Cell based therapy reduces secondary damage and increases extent of microglial activation following cortical injury.

Cortical injury elicits long-term cytotoxic and cytoprotective mechanisms within the brain and the balance of these pathways can determine the functional outcome for the individual. Cytotoxicity is exacerbated by production of reactive oxygen species, accumulation of iron, and peroxidation of cell membranes and myelin. There are currently no neurorestorative treatments to aid in balancing the cytotoxic and cytoprotective mechanisms following cortical injury. Cell based therapies are an emerging treatment that may function in immunomodulation, reduction of secondary damage, and reorganization of surviving structures. We previously evaluated human umbilical tissue-derived cells (hUTC) in our non-human primate model of cortical injury restricted to the hand area of primary motor cortex. Systemic hUTC treatment resulted in significantly greater recovery of fine motor function compared to vehicle controls. Here we investigate the hypothesis that hUTC treatment reduces oxidative damage and iron accumulation and increases the extent of the microglial response to cortical injury. To test this, brain sections from these monkeys were processed using immunohistochemistry to quantify oxidative damage (4-HNE) and activated microglia (LN3), and Prussian Blue to quantify iron. hUTC treated subjects exhibited significantly reduced oxidative damage in the sublesional white matter and iron accumulation in the perilesional area as well as a significant increase in the extent of activated microglia along white matter pathways. Increased perilesional iron accumulation was associated with greater perilesional oxidative damage and larger reconstructed lesion volume. These findings support the hypothesis that systemic hUTC administered 24 h after cortical damage decreases the cytotoxic response while increasing the extent of microglial activation.

Cell based therapy enhances activation of ventral premotor cortex to improve recovery following primary motor cortex injury.

Stroke results in enduring damage to the brain which is accompanied by innate neurorestorative processes, such as reorganization of surviving circuits. Nevertheless, patients are often left with permanent residual impairments. Cell based therapy is an emerging therapeutic that may function to enhance the innate neurorestorative capacity of the brain. We previously evaluated human umbilical tissue-derived cells (hUTC) in our non-human primate model of cortical injury limited to the hand area of primary motor cortex. Injection of hUTC 24 h after injury resulted in significantly enhanced recovery of fine motor function compared to vehicle treated controls (Moore et al., 2013). These monkeys also received an injection of Bromodeoxyuridine (BrdU) 8 days after cortical injury to label cells undergoing replication. This was followed by 12 weeks of behavioral testing, which culminated 3 h prior to perfusion in a final behavioral testing session using only the impaired hand. In this session, the neuronal activity initiating hand movements leads to the upregulation of the immediate early gene c-Fos in activated cells. Following perfusion-fixation of the brain, sections were processed using immunohistochemistry to label c-Fos activated cells, pre-synaptic vesicle protein synaptophysin, and BrdU labeled neuroprogenitor cells to investigate the hypothesis that hUTC treatment enhanced behavioral recovery by facilitating reorganization of surviving cortical tissues. Quantitative analysis revealed that c-Fos activated cells were significantly increased in the ipsi- and contra-lesional ventral premotor but not the dorsal premotor cortices in the hUTC treated monkeys compared to placebo controls. Furthermore, the increase in c-Fos activated cells in the ipsi- and contra-lesional ventral premotor cortex correlated with a decrease in recovery time and improved grasp topography. Interestingly, there was no difference between treatment groups in the number of synaptophysin positive puncta in either ipsi- or contra-lesional ventral or dorsal premotor cortices. Nor was there a significant difference in the density of BrdU labeled cells in the subgranular zone of the hippocampus or the subventricular zone of the lateral ventricle. These findings support the hypothesis that hUTC treatment enhances the capacity of the brain to reorganize after cortical injury and that bilateral plasticity in ventral premotor cortex is a critical locus for this recovery of function. This reorganization may be accomplished through enhanced activation of pre-existing circuits within ventral premotor, but it could also reflect ventral premotor projections to the brainstem or spinal cord.

Recovery of fine motor performance after ischemic damage to motor cortex is facilitated by cell therapy in the rhesus monkey.

We investigated the efficacy on recovery of function following controlled cortical ischemia in the monkey of the investigational cell drug product, CNTO 0007. This drug contains a cellular component, human umbilical tissue-derived cells, in a proprietary thaw and inject formulation. Results demonstrate significantly better recovery of motor function in the treatment group with no difference between groups in the volume or surface area of ischemic damage, suggesting that the cells stimulated plasticity.

Injection of human umbilical tissue-derived cells into the nucleus pulposus alters the course of intervertebral disc degeneration in vivo.

BACKGROUND CONTEXT: Patients often present to spine clinic with evidence of intervertebral disc degeneration (IDD). If conservative management fails, a safe and effective injection directly into the disc might be preferable to the risks and morbidity of surgery. PURPOSE: To determine whether injecting human umbilical tissue-derived cells (hUTC) into the nucleus pulposus (NP) might improve the course of IDD. DESIGN: Prospective, randomized, blinded placebo-controlled in vivo study. PATIENT SAMPLE: Skeletally mature New Zealand white rabbits. OUTCOME MEASURES: Degree of IDD based on magnetic resonance imaging (MRI), biomechanics, and histology. METHODS: Thirty skeletally mature New Zealand white rabbits were used in a previously validated rabbit annulotomy model for IDD. Discs L2-L3, L3-L4, and L4-L5 were surgically exposed and punctured to induce degeneration and then 3 weeks later the same discs were injected with hUTC with or without a hydrogel carrier. Serial MRIs obtained at 0, 3, 6, and 12 weeks were analyzed for evidence of degeneration qualitatively and quantitatively via NP area and MRI Index. The rabbits were sacrificed at 12 weeks and discs L4-L5 were analyzed histologically. The L3-L4 discs were fixed to a robotic arm and subjected to uniaxial compression, and viscoelastic displacement curves were generated. RESULTS: Qualitatively, the MRIs demonstrated no evidence of degeneration in the control group over the course of 12 weeks. The punctured group yielded MRIs with the evidence of disc height loss and darkening, suggestive of degeneration. The three treatment groups (cells alone, carrier alone, or cells+carrier) generated MRIs with less qualitative evidence of degeneration than the punctured group. MRI Index and area for the cell and the cell+carrier groups were significantly distinct from the punctured group at 12 weeks. The carrier group generated MRI data that fell between control and punctured values but failed to reach a statistically significant difference from the punctured values. There were no statistically significant MRI differences among the three treatment groups. The treated groups also demonstrated viscoelastic properties that were distinct from the control and punctured values, with the cell curve more similar to the punctured curve and the carrier curve and carrier+cells curve more similar to the control curve (although no creep differences achieved statistical significance). There was some histological evidence of improved cellularity and disc architecture in the treated discs compared with the punctured discs. CONCLUSIONS: Treatment of degenerating rabbit intervertebral discs with hUTC in a hydrogel carrier solution might help restore the MRI, histological, and biomechanical properties toward those of nondegenerated controls. Treatment with cells in saline or a hydrogel carrier devoid of cells also might help restore some imaging, architectural, and physical properties to the degenerating disc. These data support the potential use of therapeutic cells in the treatment of disc degeneration.

Intravenous administration of human umbilical tissue-derived cells improves neurological function in aged rats after embolic stroke.

Intravenous administration of human umbilical tissue-derived cells (hUTC) improves neurological function in young adult rats after stroke. However, stroke is a major cause of death and disability in the aged population, with the majority of stroke patients 65 years and older. The present study investigated the effect of hUTC on aged rats after embolic stroke. Rats at the age of 18-20 months were subjected to embolic middle cerebral artery (MCA) occlusion. Two groups of eight animals each were compared. The investigational group was injected intravenously with 1×10(7) cells/kg in serum-free culture medium (vehicle) 24 h after stroke onset, and the control group was treated with vehicle only at the same time poststroke. Intravenous administration of hUTC significantly improved neurological functional recovery without reducing infarct volume compared to vehicle-treated aged rats. Additionally, hUTC treatment significantly enhanced synaptogenesis and vessel density in the ischemic boundary zone (IBZ). Moreover, hUTC treatment resulted in a trend toward increased progenitor cell proliferation in the subventricular zone (SVZ) compared to vehicle-treated aged rats. Intravenous administration of hUTC improved functional recovery in aged rats after stroke. The enhancement of synaptogenesis and vessel density may contribute to the beneficial effects of hUTC in the treatment of stroke in the aged animal.

Different routes of administration of human umbilical tissue-derived cells improve functional recovery in the rat after focal cerebral ischemia.

Human umbilical tissue-derived cells (hUTC) are a potential neurorestorative candidate for stroke treatment. Here, we test the effects of hUTC treatment in a rat model of stroke via various routes of administration. Rats were treated with hUTC or phosphate-buffered saline (PBS) via different routes including intraarterial (IA), intravenous (IV), intra-cisterna magna (ICM), lumber intrathecal (IT), or intracerebral injection (IC) at 24h after stroke onset. Treatment with hUTC via IV and IC route led to significant functional improvements starting at day 14, which persisted to day 60 compared with respective PBS-treated rats. HUTC administered via IA, ICM, and IT significantly improved neurological functional recovery starting at day 14 and persisted up to day 49 compared with PBS-treated rats. Although IA administration resulted in the highest donor cell number detected within the ischemic brain compared to the other routes, hUTC treatments significantly increased ipsilateral bromodeoxyuridine incorporating subventricular zone (SVZ) cells and vascular density in the ischemic boundary compared with PBS-treated rats regardless of the route of administration. While rats received hUTC treatment via IA, IV, IC, and ICM routes showed greater synaptophysin immunoreactivity, significant reductions in TUNEL-positive cells in the ipsilateral hemisphere were observed in IA, IV, and IC routes compared with PBS-treated rats. hUTC treatments did not reduce infarct volume when compared to the PBS groups. Our data indicate that hUTC administered via multiple routes provide therapeutic benefit after stroke. The enhancement of neurorestorative events in the host brain may contribute to the therapeutic benefits of hUTC in the treatment of stroke.

MRI detects brain reorganization after human umbilical tissue-derived cells (hUTC) treatment of stroke in rat.

Human umbilical tissue-derived cells (hUTC) represent an attractive cell source and a potential technology for neurorestoration and improvement of functional outcomes following stroke. Male Wistar rats were subjected to a transient middle cerebral artery occlusion (tMCAo) and were intravenously administered hUTC (N = 11) or vehicle (N = 10) 48 hrs after stroke. White matter and vascular reorganization was monitored over a 12-week period using MRI and histopathology. MRI results were correlated with neurological functional and histology outcomes to demonstrate that MRI can be a useful tool to measure structural recovery after stroke. MRI revealed a significant reduction in the ventricular volume expansion and improvement in cerebral blood flow (CBF) in the hUTC treated group compared to vehicle treated group. Treatment with hUTC resulted in histological and functional improvements as evidenced by enhanced expression of vWF and synaptophysin, and improved outcomes on behavioral tests. Significant correlations were detected between MRI ventricular volumes and histological lesion volume as well as number of apoptotic cells. A positive correlation was also observed between MRI CBF or cerebral blood volume (CBV) and histological synaptic density. Neurological functional tests were also significantly correlated with MRI ventricular volume and CBV. Our data demonstrated that MRI measurements can detect the effect of hUTC therapy on the brain reorganization and exhibited positive correlation with histological measurements of brain structural changes and functional behavioral tests after stroke. MRI ventricular volumes provided the most sensitive index in monitoring brain remodeling and treatment effects and highly correlated with histological and functional measurements.

Brain-derived neurotrophic factor produced by human umbilical tissue-derived cells is required for its effect on hippocampal dendritic differentiation.

The potential for nonembryonic cells to promote differentiation of neuronal cells has therapeutic implications for regeneration of neurons damaged by stroke or injury and avoids many ethical and safety concerns. The authors have assessed the capacity of human umbilical tissue-derived cells (hUTC) and human mesenchymal stromal cells (hMSC) to enhance differentiation of rodent hippocampal neurons. Co-culture of hippocampal cells with hUTC or hMSC in transwell inserts for 3 days resulted in increase of several dendritic parameters including the number and length of primary dendrites. The effect of hUTC or hMSC on dendritic maturation was only apparent on neurons grown for 2 weeks in vitro prior to co-culture. Changes in dendritic morphology in the presence of hUTC were also accompanied by increased expression of the presynaptic marker synaptotagmin and the postsynaptic marker postsynaptic density protein 95 kDa (PSD95) suggesting that there may also be an increase in the number of synapses formed in the presence of hUTC. The effect of hUTC and hMSC on hippocampal cells in co-culture was comparable to those induced by treatment with recombinant human brain-derived neurotrophic factor (BDNF) implying that a similar factor may be released from hUTC or hMSC. Analysis of hUTC-conditioned medium by ELISA demonstrated that BDNF was indeed secreted. An antibody that blocks the actions of BDNF partially inhibited the actions of hUTC on dendritic morphology suggesting that BDNF is at least one of the factors secreted from the cells to promote dendritic maturation. These results indicate that hUTC secrete biologically active BDNF, which can affect dendritic morphology.

Adult rat bone marrow stromal cells express genes associated with dopamine neurons.

An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFRalpha1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease.

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions.

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x(L) in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7-14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3-7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10-14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.

Toxicity of glutathione depletion in mesencephalic cultures: a role for arachidonic acid and its lipoxygenase metabolites.

The contribution of arachidonic acid (AA) release and metabolism to the toxicity that results from glutathione (GSH) depletion was studied in rat mesencephalic cultures treated with the GSH synthesis inhibitor l-buthionine sulfoximine. Our data show that GSH depletion is accompanied by increased release of AA, which is phosholipase A2 (PLA2) dependent. Exogenous AA is toxic to GSH-depleted cells. This toxicity is prevented by inhibition of lipoxygenase activity, suggesting participation of toxic byproducts of AA metabolism. Hydroxyperoxyeicosatetraenoic acid (HPETE), one of the primary products of AA metabolism by lipoxygenase is also toxic to GSH-depleted cells, whereas hydroeicosatetraenoic acid (HETE) is not. Cell death caused by GSH depletion is prevented by: (i) replenishment of GSH levels with GSH-ethyl ester; (ii) inhibition of PLA2 activity; (iii) inhibition of lipoxygenase activity; and (iv), treatment with ascorbic acid. These data suggest that the following events likely contribute to cell death when GSH levels become depleted. Loss of GSH results in increased release of AA, which is PLA2 dependent. Metabolism of arachidonic acid via the lipoxygenase pathway results in generation of oxygen free radicals possibly produced during conversion of HPETE to HETE, which contribute to cellular damage and death. Our study suggests that limiting AA release and metabolism may provide benefit in conditions with an existing depletion of GSH, such as Parkinson's disease.

Glutathione depletion and oxidative stress.

Oxidative stress is believed to contribute to the pathogenesis of Parkinson's disease. One of the indices of oxidative stress is the depletion of the antioxidant glutathione (GSH), which may occur early in the development of Parkinson's disease. To study the role of GSH depletion in the survival of dopamine neurons we treated mesencephalic cultures with the GSH synthesis inhibitor L-buthionine sulfoximine. Our studies have shown that the depletion of GSH causes a cascade of events, which ultimately may result in cell death. An early event following GSH depletion is a phospholipase A(2)-dependent release of arachidonic acid. Arachidonic acid can cause damage to the GSH-depleted cells through its metabolism by lipoxygenase. The generation of superoxide radicals during the metabolism of arachidonic acid is likely to play an important role in the toxic events that follow GSH depletion.