Effects of Quercetin Supplementation on Hematological Parameters in Non-Alcoholic Fatty Liver Disease: a Randomized, Double-Blind, Placebo-Controlled Pilot Study

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs

BACKGROUND: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. METHODS: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. RESULTS: Maximum CB-CD34⁺ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34⁺ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34⁺ and CD34⁻ cells in the cytokine co-culture system was observed. CONCLUSION: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

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 [alpha, beta, gamma, epsilon and zeta] 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 [alpha, beta, gamma, epsilon and zeta] 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

immunosuppressive activity of amniotic membrane mesenchymal stem cells on T lymphocytes

Mesenchymal Stem Cells [MSCs] are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane [AM] are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells [hAM-MSCs]. The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro. Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin [PHA]. Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-gamma was examined by ELISA method. Additionally, the expression of activation markers [CD38, HLA-DR] was studied on T lymphocytes by flow cytometry technique. It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes [p

important role of FLT3-L in Ex vivo expansion of hematopoietic stem cells following co-culture with mesenchymal stem cells

Hematopoietic stem cells [HSCs] transplantation using umbilical cord blood [UCB] has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand [FLT3-L] as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell [MSC] feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count [TNC], cluster of differentiation 34[+] [CD34[+]] cell count, colony forming unit assay [CFU], long-term culture initiating cell [LTC-IC], homeobox protein B4 [HoxB4] mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines [P<0.05]. FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor [SCF], thrombopoietin [TPO] and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased [P<0.05]. FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture

[Comparison of three cord blood processing methods: hydroxyethyl starch, simple centrifugation and Sepax automated system]

Background: Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods.

Material and Methods: 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell [TNC] counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance [ANOVA] and ps less than 0.05 were considered statistically significant.

Results: The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively [p> 0.05]. The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively [p> 0.05]. Also, the colony assay recoveries were not significantly different among the three methods [p> 0.05].

Conclusion: No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.

Effects of platelet microparticles on the activation of B cells

Platelets are anucleated fragments derived from megakaryocytes. It has been demonstrated that platelets play a role in hemostasis and innate immunity. In addition, platelets have a CD40 ligand which is an important molecular marker in motivating immune cells. Thus, platelets also have a role in adaptive immunity as seen by their ability to activate B cells. Since human platelet microparticles [MPs] originate from platelets, we have chosen to examine the effects of MPs on B cell activation. Platelet MPs were isolated from platelet concentrates obtained from the Tehran Blood Transfusion Center. The MPs were co-cultured with B cells isolated from human whole blood with magnetic beads using negative selection. After seven days, the expression of activation markers CD27 and CD86, as well as IgD were evaluated by flow cytometry. In a comparison between test [B cells/MPs] and control [B cells] cells we observed that the expression of activation markers CD27 and CD86 increased during the seven-day co-culture period. However, the expression of IgD antibody decreased. As with platelets, MPs can affect B cell activation during in vitro co-culture

high yield expansion and megakaryocytic differentiation of human umbilical cord blood CD133[+] cells

Despite of many benefits, umbilical cord blood [UCB] hematopoietic stem cell [HSC] transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte [MK] progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133[+] HSCs. In this experimental study, The CD133[+] cells were separated from three cord blood samples by magnetic activated cell sorting [MACS] method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin [TPO] for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit [CFU] assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133[+] cells showed maximum expansion in the stem span medium with stem cell factor [SCF] + FMS-like tyrosine kinase 3-ligand [Flt3-L] + TPO but the maximum differentiation was seen when CD133[+] cells were expanded in stem span medium with SCF + Interleukin 3 [IL-3] + TPO for the first and in TPO for the second week. Colony Forming Unit-MK [CFU-MK] was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 +/- 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133[+] cells expansion followed by MK differentiation

[3D culture of umblical cord blood-drived CD34 + cells on a DBM scaffold coated by cord blood-derived unrestricted somatic stem cells [USSC]]

Allogeneic transplantation with umbilical cord blood [UCB] in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell [MPC] -unrestricted somatic stem cells [USSC] -was incorporated in an attempt to expand CD34+ cells from UCB. To provide a similar environment in vitro, we coated DBM scaffold with USSC cells as the matrix for support UCB-CD34+ cells growth. Human placenta USSC was isolated and characterized by morphologic and immunophenotypical analysis. UCB CD34+ cells were expanded by coculture with placental USSC in 2D and 3D environment. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture [LTC-IC] output. Ex vivo expansion of UCB hematopoietic cells, when cultured in different 2D conditions and 3D condition for 3 weeks, was significantly enhanced, the total cell count increased within the 28-day period. For total CFC, the highest CFC expansion was observed at day 14.Flow cytometry analysis of the percentage of CD34+ cells showed a decline in USSC cocultures in 2D and 3D condition at 3 weeks. These results strongly suggest that human USSC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro and USSC-coated DBM can therefore provide an ex vivo mimicry of bone marrow by enhancing of surface/ volume ratio and feeder layers, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation

Isolation of stem cells from adult rat kidneys

The kidney has an inherent ability for recovery and regeneration following acute damage. However, there has been much contention as to the source of regenerating renal cells. The aim of this study was to isolate and characterize these cells. Normal rat kidneys were minced and cells were isolated with collagenase I and were cultured in an expansion medium. Adherent cells were isolated and expanded for more than 120 days in vitro. These cells had the potential of trans-lineage differentiation into neural cells, adipocytes and osteocytes. These cells also expressed Nucleostemin, Cyclin D1, Notch1 and Survivin which are commonly expressed in stem cells. The results of the current work show that the adult kidney contains a population of multipotent stem cells.

Comparison of functional competence of umbilical cord and adult peripheral blood dendritic cells in allogenic mixed leukocyte reaction

Dendritic cells [DCs] are the most potent stimulators of primary T cell responses and play a key role in immune reactions after stem cell transplantation. Very little is known about the cord blood [CB] dendritic cells and their potential involvement in the low incidence and lower severity of acute graft-versus-host disease after CB transplantation. The aim of this study was the isolation of cord blood and peripheral blood dendritic cells and comparison of their functional competence and determination of their probable role in graft versus host disease after stem cell transplantation. In this study, fresh peripheral blood DCs [PBDCs] wereenriched as HLA-DR[+] cells, lacking the CDS, CDllb, CD14, CD16, CD19 and CD56, using immunomagnetic bead depletion. For cord blood dendritic cells [CBDCs] enrichment CD34[+] and CD66b[+] cells were needed to be depleted too. Immunomagnetically enriched PB/CB dendritic cells were co-cultured with adult Tlymphocytes and cell proliferation was measured by 3H-thymidine incorporation. Results showed that CBDCs were significantly poor stimulators of the mixed leukocyte reaction as compared with PBDCs [P < 0.05]. The demonstrated impairment of CBDCs function could be of importance in interpretation of the low incidence and milder severity of graft-versus-host disease [GVHD] in umbilical CB transplantation compared with peripheral blood or bone marrow stem cell transplantation