Membrane properties of neuron-like cells generated from adult human bone-marrow-derived mesenchymal stem cells.

Adult mesenchymal stem cells (MeSCs) isolated from human bone marrow are capable of generating neural stem cell (NSC)-like cells that can be subsequently differentiated into cells expressing molecular markers for neurons. Here we report that these neuron-like cells had functional properties similar to those of brain-derived neurons. Whole-cell patch-clamp recordings and calcium imaging experiments were performed on neuron-like cells differentiated from bone-marrow-derived NSC-like cells. The neuron-like cells were subjected to current pulses to determine if they were capable of generating depolarization-induced action potentials. We found that nearly all of the cells with neuron-like morphology exhibited active membrane properties in response to the depolarizing pulses. The most common response was a single spike-like event with an overshoot and brief afterhyperpolarization. Cells that did not generate overshooting spike-like events usually displayed rectifying current-voltage relationships. The prevalence of these active membrane properties in response to the depolarizing current pulses suggested that the human MeSCs (hMeSCs) were capable of converting to a neural lineage under defined culture conditions. The spike-like events were blocked by the voltage-gated sodium channel inhibitor lidocaine, but unaffected by another sodium channel inhibitor tetrodotoxin (TTX). In calcium imaging experiments, the neuron-like cells responded to potassium chloride depolarization and l-glutamate application with increases in the cytoplasmic calcium levels. Thus, the neuron-like cells appeared to express TTX-resistant voltage-gated sodium channels, voltage-gated calcium channels, and functional l-glutamate receptors. These results demonstrate that hMeSCs were capable of generating cells with characteristics typical of functional neurons that may prove useful for neuroreplacement therapies.

Phenotypic characteristics of hybrid cells produced by cell fusion of porcine adrenal chromaffin cells with human mesenchymal stem cells: a preliminary study.

BACKGROUND AND PURPOSE: Transplantation of adrenal chromaffin cells (CCs) that release endogenous opioid peptides and catecholamines produces significant antinociceptive effects in patients with terminal cancer pain. In clinical practice, however, obtaining a sufficient number of chromaffin cells may not be possible because of the limited availability of human adrenal tissue. Recent works have shown that fusion of bone marrow-derived cells with differentiated cells can occur spontaneously in vivo and acquire the phenotype of the recipient cells. In this study, we investigated the possibility of producing chromaffin-like cells by fusing human bone marrow-derived mesenchymal stem cells (MeSCs) with post-mitotic porcine CCs in vitro with the application of polyethylene glycol (PEG). METHODS AND RESULTS: Before cell-to-cell fusion was initiated, MeSCs and CCs were labeled by fluorescence dyes DiO (green) and Dil (red), respectively. The hybrid cells generated by PEG-mediated fusion expressed a DiO and Dil double-staining with estimated fusion efficiency at approximately 40% of the total cell population. Further immunocytochemical examination for tyrosine hydroxylase and methionine enkephalin (markers for CCs) demonstrated positive immuno-reactivity in these hybrid cells 2 weeks post-fusion. More interestingly, some of the hybrid cells showed bromodeoxyuridine (BrdU)-positive immunostaining in the nuclei. DISCUSSION: Our results show that these hybrid cells fused by CCs and MeSCs express some characteristics of the CC phenotype. A subpopulation of these hybrid cells are dividing cells with positive BrdU immunostaining, suggesting that a novel cellular production could be developed by a "reprogramming" mechanism through the application of targeted cell fusion strategies.

Genetically engineered human mesenchymal stem cells produce met-enkephalin at augmented higher levels in vitro.

We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patient's bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.

Recombinant herpes vector-mediated analgesia in a primate model of hyperalgesia.

Some chronic pain syndromes are characterized by episodes of intense burning and hyperalgesia in localized areas of skin. These sensations are thought to be mediated, at least in part, by the activity of damaged, unmyelinated C nociceptors. These phenomena were modeled by assaying responses of macaques to thermal and chemical stimuli that produced periodic activation and sensitization of C nociceptors. Upon validation of this method, a recombinant herpes simplex vector encoding human preproenkephalin was topically applied to the dorsal surface of the feet of the monkeys. Immunohistochemistry and radioimmunoassay revealed that enkephalin peptides were being produced in releasable pools in sensory neurons innervating the treated skin area. Behavioral responses evoked by periodic sensitization and activation of C nociceptors innervating the vector-treated skin area revealed a substantial and long-lasting (at least 20 weeks) antihyperalgesic and analgesic effect limited to the areas to which the virus was applied. This approach may be a viable means of treating localized cutaneous burning pain and hyperalgesia.

Oxytocin receptors in brain cortical regions are reduced in haploinsufficient (+/-) reeler mice.

OBJECTIVE: Both oxytocin (OT) and reelin are particularly significant during development and the absence of either may interfere with normal brain development. In addition, reelin is critical to the development of the GABAergic system and GABA modulates the release of OT. Availability of the reelin haploinsufficient (+/-) reeler mouse (HRM) provides a model for examining the role of reelin in the development of the OT system and especially in the expression of the OT receptor (OTR). METHODS: In this study we used immunocytochemistry and in situ hybridization in HRM versus wild-type (+/-) mice (WTM) to quantify OTR abundance in regions of the brain cortex. RESULTS: Our findings reveal that the oxytocin receptor (OTR), measured either by immunohistochemistry or in situ hybridization, is significantly lower in HRM. Areas showing significant deficits included the piriform cortex, neocortex, retrosplenial cortex and certain regions of the hippocampus. CONCLUSION: Both reelin and OT play a role in regulating affect and mood. Down-regulation of reelin has been strongly correlated with schizophrenia and it is proposed that HRM may serve as a model for neural deficits seen in both schizophrenia and autism. We report that HRM show regionally specific reductions in OTRs, especially in cortical areas, which previously have been implicated in social memory and cognitive functions. These findings offer support for the more general hypothesis that down-regulation of reelin, of either genetic or epigenetic origin, through associated reductions in the OTRs, contributes to the deficiencies in social behavior that are characteristic of both schizophrenia and autism.

Secretion without membrane fusion: porocytosis.

We have recently proposed a mechanism to describe secretion, a fundamental process in all cells. That hypothesis, called porocytosis, embodies all available data and encompasses both forms of secretion, i.e., vesicular and constitutive. The current accepted view of exocytotic secretion involves the physical fusion of vesicle and plasma membranes; however, that hypothesized mechanism does not fit all available physiological data. Energetics of apposed lipid bilayers do not favor unfacilitated fusion. We consider that calcium ions (e.g., 10(-4) to 10(-3) M calcium in microdomains when elevated for 1 ms or less), whose mobility is restricted in space and time, establish salt bridges among adjacent lipid molecules. This establishes transient pores that span both the vesicle and plasma membrane lipid bilayers; the diameter of this transient pore would be approximately 1 nm (the diameter of a single lipid molecule). The lifetime of the transient pore is completely dependent on the duration of sufficient calcium ion levels. This places the porocytosis hypothesis for secretion squarely in the realm of the physical and physical chemical interactions of calcium and phospholipids and places mass action as the driving force for release of secretory material. The porocytosis hypothesis that we propose satisfies all of the observations and provides a framework to integrate our combined knowledge of vesicular and constitutive secretion.

Porocytosis: a transient pore array secretes the neurotransmitter packet.

The porocytosis hypothesis is based on the arrayed nature of synaptic vesicles which forms the anatomical functional unit of secretion. The presynaptic array and the postsynaptic array of receptors form a synaptomere which is the unit of transmission. A transient increase in calcium ions, triggered by an action potential, activates all pores of the array to pulse transmitter. The array insures transmission while permitting a frequency dependent amount of secretion. Therefore the amount of secretion is variable which permits plasticity. Secretion from the array has the property of immediate synaptic plasticity whereas a change in array size would change synaptic strength. The robust nature of the array insures fidelity of transmission, a frequency dependent dynamic signature of transmission giving the property of immediate plasticity; and, a change in array size yields a change in synaptic strength for long term reliability.

Porcine chromaffin cells, culture, and transplant for antinociceptive effects in rodents and primates.

It has been shown that xenografts and allografts of spinally transplanted adrenal chromaffin cells produce antinociception in animals and pain relief in patients with cancer pain. As there is a very limited availability of human adrenal tissue to serve as allografts, the clinical need for xenogeneic chromaffin cells as transplants is obvious. Bovine adrenal glands as a steady source of chromaffin cells have been extensively studied. There is however concern about the possible infection in humans with retrovirus following transplantation. The purpose of this study is to use the pig as a preferred donor animal species for xenotransplantation into rat and monkey. As pigs have been cloned, this opens the door to gene-targeted technologies and allows for genetic modifications, which possibly could improve the efficacy and safety of chromaffin cell transplantation. Porcine chromaffin cells were isolated from adrenal glands of 6-8-month-old pigs. After culturing cells for 1 week in a medium containing serum, the release of met-enkephalin and norepinephrine from the cells was detected by high-performance liquid chromatography and radioimmunoassay with nicotine stimulation, lasting approximately 3 weeks. Transplantation of these cells into the subarachnoid space of rats produced antinociceptive effects on Adelta and C fiber-mediated responses lasting 2-3 weeks. Similar findings were observed in studies with macaque monkeys. Compared with the same number of bovine chromaffin cells, porcine chromaffin cells showed a more robust and longer antinociceptive effect, and could be a better source of cells for human transplantation.

The synaptic bouton acts like a salt shaker.

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.

Immunocytochemical localization of reelin in the olfactory bulb of the heterozygous reeler mouse: an animal model for schizophrenia.

Because heterozygous reeler (HR) mice share some abnormal traits with schizophrenic patients, and schizophrenia is often accompanied by impairment of olfactory function, this study examines reelin in the olfactory bulb of the HR mouse. In the WT mouse, reelin immunoreactivity is found in the extracellular matrix, and in the cytoplasm of olfactory nerve fibers, GABAergic interneurons, and glutamatergic mitral cells. Western blot analysis reveals that reelin immunoreactivity in the HR mouse is reduced by 45% compared to WT mouse. This is especially evident in the glomerular GABAergic interneurons. In WT mitral cells, reelin is found in discrete clumps near the axon hillock and within the axon. In the HR mouse, reelin axonal staining is diffuse and densely packed. In the rostral migratory stream of the HR mouse, immunolabeling shows an accumulation of reelin-containing neuronal precursors, apparently unable to shift from tangential to radial migration. These observations indicate that there is a downregulation of reelin in the HR mouse and suggest that secretion of reelin may be compromised. Further studies of the HR mouse may provide a new basis for understanding the role of reelin in the adult CNS, especially as it may relate to schizophrenia.

Functional and morphological characteristics of bovine adrenal chromaffin cells on macroporous poly(D,L-lactide-co-glycolide) scaffolds.

Adrenal chromaffin cells (ACCs) secrete several neuroactive substances that are effective in influencing pain sensitivity in the central nervous system as well as enhancing the recovery of the intrinsic nigrostriatal dopaminergic system in patients with Parkinson's disease. ACC transplantation may be upregulated by the use of three-dimensional (3-D) scaffolds. In this study, we determined whether biodegradable poly(D,L-lactic-coglycolic acid) (PLGA) (85:15) sponges could be used as support for chromaffin cells. ACCs were isolated from bovine adrenal glands by standard perfusion (95% purity) followed by additional purification (>99.5% purity). ACC (approximately 5 x 10(5) cells) suspension in collagen (type I) was seeded on prewetted sponges and cultured in DMEM-F12 (1:1) medium (5% fetal bovine serum). The catecholamine and enkephalin levels of the samples were measured by high-performance liquid chromatography and radioimmunoassay. Cell morphology was examined by transmission electron microscopy. Morphological evidence showed prolonged viability of chromaffin cells on scaffolds having pores of 250-400 microm. Cell counts and scanning electron microscopy demonstrated that the majority of seeded cells were located within the scaffold. Chromaffin cells exhibited higher levels of enkephalins and catecholamines on PLGA scaffold compared with their monolayer cultures. By the use of 3-D PLGA as support for ACCs, it is possible to upregulate metabolic function and localize a high number of morphologically healthy-looking cells. Highly purified ACCs cultured on PLGA scaffold may have promise in transplantation studies, because these cells are less immunogenic and may be applied to in vivo settings by using short-term immunosuppression.

Proliferation of a subpopulation of reactive astrocytes following needle-insertion lesion in rat.

It is well known that traumatic injuries of the CNS induce a gliotic reaction, characterized by the presence of reactive astrocytes. Reactive astrocytes exhibit enhanced expression of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), hypertrophy, and thickened processes. Recently, we have demonstrated that injuries of the CNS induce a re-expression of an embryonic intermediate filament-associated protein, IFAP-70/280 kDa. Based on IFAP-70/280 kDa immunolabeling, we have shown that reactive astrocytes, activated by stab-wound injury, can be divided into two major groups: 1. persistent IFAP+/GFAP+ cells which are close to the wound in the area of glial scar, and 2. transient IFAP-/GFAP+ cells which are farther from the wound. In this study, we use BrdU incorporation to examine proliferation in these two groups of reactive astrocytes induced by stab injury of the rat cerebrum. Triple/double-label immunofluorescence microscopy was performed using antibodies to IFAP-70/280 kDa, GFAP, and BrdU. The results showed that BrdU+ reactive astrocytes (GFAP+) were always IFAB-70/280 kDa+ as well. However, not all IFAP+ reactive astrocytes are BrdU+. BrdU+ signal was not observed in any IFAP- reactive astrocytes. At five days post-lesion, IFAP+ reactive astrocytes were increasing in the area of the wound (0-50 micrograms from the wound edge), but had reached a peak in the proximal area (50-800 micrograms away from the wound edge). At eight days post-lesion, IFAP+ reactive astrocytes achieved the highest percentage in the wound area. At the same time, BrdU-containing reactive astrocytes occupied an area closer to the wound. By 20 days post-lesion, following the formation of the gliotic scar at the stab-wound, a few IFAP+/GFAP+ cells still persisted. BrdU-containing reactive astrocytes were only observed in the scar. These results indicate that many IFAP+ reactive astrocytes close to the wound, in contrast to the IFAP- ones farther from the wound, appear to regain their proliferative potential to increase in number and participate in the formation of the gliotic scar.

Porocytosis: secretion from small and medium-diameter vesicles and vesicle arrays without membrane fusion.

We have recently proposed a mechanism to describe secretion, a fundament process in all cells. That hypothesis, called porocytosis, embodies all available data, and encompasses both forms of secretion, i.e., vesicular and constitutive. The current accepted view of exocytotic secretion involves the physical fusion of vesicle- and plasma membranes. However, that hypothesized mechanism does not fit all available physiological data (Silver et al., 2001; Kriebel et al., 2001). Energetics of apposed lipid bilayers do not favor unfacilitated fusion. Calcium ion levels are elevated in microdomains at levels of 10(-4)-10(-3)M for 1 ms or less, with the calcium ions showing limited lateral mobility at the site of secretion (Llinas et al., 1992, Silver et al., 1994). We consider that calcium ions, whose mobility is restricted in space and time, establish "salt-bridges" among adjacent lipid molecules, and establishes transient pores that span the vesicle and plasma membrane lipid bilayers; the lifetime of that transient pore being completely dependent on duration of sufficient calcium ion levels.

ALLEVIATION OF PAIN IN CANCER PATIENTS BY ADRENAL MEDULLARY TRANSPLANTS IN THE SPINAL SUBARACHNOID SPACE.

The treatment of intractable pain with currently available therapeutic regimens is often unsatisfactory due to tolerance and untoward complications. Studies in our laboratory have suggested that the transplantation of adrenal medullary tissue into the spinal subarachnoid space can significantly reduce pain in animal models, most likely via release of opioid peptides and catecholamines. The current study was an initial attempt to assess the potential for adrenal medullary transplants in the spinal subarachnoid space to alleviate pain in humans. Donor adrenal medullary tissue was prepared for transplantation in our laboratory. One cc of adrenal medullary tissue was transplanted via lumbar puncture in five patients suffering from terminal cancer pain. Pain levels were determined using a Visual Analog Pain Scale prior to and following the transplantation procedure. In addition, records of narcotic intake and activity were kept. When possible, CSF samples were collected via lumbar puncture for biochemical and cytological analysis. Four of the patients demonstrated progressive decreases in pain scores following the procedure, with concomitant reductions in narcotic intake. Three patients remained pain free, two for over 10 mo. One patient, who developed spinal cord compression secondary to metastasis, was initially pain free, but the pain returned after 10 wk. The fifth patient had no pain reduction by 1 mo following the procedure, but further information was unavailable due to poor patient compliance. In most cases, spinal cerebrospinal fluid (CSF) samples revealed increased levels of met-enkephalin and/or catecholamines following the transplants. The results of this study suggest that adrenal medullary transplants in the spinal subarachnoid space have potential as an alternative approach to the management of chronic pain in humans.

Adrenal medullary implants in the rat spinal cord reduce nociception in a chronic pain model.

Previous work in this laboratory has indicated that the transplantation of adrenal medullary tissue into the subarachnoid space of the rat spinal cord can reduce pain sensitivity to acute noxious stimuli, particularly following stimulation by nicotine. This most likely results from the stimulated release of opioid peptides and catecholamines from the transplanted chromaffin cells. However, chronic pain models may more closely resemble human clinical pain, and the arthritic rat model has been used for screening potential therapeutic strategies. The purpose of the present study was to assess the potential for adrenal medullary tissue implanted into the spinal subarachnoid space to alleviate chronic pain. Adrenal medullary tissue was implanted into adjuvant-induced arthritic rats, and changes in body weight and vocalization responses were monitored over the 10 week course of the disease. Results indicate that the severe weight reduction normally associated with this inflammatory arthritis was attenuated by adrenal medullary, but not control, implants. In addition, vocalizations were reduced in animals implanted with adrenal medullary, but not control tissue following nicotine stimulation. This reduction was blocked by the opiate antagonist, naloxone, and partially attenuated by the alpha-adrenergic antagonist, phentolamine. Together, these results suggest that the transplantation of adrenal medullary tissue into the subarachnoid space of the spinal cord may provide a local source of opioid peptides and catecholamines for the reduction of chronic pain.