The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells

MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. Materials and Methods: In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14th and 21th after transduction. Results: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, β, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084- and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). Conclusion: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokine

Simultaneous regulation of miR-451 and miR-191 led to erythroid fate decision of mouse embryonic stem cell

Objective(s): Various microRNAs (miRNAs) are expressed during development of mammalian cells, when they aid in modulating gene expression by mediating mRNA transcript cleavage and/or regulation of translation rate. miR-191 and miR-451 have been shown to be critical regulators of hematopoiesis and have important roles in the induction of erythroid fate decision. So, the aim of this study is investigation of the miR-191 and miR-451 roles in the controlling mouse embryonic stem cell (mESC) differentiation toward the erythroid lineage.Materials and Methods: mESCs were infected with either pCDH-miR-Off-191 viruses in pCDH-miR-Off-191 group or simultaneously with pCDH-miR-Off-191 and pCDH-miR-451 lentiviruses in simultaneous  group. Then, the expression profiles of erythroid specific transcription factors and globin genes were analyzed using QRT-PCR on day 14 and 21 of differentiation. Flow cytometry analysis was used to evaluate of TER119 and CD235a erythroid specific surface markers. Results: Gata-1, Klf-1, Epor and globin chains were found to be expressed in pCDH-miR-Off-191 and in simultaneous groups. The majority of globin chains showed changes in their expression levels with progression of differentiation from day 14 to day 21. Flow cytometry results showed that miR-451 up- regulation and miR-191 down-regulation is associated with the expression of TER119 and CD235a. Of these two groups analyzed, simultaneous group was most significantly potent in stimulation of erythroid fate decision of mESCs. Conclusion: Together, present data demonstrate that down-regulation of miR-191 alone can enhance the differentiation of mESCs. However, the simultaneous effect of miR-451up-regulation and miR-191 down-regulation is much stronger and can have more practical use in artificial blood production

Comparative impact of platelet rich plasma and transforming growth factor-β on chondrogenic differentiation of human adipose derived stem cells

Introduction: Transforming growth factor-beta (TGF-β) is known as standard chondrogenic differentiation agent, even though it comes with undesirable side effects such as early hypertrophic maturation, mineralization, and secretion of inflammatory/angiogenic factors. On the other hand, platelet-rich plasma (PRP) is found to have a chondrogenic impact on mesenchymal stem cell proliferation and differentiation, with no considerable side effects. Therefore, we compared chondrogenic impact of TGF-β and PRP on adipose-derived stem cells (ADSCs), to see if PRP could be introduced as an alternative to TGF-β. Methods: Differentiation of ADSCs was monitored using a couple of methods including glycosaminoglycan production, miRNAs expression, vascular endothelial growth factor (VEGF)/tumor necrosis factor alpha (TNFα) secretion, alkaline phosphatase (ALP) and calcium content assays. Results: Accordingly, the treatment of differentiating cells with 5% (v/v) PRP resulted in higher glycosaminoglycan production, enhanced SOX9 transcription, and lowered TNFα and VEGF secretion compared to the control and TGF-β groups. Besides, the application of PRP to the media up-regulated miR-146a and miR-199a in early and late stages of chondrogenesis, respectively. Conclusion: PRP induces in vitro chondrogenesis, as well as TGF-β with lesser inflammatory and hypertrophic side effects

The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells

Objective MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. Materials and Methods In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14thand 21thafter transduction. Results Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, β, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). Conclusion Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines

Investigating the Relationship Between MiR210 Upregulation and Hemoglobin Gamma Chain Expression

Background and Aim: Micro RNAs are a group of small non-coding RNAs which play an important role in multiple processes such as proliferation, differentiation, apoptosis, and cancer. Recent studies indicate that mir-210 is overexpressed into erythroid linage during the differentiation of hematopoietic precursor. The main goal of the present study is to investigate the influence of mir-210 on the pattern of expression in hemoglobin gamma chain. Materials and Methods: First, K562 cell line was cultured in RPMI1640 media. Then, pre-miR-210 was transferred into K562 cell line by lipofectamin. Finally, the alterations in the pattern of gamma chain expression were analyzed in days 7 and 14 by RT-PCR and real time PCR technique. Results: It was demonstrated that the overexpression of mir-210 in K562 cell line would lead to a 25-fold increase in the expression of gamma chain in comparison with the control group. Data analysis revealed that the change in the pattern of hemoglobin gamma chain expression was meaningful (p&lt;0.002). Conclusion: Based on these data, overexpression of mir-210 can lead to a significant increase in the production of gamma chain. Therefore, more studies in the field may reveal the fact that an increase in mir-210 can be a suitable goal in the improvement of sickle cell anemia and β-thalassemia

Development of Inactivated FAKHRAVAC® Vaccine against SARS-CoV-2 Virus: Preclinical Study in Animal Models

The recent viral infection disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global public health crisis. Iran, as one of the countries that reported over five million infected cases by September 2021, has been concerned with the urgent development of effective vaccines against SARS-CoV-2. In this paper, we report the results of a study on potency and safety of an inactivated SARS-CoV-2 vaccine candidate (FAKHRAVAC) in a preclinical study so as to confirm its potential for further clinical evaluation. Here, we developed a pilot-scale production of FAKHRAVAC, a purified inactivated SARS-CoV-2 virus vaccine candidate that induces neutralizing antibodies in Balb/c mice, guinea pigs, rabbits, and non-human primates (Rhesus macaques—RM). After obtaining ethical code of IR.IUMS.REC.1399.566, immunizations of animals were conducted by using either of three different vaccine dilutions; High (H): 10 μg/dose, Medium (M): 5 μg/dose, and Low (L): 1 μg/dose, respectively. In the process of screening for viral seeds, viral strains that resulted in the most severe clinical manifestation in patients have been isolated for vaccine development. The viral seed produced the optimal immunity against SARS-CoV-2 virus, which suggests a possible broader neutralizing ability against SARS-CoV-2 strains. The seroconversion rate at the H-, M-, and L-dose groups of all tested animals reached 100% by 28 days after immunization. These data support the eligibility of FAKHRAVAC vaccine candidate for further evaluation in a clinical trial