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1.  Chemical Conversion of Human Fibroblasts into Functional Schwann Cells 
Stem Cell Reports  2014;3(4):539-547.
Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.
Graphical Abstract
•Direct conversion of human fibroblasts into Schwann cells•No introduction of ectopic genes•Induction of conversion by multikinase inhibitor
Here, Thoma and colleagues developed a strategy to convert human fibroblasts into Schwann cells solely by chemical treatment. Using a multikinase inhibitor, fibroblasts are transdifferentiated into transient neural precursors differentiating into cells with molecular and functional characteristics of Schwann cells. This study shows that cellular conversion can be achieved solely by small molecules and presents an approach to generate human Schwann cells without genetic modifications.
PMCID: PMC4223700  PMID: 25358782
2.  Multicellular Self-Assembled Spheroidal Model of the Blood Brain Barrier 
Scientific Reports  2013;3:1500.
The blood brain barrier (BBB) has evolved unique characteristics such as dense coverage of the endothelial cells by pericytes and interactions with astrocytes through perivascular endfeet. We study BBB formation in a 3-dimensional multicellular spheroid system of human primary brain endothelial cells (hpBECs), primary pericytes (hpPs) and primary astrocytes (hpAs). We show for the first time that hpBECs, hpPs and hpAs spontaneously self-organize into a defined multicellular structure which recapitulates the complex arrangement of the individual cell types in the BBB structure. Pericytes play a crucial role mediating the interaction between hpBECs and hpAs. This process is not dependent on a scaffold support demonstrating that formation and cellular architecture of the BBB is intrinsically programmed within each specific cell type. In a matrigel setup the hpBECs, hpPs and hpAs also undergo self-arrangement to form endothelial tube-like structures tightly covered by hpPs and loosely attached hpAs mainly at the junctions.
PMCID: PMC3603320  PMID: 23511305
3.  P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX’s MORFO domain 
Purinergic Signalling  2011;8(2):181-190.
In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y12 receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX’s modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y12 receptor is necessary for ATX’s MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y12 receptor can render cells responsive to the known effects of ATX’s MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y12 signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y12 receptor provides the basis for an ATX-MORFO-P2Y12 signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-011-9283-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3350589  PMID: 22139091
Oligodendrocyte; Autotaxin; Purinergic receptor; Adhesion; P2Y12
4.  Caspase-, cathepsin-, and PERK-dependent regulation of MDA-7/IL-24-induced cell killing in primary human glioma cells 
Molecular cancer therapeutics  2008;7(2):297-313.
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R–like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK−/− cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B–dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methylade-nine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.
PMCID: PMC3204355  PMID: 18281515
5.  Tumor-infiltrating, myeloid-derived suppressor cells inhibit T cell activity by nitric oxide production in an intracranial rat glioma + vaccination model 
Journal of neuroimmunology  2010;223(1-2):20-30.
In rats bearing an intracranial T9 glioma, immunization with tumor antigens induces myeloid suppressor cells, which express neutrophil (His48) and monocyte (CD11bc) markers, to infiltrate the tumors. The His48+/CD11bc+ cells were not derived from CNS microglia but were hematogenous; suppressed multiple T cell effector functions; and are myeloid-derived suppressor cells (MDSC). The glioma-infiltrating MDSC expressed arginase I, iNOS, indoleamine 2,3-dioxygenase and TGF-β; however, inhibitor/blocking studies demonstrated that NO production was the primary mechanism of suppression which induced T cell apoptosis. These findings suggest that neuro-immunomodulation by MDSC in rat gliomas maybe mediated by a pathway requiring NO production.
PMCID: PMC2883008  PMID: 20452681
brain tumor-associated immunosuppression; malignant glioma; myeloid-derived suppressor cells; nitric oxide
6.  FTY720 Reduces Inflammation and Promotes Functional Recovery after Spinal Cord Injury 
Journal of Neurotrauma  2009;26(12):2335-2344.
A robust and complex inflammatory cascade is known to be a prominent component of secondary injury following spinal cord injury (SCI). Specifically, the concept of trauma-induced autoimmunity has linked the lymphocyte population with neural tissue injury and neurologic deficit. FTY720, a sphingosine receptor modulator that sequesters lymphocytes in secondary lymphoid organs, has been shown to be effective in the treatment of a variety of experimental autoimmune disorders. Accordingly, by reducing lymphocyte infiltration into the spinal cord following SCI, this novel immunomodulator may enhance tissue preservation and functional recovery. In the present study, a moderate to severe contusion SCI was simulated in adult Long-Evans hooded rats. Using flow cytometry we showed that daily FTY720 treatment dramatically reduced T-cell infiltration into the SCI lesion site at 4 and 7 days post-injury, while other inflammatory cell populations were relatively unaltered. To assess functional recovery, three groups of injured animals (treated, vehicle, and injury only) were evaluated weekly for hindlimb recovery. Animals in the treated group consistently exhibited higher functional scores than animals in the control groups after 2 weeks post-injury. This finding was associated with a greater degree of white matter sparing at the lesion epicenter when cords were later sectioned and stained. Furthermore, treated animals were found to exhibit improved bladder function and a reduced incidence of hemorrhagic cystitis compared to control counterparts. Collectively these results demonstrate the neuroprotective potential of FTY720 treatment after experimental SCI.
PMCID: PMC2850297  PMID: 19624262
behavioral assessment; FTY720; inflammation; locomotor function; spinal cord injury
7.  Heterogeneous Pattern of Retinal Nerve Fiber Layer in Multiple Sclerosis. High Resolution Optical Coherence Tomography: Potential and Limitations 
PLoS ONE  2010;5(11):e13877.
Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique.
Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP).
Principal Findings
All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9–540 months).
RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels).
PMCID: PMC2975633  PMID: 21079732
8.  MDA-7/IL-24–induced cell killing in malignant renal carcinoma cells occurs by a ceramide/CD95/PERK–dependent mechanism 
Molecular cancer therapeutics  2009;8(5):1280-1291.
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7–induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal–regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH2-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7–induced phosphorylation of protein kinase R–like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R–like endoplasmic reticulum kinase that correlated with reduced c-jun NH2-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal–regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.
PMCID: PMC2889018  PMID: 19417161
9.  Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation 
Cancer biology & therapy  2008;7(10):1648-1662.
We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2α phosphorylation; suppression of eIF2α function abolished drug combination lethality. Cell killing was paralleled by PERK- and eIF2α-dependent lowering of c-FLIP-s protein levels and over-expression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRβ and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Over-expression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2α -↓c-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).
PMCID: PMC2674577  PMID: 18787411
Vorinostat; Sorafenib; CD95; c-FLIP-s; PDGFRβ; FLT3; autophagy; ceramide; cell death; ASMase
10.  Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation 
Purpose and Design
Mechanism(s) by which the multi-kinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal and pancreatic adenocarcinoma cells have been defined.
Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal and pancreatic adenocarcinoma cells in multiple short term viability (24–96h) and in long term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase 8, and to a lesser extent by inhibition of caspase 9. Twenty four hours after exposure, the activities of ERK1/2, AKT and NFκB were only modestly modulated by sorafenib and vorinostat treatment. However, 24h after exposure, sorafenib and vorinostat- treated cells exhibited markedly diminished expression of c-FLIP-s, full length BID, BCL-2, BCLXL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK and BAD. Expression of eIF2α S51A blocked sorafenib and vorinostat –induced suppression of c-FLIP-s levels and over-expression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase 8. Knock down of CD95 or FADD expression significantly reduced sorafenib / vorinostat -mediated lethality.
These data demonstrate that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple anti-apoptotic proteins, promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the pro-apoptotic signals from CD95 to promote mitochondrial dysfunction and death.
PMCID: PMC2561272  PMID: 18765530
Vorinostat; Sorafenib; CD95; c-FLIP-s; caspase 8; cathepsin; cell death
11.  PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells 
Autophagy  2008;4(4):513-515.
Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK-/- cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.
PMCID: PMC2674579  PMID: 18299661
autophagy; caspase; ER stress; cell death
12.  OSU-03012 Stimulates PKR-Like Endoplasmic Reticulum-Dependent Increases in 70-kDa Heat Shock Protein Expression, Attenuating Its Lethal Actions in Transformed Cells 
Molecular pharmacology  2008;73(4):1168-1184.
We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK-/-), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knockdown or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.
PMCID: PMC2674576  PMID: 18182481
The Journal of Experimental Medicine  1970;132(6):1279-1287.
Rabbits were immunized to two antigens and 18–55 days later exchange transfusion was performed using blood of rabbits immunized to one antigen only. By this means, serum antibody levels to one antigen were reduced 50–84% while maintaining serum antibody levels to the second antigen. After exchange, serum antibody levels of the removed antibody rose rapidly for 24–48 hr and then more slowly, reaching peak titers an average of 8 days later. The peak titer was 48–222% higher than the preexchange titer. The specificity of this rebound excluded as a cause nonspecific changes in Ig levels. Passive administration of antibody to a third antigen 4–7 days before the exchange indicated that re-equilibration of preformed antibody was not a major factor in the rebound. A change in the ratio of IgM to IgG antibodies as a cause of an increased neutralization titer in the postexchange sera was also excluded. It was therefore suggested that a change in the rate of antibody formation had occurred, although other changes in the quality of serum antibody were not excluded.
PMCID: PMC2180498  PMID: 5511573
The Journal of Experimental Medicine  1969;130(5):1175-1186.
Rabbits were injected intravenously with bovine serum albumin (BSA) and bacteriophage T2 (T2). 2–3 wk later, anti-BSA was removed from such animals by a procedure which involved exposure of removed plasma to an immunoadsorbent (125I-BSA bound to bromoacetyl cellulose) and return of the adsorbed plasma to the animal. This resulted in removal of the majority of antibody activity to BSA without affecting antibody levels to T2. 1–2 days later, anti-BSA levels began to rise, and reached peak levels usually 5 days after the removal of antibody. Antibody levels to T2 did not change. No evidence was obtained that BSA was released from the immunoadsorbent into the circulation of the rabbits. Thus, only trace amounts of radioactivity were released into the plasma; most of the radioactivity was equally coprecipitable with BSA or human gamma globulin and their specific antibodies; the released material was not demonstrated to be immunogenic in primed rabbits; and the released material did not elute with BSA on gel filtration. The results are interpreted as evidence that serum antibody acts as a regulatory mechanism for antibody formation during the conventional antibody response to a metabolizable antigen.
PMCID: PMC2180481  PMID: 5347697

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