Elevated expression of stemness genes in adipose-derived mesenchymal stem cells cultured on fibrin scaffold.

In regenerative medicine, MSCs need to be pluripotent for better results. In this study, the effect of fibrin scaffold on expression of stemness genes was examined. Adipose-derived MSCs were cultured in tissue culture plates (2D) and 3-dimensional (3D) fibrin scaffolds. The effect of fibrin scaffold on proliferation of adiposederived MSCs was evaluated by MTT assay. The expression of stemness genes (OCT4 and SOX2) were evaluated by qRT-PCR, and flow cytometry was done for Nanog protein level. Cultured MSCs on fibrin scaffold were able to proliferate according to data obtained by MTT assay. Expression of OCT4 and SOX2 had a significant increase in cells were cultured in 3D condition compared to 2D condition (P < 0.05). Also, increased expression of Nanog protein in 3D culture was observed (P < 0.05). OCT4 and SOX2 in 3D condition increased two-fold and three-fold respectively in 2D and 3D conditions. Moreover, expression of Nanog increased 30% more than in 2D condition. Evaluation of important pluripotency regulators such as OCT4, SOX2, and Nanog showed that fibrin scaffolds are useful instruments to maintain stemness of MSCs, which is essential in field of stem cell therapy and regenerative medicine.

Substrate topography interacts with substrate stiffness and culture time to regulate mechanical properties and smooth muscle differentiation of mesenchymal stem cells.

Substrate stiffness and topography are two powerful means by which mesenchymal stem cells (MSCs) activities can be modulated. The effects of substrate stiffness on the MSCs mechanical properties were investigated previously, however, the role of substrate topography in this regard is not yet well understood. Moreover, in vessel wall, these two physical cues act simultaneously to regulate cellular function, hence it is important to investigate their cooperative effects on cellular activity. Herein, we investigated the combined effects of substrate stiffness, substrate topography and culture time on the mechanical behavior of MSCs. The MSCs were cultured on the stiff and soft substrates with or without micro-grooved topography for 10 days and their viscoelastic properties and smooth muscle (SM) gene expression were investigated on days 2, 6 and 10. In general, substrate topography significantly interacted with substrate stiffness as well as culture time in the modulation of cell viscoelastic behavior and SM gene expression. The micro-grooved, stiff substrates resulted in the maximum cell stiffness and gene expression of α-actin and h1-calponin, and these values were detected to be minimum in the smooth, soft substrates. The findings can be helpful in the mechano-regulation of MSCs for vascular tissue engineering applications.

Comparison of miRNA Profiles of Cord Blood Stem Cells in Identical and Fraternal Twins.

OBJECTIVE: The role of epigenetic in regulating of the gene expression profile the embryo has been documented. MicroRNAs (miRNAs) are one of these epigenetic mechanisms. Twins are valuable models in determining the relative contributions of genetics and the environment. In this study, we compared differences in the expression levels of 44 miRNAs in hematopoietic stem cells (HSCs) of identical twins to that of fraternal twins as a controls. MATERIALS AND METHODS: In this experimental study, CD133+ HSCs were isolated from cord blood of identical and fraternal twins via magnetic-activated cell sorting (MACS). Variation in of gene expression levels of 44 miRNAs were evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Significant differences in expression were observed in both fraternal and identical twins to varying degrees, but variations alteration in expression of the miRNAs were higher in fraternal twins. CONCLUSION: Identical twins had a positive correlation in miRNA expression, while the correlation was not statistically significant in fraternal twins. Altogether, more differences in miRNA expression level in fraternal twins can be attributed to the both genetics and the intrauterine environment. The contribution of the intrauterine environment and genetics to miRNAs expression in HSCs was estimated 8 and 92%, respectively. By comparing of miRNA expression in identical and fraternal twins and identification of their target genes and biological pathways, it could be possible to estimate the effects of genetics and the environment on a number of biological pathways.

Evaluation of hematopoietic stem cell expansion in the presence of garcinol.

OBJECTIVE: The application of human cord blood (hCB) is limited to children by using relatively small volume of cord blood that does not contain enough hematopoietic stem cells (HSCs). So, efforts for applying cord blood stem cells in transplantation have led to establishment of some approaches for ex vivo expansion of HSCs such as garcinol. MATERIALS AND METHODS: CD133+ HSCs were separated by a magnetic-activated cell sorting (MACS) system. Isolated cells were cultured with different doses of garcinol, SCF, TPO and FLT-3L. The optimal dose of garcinol for ex vivo expansion of HSCs was determined by direct counting. Flow cytometry was used to evaluate the expression of CD133 marker to check the ability of garcinol in maintenance of HSCs. Colony forming cell (CFC) assay was performed to evaluate clonogenic capability of treated cells. The level of expression of CXCR4 gene was evaluated by RT-PCR. Data were analyzed using Student's t test. RESULTS: Our results showed that CD133+ HSCs in the presence of garcinol (5-10 µM) had high expansion activity and cell counting showed that the number of cells in treated group was higher than control group (1.9 -fold) and CFC assay showed that the number of colonies following treatment with garcinol had 1.3-fold increase. Treatment of HSCs with garcinol resulted in 9.6-fold increase in terms of CXCR4 expression in comparison to control group. CONCLUSION: The present study showed that garcinol can improve ex vivo expansion of HSCs and enhance their potential for homing to bone marrow.

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.

Erythropoietin induces production of hepatocyte growth factor from bone marrow mesenchymal stem cells in vitro.

BACKGROUND: Hepatocyte Growth Factor (HGF) plays a pivotal role in hematopoiesis, motility, growth and mobilization of hematopoietic stem/progenitor cells (HSPCs). HGF mainly is produced by bone marrow mesenchymal stem cells (BM-MSCs). MSCs express erythropoietin (EPO) receptor. In this study, we aimed to assess the effect of EPO on HGF secretion in BM-MSCs. METHODS: The BM-MSCs treated with EPO (4 IU/ml) for 6, 24 and 48 h. HGF gene expression and protein level were assessed using quantitative real time PCR (qRT-PCR) and Enzyme-linked immunosorbant Assay. In order to show the effect of secreted HGF on migration of HSPCs, hematopoietic stem cells (HSCs) were isolated from cord blood and evaluated using transwell migration assay. RESULTS: We observed a significant increase in level of HGF in cell supernatant after 48 h compared to control group (P < 0.05). Also, qRT-PCR results demonstrated a significant elevation in HGF expression level after 24 and 48 h treatment with EPO compared to control group (P < 0.05). Finally, migration assay results showed a significant increase in migration of HSCs in treated group after 48 h. CONCLUSION: Our data indicated that EPO may play an important role in stem cell mobilization through up regulating HGF in MSCs and inducing migration of HSCs.

Investigating Effects of Acidic pH on Proliferation, Invasion and Drug-Induced Apoptosis in Lymphoblastic Leukemia.

Some studies have shown that extracellular pH in tumors, which results in tumor progression, is less than that in normal tissues. The aim of this study was to investigate the effects of extracellular acidic pH on proliferation, invasion, and drug-induced apoptosis in acute lymphoblastic cells. The cells were cultured in different pH (pH 6.6 and pH 7.4) for 12 days. Cell proliferation was assessed by MTT assay and cell invasion was assayed by invasion assay and gene expression analysis of MMP-9. Drug-induced apoptosis was evaluated after exposure to doxorubicin for 24 hours by annexin V/PI staining and gene expression analysis of BAX pro-apoptotic protein. The results indicated the enhanced growth and invasion of leukemic cells at pH 6.6 (P ≤ 0.05). Furthermore, the cells at pH 6.6 were resistant to apoptosis by doxorubicin (P ≤ 0.05). It can be concluded that acidic pH increases the proliferation, invasion and reduces the drug-induced apoptosis in acute lymphoblastic leukemia. Extracellular acidity can influence the behavior of leukemic cells and therefore, the manipulation of extracellular liquid can be selected as a therapeutic strategy for leukemia, especially for acute lymphoblastic leukemia.

Possible involvement of miRNAs in tropism of Parvovirus B19.

Human Parvovirus B19 (PVB19) is one of the most important pathogens that targets erythroid lineage. Many factors were mentioned for restriction to erythroid progenitor cells (EPCs). Previous studies showed that in non-permissive cells VP1 and VP2 (structural proteins) mRNAs were detected but could not translate to proteins. A bioinformatics study showed that this inhibition might be due to specific microRNAs (miRNAs) present in non-permissive cells but not in permissive EPCs. To confirm the hypothesis, we evaluated the effect of miRNAs on VP expression. CD34(+) HSCs were separated from cord blood. Then, CD34(+) cells were treated with differentiation medium to obtain CD36(+) EPCs. To evaluate the effect of miRNAs on VP expression in MCF7 and HEK-293 cell lines (non-permissive cells) and CD36(+) EPCs, dual luciferase assay was performed in presence of shRNAs against Dicer and Drosha to disrupt miRNA biogenesis. QRT-PCR was performed to check down-regulation of Dicer and Drosha after transfection. All measurements were done in triplicate. Data means were compared using one-way ANOVAs. MicroRNA prediction was done by the online microRNA prediction tools. No significant difference was shown in luciferase activity of CD36(+) EPCs after co-transfection with shRNAs, while it was significant in non-permissive cells. Our study revealed that miRNAs may be involved in inhibition of VP expression in non-permissive cells, although further studies are required to demonstrate which miRNAs exactly are involved in regulation of PVB19 replication.

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.

Differential characteristics of CD133(+) and CD133 (-) Jurkat cells.

T cell acute lymphoblastic leukemia (T-ALL) is a hematological disease including malignancy of T cell precursors. There are some T-ALL patients that are drug-resistant. A major cause of treatment failure in cancers can be associated with the existence of cancer stem cells. The identification of these cell populations helps us to clarify resistance mechanisms and rely on special markers for recognizing cancer stem cells. CD133 is one of the markers that is used for the identification of cancer stem cells. In this study, we evaluated CD133(+) and CD133(-) characteristic cells in Jurkat cells by assay proliferation, invasion, and apoptosis. CD133(+) and CD133(-) Jurkat cells were separated and immediately analyzed for proliferation, invasion, and doxorubicin-induced apoptosis. Proliferation, invasion, and resistance to chemotherapy of CD133(+) Jurkat cells were significantly more than CD133(-) Jurkat cells. Also, our results showed that CD133(+) Jurkat cells expressed ABCG2 gene more than CD133(-) Jurkat cells. In conclusion, CD133 marker could be introduced as a specific marker of cancer stem cells in Jurkat cell line.