Publications by authors named "Fazel Sahraneshin Samani"

13 Publications

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In Vitro Differentiation of Human Umbilical Cord Blood CD133+ Cells into Insulin Producing Cells in Co-Culture with Rat Pancreatic Mesenchymal Stem Cells.

Cell J 2021 Apr 1;23(1):138-139. Epub 2021 Mar 1.

Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

In this article which was published in Cell J, Vol 17, No 2, Summer 2015, on pages 211-220, the authors found that Figures 3 and 4 had some errors that accidentally happened during organizing figures as well as because of mislabeling of some images and saving them in an incorrect folder. The following figures are corrected. The authors would like to apologies for any inconvenience caused.
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http://dx.doi.org/10.22074/cellj.2021.7935DOI Listing
April 2021

Combine effect of Chondroitinase ABC and low level laser (660nm) on spinal cord injury model in adult male rats.

Neuropeptides 2017 Oct 14;65:90-99. Epub 2017 Jul 14.

Physiology Research Center, Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran. Electronic address:

After spinal cord injury (SCI) there are many recoveries inhibiting factors such as chondroitin sulfate proteoglycan (CSPG) and inflammation. The present study investigated the combinational effect of low level laser therapy (LLLT) as anti-inflammatory agent and Chondroitinase ABC (ChABC) enzyme as CSPG digesting factor on spinal cord after injury. This study performed on 44 male Wistar rats, spinal cord injury induced by a clip compression injury. Animals received two-weeks treatment of 660nm low level laser (LLL) and intraspinal injection of 1μg ChABC. Functional recovery, cavity size, myelination, axonal projections around the cavity, fibroblast invasion and expression of glycogen synthase kinase-3β (GSk 3β), CSPG and aquaporin 4 (AQP4) expression were evaluated. In statistical evaluation p<0.05 considered significant. Result showed the combination of LLLT and ChABC have more effect on reduction of cavity size, improvement of myelination and number of axons around the cavity and decreasing the expression of GSK3β, CSPG and AQP4 expression compared to LLLT and ChABC alone. In the laser and laser+enzyme groups AQP4 expression decreased significantly after SCI. Functional recovery, improved in LLLT and ChABC treated animals, but higher recovery belonged to the combination therapy group. The current study showed combination therapy by LLLT and ChABC is more efficient than a single therapy with each of them.
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http://dx.doi.org/10.1016/j.npep.2017.06.002DOI Listing
October 2017

Static Magnetic Field Effect on Cell Alignment, Growth, and Differentiation in Human Cord-Derived Mesenchymal Stem Cells.

Cell Mol Bioeng 2017 Jun 20;10(3):249-262. Epub 2017 Mar 20.

4Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

This investigation is performed to evaluate the impact of static magnetic field on the Cell growth alignment, and differentiation potential in Human Mesenchymal Stem cells derived from human newborn cords. -cultured mesenchymal stem cells derived from human newborn cords were exposed to SMF up to 24 mT and compared with the control (unexposed) cultures. Viability was assessed Trypan Blue staining and MTT assay. Cell cycle progression was studied after flow cytometry data analysis. Sox-2, Nanong, and Oct-4 Primers used for RT-PCR experiment. Morphological studies showed that the exposed cells were significantly aligned in parallel bundles in a correlation with the magnetic field lines. Viability measurements showed a significant reduction in cell viability which was noted after exposure to static magnetic field and initiated 36 h after the end of exposure time. Flow cytometric data analysis confirmed a decrease in G1 phase cell population within the treated and cultured groups compared with the corresponding control samples. However, the induced changes were recovered in the cell cultures after the post-exposure culture recovery time which may be attributed to the cellular repair mechanisms. Furthermore, the proliferation rate and Oct-4 gene expression were reduced due to the 18 mT static magnetic field exposure. The significant proliferation rate decrease accompanied by the Sox-2, Nanong, and Oct-4 gene expression decline, suggested the differentiation inducing effects of SMF exposure. Exposure to Static Magnetic fields up to 24 mT affects mesenchymal stem cell alignment and proliferation rate as well as mRNA expression of Sox-2, Nanong, and Oct-4 genes, therefore can be considered as a new differentiation inducer in addition to the other stimulators.
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http://dx.doi.org/10.1007/s12195-017-0482-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816594PMC
June 2017

Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7.

Clin Epigenetics 2016 30;8:46. Epub 2016 Apr 30.

Eberhard Karls University Tuebingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tuebingen, Germany.

Background: 5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.

Methods: HCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated.

Results: Our results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest.

Conclusions: We conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.
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http://dx.doi.org/10.1186/s13148-016-0213-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851801PMC
December 2016

STAT3 is Overactivated in Gastric Cancer Stem-Like Cells.

Cell J 2016 17;17(4):617-28. Epub 2016 Jan 17.

Department of Stem Cells and Developmental Biology, Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Objective: Gastric cancer (GC) is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcrip- tion-3 (STAT3) signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell (ESCs) pluripotency. Here, we have investi- gated the activation status of STAT3 in GC stem-like cells (GCSLCs).

Materials And Methods: In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, character- ized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation.

Results: Spheroid cells showed higher potential for spheroid formation than the pa- rental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition (EMT) related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated (P<0.05), but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel (DTX) when compared with parental cells (P<0.05) according to the MTS assay. Al- though immunostaining and Western blotting showed expression of the STAT3 pro- tein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 (p-STAT3) in spheroid structures relative to the parent cells accord- ing to flow cytometry analysis (P<0.05).

Conclusion: The present findings point to STAT3 over activation in GCSLCs. Com- plementary experiments are required to extend the role of STAT3 in stemness fea- tures and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746412PMC
http://dx.doi.org/10.22074/cellj.2016.3834DOI Listing
February 2016

Hyaluronic Acid Binding Assay Is Highly Sensitive to Select Human Spermatozoa with Good Progressive Motility, Morphology, and Nuclear Maturity.

Gynecol Obstet Invest 2016 20;81(3):244-50. Epub 2015 Nov 20.

Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.

Objective: This study was performed to evaluate the correlation of hyaluronic acid binding assay (HBA) with conventional semen parameters, lipid peroxidation (LPO), intracellular reactive oxygen species (ROS), DNA fragmentation (DF), DNA maturity and mitochondrial membrane potential (MMP) level in human spermatozoa.

Methods: The semen samples were obtained from 98 patients. The seminal plasma was separated for the study of LPO, and the pellet was employed for evaluation of intracellular ROS, DF, nuclear maturity (sperm chromatin structure assay) and MMP through flowcytometry.

Results: The correlation and strength of HBA with respect to the studied parameters were estimated by the Pearson coefficient and multiple liner regression tests. While HBA indicated a positive correlation with progressive motility (β-coefficients = 0.449, p < 0.05) and normal morphology (β-coefficients = 2.722, p < 0.01), it had only negative relationship with DNA integrity (high DNA stain ability; β-coefficients = -0.517, p < 0.05). HBA also did not show any important correlation with other conventional and intracellular sperm parameters.

Conclusions: The HBA is sensitive to morphological integrity, high progressive motility and nuclear maturation. Nonetheless, HBA is not a reliable test for prediction of sperm intracellular ROS, DF and MMP risks and healthy spermatozoa selection.
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http://dx.doi.org/10.1159/000439530DOI Listing
April 2017

Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells.

Cell J 2015 7;17(3):438-50. Epub 2015 Oct 7.

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Objective: Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor.

Materials And Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after trans- fection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene.

Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation ef- ficiency revealed that the vector concentrations from 20-60 μg and the density of the sub- jected cells (5×10(5)and 1×10(6)cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies.

Conclusion: Electroporation was an efficient tool for genetic engineering of hESCs compared to the chemical method. The genetic background of the subjected cell line for transfection seemed to be a fundamental factor in each gene delivery method. For each cell line, optimum voltage rate should be calculated as it has been shown to play a crucial role in cell death and rate of gene delivery.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601864PMC
http://dx.doi.org/10.22074/cellj.2015.5DOI Listing
October 2015

Evaluation of the circulating CD34(+), CD309(+), and endothelial progenitor cells in patients with first attack of optic neuritis.

Adv Biomed Res 2015 27;4:151. Epub 2015 Jul 27.

Isfahan Neurosciences Research Center, AlZahra Hospital, Isfahan, Iran.

Background: Endothelial progenitor cells (EPCs) are present in circulation and contribute to vasculogenesis in adults. The aim of the present study was to determine the number of circulating EPCs in patients with optic neuritis (ON).

Materials And Methods: Fifty patients with ON were diagnosed by expert neurologist and optometrist at the Feiz Hospital, Isfahan, Iran (2012-2013). Blood samples were collected from ON patients in the first attack. The number of EPCs was measured by flow cytometry through the assessment of CD34(+) and CD309(+) in patients and healthy individuals.

Results: With using flow cytometry, CD34(+) and CD309(+) cells detected in peripheral blood cells of patients (n = 50) with ON, and healthy individuals (n = 30). Patients with ON had (mean = 66.71 ± 17.82) CD34(+) and CD309(+) cells compared with healthy controls (mean = 28.72 ± 22.46). In addition, there was no significant difference in CD309(+) cells in both groups.

Conclusion: This study showed elevated CD34(+) and CD309(+) cells in the early stage of the disease. Regarded to EPC increment in neural repair, it expected the EPC level be increased in these patients, but no detectable differences were observed among both markers in healthy and patient with first attack.
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http://dx.doi.org/10.4103/2277-9175.161578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550950PMC
September 2015

In Vitro Differentiation of Human Umbilical Cord Blood CD133(+)Cells into Insulin Producing Cells in Co-Culture with Rat Pancreatic Mesenchymal Stem Cells.

Cell J 2015 11;17(2):211-20. Epub 2015 Jul 11.

Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Objective: Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells (PMCs) as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133(+) (UCB-CD133(+)) cells into newly-formed β-cells in vitro.

Materials And Methods: This study is an experimental research. The UCB-CD133(+)cells were purified by magnetic activated cell sorting (MACS) and differentiated into insulin producing cells (IPCs) in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay (ELISA) were used to determine expression and production of insulin and C-peptide at the protein level.

Results: Our results demonstrated that UCB-CD133(+)differentiated into IPCs. Cells in islet-like clusters with (out) co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges.

Conclusion: Rat PMCs possibly affect differentiation of UCB-CD133(+)cells into IPCs by increasing the number of immature β-cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503835PMC
http://dx.doi.org/10.22074/cellj.2016.3717DOI Listing
July 2015

Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients.

J Proteome Res 2015 Sep 29;14(9):3595-605. Epub 2015 Jul 29.

Department of Molecular Systems Biology, ‡Stem Cells and Developmental Biology Group, and ∇Department of Stem Cells and Developmental Biology at Cell Science Research Center, §Department of Andrology and ⊥Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, and ○Department of Developmental Biology, University of Science and Culture, ACECR , Tehran, Iran.

The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.
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http://dx.doi.org/10.1021/acs.jproteome.5b00520DOI Listing
September 2015

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549.

Cell Mol Biol Lett 2014 Mar 23;19(1):23-36. Epub 2013 Dec 23.

Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44(+)/24(+) and CD44(+)/CD24(-/low) subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44(+)/CD24(+) and CD44(+)/CD24(-/low) subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.
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http://dx.doi.org/10.2478/s11658-013-0112-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275711PMC
March 2014

Features of free software packages in flow cytometry: a comparison between four non-commercial software sources.

Cytotechnology 2014 Aug 10;66(4):555-9. Epub 2013 Jul 10.

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran,

Flow cytometers designed to analyze large particles are enabling new applications in biology. Data analysis is a critical component of the process FCM. In this article we compare features of four free software packages including WinMDI, Cyflogic, Flowing software, and Cytobank.
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http://dx.doi.org/10.1007/s10616-013-9609-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082777PMC
August 2014

Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly.

In Vitro Cell Dev Biol Anim 2012 Feb 25;48(2):75-83. Epub 2012 Jan 25.

Institute of Bioscience, University Putra Malaysia, Kuala Lumpur, Malaysia.

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.
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http://dx.doi.org/10.1007/s11626-011-9480-xDOI Listing
February 2012