Gangliosides in nervous system development, regeneration, and pathologies.

Gangliosides, sialic acid-containing sphingolipids, are major constituents of neuronal membranes. According to the number of sialic acids and the structure of the oligosaccharide chain, gangliosides can be classified as simple or complex and grouped in different ganglio-series. Hundreds of gangliosides have been identified in vertebrate cells, with different expression patterns during development and related to several physiological processes, especially in the nervous system. While GD3 and its O-acetylated form, 9acGD3, are highly expressed in early developmental stages, GM1, GD1a, GD1b, and GT1b are the most abundant ganglioside species in the mature nervous system. Mutations in enzymes involved in ganglioside metabolism can lead to the accumulation of specific species, a condition termed gangliosidosis and usually marked by severe neurological impairment. Changes in ganglioside levels have also been described in several neurodegenerative diseases, such as Alzheimer's and Parkinson's. In this review, we summarized recent information about the roles of GD3, 9acGD3, GM1, GD1a, GD1b, GT1b, and other ganglioside species in nervous system development and regeneration, as well as clinical trials evaluating possible therapeutic applications of these molecules.

Current Status of Mesenchymal Stem/Stromal Cells for Treatment of Neurological Diseases.

Neurological disorders include a wide spectrum of clinical conditions affecting the central and peripheral nervous systems. For these conditions, which affect hundreds of millions of people worldwide, generally limited or no treatments are available, and cell-based therapies have been intensively investigated in preclinical and clinical studies. Among the available cell types, mesenchymal stem/stromal cells (MSCs) have been widely studied but as yet no cell-based treatment exists for neurological disease. We review current knowledge of the therapeutic potential of MSC-based therapies for neurological diseases, as well as possible mechanisms of action that may be explored to hasten the development of new and effective treatments. We also discuss the challenges for culture conditions, quality control, and the development of potency tests, aiming to generate more efficient cell therapy products for neurological disorders.

Mesenchymal Stem Cell Therapy in Diabetic Cardiomyopathy.

The incidence and prevalence of diabetes mellitus (DM) are increasing worldwide, and the resulting cardiac complications are the leading cause of death. Among these complications is diabetes-induced cardiomyopathy (DCM), which is the consequence of a pro-inflammatory condition, oxidative stress and fibrosis caused by hyperglycemia. Cardiac remodeling will lead to an imbalance in cell survival and death, which can promote cardiac dysfunction. Since the conventional treatment of DM generally does not address the prevention of cardiac remodeling, it is important to develop new alternatives for the treatment of cardiovascular complications induced by DM. Thus, therapy with mesenchymal stem cells has been shown to be a promising approach for the prevention of DCM because of their anti-apoptotic, anti-fibrotic and anti-inflammatory effects, which could improve cardiac function in patients with DM.

Mesenchymal Stem Cells Therapies on Fibrotic Heart Diseases.

Stem cell therapy is a promising alternative approach to heart diseases. The most prevalent source of multipotent stem cells, usually called somatic or adult stem cells (mesenchymal stromal/stem cells, MSCs) used in clinical trials is bone marrow (BM-MSCs), adipose tissue (AT-MSCs), umbilical cord (UC-MSCs) and placenta. Therapeutic use of MSCs in cardiovascular diseases is based on the benefits in reducing cardiac fibrosis and inflammation that compose the cardiac remodeling responsible for the maintenance of normal function, something which may end up causing progressive and irreversible dysfunction. Many factors lead to cardiac fibrosis and failure, and an effective therapy is lacking to reverse or attenuate this condition. Different approaches have been shown to be promising in surpassing the poor survival of transplanted cells in cardiac tissue to provide cardioprotection and prevent cardiac remodeling. This review includes the description of pre-clinical and clinical investigation of the therapeutic potential of MSCs in improving ventricular dysfunction consequent to diverse cardiac diseases.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.

Human mesenchymal stem cell therapy promotes retinal ganglion cell survival and target reconnection after optic nerve crush in adult rats.

BACKGROUND: Optic-nerve injury results in impaired transmission of visual signals to central targets and leads to the death of retinal ganglion cells (RGCs) and irreversible vision loss. Therapies with mesenchymal stem cells (MSCs) from different sources have been used experimentally to increase survival and regeneration of RGCs. METHODS: We investigated the efficacy of human umbilical Wharton's jelly-derived MSCs (hWJ-MSCs) and their extracellular vesicles (EVs) in a rat model of optic nerve crush. RESULTS: hWJ-MSCs had a sustained neuroprotective effect on RGCs for 14, 60, and 120 days after optic nerve crush. The same effect was obtained using serum-deprived hWJ-MSCs, whereas transplantation of EVs obtained from those cells was ineffective. Treatment with hWJ-MSCs also promoted axonal regeneration along the optic nerve and reinnervation of visual targets 120 days after crush. CONCLUSIONS: The observations showed that this treatment with human-derived MSCs promoted sustained neuroprotection and regeneration of RGCs after optic nerve injury. These findings highlight the possibility to use cell therapy to preserve neurons and to promote axon regeneration, using a reliable source of human MSCs.

Therapeutic Benefit of the Association of Lodenafil with Mesenchymal Stem Cells on Hypoxia-induced Pulmonary Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. This work investigated the benefit of the association of human umbilical cord mesenchymal stem cells (hMSCs) with lodenafil, a phosphodiesterase-5 inhibitor, in an animal model of PAH. Male Wistar rats were exposed to hypoxia (10% O2) for three weeks plus a weekly i.p. injection of a vascular endothelial growth factor receptor inhibitor (SU5416, 20 mg/kg, SuHx). After confirmation of PAH, animals received intravenous injection of 5.105 hMSCs or vehicle, followed by oral treatment with lodenafil carbonate (10 mg/kg/day) for 14 days. The ratio between pulmonary artery acceleration time and RV ejection time reduced from 0.42 ± 0.01 (control) to 0.24 ± 0.01 in the SuHx group, which was not altered by lodenafil alone but was recovered to 0.31 ± 0.01 when administered in association with hMSCs. RV afterload was confirmed in the SuHx group with an increased RV systolic pressure (mmHg) of 52.1 ± 8.8 normalized to 29.6 ± 2.2 after treatment with the association. Treatment with hMSCs + lodenafil reversed RV hypertrophy, fibrosis and interstitial cell infiltration in the SuHx group. Combined therapy of lodenafil and hMSCs may be a strategy for PAH treatment.

Generation of patient-specific pluripotent induced stem cell line UFRJi007-A from a Brazilian familial amyotrophic lateral sclerosis patient.

Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Generation of four patient-specific pluripotent induced stem cell lines from two Brazilian patients with amyotrophic lateral sclerosis and two healthy subjects.

Induced pluripotent stem cell (iPSC) lines were generated from erythroblasts of two patients with amyotrophic lateral sclerosis (ALS) and two healthy individuals. One familial and one sporadic ALS patients were used, both with genetic alterations in VAPB gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the iPSC cell lines. The four iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Long-term neuronal survival, regeneration, and transient target reconnection after optic nerve crush and mesenchymal stem cell transplantation.

BACKGROUND: Retina and/or optic nerve injury may cause irreversible blindness, due to degeneration of retinal ganglion cells. We and others have previously shown that the intravitreal injection of mesenchymal stem cells (MSCs) protects injured retinal ganglion cells and stimulates their regeneration after optic nerve injury, but the long-term effects of this therapy are still unknown. METHODS: We injected rat MSC (rMSC) intravitreally in adult (3-5 months) Lister Hooded rats of either sex after optic nerve crush. Retinal ganglion cell survival, axonal regeneration, and reconnection were analyzed 60 and 240 days after crush by immunohistochemistry for Tuj1, anterograde labeling with cholera-toxin B and by immunohistochemistry for nerve growth factor-induced gene A (NGFI-A, driven by light stimulation) in the superior colliculus after a cycle of light deprivation-stimulation. Visual behaviors (optokinetic reflex, looming response, and preference for dark) were analyzed 70 days after crush. RESULTS: rMSC treatment doubled the number of surviving retinal ganglion cells, preferentially of a larger subtype, and of axons regenerating up to 0.5 mm. Some axons regenerated to the lateral geniculate nucleus and superior colliculus. NGFI-A+ cells were doubled in rMSC-treated animals 60 days after crush, but equivalent to vehicle-injected animals 240 days after crush, suggesting that newly formed synapses degenerated. Animals did not recover visual behaviors. CONCLUSIONS: We conclude that rMSC-induced neuroprotection is sustained at longer time points. Although rMSCs promoted long-term neuroprotection and long-distance axon regeneration, the reconnection of retinal ganglion cells with their targets was transitory, indicating that they need additional stimuli to make stable reconnections.