Differentiation of inflammation-responsive astrocytes from glial progenitors generated from human induced pluripotent stem cells

WOS: 000402964700027PubMed ID: 28591655Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1 beta or tumor necrosis factor a elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1 beta. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.Paul G. Allen Family Foundation; JPB Foundation; Leona M. and Harry B. Helmsley Charitable Trust [2012-PG-MED002]; Annette C. Merle-Smith [R01 MH095741, U19MH106434]; G. Harold & Leila Y. Mathers Foundation; Flow Cytometry Core Facility of the Salk Institute; NIH-NCI CCSG [P30 014195]; Next Generation Sequencing Core Facility of the Salk Institute; Chapman Foundation; Helmsley Charitable Trust; Razavi Newman Integrative Genomics and Bioinformatics Core Facility of the Salk Institute; Swiss-NSF outgoing PD fellowship; Lynn and Edward Streim fellowship; EMBO long-term fellowship; Bettencourt Schueller Foundation; Philippe Foundation; Bob and Mary Jane EngmanFor the production of the iPSCs, the authors would like to acknowledge financial support from Janssen Pharmaceuticals. This work was supported by the Paul G. Allen Family Foundation, Bob and Mary Jane Engman, The JPB Foundation, The Leona M. and Harry B. Helmsley Charitable Trust grant # 2012-PG-MED002, Annette C. Merle-Smith, R01 MH095741 (F.H.G.), U19MH106434 (F.H.G.), and The G. Harold & Leila Y. Mathers Foundation. This work was supported by the Flow Cytometry Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195; the Next Generation Sequencing Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195; the Chapman Foundation and the Helmsley Charitable Trust and by The Razavi Newman Integrative Genomics and Bioinformatics Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195. This research was also supported by the Swiss-NSF outgoing PD fellowship (K.C.V.), Lynn and Edward Streim fellowship (K.C.V.), EMBO long-term fellowship (B.N.J.), the Bettencourt Schueller Foundation (B.N.J.), and the Philippe Foundation (B. N. J.). The authors would like to thank M. L. Gage for editorial comments

Single-cell transcriptomics reveal that PD-1 mediates immune tolerance by regulating proliferation of regulatory T cells

Abstract Background We have previously reported an antigen-specific protocol to induce transplant tolerance and linked suppression to human embryonic stem cell (hESC)-derived tissues in immunocompetent mice through coreceptor and costimulation blockade. However, the exact mechanisms of acquired immune tolerance in this model have remained unclear. Methods We utilize the NOD.Foxp3 hCD2 reporter mouse line and an ablative anti-hCD2 antibody to ask if CD4+FOXP3+ regulatory T cells (Treg) are required for coreceptor and costimulation blockade-induced immune tolerance. We also perform genome-wide single-cell RNA-sequencing to interrogate Treg during immune rejection and tolerance and to indicate possible mechanisms involved in sustaining Treg function. Results We show that Treg are indispensable for tolerance induced by coreceptor and costimulation blockade as depletion of which with an anti-hCD2 antibody resulted in rejection of hESC-derived pancreatic islets in NOD.Foxp3 hCD2 mice. Single-cell transcriptomic profiling of 12,964 intragraft CD4+ T cells derived from rejecting and tolerated grafts reveals that Treg are heterogeneous and functionally distinct in the two outcomes of transplant rejection and tolerance. Treg appear to mainly promote chemotactic and ubiquitin-dependent protein catabolism during transplant rejection while seeming to harness proliferative and immunosuppressive function during tolerance. We also demonstrate that this form of acquired transplant tolerance is associated with increased proliferation and PD-1 expression by Treg. Blocking PD-1 signaling with a neutralizing anti-PD-1 antibody leads to reduced Treg proliferation and graft rejection. Conclusions Our results suggest that short-term coreceptor and costimulation blockade mediates immune tolerance to hESC-derived pancreatic islets by promoting Treg proliferation through engagement of PD-1. Our findings could give new insights into clinical development of hESC-derived pancreatic tissues, combined with immunotherapies that expand intragraft Treg, as a potentially sustainable alternative treatment for T1D

Mitochondrial Aging Defects Emerge in Directly Reprogrammed Human Neurons due to Their Metabolic Profile

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain, but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging, in contrast to iPSC differentiation. Here, we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression, impaired axonal mitochondrial morphologies, lower mitochondrial membrane potentials, reduced energy production, and increased oxidized proteins levels. In contrast, the fibroblasts from which iNs were generated show only mild age-dependent changes, consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed, OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging

Mapping and Analysis of Chromatin State Dynamics in Nine Human Cell Types

Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell-type specificities and their functional interactions. Focusing on cell-type-specific patterns of promoters and enhancers, we define multicell activity profiles for chromatin state, gene expression, regulatory motif enrichment and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell-type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for the interpretation of genome-wide association studies. Top-scoring disease single nucleotide polymorphisms are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus suggesting a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.National Human Genome Research Institute (U.S.) (R01HG004037)National Human Genome Research Institute (U.S.) (RC1HG005334)National Science Foundation (U.S.). (Award 0644282)National Science Foundation (U.S.). (Award 0905968)Alfred P. Sloan Foundatio