Data defining markers of human neural stem cell lineage potential

Neural stem cells (NSCs) and neural progenitor cells (NPCs) are self-renewing and multipotent cells, however, NPCs are considered to be more lineage-restricted with a reduced self-renewing capacity. We present data comparing the expression of 21 markers encompassing pluripotency, self-renewal (NSC) as well as neuronal and glial (astrocyte and oligodendrocyte) lineage specification and 28 extracellular proteoglycan (PG) genes and their regulatory enzymes between embryonic stem cell (ESC)-derived human NSCs (hNSC H9 cells, Thermo Fisher) and human cortex-derived normal human NPCs (nhNPCs, Lonza). The data demonstrates expression differences of multiple lineage and proteoglycan-associated genes between hNSC H9 cells and nhNPCs. Data interpretation of markers and proteoglycans defining NSC and neural cell lineage characterisation can be found in “Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination” (Oikari et al. 2015) [1].


Specifications
Cell biology More specific subject area Human neural stem cell (hNSC) and human neural progenitor cell (hNPC) marker characterisation Type of data Text file, graphs and immunofluorescence images How data was acquired in vitro culture/expansion and phase-contrast fluorescence microscopy data for phenotypic analysis was obtained on an Olympus IX81 inverted fluorescent microscope via Volocity Imaging package; raw Q-PCR data was obtained on Applied Biosystems 7900HT Fast Real-Time PCR system Data format The data provides an extensive panel of markers for better characterisation of human NSCs and NPCs. The data demonstrates significant and specific differences in expression of pluripotency, NSC selfrenewal and neural cell lineage markers between hNSCs and hNPCs.
The marker profile data could be used to identify and differentiate between the two cell types to improve their efficacy in research or therapeutic applications.
The data provides information on the proteoglycan profile of human NSCs and NPCs providing potential new additional markers defining lineage progression of NSCs to NPCs.

Data
We compared the expression of 49 selected genes between human NSCs (hESC-derived hNSC H9 cells, Thermo Fisher) and normal human progenitor cells (nhNPCs, Lonza) following short-term culture under basal growth conditions. Q-PCR data was obtained for pluripotency genes, NSC, neuronal, astrocyte and oligodendrocyte lineage defining genes (n¼21; Table 1.) ( Fig. 1) with several of these markers also detected through immunofluorescence (IF) (Fig. 2) using specific antibodies (Table 3). In addition, Q-PCR data was obtained for 28 heparan and chondroitin sulphate proteoglycan biosynthesis enzymes and core protein genes ( Table 2) ubiquitous to the neural niche [1][2][3][4][5][6][7] in hNSC H9 cells and nhNPCs (Figs. 3 and 4). The data presented provides information on self-renewal and multilineage potential as well as proteoglycan expression differences between the two neural stem/progenitor cell types.

Cell culture
Gibco s human neural stem cells derived from NIH-approved H9 (WA09) embryonic stem cells (hNSC H9 cells) were cultured as a monolayer on Geltrex s coated culture dishes in StemPro s NSC serum-free medium (NSC SFM) containing KnockOUT ™ DMEM/F-12 supplemented with 2% StemPro s Neural Supplement, 20 ng/ml FGFb and EGF and 2 mM GlutaMAX ™ (cells and culture reagents obtained from Thermo     hNSC H9 and nhNPCs cultures were maintained in 5% CO 2 at 37°C in a humidified atmosphere with phenotype of the cells monitored under an Olympus IX81 inverted phase-contrast microscope.

RNA extraction
RNA was harvested from cultured cells using TRIzol s reagent (Invitrogen) using the Direct-zol ™ RNA miniprep kit (Zymo Research) according to the manufacturer's instructions with samples treated in-column with DNase I (Zymo Research). RNA was eluted in RNase-free H 2 O and concentration and quality of RNA determined with a NanoDrop spectrophotometer (Thermo Scientific). for 30 min and finally at 85°C for 5 min. Concentration and quality of cDNA was measured on a NanoDrop spectrophotometer and cDNA was diluted to 40 ng/mL working concentrations.

Quantitative real-time PCR
Relative gene expression was detected using quantitative real-time PCR (Q-PCR). The 10 μl reaction volume contained 5 μl of SYBR s -Green PCR Master Mix (Promega), 200 ng of forward and reverse primer, 0.1 μl CXR reference dye (Promega) and 120 ng cDNA template. Amplification was monitored on an Applied Biosystems 7900HT Fast Real-Time PCR system with an enzyme activation of 2 min at 50°C and 3 min at 95°C followed by 50 cycles of 3 s at 95°C and 30 s at 60°C. The cycle included in each run, and relative gene expression was determined by the ΔΔCt value (2 ( À ΔCt) ). For ease of graphic presentation of relative gene expression, ΔΔCt values were multiplied by 10 6 . Primer sequences for detected NSC and neural lineage genes are presented in Table 1 and primer sequences for heparan and chondroitin sulphate proteoglycan associated genes are presented in Table 2.

Immunofluorescence (IF)
Expression of selected NSC and neural lineage marker proteins were detected via IF using an Olympus IX81 inverted phase-contrast fluorescent microscope and images acquired using Volocity software (Perkin-Elmer) on a Hamamatsu Orca camera. For imaging, cells were plated on 8-well CC2-coated chamber slides (Lab-Tek) at 20-30 Â 10 4 cells/well and cells were cultured for 3-4 days before fixing and staining. Briefly, culture medium was removed, cells rinsed with 1 Â PBS with Ca 2þ and Mg 2 þ and fixed with 4% paraformaldehyde. After this cells were blocked (5% Donkey serum, 1% BSA in PBS with or without 0.1% Triton-X to allow permeabilisation) and primary antibodies were incubated overnight at 4°C. Isotype control antibodies were used as a negative control. After 24 h incubation, primary antibodies were removed, cells rinsed with 1 Â PBS with Ca 2þ and Mg 2 þ and cells incubated with secondary antibodies for 2 h at room temperature. Finally, cells were rinsed with 1 Â PBS with Ca 2 þ and Mg 2 þ and slides mounted with DAPI (ab104139, Abcam). Antibodies and dilutions used are presented in Table 3.

Statistical analysis
For Q-PCR analysis each gene was detected in quadruplicate per sample. Paired t-test was used to determine statistical significance and defined as * po 0.5, ** p o0.01 and *** p o0.001. Error bars represent SD.