Adult Human Dermal Fibroblasts Research Articles

Complete Artificial Saliva Alters Expression of Proinflammatory Cytokines in Human Dermal Fibroblasts

Complete artificial saliva (CAS) is a saliva substitute often used as a vehicle for test articles, including smokeless tobacco products. In the course of a study employing normal adult human dermal fibroblasts (HDFa) as a model in vitro, we discovered that CAS as a vehicle introduced a significant change in the expression of proinflammatory cytokines. To determine the effects of CAS on gene expression, real-time quantitative reverse-transcriptase PCR gene array analysis was used. Results indicate that robust changes in the expression of the proinflammatory cytokine interleukin 8 (IL8) and the vascular cell adhesion molecule 1 (VCAM1) occur within 5h of exposure to CAS. To determine whether CAS also alters cytokine release into the culture media, cytometric bead array assays for human inflammatory cytokines were performed. Analysis shows that CAS induced the release of IL8 and IL6. This study focused on determining which components in CAS were responsible for the proinflammatory response in HDFa. The following components were investigated: α-amylase, lysozyme, acid phosphatase, and urea. Results demonstrated that enzymatically active α-amylase induced gene expression for proinflammatory cytokines IL8, IL6, tumor necrosis factor-α, and IL1α and for VCAM1. Therefore, it is important to carefully evaluate the "vehicle effects" of CAS and its components in in vitro toxicology research.

Heart repair by cardiac reprogramming

Heart repair by cardiac reprogramming

Human fibroblast‐derived extracellular matrix constructs for bone tissue engineering applications

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications.

Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy.

Contact and Encirclement of Glioma Cells In Vitro Is an Intrinsic Behavior of a Clonal Human Neural Stem Cell Line

Pathotropic neural stem and/or progenitor cells (NSCs) can potentially deliver therapeutic agents to otherwise inaccessible cancers. In glioma, NSCs are found in close contact with tumor cells, raising the possibility that specificity of NSC contact with glioma targets originates in the tumor cells themselves. Alternatively, target preferences may originate, at least in part, in the tumor microenvironment. To better understand mechanisms underlying NSC interactions with glioma cells, we examined NSC-target cell contacts in a highly simplified 3-dimensional peptide hydrogel (Puramatrix) in which cell behaviors can be studied in the relative absence of external cues. HB1.F3 is an immortalized clonal human NSC line extensively characterized in preclinical investigations. To study contact formation between HB1.F3 NSCs and glioma cells, we first examined co-cultures of eGFP-expressing HB1.F3 (HB1.F3.eGFP) NSCs and dsRed-expressing U251 glioma (U251.dsRed) cells. Using confocal microscopy, HB1.F3.eGFP cells were observed contacting or encircling U251.dsRed glioma cells, but never the reverse. Next, examining specificity of these contacts, no significant quantitative differences in either percentages of HB1.F3 NSCs contacting targets, or in the extent of target cell encirclement, were observed when HB1.F3.eGFP cells were presented with various potential target cells (human glioma and breast cancer cell lines, patient-derived brain tumor lines, non-tumor fibroblasts, primary mouse and human astroglial cells, and primary adult and newborn human dermal fibroblasts) except that interactions between HB1.F3 cells did not progress beyond establishing contacts. Finally cytoskeletal mechanisms employed by HB1.F3.eGFP cells varied with the substrate. When migrating in Puramatrix, HB1.F3 NSCs exhibited intermittent process extension followed by soma translocation, while during encirclement their movements were more amoeboid. We conclude that formation of contacts and subsequent encirclement of target cells by HB1.F3 NSCs is an intrinsic property of these NSCs, and that preferential contact formation with tumor cells in vivo must therefore be highly dependent on microenvironmental cues.

Generation and Characterization of Multipotent Stem Cells from Established Dermal Cultures

Human multipotent skin derived precursor cells (SKPs) are traditionally sourced from dissociated dermal tissues; therefore, donor availability may become limiting. Here we demonstrate that both normal and diseased adult human dermal fibroblasts (DF) pre-cultured in conventional monolayers are capable of forming SKPs (termed m-SKPs). Moreover, we show that these m-SKPs can be passaged and that cryopreservation of original fibroblast monolayer cultures does not reduce m-SKP yield; however, extensive monolayer passaging does. Like SKPs generated from dissociated dermis, these m-SKPs expressed nestin, fibronectin and versican at the protein level. At the transcriptional level, m-SKPs derived from normal adult human DF, expressed neural crest stem cell markers such as p75NTR, embryonic stem cell markers such as Nanog and the mesenchymal stem cell marker Dermo-1. Furthermore, appropriate stimuli induced m-SKPs to differentiate down either mesenchymal or neural lineages resulting in lipid accumulation, calcification and S100β or β-III tubulin expression (with multiple processes). m-SKP yield was greater from neonatal foreskin cultures compared to those from adult DF cultures; however, the former showed a greater decrease in m-SKP forming capacity after extensive monolayer passaging. m-SKP yield was greater from adult DF cultures expressing more alpha-smooth muscle actin (αSMA). In turn, elevated αSMA expression correlated with cells originating from specimens isolated from biopsies containing more terminal hair follicles; however, αSMA expression was lost upon m-SKP formation. Others have shown that dissociated human hair follicle dermal papilla (DP) are a highly enriched source of SKPs. However, conversely and unexpectedly, monolayer cultured human hair follicle DP cells failed to form m-SKPs whereas those from the murine vibrissae follicles did. Collectively, these findings reveal the potential for using expanded DF cultures to produce SKPs, the heterogeneity of SKP forming potential of skin from distinct anatomical locations and ages, and question the progenitor status of human hair follicle DP cells.

Derivation of Hematopoietic Progenitor Cells From Vector-Free Human Induced Pluripotent Stem Cells

Abstract 4746Induced pluripotent stem cells (iPSCs), due to their self-renewal and differentiation capability, have tremendous potential in regenerative medicine. Differentiation of IPSCs in vitro to obtain sufficient number of hematopoietic stem cells (HSCs) and their progenitors (HPCs) from iPSCs for therapeutic purposes is a holy grail of cellular therapy. To this end, we are comparing different in vitro differentiation approaches for generation of HSCs/HPCs from IPSCs.We have generated iPSCs from human adult dermal fibroblasts using two different reprogramming methods: 1) Transduction with retroviral vectors encoding human Klf4, Oct3/4, Sox2 and cMyc or 2) Electroporation with Epstein–Barr virus (EBV) based episomal plasmid vectors encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA. The transduced/electroporated cells were reprogrammed on SNL5 mouse feeder cells. Putative iPSC-like colonies were cloned and adapted to grow under feeder-free conditions on Matrigel (BD) in mTeSR1 (Stem Cell Technologies) medium. From over 30 individual clones isolated, six were further characterized for: 1) expression of pluripotency markers (Tra-1–60, SSEA-3, SSEA-4, Nanog and Oct3/4) by immunofluorescence; 2) endogenous and total mRNA expression by quantitative real-time reverse-transcriptase PCR (RT-qPCR) for Klf4, Oct3/4, Sox2 and cMyc to distinguish between cellular and vector derived expression of reprogramming factors; 3) RT-qPCR to determine expression of other markers of pluripotency such as Nanog and DNA methyl transferease; 4) karyotype analysis to determine chromosomal anomalies. The vector-free IPSC clones were also tested for residual integrated EBV plasmid DNA by qPCR. Trilineage differentiation ability of the clones was determined through embryoid body formation in suspension cultures, and subsequent staining of resulting embryoid bodies after adherence to gelatin coated dishes for makers of ectoderm, mesoderm and endoderm.HSCs/HPCs were obtained from IPSCs by 1) coculture with OP9 stromal cells, or 2) step-wise differentiation in feeder-free conditions on Matrigel under defined conditions in the presence of appropriate growth factors [Niwa A et al. PLoS One. (2011); 6(7):e22261.]. The resultant HSCs/HPCs were subjected to colony forming assays in semi-solid medium containing hematopoietic cytokines. Both erythroid and myelomonocytic colonies could be readily identified. The influence of ambient oxygen concentration on the HSC/HPC derivation procedure is being investigated. The results of these studies will be presented. Disclosures:No relevant conflicts of interest to declare.

Interleukin-1 Beta Promotes the Expression of Prox-1 and Vascular Endothelial Growth Factor Receptor Type-3 in Adult Human Dermal Fibroblasts

Abstract 4738 Background and Aims:We recently reported that human interleukin 1 beta (IL-1 beta) stimulates bone-marrow stromal myofibroblasts to express a hematopoietic stem cell marker, CD34 (16th EHA, and 53rdASH meeting). To characterize precisely the effects of IL-1 beta to the stromal cells, adult human dermal fibroblasts (HDF) were cultured with IL-1 beta, and the morphological changes and molecular expressions were observed. Materials and Methods:HDF were purchased, and cultured in knockout DMEM medium with 15% KSR with or without recombinant human IL-1 beta for 3 weeks. The morphological changes and the expression of specific hematopoiesis-related genes were analyzed time-dependently. The concentration of hematopoietic growth factors in the culturing supernatants was also measured. Results:When HDF were cultured with human IL-1 beta for 3 weeks, the cellular morphology changed to the filamentous appearance. RT-PCR analyses revealed that cells expressed Prox-1, a master molecule for lymphatic duct neogenesis, and also vascular endothelial growth factor receptor (VEGFR) type-3. Smad7 and PAI1 were also expressed; however, LYVE1, a marker for lymphatic vascular endothelial cell, was not induced. VEGF-C production was also demonstrated at RNA and protein levels, and VEGF-C and VEGFR type-3 system played an important role in morphological changes and cell-proliferation. Discussion:We previously reported that when HDF were cultured in DMEM/F12 medium with IL-1 beta and EPO, hematopoietic markers were induced. When HDF were cultured in knockout DMEM, a condition for immature cell-growth at ES cell levels, and IL-1 beta, lymphatic duct neogenesis-related genes were activated, and HDF converted their morphology without an introduction of any genes externally. We are now making clear the precise action of IL-1 beta to HDF using microarray analysis. Disclosures:No relevant conflicts of interest to declare.

Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf

AbstractDeterminant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid–derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

Generation of Corneal Epithelial Cells from Induced Pluripotent Stem Cells Derived from Human Dermal Fibroblast and Corneal Limbal Epithelium

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6+/K12+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

Human Mesenchymal Stem/Stromal Cells Cultured as Spheroids are Self‐activated to Produce Prostaglandin E2 that Directs Stimulated Macrophages into an Anti‐inflammatory Phenotype

Culturing cells in three dimension (3D) provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti-inflammatory properties. Here, we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium (CM) from hMSC spheroids inhibited lipopolysaccharide-stimulated macrophages from secreting proinflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. CM also increased the secretion of anti-inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti-inflammatory activity in CM had a small molecular weight, and microarray data suggested that it was prostaglandin E2 (PGE2). This was confirmed by the observations that PGE2 levels were markedly elevated in hMSC spheroid-CM, and that the anti-inflammatory activity was abolished by an inhibitor of cyclooxygenase-2 (COX-2), a silencing RNA for COX-2, and an antibody to PGE2. The anti-inflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti-inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFκB activation in the hMSCs. The results indicated that hMSCs in 3D-spheroid cultures are self-activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily proinflammatory M1 phenotype to a more anti-inflammatory M2 phenotype.

Zippered release from polymer-gated carbon nanotubes

A thermosensitive drug delivery system based on polymer-gated carbon nanotubes (CNTs) that are loaded with the anticancer drug doxorubicin (DOX) is herein reported. The development of carbon nanotubes for various biomedical applications is the research focus of many research groups and holds great promise. The major drawback of these materials is the toxicity that is associated with conjugated carbon systems. Functionalization of CNTs with polymers has proved very successful in lowering the toxicity and improving the pharmacokinetic profile. In this work, CNTs are coated with polyethylenimine (PEI) and polyvinyl alcohol (PVA) via the “zipper effect” that provides both support and control over drug release. PEI/PVA provides excellent support to increase DOX loading on the nanocarrier. The system is controlled by changes in temperature due to the complexation (low temperature) and decomplexation (high temperature) of PEI and PVA via hydrogen bonding. The release of DOX was tested in three cell lines (Lung fibroblast (LF), Breast Adenocarcinoma (BA), and HeLa). It was further tested in primary cell lines (Human Dermal Fibroblast adult (HDFa) and Human Dermal Fibroblast neonatal (HDFn)). When the bonds between PEI and PVA are decomplexed at high temperature (≥40 °C), drug release was observed as verified by fluorescence microscopy. There was no drug release at room temperature (25 °C) and a slow release at normal body temperature (37 °C). This system represents a promising method for incorporating stimuli triggered polymer-gated CNTs in future controlled release applications.

Cytotoxicity Comparison of Three Current Direct Pulp-capping Agents with a New Bioceramic Root Repair Putty

IntroductionThe purpose of this in vitro study was to compare the cytotoxicity of white mineral trioxide aggregate cement (AMTA, MTA-Angelus), Brasseler Endosequence Root Repair Putty (ERRM), Dycal, and Ultra-blend Plus (UBP) by using human dermal fibroblasts and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MethodsCultured adult human dermal fibroblasts were exposed to multiple concentrations of material elutes. The test material samples were immersed and incubated in the culture medium for 2, 5, or 8 days at 37°C. The cytotoxic effects were recorded by using an MTT-based colorimetric assay. Positive and negative controls were used. The results were statistically examined by one-way analysis of variance and Tukey post tests. ResultsThe cell viability of cultures exposed to all dilutions of AMTA, ERRM, and UBP was statistically similar to the negative control at 2 and 5 days. Only the Dycal-exposed specimens exhibited a statistically significant increase in cytotoxicity at the 2 initial evaluation periods. After exposure to the 8-day elutes, the respective percentage of cell survivability was 91% (Brasseler), 88% (MTA-Angelus), 76% (Ultra-blend Plus), and 37% (Dycal). ConclusionsFrom the data in this in vitro study, AMTA, ERRM, and UBP had statistically similar adult human dermal fibroblast cytotoxicity levels. Relative to the negative control, only Dycal was shown to have a statistically significant cytotoxic effect to adult human dermal fibroblasts at all tested intervals.

Targeting of Mitochondria-Endoplasmic Reticulum by Fluorescent Macrocyclic Compounds

BackgroundUseful probes of the intracellular environment that target a specific organelle in order to allow direct observation of the changes in these regions is of high current interest. Macrocyclic ligands have already revealed themselves as important selective hosts in some biological applications, forming stable and specific complexes. Therefore, in this paper, several macrocyclic ligands are evaluated as potential molecular probes.MethodologyFour polyammonium macrocycles and one macrotricyclic bearing pyridine and phenanthroline chromophores have been synthesised and evaluated as molecular probes. The cytotoxicity of the compounds has been analyzed using human breast cancer cells (MCF-7), non-cancerous human dermal fibroblasts (NHDF) and human adult dermal skin fibroblasts from a breast cancer patient (P14). All the compounds showed low toxicity at concentrations ranging from 10 nM to 10 µM, except for [32]phen2N4 which proved to be highly cytotoxic for MCF-7 cells. Flow cytometry studies evidenced that the percentage of apoptotic and necrotic MCF-7 and NHDF cells induced by the compounds is considerably low. Also, flow cytometry analysis showed that some compounds seem to modify the mitochondrial membrane potential (MMP) of the cells. Fluorescence microscopy evidenced that compounds easily cross the plasma membrane (5 min) and accumulated into the mitochondria, as confirmed by co-localization with MitoTracker Green™. The fluorescence images also evidenced an intact mitochondria structure after 48 h. Moreover, reticular staining suggestive of endoplasmic reticulum (ER) localization, in addition to the mitochondrial one, has been found by confocal microscopy.ConclusionOur study reveals that compounds Me2[28]py2N6, cryptphen, [16]phenN2, [30]phen2N6, have low toxicity and localize in mitochondria and ER. The ability of these compounds for translocating the cellular membrane (5 min) without special conditioning of the cells or derivatization of the probe, the time-dependent localization (48 h) and the cellular viability provide a proof-of-concept towards their use as promising probes towards biomedical studies.

Induction of Megakaryocytes From Fibroblasts by p45NF-E2/Maf

Abstract 908The determinant factors leading from stem cells to megakaryocytes (MK) and the subsequent release of platelets have yet to be identified. Here we report that a combination of transcription factors p45NF-E2, MafG, and MafK converts mouse fibroblast cell line 3T3 and adult human dermal fibroblasts into MKs. We first performed screening for MK-inducing factors using the preadipocyte cell line 3T3-L1 known to be able to differentiate into MKs in a previously defined MK lineage induction media containing thrombopoietin (Zauli G et al, Blood 1997). Gene expression levels of candidate transcription factors were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells using quantitative real-time PCR. The expressions of p45NF-E2 and CEBPα were undetectable in 3T3 cells, whereas expression was seen in 3T3-L1 cells. Both cell lines had similar expression levels of GATA2, RUNX1, FOG1, and PPARγ and undetectable levels of GATA1. 3T3 cells transfected with p45NF-E2, its binding protein Maf, or CEBPα, and 3T3 cells transfected with any combination of these genes, were cultured in MK lineage induction media for 8 days. MK differentiation was assessed using CD41 expression in MK-sized cells by flow cytometry analysis. Frequency of CD41 expression in the 3T3 cells varied based on transgenic factors expressed: 19 ± 12% (p45NF-E2); 25 ± 13% (p45NF-E2/MafG/MafK); 18 ± 3% (p45NF-E2/MafG/MafK/CEBPα); 13 ± 2% (p45NF-E2/CEBPα); and 4 ± 1% (CEBPα). These results suggest that p45NF-E2 with or without Maf are defined factors for reprogramming of 3T3 fibroblast to MKs. We then tested whether other adult non-hematopoietic tissues could be forced into megakaryopoiesis by ectopic expression of p45NF-E2 and Maf. Adult human dermal fibroblasts (Cell Applications) were transfected with p45NF-E2/Maf genes and cultured in MK lineage induction media for 12 days. Large-sized cells were isolated using a 2-step density BSA gradient. By flow cytometry analysis, >99% of these cells expressed CD41. DNA ploidy of the induced MKs (iMKs) ranged from 2N to 16N with a mean of 4N. We then infused 5 × 105 large-sized iMKs into NOG mouse after induction of mild thrombocytopenia in them at day 7 after irradiation with 2.0 Gy. Tail vein samples were obtained from the tested female recipient NOG mice before and after 5 min, 30 min, 90 min, and 3h post iMK infusion. Flow cytometry analysis was performed on each sample stained with PE-conjugated anti-mouse CD41 and FITC-conjugated anti-human CD41 antibody. Human CD41-positive platelet-sized cells in the sample from before and after 5 min, 30 min, 90 min, and 3h represented 0.9%, 0.5%, 2.1%, 3.3%, and 4.0%, respectively, of the entire platelet pool. To examine whether the iMK-derived platelets were incorporated into ex vivo thrombus formation, FITC-anti human CD41 antibody-labeled blood sample from iMK-infused thrombocytopenic NOG mouse was perfused on type I collagen-coated chip under flow condition (Total Thrombus-formation Analyzing System). After perfusion for 10 min, we observed that cells expressing human CD41 were incorporated into thrombi on collagen surface. These findings demonstrate that p45NF-E2 is a key determinant factor for megakaryopoiesis and thrombopoiesis, and is sufficient to lead to platelet formation from a number of non-hematopoietic adult tissues. Generation of iMKs from fibroblasts could have important implications for studying the mechanisms of MK differentiation and platelet production. Disclosures:No relevant conflicts of interest to declare.

Interleukin-1β Promotes the Expression of CD34 and Granulocyte Colony-Stimulating Factor-Receptor in Adult Dermal Fibroblasts

Abstract 4815 Background and Aims:We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF). Materials and Methods:cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed. Results and Discussion:Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo. Disclosures:No relevant conflicts of interest to declare.

Influence of Silicification on the Structural and Biological Properties of Buffer‐Mediated Collagen Hydrogels

AbstractA buffer‐mediated gelation route for collagen hydrogels that allows the formation of homogeneous composite and hybrid materials with various silica sources (i.e., colloidal silica and soluble silicates) at high concentration (up to 25 × 10−3 M) is described. Most significant improvement in rheological properties and proliferation of primary adult human dermal fibroblasts was obtained for the silicate‐based hybrid materials. A similar trend was observed in composite materials incorporating 14 nm SiO2 nanoparticles, although to a much lesser extent, whereas larger colloids (80 and 390 nm) did not significantly impact mechanical stability and cell behavior. Modification of 80 nm particles surface with amine groups weakens the collagen‐mineral interface, resulting in the decrease of material stability and leading to particle aggregation during the course of cell proliferation experiments.

Specific survivin dual fluorescence resonance energy transfer molecular beacons for detection of human bladder cancer cells

Survivin molecular beacons can be used to detect bladder cancer cells in urine samples non-invasively. The aim of this study is to improve the specificity of detection of bladder cancer cells using survivin dual fluorescence resonance energy transfer molecular beacons (FRET MBs) that have fluorophores forming one donor-acceptor pair. Survivin-targeting dual fluorescence resonance energy transfer molecular beacons with unique target sequences were designed, which had no overlap with the other genes in the apoptosis inhibitor protein family. Human bladder cancer cell lines 5637, 253J and T24, as well as the exfoliated cells in the urine of healthy adults and patients with bladder cancer were examined. Images of cells were taken using a laser scanning confocal fluorescence microscope. For assays using dual FRET MBs, the excitation wavelength was 488 nm, and the emission detection wavelengths were 520±20 nm and 560±20 nm, respectively. The human bladder cancer cell lines and exfoliated cells in the urine of patients with bladder cancer incubated with the survivin dual FRET MBs exhibited strong fluorescence signals. In contrast, no fluorescence was detected in the survivin-negative human dermal fibroblasts-adult (HDF-a) cells or exfoliated cells in the urine of healthy adults incubated with the survivin dual FRET MBs. The results suggest that the survivin dual FRET MBs may be used as a specific and non-invasive method for early detection and follow-up of patients with bladder cancer.

Abstract B50: Supercooling under magnetic field can control the risk for malignant transformations of human pluripotent stem (iPS) cells

Abstract Background: The solution in order to control the risk for malignant transformations of human pluripotent stem (iPS) cells are still unclear. Therefore, to seek the solution in order to control the tumorigenicity of human iPS cells is needed in the clinical applications of human iPS cells. Methods: By using the technology of supercooling under magnetic field (2 kHz), integration-free human iPS cell lines from adult human dermal fibroblasts were supercooled and stored until the use at −80° C at a fixed −1°C/min with ROCK inhibitor with serum replacement medium and 10% DMSO. As for the control groups, the conventional cryopreservation method group (Nature Protocols, 2010) and the conventional cultivation method group (Takahashi K et al, Cell, 2007) were settled. Furthermore, mRNA expression levels of Oct 3/4 and Nanog in the human iPS cell lines after thawing or during the conventional cultivation were investigated by using real-time quantitative PCR (Notes: mRNA expression levels of Oct3/4 and Nanog in the human iPS cell lines of prefreezing were defined as 1.0 [arbitrary units.]). Moreover, the occurrences of mutant p53 as a risk factor for malignant transformations of mouse or human iPS cells (Sarig R et al, J Exp Med, 2010, and Mizuno H et al, PNAS, 2010) were evaluated. In addition, all integration-free human iPS cell lines have been compared at the same timepoint/passage number after generation. Results: The survival rates of the human iPS cell lines for the supercooling under magnetic field group in 1 passage after thawing was higher than the conventional cryopreservation method group (96% in the supercooling under magnetic field group and 80% in the conventional cryopreservation method group: p<0.0001, t-test). Furthermore, mRNA expression levels of Oct 3/4 and Nanog in the human iPS cell lines of 40 passages after thawing were higher in the conventional cryopreservation method group compared with the supercooling under magnetic field group (p<0.0001, t-test). Moreover, in 40 passages after thawing, the human iPS cell lines which carry mutant p53 were observed in the conventional cryopreservation method group more than the supercooling under magnetic field group (11/24 in the conventional cryopreservation method group, 0/24 in the supercooling under magnetic field group; p<0.0001, Mann-Whitney U test). Also, in 40 passages of the human iPS cell lines in the conventional cultivation method group, the human iPS cell lines which carry mutant p53 were 17.4 % (4/23). Therefore, the human iPS cell lines which carry mutant p53 were observed in the conventional cultivation method group more than the supercooling under magnetic field group (p=0.035, Mann-Whitney U test). In addition, the cases of the human iPS cell lines which carry mutant p53 were observed when mRNA expression levels of Oct 3/4 was 2.3 (arbitrary units) and more or mRNA expression levels of Nanog was 2.0 (arbitrary units) and more in the human iPS cell lines of 40 passages. Discussion and Conclusion: In the current study, we have found that the supercooling under magnetic field can avoid the occurrences of mutant p53 as a risk factor for malignant transformations of human iPS cells by controlling the overexpressions of Oct 3/4 and/or Nanog. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B50.

Direct Reprogramming of Adult Human Fibroblasts to Functional Neurons under Defined Conditions

Human induced pluripotent stem cells (hiPSCs) have been generated by reprogramming a number of different somatic cell types using a variety of approaches. In addition, direct reprogramming of mature cells from one lineage to another has emerged recently as an alternative strategy for generating celltypes of interest. Here we show that a combination of a microRNA (miR-124) and two transcriptionfactors (MYT1L and BRN2) is sufficient to directlyreprogram postnatal and adult human primary dermal fibroblasts (mesoderm) to functional neurons (ectoderm) under precisely defined conditions. These human induced neurons (hiNs) exhibit typical neuronal morphology and marker gene expression, fire action potentials, and produce functional synapses between each other. Our findings have major implications for cell-replacement strategies in neurodegenerative diseases, disease modeling, and neural developmental studies.