Expansion of cord blood stem cells in fibronectin-coated microfluidic bioreactor

ABSTRACT Background: Hematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methods: A microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. Results: The expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. Conclusion: Utilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.

Expression of hsa-MIR-204, RUNX2, PPAR gamma, and BCL2 in bone marrow derived mesenchymal stem cells from multiple myeloma patients and normal individuals

Objective: Multiple Myeloma [MM] is a heterogeneous cytogenetic disorder in which clonal plasma cells proliferate in the bone marrow [BM] and cause bone destruction. The BM microenvironment plays a crucial role in pathogenesis of this disease, and mesenchymal stem cells [MSCs] are one of the key players. Herein, we propose to investigate the expressions of hsa-MIR-204, runt-related transcription factor 2 [RUNX2], peroxisome proliferator-activated receptor gamma [PPAR gamma], and B-cell lymphoma 2 [BCL2] as factors involved in osteogenesis, adipogenesis, and MSC survival in BM-MSCs from MM patients and normal individuals

Materials and Methods: In this experimental study, we isolated MSCs from BM aspirates of MM patients and healthy donors. Total RNA were extracted before and after co-culture with L363 myeloma cells. Gene expressions of RUNX2, PPAR gamma, BCL2, and hsa-MIR-204 were assessed by quantitive real time polymerase chain reaction [qRT-PCR]

Results: Higher levels of RUNX2, PPAR gamma, and hsa-MIR-204 expressions existed in MM-MSCs compared to normally derived [ND]-MSCs. BCL2 expression decreased in MM-MSCs. We observed different results in the co-culture model

Conclusion: In general, the MM-MSCs gene expression profile differed compared to ND-MSCs. Upregulation of RUNX2, PPAR gamma, and hsa-MIR-204 in MM-MSCs compared to ND-MSCs would result in formation of bone defects. Downregulation of BCL2 would lead to MM-MSC cell death

16 month survey of cyclosporine utilization evaluation in allogeneic hematopoietic stem cell transplant recipients

Objectives: Graft versus host disease [GVHD] is a life threatening reaction in the stem cell transplantation process. Nowadays Cyclosporine is the most commonly utilized agent for GVHD prophylaxis and it has a major role in successful transplantation. Cyclosporine has been applied for many years in this field but it could be stated that currently no general consensus is available for its optimal method of administration. Conditions related to cyclosporine administration and possible related adverse reactions observed closely in our patients with the aim of constructing a comprehensive practice guideline in the future

Patients and Methods: Allogeneic stem cell transplant recipients who have been taking cyclosporine were monitored during and after their hospitalization while recording all observations on predefined questionnaires on the basis of periodic clinical and laboratory examinations for a 16 month period

Results: Mean recorded duration of infusions was 1.44 +/- 0.68 h and by twice daily administration, means intravenous and oral dose was 101.85 +/- 22.03 mg and 219.28 +/- 63.9 mg, respectively. A mean CsA trough level after about 12 h of specified unique doses was 223 +/- 65 ng/mL. We found hypertension, nephrotoxicity, neurotoxicity, hypertension, and dyslipidemia in about 14, 20, 48, and 94 percent of patients

Conclusions: This study proposed that permanent guidance of healthcare team according to a fixed and standard method of cyclosporine administration routine with using efficient facilities and protocols would be helpful considerably for an optimal pharmacotherapy

Plerixafor in the treatment of stem cell mobilization failure; first experience in Iran

High-dose chemotherapy and autologous stem cell transplantation [SCT] have become an effective care for many patients with hematological malignancies. Harvesting the stem cells is one the most important parts of SCT. The two most commonly used mobilization regimens are the use of granulocyte colony-stimulating factor [G-CSF] or G-CSF plus chemotherapy. However, about 10-30% of patients are unable to collect enough cells to support HSCT due to previous chemotherapies, radiation, marrow involvement or fibrosis. In multiple myeloma patients, it is hard to collect enough stem cells when the bone marrow is extensively involved. Plerixafor has emerged as a novel mobilizing agent and its efficacy has been shown in two phase III studies. Considering the importance of autologous SCT in patients with multiple myeloma, we report the first successful Iranian experience at Tehran Taleghani bone marrow transplantation center using plerixafor to mobilize stem cells in a patient with refractory multiple myeloma with extensive bone marrow involvement who failed mobilization with G-CSF

Osteogenic inhibition in multiple Myeloma

Multiple myeloma [MM] is a plasma cell malignancy where plasma cells are increased in the bone marrow [BM] and usually do not enter peripheral blood, but produce harmful factors creating problems in these patients [e.g. malignant plasma cells over activate osteoclasts and inhibit osteoblasts with factors like RANKL and DKK]. These factors are a main cause of bone lesion in MM patients. Recently SOST gene which responsible to encodes the sclerostin protein was identify. This protein specifically inhibits Wnt signaling in osteoblasts [inhibition of osteoblast differentiation and proliferation] and decrease bone formation and can also cause bone lesion in MM patients. In this experimental study, human myeloma cell lines [U266 b1] were purchased from Pasteur Institute of Iran. Samples consisted of BM aspirates from the iliac crest of MM patients. BM with more than 70% plasma cell were selected for our study [6 patients] and one healthy donor. RNA extraction was done with Qiagen kit. was undertaken on mRNA of samples and cell lines. Also we purchased unrestricted somatic stem cells from Bonyakhte Company to evaluate the effect of soluble factors from myeloma cell lines on osteogenic differentiation medium. Our results showed that SOST is expressed significantly in primary myeloma cells derived from MM patients and myeloma cell lines. In other words, patients with more bone problems, express SOST in their plasma cells at a higher level. In addition, myeloma cells inhibit osteoblast differentiation in progenitor cells from umbilical cord blood stem cell [UCSC] in osteogenic inducing medium. There are many osteoblast maturation inhibitory factors such as DKK, Sfrp and Sclerostin that inhibit maturation of osteoblast in bone. Among osteoblast inhibitory agents [DKK, Sfrp, Sclerostin] sclerostin has the highest specificity and therefore will have less side effect versus non-specific inhibitory agents. Our results also show that based on SOST expression in MM, there is a potential to inhibit sclerostin with antibody or alternative methods and prevent bone lesion in MM patients with the least side effect

Production of recombinant lentiviruses expressing mir-16 by transient transfection of 293t cells

The aim of the present study was the production of recombinant lentviruses that express miR-16. After transduction, altered expression levels of miRNA and its target protein were analyzed. A DNA fragment that contained the miR-16 precursor was cloned in a lentiviral plasmid. Lentiviral vector particles were produced by transient calcium phosphate co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids. Viral supernatants were harvested and concentrated by ultracentrifuge. Virus titration was determined by fluorescent microscopy and flow cytometry. Altered expression levels of miR-16 were evaluated by real-time PCR; its protein target was evaluated by Western blot. The identity of DNA was established by colony-PCR, enzymatic digestion of positive clones, and DNA sequencing. After co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids, viral particles were concentrated and the virus titer determined. Maximum expression of the GFP reporter gene was obtained in more than 80% of the cells transduced with lentivirus at MOI=1. Real-time PCR assay showed that miR-16 expression levels significantly increased in transduced cells compared with the control group. As shown by Western blot analysis, miR-16 overexpression downregulated Bcl-2 expression at the protein level. This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells

Geographic heterogeneity of cytogenetic characteristics of acute myeloid leukemia in the early detection: a comparative study of Iranian and Indian adult patients

Acute Meyloid Leukemia [AML] in adults is known to be a heterogeneous disease with diverse chromosome abnormalities. Some of these chromosome abnormalities are found with a high incidence in populations from specific geographical areas and ethnic societies. Therefore, we studied the cytogenetic features of AML cases in contrasting societies of Iran and India. Cytogenetic investigation was performed in various subtypes of AML with unstimulated short-term culture and High Resolution Cell Synchronization with some modification. Cytogenetically, Iranian M3 displayed a higher frequency of t[15;17] than Indian M3 [33.8% vs 19.3%] followed by M2 [t[8;21] [27.7% vs 16.2%]] and M1 [t[9;22] [16.0% vs 11.3%]]; whereas, inv[16]11q23 and numerical chromosomal aberrations in chromosome 5,7,8 occurred more frequently in Indian than Iranian. These findings represented different cytogenetic characteristics of t[15;17] between the two populations. This is the first systematic cytogenetic study of an ethnic Iranian population. Extensive biological studies of AML in Iran and India and various countries to be needed to clarify the role of genetic as well as geographic heterogeneity in the pathogenesis of AML

In-patient satisfaction and its related factors in Taleghani University Hospital, Tehran, Iran

Patient satisfaction survey is an instrumental component in hospital's quality of care monitoring in relation to cost and services. This study was conducted to evaluate patient satisfaction and its related factors. A cross sectional study was performed between April 2006 and August 2006. Sample size was determined as 476 from 5021 by randomized sampling in several phases according to the proportion of hospitalized patient. Participants were interviewed privately face to face in the hospital at discharge time. Interviews were conducted by trained interviewers using pre tested questionnaires. Correlation between variables was estimated by using Pearson's Correlation. The majority, 83% of patient was quite satisfied with their care and 1% was dissatisfied. About 91% of patients were most satisfied with physician communication and treatment. Only 27% of patients were satisfied with nutrition status. There was no relationship between age, education and total satisfaction. Percentage of patient faithfulness and recommendation for this hospital to their friends was 66% and 65% respectively. Both male and female patients whose hospital stay was between 11-15 days were more satisfied with the service provided. In general, patients were quite satisfied with their hospital care. More studies such as this survey are required to improve the quality of care and overall health cares outcome

[Patient satisfaction and its related factors in Ayatollah Taleghani Hospital in 2006]

Patient satisfaction survey is an instrumental component in monitoring the hospital's quality of care, in relation to cost and services. Many studies have been conducted throughout the world to determine the patient satisfaction and its related factors. This study was carried out to evaluate patient satisfaction and its related factors. A cross sectional study was based on in-hospital patient who attended the Taleghni hospital between April 2006 and August 2006. Sample size was determined as 476 from 5021 by randomized sampling. Sample sizes in each ward were according to the proportion of hospitalized patient. Participants were interviewed privately face to face in the hospital at discharge time. Interviews were conducted by trained interviewers using pre tested questionnaires [Verona Service Satisfaction Scale- VSSS 32]. Correlation between total satisfaction and Patient's age, education] was estimated by using Pearson's Correlation. Prevalence and confidence interval [CI] of unsatisfied patient was calculated. In majority, 83% of patients were quite satisfied with their care and 1.5% was dissatisfied [CI=0.7-2.2]. 93% of patient was most satisfied with physician communication and treatment. Only 49% of patients were satisfied with nutrition status. There was no relationship between age, educational status and total satisfaction. Percentage of patient-faithfulness was 66%. 65% of admitted subjects were recommending this hospital to their friends. Patients whose in-hospital course was between 11-15 days were more satisfied with hospitalization among both sexes. In general, patients were quite satisfied with their hospital care. Further studies similar to this survey is offer to improve the quality of care and overall health-care outcomes

[Effects of leukocyte-depletion filters on alluimunization derived reactions of platelet transfusion in acute leukemic patients]

Leukemia is the most common malignancy among adults and children which may develop several side affects such as anemia, infection and thrombocytopenia-related hemorrage. Many modalities have been suggested for prevention of Alluimunization and one of them is using of leukocytes free products. In fact, platelets could not solely stimulate immune reaction. This study is conducted at two hospials of "Shohada" and " Taleghani" to demonstrate the usefulness of such products. This research is a single- blinded clinical trial. All the patients had acute leukemia. In control group [24 subjects] we used a routin infusion set for transfusion and in experimental group [20 subjects] a biofilter was applied during the transfusion, platelet were counted one hour and 24 hr after transfusion. CCI formulate was used to represent a signs for Alluimunization. If CCI was less than 10x10 L/M[2] after transfusion or it became below 5-7x10 L/M[2] after 18-24 hr, the subject was considered alluimmune. After one hour, 21 cases out of 24 were alluimmuned in control group whereas in experimental group 13 cases from 20 patients became alluimmune. After 24 hours, 5 cases in control group and 5 ones in experiments were found to be Alluimmune. We did not prove any preventive properties from leukocyte-depletion filters in reducing the risk factors of Alluimunization amongs patients with acute leukemia

Analysis of HLA-G gene expression in b-lymphocytes from chronic lymphocytic leukemia patients

The human leukocyte antigen G [HLA-G] molecule exhibits limited tissue distribution, low polymorphism and alternative splicings that generate seven HLA-G isoforms. HLA-G exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells that may favor their escape from anti-tumor immune responses. This study it is an effort to clarify the presence of HLA-G in B-cell chronic lymphocytic leukemia [B-CLL] patients. HLA-G mRNA expression was studied in a pilot study in circulating B-CLL and also healthy controls by reverse transcription [RT]-PCR using a set of pan-HLA-G primers. RT-PCR was performed on B-cells from 74 B-CLL patients and 12 healthy controls. The data showed HLA-G gene expression in 20% of the B-CLL patients. No expression of HLA-G could be detected in the healthy control group. These data suggest that HLA-G is expressed at the gene level in B cells from B-CLL patients but not in B cells from healthy controls. Further study is required to clarify the role of HLA-G as a regulatory factor that could affect immune response in B-CLL patients

External evaluation: a requirement for accountability and responsiveness

Quality assurance as a comprehensive term encompasses all policies, processes and actions maintaining and developing higher education quality. Quality assurance emphasizes on external goals of evaluation, one of which is to assure learners, public and government that each unit, department, program or institution manages its quality, thus quality assurance focuses on accountability. This article first describes importance of external evaluation and necessity to conduct it, then considering the fact that two educational programs leading to bachelor degrees of nursing and obstetrics, which their internal evaluation have already completed, are volunteers to go under external evaluation. Explains steps of external evaluation and focuses on the external aspect of evaluation to assure quality of educational programs following internal evaluation

Increase of CXCR4 expression on expanded non-enriched cord blood CD34+cells using MSCs

A number of potential cell adhesion molecules, which mediate essential cell-to-cell or cell-to-matrix interactions, are expressed on the surface of CD34+ hematopoietic progenitor cells [HPCs], including integrins, CD44, and CXCR4. These molecules are essential for homing process. In this study, we compared the changes of expression of CD44 and CXCR4 on the CD34+ hematopoietic progenitor cells expanded on MSCs in the presence of cytokines. Cord blood CD34+ cells were expanded using human bone marrow mesenchymal stem cells and cytokines [TPO, SCF, FLt-3, IL-6, and IL-3], and then expression of CD44 and CXCR4 on CD34+ cells were evaluated by flow cytometric analysis. After 2 weeks of serum free culture of CD34+ cells in the presence of cytokines, the expression of CXCR4 on CD34+ cells was decreased 3.4 fold [p<0.05]. In contrast, the expression of CXCR4 on CD34+ cells expanded on hMSCs was increased [p<0.05]. The expression of CD44 on expanded CD34+ cells in both methods did not differ significantly. Our results indicated that co-culture of cord blood stem cells on hMSCs significantly increased CXCR4 expression on cord blood CD34+ cells

preliminary study on the effect of two therapeutic procedures on the expression of BCR-ABL gene in CML patients

Chronic myelogensis leukemia [CML] is a chronic myeloproliferative disorder resulting from a specific mutation in a pluripotent stem cell. The association of Philadelphia chromosome with this disorder was described in 1973. Subsequently, the BCR-ABL fusion gene and its product which is a tyrosin kinase inhibiting apoptosis were introduced. The treatment of choice for these patients is BMT as well as the new molecular treatment of imatinib mesylate. The present study was designed in order to compare the rate of expression of the BCR-ABL gene in patients who had undergone treatment by bone marrow transplantation with those treated by imatimib. Expression of BCR-ABL gene was measured in 34 patients 17 patients were treated with BMT and 17 with imatinib mesylate using quantitative PCR on a light cycler instrument. The results obtained in this preliminary study demonstrates that rate of gene expression patients treated with imatinib mesylate for more than 8 month was similar to that found in patients treated with 8 BMT who were in relatively stable conditions. This finding may be an indication that imatinib mesylate as a molecular inhibitor can have the same effect as BMT but with less adverse effects. Clearly definite conclusions require more extensive studies on larger number of patients