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INTRODUCTION The stem cell portion of the dental pulp derived cultures (DPSCs) showed a higher resistance to cytotoxic effect of restorative dental materials compared to pulpal fibroblasts (DPFs). Here,we aimed to compare the expression of some drug resistant genes between these cells. METHODS AND MATERIALS To separate DPSCs from DPFs,we used magnetic cell sorting technique based on CD146 expression. To assess the stem cell properties,the positive and negative portions underwent colony forming assays and were induced to be differentiated into the adipocytes,osteoblasts,hepatocytes,and neural cells. Cell surface antigen panels were checked using immune fluorescence and flow-cytometry techniques. The mRNA expression of 14 ABC transporters including ABCA2,ABCB1,ABCB11,ABCC1,ABCC2,ABCC3,ABCC4,ABCC5-2,ABCC5-4,ABCC5-13,ABCC6,ABCC10,ABCC11,and ABCG2 genes was assessed,using quantitative RT-PCR technique. RESULTS Only the CD146 positive portion could be differentiated into the desired fates,and they formed higher colonies (16.7 ± 3.32 vs. 1.7 ± 1.67,p {\textless} .001). The cell surface antigen panels were the same,except for CD146 and STRO-1 markers which were expressed only in the positive portion. Among the ABC transporter genes studied,the positive portion showed a higher expression (approximately two-fold) of ABCA2,ABCC5-13,and ABCC5-2 genes. CONCLUSION Dental pulp stem cells which can be separated from dental pulp fibroblasts based on CD146 expression,express higher levels of some drug resistance genes which probably accounts for their features of more resistance to cytotoxic effects of some dental materials. This needs to be more validated in future. View Publication

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells,making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis,Nampt,in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells,but not normal donor as well as MM patients PBMCs. Importantly,FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6,IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly,cytotoxicity of FK866 triggered autophagy,but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally,FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model,associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology,providing the basis for a novel targeted therapeutic approach. View Publication

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4,Klf4,Sox2,cMyc,and Lin28 resulted in formation of five iPSC colonies,from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro,$$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs,whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%,but with a decreased hematopoietic colony-forming capability. In conclusion,we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover,it was demonstrated that the mRNA-based reprogramming method,used mainly in fibroblasts,is also suitable for reprogramming of human BM-MSCs. View Publication

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here,we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth,chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration,altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development. Zhang et al. apply metabolic flux analysis to comprehensively characterize the metabolism of human pluripotent stem cells cultured in different media. Cells maintained in chemically defined media significantly upregulate lipid biosynthesis and redox pathways to compensate for medium lipid deficiency while downregulating oxidative mitochondrial metabolism. View Publication

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses,host defense mechanisms,and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system,DCs are very rare in blood,accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore,alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity,affordability,high purity,and high yield of cells is imperative to consider. In the current study,two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability,proliferation,and phenotype were assessed using viability dyes,MTT assay,and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method,the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded textgreater 70% CD11c+ MDDCs. Therefore,our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs. View Publication

The bcr-abl oncogene,present in 95% of patients with chronic myelogenous leukemia (CML),has been implicated as the cause of this disease. A compound,designed to inhibit the Abl protein tyrosine kinase,was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML,there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. View Publication

Resident host microflora condition and prime the immune system. However,systemic and mucosal immune responses to bacteria may be divergent. Our aim was to compare,in vitro,cytokine production by human mononuclear and dendritic cells (DCs) from mesenteric lymph nodes (MLNs) and peripheral blood mononuclear cells (PBMCs) to defined microbial stimuli. Mononuclear cells and DCs isolated from the MLN (n = 10) and peripheral blood (n = 12) of patients with active colitis were incubated in vitro with the probiotic bacteria Lactobacillus salivarius UCC118 or Bifidobacterium infantis 35624 or the pathogenic organism Salmonella typhimurium UK1. Interleukin (IL)-12,tumor necrosis factor (TNF)-alpha,transforming growth factor (TGF)-beta,and IL-10 cytokine levels were quantified by ELISA. PBMCs and PBMC-derived DCs secreted TNF-alpha in response to the Lactobacillus,Bifidobacteria,and Salmonella strains,whereas MLN cells and MLN-derived DCs secreted TNF-alpha only in response to Salmonella challenge. Cells from the systemic compartment secreted IL-12 after coincubation with Salmonella or Lactobacilli,whereas MLN-derived cells produced IL-12 only in response to Salmonella. PBMCs secreted IL-10 in response to the Bifidobacterium strain but not in response to the Lactobacillus or Salmonella strain. However,MLN cells secreted IL-10 in response to Bifidobacteria and Lactobacilli but not in response to Salmonella. In conclusion,commensal bacteria induced regulatory cytokine production by MLN cells,whereas pathogenic bacteria induce T cell helper 1-polarizing cytokines. Commensal-pathogen divergence in cytokine responses is more marked in cells isolated from the mucosal immune system compared with PBMCs. View Publication

The aims of our study were to verify whether it was possible to generate in vitro,from different adult human tissues,a population of cells that behaved,in culture,as multipotent stem cells and if these latter shared common properties. To this purpose,we grew and cloned finite cell lines obtained from adult human liver,heart,and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs,obtained from the 3 different tissues,expressed the pluripotent state-specific transcription factors Oct-4,NANOG,and REX1,displayed telomerase activity,and exhibited a wide range of differentiation potential,as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content,and shared a common gene expression signature,compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular,the pathways regulating stem cell self-renewal/maintenance,such as Wnt,Hedgehog,and Notch,were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin. View Publication

Human embryonic stem cells and induced pluripotent stem cells have great potential in research and thera-pies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here,we demonstrate,for the first time,that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4,NANOG,and SSEA-4,in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme,cellulase,enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly,the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus,the NFC hydrogel represents a flexible,xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine. View Publication

Type 1 diabetes mellitus (T1DM) is the most common type of diabetes in children and adolescents. Diabetic subjects are more likely to experience a myocardial infarction compared to nondiabetic subjects. In recent years,induced pluripotent stem cells (iPSCs) have received increasing attention from basic scientists and clinicians and hold promise for myocardial regeneration due to their unlimited proliferation potential and differentiation capacity. However,cardiomyogenesis of type 1 diabetic donor-derived iPSCs (T1DM-iPSCs) has not been investigated yet. The aim of the study was to comparatively analyze cardiomyocyte (CM) differentiation capacity of nondiabetic donor-derived iPSCs (N-iPSCs) and T1DM-iPSCs. The differentiated CMs were confirmed by both expression of cardiac-specific markers and presence of cardiac action potential. Since mitochondrial bioenergetics is vital to every aspect of CM function,extracellular acidification rates and oxygen consumption rates were measured using Seahorse extracellular flux analyzer. The results showed that N-iPSCs and T1DMiPSCs demonstrated similar capacity of differentiation into spontaneously contracting CMs exhibiting nodal-,atrial-,or ventricular-like action potentials. Differentiation efficiency was up to 90%. In addition,the CMs differentiated from N-iPSCs and T1DM-iPSCs (N-iPSC-CMs and T1DM-iPSC-CMs,respectively) showed 1) well-regulated glucose utilization at the level of glycolysis and mitochondrial oxidative phosphorylation and 2) the ability to switch metabolic pathways independent of extracellular glucose concentration. Collectively,we demonstrate for the first time that T1DM-iPSCs can differentiate into functional CMs with well-regulated glucose utilization as shown in N-iPSCs,suggesting that T1DM-iPSC-CMs might be a promising autologous cell source for myocardial regeneration in type 1 diabetes patients. View Publication

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end,we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells,and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo,while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence,we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase,the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings,two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly,these cocktails were either deprived of thrombopoietin or of stem cell factor,two cytokines known to favor megakaryopoiesis and HPC expansion,respectively. Based on these results,a short resource-efficient two-phase culture protocol for the production of Mks near purity (textgreater95%) from human CD34+ CB cells has been established. View Publication