210 research outputs found
Induced Pluripotent Stem Cell (iPSC)-derived Microglia and Organoids for neuronal disease modeling
No full textMicroglia are the principal resident immune cells of the central nervous system (CNS) representing 5-12% of the total cell population in the brain. They are of myeloid origin and their survival and maintenance depend on several cytokines. Microglia play a role in development of the CNS and are constantly engaged in detecting changes in their environment, maintaining homeostasis and protecting against endogenous or environmental injurious agents. Microglia has emerged as an interesting target in many neurodegenerative diseases which deserve to be studied in more detail. Indeed, the mechanisms by which aberrant microglia activation causes neurodegeneration remains unclear. In its activated state, microglia play a role in neurodegeneration of most CNS disorders such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease and Parkinson’s disease. Microglia is implicated in the initiation and in the progress of these diseases, switching from a neuroprotective activated state to a neurotoxic one. Phagocytosis, which involves engulfment and elimination of large cellular structures, is carried out by microglia and can be regulated by an autophagic process which impairment is associated with many neurodegenerative disorders, in part related to an increased release of inducers of the inflammatory response. We preliminary optimized a protocol to differentiate microglia from iPSCs generated from both healthy donor and patients affected by neurodegenerative diseases such as ALS and FTD. Microglia cells were characterized for the expression of specific markers (CD11b, Vimentin, TREM2, TMEM119, IBA1) by immunofluorescence, while their functionality was evaluated by assessing fluorescent latex-beads internalization which confirmed their phagocytic activity. We also evaluated the presence of lipid droplets, consisting in organelles that contain neutral lipids, increasingly being accepted as structural markers of inflammation. In addition, both neural and spinal cord organoids were obtained from iPSCs. Given the important modulatory role in neural death in neurodegenerative diseases, we will establish co-cultures of microglia and iPSC derived organoids combining microglia obtained from patients with organoids derived from controls and vice-versa in order to analyze engraftment of microglia on organoids and to study the interactions between microglial and neural cells
Induced Pluripotent Stem Cell (iPSC)-derived Microglia for ALS modeling
No full textAmyotrophic Lateral Sclerosis (ALS) is a progressive and fatal disease, primarily characterized by the degeneration of upper/lower motoneurons. ALS has a still incompletely understood etiopathology, but it is thought to be caused by the combination of neuronal cell-autonomous and non-cell autonomous mechanisms involving microglia. Microglia, the innate immune cells of the central nervous system, regulate neuroinflammation and their uncontrolled activation, consisting in the aberrant persistence of a pro-inflammatory state, can promote neurotoxicity in ALS.
Aim of our work is to investigate the possible effects of ALS patient-derived microglia in promoting neurotoxicity and the potential neuroprotective role of healthy donor-derived microglia by co-culturing microglial cells obtained from induced pluripotent stem cells (iPSC-microglia) with iPSC-derived motoneurons (iPSC-MNs) and iPSC-derived spinal cord organoids (iPSC-spORGs).
Our work first focused on the preliminary optimization of the differentiation protocols to obtain all the iPSC-derived models to be further used in co-culture conditions. In particular, iPSC-microglia were obtained from healthy donor iPSCs after 55 days of differentiation. Microglia cells were characterized for the expression of specific markers (CD11b, Vimentin, TREM2, TMEM119, IBA1) by immunofluorescence, while their functionality was evaluated by assessing fluorescent latex-beads internalization which confirmed their phagocytic activity. iPSC-MNs were differentiated from embryoid bodies for 34 days and iPSC-spORGs were obtained in 21 days. Both iPSC-MNs and iPSC-spORGs tested positive for the expression of neuronal and motoneuronal markers (SMI-312, Choline Acetyltransferase and HB9) by immunofluorescence and RT-PCR.
Our data indicate that we can efficiently obtain all the iPSC-derived in vitro models to be now used in co-culture systems also from ALS patients’ cells. This will allow to study the possible neurotoxicity of human ALS iPSC-microglia in triggering neuronal death in iPSC-MNs/spORGs and the neuroprotective role of healthy microglia in counteracting neurodegeneration, further contributing to elucidate the interplay of cell-autonomous and non-cell autonomous mechanisms in ALS pathogenesis
Induced Pluripotent Stem Cell (iPSC)-derived Microglia for ALS modeling
No full textAmyotrophic Lateral Sclerosis (ALS) is a progressive and fatal disease, primarily characterized by the degeneration of upper/lower motoneurons. ALS has a still incompletely understood etiopathology, but it is thought to be caused by the combination of neuronal cell-autonomous and non-cell autonomous mechanisms involving microglia. Microglia, the innate immune cells of the central nervous system, regulate neuroinflammation and their uncontrolled activation, consisting in the aberrant persistence of a pro-inflammatory state, can promote neurotoxicity in ALS.
Aim of our work is to investigate the possible effects of ALS patient-derived microglia in promoting neurotoxicity and the potential neuroprotective role of healthy donor-derived microglia by co-culturing microglial cells obtained from induced pluripotent stem cells (iPSC-microglia) with iPSC-derived motoneurons (iPSC-MNs) and iPSC-derived spinal cord organoids (iPSC-spORGs).
Our work first focused on the preliminary optimization of the differentiation protocols to obtain all the iPSC-derived models to be further used in co-culture conditions. In particular, iPSC-microglia were obtained from healthy donor iPSCs after 55 days of differentiation. Microglia cells were characterized for the expression of specific markers (CD11b, Vimentin, TREM2, TMEM119, IBA1) by immunofluorescence, while their functionality was evaluated by assessing fluorescent latex-beads internalization which confirmed their phagocytic activity. iPSC-MNs were differentiated from embryoid bodies for 34 days and iPSC-spORGs were obtained in 21 days. Both iPSC-MNs and iPSC-spORGs tested positive for the expression of neuronal and motoneuronal markers (SMI-312, Choline Acetyltransferase and HB9) by immunofluorescence and RT-PCR.
Our data indicate that we can efficiently obtain all the iPSC-derived in vitro models to be now used in co-culture systems also from ALS patients’ cells. This will allow to study the possible neurotoxicity of human ALS iPSC-microglia in triggering neuronal death in iPSC-MNs/spORGs and the neuroprotective role of healthy microglia in counteracting neurodegeneration, further contributing to elucidate the interplay of cell-autonomous and non-cell autonomous mechanisms in ALS pathogenesis
Induced Pluripotent Stem Cell (iPSC)-derived Microglia for ALS modeling
No full textAmyotrophic Lateral Sclerosis (ALS) is a progressive and fatal disease, primarily characterized by the degeneration of upper/lower motoneurons. ALS has a still incompletely understood etiopathology, but it is thought to be caused by the combination of neuronal cell-autonomous and non-cell autonomous mechanisms involving microglia. Microglia, the innate immune cells of the central nervous system, regulate neuroinflammation and their uncontrolled activation, consisting in the aberrant persistence of a pro-inflammatory state, can promote neurotoxicity in ALS.
Aim of our work is to investigate the possible effects of ALS patient-derived microglia in promoting neurotoxicity and the potential neuroprotective role of healthy donor-derived microglia by co-culturing microglial cells obtained from induced pluripotent stem cells (iPSC-microglia) with iPSC-derived motoneurons (iPSC-MNs) and iPSC-derived spinal cord organoids (iPSC-spORGs).
Our work first focused on the preliminary optimization of the differentiation protocols to obtain all the iPSC-derived models to be further used in co-culture conditions. In particular, iPSC-microglia were obtained from healthy donor iPSCs after 55 days of differentiation. Microglia cells were characterized for the expression of specific markers (CD11b, Vimentin, TREM2, TMEM119, IBA1) by immunofluorescence, while their functionality was evaluated by assessing fluorescent latex-beads internalization which confirmed their phagocytic activity. iPSC-MNs were differentiated from embryoid bodies for 34 days and iPSC-spORGs were obtained in 21 days. Both iPSC-MNs and iPSC-spORGs tested positive for the expression of neuronal and motoneuronal markers (SMI-312, Choline Acetyltransferase and HB9) by immunofluorescence and RT-PCR.
Our data indicate that we can efficiently obtain all the iPSC-derived in vitro models to be now used in co-culture systems also from ALS patients’ cells. This will allow to study the possible neurotoxicity of human ALS iPSC-microglia in triggering neuronal death in iPSC-MNs/spORGs and the neuroprotective role of healthy microglia in counteracting neurodegeneration, further contributing to elucidate the interplay of cell-autonomous and non-cell autonomous mechanisms in ALS pathogenesis
Brisk walking improves fitness and metabolic markers in combination antiretroviral therapy (cART)-treated persons
No full textThe potential impact of splenectomy in treatment of visceral Leishmaniasis in a multi-experienced HIV-coinfected patient
No full textMotor neuron differentiation of iPSCs obtained from peripheral blood of a mutant TARDBP ALS patient
No full textAmyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease, mainly affecting the motor neurons (MNs) and without effective therapy. Drug screening is hampered by the lack of satisfactory experimental and pre-clinical models. Induced pluripotent stem cells (iPSCs) could help to define disease mechanisms and therapeutic strategies as they could be differentiated into MNs, otherwise inaccessible from living humans. In this study, given the seminal role of TDP-43 in ALS pathophysiology, MNs were obtained from peripheral blood mononuclear cells-derived iPSCs of an ALS patient carrying a p.A382T TARDBP mutation and a healthy donor. Venous samples were preferred to fibroblasts for their ease of collection and no requirement for time consuming extended cultures before experimentation. iPSCs were characterized for expression of specific markers, spontaneously differentiated into primary germ layers and, finally, into MNs. No differences were observed between the mutated ALS patient and the control MNs with most of the cells displaying a nuclear localization of the TDP-43 protein. In conclusion, we here demonstrated for the first time that human TARDBP mutated MNs can be successfully obtained exploiting the reprogramming and differentiation ability of peripheral blood cells, an easily accessible source from any patient
A case of relapse of progressive multifocal leukoencephalopathy (PML) in an HIV-infected patient.
No full textRapamycin reverts TDP-43 splicing defects and mislocalization in human in vitro models of TDP-43 proteinopathy
No full textAggregates of phosphorylated TDP-43 protein in the cytoplasm of neurons are an ALS neuropathological hallmark. Response to stress and formation of stress granules (SGs) have been proposed as initiators of TDP-43 pathological aggregation. We previously showed that chronic oxidative stress by arsenite (ARS) induces the formation of SGs and phospho-TDP-43 aggregates in primary fibroblasts and iPSC-motor neurons from ALS patients.
Aim of our study was to generate a robust and reproducible in vitro model of TDP-43 pathology to be used for drug screening. We induced a chronic oxidative insult in human neuroblastoma SK-N-BE cells by exposure to low doses of ARS for 9-24 hours. Our data showed TDP-43 mislocalization from the nucleus to the cytoplasm in both a dose- and time-dependent manner and an increase of P62. We also observed a defective splicing activity of TDP-43 towards its target genes UNC13A and POLDIP3, used as readouts of TDP-43 nuclear loss-of-function. We tested candidate drugs involved in promoting autophagy, namely rapamycin, lithium carbonate and metformin in our in vitro model of TDP-43 proteinopathy. Only rapamycin was able to rescue ARS-induced loss of TDP-43 splicing activity on its target gene UNC13A and to reduce P62 accumulation upon chronic ARS treatment. We also tested rapamycin in C9ORF72 patients-derived fibroblasts and iPSC-motor neurons, where its efficacy in rescuing ARS-induced loss of TDP-43 splicing activity was confirmed. Importantly, rapamycin also significantly reduced phospho-TDP-43 aggregates and SGs formation in both patients-derived cell models.
In conclusion, we have set up human cell models of TDP-43 pathology in which rapamycin was proven to be beneficial in rescuing chronic oxidative stress-induced alterations in TDP-43 splicing activity and cytoplasmic mislocalization by modulating autophagy. Human SK-N-BE and ALS patient-derived cells chronically treated with ARS can therefore be exploited as valuable in vitro platforms for future drug screening approaches
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