arun a neuromics hca hts overview v3 04 26 2011
DESCRIPTION
Testing ATP levels in hN2™ , hNP1™ and iPS-NP1™cells using HemoGenix HTS LUMISTEM™ assay kitTRANSCRIPT
ArunA BiomedicalSTEMEZ™ hNP1™, hN2™ and iPS-NP1™
Demonstrated use in HTS and HCA assay systems
April 26, 2011
PROPRIETARY AND CONFIDENTIAL
Overview of ArunA’s neural cell products
STEMEZ™ hNP1™ Human Neural Progenitor Cells
Tissue source: Human embryonic stem cells
Culture system: Highly proliferative adherent mono-layer
Feeder system: Feeder and serum free cultures
Differentiation potential: Dopaminergic, cholinergic, glutamatergic, GABA-ergic and astrocytic lineages
STEMEZ™ hN2 Human Neural CellsTissue source: Human embryonic stem cellsCulture system: Adherent mono-layerFeeder system: Feeder and serum free culturesDifferentiation potential: hN2 cells are a mixed population of differentiated neuronal cells
STEMEZ™ iPS-NP1™
Tissue source: Human induced pluripotent stem cells from lung fibroblasts
Culture system: Highly proliferative adherent mono-layer
Feeder system: Feeder and serum free cultures
Differentiation potential: Mixed population of neuronal cells. Further studies are underway.
PROPRIETARY AND CONFIDENTIAL 2
Application of neural cells to HTS ATP assay
Objective:
To test ATP levels in hN2™ , hNP1™ and iPS-NP1™cells using HemoGenix HTS LUMISTEM™ assay kit
Procedure:• 96-well plates were coated with Matrigel (1:200) for ~1 hour at 4°C and washed
twice with PBS.• Cells were plated at 10,000 cells per well and incubated at 37°C for ~48 hrs then
assayed for ATP levels, as directed in the assay kit protocol.
PROPRIETARY AND CONFIDENTIAL 3
Cellular ATP levels in ArunA’s neural cells
• STEMEZ™ hNP1™ and STEMEZ™ iPS-NP1™ STEMEZ neural progenitor cells and hN2™ differentiated neural cells are amenable to HTS ATP analysis using the LUMISTEM assay.
Cell Type Mean [ATP] SD %CV
hNP1™ 1.15 µM 0.03 µM 2.4%
iPS-NP1™ 1.11 µM 0.02 µM 2.1%
hN2™ 0.68 µM 0.01 µM 2.2%
PROPRIETARY AND CONFIDENTIAL 4
Data in collaboration with HemoGenix
STEMEZ™ hNP1™ and hN2™ use in HCA assays
Objective: • Evaluate the applicability of STEMEZ™ hNP1™ and hN2™ cells in HCA
neurite outgrowth assay to assess:– Neurotoxicity (hN2™)– Neuronal differentiation
Procedure and outcomes:• Molecular Devices’ ImageXpress platform and associated neurite outgrowth
scoring modules were used for image acquisition and analysis• Both cell types proved to be effective choices for automated HTS formatted
compound testing
PROPRIETARY AND CONFIDENTIAL 5
Automated measurement of neurite outgrowth in hN2™. Neuritesemerging from accepted cell bodies are traced (light blue, green and purple lines) and quantified while cells without process (red) are not counted
PROPRIETARY AND CONFIDENTIAL 6
Harrill JA, Freudenrich TM, Machacek DW, Stice SL, Mundy WR. (2010) Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis. Neurotoxicology. 31(3):277-90.
Application of hN2™ neural cells to HCA neurite outgrowth assay
Green, beta III-tubulin (neurites)Blue, Hoechst (nuclei)
Image analysis of neurite outgrowth in hN2™ cells using ImageXpress
Nuclei and neurite traces
PROPRIETARY AND CONFIDENTIAL 7
Data in collaboration with Molecular Devices
Effect of Antimycin A on hN2™ neurite outgrowth
0 µM
1 µM
10 µM
100 µM
PROPRIETARY AND CONFIDENTIAL 8
Compound effects on hN2™ neurite outgrowth
PROPRIETARY AND CONFIDENTIAL 9
Differentiation of hNP1™ for 14 days in 96-well format
PROPRIETARY AND CONFIDENTIAL 10
Application of differentiated hNP1™ cells to HCA neurite outgrowth assay
Nuclei and neurite traces
PROPRIETARY AND CONFIDENTIAL 11
Green, beta III-tubulin (neurites)Blue, Hoechst (nuclei)
Data in collaboration with Molecular Devices
Effect of LIF on neurite outgrowth in 14-day differentiated hNP1™ cells
0 50 100 150 200 250
max process length
mean process length
total outgrowth
Length (µm)
-LIF
+LIF
PROPRIETARY AND CONFIDENTIAL 12
Data in collaboration with Molecular Devices