Ex vivo expansion of hematopoietic stem cells in two/ three-dimensional co-cultures with various source of stromal cells.

The ex vivo expansion of hematopoietic stem cells, with both high quantities and quality, is considered a paramount issue in cell and gene therapy for hematological diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells reveal the importance of using 2D and 3D coculture as a physiological system simulator in the proliferation, differentiation, and homeostasis of HSCs. Herein, the capacity of mesenchymal stem cells derived from different sources to support the expansion and maintenance of HSPC was compared with each other. We evaluated the fold increase of HSPC, CD34 marker expression, cytokine secretion profile of different MSCs, and the frequency of hematopoietic colony-forming unit parameters. Our results show that there was no significant difference between adipose tissue-MSC, Wharton jelly-MSC, and Endometrial-MSCs in HSPC expansion (fold increase: 34.74±4.38 in Wj-MSC, 32.22±5.07 in AD-MSC, 25.9±1.27 in En-MSCs); However, there were significantly more than the expansion media alone (4.4±0.69). The results obtained from the cytokine secretion analysis also confirm these results. Also, there were significant differences in the clonogenicity of Wj-MSC, En-MSCs, and expansion media (CFU-GEMM: 7±1.73, 2.3±1.15, and 2.3±1.52), which indicated that Wj-MSC could significantly maintain the primitive state. As a result, using Wj-mesenchymal stem cells on a 3D coculture system effectively increases the HSPC expansion and maintains the colonization potential of hematopoietic stem cells.

Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold.

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin.

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB-HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

Upregulation of Pro-inflammatory Cytokine Genes by Parvovirus B19 in Human Bone Marrow Mesenchymal Stem Cells.

Chronic inflammation plays a prominent role in cancer initiation and development. On the other hand, the Inflammation can be established by a number of factors such as viral infections. Parvovirus B19 (B19V) is a pathogen with widespread infection, which infects bone marrow erythroid progenitor cells. It has been shown that B19V can also enter human bone marrow mesenchymal stem cells (BM-MSCs). In this study, we hypothesized that BM-MSCs as the main cellular component of bone marrow niche may be induced to secret pro-inflammatory cytokines after B19V infection. BM-MSCs were cultured up to passage 3. The cells were then subjected to nucleofection to transfer a plasmid containing B19V genome. After 36 h, total RNA was extracted and the expression levels of IL-1ß, IL-6, TNF-α and NF-κB genes were examined using qRT-PCR. Data analysis showed the significant increase in expression levels of all studied genes in the B19V-transfected cells (P < 0.05). Although further researches are required, our findings for the first time suggest the importance of B19V infection to establish an inflammatory microenvironment in the bone marrow and its involvement in inflammation-related diseases. Finally, based on our results, molecular assay to diagnose B19V infection of BM-MSCs prior to stem cell therapy is strongly recommended.

Generation of an in vitro model of ß-thalassemia using the CRISPR/Cas9 genome editing system.

ß-Thalassemia is a common monogenic disease characterized by defective ß-globin chains synthesis. In vitro ß-thalassemia-related research on increasing ß-like globin genes or identification of factors reducing the severity of the disease, has been performed on mouse erythroleukaemia or K562 cell lines. The aim of this study was the production of an in vitro model of ß-thalassemia using the highly efficient CRISPR-Cas9 system. Embryonic stem (ES) cells were nucleofected with guide RNA (gRNA)-Cas9 expression vectors. Molecular testing was done on extracted DNA to assess Hbb-b1 mutation. Analysis of transcription factors and hemoglobin genes were evaluated using quantitative reverse transcription-polymerase chain reaction following erythroid differentiation of ES cells. Sequencing data confirmed Hbb-b1 knockout alleles. Significant expression of erythroid transcription factors was observed in wild-type, Hbb-b1+/- and Hbb-b1-/- groups (P < .001). Compared with the wild-type group, the absolute number of Hbb-b1 mRNA in Hbb-b1+/- group significantly decreased from 6.44 × 106 to 3.23 × 106 copy number (P < .01), whereas in Hbb-b1-/- group had zero expression. The CRISPR/Cas9-mediated Hbb-b1 knockout in ES cells provides accessibility to an in vitro thalassemia model following erythroid differentiation. Considering the need for in vitro and mouse models to investigate the molecular basis of ß-thalassemia which also enables testing of therapeutic approaches, this method can be utilized to produce a mouse model of ß-thalassemia intermedia (Hbbth1/th1).

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.

The anticancer effects of pharmacological inhibition of autophagy in acute erythroid leukemia cells.

Although recent studies have reported different aspects of autophagy, from pro-survival to pro-death roles of this process in malignant cells, the underlying mechanisms by which autophagy inhibitors contribute toward the induction of programmed cell death in cancerous cells are still unclear. In the present study, we have attempted to explore some of the molecular features of pharmacological inhibition of autophagy in TF-1 cells (an acute erythroid leukemia model). Our findings indicated that ara-C induces autophagy (with alteration of LC3B, p62, and Beclin-1) in the cells; however, targeting autophagy by 3-methyladenine and chloroquine significantly increased caspase-dependent apoptosis and the sub-G1 compartment in ara-C-treated cells. Moreover, cell cycle analysis showed that 3-MA, as an early-stage autophagy inhibitor, could elevate the cell population in the G0/G1 cell cycle phase, which was associated with upregulation of p21 and p27 expressions. Interestingly, autophagy inhibition was also accompanied by downregulation of c-Myc gene and protein expression levels and upregulated levels of Bax and Bak gene expressions. In addition, following inhibition of autophagy, the levels of tumor-suppressive miRNA (i.e. miR-204) increased, whereas the values of oncogenic miRNAs (including miR-21, miR-221, miR-30a, and miR-17) decreased. Overall, our experiments indicate that autophagy inhibitors (especially chloroquine) seem to be promising agents for combination therapy in acute erythroid leukemia.

Modulation of microRNAs expression in hematopoietic stem cells treated with sodium butyrate in inducing fetal hemoglobin expression.

Context Inherited hemoglobin diseases are the most common single-gene disorders. Induction of fetal hemoglobin in beta hemoglobin disorders compensate for abnormal chain and ameliorate the clinical complications. Sodium butyrate is used conventionally for fetal hemoglobin induction; it can be replaced by safer therapeutic tools like microRNAs, small non-coding RNAs that control number of epigenetic mechanisms. Objective In this study, we compared the changes in the microRNAs of differentiated erythroid cells between control and sodium butyrate treated groups. The objective is to find significant association between these changes and gamma chain up regulation. Materials and methods First, CD133+ hematopoietic stem cells were isolated from cord blood by magnetic cell sorting (MACS) technique. After proliferation, the cells were differentiated to erythroid lineage in culture medium by EPO, SCF, and IL3. Meanwhile, the test group was treated with sodium butyrate. Then, gamma chain upregulation was verified by qPCR technique. Finally, microRNA profiling was performed through microarray assay and some of them confirmed by qPCR. Result Results demonstrated that gamma chain was 5.9-fold upregulated in the treated group. Significant changes were observed at 76 microRNAs, in which 20 were up-regulated and 56 were down-regulated. Discussion Five of these microRNAs including U101, hsa-miR-4726-5p, hsa-miR7109 5p, hsa-miR3663, and hsa-miR940 had significant changes in expression and volume. Conclusion In conclusion, it can be assumed that sodium butyrate can up-regulate gamma chain gene, and change miRNAs expression. These results can be profitable in future studies to find therapeutic goal suitable for such disorders.

Deregulation of miR-1, miR486, and let-7a in cytogenetically normal acute myeloid leukemia: association with NPM1 and FLT3 mutation and clinical characteristics.

Cytogenetically normal acute myeloid leukemia (CN-AML) constitutes the largest subgroup of AML patients that is associated with molecular alteration. MiRNAs have been shown to be aberrantly expressed in CN-AML. In addition, specific miRNA (miR) expression patterns were found to be associated with certain genetic alterations in these patients. This study investigated the expression level of miR-1, miR-486, and let-7a in 45 CN-AML patients well characterized for FLT3 and/or NPM1 mutations using real-time quantitative RT-PCR and evaluated the association between candidate miRs expression and clinical features. Our data revealed that miR-1 was significantly overexpressed in CN-AML patients, and increasing expression of miR-1 correlated with NPM1 mutation (P < 0.05) and lower hemoglobin level was also observed in patients with miR-1 overexpression (P < 0.05). The expression of miR-1 was much higher in AML-M2 compared with other subtypes. Further, we found significantly increasing miR-486 expression in 40 of 45 (89 %) CN-AML patients. There was no significant association of upregulation of miR-486 with clinical parameters. The expression level of miR-486 was increased in AML-M2 subtype. The levels of let-7a were significantly increased in CN-AML patients compared to the healthy control and significantly higher in the NPM1 ± CN-AML patients. There was no correlation detected between the level of let-7a and FLT3+. An increasing expression level of let-7a was demonstrated in M2 subtype. In addition, our data showed no significant association between increasing let-7a and clinical characteristic. Comparison of peripheral blood and bone marrow results in 30 CN-AML patients showed that there is a considerable concordance between PB and BM in the results of candidate miR levels (P < 0.001). In conclusion, further studies should also be performed to detect functional mechanism of these miRs.