Three-dimensional video reconstruction of iCtx cell ex vivo transplanted onto adult human-derived... more Three-dimensional video reconstruction of iCtx cell ex vivo transplanted onto adult human-derived cortical tissue. Fly-through animation of three-dimensional data illustrating pyramidal shape of iCtx cell (in red) with elaborated neurites extending throughout the whole depth of the adult human cortical slice at 3Â weeks after ex vivo transplantation. Note many spines of different size and shape including mushroom-like ones. Many Hoechst-stained cell nuclei of human cortex (in blue) are distributed throughout the slice. Scale bar in left bottom corner. (MOV 16146 kb)
CBL is a multifunctional protein engaged in the regulation of intracellular signaling pathways. R... more CBL is a multifunctional protein engaged in the regulation of intracellular signaling pathways. Recent studies have found CBL mutations and deletions in a variety of myeloid diseases. In acute myeloid leukemia (AML), CBL mutations are associated with core binding factor (CBF) leukemia, a subset of AML characterized by the presence of AML1-ETO or CBFB-MYH11 fusion genes. However, the potential cooperation between mutant CBL and CBF-fusion proteins has not been demonstrated experimentally. We have developed an experimental system to model myeloid leukemogenesis using primary human cord blood (CB) cells, and have shown that CBF-fusion proteins promote self-renewal and long-term proliferation of human CB CD34+ cells in vitro. Using the human cell-based model, we demonstrated that CBL mutations/depletion promote cytokine-dependent growth, but do not increase self-renewal, of human CB CD34+ progenitors. We also showed that CBL mutations/depletion collaborate with AML1-ETO to expand CB cells both in vitro and in a xenograft model. CBL inactivation promoted cell cycle progression, conferred hyperresponsiveness to thrombopoietin (THPO), and enhanced STAT5/ERK/Src signaling pathways in human AML1-ETO cells. The CBL-inactivated AML1-ETO cells did not show addiction to a specific pathway, but JAK inhibitors suppressed the growth of AML1-ETO cells including those with CBL mutations/depletion. Furthermore, we found that a protein tyrosine phosphatase UBASH3B (also called STS-1), which is known to inhibit CBL function, is highly expressed in CBF leukemia. Knockdown of UBASH3B inhibited the growth of AML1-ETO leukemia cells, and the growth-inhibitory effect of UBASH3B depletion was reversed by overexpression of oncogenic CBL mutants. Thus, the oncogenic potential of UBASH3B is largely dependent on inactivation of CBL. In summary, we established the first human cell-based model for AML with CBL dysfunction. The UBASH3B-CBL axis plays a crucial role to control multiple signaling pathways and myeloid proliferation in human AML1-ETO leukemia, thereby providing potential therapeutic targets.
In the field of developmental biology, there is compelling evidence for a network of activity of ... more In the field of developmental biology, there is compelling evidence for a network of activity of pluripotency and stem-associated genes comprising of Oct4, Nanog and nestin. During neurogenesis, the choice between enhancement versus suppression of transcriptional modulation of these identified genes determines the balance between self-renewal neural stem cells (NSC) and immature neuronal phenotypes. By using immunocytochemistry and RT-PCR techniques, our study aims to address the question whether and to what extent mRNA and protein profiles are expressed in human fetal neurospheres obtained from cortical and striatal brain regions, both in expansion (undifferentiated cells) and differentiation conditions monitored after 1 and 4 weeks in vitro culturing. Our results clearly demonstrate the sustained presence of opposite signals: strong downregulation of Oct4 and Nanog genes in cortical differentiating cells and significant up-regulation for nestin gene both in cortical and striatal d...
Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation... more Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk Ϫ/Ϫ mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain.
Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel a... more Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells survive long-term, differentiate to functional neurons, and induce recovery in the stroke-injured brain are unclear. We have transplanted long-term self-renewing neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs, into the stroke-damaged mouse and rat striatum or cortex. Recovery of forepaw movements was observed already at 1 week after transplantation. Improvement was most likely not due to neuronal replacement but was associated with increased vascular endothelial growth factor levels, probably enhancing endogenous plasticity. Transplanted cells stopped proliferating, could survive without forming tumors for at least 4 months, and differentiated to morphologically mature neurons of different subtypes. Neurons in intrastriatal grafts sent axonal projections to t...
Neural stem/progenitor cells (NSPCs) in subventricular zone (SVZ) produce new striatal neurons du... more Neural stem/progenitor cells (NSPCs) in subventricular zone (SVZ) produce new striatal neurons during several months after stroke, which may contribute to recovery. Intracerebral grafts of NSPCs can exert beneficial effects after stroke through neuronal replacement, trophic actions, neuroprotection, and modulation of inflammation. Here we have explored whether human fetal striatum-derived NSPC-grafts influence striatal neurogenesis and promote recovery in stroke-damaged brain. T cell-deficient rats were subjected to 1 h middle cerebral artery occlusion (MCAO). Human fetal NSPCs or vehicle were implanted into ipsilateral striatum 48 h after MCAO, animals were assessed behaviorally, and perfused at 6 or 14 weeks. Grafted human NSPCs survived in all rats, and a subpopulation had differentiated to neuroblasts or mature neurons at 6 and 14 weeks. Numbers of proliferating cells in SVZ and new migrating neuroblasts and mature neurons were higher, and numbers of activated microglia/macrophages were lower in the ischemic striatum of NSPC-grafted compared to vehicle-injected group both at 6 and 14 weeks. A fraction of grafted NSPCs projected axons from striatum to globus pallidus. The NSPC-grafted rats showed improved functional recovery in stepping and cylinder tests from 6 and 12 weeks, respectively. Our data show, for the first time, that intrastriatal implants of human fetal NSPCs exert a long-term enhancement of several steps of striatal neurogensis after stroke. The grafts also suppress striatal inflammation and ameliorate neurological deficits. Our findings support the idea that combination of NSPC transplantation and stimulation of neurogenesis from endogenous NSPCs may become a valuable strategy for functional restoration after stroke.
Journal of Cerebral Blood Flow & Metabolism, 2010
Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survi... more Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survive in stroke-damaged striatum, migrate from the implantation site, and differentiate into mature neurons. Here, we investigated how various steps of neurogenesis are affected by intrastriatal transplantation of human NSCs at different time points after stroke and with different numbers of cells in each implant. Rats were subjected to middle cerebral artery occlusion and then received intrastriatal transplants of NSCs. Transplantation shortly after stroke (48 hours) resulted in better cell survival than did transplantation 6 weeks after stroke, but the delayed transplantation did not influence the magnitude of migration, neuronal differentiation, and cell proliferation in the grafts. Transplanting greater numbers of grafted NSCs did not result in a greater number of surviving cells or increased neuronal differentiation. A substantial number of activated microglia was observed at 48 hours ...
Stem cell-based approaches to restore function after stroke through replacement of dead neurons r... more Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term selfrenewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated celltransplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our findings show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intracortical implantation in a rat stroke model.
Induced pluripotent stem cells (iPSCs) improve behavior and form neurons after implantation into ... more Induced pluripotent stem cells (iPSCs) improve behavior and form neurons after implantation into the stroke-injured adult rodent brain. How the aged brain responds to grafted iPSCs is unknown. We determined survival and differentiation of grafted human fibroblast-derived iPSCs and their ability to improve recovery in aged rats after stroke. Twenty-four months old rats were subjected to 30 min distal middle cerebral artery occlusion causing neocortical damage. After 48 h, animals were transplanted intracortically with human iPSC-derived long-term neuroepithelial-like stem (hiPSC-lt-NES) cells. Controls were subjected to stroke and were vehicle-injected. Cell-grafted animals performed better than vehicle-injected recipients in cylinder test at 4 and 7 weeks. At 8 weeks, cell proliferation was low (0.7 %) and number of hiPSC-lt-NES cells corresponded to 49.2% of that of implanted cells. Transplanted cells expressed markers of neuroblasts and mature and GABAergic neurons. Cell-grafted r...
The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)‐2α... more The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)‐2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development.
Stem cell transplantation can improve behavioral recovery after stroke in animal models but wheth... more Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell–derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell–derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus–based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neuro...
The neural crest is a stem cell population that gives rise to sympathetic ganglia, the cell type ... more The neural crest is a stem cell population that gives rise to sympathetic ganglia, the cell type of origin of neuroblastoma. Hypoxia Inducible Factor (HIF)-2α is associated with high risk neuroblastoma, however, little is known about its role in normal neural crest development. To address this important question, here we show that HIF-2α is expressed in trunk neural crest cells of human, murine and avian embryos. Modulating HIF-2α in vivo not only causes developmental delays but also induces proliferation and stemness of neural crest cells while altering the number of cells migrating ventrally to sympathoadrenal sites. Transcriptome changes after loss of HIF-2α reflect the in vivo phenotype. The results suggest that expression levels of HIF-2α must be strictly controlled and abnormal levels increase stemness and may promote metastasis. Our findings help elucidate the role of HIF-2α during normal development with implications also in tumor initiation at the onset of neuroblastoma.
Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols ... more Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.
Mutations of Fused in sarcoma (FUs), a ribonucleoprotein involved in RNA metabolism, have been fo... more Mutations of Fused in sarcoma (FUs), a ribonucleoprotein involved in RNA metabolism, have been found associated with both familial and sporadic cases of amyotrophic lateral sclerosis (ALs). Notably, besides mutations in the coding sequence, also mutations into the 3′ untranslated region, leading to increased levels of the wild-type protein, have been associated with neuronal death and ALs pathology, in ALs models and patients. the mechanistic link between altered FUs levels and ALsrelated neurodegeneration is far to be elucidated, as well as the consequences of elevated FUs levels in the modulation of the inflammatory response sustained by glial cells, a well-recognized player in ALS progression. Here, we studied the effect of wild-type FUS overexpression on the responsiveness of mouse and human neural progenitor-derived astrocytes to a pro-inflammatory stimulus (IL1β) used to mimic an inflammatory environment. We found that astrocytes with increased FUS levels were more sensitive to IL1β, as shown by their enhanced expression of inflammatory genes, compared with control astrocytes. Moreover, astrocytes overexpressing FUS promoted neuronal cell death and pro-inflammatory microglia activation. We conclude that overexpression of wild-type FUS intrinsically affects astrocyte reactivity and drives their properties toward pro-inflammatory and neurotoxic functions, suggesting that a non-cell autonomous mechanism can support neurodegeneration in FUsmutated animals and patients.
Three-dimensional video reconstruction of iCtx cell ex vivo transplanted onto adult human-derived... more Three-dimensional video reconstruction of iCtx cell ex vivo transplanted onto adult human-derived cortical tissue. Fly-through animation of three-dimensional data illustrating pyramidal shape of iCtx cell (in red) with elaborated neurites extending throughout the whole depth of the adult human cortical slice at 3Â weeks after ex vivo transplantation. Note many spines of different size and shape including mushroom-like ones. Many Hoechst-stained cell nuclei of human cortex (in blue) are distributed throughout the slice. Scale bar in left bottom corner. (MOV 16146 kb)
CBL is a multifunctional protein engaged in the regulation of intracellular signaling pathways. R... more CBL is a multifunctional protein engaged in the regulation of intracellular signaling pathways. Recent studies have found CBL mutations and deletions in a variety of myeloid diseases. In acute myeloid leukemia (AML), CBL mutations are associated with core binding factor (CBF) leukemia, a subset of AML characterized by the presence of AML1-ETO or CBFB-MYH11 fusion genes. However, the potential cooperation between mutant CBL and CBF-fusion proteins has not been demonstrated experimentally. We have developed an experimental system to model myeloid leukemogenesis using primary human cord blood (CB) cells, and have shown that CBF-fusion proteins promote self-renewal and long-term proliferation of human CB CD34+ cells in vitro. Using the human cell-based model, we demonstrated that CBL mutations/depletion promote cytokine-dependent growth, but do not increase self-renewal, of human CB CD34+ progenitors. We also showed that CBL mutations/depletion collaborate with AML1-ETO to expand CB cells both in vitro and in a xenograft model. CBL inactivation promoted cell cycle progression, conferred hyperresponsiveness to thrombopoietin (THPO), and enhanced STAT5/ERK/Src signaling pathways in human AML1-ETO cells. The CBL-inactivated AML1-ETO cells did not show addiction to a specific pathway, but JAK inhibitors suppressed the growth of AML1-ETO cells including those with CBL mutations/depletion. Furthermore, we found that a protein tyrosine phosphatase UBASH3B (also called STS-1), which is known to inhibit CBL function, is highly expressed in CBF leukemia. Knockdown of UBASH3B inhibited the growth of AML1-ETO leukemia cells, and the growth-inhibitory effect of UBASH3B depletion was reversed by overexpression of oncogenic CBL mutants. Thus, the oncogenic potential of UBASH3B is largely dependent on inactivation of CBL. In summary, we established the first human cell-based model for AML with CBL dysfunction. The UBASH3B-CBL axis plays a crucial role to control multiple signaling pathways and myeloid proliferation in human AML1-ETO leukemia, thereby providing potential therapeutic targets.
In the field of developmental biology, there is compelling evidence for a network of activity of ... more In the field of developmental biology, there is compelling evidence for a network of activity of pluripotency and stem-associated genes comprising of Oct4, Nanog and nestin. During neurogenesis, the choice between enhancement versus suppression of transcriptional modulation of these identified genes determines the balance between self-renewal neural stem cells (NSC) and immature neuronal phenotypes. By using immunocytochemistry and RT-PCR techniques, our study aims to address the question whether and to what extent mRNA and protein profiles are expressed in human fetal neurospheres obtained from cortical and striatal brain regions, both in expansion (undifferentiated cells) and differentiation conditions monitored after 1 and 4 weeks in vitro culturing. Our results clearly demonstrate the sustained presence of opposite signals: strong downregulation of Oct4 and Nanog genes in cortical differentiating cells and significant up-regulation for nestin gene both in cortical and striatal d...
Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation... more Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk Ϫ/Ϫ mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain.
Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel a... more Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells survive long-term, differentiate to functional neurons, and induce recovery in the stroke-injured brain are unclear. We have transplanted long-term self-renewing neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs, into the stroke-damaged mouse and rat striatum or cortex. Recovery of forepaw movements was observed already at 1 week after transplantation. Improvement was most likely not due to neuronal replacement but was associated with increased vascular endothelial growth factor levels, probably enhancing endogenous plasticity. Transplanted cells stopped proliferating, could survive without forming tumors for at least 4 months, and differentiated to morphologically mature neurons of different subtypes. Neurons in intrastriatal grafts sent axonal projections to t...
Neural stem/progenitor cells (NSPCs) in subventricular zone (SVZ) produce new striatal neurons du... more Neural stem/progenitor cells (NSPCs) in subventricular zone (SVZ) produce new striatal neurons during several months after stroke, which may contribute to recovery. Intracerebral grafts of NSPCs can exert beneficial effects after stroke through neuronal replacement, trophic actions, neuroprotection, and modulation of inflammation. Here we have explored whether human fetal striatum-derived NSPC-grafts influence striatal neurogenesis and promote recovery in stroke-damaged brain. T cell-deficient rats were subjected to 1 h middle cerebral artery occlusion (MCAO). Human fetal NSPCs or vehicle were implanted into ipsilateral striatum 48 h after MCAO, animals were assessed behaviorally, and perfused at 6 or 14 weeks. Grafted human NSPCs survived in all rats, and a subpopulation had differentiated to neuroblasts or mature neurons at 6 and 14 weeks. Numbers of proliferating cells in SVZ and new migrating neuroblasts and mature neurons were higher, and numbers of activated microglia/macrophages were lower in the ischemic striatum of NSPC-grafted compared to vehicle-injected group both at 6 and 14 weeks. A fraction of grafted NSPCs projected axons from striatum to globus pallidus. The NSPC-grafted rats showed improved functional recovery in stepping and cylinder tests from 6 and 12 weeks, respectively. Our data show, for the first time, that intrastriatal implants of human fetal NSPCs exert a long-term enhancement of several steps of striatal neurogensis after stroke. The grafts also suppress striatal inflammation and ameliorate neurological deficits. Our findings support the idea that combination of NSPC transplantation and stimulation of neurogenesis from endogenous NSPCs may become a valuable strategy for functional restoration after stroke.
Journal of Cerebral Blood Flow & Metabolism, 2010
Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survi... more Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survive in stroke-damaged striatum, migrate from the implantation site, and differentiate into mature neurons. Here, we investigated how various steps of neurogenesis are affected by intrastriatal transplantation of human NSCs at different time points after stroke and with different numbers of cells in each implant. Rats were subjected to middle cerebral artery occlusion and then received intrastriatal transplants of NSCs. Transplantation shortly after stroke (48 hours) resulted in better cell survival than did transplantation 6 weeks after stroke, but the delayed transplantation did not influence the magnitude of migration, neuronal differentiation, and cell proliferation in the grafts. Transplanting greater numbers of grafted NSCs did not result in a greater number of surviving cells or increased neuronal differentiation. A substantial number of activated microglia was observed at 48 hours ...
Stem cell-based approaches to restore function after stroke through replacement of dead neurons r... more Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term selfrenewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated celltransplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our findings show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intracortical implantation in a rat stroke model.
Induced pluripotent stem cells (iPSCs) improve behavior and form neurons after implantation into ... more Induced pluripotent stem cells (iPSCs) improve behavior and form neurons after implantation into the stroke-injured adult rodent brain. How the aged brain responds to grafted iPSCs is unknown. We determined survival and differentiation of grafted human fibroblast-derived iPSCs and their ability to improve recovery in aged rats after stroke. Twenty-four months old rats were subjected to 30 min distal middle cerebral artery occlusion causing neocortical damage. After 48 h, animals were transplanted intracortically with human iPSC-derived long-term neuroepithelial-like stem (hiPSC-lt-NES) cells. Controls were subjected to stroke and were vehicle-injected. Cell-grafted animals performed better than vehicle-injected recipients in cylinder test at 4 and 7 weeks. At 8 weeks, cell proliferation was low (0.7 %) and number of hiPSC-lt-NES cells corresponded to 49.2% of that of implanted cells. Transplanted cells expressed markers of neuroblasts and mature and GABAergic neurons. Cell-grafted r...
The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)‐2α... more The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)‐2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development.
Stem cell transplantation can improve behavioral recovery after stroke in animal models but wheth... more Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell–derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell–derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus–based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neuro...
The neural crest is a stem cell population that gives rise to sympathetic ganglia, the cell type ... more The neural crest is a stem cell population that gives rise to sympathetic ganglia, the cell type of origin of neuroblastoma. Hypoxia Inducible Factor (HIF)-2α is associated with high risk neuroblastoma, however, little is known about its role in normal neural crest development. To address this important question, here we show that HIF-2α is expressed in trunk neural crest cells of human, murine and avian embryos. Modulating HIF-2α in vivo not only causes developmental delays but also induces proliferation and stemness of neural crest cells while altering the number of cells migrating ventrally to sympathoadrenal sites. Transcriptome changes after loss of HIF-2α reflect the in vivo phenotype. The results suggest that expression levels of HIF-2α must be strictly controlled and abnormal levels increase stemness and may promote metastasis. Our findings help elucidate the role of HIF-2α during normal development with implications also in tumor initiation at the onset of neuroblastoma.
Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols ... more Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.
Mutations of Fused in sarcoma (FUs), a ribonucleoprotein involved in RNA metabolism, have been fo... more Mutations of Fused in sarcoma (FUs), a ribonucleoprotein involved in RNA metabolism, have been found associated with both familial and sporadic cases of amyotrophic lateral sclerosis (ALs). Notably, besides mutations in the coding sequence, also mutations into the 3′ untranslated region, leading to increased levels of the wild-type protein, have been associated with neuronal death and ALs pathology, in ALs models and patients. the mechanistic link between altered FUs levels and ALsrelated neurodegeneration is far to be elucidated, as well as the consequences of elevated FUs levels in the modulation of the inflammatory response sustained by glial cells, a well-recognized player in ALS progression. Here, we studied the effect of wild-type FUS overexpression on the responsiveness of mouse and human neural progenitor-derived astrocytes to a pro-inflammatory stimulus (IL1β) used to mimic an inflammatory environment. We found that astrocytes with increased FUS levels were more sensitive to IL1β, as shown by their enhanced expression of inflammatory genes, compared with control astrocytes. Moreover, astrocytes overexpressing FUS promoted neuronal cell death and pro-inflammatory microglia activation. We conclude that overexpression of wild-type FUS intrinsically affects astrocyte reactivity and drives their properties toward pro-inflammatory and neurotoxic functions, suggesting that a non-cell autonomous mechanism can support neurodegeneration in FUsmutated animals and patients.
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Papers by Emanuela Monni