Engineered adenosine-releasing cells for epilepsy therapy: Human mesenchymal stem cells and human embryonic stem cells

Adenosine is a modulator of neuronal activity with anticonvulsant and neuroprotective properties. Conversely, focal deficiency in adenosine contributes to ictogenesis. Thus, focal reconstitution of adenosine within an epileptogenic brain region constitutes a rational therapeutic approach, whereas systemic augmentation of adenosine is precluded by side effects. To meet the therapeutic goal of focal adenosine augmentation, genetic disruption of the adenosine metabolizing enzyme, adenosine kinase (ADK) in rodent cells was used as a molecular strategy to induce adenosine release from cellular brain implants, which demonstrated antiepileptic and neuroprotective properties. Currently, the second generation of adenosine-releasing cells is under development based on the rationale to use human stem cell-derived brain implants to avoid xenotransplantation. To effectively engineer human stem cells to release adenosine, a lentiviral vector was constructed to express inhibitory micro-RNA directed against ADK. Lentiviral knockdown of ADK induced therapeutic adenosine release in human mesenchymal stem cells, which reduced acute injury and seizures, as well as chronic seizures, when grafted into the mouse hippocampus. The therapeutic potential of this approach suggests the feasibility to engineer autologous adenosine-releasing stem cells derived from a patient. Human embryonic stem cells (hESCs) have a high proliferative capacity and can be subjected to specific cellular differentiation pathways. hESCs, differentiated in vitro into neuroepithelial cells and grafted into the mouse brain, displayed intrahippocampal location and neuronal morphology. Using the same lentiviral micro-RNA vector, we demonstrated knockdown of ADK in hESCs. New developments and therapeutic challenges in using human mesenchymal stem cells and hESCs for epilepsy therapy will be critically evaluated.     

Suppression of kindled seizures by paracrine adenosine release from stem cell-derived brain implants

PURPOSE: Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery because of (a) their unique ability to differentiate into various somatic cell types, (b) the virtually unlimited donor source for transplantation, and (c) the advantage of being amenable to a wide spectrum of genetic manipulations. Previously, adenosine-releasing embryonic stem (ES) cells have been generated by disruption of both alleles of adenosine kinase (Adk-/-). Lack of ADK did not compromise the cells' differentiation potential into embryoid bodies or glial precursor cells. The aim of the present study was to investigate the potential of differentiated Adk-/- ES cell progeny for seizure suppression by paracrine adenosine release. METHODS: To isolate paracrine effects of stem cell-derived implants from effects caused by network integration, ES cell-derived embryoid bodies and glial precursor cells were encapsulated into semipermeable polymer membranes and grafted into the lateral brain ventricles of kindled rats. RESULTS: While seizure activity in kindled rats with wild-type Adk+/+ implants remained unaltered, rats with adenosine-releasing Adk-/- ES cell-derived implants displayed transient protection from convulsive seizures and a profound reduction of afterdischarge activity in EEG recordings. Long-term seizure suppression was precluded by limited viability of the encapsulated cells. CONCLUSIONS: We thereby provide a proof-of-principle that Adk-/- ES cell-derived brain implants can suppress seizure activity by a paracrine mode of action. Adk-deficient stem cells therefore represent a potential tool for the treatment of epileptic disorders.     

Basic Science

Martin Güttinger , Denise Fedele , Peter Koch , Vivianne Padrun , William F. Pralong , Oliver Brüstle , and Detlev Boison
Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery because of (a) their unique ability to differentiate into various somatic cell types, (b) the virtually unlimited donor source for transplantation, and (c) the advantage of being amenable to a wide spectrum of genetic manipulations. Embryonic stem (ES) cells have been engineered to release adenosine, an endogenous antiepileptic substance of the brain. This has been achieved by a genetic disruption of the gene coding for adenosine kinase (ADK), the major adenosine-removing enzyme. Lack of ADK did not compromise the potential of the cells to differentiate into specialized cell types. The aim of the present study was to investigate the potential of adenosine releasing ES cell–derived brain implants for seizure suppression. To isolate local adenosine-mediated effects from effects caused by integration of the transplanted cells into preexisting networks, ES cell–derived embryoid bodies and glial precursor cells were encapsulated into semipermeable polymer membranes and grafted into the lateral brain ventricle of kindled rats, a widely used model of temporal lobe epilepsy. Although seizure activity in kindled rats with normal control implants remained unaltered, rats with adenosine-releasing ES cell–derived implants displayed transient protection from convulsive seizures and a profound reduction of seizure activity in recordings of electrical brain activity. We thereby provide a proof-of-principle that adenosine-releasing ES cell–derived brain implants can suppress seizure activity by a local, paracrine mode of action. ADK-deficient stem cells therefore represent a potential tool for the treatment of epileptic disorders.



     

Adenosine kinase as a target for therapeutic antisense strategies in epilepsy

Purpose: Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral‐mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures have received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte‐based key negative regulator of the brain’s endogenous anticonvulsant adenosine, as a potential therapeutic target for antisense‐mediated seizure suppression. Methods: We developed adenoassociated virus 8 (AAV8)‐based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk‐cDNA in sense or antisense orientation under the control of an astrocyte‐specific gfaABC1D promoter. Viral vectors where injected into the CA3 of wild‐type mice or spontaneously epileptic Adk‐tg transgenic mice that overexpress ADK in brain. After virus injection, ADK expression was assessed histologically and biochemically. In addition, intracranial electroencephalography (EEG) recordings were obtained. Key Findings: We demonstrate in wild‐type mice that viral overexpression of ADK within astrocytes is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event, whereas ADK downregulation via AAV8‐mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk‐tg mice. Significance: Our data demonstrate that modulation of astrocytic ADK expression can trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy.     

Local disruption of glial adenosine homeostasis in mice associates with focal electrographic seizures: A first step in epileptogenesis?

Astrogliosis and associated dysfunction of adenosine homeostasis are pathological hallmarks of the epileptic brain and thought to contribute to seizure generation in epilepsy. The authors hypothesized that astrogliosis-an early component of the epileptogenic cascade-might be linked to focal seizure onset. To isolate the contribution of astrogliosis to ictogenesis from other pathological events involved in epilepsy, the authors used a minimalistic model of epileptogenesis in mice, based on a focal onset status epilepticus triggered by intra-amygdaloid injection of kainic acid. The authors demonstrate acute neuronal cell loss restricted to the injected amygdala and ipsilateral CA3, followed 3 weeks later by focal astrogliosis and overexpression of the adenosine-metabolizing enzyme adenosine kinase (ADK). Using synchronous electroencephalographic recordings from multiple depth electrodes, the authors identify the KA-injected amygdala and ipsilateral CA3 as two independent foci for the initiation of non-synchronized electrographic subclinical seizures. Importantly, seizures remained focal and restricted to areas of ADK overexpression. However, after systemic application of a non-convulsive dose of an adenosine A(1) -receptor antagonist, seizures in amygdala and CA3 immediately synchronized and spread throughout the cortex, leading to convulsive seizures. This focal seizure phenotype remained stable over at least several weeks. We conclude that astrogliosis via disruption of adenosine homeostasis per se and in the absence of any other overt pathology, is associated with the emergence of spontaneous recurrent subclinical seizures, which remain stable over space and time. A secondary event, here mimicked by brain-wide disruption of adenosine signaling, is likely required to turn pre-existing subclinical seizures into a clinical seizure phenotype.     

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.     

Inhibitory RNA in epilepsy: Research tools and therapeutic perspectives

Since its discovery a decade ago, RNA interference (RNAi) has been developed not only into powerful experimental tools but also into promising novel therapeutics. In contrast to conventional antiepileptic drugs (AEDs) that target specific proteins such as ion channels or receptors, RNAi‐based therapeutics exploit an endogenous regulatory mechanism of gene expression and thereby are poised to prevent or reverse pathogenetic mechanisms involved in seizure development. Therapeutic RNAi has been widely explored for dominant targets involved in neurodegenerative diseases; however, their use for epilepsy therapy has received less attention. This review discusses potential RNAi‐based targets that are of interest for epilepsy therapy, including adenosine kinase (ADK), the key negative regulator of the brain’s endogenous anticonvulsant adenosine. Overexpression of ADK, and the resulting adenosine deficiency, are pathologic hallmarks of the sclerotic epileptic brain, and have been implicated in seizure generation. Therefore, RNAi‐strategies aimed at reducing ADK (and increasing adenosine) are based on a direct neurochemical rationale that has recently been explored experimentally using ex vivo and in vivo gene therapy approaches. Technical issues and challenges remain before those promising tools can be developed into future therapeutics for epilepsy.     

Seizure suppression by adenosine A1 receptor activation in a mouse model of pharmacoresistant epilepsy

PURPOSE: Because of the high incidence of pharmacoresistance in the treatment of epilepsy (20-30%), alternative treatment strategies are needed. Recently a proof-of-principle for a new therapeutic approach was established by the intraventricular delivery of adenosine released from implants of engineered cells. Adenosine-releasing implants were found to be effective in seizure suppression in a rat model of temporal lobe epilepsy. In the present study, activation of the adenosine system was applied as a possible treatment for pharmacoresistant epilepsy. METHODS: A mouse model for drug-resistant mesial temporal lobe epilepsy was used, in which recurrent spontaneous seizure activity was induced by a single intrahippocampal injection of kainic acid (KA; 200 ng in 50 nl). RESULTS: After injection of the selective adenosine A1-receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA; either 1.5 or 3 mg/kg, i.p.), epileptic discharges determined in EEG recordings were completely suppressed for a period of 相似文献   

Antiepileptic effects of silk-polymer based adenosine release in kindled rats

Pharmacotherapy for epilepsy is limited by high incidence of pharmacoresistance and failure to prevent development and progression of epilepsy. Using the rat hippocampal kindling model, we report on the therapeutic potential of novel silk-based polymers engineered to release the anticonvulsant adenosine. Polymers were designed to release 1000 ng adenosine per day during a time span of ten days. In the first experiment rats were kindled by hippocampal electrical stimulation until all animals reacted with stage 5 seizures. Adenosine-releasing or control polymers were then implanted into the infrahippocampal fissure ipsilateral to the site of stimulation. Subsequently, only recipients of adenosine-releasing implants were completely protected from generalized seizures over a period of ten days corresponding to the duration of sustained adenosine release. To monitor seizure development in the presence of adenosine, adenosine-releasing or control polymers were implanted prior to kindling. After 30 stimulations – delivered from days 4 to 8 after implantation – control animals had developed convulsive stage 5 seizures, whereas recipients of adenosine-releasing implants were still protected from convulsive seizures. Kindling was resumed after nine days to allow expiration of adenosine release. During additional 30 stimulations, recipients of adenosine-releasing implants gradually resumed kindling development at seizure stages corresponding to those when kindling was initially suspended, while control rats resumed kindling development at convulsive seizure stages. Blockade of adenosine A₁ receptors did not exacerbate seizures in protected animals. We conclude that silk-based adenosine delivery exerts potent anti-ictogenic effects, but might also have at least partial anti-epileptogenic effects. Thus, silk-based adenosine augmentation holds promise for the treatment of epilepsy.     

Transplantation of neuronal cells induced from human mesenchymal stem cells improves neurological functions after stroke without cell fusion

The options for treating stroke are limited, but stem cells hold promise as a therapy because of their multipotency. Neuronal cells derived from mesenchymal stem cells (MSC) were reported to have more therapeutic effect than MSCs. For elucidating the therapeutic mechanism of neuronal cells, here we generated a model of focal cerebral infarction by performing left common carotid artery occlusion in adult gerbils. We transfected human trabecular bone-derived MSCs (hMSCs) with the Notch intracellular domain to induce their differentiation into neuronal cells (hN-MSCs). These cells were stereotaxically transplanted into the local ischemic hemisphere 4 days after the occlusion. Behavioral analyses were conducted 28 days after transplantation, and then fluorescence in situ hybridization (FISH) and a histological evaluation were performed. Histologically, transplanted cells were distributed around the periinfarct region, and approximately 8.5% and 4.2% of hN-MSCs and hMSCs survived, respectively; 53.2% ± 9.6% of hN-MSCs were microtubule-associated protein 2(+) (MAP-2(+) ) and extended neurites, whereas only 0.9% ± 0.3% of hMSCs were MAP-2(+) . In FISH, human nucleus-specific signals were detected in both hN-MSCs and hMSCs grafted brains, but no transplanted cell had a merged gerbil-specific nuclear signals. hN-MSC-transplanted animals showed significantly better recovery than animals given control vehicle in the T-maze, bilateral asymmetry, and open field tests. These findings suggested that hN-MSCs have greater therapeutic potential than hMSCs for stroke and that cell fusion does not primarily contribute to the therapeutic mechanism of MSC transplantation.     

Adenosine dysfunction in epilepsy

Extracellular levels of the brain's endogenous anticonvulsant and neuroprotectant adenosine largely depend on an astrocyte-based adenosine cycle, comprised of ATP release, rapid degradation of ATP into adenosine, and metabolic reuptake of adenosine through equilibrative nucleoside transporters and phosphorylation by adenosine kinase (ADK). Changes in ADK expression and activity therefore rapidly translate into changes of extracellular adenosine, which exerts its potent anticonvulsive and neuroprotective effects by activation of pre- and postsynaptic adenosine A(1) receptors. Increases in ADK increase neuronal excitability, whereas decreases in ADK render the brain resistant to seizures and injury. Importantly, ADK was found to be overexpressed and associated with astrogliosis and spontaneous seizures in rodent models of epilepsy, as well as in human specimen resected from patients with hippocampal sclerosis and temporal lobe epilepsy. Several lines of evidence indicate that overexpression of astroglial ADK and adenosine deficiency are pathological hallmarks of the epileptic brain. Consequently, adenosine augmentation therapies constitute a powerful approach for seizure prevention, which is effective in models of epilepsy that are resistant to conventional antiepileptic drugs. The adenosine kinase hypothesis of epileptogenesis suggests that adenosine dysfunction in epilepsy undergoes a biphasic response: an acute surge of adenosine that can be triggered by any type of injury might contribute to the development of astrogliosis via adenosine receptor-dependent and -independent mechanisms. Astrogliosis in turn is associated with overexpression of ADK, which was shown to be sufficient to trigger spontaneous recurrent electrographic seizures. Thus, ADK emerges as a promising target for the prediction and prevention of epilepsy.     

Overexpression of ADK in human astrocytic tumors and peritumoral tissue is related to tumor-associated epilepsy

Purpose: Adenosine kinase (ADK), a largely astrocyte‐based metabolic enzyme, regulates adenosine homeostasis in the brain. Overexpression of ADK decreases extracellular adenosine and consequently leads to seizures. We hypothesized that dysfunction in the metabolism of tumor astrocytes is related to changes in ADK expression and that those changes might be associated with the development of epilepsy in patients with tumors. Methods: We compared ADK expression and cellular distribution in surgically removed tumor tissue (n = 45) and peritumoral cortex (n = 20) of patients with glial and glioneuronal tumors to normal control tissue obtained at autopsy (n = 11). In addition, we compared ADK expression in tumor patients with and without epilepsy. To investigate ADK expression, we used immunohistochemistry and Western blot analysis. ADK activity measurement was performed in surgical specimens of astrocytomas World Health Organization (WHO) grade III (n = 3), peritumoral cortex (n = 3), and nonepileptic cortex (n = 3). Key Findings: Immunohistochemistry predominantly showed cytoplasmic labeling in tumors and peritumoral tissue containing infiltrating tumor cells. ADK immunoreactivity was significantly stronger in tumor and peritumoral tissue compared to normal white matter and normal cortex, especially in astrocytoma WHO grade III, as confirmed by Western blot analysis and ADK activity measurements. Importantly, we found a significantly higher expression of ADK in the peritumoral infiltrated tissue of patients with epilepsy than in patients without epilepsy. Significance: These results suggest a dysregulation of ADK in astrocytic brain tumors. Moreover, the upregulation of ADK observed in peritumoral infiltrated tissue of glioma patients with epilepsy supports the role of this enzyme in tumor‐associated epilepsy.     

Loss of gene expression in lentivirus- and retrovirus-transduced neural progenitor cells is correlated to migration and differentiation in the adult spinal cord

Gene transfer into multipotent neural progenitor cells (NPC) and stem cells may provide for a cell replacement therapy and allow the delivery of therapeutic proteins into the degenerating or injured nervous system. Previously, murine leukemia virus-based retroviral vectors expressing GFP from an internal EF-1alpha promoter and lentiviral vectors expressing GFP from a hybrid CMV/beta-actin promoter have been described to be resistant to stem cell specific gene silencing. Therefore, we investigated whether these viral vectors allow stable in vivo gene expression in genetically modified NPC isolated from the adult rat spinal cord. In vitro, NPC genetically modified to express GFP using the described retroviral vector showed strong GFP expression in undifferentiated NPC. However, in vitro differentiation resulted in the loss of GFP expression in 50% of cells. Grafting of BrdU-prelabeled NPC to the spinal cord resulted in a loss of GFP expression in 70% and 95% of surviving NPC at 7 and 28 days post-grafting, respectively. The loss in gene expression was paralleled by the differentiation of NPC into a glial phenotype. Transgene downregulation although less profound was also observed in cells modified with lentiviral vectors, whereas in vivo lentiviral gene transfer resulted in stable transgene expression for up to 16 months. Thus, in vivo gene expression in genetically engineered neural progenitor cells is temporally limited and mostly restricted to undifferentiated NPC using the viral vectors tested.     

Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro

We found the expression of neurofilament was very low in undifferentiated human adult bone marrow mesenchymal stem cells (hMSCs), but its expression could be significantly induced after treatment with combination of growth factors. Approximately 16% of hMSCs differentiated into cells expressing neurofilament after treatment with a combination of FGF and RA, retinoic acid. We also examined the effect of five different cell culture substrates on the expression of neurofilament. One specific combination that was particularly effective in provoking pre-neuronal differentiation was culturing hMSCs on fibronectin-coated dishes and stimulating them with FGF and RA; 40% of cells expressed neurofilament. These results suggest that growth factors and substrates, in combination, can effectively initiate differentiation of hMSCs along a neuronal pathway.     

Downregulation of hippocampal adenosine kinase after focal ischemia as potential endogenous neuroprotective mechanism.

The rate of ischemic brain injury varies with the brain region, requiring only hours in striatum but days in hippocampus. Such maturation implies the existence of endogenous neuroprotective mechanisms. Adenosine is an endogenous neuroprotectant regulated by adenosine kinase (ADK). To investigate, whether adenosine might play a role in protecting the hippocampus after focal ischemia, we subjected transgenic mice, which overexpress ADK in hippocampal neurons (Adk-tg mice) to transient middle cerebral artery occlusion (MCAO). Although the hippocampus of wild-type (wt) mice was consistently spared from injury after 60 mins of MCAO, hippocampal injury became evident in Adk-tg mice after only 15 mins of MCAO. To determine, whether downregulation of hippocampal ADK might qualify as candidate mechanism mediating endogenous neuroprotection, we evaluated ADK expression in wt mice after several periods of reperfusion after 15 or 60 mins of MCAO. After 60 mins of MCAO, hippocampal ADK was significantly reduced in both hemispheres after 1, 3, and 24 h of reperfusion. Reduction of ADK-immunoreactivity corresponded to a 2.2-fold increase in hippocampal adenosine at 3 h of reperfusion. Remarkably, a significant reduction of ADK immunoreactivity was also found in the ipsilateral (stroked) hippocampus after 15 mins of MCAO and 3 h of reperfusion. Thus, transient downregulation of hippocampal ADK after stroke might be a protective mechanism during maturation hippocampal cell loss.     

Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy

Purpose: Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphologic feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathologic hallmark of TLE. Methods: Using immunocytochemistry and Western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). Key Findings: In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up‐regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3–4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of patients with TLE. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several proinflammatory molecules (interleukin‐1β and lipopolysaccharide). Significance: These results suggest that dysregulation of ADK in astrocytes is a common pathologic hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte‐based adenosine cycle and inflammation. Whether this interaction also can play a role in vivo needs to be further investigated.     

Cyclooxygenase-2 inhibitor, celecoxib, inhibits the altered hippocampal neurogenesis with attenuation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus

Recent evidences suggest key roles of abnormal neurogenesis and astrogliosis in the pathogenesis of epilepsy. Alterations in the microenvironment of the stem cell, such as microglial activation and cyclooxygenase-2 induction may cause ectopic neurogenesis or astrogliosis. Here, we examined if inflammatory blockade with celecoxib, a selective cyclooxygenase-2 inhibitor, could modulate the altered microenvironment in the epileptic rat brain. Celecoxib attenuated the likelihood of developing spontaneous recurrent seizures after pilocarpine-induced prolonged seizure. During the latent period, celecoxib prevented neuronal death and microglia activation in the hilus and CA1 and inhibited the generation of ectopic granule cells in the hilus and new glia in CA1. The direct inhibition of precursor cells by celecoxib was further demonstrated in human neural stem cells culture. These findings raise the evidence of COX-2 induction to act importantly on epileptogenesis and suggest a potential therapeutic role for COX-2 inhibitors in chronic epilepsy.     

Rac1b regulates NT3-stimulated Mek-Erk signaling, directing marrow-isolated adult multilineage inducible (MIAMI) cells toward an early neuronal phenotype

Due to the limitations of neural stem cells to repair neuronal damage in the human brain, alternative approaches of repair using autologous adult stem cells have been examined for direct cell-replacement, or paracrine mediated neuroprotective effects. Human bone marrow-derived stromal cells (hMSCs) are a heterogeneous adult stem cell population with diverse immunomodulatory properties and the potential to differentiate into cells characteristic of all three germ layers. hMSCs are a renewable source of progenitor cells suitable for cell-based tissue repair. The marrow isolated adult multilineage inducible (MIAMI) cells developed by our laboratory are a developmentally immature homogeneous subpopulation of hMSCs that maintain self-renewal potential during ex vivo expansion, efficient differentiation capacity into neuron-like cells in vitro, as well as direct in vivo neuroprotection and functional recovery in animal models of neurological diseases.We now address the early signaling mechanisms regulating the neuron-like differentiation of MIAMI cells in vitro, in response to activation of the neurotrophic tyrosine-kinase receptor, type 3 (NTRK3) via neurotrophin 3 (NT3). We molecularly characterize a novel role for Rac1b mediating the neurogenic potential of MIAMI cells. Rac1b had an overall negative modulatory effect on the NT3-stimulated Mek1/2-Erk1/2 signaling pathway, proneuronal gene expression and neurite-like extensions. Rac1b was required for NT3-stimulated cell proliferation of MIAMI cells, yet was found to repress CCND1 and CCNB1 mRNA expression independent of NT3 stimulation, suggesting a dual neurotrophin dependent/independent function. Differential levels of Rac1b activity in hMSCs may explain the apparent contradictory reports regarding their neurogenic potential. These findings demonstrate the in vitro neurogenic potential of hMSCs as governed by Rac1b during NT3 stimulation.     

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols

Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, beta-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs.