Rapid Screening of \u3b2-Globin Gene Mutations by Real-Time PCR in Egyptian Thalassemic Children

Thalassemia is one of the most common genetic disorders in Egypt. With the total population of 70 million, there are approximately 600,000 affected individuals and more than 20 million thalassemia carriers. Thalassemia is therefore one of the major health problems in Egypt. B-Thalassemias are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. Introduction of the real time PCR has made a revolution in the time taken for the PCR reactions. We present a method for the diagnosis of the common mutations of the B-thalassemia in Egyptian children & families. The procedure depends on the real-time PCR using specific fluorescently labeled hybridization probes. The melting temperature for each of the specific probes obtained after the PCR reaction permits the identification of the specific mutation. Genotyping of 20 thalassemic children attending the hematology clinic of the children specialized hospital and 10 controls was done using Real-time PCR and the conventional Amplification Refractory Mutation System (ARMS) technique. Analysis revealed identical results to most of the patients and they were further checked by the sequencing results of the DNA samples. The established method is a robust, fast and straight forward assay that allows the detection of the common B-thalassemia mutations in Egypt. The described LightCycler system protocol can rapidly screen for many B-globin gene mutations

Detection of BCR/ABL Translocation in Bone Marrow Derived Mesenchymal Stem Cells in Egyptian CML Patients

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. It is characterized at the cytogenetic level by Philadelphia (ph) chromosome and at the molecular level by the BCR/ABL gene rearrangement. Bone marrow derived mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate into several mesenchymal tissues.AIM: To observe the biological characteristics of MSCS from CML patients and to determine whether MSCs harbor the abnormal BCR/ABL translocation similar to CML bone marrow cells.SUBJECTS AND METHODS: Characterized MSCs were isolated from 12 newly diagnosed Philadelphia positive untreated CML patients.RESULTS: MSCs can be readily isolated from CML marrow and exhibit major expansion. Flow cytometry analysis revealed the typical MSC phenotype. Moreover; MSCs do not harbor the BCR/ABL translocation confirmed by karyotype and real time PCR.CONCLUSION: MSCs from CML patients express the typical MSC phenotype; and do not express the BCR/ABL gene. Since; MSCs are able to support engraftment of hematopoietic stem cells in stem cell transplantation(SCT) as well as suppress alloreactive T cells causing graft versus –host disease, this current study provides evidence that in a SCT setting of CML patients, autologous MSCs could be a source of stem cell support in future cell therapy applications

Comparative characteristics of amniotic membrane, endometrium and ovarian derived mesenchymal stem cells: A role for amniotic membrane in stem cell therapy

Objectives: Isolation and comparison of cellular and molecular characteristics of MSCs derived from full term amniotic membrane (AM), endometrium and ovarian tissues. Investigation of a possible therapeutic potential for AM MSCs in the female reproductive tract. Study design & methods: Human amniotic membranes (AMs) from term placentas (N = 8), endometrial/stromal (by scrapping endometrium) and ovarian tissues from premenopausal hysterectomies (N = 6) after mincing and enzymatic digestion were included in the study. Immunophenotyping by flow cytometry, Karyotyping, differentiation to mesoderm, RT-PCR analysis for transcription factors, WNT signaling pathways, pluripotency markers and HOXA genes were performed. Results: Generated cells showed morphologically and phenotypically the characteristics of MSCs from other sources with fairly comparable results. The cumulative population doubling was significantly higher in endometrium MSCs 25 ± 5.2 compared to 17.2 ± 4.5 for AM MSCs and 10.7 ± 2.4 for ovarian MSCs (P < 0.001). They differentiated into osteogenic, adipogenic and chondrogenic lineages till passage 9. They all possessed normal karyotype by passage 6. They all expressed HOXA genes and differentially pleuripotency markers. Intrauterine injection of GFP transfected AM MSCs into female Wistar rats (N = 10), demonstrated the presence of AM cells with GFP expression after 14 days within several mice uterine and ovarian cryosections. AM MSCs highly expressed genes involved in patterning of the female reproductive tract. Conclusions: AM, endometrium and ovarian derived MSCs share fairly similar morphological, cellular and molecular characteristics. They possess mesenchymal stemmness potentials and endometrial MSCs possess the highest expansion capacity. AM MSCs may have a potential for stem cell therapy in the female reproductive tract

Comparative characteristics of endothelial-like cells derived from human adipose mesenchymal stem cells and umbilical cord blood-derived endothelial cells

Adult peripheral blood contains a limited number of endothelial progenitor cells that can be isolated for treatment of ischemic diseases. The adipose tissue became an interesting source of stem cells for regenerative medicine. This study aimed to investigate the phenotype of cells obtained by culturing adipose-derived mesenchymal stem cells (ad-MSCs) in the presence of endothelial growth supplements compared to endothelial cells obtained from umbilical cord blood (UCB). Passage 3 ad-MSCs and mononuclear layer from UCB were cultured in presence of endothelial growth media for 3 weeks followed by their characterization by flow cytometry and polymerase chain reaction. After culture in endothelial inductive media, ad-MSCs expressed endothelial genes and some endothelial marker proteins as CD31 and CD34, respectively. Adipose tissue could be a reliable source for easy obtaining, expanding and differentiating MSCs into endothelial-like cells for autologous cell-based therapy