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.

Molecular Spectrum of Beta-Globin Mutations in Transfusion-Dependent Patients with Thalassemia in Qazvin Province, Iran

Background: Beta thalassemia is a common inherited disease,resulting from one or more of 200 different mutations in the betaglobingene. Qazvin province has attracted migrations of severaldifferent populations due to industrialization during the past fivedecades. The aim of this study was to define the molecular spectrumofbeta-thalassemia mutations in Qazvin province.Methods: Ethylen diamin acetic acid-containing venous bloodsamples were collected from 100 patients with transfusiondependentbeta-thalassemia from the department of Pediatricsin Qods hospital. Age, sex, history, and consanguinity betweenthe parents were recorded by reviewing the patients’files. DNA was isolated from leukocytes using the standardprocedure. Amplification refractory mutation system (ARMS)technique was used for molecular detection of mutations. Directsequencing analysis was applied for DNA samples whenno mutation was detected with ARMS.Results: Of the 200 chromosomes investigated, 11 types of mutationswere identified by ARMS technique while direct sequencingrevealed the remaining alleles (9 types of mutations).Total 20 different mutations discovered by this two-step approach.Abundant alleles (IVS II-1, IVS I-10, FSC 8/9) accountedfor 59.3% of the mutations. IVS II-1 with a frequencyof 31.3 % was the most common while HbS, Cd 74/75 and Cd15, each with a frequency of 0.55%, had the least frequencies.Conclusion: Beta thalassemia mutations are very heterogeneousin Qazvin province. Extensive ethnic and genetic admixture hasresulted in unexpectedly high number of different mutations,most of them similar to that of north and north-western provincesof Iran. Different mutations in this region suggest migration ofchromosomes fromdistant places and genetic admixture

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 Rh blood group system and its role in alloimmunization rate among sickle cell disease and sickle thalassemia patients in Iran

Abstract Introduction The alloimmunization following blood transfusion can be life‐threatening. The Rh alloantibodies are one of the most common causes contributing to alloimmunization. This study aimed to evaluate the rate and causes of alloimmunization and to determine the Rh phenotypes and genotypes among sickle cell disease (SCD) and sickle thalassemia (Sβ). Materials and Methods Our study included 104 SCD and Sβ patients referring to Baghaei 2 Hospital of Ahvaz in 2019 using a non‐random simple sampling method. The blood samples were collected for Rh phenotypes, alloantibody screening and identification, and molecular tests. The SSP‐PCR and RFLP methods with the Pst 1 enzyme were used. Results The alloimmunization rate was 9.6% and 13.2% based on immunohematological tests and medical records, respectively. The main alloantibodies (90%) were anti‐Rh, and 40% of the patients had multiple alloantibodies. A significant correlation was found between gender and alloimmunization. The phenotypes of DCce (37.5%), DCcEe (24%), Dce (20.2%), and dce (5.8%) and genotypes of R1r (25%), R1R2 (20.2%), R1R1 (18.3%), and R1R0 (10.6%) were the most prevalent. The R1R2 was a frequent genotype in Sβ. Conclusion R0r′ and R1R0 genotypes were limited to our population in Iran. Due to the differences in RH genotypes between our population and others, the blood transfusion from other ethnicities increased our total alloimmunization rate

Analysis of Alterations in Morphologic Characteristics of Mesenchymal Stem Cells by Mechanical Stimulation during Differentiation into Smooth Muscle Cells

Objective: Mesenchymal stem cells (MSCs) can be expanded and differentiated intomany mature cell types including smooth muscle cells (SMCs). In addition to growth factor,cyclic stretch contributes to differentiation of stem cells. Mechanical stimuli are criticalto morphological changes, development, regeneration, differentiation and pathology ofmesenchymal tissues. The aim of this study is to investigate effects of cyclic stretch withdiffering amplitudes on morphology and differentiation of mesenchymal stem cells.Materials and Methods: Mesenchymal stem cells are extracted from human bone marrow.Cells are cultured on silicone membrane and exposed to cyclic stretch by a custommade device. Cellular images are captured before and after tests. Effects of 5% and 15%uniaxial strain with 1Hz frequency and 1-8 hour durations on morphology of human mesenchymalstem cells are investigated. It is assumed that environmental factors such asmechanical loading regulate MSCs differentiation to SMCs. Fractal analysis is used toquantify alterations in cellular morphology. An image processing method with a designedcode is used for evaluation of fractal dimension parameter.Results: Results demonstrate statistically significant change in cell morphology due tomechanical stretch. By elevation of strain amplitude and number of load cycles, fractaldimensions of cell images decrease. Such decrease is equivalent to alignment of cells bymechanical stimulus. Cells are differentiated to SMCs purely by cyclic stretch. The initiationand rate of differentiation depend on mechanical conditions.Conclusion: To produce functional SMCs for engineered tissues, MSCs can be exposed to uniaxialcyclic stretch. The functionality of differentiated SMCs depends on loading conditions