Role for cell death pathway in Alzheimer's disease.

Human neurons transplanted into mice with amyloid plaques die by necroptosis.

Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications.

Neural stem cells (NSCs) are a valuable tool for the study of neural development and function as well as an important source of cell transplantation strategies for neural disease. NSCs can be used to study how neurons acquire distinct phenotypes and how the interactions between neurons and glial cells in the developing nervous system shape the structure and function of the CNS. NSCs can also be used for cell replacement therapies following CNS injury targeting astrocytes, oligodendrocytes, and neurons. With the availability of patient-derived induced pluripotent stem cells (iPSCs), neurons prepared from NSCs can be used to elucidate the molecular basis of neurological disorders leading to potential treatments. Although NSCs can be derived from different species and many sources, including embryonic stem cells (ESCs), iPSCs, adult CNS, and direct reprogramming of nonneural cells, isolating primary NSCs directly from fetal tissue is still the most common technique for preparation and study of neurons. Regardless of the source of tissue, similar techniques are used to maintain NSCs in culture and to differentiate NSCs toward mature neural lineages. This chapter will describe specific methods for isolating and characterizing multipotent NSCs and neural precursor cells (NPCs) from embryonic rat CNS tissue (mostly spinal cord) and from human ESCs and iPSCs as well as NPCs prepared by reprogramming. NPCs can be separated into neuronal and glial restricted progenitors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSCs and NPCs isolated from rat and mouse spinal cords for subsequent in vitro or in vivo studies as well as new methods associated with ESCs, iPSCs, and reprogramming.

LRP10 interacts with SORL1 in the intracellular vesicle trafficking pathway in non-neuronal brain cells and localises to Lewy bodies in Parkinson's disease and dementia with Lewy bodies.

Loss-of-function variants in the low-density lipoprotein receptor-related protein 10 (LRP10) gene have been associated with autosomal-dominant Parkinson's disease (PD), PD dementia, and dementia with Lewy bodies (DLB). Moreover, LRP10 variants have been found in individuals diagnosed with progressive supranuclear palsy and amyotrophic lateral sclerosis. Despite this genetic evidence, little is known about the expression and function of LRP10 protein in the human brain under physiological or pathological conditions. To better understand how LRP10 variants lead to neurodegeneration, we first performed an in-depth characterisation of LRP10 expression in post-mortem brains and human-induced pluripotent stem cell (iPSC)-derived astrocytes and neurons from control subjects. In adult human brain, LRP10 is mainly expressed in astrocytes and neurovasculature but undetectable in neurons. Similarly, LRP10 is highly expressed in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons. In astrocytes, LRP10 is present at trans-Golgi network, plasma membrane, retromer, and early endosomes. Interestingly, LRP10 also partially co-localises and interacts with sortilin-related receptor 1 (SORL1). Furthermore, although LRP10 expression and localisation in the substantia nigra of most idiopathic PD and DLB patients and LRP10 variant carriers diagnosed with PD or DLB appeared unchanged compared to control subjects, significantly enlarged LRP10-positive vesicles were detected in a patient carrying the LRP10 p.Arg235Cys variant. Last, LRP10 was detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients and in LRP10 variant carriers. In conclusion, high LRP10 expression in non-neuronal cells and undetectable levels in neurons of control subjects indicate that LRP10-mediated pathogenicity is initiated via cell non-autonomous mechanisms, potentially involving the interaction of LRP10 with SORL1 in vesicle trafficking pathways. Together with the specific pattern of LRP10 incorporation into mature LBs, these data support an important mechanistic role for disturbed vesicle trafficking and loss of LRP10 function in neurodegenerative diseases.

Lipocalin-2 mediates the rejection of neural transplants.

Lipocalin-2 (LCN2) has been implicated in promoting apoptosis and neuroinflammation in neurological disorders; however, its role in neural transplantation remains unknown. In this study, we cultured and differentiated Lund human mesencephalic (LUHMES) cells into human dopaminergic-like neurons and found that LCN2 mRNA was progressively induced in mouse brain after the intrastriatal transplantation of human dopaminergic-like neurons. The induction of LCN2 protein was detected in a subset of astrocytes and neutrophils infiltrating the core of the engrafted sites, but not in neurons and microglia. LCN2-immunoreactive astrocytes within the engrafted sites expressed lower levels of A1 and A2 astrocytic markers. Recruitment of microglia, neutrophils, and monocytes after transplantation was attenuated in LCN2 deficiency mice. The expression of M2 microglial markers was significantly elevated and survival of engrafted neurons was markedly improved after transplantation in LCN2 deficiency mice. Brain type organic cation transporter (BOCT), the cell surface receptor for LCN2, was induced in dopaminergic-like neurons after differentiation, and treatment with recombinant LCN2 protein directly induced apoptosis in dopaminergic-like neurons in a dose-dependent manner. Our results, therefore, suggested that LCN2 is a neurotoxic factor for the engrafted neurons and a modulator of neuroinflammation. LCN2 inhibition may be useful in reducing rejection after neural transplantation.

Immune-tolerance to human iPS-derived neural progenitors xenografted into the immature cerebellum is overridden by species-specific differences in differentiation timing.

We xeno-transplanted human neural precursor cells derived from induced pluripotent stem cells into the cerebellum and brainstem of mice and rats during prenatal development or the first postnatal week. The transplants survived and started to differentiate up to 1 month after birth when they were rejected by both species. Extended survival and differentiation of the same cells were obtained only when they were transplanted in NOD-SCID mice. Transplants of human neural precursor cells mixed with the same cells after partial in vitro differentiation or with a cellular extract obtained from adult rat cerebellum increased survival of the xeno-graft beyond one month. These findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts induction of immune-tolerance to human antigens expressed before completion of maturation of the immune system. With further maturation the transplanted neural precursors expressed more mature antigens before the graft were rejected. Supplementation of the immature cells suspensions with more mature antigens may help to induce immune-tolerance for those antigens expressed only later by the engrafted cells.

Cellular complexity in brain organoids: Current progress and unsolved issues.

Brain organoids are three-dimensional neural aggregates derived from pluripotent stem cells through self-organization and recapitulate architectural and cellular aspects of certain brain regions. Brain organoids are currently a highly exciting area of research that includes the study of human brain development, function, and dysfunction in unprecedented ways. In this Review, we discuss recent discoveries related to the generation of brain organoids that resemble diverse brain regions. We provide an overview of the strategies to complement these primarily neuroectodermal models with cell types of non-neuronal origin, such as vasculature and immune cells. Recent transplantation approaches aiming to achieve higher cellular complexity and long-term survival of these models will then be discussed. We conclude by highlighting unresolved key questions and future directions in this exciting area of human brain organogenesis.

Bioengineering tissue morphogenesis and function in human neural organoids.

Over the last decade, scientists have begun to model CNS development, function, and disease in vitro using human pluripotent stem cell (hPSC)-derived organoids. Using traditional protocols, these 3D tissues are generated by combining the innate emergent properties of differentiating hPSC aggregates with a bioreactor environment that induces interstitial transport of oxygen and nutrients and an optional supportive hydrogel extracellular matrix (ECM). During extended culture, the hPSC-derived neural organoids (hNOs) obtain millimeter scale sizes with internal microscale cytoarchitectures, cellular phenotypes, and neuronal circuit behaviors mimetic of those observed in the developing brain, eye, or spinal cord. Early studies evaluated the cytoarchitectural and phenotypical character of these organoids and provided unprecedented insight into the morphogenetic processes that govern CNS development. Comparisons to human fetal tissues revealed their significant similarities and differences. While hNOs have current disease modeling applications and significant future promise, their value as anatomical and physiological models is limited because they fail to form reproducibly and recapitulate more mature in vivo features. These include biomimetic macroscale tissue morphology, positioning of morphogen signaling centers to orchestrate appropriate spatial organization and intra- and inter-connectivity of discrete tissue regions, maturation of physiologically relevant neural circuits, and formation of vascular networks that can support sustained in vitro tissue growth. To address these inadequacies scientists have begun to integrate organoid culture with bioengineering techniques and methodologies including genome editing, biomaterials, and microfabricated and microfluidic platforms that enable spatiotemporal control of cellular differentiation or the biochemical and biophysical cues that orchestrate organoid morphogenesis. This review will examine recent advances in hNO technologies and culture strategies that promote reproducible in vitro morphogenesis and greater biomimicry in structure and function.

Stem Cell-Based Disease Modeling and Cell Therapy.

Stem cell science is among the fastest moving fields in biology, with many highly promising directions for translatability. To centralize and contextualize some of the latest developments, this Special Issue presents state-of-the-art research of adult stem cells, induced pluripotent stem cells (iPSCs), and embryonic stem cells as well as cancer stem cells. The studies we include describe efficient differentiation protocols of generation of chondrocytes, adipocytes, and neurons, maturation of iPSC-derived cardiomyocytes and neurons, dynamic characterization of iPSC-derived 3D cerebral organoids, CRISPR/Cas9 genome editing, and non-viral minicircle vector-based gene modification of stem cells. Different applications of stem cells in disease modeling are described as well. This volume also highlights the most recent developments and applications of stem cells in basic science research and disease treatments.

The Fate of Transplanted Olfactory Progenitors Is Conditioned by the Cell Phenotypes of the Receiver Brain Tissue in Cocultures.

Among the numerous candidates for cell therapy of the central nervous system (CNS), olfactory progenitors (OPs) represent an interesting alternative because they are free of ethical concerns, are easy to collect, and allow autologous transplantation. In the present study, we focused on the optimization of neuron production and maturation. It is known that plated OPs respond to various trophic factors, and we also showed that the use of Nerve Growth Factor (NGF) allowed switching from a 60/40 neuron/glia ratio to an 80/20 one. Nevertheless, in order to focus on the integration of OPs in mature neural circuits, we cocultured OPs in primary cultures obtained from the cortex and hippocampus of newborn mice. When dissociated OPs were plated, they differentiated into both glial and neuronal phenotypes, but we obtained a 1.5-fold higher viability in cortex/OP cocultures than in hippocampus/OP ones. The fate of OPs in cocultures was characterized with different markers such as BrdU, Map-2, and Synapsin, indicating a healthy integration. These results suggest that the integration of transplanted OPs might by affected by trophic factors and the environmental conditions/cell phenotypes of the host tissue. Thus, a model of coculture could provide useful information on key cell events for the use of progenitors in cell therapy.

In vitro modeling for inherited neurological diseases using induced pluripotent stem cells: from 2D to organoid.

Stem cells are characterized by self-renewal and by their ability to differentiate into cells of various organs. With massive progress in 2D and 3D cell culture techniques, in vitro generation of various types of such organoids from patient-derived stem cells is now possible. As in vitro differentiation protocols are usually made to resemble human developmental processes, organogenesis of patient-derived stem cells can provide key information regarding a range of developmental diseases. Human stem cell-based in vitro modeling as opposed to using animal models can particularly benefit the evaluation of neurological diseases because of significant differences in structure and developmental processes between the human and the animal brain. This review focuses on stem cell-based in vitro modeling of neurodevelopmental disorders, more specifically, the fundamentals and technical advancements in monolayer neuron and brain organoid cultures. Furthermore, we discuss the drawbacks of the conventional culture method and explore the advanced, cutting edge 3D organoid models for several neurodevelopmental diseases, including genetic diseases such as Down syndrome, Rett syndrome, and Miller-Dieker syndrome, as well as brain malformations like macrocephaly and microcephaly. Finally, we discuss the limitations of the current organoid techniques and some potential solutions that pave the way for accurate modeling of neurological disorders in a dish.

Glial cells in Parkinson´s disease: protective or deleterious?

Glial cells have been identified more than 100 years ago, and are known to play a key role in the central nervous system (CNS) function. A recent piece of evidence is emerging showing that in addition to the capacity of CNS modulation and homeostasis, glial cells are also being looked like as a promising cell source not only to study CNS pathologies initiation and progression but also to the establishment and development of new therapeutic strategies. Thus, in the present review, we will discuss the current evidence regarding glial cells' contribution to neurodegenerative diseases as Parkinson's disease, providing cellular, molecular, functional, and behavioral data supporting its active role in disease initiation, progression, and treatment. As so, considering their functional relevance, glial cells may be important to the understanding of the underlying mechanisms regarding neuronal-glial networks in neurodegeneration/regeneration processes, which may open new research opportunities for their future use as a target or treatment in human clinical trials.

Orexin cell transplant reduces behavioral arrest severity in narcoleptic mice.

Narcolepsy is a sleep disorder that has been associated with the loss of orexinergic neurons from the lateral hypothalamic area. This loss leads to dysregulated sleep and cataplexy attacks. Therapeutic options are currently limited to symptom management with pharmacotherapy and nonpharmacological approaches. Nonetheless, cell replacement therapy could offer relief, and research in the field has yielded positive results for other neurodegenerative disorders, such as Parkinson's disease. Thus, we propose that orexin cell rich grafts could help improve narcoleptic symptoms in the orexin/ataxin-3 mouse model of narcolepsy. For this purpose, we isolated EGFP+ cells from either orexin/EGFP or CAG-EGFP mice with the use of a flow cytometer and grafted them into the pedunculopontine and laterodorsal tegmentum nuclei (PPT/LDDT) of orexin/ataxin-3 mice. Our results show that even small orexinergic grafts can reduce the severity of behavioral arrests, with a median reduction of 30.31% in episode duration, 51.35% for number of events and 69.73% in time spent in the behavioral arrest state and help with sleep fragmentation measured in number of bouts per behavioral state. Surprisingly, control grafts made from cerebellar tissue also reduced behavioral arrest severity, but to a lesser degree. Although still at a very early stage, these results show that there is potential in cell grafts for improving aspects of the narcoleptic phenotype and further research could help elucidate realistic expectations of an orexin cell replacement therapy for narcolepsy.

Human Pluripotent Stem Cell-Derived Neurons Are Functionally Mature In Vitro and Integrate into the Mouse Striatum Following Transplantation.

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely "high", "intermediate" and "low" firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology.

Injured adult neurons regress to an embryonic transcriptional growth state.

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.

Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior.

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 neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.

3D Bioprinting Electrically Conductive Bioink with Human Neural Stem Cells for Human Neural Tissues.

Bioprinting cells with an electrically conductive bioink provides an opportunity to produce three-dimensional (3D) cell-laden constructs with the option of electrically stimulating cells in situ during and after tissue development. We and others have demonstrated the use of electrical stimulation (ES) to influence cell behavior and function for a more biomimetic approach to tissue engineering. Here, we detail a previously published method for 3D printing an electrically conductive bioink with human neural stem cells (hNSCs) that are subsequently differentiated. The differentiated tissue constructs comprise functional neurons and supporting neuroglia and are amenable to ES for the purposeful modulation of neural activity. Importantly, the method could be adapted to fabricate and stimulate neural and nonneural tissues from other cell types, with the potential to be applied for both research- and clinical-product development.

Optogenetics for neural transplant manipulation and functional analysis.

Transplantation of neural stem cells (NSCs) or NSC-derived neurons into the brain is a promising therapeutic approach to restore neuronal function. Rapid progress in the NSCs research field, particularly due to the exploitation of induced pluripotent stem cells (iPSCs), offers great potential and an unlimited source of stem cell-derived neural grafts. Studying the functional integration of these grafts into host brain tissues and their effects on each other have been boosted by the implementation of optogenetic technologies. Optogenetics provides high spatiotemporal functional manipulations of grafted or host neurons in parallel. This review aims to highlight the impact of optogenetics in neural stem cell transplantations.

Enteric neuronal cell therapy reverses architectural changes in a novel diphtheria toxin-mediated model of colonic aganglionosis.

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

Transplantation of neural scaffolds consisting of dermal fibroblast-reprogrammed neurons and 3D silk fibrous materials promotes the repair of spinal cord injury.

Transplantation of tissue-engineered neural scaffolds bears great potential for reconstructing neural circuits after spinal cord injury (SCI). In this study, a 3D porous silk fibrous scaffold (3D-SF) with biomimetic interconnected micro- to nanofibrous structure and good biocompatibility is fabricated. Then, a small-molecule combination CFLSSVY (CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA, and Y27632) that efficiently reprograms rat dermal fibroblasts into neurons is screened, and these chemically induced neurons (CiNs) are shown to readily communicate on the 3D-SF and form neural scaffolds. After transplantation of these silk-based neural scaffolds into the stumps of transected spinal cords in rats, the damaged tissue is repaired significantly, as indicated by the reduced cavity areas, decreased GFAP expression, and improved axonal regeneration and myelination in the injury site. Moreover, the hindlimb movement and motor-nerve conductivity are greatly improved as indicated by the elevated BBB score, the alternate movement of two hindlimbs during the 45° inclined grid test, and the shortened latency and enhanced amplitude in cMEP detection. Together, these results demonstrate that transplantation of neural scaffolds consisting of 3D-SF and dermal fibroblast-reprogrammed neurons leads to significant nerve regeneration and functional recovery, providing a promising therapeutic strategy for SCI.