Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-Myc.

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133(+) cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.

Bone marrow-derived nonreactive astrocytes in the mouse brain after permanent middle cerebral artery occlusion.

We studied the effect of permanent unilateral middle cerebral artery occlusion (PMCAO) on the generation of bone marrow (BM)-derived astrocytes in female mice previously transplanted with enhanced green fluorescent protein-expressing BM from male donors. In addition to an untreated PMCAO group, one group of mice also received intracerebral infusion of transforming growth factor-alpha, resulting in a decrease in the size of the infarct. Two months after PMCAO, we found a specific type of astrocyte of BM origin in the side of the injury, near the lesion. These astrocytes did not express glial fibrillary acidic protein (GFAP) by conventional fluorescence immunostaining; however, GFAP was easily detectable by tyramide signal amplification. These cells also expressed S100ß, confirming their astrocytic character. Unlike the endogenous reactive astrocytes, these BM-derived astrocytes did not proliferate during the first week of ischemia and did not contribute to the glial scar formation. Transforming growth factor-alpha infusion increased the number of BM-derived astrocytes, without affecting their distribution. Interestingly, exclusively by tyramide signal amplification staining, we found that endogenous astrocytes displaying an identical morphology were also present in control mouse and human brains. Our data demonstrate that a subpopulation of nonreactive astrocytes expressing low levels of GFAP can originate from transplanted BM in the ischemic brain. We believe that these cells represent a subpopulation of astrocytes earlier considered to be GFAP negative. The high number of astrocytes with identical morphology and chemical character in control brains suggest that these type of astrocytes may have important functional role in the central nervous system that calls for further studies.

Resolution of cavitational osteonecrosis through NeuroModulation Technique, a novel form of intention-based therapy: a clinical case study.

OBJECTIVES: This study evaluated the possibility of using NeuroModulation Technique (NMT), a form of intention-based medicine, to induce osteogenesis and healing of cavitational osteonecrosis, a common progressive form of ischemic disease of the alveolar arch. DESIGN: Eleven (11) adult patients were enrolled based on the presence of lesions in the jawbone. Ten (10) subjects underwent NMT therapy for up to 10 months, while 1 subject received no treatment. OUTCOME MEASURES: A sensitive analysis of bone density in the alveolar processes of maxilla and mandible was performed before and after therapy using the U.S. Food and Drug Administration-approved Cavitat system of through-transmission ultrasonography and computer imaging. RESULTS: All subjects presented between one and six cavitational lesions at the first scan, most of which (92%) were associated with sites of previous tooth extraction. NMT-treated patients demonstrated significant improvement in bone density in 27 of the 34 lesions analyzed (79%). The median number of lesions per patient was 4 pretreatment and 0 post-treatment (p < 0.01). One NMT-treated patient, 1 surgically treated patient, and the control subject were also imaged at later time points, showing a durable healing of the lesions through NMT comparable to that of surgery, as opposed to disease persistence in the untreated control. CONCLUSIONS: NMT therapy provides a safe and potentially effective treatment for jawbone osteonecrosis. Preclinical placebo-controlled trials are encouraged to investigate in depth the potential of NMT for treating inflammatory and degenerative pathologies.

CD45-positive blood cells give rise to uterine epithelial cells in mice.

The uterine endometrium is composed of epithelial and stromal cells, which undergo extensive degeneration and regeneration in every estrous cycle, and dramatic changes occur during pregnancy. The high turnover of cells requires a correspondingly high level of cell division by progenitor cells in the uterus, but the character and source of these cells remain obscure. In the present study, using a novel transgenic mouse, we showed that CD45-positive hematopoietic progenitor cells colonize the uterine epithelium and that in pregnancy more than 80% of the epithelium can derive from these cells. Since we also found green fluorescent protein (GFP)-positive uterine endothelial cells in long-term GFP bone marrow-transplanted mice, we conclude that circulating CD45+ cells play an important role in regenerating the uterine epithelium.

Disease progression after bone marrow transplantation in a model of multiple sclerosis is associated with chronic microglial and glial progenitor response.

Multiple sclerosis (MS), the most common nontraumatic cause of neurologic disability in young adults in economically developed countries, is characterized by inflammation, gliosis, demyelination, and neuronal degeneration in the CNS. Bone marrow transplantation (BMT) can suppress inflammatory disease in a majority of patients with MS but retards clinical progression only in patients treated in the early stages of the disease. Here, we applied BMT in a mouse model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE), and investigated the kinetics of reconstitution of the immune system in the periphery and in the CNS using bone marrow cells isolated from syngeneic donors constitutively expressing green fluorescent protein. This approach allowed us to dissect the contribution of donor cells to the turnover of resident microglia and to the pathogenesis of observed disease relapses after BMT. BMT effectively blocked or delayed EAE development when mice were treated early in the course of the disease but was without effect in mice with chronic disease. We found that there is minimal overall replacement of host microglia with donor cells in the CNS and that newly transplanted cells do not appear to contribute to disease progression. In contrast, EAE relapses are accompanied by the robust activation of endogenous microglial and macroglial cells, which further involves the maturation of endogenous Olig2 glial progenitor cells into reactive astrocytes through the cytoplasmic translocation of Olig2 and the expression of CD44 on the cellular membrane. The observed maturation of large numbers of reactive astrocytes from glial progenitors and the chronic activation of host microglial cells have relevance for our understanding of the resident glial response to inflammatory injury in the CNS. Our data indicate that reactivation of a local inflammatory process after BMT is sustained predominantly by endogenous microglia/macrophages.

Evidence that nucleocytoplasmic Olig2 translocation mediates brain-injury-induced differentiation of glial precursors to astrocytes.

The mechanisms by which neural and glial progenitor cells in the adult brain respond to tissue injury are unknown. We studied the responses of these cells to stab wound injury in rats and in two transgenic mouse models in which Y/GFP is driven either by Sox2 (a neural stem cell marker) or by Talpha-1 (which marks newly born neurons). The response of neural progenitors was low in all nonneurogenic regions, and no neurogenesis occurred at the injury site. Glial progenitors expressing Olig2 and NG2 showed the greatest response. The appearance of these progenitors preceded the appearance of reactive astrocytes. Surprisingly, we found evidence of the translocation of the transcription factor Olig2 into cytoplasm in the first week after injury, a mechanism that is known to mediate the differentiation of astrocytes during brain development. Translocation of Olig2, down-regulation of NG2, and increased glial fibrillary acidic protein expression were recapitulated in vitro after exposure of glial progenitors to serum components or bone morphogentic protein by up-regulation of Notch-1. The glial differentiation and Olig2 translocation could be blocked by inhibition of Notch-1 with the gamma-secretase inhibitor DAPT. Together, these data indicate that the prompt maturation of numerous Olig2(+) glial progenitors to astrocytes underlies the repair process after a traumatic injury. In contrast, neural stem cells and neuronal progenitor cells appear to play only a minor role in the injured adult CNS.

Revisiting the astrocyte-oligodendrocyte relationship in the adult CNS.

The lineages of both astrocytes and oligodendrocytes have been popular areas of research in the last decade. The source of these cells in the mature CNS is relevant to the study of the cellular response to CNS injury. A significant amount of evidence exists to suggest that resident precursor cells proliferate and differentiate into mature glial cells that facilitate tissue repair and recovery. Additionally, the re-entry of mature astrocytes into the cell cycle can also contribute to the pool of new astrocytes that are observed following CNS injury. In order to better understand the glial response to injury in the adult CNS we must revisit the astrocyte-oligodendrocyte relationship. Specifically, we argue that there is a common glial precursor cell from which astrocytes and oligodendrocytes differentiate and that the microenvironment surrounding the injury determines the fate of the stimulated precursor cell. Ideally, better understanding the origin of new glial cells in the injured CNS will facilitate the development of therapeutics targeted to alter the glial response in a beneficial way.

Cytoplasmic translocation of Olig2 in adult glial progenitors marks the generation of reactive astrocytes following autoimmune inflammation.

The injury response in the brain involves complex interplay between neural and immune components. Following inflammatory insults to the adult CNS, formation of an astroglial scar often impedes functional repair. Glial progenitor cells expressing the nuclear transcription factor Olig2 possibly generate astrocytes in response to various types of injuries; however, the mechanisms underlying this differentiation are unclear. In a model of immune-mediated injury (MOG(35-55)-experimental autoimmune encephalomyelitis), we show that the conversion from progenitor to reactive astrocyte is marked by the translocation of Olig2 into the cytoplasm. Evidence of this process is found for months after disease initiation in the absence of new inflammatory infiltrates. A proportion of cells with cytoplasmic Olig2 was found to express NG2 or Nkx2.2, but only Nkx2.2 was occasionally retained by GFAP+ cells. We further show that differentiation to astrocytes is induced in glial progenitors in vitro through exposure to the pro-inflammatory cytokine IFN-gamma, but not to TNF-alpha. Together, these data ascribe a pivotal role to Olig2+ glial precursor cells in the adult CNS, linking autoimmune inflammation and glial scar formation.

Borrelia burgdorferi Induces TLR1 and TLR2 in human microglia and peripheral blood monocytes but differentially regulates HLA-class II expression.

The spirochete Borrelia burgdorferi is the agent of Lyme disease, which causes central nervous system manifestations in up to 20% of patients. We investigated the response of human brain microglial cells, glial progenitors, neurons, astrocytes, as well as peripheral blood monocytes to stimulation with B. burgdorferi. We used oligoarrays to detect changes in the expression of genes important for shaping adaptive and innate immune responses. We found that stimulation with B. burgdorferi lysate increased the expression of Toll-like receptors (TLRs) 1 and 2 in all cell types except neurons. However, despite similarities in global gene profiles of monocytes and microglia, only microglial cells responded to the stimulation with a robust increase in HLA-DR, HLA-DQ, and also coexpressed CD11-c, a dendritic cell marker. In contrast, a large number of HLA-related molecules were repressed at both the RNA and the protein levels in stimulated monocytes, whereas secretion of IL-10 and TNF-alpha was strongly induced. These results show that signaling through TLR1/2 in response to B. burgdorferi can elicit opposite immunoregulatory effects in blood and in brain immune cells, which could play a role in the different susceptibility of these compartments to infection.

Clonotypic analysis of cerebrospinal fluid T cells during disease exacerbation and remission in a patient with multiple sclerosis.

Migration of autoreactive T cells into the central nervous system (CNS) compartment is thought to be an important step in the pathogenesis of multiple sclerosis (MS). To follow the evolution of T cell repertoire in the CNS of a patient with relapsing-remitting MS, we analyzed cerebrospinal fluid (CSF) cells obtained during an acute clinical exacerbation, and subsequent disease remission after 13 months of immunomodulatory therapy. T cell receptor CDR3 region length distribution was significantly altered during the relapse, demonstrating the presence of clonally expanded T cells in the CSF. CDR3 spectratyping is a valuable approach to identify disease-associated T cells in the CNS.

Variant-specific tropism of human herpesvirus 6 in human astrocytes.

Though first described as a lymphotropic virus, human herpesvirus 6 (HHV-6) is highly neuropathogenic. Two viral variants are known: HHV-6A and HHV-6B. Both variants can infect glial cells and have been differentially associated with central nervous system diseases, suggesting an HHV-6 variant-specific tropism for glial cell subtypes. We have performed infections with both viral variants in human progenitor-derived astrocytes (HPDA) and monitored infected cell cultures for cytopathic effect (CPE), intra- and extracellular viral DNA load, the presence of viral particles by electronic microscopy, mRNA transcription, and viral protein expression. HHV-6A established a productive infection with CPE, visible intracellular virions, and high virus DNA loads. HHV-6B-infected HPDA showed no morphological changes, intracellular viral particles, and decreasing intra- and extracellular viral DNA over time. After long-term passage, HHV-6B-infected HPDA had stable but low levels of intracellular viral DNA load with no detectable viral mRNA. Our results demonstrate that HHV-6A and HHV-6B have differential tropisms and patterns of infection for HPDA in vitro, where HHV-6A results in a productive lytic infection. In contrast, HHV-6B was associated with a nonproductive infection. These findings suggest that HHV-6 variants might be responsible for specific infection patterns in glial cells in vivo. Astrocytes may be an important reservoir for this virus in which differential tropism of HHV-6A and HHV-6B may be associated with different disease outcomes.

CD46 on glial cells can function as a receptor for viral glycoprotein-mediated cell-cell fusion.

Membrane cofactor protein (CD46) is a regulator of complement activation that also serves as the entry receptor for human herpes virus 6 (HHV-6) and measles virus (MV) into human cells. While it is clear that oligodendrocytes and astrocytes are cell types commonly infected by these viruses, it is unclear whether oligodendrocytes express CD46, or which are the cellular mechanisms underlying the infection. We show that adult oligodendrocytes, as well as astrocytes and microglial cells, express CD46 on the cellular surface. Moreover, we employed a quantitative fusion assay to demonstrate that HHV-6A infection of T lymphocytes enables cell-cell fusion of these cells to astrocytes or to oligodendroglial cells. This fusion is mediated by the interaction between viral glycoproteins expressed on the membrane of the infected cells and CD46 on the glial targets, and is also observed using cells expressing recombinant MV glycoproteins. These data suggest a mechanism that involves cell-cell fusion by which certain viruses could spread the infection from the periphery to the cells in the nervous system.

Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients.

Clinical trials have indicated that autologous hematopoietic stem cell transplantation (HSCT) can persistently suppress inflammatory disease activity in a subset of patients with severe multiple sclerosis (MS), but the mechanism has remained unclear. To understand whether the beneficial effects on the course of disease are mediated by lympho-depletive effects alone or are sustained by a regeneration of the immune repertoire, we examined the long-term immune reconstitution in patients with MS who received HSCT. After numeric recovery of leukocytes, at 2-yr follow-up there was on average a doubling of the frequency of naive CD4(+) T cells at the expense of memory T cells. Phenotypic and T cell receptor excision circle (TREC) analysis confirmed a recent thymic origin of the expanded naive T cell subset. Analysis of the T cell receptor repertoire showed the reconstitution of an overall broader clonal diversity and an extensive renewal of clonal specificities compared with pretherapy. These data are the first to demonstrate that long-term suppression of inflammatory activity in MS patients who received HSCT does not depend on persisting lymphopenia and is associated with profound qualitative immunological changes that demonstrate a de novo regeneration of the T cell compartment.

Hematopoietic stem cell transplantation for multiple sclerosis: current status and future challenges.

PURPOSE OF REVIEW: This article reviews recent advances in clinical trials of hematopoietic stem cell transplantation as a therapy for multiple sclerosis, and progress in exploring the potential for neural repair of hematopoietic-derived precursors. RECENT FINDINGS: Important recent findings are that hematopoietic stem cell transplantation can completely suppress the inflammatory component of multiple sclerosis, hematopoietic stem cells can migrate into the central nervous systems of rodents and humans, and can differentiate into cells expressing neural and glial markers. Hematopoietic stem cells also have neural and myelin repair potential. The heterogeneity of transplant regimens, the selection of patients at different stages of disease in clinical trials, and the limited duration of follow-up all currently preclude the evaluation of the long-term clinical benefits of hematopoietic stem cell transplantation for multiple sclerosis. SUMMARY: Hematopoietic stem cell transplantation is an experimental treatment that shows strong effects on the inflammatory component of multiple sclerosis. On the basis of experience acquired from initial pilot studies, controlled clinical trials are now being designed to verify long-term clinical efficacy. Selecting patients at high risk in the earlier stages of the disease that is dominated by inflammation, and monitoring objectively disease activity by magnetic resonance imaging will be critically important in these studies. Recent advances on the migratory potential and on the differentiation plasticity of hematopoietic stem cells have opened new opportunities for remyelination and axonal repair strategies for multiple sclerosis.