Menstrual blood stem cells (MensSCs) have enormous potential as a source for cell replacement therapies. Since there is a major concern in utilization of nanofibers in tissue engineering of stem cells, we examined the potential of MensSCs to differentiate into hepatocytes, using different protocols and compare cells, with two-dimensional (2D) and three-dimensional (3D) culture systems. Cell characterization experiments of MensSCs have demonstrated that they are multipotent stem cells similar to mesenchymal stem cells, which can successfully differentiate into osteogenic and adipogenic lineages. The efficiency of the cells on the scaffold was appraised by scanning electron microscopy (SEM), MTT assay, and hematoxylin and eosin (H&E) staining. Thereafter, the differentiation protocols were developed by hepatocyte growth factor (HGF) and oncostatin M (OSM) with serum-supplemented or serum-free culture media up to 30 days. Immunofluorescence analysis and ELISA assay revealed the expression of albumin (ALB) in differentiated cells. Hepatocyte-like cells expressed liver-specific gene such as albumin(ALB), α-fetoprotein (AFP), tyrosine aminotransferase (TAT) and cytochrome P450 subunit 7a1 (Cyp7a1) at mRNA levels. In conclusion, the evidences presented in this study show that the nanofiber scaffold and MensSCs may provide a source of differentiated cells for treatment of liver diseases.
Hepatocyte transplantation could be a superseded solution for people on transplant waiting lists as a replacement for whole organ transplantation [
Dulbecco’s Modified Eagle’s Medium (DMEM, pH 7.4), fetal bovine serum (FBS), 2 mM l-glutamine, penicillin and streptomycin, non-essential amino acids, fangizone, 0.05% trypsin-EDTA were obtained from Gibco Life Technologies Co.PCL (70,000 - 90,000 KDa), 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), insulin-transferrin-selenium + 1 (ITS + 1) premix, Epithelial growth factor (EGF), basic fibroblast growth factor (bFGF), nicotine amid (NTA), Hepatocyte growth factor (HGF), dexamethasone (Dex) were purchased from Sigma-Aldrich Co. and Ficoll-Paque was provided from Amersham Bioscience Co. (Sweden). Antibodies for flowcytometric and Immunofluorescence analysis were prepared from Abcam, UK, Cambridge.
Human MensSCs were obtained from menstrual blood of several healthy female donors ranging in age from 20 to 32 years, by using sterile Divacup during the first few days of the menstrual cycle. Samples were only taken after obtaining informed consent from individuals, under an approved institutional review board protocol according to guidelines by the Ethics Committee of Avicenna Research Institute for providing menstrual blood sample. The specimens were quickly illustrated into the falcon tube containing 2.5 µg/ml fungizone, 100 µg/ml streptomycin, 100 U/ml penicillin and 0.5 mM EDTA in phosphate buffered saline without Ca2+, Mg2+ (PBS). The mononuclear cell fraction was separated by centrifugation over a Ficoll-Paque gradient; centrifuge at 600 g for 20 min at room temperature (RT). Cells at the interface were recovered and washed three times with PBS by centrifugation, 600 g for 20 min at room temperature. Cells resuspended in DMEM-F12 and plated in polystyrene plastic 75-cm2 tissue culture flasks. After 3 days, the non-adherent cell was removed by washing with PBS. Contaminated cells were eliminated by sequential passaging.
Nanofibers scaffold should be sterilized before use so the samples were soaked infiltrated 70% ethanol for 2 h at room temperature (RT), then washed twice with media and left overnight in DMEM. After trypsinized and counted, 5 × 104 cells suspended in 50 µl DMEM transferred to the surface of each prepared scaffolds. To adhere the cell on the surface of PCL, plate was incubated at 37˚C for 45 minutes. After this time 50 µl of culture medium was applied to each cellular scaffold every 30 minutes until 3 h (300 µL).
To study the morphology and cell adhesion on the surface of the scaffold electron Microscopic studies were performed. In brief, after washing non-adherent cells with PBS, MensSCs has grown on scaffolds fixed in 3% glutaraldehyde for 3 h at RT. The dehydration process is accomplished by passing the scaffold through a series of graded alcohol solutions (diluted from 100% pure ethanol, Merk). All specimens were then coated with palladium-gold (Au-Pd) and viewed under scanning electron microscope (Philips XL30, Amsterdam, The Netherlands).
For observation of the diffusion of cell-seeded samples, hematoxylin and eosin staining was performed. Specimens were fixed at 10% v/v buffered formalin. Dehydration of the scaffold was done by a graded ethanol series, and then the scaffold embedded in paraffin. The specimens were cut into 10 μm-thick sections and stained with hematoxylin and eosin (H&E) [
To compare proliferate capability of MensSCs in 3D with 2D, cells were seeded at a concentration of 5 × 104 cells per well in 24-well plates on both scaffold and 2D culture systems. Cell proliferation was analyzed at days 4, 7 and 10 by MTT reduction test [
Hepatic differentiation was performed with two protocols. For 2 days cultured cells were treated with 20 ng/ml EGF and 10 ng/ml bFGF, this is the same for both. Protocol 1: the cells were cultured in a culture medium consisting of DMEM-F12 supplemented with, 40 ng/ml HGF, 10 ng/ml bFGF, and 50 μg/ml NTA for 14 days. The second step also continued until 14 days with 20 ng/mL OSM, 10−7 Mol/L DEX, 1X ITS+1 premix and 50 μg/ml NTA. Protocol 2: in this protocol culture medium consisting of DMEM-F12 supplemented with, 40 ng/ml HGF, 10−7 Mol/l DEX, 1X ITS+1 premix and 50 μg/ml NTA for 14 days. In the second phase, OSM was replaced by HGF at a concentration of 10 ng/ml and continued until 28 days of differentiation. The plate was incubated at 37˚C and culture media were replaced every second day for up to 30 days.
After 30 days differentiation the culture, cells on the scaffold and 2D culture were fixed with 3% glutaraldehyde for 1 hour at room temperature then washed with PBS. In the next step cell Permeabilized with 0.4% triton X-100 for 20 min. Washed cells were incubated overnight at 4˚C with primary antibodies, including monoclonal mouse anti-human albumin (1:100), the cells were washed five times with PBS and incubated with fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse IgG at 37˚C for 45 minutes in the dark. After washing with PBS, cells were incubated with DAPI (4¢, 6-diamidino-2-phenylindole; 1:1000) for nuclear staining. The cells were visualized and observed by using a fluorescence microscope (Nikon, TE-2000, Tokyo, Japan).
Study of albumin production was performed with ELISA assay. Conditioned media from the differentiated MensSCs cultured in 3D and 2D culture’s system were collected at days14and 30 then frozen at -20˚C until assay. The conditioned media were assayed for ALB production using a quantitative enzyme-linked immunosorbent assay kit (ELISA) according to manufacturer’s recipes. Finally, albumin concentration was calculated from a standard curve prepared from different dilutions of albumin standard. The conditioned media of undifferentiated MensSCs (day 0) was used as a negative control.
RT-PCR was used to evaluate the expression of albumin (ALB), Alpha-fetoprotein (AFP), Tyrosine aminotransferase (TAT), and cytochrome P4507A1 (CYP7A1) in differentiated cells on the scaffold. Total RNA was isolated from cultured cells. Genomic DNA was digested from RNA samples by DNase I. Standard RT was performed using a Reverse RevertAid™ First Strand cDNA Synthesis Kit and 2 µg of total RNA, 0.5 µg oligo (dt18) per reaction, according to the manufacturer’s instructions. The cDNA samples were subjected to polymerase chain reaction (PCR) amplification using human specific primers designed using different exons (
The data were analyzed by the Mann-Whitney test. The further analyses were performed by LSD. All grafts were depicted by a prism. A p-value less than 0.05 were considered as significant.
MensSCs adhered to the tissue culture flask a day after isolation. Expanded MensSCs had fibroblast-like shapes and morphologically conserved during passages. In order to find the cell characterization of the cultured cells, the surface CD markers were analyzed. To eliminate of contamination, the cells were passaged 3 times prior to experiments. Flow cytometric analysis of the cultured cells showed MensSCs regularly express the mesenchymal stem cell markers like CD73 (95.5% ± 0.4%), CD90 (97.7% ± 2.1%), CD44 (99.4% ± 0.55%), CD105 (97.6% ± 0.9%), while lacking surface expression of CD34 (0.165% ± 1.3%), CD45 (0.940% ± 0.6%) as hematopoietic markers. To evaluate the potential of the MensSCs, cells were differentiated into adipogenic and oesteogenic lineage. They were exposed to adipogenic and osteogenic media. The morphology of cells treated by adipogenic
Primer | Sequence | Annealing temperature (˚C) | Product size (bp) |
---|---|---|---|
ALB | F 5'-GATGAGATGCCTGCTGACTTGC-3' R 5'-CACGACAGAGTAATCAGGATGCC-3' | 60 | 147 |
AFP | F 5'-CATGAGCACTGTTGCAGAGGAGA-3' R 5'-CGTGGTCAGTTTGCAGCATTCTG-3' | 60 | 113 |
TAT | F 5'-CTTCTGGGGCTATGTACCTCA-3' R 5'-GGACTGTGATGACCACTCGGAT-3' | 55 | 165 |
CYP7A1 | F 5'-CATGAGCACTGTTGCAGAGGAGA-3 R 5'-ACTCGGTAGCAGAAAGAATACATC-3' | 60 | 388 |
GAPDH | F5'-CTCTCTGCTCCTCCTGTTCG-3' R 5'-ACGACCAAATCCGTTGACTC-3' | 60 | 142 |
media was changed and intra-cytoplasmic lipid droplets which were positive stained with Oil Red O. Alizarin red staining demonstrated the capability of the cells into osteoblasts. In both staining procedures undifferentiated MensSCs were negative controls (
The scanning electron microscopy (SEM) micrographs of the scaffolds before and after cell culture are shown in
To confirm in vitro hepatic differentiation from MensSCs, we analyzed expression of ALB with two protocols in 2D and 3D culture systems. The differentiated MensSCs stained positively for ALB at day30. There was no difference between expression levels of ALB of cells differentiated under both protocols in 2D culture and also no difference between two groups of hepatocyte-like cells on scaffolds. However, the cells differentiated on
scaffolds showed significantly higher expression levels of this protein compared with other groups. The percentages of the differentiated cells and the intensity of the reactions of the antibodies were increased significantly compared with those in undifferentiated cultures. The expression of these protein markers was detected in human hepG2 hepatoma cells, used as positive control (
Albumin was secreted into the culture media and significantly increased on day 30 of differentiation. As shown in
Hepatocyte-like cell differentiation was confirmed by RT-PCR hepatocyte-specific genes, namely albumin (ALB), α-Fetoprotein (AFP), cytochrome P450 subunits 7a1 (Cyp7a1), tyrosine aminotransferase (TAT) and GAPDH was used as an internal control. In this study, the undifferentiated MensSCs did not express mRNA of the hepatocyte lineage genes. As shown in
Many researchers are experimenting different adult stem cells to be used for tissue regeneration. Recently a
novel adult stem cell described that can be obtained from women’s menstrual blood derived from the endometrium. This source of stem cells non-invasively isolated to provide a renewable source of stem cells from childbearing women [
Characterization of MensSCs prior to assessment of the potential of MensSCs to differentiate into hepatocytes was evaluated. High levels of mesenchymal stem cell markers [
The evidences presented in this paper clearly show that the PCL scaffold can support proliferation and hepatogenic differentiation of MensSCs. However, very little is known about the stimulating effects of this hybrid polymer on hepatic differentiation. In summary, our study demonstrates that a unique population of stromal stem cells can be collected, isolated, characterized, expanded, from human menstrual blood and differentiation into hepatocyte-like cell on three dimensional nanofibrous scaffolds.
We would like to thank Avicenna Research Institute for offering the grant.
The authors indicate no potential conflicts of interest.
Farnaz Sani,Giti Borzooeian,Somayeh Kazemnejad,Sepideh Ebrahimi,Masoomeh Mohamadpour,Sayeh Khanjani,Mona Latifi,Seyed Mojtaba Hosseini,Mahin Salmannejad,Fatemeh Aleahmad,Hossein Mehraban Jahromi,Mahsa Sani, (2016) Differentiation of Menstrual Blood Derived Stem Cell (MensSCs) to Hepatocyte-Liked Cell on Three Dimensional Nanofiberscaffold: Poly Caprolacton (PCL). Journal of Biomedical Science and Engineering,09,216-225. doi: 10.4236/jbise.2016.94016