@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2017;1(1):9-13

Background
Amniotic Fluid stem cells (AFSC), are multi-potent stem cells that have high reproduction and self-‎healing powers [1]. It also has the potential for differentiation to various cell types, including ‎osteoblast cells [2], fat cells [3], and myositis cells [4].‎
Previous Studies
The power of differentiation of these cells has been observed in vivo and in vitro conditions as well as ‎Ex vivo cultures [5]. … [6].This ability has led to the use of this cell in the treatment of diseases that ‎are mainly associated with the removal of cells (such as degenerative diseases). Meanwhile, stem cells ‎derived from amniotic fluid have special place because they have less immunologic effect, and after ‎injection to mice, they did not show tumorigenecity effect like embryonic stem cells [7]. For this ‎reason, many studies have emphasized their impact on cell therapy because they are not applied in ‎medical problems, and usually, amniocentesis does not pose a risk to the fetus and mother [8, 9].‎ ‎… [10].Recently, osteoblast cells derived from stem cell differentiation have been proposed as a cell ‎source for the treatment of degenerative bone diseases [11, 12]. In the process of osteoblast ‎differentiation, the primary division of mesenchymal stem cells is asymmetric and produce two types ‎of cells, one stem cells that retains the stem cell (self-renewal feature), and the other is a committed ‎progenitor cells that has ability to become osteoblast (Potency). There are two important steps in ‎differentiating osteoblasts from MSCs. The first step is the passage of the mesenchymal stem cell into ‎the progenitor cells committed to osteoblast or the Transition stage, and the second step is the end ‎differentiation and the end of the cell cycle. Osteoblasts are distinct cells expressing osteocalcin, ‎osteopontin, and bone sialoprotein. These cells have limited proliferative power, and after the ‎introduction of osteoblast cells into end-stage of differentiation, the cell cycle is stopped and post ‎mitotic osteocytes are obtained. Osteocalcin is a marker of end stage of differentiation of osteoblast ‎cells. The role of the transit amplifying in bone formation is very important, as increased proliferation ‎at this stage lead to an increase in bone mass [13]. ‎ One of the characteristics of osteoblast differentiation is the formation of calcium as well as porous ‎areas even in vitro, which in some cases leads to the removal of the cells from the flask floor. In this ‎study, a method was developed to advance differentiation and to prevent cell separation, so that half of ‎the supernatant medium of the cells was slowly expelled in each medium change and the severe shakes ‎of the flask were avoided. Thus, the amount of calcification of cells was very significant. ‎
Aim(s)
The purpose of this study was to isolate stem cells from amniotic fluid and to differentiate them into ‎osteoblast cells.‎
Research type

Research society, place and time

Sampling method and number

Used devices&materials
About 10 ml of amniotic acid from a healthy donor prepared from Sarem Hospital, after receiving the ‎consent of the individual, was transferred to a cell culture lab in a sterile tube. ‎ Cell culture: After centrifugation, the cell plate was suspended with DMEM-low glucose culture ‎medium (Gibco) containing 20% FBS (Gibco) and penicillin-streptomycin (Gibco). Cell counting and ‎determination of viability percentage was carried out by using trypan with Neobar slide. For ‎differentiation, mesenchymal stem cells were treated for 21 days with an osteoblast differentiation ‎medium including DMEM high glucose medium enriched with 10% serum and 2 mM L-glutamine, and ‎‎100 units of penicillin-streptomycin (Sigma) and 50 μg / ml Ascorbate-2-phosphate and 5 mM beta-‎glycerol phosphate and 10 nM dexamethasone. During the period of differentiation, the cells` medium ‎was changed twice a week. ‎ Alizarin Red staining: After 21 days, differentiated cells and mesanchylmal stem cell were used as ‎control of differentiation for Alizarin Red staining. The slides were washed twice with PBS and then ‎fixed with 40% formaldehyde. The staining with 1% solvent was performed for 30 minutes at room ‎temperature and after washing under a microscope, the staining of osteoblast cells was compared with ‎MSC RT-PCR for markers of alkaline phosphatase and osteocalcin: The Rima Zol kit (Teyf Ara ‎Farayand) was used to extract RNA. According to the kit protocol, lysis of cells was performed. After ‎cell lysis, extraction is performed using chloroform and isopropanol to obtain RNA deposition. Finally, ‎after washing with ethanol 75%, the obtained RNA is dissolved in water. To prepare the cDNA, ‎according to the protocol of Sinagen Co. (Tehran, Iran), 10 μl of RNA solution was incubated at 65 ° C ‎for 5-10 minutes and then a mixture of 3 μl buffer (10X PCR), random primer 6, dNTP(10 mM 0.2), 6 μl ‎MgCl 2 (25 Mm ) And 0.5 μl Reverse Transcriptase enzyme were added. The resulting solution was ‎incubated for 10 min at 25 ° C and then incubated for an hour at 42 ° C, and then, for the stopping of the ‎reaction, the sample was placed at 70 ° C for 10 minutes. The resulting cDNA was used for PCR. The ‎cDNA sample can be freeze at minus 70 ° C. Alkaline phosphatase is a primary indicator off osteoblast ‎cells. In this experiment, cDNA prepared from RNA extracted from osteoblast cells, and cDNA prepared ‎from RNA extracted from mesenchymal stem cells were tested as test sample and control sample ‎respectively. PCR test was used for each primer (100ng) of Taq DNA Polymerase for each reaction with ‎a final volume of 25 μL using a mixture of MgCl2, PCR, l0X, dNTP (200μM) and primer (1mm) buffer. ‎After the initial denaturation step, for 3 minutes at 94 ° C, the replication of components performed In ‎‎35 cycles at 94 ° C for 30 seconds, 58 ° C for 20 seconds and 72 ° C for 30 seconds. The final extension ‎was performed at 72 ° C for 7 minutes. The PCR product was electrophoresed on agarose gel 1.5%, and ‎after staining with ethidium bromide, its photo was taken using UV rays (Table 1).‎
Findings by Text
The percentage of viability of the cells was about 80%. The observation of cells with inverted ‎microscopy showed that the morphology of these cells was similar to that of fibroblast cells (Fig. 1).‎ Also, the results of flow cytometry indicate the expression of mesenchymal stem cell markers in ‎isolated bone marrow cells, confirming the quality of the isolation of MSCs. By using the osteoblast ‎differentiation medium, mesenchymal stem cells were differentiated into osteoblast cells, and ‎morphological changes were noticeable on different days of microscopic observation (Fig. 2). ‎ Alizarin Red staining indicated calcium sedimentation in the culture medium, confirming the ‎differentiation of osteoblasts. The color of Alizarin red showed calcium sedimentation and confirmed ‎the differentiation of mesenchymal cells in to osteoblasts, which were not differentiated from ‎mesenchymal stem cells and were considered as negative control after staining with Alizan Red ‎‎(Fig.3).‎ To confirm the differentiation of osteoblast cells, besides the staining of Alizarin Red, transcriptome ‎markers were also used. For this purpose, the expression of alkaline phosphatase index from the early ‎indices of osteoblast differentiation and osteocalcin markers from endpoint of osteoblast ‎differentiation were studied. RT-PCR results of both markers showed that these indices were expressed ‎in osteoblast cells, but not expressed in amniotic fluid stem cells, which confirmed the differentiation ‎of osteoblast cells (Figs. 4 and 5).‎
Main comparison to the similar studies
In the studies, isolation of MSC from human amniotic fluid has been carried out in the second trimester ‎of pregnancy as well as from the amniotic fluid of C57BL / 6 mice [14]. Osteoblasts differentiation is ‎osteogenesis of a highly controlled evolutionary process that involves a large number of external ‎factors, such as hormones, growth factors, messenger pathways, and transcription factors. ‎ Studies have shown that osteoblast cells derived from amniotic fluid cells are effective in bone ‎regeneration of destructive injuries [15, 16]. Confirmation of differentiation using Alizarin red and RT-‎PCR staining in this study, using AR staining revealed that the cells used can produce calcium. This ‎staining has been used as a standard marker for confirmation of distinction in previous studies [17, ‎‎18]. Of course, in some studies it is suggested that using AR staining, the use of expression of ‎osteocalcin and alkaline phosphatase genes can be very helpful [19]. Of course, many other markers, ‎such as osteopontin, and sialoprotein, as well as transcription factors such as Ets1, Runx2 / Cbfa1, ‎have been used to confirm the differentiation of osteoblast cells [20].‎
Suggestions

Limitations

Conclusions
Amniotic fluid cells have the ability to differentiate into osteoblast cells using an osteoblast ‎differentiation medium. ‎
Acknowledgments

Conflict of interest

Ethical Permissions

Funding Sources

TABLES and CHARTS

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