In Vivo Micro-CT Imaging of Human Mesenchymal Stem Cells Labeled with Gold-Poly-L-Lysine Nanocomplexes.

Developing in vivo cell tracking is an important prerequisite for further development of cell-based therapy. So far, few computed tomography (CT) cell tracking studies have been described due to its notoriously low sensitivity and lack of efficient labeling protocols. We present a simple method to render human mesenchymal stem cells (hMSCs) sufficiently radiopaque by complexing 40 nm citrate-stabilized gold nanoparticles (AuNPs) with poly-L-lysine (PLL) and rhodamine B isothiocyanate (RITC). AuNP-PLL-RITC labeling did not affect cellular viability, proliferation, or downstream cell differentiation into adipocytes and osteocytes. Labeled hMSCs could be clearly visualized in vitro and in vivo with a micro-CT scanner, with a detection limit of approximately 2×104 cells/µl in vivo. Calculated HU values were 2.27 /pg of intracellular Au as measured with inductively coupled plasma mass spectrophotometry (ICP-MS), and were linear over a wide range of cell concentrations. This linear CT attenuation was observed for both naked AuNPs and those that were taken up by hMSCs, indicating that the number of labeled cells can be quantified similar to the use of radioactive or fluorine tracers. This approach for CT cell tracking may find applications in CT image-guided interventions and fluoroscopic procedures commonly used for the injection of cellular therapeutics.

IL-6 induces regionally selective spinal cord injury in patients with the neuroinflammatory disorder transverse myelitis.

Transverse myelitis (TM) is an immune-mediated spinal cord disorder associated with inflammation, demyelination, and axonal damage. We investigated the soluble immune derangements present in TM patients and found that IL-6 levels were selectively and dramatically elevated in the cerebrospinal fluid and directly correlated with markers of tissue injury and sustained clinical disability. IL-6 was necessary and sufficient to mediate cellular injury in spinal cord organotypic tissue culture sections through activation of the JAK/STAT pathway, resulting in increased activity of iNOS and poly(ADP-ribose) polymerase (PARP). Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage, which were blocked by PARP inhibition. Addition of IL-6 to brain organotypic cultures or into the cerebral ventricles of adult rats did not activate the JAK/STAT pathway, which is potentially due to increased expression of soluble IL-6 receptor in the brain relative to the spinal cord that may antagonize IL-6 signaling in this context. The spatially distinct responses to IL-6 may underlie regional vulnerability of different parts of the CNS to inflammatory injury. The elucidation of this pathway identifies specific therapeutic targets in the management of CNS autoimmune conditions.

High resolution diffusion tensor imaging of axonal damage in focal inflammatory and demyelinating lesions in rat spinal cord.

Inflammation, demyelination, gliosis and axonal degeneration are pathological hallmarks of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis. Axonal damage is thought to contribute to irreversible damage and functional impairment, but is difficult to quantify. Conventional MRI has been used to assess the inflammatory and demyelinating aspects of MS lesions, but more sensitive and specific methods are needed to identify axonal damage to monitor disease progression and to determine efficacy of putative neuroprotective agents. We used high resolution diffusion tensor imaging (DTI) and fibre tracking to examine the spinal cord in rats with focal dorsal column inflammatory or demyelinating lesions to determine whether DTI measures can be used to detect pathology at the site of the focal lesion and to measure axonal damage in tracts distal to the focal lesion. Distant from the focal lesion, total axon counts, degenerating axon counts and SMI-31 staining, but not Luxol fast blue staining, were significantly correlated with fractional anisotropy, axial diffusivity and radial diffusivity, all of which are derived from the DTI data. These data suggest that high resolution DTI may be a more sensitive method than conventional imaging for detecting axonal damage at sites distant from inflammation.

Co-transplantation of syngeneic mesenchymal stem cells improves survival of allogeneic glial-restricted precursors in mouse brain.

Loss of functional cells from immunorejection during the early post-transplantation period is an important factor that reduces the efficacy of stem cell-based therapies. Recent studies have shown that transplanted mesenchymal stem cells (MSCs) can exert therapeutic effects by secreting anti-inflammatory and pro-survival trophic factors. We investigated whether co-transplantation of MSCs could improve the survival of other transplanted therapeutic cells. Allogeneic glial-restricted precursors (GRPs) were isolated from the brain of a firefly luciferase transgenic FVB mouse (at E13.5 stage) and intracerebrally transplanted, either alone, or together with syngeneic MSCs in immunocompetent BALB/c mice (n=20) or immunodeficient Rag2(-/-) mice as survival control (n=8). No immunosuppressive drug was given to any animal. Using bioluminescence imaging (BLI) as a non-invasive readout of cell survival, we found that co-transplantation of MSCs significantly improved (p<0.05) engrafted GRP survival. No significant change in signal intensities was observed in immunodeficient Rag2(-/-) mice, with transplanted cells surviving in both the GRP only and the GRP+MSC group. In contrast, on day 21 post-transplantation, we observed a 94.2% decrease in BLI signal intensity in immunocompetent mice transplanted with GRPs alone versus 68.1% in immunocompetent mice co-transplanted with MSCs and GRPs (p<0.05). Immunohistochemical analysis demonstrated a lower number of infiltrating CD45, CD11b(+) and CD8(+) cells, reduced astrogliosis, and a higher number of FoxP3(+) cells at the site of transplantation for the immunocompetent mice receiving MSCs. The present study demonstrates that co-transplantation of MSCs can be used to create a microenvironment that is more conducive to the survival of allogeneic GRPs.

Derivation of glial restricted precursors from E13 mice.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

Recovery from paralysis in adult rats using embryonic stem cells.

OBJECTIVE: We explored the potential of embryonic stem cell-derived motor neurons to functionally replace those cells destroyed in paralyzed adult rats. METHODS: We administered a phosphodiesterase type 4 inhibitor and dibutyryl cyclic adenosine monophosphate to overcome myelin-mediated repulsion and provided glial cell-derived neurotrophic factor within the sciatic nerve to attract transplanted embryonic stem cell-derived axons toward skeletal muscle targets. RESULTS: We found that these strategies significantly increased the success of transplanted axons extending out of the spinal cord into ventral roots. Furthermore, transplant-derived axons reached muscle, formed neuromuscular junctions, were physiologically active, and mediated partial recovery from paralysis. INTERPRETATION: We conclude that restoration of functional motor units by embryonic stem cells is possible and represents a potential therapeutic strategy for patients with paralysis. To our knowledge, this is the first report of the anatomical and functional replacement of a motor neuron circuit within the adult mammalian host.

Axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats.

We generated spinal motoneurons from embryonic stem (ES) cells to determine the developmental potential of these cells in vitro and their capacity to replace motoneurons in the adult mammalian spinal cord. ES cell-derived motoneurons extended long axons, formed neuromuscular junctions, and induced muscle contraction when cocultured with myoblasts. We transplanted motoneuron-committed ES cells into the spinal cords of adult rats with motoneuron injury and found that approximately 3,000 ES cell-derived motoneurons (25% of input) survived for >1 month in the spinal cord of each animal. ES cell-derived axonal growth was inhibited by myelin, and this inhibition was overcome by administration of dibutyryl cAMP (dbcAMP) or a Rho kinase inhibitor in vitro and in vivo. In transplanted rats infused with dbcAMP, approximately 80 ES cell-derived motor axons were observed within the ventral roots of each animal, whereas none were observed in transplanted rats not treated with dbcAMP. Because these cells replicate many of the developmental and mature features of true motoneurons, they are an important biological tool to understand formation of motor units in vitro and a potential therapeutic tool to reconstitute neural circuits in vivo.