Growth kinetics, characterization, and plasticity of human menstrual blood Stem Cells

Background: Men-SCs are widely available (about 12 times a year from a regular-cycle woman) and are capable of long-term proliferation; they can also be obtained via a noninvasive procedure easily. In recent years, MenSCs have been deemed an easily accessible and refreshing stem cell source with no ethical considerations in the field of regenerative medicine. There are several reports on the differentiation of MenSCs into osteoblasts,7 nucleus cardiomyocytes,8 glial-like cells,9 pulposus-like cells,10 hepatocyte-like cells,11 adipocytes,12 and chondrocytes.13 Men-SCs are widely regarded as a new source of MSCs with several potential therapeutic applications; nonetheless, there is a dearth of data in the existing literature on their growth kinetics. The present study was designed to isolate, culture, and determine the growth kinetics and characterization of Men-SCs in women in 2 age groups of 30 to 40 and 40 to 50 years old.Methods: During spring 2014, menstrual blood specimens were collected from 10 volunteer women who referred to the department of obstetrics and gynecology of hospitals affiliated to Shiraz University of Medical Sciences, Shiraz, Southern Iran (5 aged between 30 and 40 years and 5 aged between 40 and 50 years). All the women were on their third day of bleeding period, and the samples were collected with menstrual cups (Diva Cup Co., U.S.A.) inserted deeply into the vagina. All the candidates were healthy and without any history of previous genital diseases. Before the insertion of the cups, sanitary disinfectant pads were used to prevent the local bacterial flora from contaminating the samples. The collection procedure was approved only for research purposes by the institutional ethics committee, and the donors signed a written informed consent form. The sample volume obtained from each participant was 5 mL, and they were separately added to falcons containing 10% sodium citrate and 1% penicillin/streptomycin (Gibco, Germany). Subsequently, the samples were transferred to the Stem Cell Laboratory of The Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, for further evaluation. Isolation of Mononuclear Cells Menstrual blood was gently added to an equal volume of Ficoll under laminar flow hood condition (Class II, Jal Tajhiz, Iran). Ficoll-Paque (Biowest, France) density-gradient centrifugation was performed at 1,200 rpm for 30 minutes at 4.C, resulting in the separation of the contents in four fractions (i.e., red blood cells, Ficoll, mononuclear cells, and plasma). In brief, the transparent fraction of the mononuclear cells was separated and added to a threefold volume of Dulbecco’s Modified Eagle’s Medium-F12 (DMEM-F12, Biowest, France), supplemented with 10% fetal bovine serum (FBS) (BioIdea, Iran), 1% penicillin/streptomycin (Biowest, France), and 1% L-glutamine (BioIdea, Iran). Culturing the menstrual blood-derived stem cells after centrifugation and removal of the supernatant, the cell pellet was suspended in 6 mL of fresh DMEM-F12 media, supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, and was then transferred into T25 flasks. The flasks were kept in a CO2 incubator (Memmert, Germany) at 37°C, 5% CO2, and saturated humidity. The media were replaced twice a week.The primary culture (P0) was trypsinized (Trypsin, Sigma, U.S.A.) after 10 days and was transferred into new T25 culture flasks (P1, the first passage). After 3 days, the cells were evaluated for adherence, confluence, and morphology using an inverted light microscope (Nikon, Japan). When the adherent spindle- shaped cells attained 70–80% confluence, the time was considered optimum for harvesting the cells by trypsin and starting subculturing. Passaging the cells of each of the 10 samples was continued up to P4; and at the end of each passage, the number of live and dead cells was determined via the dye-exclusion method. The cell-counting and cell-staining procedures were carried out using a Neubauer chamber and trypan blue (Sigma, U.S.A.), respectively. The passage 4 cells of both age groups were seeded into 12- and 24-well culture plates at a density of 5×104 cells per well to evaluate the growth kinetics and study the in vitro behavior of the Men-SCs. The cells were counted every 24 hours (each time 3 wells/group). This procedure was continued for 8 days, and the mean number of the cells at each time point was depicted using GraphPad Prism (version 5.01; GraphPad Software Inc., San Diego, CA, U.S.A.). The following formula was used to determine population doubling time (PDT): T×ln2/ln (Xe/Xb), while Xe, Xb, and T were defined as the final cell number, the initial cell number, and the incubation time in any unit, respectively.Result: Following cell expansion, the adherent Men-SCs obtained from both groups of women revealed a spindle-shaped, fibroblast-like morphology similar to the typical appearance of MSCs in the primary culture and passage 2 (Figures 1a and b). About 3 to 5 days were needed for the cells of each passage to reach the confluence of 70 to 80%, except for the primary passage, which took 10 days. The results from the culture of the Men-SCs of the women aged between 30 and 40 years when seeding 5×104 cells into 12- and 24-well culture plates revealed PDT values of 55.5 and 62 hours, respectively (Figures 2a, g, and H). However, for the women aged between 40 and 50 years, PDT values were 70.4 and 72.4 hours, correspondingly (Figures 2b, e, and f). According to the cell enumeration findings in either the 12- or the 24-well plates, the growth of the Men-SCs in the women aged between 30 and 40 years was significantly more than that in the women aged between 40 and 50 years (Figures 2c and 2d; PConclusion: MSCs can be isolated from several adult tissue sources. They show progenitor cell-like features such as proliferation and differentiation capacities. One of the most historically prominent sources of MSCs has been the bone marrow, while other sources recently includethe adipose tissue, bone, muscle, liver, pancreas, tooth, umbilical cord, placenta, and cord blood. The isolation of these progenitor cells requires traumatic procedures that are poorly feasible and associated with patient discomfort.2 Menstrual blood has been considered one of the most accessible sources for obtaining MSCs noninvasively. 14 Recently, there has been a great deal of interests in the application of these cells in regenerative medicine due to their multilineage and highly proliferative features.14 Men-SCs have been introduced as a good source of cell transplantation for various therapeutic procedures such as the treatment of premature ovarian failure.14 In cell expansion, one of the most prominent criteria is the morphology of cultured cells.15 In our study, similar to previous findings, during subcultures, the Men-SCs displayed a spindle-shaped, fibroblast-like morphology under light microscopy, resembling the typical appearance of MSCs derived from other adult tissues.14 It has been previously shown that Men-SCs are more uniform in shape and size (10 to 100 .) than are the cells obtained from other tissues.

کلیدواژه ها:

Mesenchymal stem cells ، Plasticity ، Reverse transcriptase polymerase chain reaction ، Menstrual blood