Figure 2. (A) Gene expression profiles of iPSCs and Quick-Glia™ – Astrocyte SeV Culture on day 28 were compared with the profile of human primary astrocytes and the results are shown as scatter plots. The horizontal axis indicates the expression levels of genes in human primary astrocytes purchased from ScienCell (Catalog Number: 1800-5), whereas the vertical axis indicates the expression levels of genes in iPSCs (left) and in Quick-Glia™ – Astrocyte SeV Culture on day 28 (right). The levels of gene expression are shown based on transcripts per million (TPM) in the log10 scale. Blue and green dots represent upregulated and downregulated genes (FDR<0.05), respectively, relative to their levels in human primary astrocytes. (B) Similarities of gene expression profiles of human iPSCs and Quick-Glia™ – Astrocyte SeV Culture on days 14, 28 and 42 to the profile of human primary astrocytes are shown as a bar chart. The vertical axis indicates Pearson correlation (r) based on median-subtracted logTPM.

Figure 3. Real-time quantitative PCR analysis of expression levels of astrocyte-associated genes CD44, GFAP, S100β and ALDH1L1 were examined. Graphs show comparison of Quick-Glia™ – Astrocyte SeV Culture on  day 28 (A) and day 42 (B) with human brain total RNA (TaKaRa, Catalog Number: 636530). The relative gene expression is normalized to phosphoglycerate kinase 1 (PGK1), and then calculated as a fold induction relative to undifferentiated hPSCs as a control. Error bars show standard deviation.

Figure 4. Human iPSCs (negative control), human primary astrocytes (hpA, positive control) and Quick-Glia™ Astrocytes  at days 42 and 56 post-differentiation were exposed to 100µM L-glutamate for 60 minutes. The amount of glutamate cleared was determined using a Promega Glutamate-Glo assay kit. Bar graph represents 3 biological replicates. Error bars show SD.

Human primary astrocytes (ScienCell Research Laboratories, Catalog number: 1800) and Quick-Glia - Astrocyte SeV kit cultures, harvested at either 28 days or 42 days post SeV infection, were plated at 7,800 cells/cm2 in a Geltrex-coated 96-well plate (Corning, Catalog number: 353072) and grown in ScienCell Astrocyte medium (ScienCell Research Laboratories, without FBS, Catalog number: 1801) for 14 days. As a negative control, human iPSCs were plated in StemFit Basic04 medium (Ajinomoto, Catalog number: ASB04-C) two days before assay measurement using the plating density stated above. 30 minutes prior to assay execution, culture medium was replaced with Hanks' Balanced Salt Solution (HBSS) without phenol red (Fisher Scientific, Catalog number: 14025092). Subsequently, cells were exposed to 100µM L-glutamate (Tocris, Catalog number: 0218) in HBSS for 60 minutes. Immediately after the assay, solution was collected and cells were dislodged using a 1:1 mixture of TrypLE Select Enzyme (Fisher Scientific, Catalog number: 12563029) and 0.02% EDTA (Lonza, Catalog number: 17-711E). The numbers of live cells were determined using a NucleoCounter NC-200 (Chemometec, Catalog number: 900-0200) cell counter. Glutamate concentration was measured by a bioluminescence-based Glutamate-Glo assay kit (Promega, Catalog number: J7021) and a SPECTRAFluor Plus microplate reader (Tecan). To determine the amount of glutamate cleared, all values were first background signal-subtracted using a HBSS only sample and concentrations were thereafter acquired from the glutamate titration curve according to manufacturer’s instructions.