31 research outputs found
Using Human Pluripotent Stem Cell-Derived Neural Cultures to Assess Safety of New Drugs and Chemicals
The central nervous system (CNS) is a central pillar in safety pharmacology studies of new drugs. Characterization of serious adverse drug reactions to a new chemical entity involves extensive investigation using in vitro and in vivo models. However, primary culture of human neurons in vitro can be challenging, giving limited sample availability. Additionally, the inter-species differences between humans and current animal models impose a considerable obstacle to successfully predict the outcome of new drugs. New technologies also need to help address the 3Rs principles in animal research. Human pluripotent stem cells (hPSC) have the potential to change the current paradigm in pharmacological research. By using hPSCs and state-of-the-art differentiation protocols, researchers now have available an unlimited source of neural cells, able to mimic early and late stage of human CNS development. Moreover, hPSC-derived cells can be used at early stages of drug development, improving clinical predictability and reducing overall drug development costs. This chapter covers the advancements that resulted in hPSC-derived models intended to enable neurotoxicity assessment and drug screening. Finally, this chapter will also reveal the bottlenecks and the challenges to overcome of using hPSC as a predictive tool in research
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Exosomes regulate neurogenesis and circuit assembly.
Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development
Proteomic analyses reveal misregulation of LIN28 expression and delayed timing of glial differentiation in human iPS cells with MECP2 loss-of-function.
Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation
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Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA.
Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise when dysregulated. Here we consider X-linked spinal and bulbar muscular atrophy (SBMA), a repeat disorder caused by polyglutamine-expanded androgen receptor (polyQ-AR). We found that polyQ-AR reduced long-term protein turnover and impaired autophagic flux in motor neuron-like cells. Ultrastructural analysis of SBMA mice revealed a block in autophagy pathway progression. We examined the transcriptional regulation of autophagy and observed a functionally significant physical interaction between transcription factor EB (TFEB) and AR. Normal AR promoted, but polyQ-AR interfered with, TFEB transactivation. To evaluate physiological relevance, we reprogrammed patient fibroblasts to induced pluripotent stem cells and then to neuronal precursor cells (NPCs). We compared multiple SBMA NPC lines and documented the metabolic and autophagic flux defects that could be rescued by TFEB. Our results indicate that polyQ-AR diminishes TFEB function to impair autophagy and promote SBMA pathogenesis
Survival of syngeneic and allogeneic iPSC–derived neural precursors after spinal grafting in minipigs
The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)–mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis
Supplemental Figures and Tables
Supplemental figures and tables for neurospheroid articles. Shows examples of primary data, images, also provides tables with correlation coefficient
Assay optimization data_
Assay optimization data_ the table contains optimization data for selected compounds and control
SupplementaryTable 3. Benchmark Concentrations
SupplementaryTable 3. Benchmark Concentrations.The table contains benchmark concentrations and other statistical data generated from the dataset presented in the Supplementary Table 2
Supplemental Table 2
Supplemental Table 2 presents PRIMARY DATA for calcium oscillation and viability read-outs for effects of compounds from neurotoxicity librar
Supplemental Table 1
Supplemental Table 1 for neurospheroid paper. Table contains the list of tested chemicals