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1.
Cell Journal [Yakhteh]. 2019; 20 (4): 469-476
in English | IMEMR | ID: emr-199615

ABSTRACT

Objective: The ability to generate lung alveolar epithelial type II [ATII] cells from pluripotent stem cells [PSCs] enables the study of lung development, regenerative medicine, and modeling of lung diseases. The establishment of defined, scalable differentiation methods is a step toward this goal. This study intends to investigate the competency of small molecule induced mouse embryonic stem cell-derived definitive endoderm [mESC-DE] cells towards ATII cells


Materials and Methods: In this experimental study, we designed a two-step differentiation protocol. mESC line Royan B20 [RB20] was induced to differentiate into DE [6 days] and then into ATII cells [9 days] by using an adherent culture method. To induce differentiation, we treated the mESCs for 6 days in serum-free differentiation [SFD] media and induced them with 200 nM small molecule inducer of definitive endoderm 2 [IDE2]. For days 7-15 [9 days] of induction, we treated the resultant DE cells with new differentiation media comprised of 100 ng/ml fibroblast growth factor [FGF2] [group F], 0.5 micro g/ml hydrocortisone [group H], and A549 conditioned medium [A549 CM] [group CM] in SFD media. Seven different combinations of factors were tested to assess the efficiencies of these factors to promote differentiation. The expressions of DE- and ATII-specific markers were investigated during each differentiation step


Results: Although both F and H [alone and in combination] promoted differentiation through ATII-like cells, the highest percentage of surfactant protein C [SP-C] expressing cells [37%] were produced in DE-like cells treated by F+H+CM. Ultrastructural analyses also confirmed the presence of lamellar bodies [LB] in the ATII-like cells


Conclusion: These results suggest that hydrocortisone can be a promoting factor in alveolar fate differentiation of IDE2- induced mESC-DE cells. These cells have potential for drug screening and cell-replacement therapies

2.
Cell Journal [Yakhteh]. 2019; 20 (4): 496-504
in English | IMEMR | ID: emr-199618

ABSTRACT

Objective: Cardiovascular progenitor cells [CPCs] are introduced as one of the promising cell sources for preclinical studies and regenerative medicine. One of the earliest type of CPCs is cardiogenic mesoderm cells [CMCs], which have the capability to generate all types of cardiac lineage derivatives. In order to benefit from CMCs, development of an efficient culture strategy is required. We aim to explore an optimized culture condition that uses human embryonic stem cell [hESC]-derived CMCs


Materials and Methods: In this experimental study, hESCs were expanded and induced toward cardiac lineage in a suspension culture. Mesoderm posterior 1-positive [MESP1+] CMCs were subjected to four different culture conditions: i. Suspension culture of CMC spheroids, ii. Adherent culture of CMC spheroids, iii. Adherent culture of single CMCs using gelatin, and iv. Adherent culture of single CMCs using Matrigel


Results: Although, we observed no substantial changes in the percentage of MESP1+ cells in different culture conditions, there were significantly higher viability and total cell numbers in CMCs cultured on Matrigel [condition iv] compared to the other groups. CMCs cultivated on Matrigel maintained their progenitor cell signature, which included the tendency for cardiogenic differentiation


Conclusion: These results showed the efficacy of an adherent culture on Matrigel for hESC-derived CMCs, which would facilitate their use for future applications

3.
Cell Journal [Yakhteh]. 2018; 20 (1): 61-72
in English | IMEMR | ID: emr-191497

ABSTRACT

Objective: Embryonic stem cells [ESCs] are regulated by a gene regulatory circuitry composed of transcription factors, signaling pathways, metabolic mediators, and non-coding RNAs [ncRNAs]. MicroRNAs [miRNAs] are short ncRNAs which play crucial roles in ESCs. Here, we explored the impact of miR-302b-3p on ESC self-renewal in the absence of leukemia inhibitory factor [LIF]


Materials and Methods: In this experimental study, ESCs were cultured in the presence of 15% fetal bovine serum [FBS] and induced to differentiate by LIF removal. miR-302b-3p overexpression was performed by transient transfection of mature miRNA mimics. Cell cycle profiling was done using propidium iodide [PI] staining followed by flow cytometry. miRNA expression was quantified using a miR-302b-3p-specific TaqMan assay. Data were analyzed using t test, and a P<0.05 was considered statistically significant


Results: We observed that miR-302b-3p promoted the viability of both wild-type and LIF-withdrawn ESCs. It also increased ESC clonogenicity and alkaline phosphatase [AP] activity. The defective cell cycling of LIF-deprived ESCs was completely rescued by miR-302b-3p delivery. Moreover, miR-302b-3p inhibited the increased cell death rate induced by LIF removal


Conclusion: miR-302b-3p, as a pluripotency-associated miRNA, promotes diverse features of ESC self-renewal in the absence of extrinsic LIF signals

4.
Cell Journal [Yakhteh]. 2018; 20 (3): 294-301
in English | IMEMR | ID: emr-197606

ABSTRACT

Type 1 diabetes mellitus [T1DM] is a disease where destruction of the insulin producing pancreatic beta-cells leads to increased blood sugar levels. Both genetic and environmental factors play a part in the development of T1DM. Currently, numerous loci are specified to be the responsible genetic factors for T1DM; however, the mechanisms of only a few of these genes are known. Although several environmental factors are presumed responsible for progression of T1DM, to date, most of their mechanisms remain undiscovered. After several years of hyperglycemia, late onsets of macrovascular [e.g., cardiovascular] and microvascular [e.g., neurological, ophthalmological, and renal] complications may occur. This review and accompanying figures provides an overview of the etiological factors for T1DM, its pathogenesis at the cellular level, and attributed complications

5.
Cell Journal [Yakhteh]. 2018; 20 (3): 361-368
in English | IMEMR | ID: emr-197615

ABSTRACT

Objective: Dual inhibition of mitogen-activated protein kinase [MAPK] kinase [also known as MEK] and transforming growth factor beta [TGFbeta] type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells [ESC]. In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition


Materials and Methods: In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast [MEF] feeder cells in both R2i and serum conventional media. The isolated inner cell mass [ICM], ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real timepolymerase chain reaction [qRT-PCR] analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC


Results: qRT-PCR revealed a significantly higher expression of the pluripotency-related genes [Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and beta-Catenin] and lower expression of early differentiation genes [Gata6, Lefty2 and Cdx2] in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition


Conclusion: Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-beta signaling pathways in the first 5 days of ESC derivation

6.
Cell Journal [Yakhteh]. 2018; 20 (3): 377-387
in English | IMEMR | ID: emr-197617

ABSTRACT

Objective: Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells [MSCs] to improve in vitro maintenance conditions for hepatocytes


Materials and Methods: In this experimental study, following serum deprivation, human adipose tissue-derived MSCs [hAT-MSCs] were cultured for 24 hours under normoxic [N] and hypoxic [H] conditions. Their conditioned media [CM] were subsequently collected and labeled as N-CM [normoxia] and H-CM [hypoxia]. Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal [William's] medium supplemented with 4% N-CM or H-CM. Untreated William's and hepatocyte-specific media [HepZYM] were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells


Results: We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS [3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]- 2H-tetrazolium] assay. Indocyanine green [ICG] uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin [Alb] and urea secretion compared to the other groups [P<0.0001]. However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor [VEGF] in the H-CM group compared to the N-CM group [P=0.063]


Conclusion: The enrichment of William's basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture

7.
Cell Journal [Yakhteh]. 2018; 20 (3): 388-395
in English | IMEMR | ID: emr-197618

ABSTRACT

Objective: Pluripotent stem cells [PSCs], with the capacity to self-renew and differentiate into all other cell types, are of benefit in regenerative medicine applications. Tightly controlled gene expression networks and epigenetic factors regulate these properties. In this study, we aim to evaluate the metabolic signature of pluripotency under 2i and R2i culture conditions versus serum condition


Materials and Methods: In this experimental study, we investigated bioinformatics analysis of the shotgun proteomics data for cells grown under 2i, R2i, and serum culture conditions. The findings were validated by cell cycle analysis and gene expressions of the cells with flow cytometry and quantitative reverse transcription-polymerase chain reaction [qRT-PCR], respectively


Results: Expressions of 163 proteins increased in 2i-grown cells and 181 proteins increased in R2i-grown cells versus serum, which were mostly involved in glycolysis signaling pathway, oxidation-reduction, metabolic processes, amino acid and lipid metabolism. Flow cytometry analysis showed significant accumulation of cells in S phase for 2i [70%] and R2i [61%] grown cells


Conclusion: This study showed that under 2i and R2i conditions, glycolysis was highlighted for energy production and used to maintain high levels of glycolytic intermediates to support cell proliferation. Cells grown under 2i and R2i conditions showed rapid cell cycling in comparison with the cells grown under serum conditions

8.
Cell Journal [Yakhteh]. 2018; 20 (2): 267-277
in English | IMEMR | ID: emr-198738

ABSTRACT

Objective: The regenerative potential of bone marrow-derived mononuclear cells [MNCs] and CD133+ stem cells in the heart varies in terms of their pro-angiogenic effects. This phase II/III, multicenter and double-blind trial is designed to compare the functional effects of intramyocardial autologous transplantation of both cell types and placebo in patients with recent myocardial infarction [RMI] post-coronary artery bypass graft


Materials and Methods: This was a phase II/III, randomized, double-blind, placebo-controlled trial COMPARE CPM-RMI [CD133, Placebo, MNCs - recent myocardial infarction] conducted in accordance with the Declaration of Helsinki that assessed the safety and efficacy of CD133 and MNCs compared to placebo in patients with RMI. We randomly assigned 77 eligible RMI patients selected from 5 hospitals to receive CD133+ cells, MNC, or a placebo. Patients underwent gated single photon emission computed tomography assessments at 6 and 18 months post-intramyocardial transplantation. We tested the normally distributed efficacy outcomes with a mixed analysis of variance model that used the entire data set of baseline and between-group comparisons as well as within subject [time] and group×time interaction terms


Results: There were no related serious adverse events reported. The intramyocardial transplantation of both cell types increased left ventricular ejection fraction by 9% [95% confidence intervals [CI]: 2.14% to 15.78%, P=0.01] and improved decreased systolic wall thickening by -3.7 [95% CI: -7.07 to -0.42, P=0.03]. The CD133 group showed significantly decreased non-viable segments by 75% [P=0.001] compared to the placebo and 60% [P=0.01] compared to the MNC group. We observed this improvement at both the 6- and 18-month time points


Conclusion: Intramyocardial injections of CD133+ cells or MNCs appeared to be safe and efficient with superiority of CD133+ cells for patients with RMI. Although the sample size precluded a definitive statement about clinical outcomes, these results have provided the basis for larger studies to confirm definitive evidence about the efficacy of these cell types

9.
Cell Journal [Yakhteh]. 2017; 19 (2): 194-203
in English | IMEMR | ID: emr-186889

ABSTRACT

Signaling in pluripotent stem cells is a complex and dynamic process involving multiple mediators, finely tuned to balancing pluripotency and differentiation states. Characterizing and modifying the necessary signaling pathways to attain desired cell types is required for stem-cell applications in various fields of regenerative medicine. These signals may help enhance the differentiation potential of pluripotent cells towards each of the embryonic lineages and enable us to achieve pure in vitro cultures of various cell types. This review provides a timely synthesis of recent advances into how maintenance of pluripotency in hPSCs is regulated by extrinsic cues, such as the fibroblast growth factor [FGF] and ACTIVIN signaling pathways, their interplay with other signaling pathways, namely, wingless-type MMTV integration site family [WNT] and mammalian target of rapamycin [mTOR], and the pathways governing the determination of multiple lineages

10.
Cell Journal [Yakhteh]. 2017; 19 (3): 386-402
in English | IMEMR | ID: emr-193046

ABSTRACT

Objective: Due to recent progress in production of human embryonic stem cell-derived oligodendrocyte progenitor cells [hESC-OPCs] for ameliorating myelin disease such as multiple sclerosis [MS] and the role of purinergic signaling in OPCs development, we avaluated the profile of purinergic receptors expression during development of OPCs from hESC


Materials and Methods: In this experimental study, we used reverse transcription and quantitative polymerase chain reaction [RT-qPCR] to obtain more information about potential roles of purinergic receptors during in vitro production of hESC-OPCs. We first determined the expression level of different subtypes of purinergic receptors in hESCs, embryoid bodies [EBs], and hESC-OPCs. The effects of A1adenosine receptor [A1AR] activation on hESC-OPCs development were subsequently examined


Results: hESCs and OPCs had different mRNA expression levels of the AR subtypes. ARs mRNA were expressed in the EB stage, except for A2AAR. We observed expressions of several P2X [P2X1, 2, 3, 4, 5, 7] and P2Y [P2Y1, 2, 4, 6, 11-14] genes in hESCs. hESC-OPCs expressed different subtypes of P2X [P2X1, 2, 3,4,5,7] and P2Y [P2Y1, 2, 4, 6, 11-14]. Except for P2X1 and P2X6, all other P2X and P2Y purinergic receptor subtypes expressed in EBs. We also indicate that A1AR might be involved in modulating gene expression levels of cell cycle regulators in an agonist and/or dose-dependent manner


Conclusion: Elucidation of the expression pattern of purinergic receptors and the effects of different subtypes of these receptors in hESC-OPCs may have a promising role in future cell-based therapy or drug design for demyelinating disease

11.
Cell Journal [Yakhteh]. 2017; 18 (4): 532-539
in English | IMEMR | ID: emr-185778

ABSTRACT

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid


Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA [sgRNA] specific to the homology region, and either Cas9 or its mutant nickase variant [Cas9n]. Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells [ESCs]


Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus


Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies


Subject(s)
Gene Knock-In Techniques , Embryonic Stem Cells , Gene Targeting , Genetic Engineering/methods , Homologous Recombination/genetics , Mice
12.
Cell Journal [Yakhteh]. 2017; 18 (4): 565-581
in English | IMEMR | ID: emr-185782

ABSTRACT

Objective: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector


Materials and Methods: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells


Results: In the present study, we developed a nonviral episomal vector named pLENSO/Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming


Conclusion: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future


Subject(s)
Induced Pluripotent Stem Cells , CpG Islands , Plasmids/genetics , Bacteria/genetics
13.
IJB-Iranian Journal of Biotechnology. 2016; 14 (3): 169-176
in English | IMEMR | ID: emr-193919

ABSTRACT

Background: Promyelocytic leukemia protein [PML] is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid [RA]. In addition, RA acts as a natural morphogen in neural development


Objectives: This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development


Materials and Methods: RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR [QRT-PCR] and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR [BSP] was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells [NT2.NPCs]


Results: QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells [NT2.NCs]. Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell


Conclusions: The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development

14.
Cell Journal [Yakhteh]. 2015; 17 (1): 37-48
in English | IMEMR | ID: emr-161616

ABSTRACT

The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells [NCCs]. These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. In this experimental study, we cultured human embryonic stem cells [hESCs] on stromal stem cells from human exfoliated deciduous teeth [SHED] for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells [NPCs] and NCCs. In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity [SDIA] and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development

15.
Cell Journal [Yakhteh]. 2015; 17 (1): 153-158
in English | IMEMR | ID: emr-161628

ABSTRACT

In recent years transdifferentiation technology has enabled direct conversion of human fibroblasts to become a valuable, abundant and accessible cell source for patient-specific induced cell generation in biomedical research. The majority of transdifferentiation approaches rely upon viral gene delivery which due to random integration with the host genome can cause genome instability and tumorigenesis upon transplantation. Here, we provide a simple way to induce neural progenitor-like cells from human fibroblasts without genetic manipulation by changing physicochemical culture properties from monolayer culture into a suspension in the presence of a chemical DNA methyltransferase inhibitor agent, Azacytidine. We have demonstrated the expression of neural progenitor-like markers, morphology and the ability to spontaneously differentiate into neural-like cells. This approach is simple, inexpensive, lacks genetic manipulation and could be a foundation for future chemical neural transdifferentiation and a safe induction of neural progenitor cells from human fibroblasts for clinical applications

16.
Cell Journal [Yakhteh]. 2015; 17 (2): 211-220
in English | IMEMR | ID: emr-166902

ABSTRACT

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 beta-cells in vitro. 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. 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. Rat PMCs possibly affect differentiation of UCB-CD133[+] cells into IPCs by increasing the number of immature beta-cells

17.
Cell Journal [Yakhteh]. 2015; 17 (2): 288-295
in English | IMEMR | ID: emr-166909

ABSTRACT

Embryonic germ [EG] cells are the results of reprogramming primordial germ cells [PGC] in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-beta [TGFbeta] and mitogen-activated protein kinase kinase [MEK] signaling pathways. In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor [SCF] as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through G-banding technique. Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 [GSK3beta] and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3beta molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFbeta signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFbeta instead of GSK3beta significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities. Rat EG cells with higher karyotype stability establish from PGCs by inhibiting TGFbeta and MEK signaling pathways

18.
Cell Journal [Yakhteh]. 2015; 17 (3): 438-450
in English | IMEMR | ID: emr-174877

ABSTRACT

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 reports 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 transient expression of green fluorescent protein in two genetically different hESC lines, Royan 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 transfection 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 efficiency revealed that the vector concentrations from 20-60 Mug and the density of the subjected cells [5×105 and 1×106 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

19.
Cell Journal [Yakhteh]. 2015; 16 (4): 426-439
in English | IMEMR | ID: emr-154845

ABSTRACT

There is constant difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of "universal" red blood cells. Advances in somatic cell reprogramming of human-induced pluripotent stem cells [hiPSCs] have provided a valuable alternative source to differentiate into any desired cell type as a therapeutic promise to cure many human disease. In this experimental study, we examined the erythroid differentiation potential of normal Bombay hiPSCs [B-hiPSCs] and compared results to human embryonic stem cell [hESC] lines. Because of lacking ABO blood group expression in B-hiPSCs, it has been highlighted as a valuable source to produce any cell type in vitro. Similar to hESC lines, hemangioblasts derived from B-hiPSCs expressed approximately 9% KDR+CD31+ and approximately 5% CD31+CD34+. In semisolid media, iPSC and hESC-derived hemangioblast formed mixed type of hematopoietic colony. In mixed colonies, erythroid progenitors were capable to express CD71+GPA+HbF+ and accompanied by endothelial cells differentiation. Finally, iPS and ES cells have been directly induced to erythropoiesis without hemangioblast formation that produced CD71+HbF+erythroid cells. Although we observed some variations in the efficiency of hematopoietic differentiation between iPSC and ES cells, the pattern of differentiation was similar among all three tested lines


Subject(s)
Humans , Embryonic Stem Cells , Erythroid Cells
20.
Cell Journal [Yakhteh]. 2014; 16 (1): 53-62
in English | IMEMR | ID: emr-148447

ABSTRACT

Biomaterial technology, when combined with emerging human induced pluripotent stem cell [hiPSC] technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of hiPSC-derived neural progenitors [hiPSC-NPs]. In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells. Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, hiPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on hiPSC-NPs behavior were assessed. Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 Micro m for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was >98% with more than 94% swelling ratio. hiPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated hiPSC-NPs on these scaffolds. The results have suggested that the pore structure and laminin coating of collagen scaffolds significantly impact cell behavior. These biocompatible three-dimensional laminin-coated collagen scaffolds are good candidates for future hiPSC-NPs biomedical nerve tissue engineering applications


Subject(s)
Humans , Animals, Laboratory , Pluripotent Stem Cells , Laminin , Tissue Engineering , Temperature
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