Reestablishment of the endothelial lining by endothelial cell therapy stabilizes experimental abdominal aortic aneurysms.

BACKGROUND: Loss of the endothelium and its replacement by a thick thrombus are structural features of human abdominal aortic aneurysms (AAAs). In AAAs, the relationship between aortic diameter expansion, the presence of thrombus, and the lack of endothelial cells (ECs) remains unexplored. We hypothesized that reendothelialization by cell therapy would modulate aortic wall destruction and ultimately stabilize AAAs. We evaluated the impact of local seeding of rat aortic ECs or peripheral blood-derived outgrowth ECs on AAA evolution. METHODS AND RESULTS: Rat aortic ECs (n=30) or serum-free medium (controls; n=29) were seeded endovascularly immediately (day 0) or 14 days after surgery in the rat xenograft model. Rat aortic EC seeding prevented AAA formation and stabilized formed AAAs at 28 days (diameter increase at day 0+28, 51±6% versus 83±6%; day 14+28, -1±4% versus 22±6% in rat aortic ECs and controls, respectively; P<0.01). This stabilizing effect was associated with the reestablishment of the endothelial lining, the suspension of proteolysis, and the reconstitution of new aortic wall rich in smooth muscle cells and extracellular matrix. Transplanted rat aortic ECs did not participate directly in aortic wall repair but exerted their healing properties through paracrine mechanisms involving the upregulation of endothelium-derived stabilizing factors and the recruitment of resident vascular cells. In rats, the transplantation of outgrowth ECs (n=7) significantly reduced by 30% the progression of AAAs and restored the abluminal endothelium at 28 days compared with controls (n=9). CONCLUSION: Our study demonstrates the potential of restoring the endothelial lining to control AAA dynamics and designates ECs as an efficient therapy to stop AAA expansion.

Skilled motor control for the preclinical assessment of functional deficits and recovery following nigral and striatal cell transplantation.

Neural transplantation holds the promise for restoring behavioral function following brain injury. Substantial evidence indicates that fetal neurons transplanted to the adult brain survive and incorporate into remaining neural tissue and produce positive behavioral effects. A yet-unanswered question is whether the integration of new tissue can restore complex neural circuits that connect the neocortex, basal ganglia, and brainstem, and restore functions that are mediated by these circuits. This chapter describes the skilled reaching task, a task that requires transport of the arm and hand to grasp a food item and withdrawal to place the food item in the mouth for eating. It is a movement that is readily expressed in rodents and is fundamental to both nonhuman primates and humans. Methods for analyzing skilled reaching have been developed for preclinical rodent and mouse models of Parkinson's disease and Huntington's disease that are generalizable to humans with those clinical disorders. It is suggested that the task provides a motor benchmark for assessing the restoration of function produced by neural transplantation.

Suppression of ß1-integrin in gonadotropin-releasing hormone cells disrupts migration and axonal extension resulting in severe reproductive alterations.

Reproduction in mammals is dependent on the function of hypothalamic neurons whose axons project to the hypothalamic median eminence (ME) where they release gonadotropin-releasing hormone (GnRH) into a specialized capillary network for delivery to the anterior pituitary. These neurons originate prenatally in the nasal placode and migrate into the forebrain along the olfactory-vomeronasal nerves. The complex developmental events leading to the correct establishment of the GnRH system are tightly regulated by the specific spatiotemporal expression patterns of guidance cues and extracellular matrix molecules, the functions of which, in part, are mediated by their binding to ß1-subunit-containing integrins. To determine the biological role of these cell-surface proteins in reproduction, Cre/LoxP technology was used to generate GnRH neuron-specific ß1-integrin conditional KO (GnRH-Itgb1(-/-)) mice. Loss of ß1-integrin signaling impaired migration of GnRH neurons, their axonal extension to the ME, timing of pubertal onset, and fertility in these mice. These results identify ß1-integrin as a gene involved in normal development of the GnRH system and demonstrate a fundamental role for this protein in acquisition of normal reproductive competence in female mice.

Schwann cell transplantation: a repair strategy for spinal cord injury?

Schwann cells (SCs), when implanted in the injured spinal cord, support regeneration of axons, myelinate or ensheathe regenerated axons in a normal way, reduce cyst formation in the injured tissue, reduce secondary damage of tissue around the initial injury site, and modestly improve limb movements. If SC transplantation is combined with additional treatments such as methylprednisolone, neurotrophins, GDNF, olfactory ensheathing cells, chondroitinase, or elevation of cAMP levels, more axons (including those from neurons in the brainstem) regenerate into and out of the SC implant and further improve locomotion. Recent work to improve SC migration from the implant into the spinal cord by polysialylating NCAM on the SC surface has led to the novel finding that corticospinal axon growth is promoted by SCs. Recent studies are cited showing that when astrocytes extend slender processes into an implant instead of forming a sharp boundary they are permissive rather than inhibitory to axonal regrowth. The interfaces that comprise the "on-ramps" and the "off-ramps" are key to the success of a SC implant to span the injury site and to foster axon regeneration across the injury.

Feasibility of localized immunosuppression: 3. Preliminary evaluation of organosilicone constructs designed for sustained drug release in a cell transplant environment using dexamethasone.

As part of our ongoing effort to develop biohybrid devices for pancreatic islet transplantation, we are interested in establishing the feasibility of a localized immune-suppressive approach to avoid or minimize the undesirable side effects of existing systemic treatments. Since biohybrid devices can also incorporate biocompatible scaffold constructs to provide a support environment for the transplanted cells that enhances their engraftment and long-term function, we are particularly interested in an approach that would use the same three-dimensional construct, or part of the same construct, to also provide sustained release of therapeutic agents to modulate the inflammatory and immune responses locally. Within this framework, here, we report preliminary results obtained during the investigation of the suitability of organosilicone constructs for providing sustained localized drug release using small, matrix-type polydimethylsiloxane (PDMS) disks and dexamethasone as a model hydrophobic drug. Following a short burst, long-term steady sustained release was observed under in vitro conditions at levels of 0.1-0.5 microg/day/disk with a profile in excellent agreement with that predicted by the Higuchi equation. To verify that therapeutic levels can be achieved, suppression of LPS-induced activation has been shown in THP-1 cells with disks that have been pre-soaked for up to 28 days. These preliminary results prove the feasibility of this approach where an integral part of the biomaterial construct used to enhance cell engraftment and long-term function also serves to provide sustained local drug release.

Islet graft survival and function: concomitant culture and transplantation with vascular endothelial cells in diabetic rats.

BACKGROUND: Human islet transplantation is a great potential therapy for type I diabetes. To investigate islet graft survival and function, we recently showed the improved effects after co-culture and co-transplantation with vascular endothelial cells (ECs) in diabetic rats. METHODS: ECs were isolated, and the viability of isolated islets was assessed in two groups (standard culture group and co-culture group with ECs). Then streptozotocin-induced diabetic rats were divided into four groups before islet transplantation as follows: group A with infusion of islet grafts; group B with combined vascular ECs and islet grafts; groups C and D as controls with single ECs infusion and phosphate-buffered saline injection, respectively. Blood glucose and insulin concentrations were measured daily. Expression of vascular endothelial growth factor was investigated by immunohistochemical staining. The mean microvascular density was also calculated. RESULTS: More than 90% of acridine orange-propidium iodide staining positive islets demonstrated normal morphology while co-cultured with ECs for 7 days. Compared with standard control, insulin release assays showed a significantly higher simulation index in co-culture group except for the first day (P<0.05). After transplantation, there was a significant difference in concentrations of blood glucose and insulin among these groups after 3 days (P<0.05). The mean microvascular density in co-culture group was significantly higher than that in single islet group (P=0.04). CONCLUSION: Co-culture with ECs in vitro could improve the survival and function of isolated rat islet, and co-transplantation of islets with ECs could effectively prolong the islet graft survival in diabetic rats.

Cell replacement therapy for Parkinson's disease: how close are we to the clinic?

Cell replacement therapy (CRT) offers great promise as the future of regenerative medicine in Parkinson´s disease (PD). Three decades of experiments have accumulated a wealth of knowledge regarding the replacement of dying neurons by new and healthy dopaminergic neurons transplanted into the brains of animal models and affected patients. The first clinical trials provided the proof of principle for CRT in PD. In these experiments, intrastriatal transplantation of human embryonic mesencephalic tissue reinnervated the striatum, restored dopamine levels and showed motor improvements. Sequential controlled studies highlighted several problems that should be addressed prior to the wide application of CRT for PD patients. Moreover, owing to ethical and practical problems, embryonic stem cells require replacement by better-suited stem cells. Several obstacles remain to be surpassed, including identifying the best source of stem cells for A9 dopaminergic neuron generation, eliminating the risk of tumor formation and the development of graft-induced dyskinesias, and standardizing dopaminergic cell production in order to enable clinical application. In this article, we present an update on CRT for PD, reviewing the research milestones, various stem cells used and tailored differentiation methods, and analyze the information gained from the clinical trials.

Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1.

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance.

Compensation of depleted neuronal subsets by new neurons in a local area of the adult olfactory bulb.

In the olfactory bulb (OB), loss of preexisting granule cells (GCs) and incorporation of adult-born new GCs continues throughout life. GCs consist of distinct subsets. Here, we examined whether the loss and incorporation of GC subsets are coordinated in the OB. We classified GCs into mGluR2-expressing and -negative subsets and selectively ablated mGluR2-expressing GCs in a local area of the OB with immunotoxin-mediated cell ablation method. The density of mGluR2-expressing GCs showed considerable recovery within several weeks after the ablation. During recovery, an mGluR2-expressing new GC subset was preferentially incorporated over an mGluR2-negative new GC subset in the area of ablation, whereas the preferential incorporation was not observed in the intact area. The area-specific preferential incorporation of mGluR2-expressing new GCs occurred for BrdU analog- and retrovirus-labeled adult-born cells as well as for neonate-derived transplanted cells. The mGluR2-expressing new GCs in the ablated area were synaptically incorporated into the local bulbar circuit. The spine size of mGluR2-expressing new GCs in the ablated area was larger than that of those in the intact area. In contrast, mGluR2-negative new GCs did not show ablated area-specific spine enlargement. These results indicate that local OB areas have a mechanism to coordinate the loss and incorporation of GC subsets by compensatory incorporation of new GC subsets, which involves subset-specific cellular incorporation and subset-specific regulation of spine size.

Concise review: making a retina--from the building blocks to clinical applications.

The retina is the neural tissue located at the back of the eye that captures and processes light and transmits this information to visual processing centers in the brain, which enables us to see. Basic research in retinal development has provided important insight on the control of cell fate, proliferation, and neurogenesis in the central nervous system. This review summarizes the major cellular and molecular events that occur during retinal development and highlights how this knowledge may be harnessed for new therapeutic strategies to treat retinal disease.

Remyelination after olfactory ensheathing cell transplantation into diverse demyelinating environments.

Olfactory ensheathing cells (OECs) can remyelinate demyelinated spinal cord axons when transplanted into chemically induced demyelinated lesions. Cell transplantation is typically performed within a few days after lesion induction, i.e. during active demyelination when myelin debris, cytokine level increases and macrophage/microglia activation is extensive. Inflammatory signaling has been suggested to facilitate remyelination in cell transplant studies. In this review we discuss the migration and remyelination properties of OECs transplanted into various demyelinating lesion environments including conditions when inflammation is active and when it is largely subsided. While sharing many common properties, comparisons of the in vivo fate between OECs and SCs suggest unique properties of OECs as compared to SCs. A complicating factor in the assessment of experimental remyelination by transplantation of myelin-forming cells in general is the rapidity of endogenous myelin repair in most rodent models of demyelination. Alternative persistent demyelination models are discussed as potential tools to study both the competency of chronic demyelinated axons for remyelination and the remyelination potential of cells such as human progenitors that require longer times to mobilize and remyelinate axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection.

Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT(2A) receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Olfactory ensheathing glia: repairing injury to the mammalian visual system.

The visual system is widely used as a model in which to study neurotrauma of the central nervous system and to assess the effects of experimental therapies. Adult mammalian retinal ganglion cell axons do not normally regenerate their axons for long distances following injury. Trauma to the visual system, particularly damage to the optic nerve or central visual tracts, causes loss of electrical communication between the retina and visual processing areas in the brain. After optic nerve crush or transection, axons degenerate and retinal ganglion cells (RGCs) are lost over a period of days. To promote and maintain axonal growth and connectivity, strategies must be developed to limit RGC death and provide regenerating axons with permissive substrates and a sustainable growth milieu that will ultimately provide long term visual function. This review explores the role olfactory glia can play in this repair. We describe the isolation of these cells from the olfactory system, transplantation to the brain, gene therapy and the possible benefits that these cells may have over other cellular therapies to initiate repair, in particular the stimulation of axonal regeneration in visual pathways. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Clinical prospects for transplantation of OECs in the repair of brachial and lumbosacral plexus injuries: opening a door.

The reparative effects of olfactory ensheathing cells have largely been examined in lesions entirely within the CNS. There is, however, evidence that they can induce the ingrowth of severed dorsal root axons and increase the outgrowth of severed ventral root axons. The ingrowth of dorsal root axons results in reinnervation of appropriate regions in the spinal cord and dorsal column nuclei with restoration of electrical transmission and muscular control. This article discusses the further possibilities of these observations in rat studies and their potential translation to clinical injuries. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Nasal OEC transplantation promotes respiratory recovery in a subchronic rat model of cervical spinal cord contusion.

Engraftment of nasal olfactory ensheathing cells (OEC) is considered as a promising therapeutic strategy for spinal cord repair and one clinical trial has already been initiated. However, while the vast majority of fundamental studies were focused on the recovery of locomotor function, the efficiency of this cellular tool for repairing respiratory motor dysfunction, which affects more than half of paraplegic/tetraplegic patients, remains unknown. Using a rat model that mimics the mechanisms encountered after a cervical contusion that induces a persistent hemi-diaphragmatic paralysis, we assessed the therapeutic efficiency of a delayed transplantation (2 weeks post-contusion) of nasal OECs within the injured spinal cord. Functional recovery was quantified with respiratory behavior tests, diaphragmatic electromyography and neuro-electrophysiological recording of the phrenic motoneurons while axogenesis was evaluated using immunohistochemistry. We show that 3 months post-transplantation, nasal OECs improve i) breathing movements, ii) activities of the ipsilateral diaphragm and corresponding phrenic nerve, and iii) axonal sprouting in the injury site. We also demonstrate that this functional partial recovery is mediated by the restoration of ipsilateral supraspinal command. Our study brings further evidence that olfactory ensheathing cells could have clinical application especially in tetraplegic patients with impaired breathing movements. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Survival and differentiation of neuroectodermal cells with stem cell properties at different oxygen levels.

Freeze-lesioned regions of the forebrain cortex provide adequate environment for growth of non-differentiated neural progenitors, but do not support their neuron formation. Reduced oxygen supply, among numerous factors, was suspected to impair neuronal cell fate commitment. In the present study, proliferation and differentiation of neural stem/progenitor cells were investigated at different oxygen levels both in vitro and in vivo. Low (1% atmospheric) oxygen supply did not affect the in vitro viability and proliferation of stem cells or the transcription of "stemness" genes but impaired the viability of committed neuronal progenitors and the expression of proneural and neuronal genes. Consequently, the rate of in vitro neuron formation was markedly reduced under hypoxic conditions. In vivo, neural stem/progenitor cells survived and proliferated in freeze-lesioned adult mouse forebrains, but did not develop into neurons. Hypoperfusion-caused hypoxia in lesioned cortices was partially corrected by hyperbaric oxygen treatment (HBOT). HBOT, while reduced the rate of cell proliferation at the lesion site, resulted in sporadic neuron formation from implanted neural stem cells. The data indicate that in hypoxic brain areas, neural stem cells survive and proliferate, but neural tissue-type differentiation can not proceed. Oxygenation renders the damaged brain areas more permissive for tissue-type differentiation and may help the integration of neural stem/progenitor cells.

Manual vs automated delivery of cells for transplantation: accuracy, reproducibility, and impact on viability.

BACKGROUND: Cellular transplantation holds promise for the management of a variety of neurological disorders. However, there is great variability in cell type, preparation methods, and implantation technique, which are crucial to clinical outcomes. OBJECTIVE: We compared manual injection with automated injection using a prototype device to determine the possible value of a mechanized delivery system. METHODS: Neural progenitor cells and bone marrow stromal cells were injected using manual or automated methods. Consistency of injection volumes and cell number and viability were evaluated immediately or 1 day after injection. RESULTS: When cells were delivered as a series of 3 manual injections from the same syringe, the variation in fluid volume was greater than for single manual injections. Automated delivery of a series of 3 injections resulted in a lower variability in the amount of delivery than manual injection for both cell lines (1.2%-2.6% coefficient of variability for automated delivery vs 4.3%-24.0% for manual delivery). The amount delivered from injection 1 to injection 3 increased significantly with manual injections, whereas the amount injected did not vary over the 3 injections for the automated unit. Cell viability 1 day after injection was typically 30% to 40% of the value immediately after injection for the bone marrow stromal cells and 30% to 70% for the neural progenitor cells. There were no significant differences in viability attributed to the method of injection. CONCLUSION: The automated delivery device led to enhanced consistency of volumetric cell delivery but did not improve cell viability in the methods tested. Automated techniques could be useful in standardizing reproducible procedures for cell transplantation and improve both preclinical and clinical research.

Effect of cryopreservation on cell proliferation and immunogenicity of transplanted human heart cells.

BACKGROUND: Cell preservation is essential for successful cell transplantation and/or tissue engineering. We examined the effects of cryopreservation on the transplantation of human heart cells. METHODS: Cells isolated from human atrial tissues were cultured for 15 days (control group), cryopreserved for 1 week, and rapidly thawed and cultured for 15 days. Proliferation was compared among control and cryopreserved cells or tissues by constructing growth curves. Growth factors, cytokines, biochemical features, and cell cycle phase were measured immediately before and after cryopreservation, and immunogenicity was evaluated from growth curves generated from heart cells after 7 days in mixed-lymphocyte culture. Control or cryopreserved cells were transplanted into rat connective tissues and evaluated histologically 2 weeks later. RESULTS: Cryopreserved cells proliferated more effectively than control cells. Levels of basic fibroblast growth factor and transforming growth factor-ß1 were significantly higher, and those of interleukin (IL)-6 and IL-8 were significantly lower after cryopreservation. Fewer peripheral blood lymphocytes were produced in cryopreserved cells than in noncryopreserved cells, and the cell cycle phase of cryopreserved heart cells shifted primarily to G2 + M from G1 + G0. Noncryopreserved and cryopreserved cells both survived in connective tissue. CONCLUSION: Human atrial cells can be cultured, cryopreserved, and transplanted. Cryopreservation might increase the proliferation of human cells and tissues and also reduce the immunogenicity of heart cells.

Lack of effect of intranigral transplants of a GABAergic cell line on absence seizures.

The substantia nigra pars reticulata (SNpr) is involved in controlling a variety of seizure phenomena. Intranigral transplants of GABAergic cells have been shown to decrease the severity of already established epileptic seizures, but the effects observed have been short-lived. This study evaluated the ability of intranigral transplants of GABA-producing cells to reduce spontaneous absence seizures in a genetic animal model for periods up to 3 months after transplantation. Intranigral transplants did not induce any behavioral deficits in the animals, and they did not form tumors; however, the transplants failed to decrease absence seizures in the genetic model. The assumed increase in intranigral levels of GABA after the transplants may be insufficient to counteract all the factors involved in generating the absence seizures; in this animal model, it may be necessary to further decrease nigral activity by implanting GABAergic cells in another area. These results bear down on the fact that cell transplants need to be tailored for each type of convulsive disorder in terms of the type of cells delivered and the location of the transplants.

Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.

Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11-30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.