Illumina HiSeq 2500 sequencing; GSM3048096: Cell_AAAGAAAAAGAATGC; Homo sapiens; RNA-Seq

Cell_AAAGAAAAAGAATGC

Despite widespread interest in using human stem cells in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we report a protocol for efficient differentiation of hippocampal pyramidal neurons and an in vitro model for hippocampal neuronal connectivity. We developed an embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-based protocol to differentiate human CA3 pyramidal neurons from patterned hippocampal neural progenitor cells (NPCs). This differentiation induces a comprehensive patterning and generates multiple CA3 neuronal subtypes. The differentiated CA3 neurons are functionally active and readily form neuronal connection with dentate granule (DG) neurons in vitro, recapitulating the synaptic connectivity within the hippocampus. When we applied this neuronal co-culture approach to study connectivity in schizophrenia, we found deficits in spontaneous activity in patient iPSC derived DG–CA3 co-culture by multi-electrode array recording. In addition, both multi-electrode array recording and whole cell patch clamp electrophysiology revealed a reduction in spontaneous and evoked neuronal activity in CA3 neurons derived from schizophrenia patients. Altogether these results underscore the relevance of this new model in studying diseases with hippocampal vulnerability. Overall design: 4 technical replicates were used and later pooled together for the bioinformatic analysis.

Efficient generation of human CA3 neurons and modeling hippocampal neuronal connectivity in vitro (single cells)

Submitted by Gene Expression Omnibus on 04-MAY-2018

Despite widespread interest in using human stem cells in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we report a protocol for efficient differentiation of hippocampal pyramidal neurons and an in vitro model for hippocampal neuronal connectivity. We developed an embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-based protocol to differentiate human CA3 pyramidal neurons from patterned hippocampal neural progenitor cells (NPCs). This differentiation induces a comprehensive patterning and generates multiple CA3 neuronal subtypes. The differentiated CA3 neurons are functionally active and readily form neuronal connection with dentate granule (DG) neurons in vitro, recapitulating the synaptic connectivity within the hippocampus. When we applied this neuronal co-culture approach to study connectivity in schizophrenia, we found deficits in spontaneous activity in patient iPSC derived DGâ€“CA3 co-culture by multi-electrode array recording. In addition, both multi-electrode array recording and whole cell patch clamp electrophysiology revealed a reduction in spontaneous and evoked neuronal activity in CA3 neurons derived from schizophrenia patients. Altogether these results underscore the relevance of this new model in studying diseases with hippocampal vulnerability. Overall design: 4 technical replicates were used and later pooled together for the bioinformatic analysis.

Differentiating hCA3 population were dissociated into a single cell suspension by enzymatic dissociation by treating with Accutase (Stem cell technologies). Cells were collected in PBS containing 0.1%BSA on ice. The cell suspension obtained was filtered with 22µm filter (Millipore) and kept in cold PBS-BSA solution. Before counting, trypan blue was added to the cell suspension and the viability, cell density and presence of clumps were assayed on a TC20™ Automated Cell Counter. Cell suspension at a final dilution of 2500 cells/µL and containing no less than 85% live cells were taken for the encapsulation and later steps. All reagents for encapsulation, cDNA synthesis, library preparation and sequencing were from Illumina inc. Cell suspensions were loaded into a ddSEQ catridge in a ratio of 21.5 µL Cell enzyme mix to every 4.5 µL cell suspension. 60ul of 3' barcode suspension mix and encapsulation oil were also loaded on the ddSEQ cartridge. The chip was then processed on a ddSEQ™ Single-Cell Isolator for about 5 minutes to generate single cell droplets. Encapsulated samples were transferred to a 96 well plate and reverse transcription mix was added and followed by reverse transcription on a thermal cycler. When the reverse transcription was done droplet disruptor was added to the product. This was followed by the first strand purification using magnetic beads. The beads were immobilized using magnetic peg stand and dynamag and supernatant was discarded. The beads were washed twice with 80% EtOH. 30uls of resuspension buffer was added for elution and supernatant was transferred to a new tube. The purified first strand was used to synthesize the second strand to generate the double strand cDNA. Again the cDNA was purified using magnetic beads, followed by 80% EtOH wash and elution. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tagment enzyme on thermal cycler. DNA adapters were added for cluster formation. Followed by the index addition, libraries were cleaned up using magnetic beads as before. Purified libraries were eluted with 20ul resuspension buffer. 1ul of eluted product was assessed on an Agilent BioAnalyzer. Library size was about 500bp. Sequencing was performed on an Illumina HiSeq 2500 instrument using surecell-sequencing primers.

Efficient generation of human CA3 neurons and modeling hippocampal neuronal connectivity in vitro (single cells)

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