Differential Expression of the hTERT Gene in Umbilical Cord-Derived Mesenchymal Stem Cells Cocultured with B Cell Precursor Leukemia Cell Microparticles or CD41+/CD61+ Platelet Microparticles.

Several investigations are being done to increase the short lifetime of mesenchymal stem cells (MSCs). One of the crucial genes involved in the immortalization of MSCs, hTERT (human telomerase reverse transcriptase), is activated in most publications using viral-based techniques. In this work, we investigated the use of platelet-derived (PMPs) and B cell precursor leukemia-derived microparticles as a nonviral method to trigger and compare the expression of the hTERT gene in MSCs. MSCs were extracted from the umbilical cord for the current investigation and identified using a flow cytometry approach and an inverted microscope. The Nalm-6 cell line and platelet concentrate were used to isolate microparticles (MPs). MSCs and MPs were cocultured for 14 days at 25-, 50-, and 100 µg/ml concentrations. qRT-PCR was used to research the expression of the hTERT gene. SPSS 26.0's t test was used to compare the outcomes. After coculture with platelet MPs, MSCs had higher levels of hTERT gene expression than the control group. In contrast, this gene's expression was concurrently decreased in MSCs exposed to MPs generated from Nalm-6. We demonstrated that following 14-day treatment, PMP significantly boosted the hTERT gene expression in MSCs, while the Nalm-6 MPs lowered the gene expression. However, additional studies are necessary due to the stability of hTERT gene expression and the immortalization of MSCs following exposure.

An open-label phase 1 clinical trial of the allogeneic side population adipose-derived mesenchymal stem cells in SMA type 1 patients.

INTRODUCTION: Spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder of alpha motor neurons of spinal cord associated with progressive muscle weakness and hypotonia, is the most common genetic cause of infant mortality. Although there is few promising treatment for SMA, but the field of translational research is active in it, and stem cell-based therapy clinical trials or case studies are ongoing. Combination of different therapeutic approaches for noncurative treatments may increase their effectiveness and compliance of patients. We present a phase 1 clinical trial in patients with SMA1 who received side population adipose-derived mesenchymal stem cells (SPADMSCs). METHODS: The intervention group received three intrathecal administrations of escalating doses of SPADMSCs and followed until 24 months or the survival time. The safety analysis was assessed by controlling the side effects and efficacy evaluations performed by the Hammersmith Infant Neurological Examination (HINE), Ballard score, and electrodiagnostic (EDX) evaluation. These evaluations were performed before intervention and at the end of the follow-up. RESULTS: The treatment was safe and well tolerated, without any adverse event related to the stem cell administration. One of the patients in the intervention group was alive after 24 months of study follow-up. He is a non-sitter 62-month-old boy with appropriate weight gain and need for noninvasive ventilation (NIV) for about 8 h per day. Clinical scores, need for supportive ventilation, and number of hospitalizations were not meaningful parameters in the response of patients in the intervention and control groups. All five patients in the intervention group showed significant improvement in the motor amplitude response of the tibial nerve (0.56mV; p: 0.029). CONCLUSION: This study showed that SPADMSCs therapy is tolerable and safe with promising efficacy in SMA I. Probably same as other treatment strategies, early intervention will increase its efficacy and prepare time for more injections. We suggest EDX evaluation for the follow-up of treatment efficacy.

Comparison of the effects of intramyocardial and intravenous injections of human mesenchymal stem cells on cardiac regeneration after heart failure.

OBJECTIVES: Existing studies have demonstrated that intravenous and intramyocardial-administrated mesenchymal stem cells (MSCs) lead to tissue repair after cardiac disorders. We compared the efficiency of both administration methods. MATERIALS AND METHODS: A rat model of isoproterenol-induced heart failure (ISO-HF) was established to compare the effects of intravenous and intramyocardial-administrated MSCs on cardiac fibrosis and function. The animals were randomly assigned into six groups: i) control or normal, ii) ISO-HF (HF) iii) ISO-HF rats treated with intramyocardial administration of culture medium (HF+IM/CM), iv) ISO-HF rats treated with intravenous administration of culture medium ( HF+IV/CM), v) ISO-HF rats treated with intravenous administration of MSCs (HF+IV/MSCs), vi) ISO-HF rats treated with intramyocardial administration of MSCs ( HF+IM/MSCs). Cultured MSCs and culture medium were administrated at 4 weeks after final injection of ISO. Heart function, identification of MSCs, osteogenic differentiation, adipogenic differentiation, cardiac fibrosis and tissue damage were evaluated by echocardiography, flow-cytometery, von Kossa, oil red O, Masson's trichrome and H & E staining, respectively. RESULTS: Both intravenous and intramyocardial MSCs therapy significantly improved heart function and reduced cardiac fibrosis and tissue damage (P<0.05), whereas the cultured medium had no beneficial effects. CONCLUSION: In sum, our results confirm the validity of both administration methods in recovery of HF, but more future research is required.

Platelets Apoptosis and Clearance in The Presence of Sodium Octanoate during Storage of Platelet Concentrate at 4˚C.

OBJECTIVE: Platelet (PLT) storage at 4˚C has several benefits, however, it is accompanied by increased clearance of PLTs after transfusion. In this study, we evaluated the potential of sodium octanoate (SO) for reducing apoptosis and clearance rate of PLTs after long-term storage in cold. MATERIALS AND METHODS: In this experimental study, PLT concentrates (PCs) were stored for 5 days under the following three conditions: 20-24˚C, 4˚C, and 4˚C in the presence of SO. To measure the viability of PLTs, the water-soluble tetrazolium salt (WST-1) assay was performed. Phosphatidylserine (PS) exposure was determined on PLTs using flow cytometry technique. The amount of human active caspase-3 was determined in PLTs using an enzyme-linked immunosorbent assay. Additionally, the amount of PLT ingestion or clearance was determined by using HepG2 cell line. RESULTS: The viability was higher in the SO-treated PLTs compared to the other groups. The level of PS exposure on PLTs was lower in the SO-treated PLTs compared to the other groups. The amount of active caspase-3 increased in all groups during 5-day storage. The highest increase in the amount of caspase-3 levels was observed at cold temperature. However, PLTs kept at 4˚C in the presence of SO had a lower amount of active caspase-3 compared to PLTs kept at 4˚C. The amount of PLTs removal by HepG2 cells was increased for 4˚C-kept PLTs but it was lower for PLTs kept at 4˚C in the presence of SO but, the differences were not significant (P>0.05). CONCLUSION: SO could partially moderate the effects of cold temperature on apoptosis and viability of platelets. It also decreases the ingestion rate of long-time refrigerated PLTs in vitro. Further studies using higher numbers of samples are required to demonstrate the effect of SO on reducing the clearance rate of PLTs.

Procoagulant Activity of Red Blood Cell-Derived Microvesicles during Red Cell Storage.

BACKGROUND: Red blood cells (RBCs) undergo structural and biochemical alterations during storage which are collectively called RBC storage lesion and cause a decrease in RBC recovery and survival. During storage, erythrocytes release an increasing number of microvesicles (MVs) that have key roles in biological processes. We aimed to investigate the procoagulant activity (PCA) of RBC-derived MVs during storage. METHODS: 20 packed RBCs were stored for up to 42 days. Samples were taken at seven different times and evaluated for the presence of RBC-MVs. MVs were separated, and following filtration flow cytometry was used to characterize RBC-MVs based on the expression of glycophorin A (Gly.A) and annexin V (AnnV) antigens. The coagulant activity of RBC-MVs was tested by clotting time (CT) and PCA assays. Results were compared before and after filtration. RESULTS: Flow cytometry revealed a 17.6-fold increase in RBC-MVs after 6 weeks of storage. Significant correlations were found between AnnV+ MVs and PCA (r = 0.96; p < 0.001), and CT (r = -0.77; p < 0.001) which was associated with increased PCA and shortened CT with RBC aging. Filtration of samples efficiently removed MVs (p < 0.001) and also reduced in vitro PCA of MVs (p < 0.001). CONCLUSION: RBC-MVs are procoagulant (particularly AnnV+ MVs) Reduction of MVs from RBC concentrates may reduce the risk of transfusion-induced thrombotic complications.

Conditioned medium obtained from human amniotic mesenchymal stem cells attenuates focal cerebral ischemia/reperfusion injury in rats by targeting mTOR pathway.

Conditioned medium obtained from human amniotic mesenchymal stem cells (hAMSC-CM) was recently shown to have many antioxidant, antiapoptotic and proangiogenic growth factors. The present study was performed to investigate whether protective effects of hAMSC-CM against focal cerebral ischemia/ reperfusion (I/R) injury is associated with modulation of the mammalian target of rapamycin (mTOR) pathway. A rat model of middle cerebral artery occlusion (MCAO) was created and the animals were divided into three groups including sham, MCAO and MCAO + hAMSC-CM. Drug was administrated immediately after cerebral reperfusion (i.v). The expressions of mTOR, p-mTOR and LC3 were measured using Western blotting and real time-PCR, respectively. Apoptosis and neuronal loss were determined using TUNEL and Nissl staining, respectively. Infarct volume and the blood-brain barrier (BBB) damage were evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans Blue (EB) uptake, respectively. Compared with sham, significant infarct volume, apoptotic cell death, and neuronal loss were found in MCAO rats that reversed by hAMSC-CM (P < 0.05). Likewise, MCAO rats exhibited increased mRNA level of light-chain 3 (LC3) and the LC3II/LC3I ratio as well as decreased expression level of p-mTOR that reversed by hAMSC-CM (P < 0.05). There were no significant differences in the expression of total mTOR among the experimental groups. In summary, our results demonstrate that hAMSC-CM gives rise to neuroprotection following ischemic stroke by restoring mTOR activity and inhibiting autophagy.

High Glucose Affects the Cytotoxic Potential of Rapamycin, Metformin and Hydrogen Peroxide in Cultured Human Mesenchymal Stem Cells.

BACKGROUND: Oxidative stress and chronic hyperglycemia are two major side effects of type 2 diabetes affecting all cell types including mesenchymal stem cells (MSCs). As a cell therapy choice, understanding the behavior of MSCs will provide crucial information for efficient treatment. METHODS: Placental mesenchymal stem cells were treated with various concentrations of glucose, metformin, rapamycin, and hydrogen peroxide to monitor their viability and cell cycle distribution. Cellular viability was examined via the MTT assay. Cell cycle distribution was studied by propidium iodide staining and apoptosis was determined using Annexin Vpropidium iodide staining and flow cytometry. Involvement of potential signaling pathways was evaluated by Western blotting for activation of Akt, P70S6K, and AMPK. RESULTS: The results indicated that high glucose augmented cell viability and reduced metformin toxic potential. However, the hydrogen peroxide and rapamycin toxicities were exacerbated. CONCLUSION: Our findings suggest that high glucose concentration has a major effect on placental mesenchymal stem cell viability in the presence of rapamycin, metformin and hydrogen peroxide in culture.

microRNA expression profiles in two- and three-dimensional culture conditions of human-umbilical-cord blood-derived CD34+ cells.

Human umbilical cord blood (HUCB) is a suitable source of hematopoietic stem cells (HSCs) for therapeutic transplantation. Different approaches have been used to expand the number of HSCs to increase the rate of HSC transplantation success in patients, such as using different cocktails of cytokines, feeder cell layers, and biocompatible scaffolds. microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. They play crucial roles in hematopoiesis including stem cell proliferation, differentiation, stemness, and self-renewal properties. Here, we studied the UCB-derived CD34+ cell expansion and the miRNA signatures of CD34+ cells on two- and three-dimensional (2D and 3D) culture conditions. We successfully expanded the UCB-derived CD34+ cells in both liquid culture (2D) and on aminated polyethersulfone nanofiber scaffolds (3D). Next, we identified the miRNA signature of CD34+ cells and their target genes. We found 58 dysregulated miRNAs in 3D culture condition and 34 dysregulated miRNAs in 2D culture condition when compared to the freshly isolated CD34+ cells. Various types of target genes were also predicted in both conditions using two online databases.

Overexpression of SMN2 Gene in Motoneuron-Like Cells Differentiated from Adipose-Derived Mesenchymal Stem Cells by Ponasterone A.

Cell therapy and stem cell transplantation strategies have provided potential therapeutic approaches for the treatment of neurological disorders. Adipose-derived mesenchymal stem cells (ADMSCs) are abundant adult stem cells with low immunogenicity, which can be used for allogeneic cell replacement therapies. Differentiation of ADMSCs into acetylcholine-secreting motoneurons (MNs) is a promising treatment for MN diseases, such as spinal muscular atrophy (SMA), which is associated with the level of SMN1 gene expression. The SMN2 gene plays an important role in MN disorders, as it can somewhat compensate for the lack of SMN1 expression in SMA patients. Although the differentiation potential of ADMSCs into MNs has been previously established, overexpression of SMN2 gene in a shorter period with a longer survival has yet to be elucidated. Ponasterone A (PNA), an ecdysteroid hormone activating the PI3K/Akt pathway, was studied as a new steroid to promote SMN2 overexpression in MNs differentiated from ADMSCs. After induction with retinoic acid, sonic hedgehog, forskolin, and PNA, MN phenotypes were differentiated from ADMSCs, and immunochemical staining, specific for ß-tubulin, neuron-specific enolase, and choline acetyltransferase, was performed. Also, the results of real-time PCR assay indicated nestin, Pax6, Nkx2.2, Hb9, Olig2, and SMN2 expression in the differentiated cells. After 2 weeks of treatment, cultures supplemented with PNA showed a longer survival and a 1.2-fold increase in the expression of SMN2 (an overall 5.6-fold increase; *P ≤ 0.05), as confirmed by the Western blot analysis. The PNA treatment increased the levels of ChAT, Isl1, Hb9, and Nkx2 expression in MN-like cells. Our findings highlight the role of PNA in the upregulation of SMN2 genes from MSC-derived MN-like cells, which may serve as a potential candidate in cellular therapy for SMA patients.

The membrane mesenchymal stem cell derived conditioned medium exerts neuroprotection against focal cerebral ischemia by targeting apoptosis.

OBJECTIVE: The mesenchymal stem cells derived from human amniotic membrane have the ability to secrete and release some factors that can promote the repair of damaged tissues. This secretome contains proteins and factors that reduce apoptosis and increase angiogenesis in the ischemia/reperfusion models. The present study was conducted to determine whether this secretome provides protection against transient focal cerebral ischemia. MATERIALS AND METHODS: A rat model of focal cerebral ischemia was established through middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion. The amniotic mesenchymal stem cells-conditioned medium (AMSC-CM) at the dose of 0.5 µl was injected intracerebroventriculary (ICV) 30 min after reperfusion. Infarct volume, brain edema, neurobehavioral functions, and blood brain barrier (BBB) integrity were assessed 24 h after reperfusion. Neuronal loss and expression of caspase-3, Bax and Bcl-2 in motor cortex were evaluated by nissl staining and immunohistochemistry assay respectively. RESULTS: ICV administration of AMSC-CM markedly reduced infarct volume, brain edema and the evans blue penetration rate compared with MCAO group (P < 0.05). Additionally, post-treatment with AMSC-CM significantly reduced neuronal loss, neurological motor disorders and expression of caspase-3, Bax and Bcl-2 in motor cortex compared with MCAO group (P < 0.05). CONCLUSION: The results of this study indicate that treatment with AMSC-CM improves the pathological effects in the acute phase of cerebral ischemia. These findings establish a substantial foundation for stroke therapy and future research.

Interaction of Platelet-Derived Microparticles with a Human B-Lymphoblast Cell Line: A Clue for the Immunologic Function of the Microparticles.

BACKGROUND: Platelets are blood cells with extensive capabilities in hemostasis. They also play a central role in the development of innate and adaptive immune responses. Little information exists about the immunostimulatory role of platelet-derived microparticles (Plt-MPs). To further elucidate this issue, we conducted this study using the B-lymphoblast cell line 'Daudi' as an available surrogate cell line for peripheral blood B lymphocytes. This cell line does not produce immunoglobulins (Igs) and has low expression of activation markers. METHODS: Plt-MPs were isolated from platelet concentrate (PC) using a multi-step centrifugation method. Daudi cells were treated with Plt-MPs in the culture medium while no treatment was given to the control cells. During 5-day co-culture, Daudi cells were evaluated for the Ig production and the expression of the cell surface markers CD86, CD27, and IgD. RESULTS: An increase was observed for the production of IgG and the expression of CD27 and CD86 on Daudi cells in response to Plt-MPs, whereas the IgD level was decreased. The response of Daudi cells was dependent on the concentration of Plt-MPs and the time of their isolation from PCs during storage. The differences of the variables were significant between the treatment and control groups. CONCLUSION: Plt-MPs could induce the activation and differentiation of immortalized cells of B-cell origin. Thus it is conceivable that Plt-MPs may play a significant role as immortalized cell activators in human monoclonal antibody technology in near future.

Harmine, a Novel DNA Methyltransferase 1 Inhibitor in the Leukemia Cell Line.

DNA methylation followed by tumor suppressor gene repression plays a critical role in the leukemia development. So, DNA methyl transferase inhibitors have great importance in treatment of theses malignancies. Harmine, A beta carboline alkaloid derivative of Peganum harmala, had shown anti- proliferative effects on leukemic cell line. This study aimed to evaluate the effect of Harmine on DNMT1 (DNA methyl transferase 1) expression in a leukemic cell line. Cell proliferation and cell cycle analysis were studied in NB4 cell line after treatment with Harmine for 72 h. DNMT1 expression in treated cells was analyzed by real time PCR. Tumor suppressor gene hypometylation and reactivation was evaluated via MSP analysis and also real time PCR. Harmine reduced cell proliferation in NB4 cell line in a time and dose-dependent manner. 102 µg/ml of Harmine was increased amount of cells in G1 Phase of cell cycle (p < 0.05). Anti proliferative doses of Harmine, has suppressed DNMT1 gene in NB4 cell line. Down-regulated DNMT1 induced p15 tumor suppressor promoter hypomethylation and reactivation. Our data indicate that Harmine can be considered as a potential treatment for AML (Acute Myeloid Leukemia), and future studies are required to test the clinical efficacy of Harmine-whether used as a single agent or as an adjuvant-for AML treatment.

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs.

BACKGROUND: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. METHODS: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. RESULTS: Maximum CB-CD34+ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34+ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34+ and CD34- cells in the cytokine co-culture system was observed. CONCLUSION: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

Evaluation of Plasma Platelet Microparticles in Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Platelet microparticles (PMPs) have a procoagulant activity about 50-100 times greater than active platelets due to high expression of negatively charged phospholipids on their surfaces. In this study, we evaluated microparticle immunophenotyping and also plasma PMPs level in patients with Thrombotic Thrombocytopenic Purpura (TTP) in Southern Iran. METHOD: We had two study groups: 15 TTP patients and 15 healthy control group and PMPs from platelet concentrate (PC) at the 5th day of storage. Microparticles were prepared in two steps, by low and high centrifugation followed by size confirmation via 'Dynamic Light Scattering (DLS)' Zetasizer. Immunophenotyping of PMPs was done via flow cytometry, using a FACS Calibur flow cytometer (BD, USA). PMPs counts were obtained using Partec-cyflow and Polysciences Microbeads (1 micron in diameter). Results were analyzed using FlowJo 7.6 (Treestar, USA) and Partec FlowMax software. RESULTS: Our results showed that the majority of microparticles in TTP patients and normal individuals were PMPs and also demonstrated that the plasma PMPs level in TTP patients was higher than the normal control group (P-value<0.001). CONCLUSION: It seems that elevated PMPs level in TTP patients could be related to thrombotic events. Nevertheless, more studies are needed to confirm these results.

Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-ß receptorII in co-culture with bone marrow mesenchymal stromal cells.

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-ß signaling pathway through reduction of TGF-ßRII expression. MATERIALS AND METHODS: CD34(+) cells were isolated from UCB and transfected by SiRNA targeting TGF-ßRII mRNA. CD34(+) cells were expanded in four culture media with different conditions, including 1) expansion of CD34(+) cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-ßRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-ßRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture. RESULTS: The fold increase in CD34(+) cells, TNCs, and colony numbers (71.8±6.9, 93.2±10.2 and 128±10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0day) before expansion was statistically significant (P<0.05). CONCLUSION: The results showed that transfection of CD34(+) cells with SiRNA targeting TGF-ßRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion.

Apoptosis, DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells.

Expansion of umbilical cord blood-derived CD34(+) cells can potentially provide them in numbers sufficient for clinical applications in adult humans. In this study apoptosis rate of expanded cells, mRNA expression and promoter methylation status of DAPK1 were evaluated during cord blood hematopoietic stem cell (CB-HSC) ex vivo expansion using cytokines and a co-culture system with mesenchymal stromal cells (MSCs). Ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs feeder layer without cytokine. Total number of cells, CD34(+) cells and colony forming unit assay were performed during expansion. Flow cytometric analysis by propidium iodide was performed to detection of apoptosis rate in expanded cells. Methylation status of the DAPK1 gene promoter was analyzed using methylation specific PCR, and DAPK1 mRNA expression was evaluated by real time-PCR. Maximum CB-CD34(+) cells expansion was observed in day 10 of expansion. The highest apoptosis rate was observed in cytokine culture without feeder layer that showed significant difference with co-culture condition. The data showed that ex vivo expansion of CD34(+) cells in all three culture conditions after 10 days resulted in, significant increased expression of DAPK1, also a significant difference between co-culture without cytokine and two other cytokine culture was observed (p < 0.01). DAPK1gene promoter of expanded CD34(+) cells at days 5, 10 and 14 of culture remained in unmethylated form similar to fresh CD34(+) cells. Expression of DAPK1 in hematopoietic cells was increased during 10 days expansion of CD34(+) cells. Also no methylation of DAPK1 promoter was observed; otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Evaluation of human cord blood CD34+ hematopoietic stem cell differentiation to megakaryocyte on aminated PES nanofiber scaffold compare to 2-D culture system.

CONTEXT: Recently, umbilical cord blood (UCB) has been recognized as a suitable potential source of hematopoietic stem/progenitor cells (HSPCs) for transplantation. Lengthy thrombocytopenia after UCB transplantation is a major problem because of insufficient megakaryocyte (Mk) progenitors, which results in delayed platelet recovery. Frequent allogenic platelet transfusion leads to resistance to platelet units and higher risk of transmission of pathogenic agent. OBJECTIVE: Ex vivo expansion of HSPCs and their differentiation to Mk progenitors on aminated PES nanofiber could lead to faster platelet recovery after UCB transplantation. MATERIALS AND METHODS: CD34 cells were positively enriched using the MidiMACS system. CD34(+) cells were seeded onto conventional culture and aminated PES scaffold. The proliferation of CD34(+) cells, and also their differentiation into Mk progenitors, were evaluated. We used the flow cytometric method for analyzing CD41 and CD61 markers and real-time PCR for the expression level of transcription factors, as NF-E2 and GATA-1. RESULTS: This study indicated increased CD34(+) cell population in aminated PES compared to the conventional system. After differentiation, the amount of CD41/CD61-expressing cells and the quantity of NF-E2 expression level increased in the aminated PES versus the 2-D system. The quantity of GATA-1 expression level was reduced on CD41/CD61(+) cells compared to CD34(+) cells, with no difference between the aminated PES and the conventional system. DISCUSSION: Aminated PES nanofiber could have more effect on the proliferation of CD34(+) cells and Mk differentiation than the conventional culture. CONCLUSION: Injection of the expanded cells and differentiated Mk progenitors, along with the transplantation of UCB stem cells might accelerate recovery of platelets and decrease the period of thrombocytopenia after transplantation.

The Immunosuppressive Activity of Amniotic Membrane Mesenchymal Stem Cells on T Lymphocytes.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane (AM) are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells (hAM-MSCs). The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro. METHODS: Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin (PHA). Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-γ was examined by ELISA method. Additionally, the expression of activation markers (CD38, HLA-DR) was studied on T lymphocytes by flow cytometry technique. RESULTS: It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes (p≤0.01) and significantly decrease the production of IFN-γ by T cells (p<0.05). hAM-MSCs also down regulated the expression of activation markers on the surface of T lymphocytes, CD38 and HLA-DR. The difference was significant between the case and control samples (p<0.05). All the comparisons were carried out between the case (Tcell+PHA+hAM-MSCs) and control (Tcell+PHA) groups. CONCLUSION: In conclusion, hAM-MSCs could inhibit the (mitogen-activated) T cells even in the absence of blood monocytes. Besides, hAM-MSCs-mediated inhibition of T lymphocytes was combined with down regulation of activation markers.

Biological parameters influencing the human umbilical cord-derived mesenchymal stem cells' response to retinoic acid.

Human umbilical cord-derived mesenchymal stem cells (HUCMSCs) are multipotent fetal stem cells that differentiate into various cell lineages. In recent years, they have gained attention for therapeutic applications but very little is known about their sensitivity to chemical agents such as widely used retinoic acid (RA). As a morphogen inducing differentiation of mesenchymal stem cells, RA has for a long time been known to be a potent teratogen promoting craniofacial and limb abnormality in vertebrate embryos. Here, using MTT assay and EB/AO staining as well as TUNEL assay we show that RA in a concentration-dependent manner induces apoptosis through upregulating Caspase expression and increasing Bax/Bcl2 ratio. Moreover, different biological parameters such as initial time seeding, cell density, passage number and duration of RA treatment play a major role in HUCMSCs cytotoxic response to this agent.

The immunophenotype of bone marrow lymphocytes in children with immune thrombocytopenic purpura.

PURPOSE: Primary immune thrombocytopenic purpura (ITP), caused by immune system dysfunction, is recognized as the leading cause of thrombocytopenia in pediatric population. Nonetheless, inadequate studies have been performed on bone marrow immunophenotyping of children with ITP. In this study, we aimed to investigate the immunophenotype of bone marrow lymphocytes in these children. PATIENTS AND METHODS: Between 2008 and 2012, 35 children with ITP and 26 age and sex matched healthy controls were recruited. All participants underwent bone marrow aspiration. Appropriate B-cell, T-cell, and myeloid lineage monoclonal antibodies were employed to determine the immunophenotype of these patients. RESULTS: CD10, CD19, and CD20, all indicative of premature B-cell markers, were significantly greater in children with ITP. CD22, mainly expressed on mature B cells was slightly, but not significantly reduced in the patients' group (P = .42). On the other hand, T cell markers including CD2, CD3, CD5, and CD7 were underexpressed. CD33, a specific marker for myeloid lineage, was underexpressed in the patients' group (5.6 ± 4.7 vs. 12.9 ± 7.3, P < .001). Noteworthy, the immunophenotype did not significantly differ between acute and persistent cases. CONCLUSION: Overall, a phenotype characterized by increased pre-B-cell markers along with decreased T cell immunophenotypic markers was observed in bone marrow lymphocytes of children with ITP in the present study. Further larger scale studies are recommended to confirm our findings, as precise mapping of the immunophenotype of lymphocytes in these patients would pave the road to improved diagnosis and treatment.