Testing ATP levels in hN2™ , hNP1™ and iPS-NP1™cells using HemoGenix HTS LUMISTEM™ assay kit

1. ArunA Biomedical
STEMEZ™ hNP1™, hN2™ and iPS-NP1™
Demonstrated use in HTS and HCA assay systems
April 26, 2011
PROPRIETARY AND CONFIDENTIAL

2. 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 Cells
Tissue source: Human embryonic stem cells
Culture system: Adherent mono-layer
Feeder system: Feeder and serum free cultures
Differentiation 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

3. 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

4. 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

5. 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

6. Automated measurement of neurite outgrowth in hN2™. Neurites
emerging 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

7. 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

8. Effect of Antimycin A on hN2™ neurite
outgrowth
0 µM
1 µM
10 µM
100 µM
PROPRIETARY AND CONFIDENTIAL 8

9. Compound effects on hN2™ neurite
outgrowth
PROPRIETARY AND CONFIDENTIAL 9

10. Differentiation of hNP1™ for 14 days in
96-well format
PROPRIETARY AND CONFIDENTIAL 10

11. 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

12. Effect of LIF on neurite outgrowth in 14-day
differentiated hNP1™ cells
0 50 100 150 200 250
max processlength
mean processlength
total outgrowth
Length (µm)
-LIF
+LIF
PROPRIETARY AND CONFIDENTIAL 12
Data in collaboration with Molecular Devices