Endogenous Astrocytes Research Articles

MODL-03. AN EX VIVO PLATFORM BASED ON ORGANOTYPIC BRAIN SLICE CULTURE FOR INFORMING PERSONALIZED BRAIN CANCER THERAPY

Abstract While the improved classification of CNS tumors is already enhancing the scientific rigor of clinical brain cancer treatment guidelines, the impact of these integrated diagnoses based on histopathological features or molecular insight on precision oncology medicine has yet to demonstrate widespread clinical benefit. Patient tumor-derived cancer models, including patient tumor-derived cell lines, organoids, and explants, have provided practical models based on a patient’s individual tumor for effective therapeutic drugs. Importantly, patient tumor-derived organoids have shown potential to guide personalized care and optimize clinical outcomes by functionally predicting patient response to antitumor drugs prior to in vivo treatment. Our team has built an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that allows for the ex vivo engraftment, rapid treatment, and systemic analysis of resected patient brain tumor tissue. This OBSC platform has successfully supported engraftment of each patient tumor tested to this point, including high- and low-grade adult and pediatric tumor tissue. The established patient tumor-derived organoids show the ability to activate the endogenous astrocytes within OBSCs, which have been reported in brain tumor. Whole exome sequencing analysis showed that patient tumor tissue engrafted onto OBSCs maintained a significant genetic resemblance to their parent tumor, while tumor tissue expanded in vitro displayed a distinctly different DNA profile. Additionally, our algorithm calculates dose-response relationships of both tumor kill and normal brain tissue toxicity, generating summarized drug sensitivity scores based on therapeutic window and allowing us to normalize response profiles across a panel of FDA-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment revealed positive associations to clinical outcomes, suggesting the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.

OTHR-05. A LIVING EX VIVO PLATFORM FOR FUNCTIONAL, PERSONALIZED PEDIATRIC BRAIN CANCER DIAGNOSIS

Abstract Functional precision medicine platforms are emerging as promising strategies to improve pre-clinical drug testing and guide clinical decisions. We have developed an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm which enable rapid engraftment, treatment, and analysis of fresh patient brain tumor tissue and patient-derived cell lines. The platform has supported engraftment of every patient tumor tested to this point: high- and low-grade adult and pediatric tumor tissue rapidly establish on OBSCs among endogenous astrocytes and microglia while maintaining the tumor’s original DNA profile. Our algorithm calculates dose-response relationships of both tumor kill and healthy tissue toxicity, generating summarized drug sensitivity scores based on therapeutic window and allowing us to normalize response profiles for a panel of FDA-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment revealed positive associations to clinical outcomes, suggesting the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.

A living ex vivo platform for functional, personalized brain cancer diagnosis

Functional precision medicine platforms are emerging as promising strategies to improve pre-clinical drug testing and guide clinical decisions. We have developed an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that enable rapid engraftment, treatment, and analysis of uncultured patient brain tumor tissue and patient-derived cell lines. The platform has supported engraftment of every patient tumor tested to this point: high- and low-grade adult and pediatric tumor tissue rapidly establishes on OBSCs among endogenous astrocytes and microglia while maintaining the tumor's original DNA profile. Our algorithm calculates dose-response relationships of both tumor kill and OBSC toxicity, generating summarized drug sensitivity scores on the basis of therapeutic window and allowing us to normalize response profiles across a panel of U.S. Food and Drug Administration (FDA)-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment show positive associations to clinical outcomes, suggesting that the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.

Direct Cell Reprogramming and Phenotypic Conversion: An Analysis of Experimental Attempts to Transform Astrocytes into Neurons in Adult Animals.

Central nervous system (CNS) repair after injury or disease remains an unresolved problem in neurobiology research and an unmet medical need. Directly reprogramming or converting astrocytes to neurons (AtN) in adult animals has been investigated as a potential strategy to facilitate brain and spinal cord recovery and advance fundamental biology. Conceptually, AtN strategies rely on forced expression or repression of lineage-specific transcription factors to make endogenous astrocytes become "induced neurons" (iNs), presumably without re-entering any pluripotent or multipotent states. The AtN-derived cells have been reported to manifest certain neuronal functions in vivo. However, this approach has raised many new questions and alternative explanations regarding the biological features of the end products (e.g., iNs versus neuron-like cells, neural functional changes, etc.), developmental biology underpinnings, and neurobiological essentials. For this paper per se, we proposed to draw an unconventional distinction between direct cell conversion and direct cell reprogramming, relative to somatic nuclear transfer, based on the experimental methods utilized to initiate the transformation process, aiming to promote a more in-depth mechanistic exploration. Moreover, we have summarized the current tactics employed for AtN induction, comparisons between the bench endeavors concerning outcome tangibility, and discussion of the issues of published AtN protocols. Lastly, the urgency to clearly define/devise the theoretical frameworks, cell biological bases, and bench specifics to experimentally validate primary data of AtN studies was highlighted.

Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes.

Organoids with region-specific architecture could facilitate the repair of injuries of the central nervous system. Here we show that human astrocytes can be directly reprogrammed into early neuroectodermal cells via the overexpression of OCT4, the suppression of p53 and the provision of the small molecules CHIR99021, SB431542, RepSox and Y27632. We also report that the activation of signalling mediated by fibroblast growth factor, sonic hedgehog and bone morphogenetic protein 4 in the reprogrammed cells induces them to form spinal-cord organoids with functional neurons specific to the dorsal and ventral domains. In mice with complete spinal-cord injury, organoids transplanted into the lesion differentiated into spinal-cord neurons, which migrated and formed synapses with host neurons. The direct reprogramming of human astrocytes into neurons may pave the way for in vivo neural organogenesis from endogenous astrocytes for the repair of injuries to the central nervous system.

Unexpected BrdU inhibition on astrocyte-to-neuron conversion.

5-Bromo-2'-deoxyuridine (BrdU) is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons, however side effects on neural stem cells and their progeny have been reported. In vivo astrocyte-to-neuron (AtN) conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons. The BrdU-labeling strategy has been used to trace astrocyte-converted neurons, but whether BrdU has any effect on the AtN conversion is unknown. Here, while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury, we accidentally discovered that BrdU inhibited AtN conversion. We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex. Although most NeuroD1-infected astrocytes were converted into neurons, the number of BrdU-labeled neurons was surprisingly low. To exclude the possibility that this BrdU inhibition was caused by the ischemic injury, we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU. Surprisingly, we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group. These results revealed an unexpected inhibitory effect of BrdU on AtN conversion, suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.

Safety and efficacy of first-in-human intrathecal transplantation of human astrocytes (AstroRx®) in ALS patients: Phase I/IIA clinical trial results

AstroRx® is a cell-based therapy, composed of healthy human astrocytes derived from embryonic-stem-cells. AstroRx® can protect neurons by several mechanisms-of -ction that were demonstrated in in-vitro and in vivo preclinical studies. The hypothesis is that transplantation of AstroRx® can compensate for the malfunctioning of endogenous astrocytes by restoring hampered physiological capabilities, i.e. reducing toxic compounds, reducing oxidative stress and secreting multiple neurotrophic factors.

Restoration of Visual Function and Cortical Connectivity After Ischemic Injury Through NeuroD1-Mediated Gene Therapy.

Neural circuits underlying brain functions are vulnerable to damage, including ischemic injury, leading to neuronal loss and gliosis. Recent technology of direct conversion of endogenous astrocytes into neurons in situ can simultaneously replenish the neuronal population and reverse the glial scar. However, whether these newly reprogrammed neurons undergo normal development, integrate into the existing neuronal circuit, and acquire functional properties specific for this circuit is not known. We investigated the effect of NeuroD1-mediated in vivo direct reprogramming on visual cortical circuit integration and functional recovery in a mouse model of ischemic injury. After performing electrophysiological extracellular recordings and two-photon calcium imaging of reprogrammed cells in vivo and mapping the synaptic connections formed onto these cells ex vivo, we discovered that NeuroD1 reprogrammed neurons were integrated into the cortical microcircuit and acquired direct visual responses. Furthermore, following visual experience, the reprogrammed neurons demonstrated maturation of orientation selectivity and functional connectivity. Our results show that NeuroD1-reprogrammed neurons can successfully develop and integrate into the visual cortical circuit leading to vision recovery after ischemic injury.

Conversion of Reactive Astrocytes to Induced Neurons Enhances Neuronal Repair and Functional Recovery After Ischemic Stroke.

The master neuronal transcription factor NeuroD1 can directly reprogram astrocytes into induced neurons (iNeurons) after stroke. Using viral vectors to drive ectopic ND1 expression in gliotic astrocytes after brain injury presents an autologous form of cell therapy for neurodegenerative disease. Cultured astrocytes transfected with ND1 exhibited reduced proliferation and adopted neuronal morphology within 2–3 weeks later, expressed neuronal/synaptic markers, and extended processes. Whole-cell recordings detected the firing of evoked action potentials in converted iNeurons. Focal ischemic stroke was induced in adult GFAP-Cre-Rosa-YFP mice that then received ND1 lentivirus injections into the peri-infarct region 7 days after stroke. Reprogrammed cells did not express stemness genes, while 2–6 weeks later converted cells were co-labeled with YFP (constitutively activated in astrocytes), mCherry (ND1 infection marker), and NeuN (mature neuronal marker). Approximately 66% of infected cells became NeuN-positive neurons. The majority (~80%) of converted cells expressed the vascular glutamate transporter (vGLUT) of glutamatergic neurons. ND1 treatment reduced astrogliosis, and some iNeurons located/survived inside of the savaged ischemic core. Western blotting detected higher levels of BDNF, FGF, and PSD-95 in ND1-treated mice. MultiElectrode Array (MEA) recordings in brain slices revealed that the ND1-induced reprogramming restored interrupted cortical circuits and synaptic plasticity. Furthermore, ND1 treatment significantly improved locomotor, sensorimotor, and psychological functions. Thus, conversion of endogenous astrocytes to neurons represents a plausible, on-site regenerative therapy for stroke.

Rising Stars: Astrocytes as a Therapeutic Target for ALS Disease.

Amyotrophic lateral sclerosis (ALS) is a multifactorial disease, characterized by a progressive loss of motor neurons that eventually leads to paralysis and death. The current ALS-approved drugs modestly change the clinical course of the disease. The mechanism by which motor neurons progressively degenerate remains unclear but entails a non-cell autonomous process. Astrocytes impaired biological functionality were implicated in multiple neurodegenerative diseases, including ALS, frontotemporal dementia (FTD), Parkinson’s disease (PD), and Alzheimer disease (AD). In ALS disease patients, A1 reactive astrocytes were found to play a key role in the pathology of ALS disease and death of motor neurons, via loss or gain of function or acquired toxicity. The contribution of astrocytes to the maintenance of motor neurons by diverse mechanisms makes them a promising therapeutic candidate for the treatment of ALS. Therapeutic approaches targeting at modulating the function of endogenous astrocytes or replacing lost functionality by transplantation of healthy astrocytes, may contribute to the development of therapies which might slow down or even halt the progression ALS diseases. The proposed mechanisms by which astrocytes can potentially ameliorate ALS progression and the status of ALS clinical studies involving astrocytes are discussed.

In vivo conversion of rat astrocytes into neuronal cells through neural stem cells in injured spinal cord with a single zinc-finger transcription factor

BackgroundSpinal cord injury (SCI) results in glial scar formation and irreversible neuronal loss, which finally leads to functional impairments and long-term disability. Our previous studies have demonstrated that the ectopic expression of Zfp521 reprograms fibroblasts and astrocytes into induced neural stem cells (iNSCs). However, it remains unclear whether treatment with Zfp521 also affects endogenous astrocytes, thus promoting further functional recovery following SCI.MethodsRat astrocytes were transdifferentiated into neural stem cells in vitro by ZFP521 or Sox2. Then, ZFP521 was applied to the spinal cord injury site of a rat. Transduction, real-time PCR, immunohistofluorescence, and function assessments were performed at 6 weeks post-transduction to evaluate improvement and in vivo lineage reprogramming of astrocytes.ResultsHere, we show that Zfp521 is more efficient in reprogramming cultured astrocytes compared with Sox2. In the injured spinal cord of an adult rat, resident astrocytes can be reprogrammed into neurons through a progenitor stage by Zfp521. Importantly, this treatment improves the functional abilities of the rats as evaluated by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and further by calculation of its subscores. There was enhanced locomotor activity in the hind limbs, step length, toe spread, foot length, and paw area. In addition, motor evoked potential recordings demonstrated the functional integrity of the spinal cord.ConclusionsThese results have indicated that the generation of iNSCs or neurons from endogenous astrocytes by in situ reprogramming might be a potential strategy for SCI repair.Graphical abstract

Neural Stem Cell Grafts Promote Astroglia-Driven Neurorestoration in the Aged Parkinsonian Brain via Wnt/β-Catenin Signaling

During aging-one the most potent risk factors for Parkinson's disease (PD)-both astrocytes and microglia undergo functional changes that ultimately hamper homoeostasis, defense, and repair of substantia nigra pars compacta (SNpc) midbrain dopaminergic (mDA) neurons. We tested the possibility of rejuvenating the host microenvironment and boosting SNpc DA neuronal plasticity via the unilateral transplantation of syngeneic neural stem/progenitor cells (NSCs) in the SNpc of aged mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced experimental PD. Transplanted NSCs within the aged SNpc engrafted and migrated in large proportions to the tegmental aqueduct mDA niche, with 30% acquiring an astroglial phenotype. Both graft-derived exogenous (ex-Astro) and endogenous astrocytes (en-Astro) expressed Wnt1. Both ex-Astro and en-Astro were key triggers of Wnt/β-catenin signaling in SNpc-mDA neurons and microglia, which was associated with mDA neurorescue and immunomodulation. At the aqueduct-ventral tegmental area level, NSC grafts recapitulated a genetic Wnt1-dependent mDA developmental program, inciting the acquisition of a mature Nurr1+ TH+ neuronal phenotype. Wnt/β-catenin signaling antagonism abolished mDA neurorestoration and immune modulatory effects of NSC grafts. Our work implicates an unprecedented therapeutic potential for somatic NSC grafts in the restoration of mDA neuronal function in the aged Parkinsonian brain. Stem Cells 2018;36:1179-1197.

Recovery of Neurovascular Unit Integrity by CDK5-KD Astrocyte Transplantation in a Global Cerebral Ischemia Model.

Astrocytes play metabolic and structural support roles and contribute to the integrity of the blood-brain barrier (BBB), linking communication between neurons and the endothelium. Cyclin-dependent kinase 5 (CDK5) likely exerts a dual effect on the endothelium and astrocytes due to its involvement in migration and angiogenesis; the overactivation of CDK5 is associated with dysfunction in glutamate recapture and hypoxia. Recently, we proposed that CDK5-targeted astrocytes facilitate the recovery of neurological and motor function in transplanted ischemic rats. In the current study, we treated cerebral ischemic rats and endothelial cells exposed to glutamate toxicity with CDK5 knock-down (CDK5-KD) astrocytes to determine the role of CDK5 in neurovascular integrity. We found that the effects of CDK5-KD were sustained for 4months, preventing neuronal and astrocyte loss, facilitating the recovery of the BBB via the production of BDNF by endogenous astrocytes (GFP-) surrounding vessels in the motor cortex and the corpus callosum of global ischemic rats, and improving neurological performance. These findings were supported by the in vitro findings of increased transendothelial resistance, p120-ctn+ adhesion and reduced intercellular gaps induced by a CDK5 inhibitor (roscovitine) in bEnd.3 cells in a glutamate-toxicity model. Additionally, CDK5-KD astrocytes in co-culture protected the endothelial cell viability, increased BDNF release from astrocytes, increased BDNF immunoreactivity in neighboring astrocytes and endothelial cells and enhanced cell adhesion in a glutamate-toxicity model. Altogether, these findings suggest that a CDK5 reduction in astrocytes protects the endothelium, which promotes BDNF release, endothelial adhesion, and the recovery of neurovascular unit integrity and brain function in ischemic rats.

Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures

Long-term neural differentiation of human pluripotent stem cells (hPSCs) is associated with enhanced neuronal maturation, which is a necessity for creation of representative in vitro models. It also induces neurogenic-to-gliogenic fate switch, increasing proportion of endogenous astrocytes formed from the common neural progenitors. However, the significance of prolonged differentiation on the neural cell type composition and functional development of hPSC-derived neuronal cells has not been well characterized. Here, we studied two hPSC lines, both of which initially showed good neuronal differentiation capacity. However, the propensity for endogenous astrogenesis and maturation state after extended differentiation varied. Live cell calcium imaging revealed that prolonged differentiation facilitated maturation of GABAergic signaling. According to extracellular recordings with microelectrode array (MEA), neuronal activity was limited to fewer areas of the culture, which expressed more frequent burst activity. Efficient maturation after prolonged differentiation also promoted organization of spontaneous activity by burst compaction. These results suggest that although prolonged neural differentiation can be challenging, it has beneficial effect on functional maturation, which can also improve transition to different neural in vitro models and applications.

Astrocytes in mouse models of tauopathies acquire early deficits and lose neurosupportive functions

Microtubule-associated protein tau aggregates constitute the characteristic neuropathological features of several neurodegenerative diseases grouped under the name of tauopathies. It is now clear that the process of tau aggregation is associated with neurodegeneration. Several transgenic tau mouse models have been developed where tau progressively aggregates, causing neuronal death. Previously we have shown that transplantation of astrocytes in P301S tau transgenic mice rescues cortical neuron death, implying that the endogenous astrocytes are deficient in survival support. We now show that the gliosis markers Glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100β) are elevated in brains from P301S tau mice compared to control C57Bl/6 mice whereas the expression of proteins involved in glutamine/glutamate metabolism are reduced, pointing to a functional deficit. To test whether astrocytes from P301S mice are intrinsically deficient, we co-cultured astrocytes and neurons from control and P301S mice. Significantly more C57-derived and P301S-derived neurons survived when cells were cultured with C57-derived astrocytes or astrocyte conditioned medium (C57ACM) than with P301S-derived astrocytes or astrocyte conditioned medium (P301SACM), or ACM from P301L tau mice, where the transgene is also specifically expressed in neurons. The astrocytic alterations developed in mice during the first postnatal week of life. In addition, P301SACM significantly decreased presynaptic (synaptophysin, SNP) and postsynaptic (postsynaptic density protein 95, PSD95) protein expression in cortical neuron cultures whereas C57ACM enhanced these markers. Since thrombospondin 1 (TSP-1) is a major survival and synaptogenic factor, we examined whether TSP-1 is deficient in P301S mouse brains and ACM. Significantly less TSP-1 was expressed in the brains of P301S tau mice or produced by P301S-derived astrocytes, whereas supplementation of P301SACM with TSP-1 increased its neurosupportive capacity. Our results demonstrate that P301S-derived astrocytes acquire an early functional deficiency that may explain in part the loss of cortical neurons in the P301S tau mice.

Sporadic ALS Astrocytes Induce Neuronal Degeneration InVivo.

SummaryAstrocytes from familial amyotrophic lateral sclerosis (ALS) patients or transgenic mice are toxic specifically to motor neurons (MNs). It is not known if astrocytes from sporadic ALS (sALS) patients cause MN degeneration in vivo and whether the effect is specific to MNs. By transplanting spinal neural progenitors, derived from sALS and healthy induced pluripotent stem cells (iPSCs), into the cervical spinal cord of adult SCID mice for 9 months, we found that differentiated human astrocytes were present in large areas of the spinal cord, replaced endogenous astrocytes, and contacted neurons to a similar extent. Mice with sALS but not non-ALS cells showed reduced non-MNs numbers followed by MNs in the host spinal cord. The surviving MNs showed reduced inputs from inhibitory neurons and exhibited disorganized neurofilaments and aggregated ubiquitin. Correspondingly, mice with sALS but not non-ALS cells showed declined movement deficits. Thus, sALS iPSC-derived astrocytes cause ALS-like degeneration in both MNs and non-MNs.

Endogenous astrocytes and differentiation time affect functional maturation of neuronal networks in human pluripotent stem cell -derived neural cultures

Endogenous astrocytes and differentiation time affect functional maturation of neuronal networks in human pluripotent stem cell -derived neural cultures

Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF.

Astrocytes provide neuroprotective effects against degeneration of dopaminergic (DA) neurons and play a fundamental role in DA differentiation of neural stem cells. Here we show that light illumination of astrocytes expressing engineered channelrhodopsin variant (ChETA) can remarkably enhance the release of basic fibroblast growth factor (bFGF) and significantly promote the DA differentiation of human embryonic stem cells (hESCs) in vitro. Light activation of transplanted astrocytes in the substantia nigra (SN) also upregulates bFGF levels in vivo and promotes the regenerative effects of co-transplanted stem cells. Importantly, upregulation of bFGF levels, by specific light activation of endogenous astrocytes in the SN, enhances the DA differentiation of transplanted stem cells and promotes brain repair in a mouse model of Parkinson’s disease (PD). Our study indicates that astrocyte-derived bFGF is required for regulation of DA differentiation of the stem cells and may provide a strategy targeting astrocytes for treatment of PD.

In vivo conversion of astrocytes to neurons in the injured adult spinal cord

Spinal cord injury (SCI) leads to irreversible neuronal loss and glial scar formation, which ultimately result in persistent neurological dysfunction. Cellular regeneration could be an ideal approach to replenish the lost cells and repair the damage. However, the adult spinal cord has limited ability to produce new neurons. Here we show that resident astrocytes can be converted to doublecortin (DCX)-positive neuroblasts by a single transcription factor, SOX2, in the injured adult spinal cord. Importantly, these induced neuroblasts can mature into synapse-forming neurons in vivo. Neuronal maturation is further promoted by treatment with a histone deacetylase inhibitor, valproic acid (VPA). The results of this study indicate that in situ reprogramming of endogenous astrocytes to neurons might be a potential strategy for cellular regeneration after SCI.

Cell therapy for stroke: use of local astrocytes

Stroke refers to a variety of conditions caused by the occlusion or hemorrhage of blood vessels supplying the brain, which is one of the main causes of death and the leading cause of disability worldwide. In the last years, cell-based therapies have been proposed as a new approach to ameliorate post-stroke deficits. However, the most appropriate type of cell to be used in such therapies, as well as their sources, remains a matter of intense research. A good candidate cell should, in principle, display high plasticity to generate diverse types of neurons and, at the same time, low risk to cause undesired outcomes, such as malignant transformation. Recently, a new approach grounded on the reprogramming of endogenous astrocytes toward neuronal fates emerged as an alternative to restore neurological functions in several central nervous system diseases. In this perspective, we review data about the potential of astrocytes to become functional neurons following expression of neurogenic genes and discuss the potential benefits and risks of reprogramming astrocytes in the glial scar to replace neurons lost after stroke.