The Bone Marrow Microenvironment in Immune-Mediated Inflammatory Diseases: Implications for Mesenchymal Stromal Cell-Based Therapies.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapy for several immune-mediated inflammatory diseases (IMIDs) due to their multiplicity of immunomodulatory and reparative properties and favorable safety profile. However, although preclinical data were encouraging, the clinical benefit demonstrated in clinical trials of autologous MSC transplantation in a number of conditions has been less robust. This may be explained by the growing body of evidence pointing to abnormalities of the bone marrow microenvironment in IMIDs, including impaired MSC function. However, it is not currently known whether these abnormalities arise as a cause or consequence of disease, the role they play in disease initiation and/or progression, or whether they themselves are targets for disease modification. Here, we review current knowledge about the function of the BM microenvironment in IMIDs including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and type I diabetes, focusing on MSCs in particular. We predict that an improved understanding of disease-related changes in the bone marrow microenvironment including the role of MSCs in vivo, will yield new insights into pathophysiology and aid identification of new drug targets and optimization of cell-based therapy in IMIDs.

An open-label pilot study of recombinant granulocyte-colony stimulating factor in Friedreich's ataxia.

Friedreich's ataxia (FA) is an inherited progressive neurodegenerative disease for which there is no proven disease-modifying treatment. Here we perform an open-label, pilot study of recombinant human granulocyte-colony stimulating factor (G-CSF) administration in seven people with FA (EudraCT: 2017-003084-34); each participant receiving a single course of G-CSF (Lenograstim; 1.28 million units per kg per day for 5 days). The primary outcome is peripheral blood mononuclear cell frataxin levels over a 19-day period. The secondary outcomes include safety, haematopoietic stem cell (HSC) mobilisation, antioxidant levels and mitochondrial enzyme activity. The trial meets pre-specified endpoints. We show that administration of G-CSF to people with FA is safe. Mobilisation of HSCs in response to G-CSF is comparable to that of healthy individuals. Notably, sustained increases in cellular frataxin concentrations and raised PGC-1α and Nrf2 expression are detected. Our findings show potential for G-CSF therapy to have a clinical impact in people with FA.

Repeat infusion of autologous bone marrow cells in progressive multiple sclerosis - A phase I extension study (SIAMMS II).

BACKGROUND: During the safety and feasibility 'Study of Intravenous Autologous Marrow in Multiple Sclerosis (SIAMMS)', intravenous infusion of autologous marrow was well tolerated. The efficacy of the approach is being explored in a placebo-controlled randomised controlled trial (ACTiMuS, NCT01815632) but it is not known whether repeated infusions will be required to optimise benefit. The objective of the current study was to explore the safety and feasibility of repeat treatment with intravenous autologous bone marrow for patients with progressive multiple sclerosis (MS). METHODS: 'SIAMMS II' was a prospective, single centre phase I extension study in which participants in the SIAMMS study were offered repeat bone marrow harvest and infusion of autologous, unfractionated bone marrow as a day-case procedure. The primary outcome measure was number of adverse events and secondary outcome measures included change in clinical rating scales of disability, global evoked potential and cranial magnetic resonance imaging (MRI). RESULTS: In total, 4 of the 6 participants in the SIAMMS study had repeat bone marrow harvest and infusion of filtered autologous marrow as a day case procedure which was well tolerated. There were no serious adverse effects. Additional outcome measures including clinical scales, global evoked potentials and cranial MRI were stable. CONCLUSION: SIAMMS II demonstrates the safety and feasibility of repeated, non-myeloablative autologous bone marrow-derived cell therapy in progressive MS.

Reduced expression of mitochondrial fumarate hydratase in progressive multiple sclerosis contributes to impaired in vitro mesenchymal stromal cell-mediated neuroprotection.

BACKGROUND: Cell-based therapies for multiple sclerosis (MS), including those employing autologous bone marrow-derived mesenchymal stromal cells (MSC) are being examined in clinical trials. However, recent studies have identified abnormalities in the MS bone marrow microenvironment. OBJECTIVE: We aimed to compare the secretome of MSC isolated from control subjects (C-MSC) and people with MS (MS-MSC) and explore the functional relevance of findings. METHODS: We employed high throughput proteomic analysis, enzyme-linked immunosorbent assays and immunoblotting, as well as in vitro assays of enzyme activity and neuroprotection. RESULTS: We demonstrated that, in progressive MS, the MSC secretome has lower levels of mitochondrial fumarate hydratase (mFH). Exogenous mFH restores the in vitro neuroprotective potential of MS-MSC. Furthermore, MS-MSC expresses reduced levels of fumarate hydratase (FH) with downstream reduction in expression of master regulators of oxidative stress. CONCLUSIONS: Our findings are further evidence of dysregulation of the bone marrow microenvironment in progressive MS with respect to anti-oxidative capacity and immunoregulatory potential. Given the clinical utility of the fumaric acid ester dimethyl fumarate in relapsing-remitting MS, our findings have potential implication for understanding MS pathophysiology and personalised therapeutic intervention.

Nodding syndrome: a concise review.

Nodding syndrome is an uncommon epileptic disorder of childhood onset, which appears to occur exclusively in clusters in sub-Saharan Africa. It was first reported in the 1960s, in what is now southern Tanzania, then in Liberia, and later in South Sudan and northern Uganda, with both epidemic and endemic patterns described. The cause remains unknown. Here we describe the background and development of descriptions of the disorder, review its clinical features and summarize current theories and studies concerning its cause, outlining the principal remaining research questions relating to this highly unusual disease.

Maternal micro-chimeric cells in the multiple sclerosis brain.

Maternal microchimeric cells (MMC) pass across the placenta from a mother to her baby during pregnancy. MMC have been identified in healthy adults, but have been reported to be more frequent and at a higher concentration in individuals with autoimmune diseases. MMC in brain tissue from individuals with autoimmune neurological disease has never previously been explored. The present study aims to identify and quantify MMC in adult human brain from control and multiple sclerosis (MS) affected individuals using fluorescent in situ hybridization (FISH) with a probe for the X and Y chromosomes. Post mortem brain tissue from 6 male MS cases and 6 male control cases were examined. Female cells presumed to be MMC were identified in 5/6 MS cases and 6/6 control cases. Cell specific labeling identified female cells of neuronal and immune phenotype in both control and active MS lesion tissue. This study shows that female cells presumed to be MMC are a common phenomenon in adult human brain where they appear to have embedded into brain tissue with the ability to express tissue specific markers.

The diagnosis of primary central nervous system vasculitis.

The diagnosis of primary central nervous system (CNS) vasculitis is often difficult. There are neither specific clinical features nor a classical clinical course, and no blood or imaging investigations that can confirm the diagnosis. Contrast catheter cerebral angiography is neither specific nor sensitive, yet still underpins the diagnosis in many published studies. Here we describe an approach to its diagnosis, emphasising the importance of obtaining tissue, and present for discussion a new, binary set of diagnostic criteria, dividing cases into only 'definite' primary CNS vasculitis, where tissue proof is available, and 'possible,' where it is not. We hope that these criteria will be modified and improved by discussion among experts, and that these (improved) criteria may then be adopted and used as the basis for future prospective studies of the clinical features and diagnosis of this difficult and dangerous disorder, particularly for coordinated multicentre therapeutic trials.

Dysregulation of Mesenchymal Stromal Cell Antioxidant Responses in Progressive Multiple Sclerosis.

The potential of autologous cell-based therapies including those using multipotent mesenchymal stromal cells (MSCs) is being investigated for multiple sclerosis (MS) and other neurological conditions. However, the phenotype of MSC in neurological diseases has not been fully characterized. We have previously shown that MSC isolated from patients with progressive MS (MS-MSC) have reduced expansion potential, premature senescence, and reduced neuroprotective potential in vitro. In view of the role of antioxidants in ageing and neuroprotection, we examined the antioxidant capacity of MS-MSC demonstrating that MS-MSC secretion of antioxidants superoxide dismutase 1 (SOD1) and glutathione S-transferase P (GSTP) is reduced and correlates negatively with the duration of progressive phase of MS. We confirmed reduced expression of SOD1 and GSTP by MS-MSC along with reduced activity of SOD and GST and, to examine the antioxidant capacity of MS-MSC under conditions of nitrosative stress, we established an in vitro cell survival assay using nitric oxide-induced cell death. MS-MSC displayed differential susceptibility to nitrosative stress with accelerated senescence and greater decline in expression of SOD1 and GSTP in keeping with reduced expression of master regulators of antioxidant responses nuclear factor erythroid 2-related factor 2 and peroxisome proliferator-activated receptor gamma coactivator 1-α. Our results are compatible with dysregulation of antioxidant responses in MS-MSC and have significant implications for development of autologous MSC-based therapies for MS, optimization of which may require that these functional deficits are reversed. Furthermore, improved understanding of the underlying mechanisms may yield novel insights into MS pathophysiology and biomarker identification. Stem Cells Translational Medicine 2018;7:748-758.

Advising patients seeking stem cell interventions for multiple sclerosis.

Given the intuitive potential of stem cell therapy and limitations of current treatment options for progressive multiple sclerosis (MS), it is not surprising that patients consider undertaking significant clinical and financial risks to access stem cell transplantation. However, while increasing evidence supports autologous haematopoietic stem cell transplantation (AHSCT) in aggressive relapsing-remitting MS, interventions employing haematopoietic or other stem cells should otherwise be considered experimental and recommended only in the context of a properly regulated clinical study. Understandably, most neurologists are unfamiliar with AHSCT procedures and the specific requirements for quality assurance and safety standards, as well as post-procedure precautions and follow-up. Consequently they may feel ill-equipped to advise patients. Here, we highlight important points for discussion in consultations with patients considering stem cell 'tourism' for MS.

Aberrant cerebellar Purkinje cell function repaired in vivo by fusion with infiltrating bone marrow-derived cells.

Bone marrow-derived cells are known to infiltrate the adult brain and fuse with cerebellar Purkinje cells. Histological observations that such heterotypic cell fusion events are substantially more frequent following cerebellar injury suggest they could have a role in the protection of mature brain neurons. To date, the possibility that cell fusion can preserve or restore the structure and function of adult brain neurons has not been directly addressed; indeed, though frequently suggested, the possibility of benefit has always been rather speculative. Here we report, for the first time, that fusion of a bone marrow-derived cell with a neuron in vivo, in the mature brain, results in the formation of a spontaneously firing neuron. Notably, we also provide evidence supporting the concept that heterotypic cell fusion acts as a biological mechanism to repair pathological changes in Purkinje cell structure and electrophysiology. We induced chronic central nervous system inflammation in chimeric mice expressing bone marrow cells tagged with enhanced green fluorescent protein. Subsequent in-depth histological analysis revealed significant Purkinje cell injury. In addition, there was an increased incidence of cell fusion between bone marrow-derived cells and Purkinje cells, revealed as enhanced green fluorescent protein-expressing binucleate heterokaryons. These fused cells resembled healthy Purkinje cells in their morphology, soma size, ability to synthesize the neurotransmitter gamma-aminobutyric acid, and synaptic innervation from neighbouring cells. Extracellular recording of spontaneous firing ex vivo revealed a shift in the predominant mode of firing of non-fused Purkinje cells in the context of cerebellar inflammation. By contrast, the firing patterns of fused Purkinje cells were the same as in healthy control cerebellum, indicating that fusion of bone marrow-derived cells with Purkinje cells mitigated the effects of cell injury on electrical activity. Together, our histological and electrophysiological results provide novel fundamental insights into physiological processes by which nerve cells are protected in adult life.

Bone marrow transplantation stimulates neural repair in Friedreich's ataxia mice.

OBJECTIVE: Friedreich's ataxia is an incurable inherited neurological disease caused by frataxin deficiency. Here, we report the neuroreparative effects of myeloablative allogeneic bone marrow transplantation in a humanized murine model of the disease. METHODS: Mice received a transplant of fluorescently tagged sex-mismatched bone marrow cells expressing wild-type frataxin and were assessed at monthly intervals using a range of behavioral motor performance tests. At 6 months post-transplant, mice were euthanized for protein and histological analysis. In an attempt to augment numbers of bone marrow-derived cells integrating within the nervous system and improve therapeutic efficacy, a subgroup of transplanted mice also received monthly subcutaneous infusions of the cytokines granulocyte-colony stimulating factor and stem cell factor. RESULTS: Transplantation caused improvements in several indicators of motor coordination and locomotor activity. Elevations in frataxin levels and antioxidant defenses were detected. Abrogation of disease pathology throughout the nervous system was apparent, together with extensive integration of bone marrow-derived cells in areas of nervous tissue injury that contributed genetic material to mature neurons, satellite-like cells, and myelinating Schwann cells by processes including cell fusion. Elevations in circulating bone marrow-derived cell numbers were detected after cytokine administration and were associated with increased frequencies of Purkinje cell fusion and bone marrow-derived dorsal root ganglion satellite-like cells. Further improvements in motor coordination and activity were evident. INTERPRETATION: Our data provide proof of concept of gene replacement therapy, via allogeneic bone marrow transplantation, that reverses neurological features of Friedreich's ataxia with the potential for rapid clinical translation. Ann Neurol 2018;83:779-793.

Reduced neuroprotective potential of the mesenchymal stromal cell secretome with ex vivo expansion, age and progressive multiple sclerosis.

BACKGROUND: Clinical trials using ex vivo expansion of autologous mesenchymal stromal cells (MSCs) are in progress for several neurological diseases including multiple sclerosis (MS). Given that environment alters MSC function, we examined whether in vitro expansion, increasing donor age and progressive MS affect the neuroprotective properties of the MSC secretome. METHODS: Comparative analyses of neuronal survival in the presence of MSC-conditioned medium (MSCcm) isolated from control subjects (C-MSCcm) and those with MS (MS-MSCcm) were performed following (1) trophic factor withdrawal and (2) nitric oxide-induced neurotoxicity. RESULTS: Reduced neuronal survival following trophic factor withdrawal was seen in association with increasing expansion of MSCs in vitro and MSC donor age. Controlling for these factors, there was an independent, negative effect of progressive MS. In nitric oxide neurotoxicity, MSCcm-mediated neuroprotection was reduced when C-MSCcm was isolated from higher-passage MSCs and was negatively associated with increasing MSC passage number and donor age. Furthermore, the neuroprotective effect of MSCcm was lost when MSCs were isolated from patients with MS. DISCUSSION: Our findings have significant implications for MSC-based therapy in neurodegenerative conditions, particularly for autologous MSC therapy in MS. Impaired neuroprotection mediated by the MSC secretome in progressive MS may reflect reduced reparative potential of autologous MSC-based therapy in MS and it is likely that the causes must be addressed before the full potential of MSC-based therapy is realized. Additionally, we anticipate that understanding the mechanisms responsible will contribute new insights into MS pathogenesis and may also be of wider relevance to other neurodegenerative conditions.

Reduced cellularity of bone marrow in multiple sclerosis with decreased MSC expansion potential and premature ageing in vitro.

BACKGROUND: Autologous bone-marrow-derived cells are currently employed in clinical studies of cell-based therapy in multiple sclerosis (MS) although the bone marrow microenvironment and marrow-derived cells isolated from patients with MS have not been extensively characterised. OBJECTIVES: To examine the bone marrow microenvironment and assess the proliferative potential of multipotent mesenchymal stromal cells (MSCs) in progressive MS. METHODS: Comparative phenotypic analysis of bone marrow and marrow-derived MSCs isolated from patients with progressive MS and control subjects was undertaken. RESULTS: In MS marrow, there was an interstitial infiltrate of inflammatory cells with lymphoid (predominantly T-cell) nodules although total cellularity was reduced. Controlling for age, MSCs isolated from patients with MS had reduced in vitro expansion potential as determined by population doubling time, colony-forming unit assay, and expression of ß-galactosidase. MS MSCs expressed reduced levels of Stro-1 and displayed accelerated shortening of telomere terminal restriction fragments (TRF) in vitro. CONCLUSION: Our results are consistent with reduced proliferative capacity and ex vivo premature ageing of bone-marrow-derived cells, particularly MSCs, in MS. They have significant implication for MSC-based therapies for MS and suggest that accelerated cellular ageing and senescence may contribute to the pathophysiology of progressive MS.

Cell-based therapeutic strategies for multiple sclerosis.

The availability of multiple disease-modifying medications with regulatory approval to treat multiple sclerosis illustrates the substantial progress made in therapy of the disease. However, all are only partially effective in preventing inflammatory tissue damage in the central nervous system and none directly promotes repair. Cell-based therapies, including immunoablation followed by autologous haematopoietic stem cell transplantation, mesenchymal and related stem cell transplantation, pharmacologic manipulation of endogenous stem cells to enhance their reparative capabilities, and transplantation of oligodendrocyte progenitor cells, have generated substantial interest as novel therapeutic strategies for immune modulation, neuroprotection, or repair of the damaged central nervous system in multiple sclerosis. Each approach has potential advantages but also safety concerns and unresolved questions. Moreover, clinical trials of cell-based therapies present several unique methodological and ethical issues. We summarize here the status of cell-based therapies to treat multiple sclerosis and make consensus recommendations for future research and clinical trials.

Cell Therapy for Multiple Sclerosis.

Cell therapy is considered a promising potential treatment for multiple sclerosis, perhaps particularly for the progressive form of the disease for which there are currently no useful treatments. Over the past two decades or more, much progress has been made in understanding the biology of MS and in the experimental development of cell therapy for this disease. Three quite distinct forms of cell therapy are currently being pursued. The first seeks to use stem cells to replace damaged myelin-forming oligodendrocytes within the CNS; the second aims, in effect, to replace the individual's misfunctioning immune system, making use of haematopoietic stem cells; and the third seeks to utilise endogenous stem cell populations by mobilisation with or without in vitro expansion, exploiting their various reparative and neuroprotective properties. In this article we review progress in these three separate areas, summarising the experimental background and clinical progress thus far made.