Abstract
D. J. Eve
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
A wide diversity of subjects are presented at the Annual American Society of Neural Therapy and Repair Meeting every year, and 2013 is no exception. An insight into the current research trends in regenerative medicine is provided, including studies to elucidate disease mechanisms and the means to treat them. Different methods featured this year include stem cell and tissue transplantation, gene therapy, dietary supplementation, and hydrogels as scaffold systems for the growth of stem cells. Diseases ranged from Parkinson's disease, spinal cord injury, and stroke through to traumatic brain injury, pain, and epilepsy. Traumatic brain injury was an increasingly popular topic highlighting the concerns of soldiers returning from duty overseas. A number of studies looked at more than one disorder. The studies including stem cells predominantly involved human-derived cells being transplanted, and the most common recipient of stem cells were rodents. Only one autologous transplant study that featured mouse bone marrow cells being transplanted into mice for the treatment of stroke was presented this year. The most popular stem cell studied was the neural stem cell, which in some instances were predifferentiated from induced pluripotent stem cells or embryonic stem cells. Other stem cells included the mesenchymal stem cell and adipose, amniotic fluid, and umbilical cord blood-derived cells. Many studies also looked at more than one stem cell type. Combinational studies such as gene therapy and transplantation were also commonly explored, as well as studies using fetal ventral mesencephalon or spinal cord tissue rather than stem cells. Numerous studies also featured the use of “drugs”— some naturally derived or naturally occurring as well as drug cocktails. A number of possible treatments including physical therapy and socialization were explored for a number of different diseases, as well as reports on the current status of four gene therapy clinical trials for the treatment of Parkinson's disease. Other studies assessed possible causes of specific disorders. In this way, the ASNTR provides an important snapshot of developments in the field of regenerative medicine.
H. Febinger,∗ K. Jordan,† H. Thomasy,‡ M. Opp,∗ and C. Gemma∗
∗Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA
†Department of Biology, University of Washington, Seattle, WA, USA
‡Neuroscience Graduate Program in Neurobiology and Behavior, University of Washington, Seattle, WA, USA
Neuropathology following traumatic brain injury (TBI) is the result of the immediate impact injury and the secondary injury mechanisms. Posttraumatic glia activation is a secondary injury mechanism that contributes to a chronic state of neuroinflammation. A prolonged state of inflammation after brain injury may linger for years and predispose patients to develop other neurological disorders, such as Alzheimer's disease. We have recently demonstrated that chemokine (C-X3-C motif) receptor 1 (CX3CR1; fractalkine receptor) is important in sustaining normal microglial activity in the brain. Loss or inhibition of CX3CR1 results in a neurotoxic microglial phenotype with consequent increases in neuronal damage. From these findings, we hypothesized that lack of CX3CR1 on microglia would contribute to the secondary neuro-pathologic sequelae following TBI and worsen neurological outcomes. Anesthetized adult CX3CR1–/– and CX3CR1+/+ male mice were placed on a stereotaxic frame and subjected to TBI using the controlled cortical impact (CCI) model (n = 7/genotype/time point). The skull fragment was removed without disrupting the underlying dura. A 5-mm diameter trephine was used to create a 5-mm diameter craniotomy over the left parietal cortex between lambda and bregma. CCI was induced with a 3-mm piston for which the impact velocity, dwell time, and depth could be precisely controlled (Leica Impact One, Richmond, IL, USA). Sham (control) animals (n = 4/genotype/time point) received identical anesthesia and craniotomy but were not subjected to CCI brain injury. Animals were humanely euthanized and transcardially perfused at 15 and 30 days post-TBI. All procedures involving the use of animals were approved by the University of Washington IACUC. Immunohistochemistry for cluster of differentiation 68 (CD68) and CD11b (activated microglial markers) and fluorojade-B (marker of cell death) was performed on every sixth section throughout the entire parietal cortex and dentate gyrus. Unbiased stereological analyses revealed that at 15 days post-TBI there was a significant decrease in the number of Fluorojade-positive cells in the ipsilateral cortex and dentate gyrus in the CX3CR1-deficient mice in comparison to the controls. The decrease in cell death was accompanied by a decreased number of CD68-positive cells. In contrast, at 30 days post-TBI, both Fluorojade-positive cells and CD68 were significantly increased when compared to the controls. These results demonstrate that deficiency in CX3CR1 leads to a delayed increase in chronic microglia activation after focal TBI, which results in a delayed neurotoxicity response. This suggests that the presence of the CX3CR1 is essential at earlier post-TBI time points in limiting the progression of TBI-induced neurological damage. Overall, our results suggest that CX3CR1/Fractalkine signaling receptor is a critical regulator of the inflammatory repair process following a focal TBI.
This work was supported by funds from the Department of Anesthesiology and Pain Medicine, University of Washington.
J.-P. Lee,∗†1 R. Zhang,∗1 M.-C. Yan,‡ S. Duggineni,§ D. R. Wakeman,∗¶ W. Niles,∗ Y. Feng,∗ Z. Huang,∗§ and E. Y. Snyder∗
∗Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
†Tulane University School of Medicine, New Orleans, LA, USA
‡The University of Southern California, Los Angeles, CA, USA
§SUNY Upstate Cancer Research Institute,
Department of Pharmacology, State University of New York, Syracuse, NY, USA
¶Rush University, Chicago, IL, USA
1These authors provided equal contribution to this work.
Nearly a decade ago, we published that inflammatory chemokines like stromal cell derived factor 1α (SDF1α) are pivotal for directing neural stem cell (NSC) homing to sites of pathology, a phenomenon called “pathotropism” [PNAS 101:18117; 2004]. NSCs, which constitutively express chemokine (C-X-C motif) receptor 4 (CXCR4), migrate in vivo to CNS injury niches where local reactive astrocytes and vascular endothelium upregulate the ligand SDF1α. Exposing NSCs to SDF1α triggers a series of CXCR4-mediated intracellular processes associated with migration, survival, proliferation, differentiation (and during early brain development, proper lamination). However, because there are also undesirable inflammatory actions following SDF1a–CXCR4 binding, one would not simply wish to inject this chemokine. However, if SDF1α could be stripped of its undesirable actions while preserving its tropic actions, an ideal chemoattractant would be derived. Based on the fact that CXCR4 has two different binding pockets, one mediating inflammatory signaling, and the other mediating binding, we performed chemical mutagenesis, divorcing the two actions, eliminating the former while maximizing the latter. We first tested the effects of this newly developed synthetic CXCR4 agonist peptide, “SDF-DV1” in normal mouse brain and observed extensive hNSC migration—even to intact adult cortex, typically an area nonsupportive of migration—when the cells were coadministered with SDF-DV1. Importantly, SDF-DV1 not only elicited more extensive and long-lasting migration and distribution than the natural CXCR4 inflammatory chemokine agonist SDF1α but elicited no host inflammation (important because the brains of mice and patients with most neurotraumatic and neurodegenerative processes are already characterized by excessive inflammation). Behavior was not impaired; the blood–brain barrier remained intact. Encouraged by these findings, we attempted to see whether this synthetic peptide could enhance the therapeutic efficacy of an NSC in a diseased brain. We previously reported [Nature Med 13:439, 2007; Stem Cells 27:2362, 2009] that, regardless of source or expansion method, transplanted human NSCs (hNSCs), when transplanted into the cerebral ventricles of brains of a newborn mouse model of the lysosomal storage disorder (LSD) Sandhoff disease (SD), migrated extensively throughout the brain, forestalled disease onset, preserved function, and substantially extended life. These actions pivoted on widespread dissemination and integration of the NSCs, particularly throughout the cortex. The hNSCs provided cross-correcting enzyme restoring lysosomal function, reducing ganglioside storage, blunting inflammation, and replacing degenerating neurons and glia. While hNSCs derived from the fetal CNS or from human embryonic stem cells (hESCs) possessed this degree of migration and hence efficacy, hNSCs derived from human induced pluripotent stem cells (hiPSCs) seemed, surprisingly, to be limited in their migration capacity and hence efficacy. However, when hiPSC-derived hNSCs were coadministered with the peptide in neonatal SD mouse brain, we now found enhanced dissemination of corrective donor-derived cells throughout the diseased brain and a significant therapeutic impact.
S. G. Scott
Physical Medicine and Rehabilitation Service, James A. Haley Veteran's Hospital, Tampa, FL, USA
The wars in Iraq and Afghanistan have created a new generation of servicemen with traumatic brain injury (TBI) that will need rehabilitation and lifelong care. It is estimated that about one out of every five servicemen sustained some type of TBI, which have ranged in severity from mild to severe. Most of these patients are surviving polytrauma/TBI on the battlefield from improvised explosive devices (IEDs). The role of rehabilitation in treating TBI servicemen is to provide comprehensive treatment to maximize functional independence to enhance their quality of life and assist them to regain their role in society as an active and useful member. To achieve this goal, rehabilitation has been changing to one of regeneration and recovery through repetitive long-term therapy. Individuals often attend many hours of therapy per day, 7 days per week. Specific rehabilitation programs have been developed to meet the needs of individuals with TBI. This has included programs for complex mild TBI patients and the Emerging Consciousness Program for the severe TBI patients. Locomotive programs for ambulation and repetitive hand therapy have been used with good outcomes. A multisensory rehabilitation team is providing care for the complex mild TBI patient, while a microstimulation environment is used for the severe TBI patients. A TBI Smart Home is being developed as part of the transitional and community reintegration programs. Results so far have revealed that recovery from TBI continues many years beyond the acute injury. Future research on regeneration rehabilitation is encouraged to further understand the long-term prognosis of recovery in TBI patients.
A. E. Willing,∗†‡ O. Zayko,∗ H. M. Derasari,∗ A. E. Rawls,∗ N. Kuzmin-Nichols,§ C. D. Sanberg,§ S. N. Garbuzova-Davis,∗†‡¶ and P. R. Sanberg∗†‡¶#
∗Center of Excellence for Aging and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
¶Department of Pathology and Cell Biology,
Morsani College of Medicine, University of South Florida, Tampa, FL, USA
#Department of Psychiatry, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Sanfilippo syndrome type 3 B (MPS III B) is an inherited disorder caused by mutations in the gene encoding α-N-acetylglucosaminidase (Naglu) on chromosome 17q21 that lead to a deficiency of the Naglu enzyme in the degradative pathway of heparan sulfate (HS). There are no treatments available for this disease, but we have previously shown that a single administration of human umbilical cord blood (HUCB) mononuclear cells (MNCs) into presymptomatic, early symptomatic, or late stage Naglu–/– mice had a beneficial effect, probably due to enzyme delivery into the enzyme-deficient mutant mice. In this study, we tested whether administering repeated doses of hUCB MNCs would be more beneficial than a single dose. Seventy-seven Naglu–/– (63 homozygote: 31 males, 32 females) 3 months of age at study start were randomly assigned to either a media-only group, hUCB cells in a single low dose (3×106 cells; single, low), single high dose (1.8×107 cells; single, high), or multiple doses (3×106 cells monthly for 6 months; multiple). An additional control group of wild-type mice (+/+) was also used. Fourteen wild-type mice (six males, eight females) were also used. We measured activity in an open field and cognition prior to treatment and then at monthly intervals for 6 months followed by histological and immunohistochemical analyses of the brain. Naglu–/– mice exhibited less anxiety than wild-type mice and mice that were treated with all doses of hUCB cells (p < 0.05). Similarly, hUCB cells reduced stereotyped behaviors in these mice (p < 0.05). Upon examination of the hippocampus, there was significant disruption of cytoarchitecture in dentate gyrus, CA1, CA2, and CA3 in the Naglu–/– mice characterized by cell loss and vacuolization of the neurons. The multiple dose group had the greatest hippocampal neuroprotection and decrease in activated microglia. In addition, the repeated doses significantly decreased monosialodihexosylganglioside (GM3) in the hippocampus (p < 0.05) but only tended to decrease this secondary degradation product in cortex and cerebellum. These data suggest that the neuroprotective effect of hUCB MNCs can be enhanced by repeated cell administrations, likely associated with continuous delivery of the missing enzyme.
This project was supported in part by grants from the Children's Medical Research Foundation, Inc., and The International Organization of Glutaric Acidemia. The cells were provided by Saneron CCEL Therapeutics, Inc. S.G.D. and A.E.W. are consultants to and P.R.S. is a cofounder of Saneron CCEL Therapeutics, Inc.