Mature Neuronal Populations Research Articles

How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

Neuroprotection elicited by taurine in sporadic Alzheimer-like disease: benefits on memory and control of neuroinflammation in the hippocampus of rats.

This study aimed to analyze whether taurine has a nootropic effect on short-term and long-term memory in a model of sporadic dementia of the Alzheimer's type (SDAT). Moreover, we evaluated the immunoreactivity and insulin receptor (IR) distribution and markers for neurons and glial cells in the hippocampus of rats with SDAT and treated with taurine. For this, Male Wistar rats received STZ (ICV, 3mg/kg, bilateral, 5ul per site, aCFS vehicle) and were treated with taurine (100mg/kg orally, 1 time per day, saline vehicle) for 25 days. The animals were divided into 4 groups: vehicle (VE), taurine (TAU), ICV-STZ (STZ) and ICV-STZ plus taurine (STZ + TAU). At the end of taurine treatment, short- and long-term memory were assessed by performance on object recognition and Y-maze tasks. Insulin receptor (IR) was evaluated by immunoperoxidase while mature neurons (NeuN), astrocytes (GFAP, S100B, SOX9), and microglia (Iba-1) were evaluated by immunofluorescence. STZ induced worse spatial and recognition memory (INDEX) in YM and ORT tasks. Taurine protected against STZ-induced memory impairment. SDAT reduced the population of mature neurons as well as increased astrocytic and microglial reactivity, and taurine protected against these STZ-induced effects, mainly in the CA1 region of the hippocampus. Taurine increases IR expression in the hippocampus, and protects against the reduction in the density of this receptor in CA1 induced by STZ. In conclusion, these findings demonstrate that taurine is able to enhance memory, up-regulates IR in the hippocampus, protects the neuron population, and reduces the astrogliosis found in SDAT.

Structural plasticity in the entorhinal and perirhinal cortices following hippocampal lesions in rhesus monkeys.

Immature neurons expressing the Bcl2 protein are present in various regions of the mammalian brain, including the amygdala and the entorhinal and perirhinal cortices. Their functional role is unknown but we have previously shown that neonatal and adult hippocampal lesions increase their differentiation in the monkey amygdala. Here, we assessed whether hippocampal lesions similarly affect immature neurons in the entorhinal and perirhinal cortices. Since Bcl2-positive cells were found mainly in areas Eo, Er, and Elr of the entorhinal cortex and in layer II of the perirhinal cortex, we also used Nissl-stained sections to determine the number and soma size of immature and mature neurons in layer III of area Er and layer II of area 36 of the perirhinal cortex. We found different structural changes in these regions following hippocampal lesions, which were influenced by the time of the lesion. In neonate-lesioned monkeys, the number of immature neurons in the entorhinal and perirhinal cortices was generally higher than in controls. The number of mature neurons was also higher in layer III of area Er of neonate-lesioned monkeys but no differences were found in layer II of area 36. In adult-lesioned monkeys, the number of immature neurons in the entorhinal cortex was lower than in controls but did not differ from controls in the perirhinal cortex. The number of mature neurons in layer III of area Er did not differ from controls, but the number of small, mature neurons in layer II of area 36 was lower than in controls. In sum, hippocampal lesions impacted populations of mature and immature neurons in discrete regions and layers of the entorhinal and perirhinal cortices, which are interconnected with the amygdala and provide major cortical inputs to the hippocampus. These structural changes may contribute to some functional recovery following hippocampal injury in an age-dependent manner.

Retinoic Acid-Mediated Differentiation of Mouse Embryonic Stem Cells to Neuronal Cells.

The capability of pluripotent embryonic stem cells (ESCs) to proliferate and differentiate into specific lineages makes them an important avenue of research in the field of cell therapy as well as a useful model to study patterns of differentiation and gene expression, recapitulating many events that occur during the very early stages of development of the mammalian embryo. With striking similarities that exist between inherently programmed embryonic development of the nervous system in vivo and the differentiation of ESCs in vitro, they have already been used to treat locomotive and cognitive deficits caused by brain injury in rodents. A suitable differentiation model thus empowers us with all these opportunities. In this chapter, we describe a neural differentiation model from mouse embryonic stem cells using retinoic acid as the inducer. This method is among the most commonly used one to acquire a homogeneous population of neuronal progenitor cells or mature neurons as desired. The method is scalable, efficient, and results in production of ~70% neural progenitor cells within 4-6days.

A multi-omics approach to visualize early neuronal differentiation from hESCs in 4D

SummaryNeuronal differentiation of pluripotent stem cells is an established method to study physiology, disease, and medication safety. However, the sequence of events in human neuronal differentiation and the ability of in vitro models to recapitulate early brain development are poorly understood. We developed a protocol optimized for the study of early human brain development and neuropharmacological applications. We comprehensively characterized gene expression and epigenetic profiles at four timepoints, because the cells differentiate from embryonic stem cells towards a heterogeneous population of progenitors, immature and mature neurons bearing telencephalic signatures. A multi-omics roadmap of neuronal differentiation, combined with searchable interactive gene analysis tools, allows for extensive exploration of early neuronal development and the effect of medications.

Dopaminergic Neurons Differentiated from LRRK2 I1371V-Induced Pluripotent Stem Cells Display a Lower Yield, α-Synuclein Pathology, and Functional Impairment.

Being a large multidomain protein, LRRK2 has several confirmed pathological mutant variants for PD, and the incidence of these variants shows ethnicity biases. I1371V, a mutation in the GTPase domain, has been reported in East-Asian populations, but there are no studies reported on dopaminergic (DA) neurons differentiated from this variant. The aim here was to assess the yield, function, and α-synuclein pathology of DA neurons differentiated from LRRK2 I1371V iPSCs. FACS analysis of neural progenitors (NPs) showed a comparable immunopositive population of cells for neural and glial progenitor markers nestin and S100β; however, NPs from I1371V iPSCs showed lower clonogenic and proliferative capacities than healthy control NPs as determined by the neurosphere assay and Ki67 expression. Floor plate cells obtained from I1371V NPs primed with FGF8 showed distinctly lower immunopositivity for FOXA2 and CLIC5 than healthy control FPCs and similar DOC2B expression. On SHH addition, a similar mature neuronal population was obtained from both groups; however, the yield of TH-immunopositive cells was significantly lower in I1371V, with lower expression of mature DA neuronal markers En1, Nurr1, and DAT. Vesicular dopamine release and intracellular Ca2+ response with KCl stimulation were lower in I1371V DA neurons, along with a significantly reduced expression of resting vesicle marker VMAT2. A concurrently lower expression of PSD95/Syn-I immunopositive puncta was observed in I1371V differentiated cells. Further, higher phosphorylation of α-synuclein and aggregation of oligomeric α-synuclein in I1371V DA neurons were observed. Our data demonstrated conclusively for the first time that mutations in the I1371V allele of LRRK2 showed developmental deficit from the FPC stage and generated a lower yield/number of TH-immunopositive neurons with impairment in their function and synapse density along with increased α-synuclein pathology.

Dietary Fiber and the Hippocampal Neurogenic Niche in a Model of Pelvic Radiotherapy

We sought to determine whether radiation to the colorectum had an impact on parameters of hippocampal neurogenesis and, if so, whether it could be modulated by a fiber-rich diet. Male C57BL/6J mice were fed a diet containing bioprocessed oat bran or a fiber-free diet, starting two weeks before colorectal irradiation with 4 fractions of 8 Gray or sham-irradiation. Diets were then continued for 1, 6 or 18 weeks, whereafter parameters of hippocampal neurogenesis were analyzed and correlated to serum cytokine levels. No statistically significant changes in neuronal markers or cell proliferation were found at one week post-irradiation. Six weeks post-irradiation there was a decreased cell proliferation in the subgranular zone that appeared slightly more pronounced in irradiated animals on a fiber-free diet and increased numbers of immature neurons per mm2 dentate gyrus in the irradiated mice, with a statistically significant increase in mice on a fiber-rich diet. Microglial abundancy was similar between all groups. 18 weeks post-irradiation, a fiber-free diet had reduced the number of immature neurons, whereas irradiation resulted in an increase. Despite this, the population of mature neurons was stable. Analysis of serum cytokines revealed a negative correlation between MIP1-α and the number of immature neurons one week after irradiation, regardless of diet. Our findings show that pelvic radiotherapy has the potential to cause a long-lasting impact on hippocampal neurogenesis, and dietary interventions may modulate this impact. More in-depth studies on the relationship between irradiation-induced intestinal injury and brain health are warranted.

Neuroglial heterotopia of the middle ear: A case series and systematic literature review.

Background: The differential diagnosis for mass forming lesions of the middle ear is broad. While uncommon, neuroglial heterotopias can occur in the middle ear and can be a source of diagnostic confusion for clinician, radiologist, and pathologist alike.Methods: We identified three cases of neuroglial heterotopia of the middle ear in our institutional archives from 2000 to 2020 and performed extensive histological and immunohistochemical characterization of the three lesions. We conducted a systematic literature review to identify 27 cases published in the English literature between the years 1980 and 2020. Only cases with histological verification of neuroglial heterotopia specifically involving the middle ear were included. We compiled the clinical, radiological, and histopathological findings for all 30 cases.Results: Patients most frequently presented with chronic otitis media (40%), hearing loss (40%), or prior history of ear surgery or trauma (13%). The median age at surgery was 49 years with a male predominance (M:F 2:1); however, a bimodal age distribution was noted with an earlier onset (11 years or younger) in a subset of patients. Immunohistochemical characterization showed mature neuronal and reactive glial populations with low Ki67 proliferation index and chronic inflammatory infiltrates. There was no neuronal dysplasia or glial atypia, consistent with benign, nonneoplastic, mature glioneuronal tissue.Conclusion: Immunohistochemical characterization of these lesions and clinical follow-up confirms their benign natural history. Potential etiologies include developmental misplacement, trauma, and chronic inflammation/ reactive changes resulting in sequestered encephalocele.

Murine Teratocarcinoma-Derived Neuronal Cultures.

This chapter describes the culture and propagation of murine embryonic stem cells, F9 and P19, and strategies for differentiation of these stem cells into neurons. Additional techniques are described for obtaining enriched populations of mature neurons from P19 cells and differentiation of F9 cells into serotonergic or catecholaminergic neurons. The protocols described herein can be used for dissection of the pathways such as gliogenesis and neurogenesis that are involved in differentiation of pluripotent stem cells such as F9 and P19 into glial cells or terminally differentiated neurons.

RBIO-06. IMPLEMENTATION OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED CEREBRAL ORGANOIDS TO MODEL NORMAL TISSUE RADIORESPONSE

Abstract Treatment-related sequelae following cranial irradiation have life changing impacts for patients and their caregivers. Characterization of the basic response of human brain tissue to irradiation has been difficult due to a lack of preclinical models. The direct study of human brain tissue in vitro is becoming possible due to advances in stem cell biology, neuroscience, and tissue engineering with the development of organoids as novel model systems which enable experimentation with human tissue models. We sought to establish a cerebral organoid (CO) model to study the radioresponse of normal human brain tissue. COs were grown using human induced pluripotent stem cells and a modified Lancaster protocol. Compositional analysis during development of the COs showed expected populations of neurons and glia. We confirmed a population of microglia-like cells within the model positive for the makers Iba1 and CD68. After 2-months of maturation, COs were irradiated to 0, 10, and 20 Gy using a Shepard Mark-II Cs-137 irradiator and returned to culture. Subsets of COs were prepared for immunostaining at 30- and 70-days post-irradiation. To examine the effect of irradiation on the neural stem cell (NSC) population, sections were stained for SOX2 and Ki-67 expression denoting NSCs and proliferation respectively. Slides were imaged and scored using the CellProfiler software package. The percentage of proliferating NSCs 30-days post-irradiation was found to be significantly reduced for irradiated COs (5.7% (P=0.007) and 3.4% (P=0.001) for 10 and 20 Gy respectively) compared to control (12.7%). The reduction in the proliferating NSC population subsequently translated to a reduced population of NeuN-labeled mature neurons 70 days post-irradiation. The loss of proliferating NSCs and subsequent reduction in mature neurons demonstrates the long-term effects of radiation. Our initial results indicate COs will be a valuable model to study the effects of radiation therapy on normal and diseased human tissue.

Nicotinamide restricts neural precursor proliferation to enhance catecholaminergic neuronal subtype differentiation from mouse embryonic stem cells.

Emerging evidence indicates that a strong relationship exists between brain regenerative therapies and nutrition. Early life nutrition plays an important role during embryonic brain development, and there are clear consequences to an imbalance in nutritional factors on both the production and survival of mature neuronal populations and the infant’s risk of diseases in later life. Our research and that of others suggest that vitamins play a fundamental role in the formation of neurons and their survival. There is a growing body of evidence that nicotinamide, the water-soluble amide form of vitamin B3, is implicated in the conversion of pluripotent stem cells to clinically relevant cells for regenerative therapies. This study investigated the ability of nicotinamide to promote the development of mature catecholaminergic neuronal populations (associated with Parkinson’s disease) from mouse embryonic stem cells, as well as investigating the underlying mechanisms of nicotinamide’s action. Nicotinamide selectively enhanced the production of tyrosine hydroxylase-expressing neurons and serotonergic neurons from mouse embryonic stem cell cultures (Sox1GFP knock-in 46C cell line). A 5-Ethynyl-2´-deoxyuridine (EdU) assay ascertained that nicotinamide, when added in the initial phase, reduced cell proliferation. Nicotinamide drove tyrosine hydroxylase-expressing neuron differentiation as effectively as an established cocktail of signalling factors, reducing the proliferation of neural progenitors and accelerating neuronal maturation, neurite outgrowth and neurotransmitter expression. These novel findings show that nicotinamide enhanced and enriched catecholaminergic differentiation and inhibited cell proliferation by directing cell cycle arrest in mouse embryonic stem cell cultures, thus driving a critical neural proliferation-to-differentiation switch from neural progenitors to neurons. Further research into the role of vitamin metabolites in embryogenesis will significantly advance cell-based regenerative medicine, and help realize their role as crucial developmental signalling molecules in brain development.

Deconstructing Neurogenesis, Transplantation and Genome-Editing as Neural Repair Strategies in Brain Disease.

Neural repair in injury and disease presents a pressing unmet need in regenerative medicine. Due to the intrinsically reduced ability of the brain to replace lost and damaged neurons, reversing long-term cognitive and functional impairments poses a unique problem. Over the years, advancements in cellular and molecular understanding of neurogenesis mechanisms coupled with sophistication of biotechnology tools have transformed neural repair into a cross-disciplinary field that integrates discoveries from developmental neurobiology, transplantation and tissue engineering to design disease- and patient-specific remedies aimed at boosting either native rehabilitation or delivering exogenous hypoimmunogenic interventions. Advances in deciphering the blueprint of neural ontogenesis and annotation of the human genome has led to the development of targeted therapeutic opportunities that have the potential of treating the most vulnerable patient populations and whose findings from benchside suggest looming clinical translation. This review discusses how findings from studies of adult neurogenesis have informed development of interventions that target endogenous neural regenerative machineries and how advances in biotechnology, including the use of new gene-editing tools, have made possible the development of promising, complex neural transplant-based strategies. Adopting a multi-pronged strategy that is tailored to underlying neural pathology and that encompasses facilitation of endogenous regeneration, correction of patient’s genomic mutations and delivery of transformed neural precursors and mature disease-relevant neuronal populations to replace injured or lost neural tissue remains no longer a fantasy.

Multifaceted circuit functions of adult-born neurons.

The dentate gyrus continually produces new neurons throughout life. Behavioral studies in rodents and network models show that new neurons contribute to normal dentate functions, but there are many unanswered questions about how the relatively small population of new neurons alters network activity. Here we discuss experimental evidence that supports multiple cellular mechanisms by which adult-born neurons contribute to circuit function. Whereas past work focused on the unique intrinsic properties of young neurons, more recent studies also suggest that adult-born neurons alter the excitability of the mature neuronal population via unexpected circuit interactions.

Systematic Identification of Cell-Cell Communication Networks in the Developing Brain.

SummarySince the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in ∼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable “Brain interactome explorer”, available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.

Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression

The identification of convergent phenotypes in different models of psychiatric illness highlights robust phenotypes that are more likely to be implicated in disease pathophysiology. Here, we utilize human iPSCs harboring distinct mutations in DISC1 that have been found in families with major mental illness. One mutation was engineered to mimic the consequences on DISC1 protein of a balanced translocation linked to mental illness in a Scottish pedigree; the other mutation was identified in an American pedigree with a high incidence of mental illness. Directed differentiation of these iPSCs using NGN2 expression shows rapid conversion to a homogenous population of mature excitatory neurons. Both DISC1 mutations result in reduced DISC1 protein expression, and show subtle effects on certain presynaptic proteins. In addition, RNA sequencing and qPCR showed decreased expression of UNC5D, DPP10, PCDHA6, and ZNF506 in neurons with both DISC1 mutations. Longitudinal analysis of neurite outgrowth revealed decreased neurite outgrowth in neurons with each DISC1 mutation, which was mimicked by UNC5D knockdown and rescued by transient upregulation of endogenous UNC5D. This study shows a narrow range of convergent phenotypes of two mutations found in families with major mental illness, and implicates dysregulated netrin signaling in DISC1 biology.

The Role of DNA Polymerase β in Neural Genome Stability.

Development of the nervous system requires robust expansion of progenitor cells that will generate a diverse population of long-lived mature neurons. Precise transmission of the genomic information from parent cell to daughter cells and the faithful maintenance of this information in the terminally

Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations.

IntroductionVitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington’s disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons.MethodsNicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods.ResultsResults presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons.ConclusionsOur results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.

Selective lesion of the hippocampus increases the differentiation of immature neurons in the monkey amygdala

A large population of immature neurons is present in the ventromedial portion of the adult primate amygdala, a region that receives substantial direct projections from the hippocampal formation. Here, we show the effects of neonatal (n = 8) and adult (n = 6) hippocampal lesions on the populations of mature and immature neurons in the paralaminar, lateral, and basal nuclei of the adult monkey amygdala. Compared with unoperated controls (n = 7), the number of mature neurons was about 70% higher in the paralaminar nucleus of neonate- and adult-lesioned monkeys, and 40% higher in the lateral and basal nuclei of neonate-lesioned monkeys. The number of immature neurons in the paralaminar nucleus was 40% higher in neonate-lesioned monkeys and 30% lower in adult-lesioned monkeys. Similar changes in neuron numbers were also found in two monkeys with nonexperimental, selective, bilateral hippocampal damage. These changes in neuron numbers following hippocampal lesions appear to reflect the differentiation of immature neurons present in the paralaminar nucleus. After adult lesions, the differentiation of immature neurons was essentially restricted to the paralaminar nucleus and was associated with a decrease in the population of immature neurons. In contrast, after neonatal lesions, the differentiation of immature neurons involved the paralaminar, lateral, and basal nuclei. It was associated with an increase in the population of immature neurons in the paralaminar nucleus. Such lesion-induced neuronal plasticity sheds new light on potential mechanisms that may facilitate functional recovery following focal brain injury.

Establishment of an induced pluripotent stem cell (iPSC) line from a patient with Clozapine-responder Schizophrenia

Peripheral blood mononuclear cells (PBMCs) were collected from a patient with treatment-refractory Schizophrenia who presented an exceptional clinical response to Clozapine. iPSC lines were established with a non-integrating reprogramming system based on Sendai virus. A footprint-free hiPSC line was characterized to confirm the expression of the main endogenous pluripotency markers and have a regular karyotype. Pluripotency was confirmed by differentiation into cells belonging to the three germ layers. This hiPSC line represents a valuable tool to study the molecular, biochemical and electrophysiological properties of mature neuronal populations belonging to Clozapine responder patients with a severe form of Schizophrenia.

Advanced biomaterial strategies to transplant preformed micro-tissue engineered neural networks into the brain

Objective. Connectome disruption is a hallmark of many neurological diseases and trauma with no current strategies to restore lost long-distance axonal pathways in the brain. We are creating transplantable micro-tissue engineered neural networks (micro-TENNs), which are preformed constructs consisting of embedded neurons and long axonal tracts to integrate with the nervous system to physically reconstitute lost axonal pathways. Approach. We advanced micro-tissue engineering techniques to generate micro-TENNs consisting of discrete populations of mature primary cerebral cortical neurons spanned by long axonal fascicles encased in miniature hydrogel micro-columns. Further, we improved the biomaterial encasement scheme by adding a thin layer of low viscosity carboxymethylcellulose (CMC) to enable needle-less insertion and rapid softening for mechanical similarity with brain tissue. Main results. The engineered architecture of cortical micro-TENNs facilitated robust neuronal viability and axonal cytoarchitecture to at least 22 days in vitro. Micro-TENNs displayed discrete neuronal populations spanned by long axonal fasciculation throughout the core, thus mimicking the general systems-level anatomy of gray matter—white matter in the brain. Additionally, micro-columns with thin CMC-coating upon mild dehydration were able to withstand a force of 893 ± 457 mN before buckling, whereas a solid agarose cylinder of similar dimensions was predicted to withstand less than 150 μN of force. This thin CMC coating increased the stiffness by three orders of magnitude, enabling needle-less insertion into brain while significantly reducing the footprint of previous needle-based delivery methods to minimize insertion trauma. Significance. Our novel micro-TENNs are the first strategy designed for minimally invasive implantation to facilitate nervous system repair by simultaneously providing neuronal replacement and physical reconstruction of long-distance axon pathways in the brain. The micro-TENN approach may offer the ability to treat several disorders that disrupt the connectome, including Parkinson’s disease, traumatic brain injury, stroke, and brain tumor excision.