فصلنامه خون
سال نهم شماره 3 (پیاپی 36، پاییز 1391)

Stem cell transplantation has achieved high success for treatment but the use of this source has some limitation. Cord blood transplantation is clinically limited by its low progenitor cell contents such as CD34+. The aim of this study was to evaluate the effect of mesenchymal stem cells (MSCs) on replication of CD34+ in cord blood. In this experimental study MSCs were isolated and cultured from BM mononuclear cells. MSCs were used for co-culture in two systems of 2D and 3D cultures. After isolation، umbilical cord blood CD34+ cells were cultured in two different systems. The level of expansion in these systems was evaluated by cell count، analysis of CD34+ expression by flowcytometery، and CFU assay. Expansion of cord blood HSCs was evaluated at three different culture systems at three different times (day3، day 7 and day 10): the medium with cytokine، 2D medium with cytokine + MSCs، and 3D medium with cytokine + MSCs on scaffold. The fold increase of TNC، CFU، and CD34+ cells at day 10 for the former medium was 87 ± 6، 84 ± 8، 11 ± 3، for the second 103 ± 8، 111 ± 9، 14 ± 3، and for the latter 127 ± 10، 170 ± 14، 20 ± 4، respectively. The highest expansion was observed in the 3D co-culture with MSCs after 10 days. The results have demonstrated that the co-culture of CB HSCs with mesenchymal cells could increase the rate of expansion especially in the 3D system.

Micro RNAs are a class of small non-coding RNAs which have been recently shown to play a crucial role in major cellular processes such as development and differentiation through post-transcriptional regulation. The role of these epigenetic elements has been also demonstrated in hematopoietic lineage differentiation and there is a large body of evidence that miR-150 and miR-146a are responsible for T lymphocyte differentiation. Our goal was to examine the effect of miR-150 and miR-146a overexpression in hematopoietic stem cells and its ability to differentiate these cells into a T lymphoid cell. In this experimental study CD133+ stem cells were employed as hematopoietic stem cells and permanent overexpression of miR-150 and miR-146a was established. The differentiation progress was tracked by flowcytometry for lymphocyte markers such as CD4 and CD8. Moreover، expression of miR-146a and miR-150 by RT-PCR and quantitative real time PCR after 7 days was studied. The cells showed T lymphoid characteristics 7 days after transduction. CD4 and especially CD8 expression significantly increased. In conclusion، miR-150 and miR-146a overexpression is an effective factor in T cytotoxic differentiation and they have the ability of directing CD133+ hematopoietic stem cells to express T lymphoid characteristics.

Bone marrow transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming by minimum manipulation، mineralized bone allograft (MBA) scaffold coated by UCB-USSC (unrestricted somatic stem cells) was used to expand CD34+ cells from UCB. In this experimental study، UCB-USSCs were isolated and characterized by morphologic and immunophenotypical analysis. Then they were seeded on MBA (for 3D figuration) and culture plate (for 2D figuration) as a feeder layer. CD34+ cells were isolated from the UCB (by MACS method) and were expanded in 2D and 3D conditions for 3 weeks. At the end، cell count، flow cytometry، cologenic assay، and LTC-IC were done. After 3 weeks، ex vivo expansion of UCB-CD34+ was enhanced 250 ± 13. 2 in 3D. The highest CFC expansion and LTC-IC were observed at day 14. Flow cytometry analysis showed the lowest percentage in 3D culture. USSCs by 3D MBA structure produce the hematopoietic cytokine، extracellular matrix and binding molecules; they also create the intercellular interaction that can be used as a suitable feeder layer. It provides an ex vivo mimicry of bone marrow niche by enhancing surface/volume ratio. This model could be a suitable and alternative environment for HSCs expansion and hematopoiesis.

Heme oxegenase1 (HO-1) is one of the potent cytoprotective factors. The goal of this study was to perform cloning and transient over expression of the human HO-1 gene in mesenchymal stem cells (MSCs) using the adenoviral expression system based on the gateway technology. In order to induce expression of HO-1، A549 cell lines were exposed to UV for 1 hour. The full length cDNA of HO-1 was isolated and cloned into pENTR TOPO/D vector by TOPO cloning reaction. To construct the expression clone، a LR recombination reaction was carried out between the entry clone and destination vector، pAd/CMV/V5-DEST. The recombinant virus was produced in the appropriate mammalian cell line. MSCs were infected by the recombinant virus expressing HO-1. The results showed that human recombinant HO-1 was successfully cloned and the accuracy of the gene and its frame in the vector were confirmed by DNA sequencing. Expression of HO-1 in MSCs was confirmed by RT-PCR and western blot analysis. The results indicated that the expression of HO-1 is transient. Transient expression of human HO-1 gene in MSCs by using adenovirus expression system may be considered as an efficient gene transfer strategy into MSCs in order to promote stem cell therapy.

The correlation between hematopoietic stem cells (HSCs) and the cells comprising the niche especially osteoblast cells is critical for maintaining stem cell activities. Yet little evidence supports the concept that HSCs regulate development of the niche. If true، it may explain why many hematopoietic defects are accompanied by changes in the osseous architecture and provide new therapeutic targets for regulating bone formation. In this exprimental study، we cocultured bone marrow mesenchymal stem cells (MSCs) with umbilical cord blood HSCs in stem span media for 3 days. Then MSCs were differentiated to osteoblast cells by using osteogenesis kit. Evaluation of osteogenic differentiation of MSCs in different days came out to be: expression of osteopontin and osteocalsin (RT-PCR on days 4 and 6)، expression of CD90 (flow cytometry on day 6)، expression of alkaline phosphatase enzyme (alkaline phosphatase staining) and mineralization of MSCs (alizarin red staining on day 10). Our results showed early expression of osteopontin and osteocalsin in HSCs coculture with MSCs. These findings support the role of HSCs in induction of osteoblastic differentiation of MSCs. Decrease in the expression of CD90، higher activation in cell alkaline phosphatase enzyme، and mineralization confirmed the results. In the field of human stem cells، our ex vivo findings demonstrated that HSCs can enhance differentiation of MSCs toward osteoblast cells thereby participating in formation of their niche.

Nowadays، cord blood hematopoietic stem cells (HSCs) are well known as a very valuable source of cells for cell therapy purposes. But unfortunately، the insufficient number of them is a limiting factor for their employment in adult bone marrow transplantation. Cord blood HSCs expansion is an approach for alleviating this problem. It has been shown by several studies during in vivo investigations in mouse models that HOXB4 is one of the most effective tools for inducing HSCs self-renewal and thus HSCs expansion. In this study، we tried to enhance self-renewal of cord blood HSCs by HOXB4 overexpression during ex vivo expansion period. In this fundamental-applied research، HSCs were transducted by lentiviral vectors containing HOXB4. HSCs were then cultured in IMDM medium containing 10% FBS and early acting cytokines (Flt3L، SCF، Tpo) for 8 days. Thereafter، we evaluated HOXB4 expression by RT-PCR. The CD34+ subpopulation were also examined by flow cytometry. We detected a high level of HOXB4 gene expression in HOXB4 transducted HSCs relative to the control. We observed that HOXB4 over-expression dramatically increases CD34+ subpopulation of HSCs. The increase in the number of CD34+ HSCs is an indicator of HSCs self-renewal during ex vivo expansion period. All in all، in this survey we observed that HOXB4 overexpression markedly increases CD34+ HSCs during ex vivo expansion period. This approach can be very effective for overcoming the limited number of cord blood HSCs for cell therapy purposes.

Anemia is one of the most common problems in cancer patients. There is a strong correlation between hemoglobin levels and fatigue and quality of life in these patients. The purpose of this study was to determine the effects of aerobic exercise on erythrocyte indices in cancer patients after autologous hematopoietic stem cell transplantation. In this applied and quasi experimental study، we evaluated thirteen transplant patients and compared them to an age and sex-matched control group (n=13). The intensity of cardio training was 60-70% of maximum heart rate for 4-6 weeks، 20-30 minutes each day. The variables of this study were RBC، Hct and Hb levels. The blood sample was taken in the first and last day of hospitalization. The results of this study showed that aerobic exercise has significant effects on RBC (p=0. 026)، Hct (p=0. 032) and Hb (p=0. 007) levels in cancer patients after autologous hematopoietic stem cell transplantation. These findings suggest that aerobic exercise may lead to an increased production of hematopoietic factors in cancer patients after autologous hematopoietic stem cell transplantation.

Hemoglobinopathies are heterogenic hereditary disorders in which mutations lead to abnormal production of hemoglobin chains، or change in the normal sequence of the hemoglobin amino acids. Thalassemia and sickle cell anemia (SCA) are the most prevalent hemoglobinopathies. Patients with the above mentioned disorders often require hypertransfusion regimens. However، continued blood transfusion may contribute to iron overload and consequent organ deterioration، in addition to viral infections. The aim of this review article is to evaluate recent treatment programs regarding these disorders. This paper is evaluating the newest protocols applied for the treatment of thalassemia and SCA by reviewing 51 references using Ebsco، Elsevier، Pubmed and OVID databases. Blood transfusion and medical treatments may improve the lifestyle or increase the lifespan of patients with hemoglobinopathies. However، hematopoietic stem cell transplantation (HSCT) is presented as the only curative therapy for thalassemia and SCA. On the other hand، HSCT may contribute to complications such as infertility and gonadal failure، especially in women، chronic graft-versus-host disease (GVHD)، and potential secondary malignancies. In spite of the limitations and complications attributed to HSCT، this method is proved to be the most effective way for treatment of hemoglobinopathies. Different cells and novel strategies used to modify transplantation are introduced in this article.