tretinoin and purmorphamine

Topics: Cell Differentiation; Delayed-Action Preparations; Drug Compounding; Drug Delivery Systems; Humans; Induced Pluripotent Stem Cells; Microspheres; Morpholines; Pregnenediones; Purines; Regenerative Medicine; Tissue Engineering; Tretinoin

Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

Topics: Animals; Biological Assay; Botulinum Toxins, Type A; Cell Line, Tumor; Culture Media; Embryonic Stem Cells; Gangliosides; Mice; Morpholines; Motor Neurons; Neurotoxins; Purines; Rats; Sensitivity and Specificity; Transforming Growth Factor beta1; Tretinoin

In the pMN domain of the spinal cord, Notch signaling regulates the balance between motor neuron differentiation and maintenance of the progenitor state for later oligodendrocyte differentiation. Here, we sought to study the role of Notch signaling in regulation of the switch from the pMN progenitor state to differentiated motor neurons in a human model system. Human embryonic stem cells (hESCs) were directed to differentiate to pMN-like progenitor cells by the inductive action of retinoic acid and a Shh agonist, purmorphamine. We found that the expression of the Notch signaling effector Hes5 was induced in hESC-derived pMN-like progenitors and remained highly expressed when they were cultured under conditions favoring motor neuron differentiation. Inhibition of Notch signaling by a γ-secretase inhibitor in the differentiating pMN-like progenitor cells decreased Hes5 expression and enhanced the differentiation toward motor neurons. Conversely, over-expression of Hes5 in pMN-like progenitor cells during the differentiation interfered with retinoic acid- and purmorphamine-induced motor neuron differentiation and inhibited the emergence of motor neurons. Inhibition of Notch signaling had a permissive rather than an inductive effect on motor neuron differentiation. Our results indicate that Notch signaling has a regulatory role in the switch from the pMN progenitor to the differentiated motor neuron state. Inhibition of Notch signaling can be harnessed to enhance the differentiation of hESCs toward motor neurons.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line; Embryonic Stem Cells; Hedgehog Proteins; Humans; Morpholines; Motor Neurons; Purines; Receptors, Notch; Signal Transduction; Tretinoin

Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair.

Topics: Cell Differentiation; Cell Survival; Cells, Cultured; Gelatin; Humans; Lactic Acid; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Morpholines; Motor Neurons; Nerve Tissue; Neural Stem Cells; Polyesters; Polymers; Purines; Spinal Cord Injuries; Tissue Scaffolds; Tretinoin

V2a interneurons of the ventral spinal cord and hindbrain play an important role in the central pattern generators (CPGs) involved in locomotion, skilled reaching, and respiration. However, sources of V2a interneurons for in vitro studies are limited. In this study, we developed a differentiation protocol for V2a interneurons from mouse embryonic stem cells (mESCs). Cells were induced in a 2(-)/4(+) induction protocol with varying concentrations of retinoic acid (RA) and the mild sonic hedgehog (Shh) agonist purmorphamine (Pur) in order to increase the expression of V2a interneuron transcription factors (eg, Chx10). Notch signaling, which influences the commitment of p2 progenitor cells to V2a or V2b interneurons, was inhibited in cell cultures to increase the percentage of V2a interneurons. At the end of the induction period, cell commitment was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, and flow cytometry to quantify expression of transcription factors specific to V2a interneurons and the adjacent ventral spinal cord regions. Low concentrations of RA and high concentrations of Pur led to greater expression of transcription factors specific for V2a interneurons. Notch inhibition favored V2a interneuron over V2b interneuron differentiation. The protocol established in this study can be used to further elucidate the pathways involved in V2a interneuron differentiation and help produce sources of V2a interneurons for developmental neurobiology, electrophysiology, and transplantation studies.

Topics: Animals; Biomarkers; Cell Differentiation; Dipeptides; Embryonic Stem Cells; Gene Expression Regulation, Developmental; Homeodomain Proteins; Interneurons; Mice; Morpholines; Neural Tube; Purines; Receptors, Notch; Signal Transduction; Transcription Factors; Tretinoin

Cancerous cell lines have traditionally shown low sensitivity to laboratory or pharmaceutical preparations of botulinum neurotoxin. The work presented here demonstrates that the mouse neuroblastoma/rat glioma hybrid cell line NG108-15 is capable of more sensitively detecting BoNT/A1 than any cell line previously described. This cell line has previously been described to have motor neuron like characteristics, therefore making it a good model to study BoNTs. Differentiation of NG108-15 cells in serum-free medium containing retinoic acid and purmorphamine dramatically increased sensitivity of the neurons to BoNT/A (EC(50) = ~16 LD(50) U). Additional pre-treatment with triasialoganglioside GT1B prior to toxin exposure reduced the EC(50) further to ~11 LD(50) U. Co-culture of the neurons with C2C12 myotubes also significantly increased BoNT/A sensitivity of NG108-15 cells (EC(50) = 26 U) in the absence of differentiation factors.

Topics: Animals; Apoptosis; Botulinum Toxins, Type A; Cell Differentiation; Cholinergic Neurons; Clostridium botulinum; Coculture Techniques; Gangliosides; Mice; Models, Neurological; Morpholines; Motor Neurons; Neurotoxins; Purines; Rats; Tretinoin

Transplantation of neural stem cells (NSCs) is a promising therapeutic approach for Huntington's disease (HD). HD is characterized by a progressive loss of medium-sized spiny neurons (MSNs) in the striatum. DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa) is expressed in 98% of these MSNs. To establish an effective cell therapy for HD, the differentiation of human NSCs into MSNs is essential. Enhancing differentiation of NSCs is therefore an important aspect to optimize transplant efficacy. A comparison of 5 differentiation protocols indicated that the Hedgehog agonist purmorphamine (1 μM) most significantly increased the neuronal differentiation of a human striatal NSC line (STROC05). This 3-fold increase in neurons was associated with a dramatic reduction in proliferation as well as a decrease in astrocytic differentiation. A synergistic effect between purmorphamine and cell density even further increased neuronal differentiation from 20% to 30% within 7 days. Upon long-term differentiation (21 days), this combined differentiation protocol tripled the number of DARPP-32 cells (7%) and almost doubled the proportion of calbindin cells. However, there was no effect on calretinin cells. Differential expression of positional specification markers (DLX2, MASH1, MEIS2, GSH2, and NKX2.1) further confirmed the striatal identity of these differentiated cells. Purmorphamine resulted in a significant upregulation of the Hedgehog (Hh) signaling pathway (GLI1 expression). Cyclopamine, an Hh inhibitor, blocked this effect, indicating that purmorphamine specifically acts through this pathway to increase neuronal differentiation. These results demonstrate that small synthetic molecules can play a pivotal role in directing the differentiation of NSCs to optimize their therapeutic potential in HD.

Topics: Antigens, Differentiation; Brain; Brain-Derived Neurotrophic Factor; Bucladesine; Calbindin 2; Calbindins; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Dopamine and cAMP-Regulated Phosphoprotein 32; Hedgehog Proteins; Humans; Huntington Disease; Interneurons; Morpholines; Neural Stem Cells; Purines; S100 Calcium Binding Protein G; Signal Transduction; Transcription Factors; Tretinoin; Up-Regulation; Valproic Acid; Zinc Finger Protein GLI1

This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% (previously termed hypoxia) and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over a period of 2 weeks. This timescale is comparable to that observed at 20% O(2), but survival is enhanced. Sequential application of retinoic acid and purmorphamine (PM), from day 14 to day 28, directs differentiation toward spinal motor neurons. Alternatively, addition of fibroblast growth factor-8 and PM generates midbrain dopaminergic neurons. OLIG2 (encoding oligodendrocyte lineage transcription factor 2) induction in motor neuron precursors is twofold greater than that at 20% O(2), whereas EN1 (encoding engrailed homeobox 1) expression is enhanced fivefold. NPCs (at 3% O(2)) can be differentiated into all three neural lineages, and such cultures can be maintained long term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advancement for in vitro disease modeling and potentially for cell-based therapies.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Culture Techniques; Cell Differentiation; Electrophysiology; Embryonic Stem Cells; Fibroblast Growth Factor 8; Homeodomain Proteins; Humans; Mesencephalon; Morpholines; Motor Neurons; Nerve Tissue Proteins; Neural Stem Cells; Oligodendrocyte Transcription Factor 2; Oxygen; Purines; Spinal Nerves; Tretinoin

We describe a chemically defined protocol for efficient differentiation of human embryonic stem cells (hESCs) to neural epithelial cells and then to functional spinal motor neurons. This protocol comprises four major steps. Human ESCs are differentiated without morphogens into neuroepithelial cells that form neural tube-like rosettes in the first 2 weeks. The neuroepithelial cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) in the following 2 weeks. These OLIG2 progenitors generate postmitotic, HB9 expressing motoneurons at the fifth week and mature to functional motor neurons thereafter. The protein factor SHH can be replaced by a small molecule purmorphamine in the entire process, which may facilitate potential clinical applications. This protocol has been shown equally effective in generating motor neurons from human induced pluropotent stem (iPS) cells.

Topics: Animals; Antineoplastic Agents; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Embryonic Stem Cells; Hedgehog Proteins; Humans; Morpholines; Motor Neurons; Purines; Spinal Cord; Tretinoin

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (>80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing Na(V;)1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

Topics: Animals; Cell Differentiation; Cell Line; Cytological Techniques; Electrophysiological Phenomena; Embryonic Stem Cells; Mice; Morpholines; Oligodendroglia; Purines; Tretinoin

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.

Topics: Animals; Cell Differentiation; Cell Line; Cells, Cultured; Directed Molecular Evolution; Embryonic Stem Cells; Hedgehog Proteins; Homeobox Protein Nkx-2.2; Homeodomain Proteins; Humans; Mice; Morpholines; Motor Neurons; Nuclear Proteins; Purines; Spinal Cord; Stem Cells; Transcription Factors; Tretinoin