microRNA-15b target Sall4 and diminish in vitro UCB-derived HSCs expansion

Hematopoietic Stem Cells (HSCs) are cells that have the ability to self-renewal and differentiate into all of hematopoietic lineages. The lack of donors and unavailable efficient protocols for ex vivo expansion of HSCs, are obstacles in successful cell therapies. MicroRNAs (also refer as miRNAs or miRs) have significant roles in hematopoiesis; they can effect on HSCs expansion, maintaining undifferentiated state, self-renewal and differentiation. Recently attentions have been given to these small regulatory molecules to utilize them in order to expand HSCs. Using bioinformatics analysis we identified Sall4 as putative target of miR-15b and miR-219-5p. Relative expression levels of miRNAs and Sall4 were evaluated by qRT-PCR. Here we show 247-fold and 4.2-fold increasing Sall4 expression level compared to control group in CD34+ cells nucleofected by anti-miR-15b and anti-miR-219-5p, respectively. These data showed that anti-miR-15b can promote clonogenic capacity of HSCs and also we found that miR-15b alone was able to increase the number of CD34+HSCs in vitro by more than 2 fold by targeting Sall4. Moreover, level of CD34 marker in HSCs nucleofected by anti-miR-15b increased more than 50 %. Our analysis showed no statistically difference in mRNA level of Sall4 after nucleofection of anti-miR-219-5p. Sall4 is a factor capable of enhancing HSC expansion significantly. We demonstrated that inhibition of miR-15b can enhance ex vivo expansion of UCB-derived HSCs and also expression of Sall4 allowed expansion and preserve self- renewal of CD34+ HSCs

Gene Expression Analysis of SOX2, NANOG, KLF4, OCT4, and REX1 Genes in Cord Blood Mononuclear Cells Treated with External Stresses

Background: Induced pluripotent stem cells (iPSCS) can be obtained from autologous cells for therapeutic purposes. So, far, many studies have been done to produce induced pluripotent cells by transferring specific pluripotency genes using different methods. In this study, pluripotency gene expression induced by external stresses was assessed in cord blood mononuclear cells.Methods: In this experimental study, mononuclear cells were isolated from umbilical cord blood. Isolated cells were divided into three groups. The first group had been exposed to HCL (pH 5.7) for 25 minutes and then transferred to the medium with normal pH. The second group was triturated with hamilton syringe for 15 min (external pressure), and the last group was considered the control group and did not receive treatment. Then, total RNA was extracted on Day 7. Gene expression of OCT4, SOX2, NANOG, REX1, and KLF4 was evaluated using qRT-PCR.Results: Gene expression of OCT4, NANOG, REX1, and KLF4 was increased after exposure to acidic pH and external pressures in comparison with control cells (P < 0.05). SOX2 gene expression was decreased in cells exposed to acidic pH but increased by external pressure.Conclusions: Exposure of umbilical cord blood mononuclear cells to acidic pH and external pressure lead to re-activation of pluripotency genes in mature cells. These findings indicate that mature cells may be reprogrammed with manipulation of environmental conditions

Gene Expression Analysis of SOX2, NANOG, KLF4, OCT4, and REX1 Genes in Cord Blood Mononuclear Cells Treated with External Stresses

Background: Induced pluripotent stem cells (iPSCS) can be obtained from autologous cells for therapeutic purposes. So, far, many studies have been done to produce induced pluripotent cells by transferring specific pluripotency genes using different methods. In this study, pluripotency gene expression induced by external stresses was assessed in cord blood mononuclear cells.Methods: In this experimental study, mononuclear cells were isolated from umbilical cord blood. Isolated cells were divided into three groups. The first group had been exposed to HCL (pH 5.7) for 25 minutes and then transferred to the medium with normal pH. The second group was triturated with hamilton syringe for 15 min (external pressure), and the last group was considered the control group and did not receive treatment. Then, total RNA was extracted on Day 7. Gene expression of OCT4, SOX2, NANOG, REX1, and KLF4 was evaluated using qRT-PCR.Results: Gene expression of OCT4, NANOG, REX1, and KLF4 was increased after exposure to acidic pH and external pressures in comparison with control cells (P < 0.05). SOX2 gene expression was decreased in cells exposed to acidic pH but increased by external pressure.Conclusions: Exposure of umbilical cord blood mononuclear cells to acidic pH and external pressure lead to re-activation of pluripotency genes in mature cells. These findings indicate that mature cells may be reprogrammed with manipulation of environmental conditions

Impact of Tissue Harvesting Sites on the Cellular Behaviors of Adipose-Derived Stem Cells: Implication for Bone Tissue Engineering

The advantages of adipose-derived stem cells (AdSCs) over bone marrow stem cells (BMSCs), such as being available as a medical waste and less discomfort during harvest, have made them a good alternative instead of BMSCs in tissue engineering. AdSCs from buccal fat pad (BFP), as an easily harvestable and accessible source, have gained interest to be used for bone regeneration in the maxillofacial region. Due to scarcity of data regarding comparative analysis of isolated AdSCs from different parts of the body, we aimed to quantitatively compare the proliferation and osteogenic capabilities of AdSCs from different harvesting sites. In this study, AdSCs were isolated from BFP (BFPdSCs), abdomen (abdomen-derived mesenchymal stem cells (AbdSCs)), and hip (hip-derived mesenchymal stem cells (HdSCs)) from one individual and were compared for surface marker expression, morphology, growth rate, and osteogenic differentiation capability. Among them, BFPdSCs demonstrated the highest proliferation rate with the shortest doubling time and also expressed vascular endothelial markers including CD34 and CD146. Moreover, the expression of osteogenic markers were significantly higher in BFPdSCs. The results of this study suggested that BFPdSCs as an encouraging source of mesenchymal stem cells are to be used for bone tissue engineering