. These constructions can be classified into two main groups: with and without cells [2]. MSCs can be useful in many pathological conditions, though the mechanism of their action is still unknown [6]. Some problems by these constructions are slow and insufficient vascularization of large ones [7]. Skin tissue engineering has been in the forefront of tissue engineering for many years and now some of its products are available. Tissue-engineered skin substitutes are divided into two general groups, cell based products, which can stimulate wound healing and cell-free products, which are cover for injury [8]. In most studies which have been done for differentiation into skin cells, the co culture method has been used to induce differentiation [9-15]. In this study, we examined the differentiation with chemical compounds [16, 17].

Umbilical cord blood is a source of mesenchymal and hematopoietic stem cells [18-20]. Human MSCs are self-renewal and can be differentiated into mesenchymal and non-mesenchymal cells [21, 22]. Compared to isolation of stem cells from bone marrow, isolation from this source is more simple and easier, safer and painless [10, 23]. And some shows that isolation of stem cells from Wharton’s Jelly is even easier and faster [24, 25].

Material and Methods

Induction of MSC into keratinocyte differentiation

MSCs were cultured into 6-well plate. We investigated 4 different mediums and 2 periods of times which are shown in Table 1.

The plates were checked daily under invert microscope and the changes in morphology of cells were photographed which some of them are shown in results.

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Table 1:

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Table 1. Concentration of compounds in culture medium of mesenchymal stem cells for differentiation into keratinocyte and the number of days in this medium.

Polymerase chain reaction (PCR)

After culturing in each one of mediums stated (Table 1), to identify the stem cells are differentiated or not, polymerase chain reaction was performed on total RNA extracted from cultured cells. We investigate the expression of gene cytokeratin 18. Total RNA from the cultured cells was extracted using QIAGEN RNeasy plus mini kit. This kit can be used in a wide range of cells and removes the contamination of DNA during extraction [26]. RNA was measured by using optical density method at 260 nm wavelength. Using Beer Lambert law, the amount of light absorbed can be attributed to the concentration of absorbing molecules. At the wavelength of 260 nm, the extinction coefficient for one strand RNA is 0.025 (μg/ml)-1cm-1. Thus, the optical density of 1, equals to 40 μg/ml for one strand RNA [27]. Then cDNA was synthesized by using Revertaid first strand cDNA synthesis kit [28]. Synthesized cDNAs should be proliferated by specific primers. PCR or polymerase chain reaction is a simple, meanwhile a sensitive method for replication of a particular piece of DNA in the laboratory. Primer cytokeratin 18 was as follows: (sense) 5’-CAAGGAGGAGCTGCTGCTCTTCATG-3’,(antisense), 5’-CAGCAGATTGAGGAGAGCACCA-3’. The temperatures, times and number of cycles are shown in Table 2.

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Table 2. Number of cycles, time and temperature of PCR for identification of gene cytokeratin 18 in differentiated stem cells into keratinocytes.

The PCR was done on a C 1000 thermal cycler (BIO-RAD). The reactions were done in a total volume of 25 μl containing 10 μM of each primer, DNA template 0.2 μg and Master Mix 2x 12.5 μl. And finally to examine the proliferation characteristic of differentiated keratinocyte from HUCBMSCs, cultured them on cellulose film. For this purpose, first the cellulose film was purified and then was dried. Cellulose obtained from fermentor containing impurities such as bacterial cells and compounds of culture medium. For making it purified, first the cellulose in the form of a square were cut then in NaOH solution of 0.1 M, 3 times and each time boiled for 30 min, then placed in incubator at 37°C as long were dried. After cellulose film being dried in the incubator, the cell suspension containing approximately 5 × 104 differentiated cells in 1 ml of culture medium was poured onto cellulose film in wells of a 6 well plate. After 48 h exposure to the incubator, preparation for SEM analysis was performed.

To study cell adhesion to cellulose fibers, we used scanning electron microscopy. To prepare samples, first they were washed in PBS, and then fixed in glutaraldehyde 2.5% for 90 min; dewatering by using ethanol solutions with concentration gradient of 30%, 50%, 70%, 90% and 95%, each one for 15 min was done. In the next step for making conductive, the surface of samples was covered with a thin layer of gold by using sputtering coater method.

Results

The morphology of differentiated mesenchymal stem cells

To investigate the differentiation to keratinocytes, cells were observed daily under the inverted microscope. Figure 1 shows the cells 9 days after induction of differentiation.

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Figure 1. Shows the cells 9 days after induction of differentiation.

The concentration of purified RNA was 2400 μg/ml by using Bio Photo Meter and explanations provided in the Materials and methods section. By using PCR analyzing, the gene expression of cytokeratin 18, skin cell-specific gene was examined as shown at Figure 2.

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Figure 2. Using PCR analyzing, the gene expression of cytokeratin 18, skin cell-specific gene was examined.

To study the proliferation characteristic of differentiated keratinocyte from HUCBMSCs on cellulose film, we used SEM. Figure 3 shows the cells after 48 hours of culturing them on a cellulose film.

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Figure 3. The keratinocytes after 48 hours of culturing them on a cellulose film (magnification 6000x).

Discussion

The changes in the appearance of cells can be observed in Figure 1. One of these changes is that spindle like shape cells, become wider. As shown in Figure 2, differentiated cells expressed the gene cytokeratin 18 well. However, the expression of this gene has not been observed in control cells which are undifferentiated MSCs. Previously in other studies the differentiation of stem cells to cutaneous cells was confirmed [1, 9, 29-34].

As figure shows, in order to study the expression of gene cytokeratin 18; two types of ladder one with a gap distance of 50 bp and one with 100 bp is used. After the primer was purchased from Sina Clon Company, the gene sequence was obtained from Pubmed-NCBI site. Gene length was equal to 221 bp which, as both ladders show, the band is located in the appropriate distance.

The results of SEM are comparable to results that have been reported by HL Almedia et al., about morphology of these cells which expressed they are round or polygonal [35]. The cells didn’t show a good adhesion and extension as you can see in Figure 3. If the environment is favorable for the cells, they will spread on the surface but if the environment isn’t favorable, the cells will place with distance from each other and are wrinkled and don’t make strong connections with their substrate. But about these cells, differentiated keratinocyte from stem cells, we can’t definitely say that this substrate isn’t appropriate, because these cells are extremely sensitive and the slight change in the culture medium of these cells can be consider as a risk for them. In a study by Neeracha Sanchavanakit et al., bacterial cellulose film has been introduced as an appropriate substrate for keratinocytes [36].

Conclusion

Growth factor, BMP4, is an appropriate induction factor for inducing of differentiation into keratinocytes. We can’t definitely say that cellulose film is not a suitable substrate for keratinocytes but results of our study show that it’s not a compatible substrate for differentiated keratinocytes.

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