Adipose Derived Stem Cells Affect miR-145 and p53 Expressions of Co-Cultured Hematopoietic Stem Cells.

In this article published in Cell J, Vol 19, No 4, Jan-Mar (Winter) 2018, on pages 654-659, the authors found that Figures 2 and 3 had some errors that accidentally happened during organizing figures. Because of mislabeling of some images and saving them in an incorrect folder, the following figures' legends are corrected. The authors would like to apologies for any inconvenience.

The safety and efficacy of umbilical cord blood mononuclear cells in individuals with spastic cerebral palsy: a randomized double-blind sham-controlled clinical trial.

INTRODUCTION: The current multi-center, randomized, double-blind study was conducted among children with cerebral palsy (CP) to assess the safety and efficacy of umbilical cord blood mononuclear cell (UCB-MNC). We performed the diffusion tensor imaging to assess the changes in the white matter structure. METHODS: Males and females aged 4 to 14 years old with spastic CP were included. Eligible participants were allocated in 4:1 ratio to be in the experimental or control groups; respectively. Individuals who were assigned in UCB-MNC group were tested for human leukocyte antigen (HLA) and fully-matched individuals were treated with UCB-MNCs. A single dose (5 × 106 /kg) UCB-MNCs were administered via intrathecal route in experimental group. The changes in gross motor function measure (GMFM)-66 from baseline to one year after treatment were the primary endpoints. The mean changes in modified Ashworth scale (MAS), pediatric evaluation of disability inventory (PEDI), and CP quality of life (CP-QoL) were also evaluated and compared between groups. The mean changes in fractional anisotropy (FA) and mean diffusivity (MD) of corticospinal tract (CST) and posterior thalamic radiation (PTR) were the secondary endpoints. Adverse events were safety endpoint. RESULTS: There were 72 included individuals (36 cases in each group). The mean GMFM-66 scores increased in experimental group; compared to baseline (+ 9.62; 95%CI: 6.75, 12.49) and control arm (ß: 7.10; 95%CI: 2.08, 12.76; Cohen's d: 0.62) and mean MAS reduced in individuals treated with UCB-MNCs compared to the baseline (-0.87; 95%CI: -1.2, -0.54) and control group (ß: -0.58; 95%CI: -1.18, -0.11; Cohen's d: 0.36). The mean PEDI scores and mean CP-QoL scores in two domains were higher in the experimental group compared to the control. The imaging data indicated that mean FA increased and MD decreased in participants of UCB-MNC group indicating improvements in white matter structure. Lower back pain, headaches, and irritability were the most common adverse events within 24 h of treatment that were related to lumbar puncture. No side effects were observed during follow-up. CONCLUSIONS: This trial showed that intrathecal injection of UCB-MNCs were safe and effective in children with CP. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov ( NCT03795974 ).

MicroRNA Microarray Profiling during Megakaryocyte Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells.

OBJECTIVES: In order to clarify the role of microRNAs (miRNA) in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133+ cells. MATERIALS AND METHODS: In this experimental study, human CD133+ hematopoietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis. RESULTS: A total of 10 and 14 miRNAs were upregulated (e.g. miR-1246 and miR-148-a) and down-regulated (e.g. miR- 551b and miR-10a) respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis. CONCLUSIONS: We conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment.

Adipose Derived Stem Cells Affect miR-145 and p53 Expressions of Co-Cultured Hematopoietic Stem Cells.

OBJECTIVES: Umbilical cord blood is used for transplantation purposes in regenerative medicine of hematological disorders. MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as cancer development and incidence. There is a new relation between p53 (tumor suppressor gene) and miR-145 (suppressor of cell growth) upregulation. In this study, we have assessed how adipose-derived stem cells (ADSCs) affect the expansion of hematopoietic stem cells (HSCs), as well as miR-145 and p53 expressions. MATERIALS AND METHODS: In this experimental study, we cultured passage-3 isolated human ADSCs as a feeder layer. Flow cytometry analysis confirmed the presence of ADSC surface markers CD73, CD90, CD105. Ex vivo cultures of cordblood CD34+ cells were cultured under the following 4 culture conditions for 7 days: i. Medium only supplemented with cytokines, ii. Culture on an ADSCs feeder layer, iii. Indirect culture on an ADSCs feeder layer (Thin Cert™ plate with a 0.4 µm pore size), and iv. Control group analyzed immediately after extraction. Real-time polymerase chain reaction (PCR) was used to determine the expressions of the p53 and miR-145 genes. Flow cytometry analysis of cells stained by annexin V and propidium iodide (PI) was performed to detect the rate of apoptosis in the expanded cells. RESULTS: ADSCs tested positive for mesenchymal stem cell (MSC) markers CD105, CD90, and CD73, and negative for HSC markers CD34 and CD45. Our data demonstrated the differentiation potential of ASCs to osteoblasts by alizarin red and alkaline phosphatase staining. MTT assay results showed a higher proliferation rate of CD34+cells directly cultured on the ADSCs feeder layer group compared to the other groups. Direct contact between HSCs and the feeder layer was prevented by a microporous membrane p53 expression increased in the HSCs group with indirect contact of the feeder layer compared to direct contact of the feeder layer. p53 significantly downregulated in HSCs cultured on ADSCs, whereas miR-145 significantly upregulated in HSCs cultured on ADSCs. CONCLUSIONS: ADSCs might increase HSCs proliferation and self-renewal through miR-145, p53, and their relationship.

Different Methylation Patterns of RUNX2, OSX, DLX5 and BSP in Osteoblastic Differentiation of Mesenchymal Stem Cells.

OBJECTIVE: Runt-related transcription factor 2 (RUNX2) and osterix (OSX) as two specific osteoblast transcription factors and distal-less homeobox 5 (DLX5) as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein (BSP), osteopontin and collagen type Iα1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells (MSCs). MATERIALS AND METHODS: In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR (MSP). Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction (PCR). RESULTS: MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. CONCLUSION: The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role.

G-CSF induces up-regulation of CXCR4 expression in human hematopoietic stem cells by beta-adrenergic agonist.

Introduction C-X-C chemokine receptor type 4/stromal-derived factor-1 (CXCR4/SDF-1) axis dynamically mediates hematopoietic stem cell trafficking in the bone marrow (BM). Granulocyte colony-stimulating factor (G-CSF) as the most effective mobilizing agent induces SDF-1 secretion from BM stromal cells into circulation that recruit CXCR4+ cells such as hematopoietic stem cells (HSCs) into circulation. However, the direct effect of G-CSF on CXCR4 expression of HSC remains unknown. The nervous system regulates HSC migration with effecting on adrenergic receptors. On the other hand, interaction of G-CSF and catecholamines has been demonstrated; hence, we examined the direct effect of G-CSF and catecholamine on CXCR4 expression. Material and methods After enrichment of CD34+ HSCs from the cord blood with magnetic-activated cell sorting (MACS), these cells were exposed to G-CSF (100 ng/ml), epinephrine (10 µM), isoproterenol (10 µM), and propranolol (1 µM) separately and together. Results Our results showed that G-CSF have no direct effect on CXCR4 expression on human CD34+ cells in vitro and treating HSCs with epinephrine leads to significantly increased CXCR4 in 1, 3, and 5 hours. Epinephrine and G-CSF-induced up-regulation of CXCR4 mRNA is dependent on beta receptors, so incubation of HSCs with propranolol led to inhibition of such increased expression. In addition, isoproterenol and agonist of beta receptors would significantly increase the expression of CXCR4 approximately 4- and 12-fold after 1-hour incubation, respectively. Discussion Co-stimulation of enriched HSCs with G-CSF and isoproterenol resulted to a further enhanced CXCR4 levels. In general, G-CSF-induced CXCR4 expression is the indirect mechanism and is specifically regulated through beta-adrenergic receptors.

Expansion of CD133(+) Umbilical Cord Blood Derived Hematopoietic Stem Cells on a Biocompatible Microwells.

Umbilical cord Blood (UCB) as a source of Hematopoietic Stem/Progenitor cells (HSPCs) used for Umbilical cord blood transplantation (UCBT). The main obstacle in application of this source as an appropriate source of HSPCs is low volume of this product. So ex vivo expansion of these cells in a microenvironment which mimic body condition is important. In current study we designed biocompatible microwells in which collagene type I is coated by softlitography method. Our findings designated that in 3-Dimensional (3D) microenvironment CD133(+) UCB derived HSC expanded significantly compared to 2-Dimensional (2D) microenvironment.