Preclinical assessments of safety and tumorigenicity of very high doses of allogeneic human umbilical cord mesenchymal stem cells.

Human umbilical cord-mesenchymal stem cells (hUC-MSCs) have been widely investigated as a new therapeutic agent to treat injuries and inflammatory-mediated and autoimmune diseases. Previous studies have reported on the safety of low-dose infusion of hUC-MSCs, but information on the cell behaviour at higher doses and frequency of injection of the cells remains uncertain. The aim of the present study was to demonstrate the safety and efficacy of hUC-MSCs by Cytopeutics® (Selangor, Malaysia) from low to an extremely high dose in different monitoring periods in healthy BALB/c mice as well as assessing the tumorigenicity of the cells in B-NDG SCID immunocompromised mice. Umbilical cord from two healthy human newborns was obtained and the isolation of the hUC-MSCs was performed based on previous established method. Assessment of the cells at different doses of single or multiple administrations was performed on healthy BALB/c mice in dose range finding, sub-acute (7 d and 28 d) and sub-chronic periods (90 d). Tumorigenicity potential of Cytopeutics® hUC-MSCs was also evaluated on B-NDG immunocompromised mice for 26 wk. Single or multiple administrations of Cytopeutics® hUC-MSCs up to 40 × 106 cells per kilogramme of body weight (kg BW) were found to have no adverse effect in terms of clinical symptoms, haematology and other laboratory parameters, and histology examination in healthy BALB/c mice. hUC-MSCs were also found to reduce pro-inflammatory cytokines (IL-6 and TNF-α) in a dose-dependent manner. No sign of tumor formation was observed in B-NDG mice in the 26-wk tumorigenicity assessment. Single or multiple administration of allogenic Cytopeutics® hUC-MSCs was safe even at very high doses, is non-tumorigenic and did not cause adverse effects in mice throughout the evaluation periods. In addition, Cytopeutics® hUC-MSCs exhibited immunomodulatory effect in a dose-dependent manner.

Revealing the potential role of hsa-miR-663a in modulating the PI3K-Akt signaling pathway via miRNA microarray in spinal muscular atrophy patient fibroblast-derived iPSCs.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder due to deletion or mutation of survival motor neuron 1 (SMN1) gene. Although survival motor neuron 2 (SMN2) gene is still present in SMA patients, the production of full-length survival motor neuron (SMN) protein is insufficient owing to missing or mutated SMN1. No current disease-modifying therapies can cure SMA. The aim of this study was to explore microRNA (miRNA)-based therapies that may serve as a potential target for therapeutic intervention in delaying SMA progression or as treatment. The study screened for potentially dysregulated miRNAs in SMA fibroblast-derived iPSCs using miRNA microarray. Results from the miRNA microarray were validated using quantitative reverse transcription polymerase chain reaction. Bioinformatics analysis using various databases was performed to predict the potential putative gene targeted by hsa-miR-663a. The findings showed differential expression of hsa-miR-663a in SMA patients in relation to a healthy control. Bioinformatics analysis identified GNG7, IGF2, and TNN genes that were targeted by hsa-miR-663a to be involved in the PI3K-AKT pathway, which may be associated with disease progression in SMA. Thus, this study suggests the potential role of hsa-miR-663a as therapeutic target for the treatment of SMA patients in the near future.

Effects of house-cultivated edible bird's nest on immunoglobulin and cytokine release in vitro.

Background and Aim: Edible bird's nest (EBN) is known as the "Caviar of the East" because of its high nutritional and medicinal values. Nevertheless, its effect on human immunity is yet to be explored. This study examined the effects of EBN's aqueous extract (EBNE) on human immunity through the modular immune in vitro construct (MIMIC) model consisting of peripheral tissue equivalent (PTE) and lymphoid tissue equivalent (LTE) modules. Materials and Methods: One hundred twenty mL of full blood was obtained from four healthy human volunteers. The human immune system was simulated using an in vitro model, called MIMIC. Under EBNE treatment, monocyte transendothelial migration through reversed endothelial layers was observed. Using PTE and LTE modules, monocytes were differentiated into dendritic cells with lipopolysaccharide, then co-cultured with T- and B-cells for cytokine and immunoglobulin (Ig) production. The human cytokine array G2000 and quantitative human Ig isotyping array were used to identify the cytokine profile and Ig isotypes, respectively. Results: IgE, IgA, and IgG3 levels were significantly raised by EBNE. These cytokines, including brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell line-derivative neurotrophic factor, insulin-like growth factor 1, and insulin-like growth factor binding protein 4, were generated. Conclusion: For the first time, this work uses a MIMIC model to illustrate the impact of EBNE on human immune response. This new understanding of EBN's immunoregulatory effect allows for further exploration of how EBN interacts with the human immune system.

Dynamic tracking of human umbilical cord mesenchymal stem cells (hUC-MSCs) following intravenous administration in mice model.

Introduction: In the past decades, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have sparked interest in cellular therapy due to their immunomodulatory properties. Nevertheless, the fate of hUC-MSCs in the body remains poorly understood. This study aimed to investigate the biodistribution, homing and clearance of systemically administered hUC-MSCs in healthy BALB/c mice model. Methods: hUC-MSCs were labelled with GFP-Luc2 protein, followed by characterisation with flow cytometry. Upon intravenous infusion of transduced hUC-MSCs into the healthy BALB/c mice, the cells were dynamically monitored through the bioluminescent imaging (BLI) approach. Results: Transduction of hUC-MSCs with GFP-Luc2 not only preserved the characteristics of MSCs, but also allowed live monitoring of transduced cells in the mice model. Upon systemic administration, BLI showed that transduced hUC-MSCs first localised predominantly in the lungs of healthy BALB/c mice and mainly remained in the lungs for up to 3 days before eventually cleared from the body. At terminal sacrifice, plasma chemistry biomarkers remained unchanged except for C-peptide levels, which were significantly reduced in the hUC-MSCs group. Histopathological findings further revealed that hUC-MSCs infusion did not cause any adverse effects and toxicity to lung, liver and heart tissues. Conclusions: Collectively, systemically administrated hUC-MSCs was safe and demonstrated dynamic homing capacity before eventually disappearing from the body.

Subtractive Hybridization Identifies Stem Cell-Associated Genes in an Acute Myeloid Leukemia with Poor Prognosis

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy.

Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.

Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy

Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.

Molecular responses during chemotherapy in acute myeloid leukemias in predicting poor-response to standard chemotherapy

Signal transduction pathways are constitutively expressed in leukaemic cells resulting in aberrant survival of the cells. It is postulated that in cells of chemo-sensitive patients, chemotherapy induces apoptotic signals leading to cell death while survival signals are maintained in cells of chemo-resistant patients. There is very little information currently, on the expression of these mediators in patients immediately after chemotherapy initiation. We examined the expression pattern of proinflammatory cytokines, signaling molecules of the PI3K and MAPK pathways molecules and death receptor, DR5 on paired samples at diagnosis and during chemotherapy in acute myeloid leukaemia patients treated with cytosine arabinoside and daunorubicin. The results were correlated with remission status one month after chemotherapy. We found that in chemo-sensitive patients, chemotherapy significantly increased the percentage of cases expressing TNF-alpha (p = 0.025, n = 9) and IL-6 (p = 0.002, n = 11) compared to chemo-resistant cases. We also observed an increased percentage of chemo-sensitive cases expressing DR5 and phosphorylated p38, and Jnk. Thus, expression of TNF-alpha, IL-6, DR5, phospho-p38 and phospho-Jnk may regulate cell death in chemo-sensitive cases. In contrast, a significantly higher percentage of chemo-resistant cases expressed phospho-Bad (p = 0.027, n = 9). IL-beta and IL-18 were also found to be higher in chemo-resistant cases at diagnosis and during chemotherapy. Thus, expression of various cellular molecules in leukaemic blasts during chemotherapy may be useful in predicting treatment outcome. These cellular molecules may also be potential targets for alternative therapy.

Dendritic cell distribution in lymphomas

Dendritic cells (DC) are specialized antigen presenting cells (APC) that have important roles in host defenses and in generating anti-tumour immune response. Altered frequency and maturation of DC have been reported in malignant tumours. We studied the distribution and maturation status of DC by immunohistochemistry, on the formalin-fixed, paraffin-embedded lymph node tissues of 32 histologically diagnosed lymphomas and 40 inflammatory conditions that were retrieved from the Department of Pathology, UKM Medical Centre, Kuala Lumpur. Our study showed a significant reduction in the total DC counts in the lymphoma tissues compared to the inflammatory conditions. The mature and immature DC counts were both significantly reduced (p = 0.008 and 0.001 respectively), although a greater reduction was observed in mature DC numbers. We also observed compartmentalization of DC where the immature DC were seen within the tumour tissues and the mature DC were more in peri-tumoural areas. Our findings were similar to other reports, suggesting that reduced numbers of DC appears to be a factor contributing to lack of tumour surveillance in these cases.

Transfected human mesenchymal stem cells do not lose their surface markers and differentiation properties

In this study, we evaluated the biological properties of human mesenchymal stem cells transfected (hMSC) with a plasmid vector expressing human cytokine interleukin-12 (IL-12). Surface markers were analysed by immunophenotyping using flow cytometry. Differentiation capability was evaluated towards adipogenesis and osteogenesis. We demonstrated that successfully transfected hMSC retained their surface immunophenotypes and differentiation potential into adipocytes and osteocytes. These results indicate that hMSC may be a suitable vehicle for gene transduction.