The effect of Mesenchymal Stem Cells of Amniotic Membrane on the Proliferation and Differentiation of Umbilical Cord Blood CD34+ cells

Introduction: Ex vivo proliferation of hematopoietic stem cells (HSCs) of umbilical cord is widely used by combination of cytokine and stromal mesenchymal stem cells (MSCs) as feeder layer due to increase the cell doses, adequately. However, numerous studies have shown that ex vivo proliferation of these cells impairs their functions, including reduced self-renewal ability, apoptosis induction, and disordered cell cycle. MSCs have different sources such as amniotic membrane with a stable karyotype and high quality because of isolation from embryonic tissues, so that they are considered as a useful source for MSCs.Materials and Methods: In this study, isolated mesenchymal cells from the amniotic membrane were used as feeders for the HSCs proliferation. Four different cultures with various conditions were used; first one containing cytokines (stem cell factor, thrombopoietin, and FMS-related tyrosine kinase 3 ligand), second one with MSCs co-cultured with the aforementioned cytokines, third medium co-cultured with MSCs without cytokines, and finally the control medium was without co-culture condition and cytokines. Expression of mRNAs of HOXB4, GATA2, BCL2, and Survivin genes was also investigated.Results: The findings showed that the expression of mRNAs of these genes decreased in culture with cytokine, solely; however the expression of these genes was significantly higher in co-cultured system with cytokine rather than just with cytokine.Conclusion: : In general, the findings of this study indicate that the derived MSCs from amniotic membrane is a good source for the proliferation of umbilical cord blood CD34+ cells”. Because these cells increase the UCB-CD34+ quantity and their preservation properties.

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

The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with Mesenchymal Stem Cells

Objective: 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. Materials and Methods: 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. Results: 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). Conclusion: 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

Mesenchymal stem cells as a reference cell for HLA-typing

Introduction: Recognition of human leukocyte antigens (HLA) is of importance for hematopoietic stem cell transplantation. Any HLA-mismatches between the donor and recipient can cause graft rejection or other complications. In HLA-typing experiments, usage of HLA-known reference cells accompany with HLA-unknown samples is obligatory. Some international centers represent these cells with high expenses. On the other hand, transferring of these cells is problematic and in some instances is not practical.  In this study, we introduced umbilical cord-derived mesenchymal stem cells (MSCs) as reference cells for HLA genotyping. These cells are national and can be prepared locally. Materials and methods: We isolated MSCs from three umbilical cord and after their growth and proliferation, these cells were characterized by flow cytometry technique using antibodies to CD29, CD34, CD44, CD45, CD73, CD90 and CD105. HLA-typing was then carried out by PCR-SSP kits for HLA-A, -B and -DRB allele’s identification. Results: Isolated MSCs were positive for MSCs markers; CD29, CD44, CD73, CD90, and CD105 and negative for hematopoietic stem cell markers; CD34 and CD45. HLA alleles were determined. One of the samples was homologous for HLA alleles and the others were heterologous. Conclusion: We can develop a reference panel for HLA-typing by obtaining MSCs from available sources like umbilical cord

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

MicroRNA Microarray Profiling during Megakaryocyte Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells

Objective In order to clarify the role of microRNAs (miRNA) in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133+ cells. Materials and Methods In this experimental study, human CD133+ hematopoietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis. Results A total of 10 and 14 miRNAs were upregulated (e.g. miR-1246 and miR-148-a) and down-regulated (e.g. miR- 551b and miR-10a) respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis. Conclusion We conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment

Peripheral blood CD163(+) monocytes and soluble CD163 in dry and neovascular age-related macular degeneration

Macrophages are the main infiltrating immune cells in choroidal neovascularization (CNV), a hallmark of the human wet, or neovascular age-related macular degeneration (AMD). Due to their plasticity and ability to adapt to the local microenvironment in a tissue-dependent manner, macrophages display polar functional phenotypes characterized by their cell surface markers and their cytokine profiles. We found accumulation of hemoglobin-scavenging cluster of differentiation 163 (CD163)(+) macrophages in laser-induced CNV lesions and higher expression of CD163(+) monocytes in the peripheral blood on day 7 post injury in mice. In comparison, CD80(+) macrophages did not differ with laser-injury in young or aged mice and did not significantly change in the peripheral blood of CNV mice. We examined the percentages of CD163(+), CD206(+), and CD80(+) monocytes in the peripheral blood of patients with wet AMD, patients with dry AMD, and in age-matched individuals without AMD as controls. Percentages of peripheral blood CD163(+) monocytes in both dry AMD (P < .001) and wet AMD (P < .05) were higher than in age-matched non-AMD controls, while there was no difference between the groups in the percentages of peripheral CD206(+) and CD80(+) monocytes. Further, serum level of soluble CD163 (sCD163) was elevated only in patients with wet AMD (P < .05). An examination of 40 cytokine levels across the study groups revealed that anti-VEGF treated patients with wet AMD, who showed no exudative signs on the day of blood drawing had a cytokine profile that was similar to that of non-AMD individuals. These results indicate that CD163 could be further evaluated for its potential as a useful marker of disease activity in patients with neovascular AMD. Future studies will address the origin and potential mechanistic role of CD163(+) macrophages in wet AMD pathologies of angiogenesis and leakage of blood components

Targeting Chronic Myeloid Leukemia Stem/Progenitor Cells Using Venetoclax-Loaded Immunoliposome

CML is a hematopoietic stem-cell disorder emanating from breakpoint cluster region/Abelson murine leukemia 1 (BCR/ABL) translocation. Introduction of different TKIs revolutionized treatment outcome in CML patients, but CML LSCs seem insensitive to TKIs and are detectable in newly diagnosed and resistant CML patients and in patients who discontinued therapy. It has been reported that CML LSCs aberrantly express some CD markers such as CD26 that can be used for the diagnosis and for targeting. In this study, we confirmed the presence of CD26+ CML LSCs in newly diagnosed and resistant CML patients. To selectively target CML LSCs/progenitor cells that express CD26 and to spare normal HSCs/progenitor cells, we designed a venetoclax-loaded immunoliposome (IL-VX). Our results showed that by using this system we could selectively target CD26+ cells while sparing CD26 12 cells. The efficiency of venetoclax in targeting CML LSCs has been reported and our system demonstrated a higher potency in cell death induction in comparison to free venetoclax. Meanwhile, treatment of patient samples with IL-VX significantly reduced CD26+ cells in both stem cells and progenitor cells population. In conclusion, this approach showed that selective elimination of CD26+ CML LSCs/progenitor cells can be obtained in vitro, which might allow in vivo reduction of side effects and attainment of treatment-free, long-lasting remission in CML patients