ArunA Biomedical
STEMEZ™ hNP1™, hN2™ and iPS-NP1™
Demonstrated use in HTS and HCA assay systems
April 26, 2011
PROPRIETARY...
Overview of ArunA’s neural cell
products
STEMEZ™ hNP1™ Human Neural Progenitor Cells
Tissue source: Human embryonic stem c...
Application of neural cells to HTS ATP
assay
Objective:
To test ATP levels in hN2™ , hNP1™ and iPS-NP1™cells using HemoGen...
Cellular ATP levels in ArunA’s neural
cells
• STEMEZ™ hNP1™ and STEMEZ™ iPS-NP1™ STEMEZ neural progenitor
cells and hN2™ d...
STEMEZ™ hNP1™ and hN2™ use in
HCA assays
Objective:
• Evaluate the applicability of STEMEZ™ hNP1™ and hN2™ cells in HCA
ne...
Automated measurement of neurite outgrowth in hN2™. Neurites
emerging from accepted cell bodies are traced (light blue, gr...
Green, beta III-tubulin (neurites)
Blue, Hoechst (nuclei)
Image analysis of neurite outgrowth in hN2™
cells using ImageXpr...
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 CONFIDE...
Effect of LIF on neurite outgrowth in 14-day
differentiated hNP1™ cells
0 50 100 150 200 250
max processlength
mean proces...
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Arun A Neuromics Hca Hts Overview V3 04 26 2011

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

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Arun A Neuromics Hca Hts Overview V3 04 26 2011

  1. 1. ArunA Biomedical STEMEZ™ hNP1™, hN2™ and iPS-NP1™ Demonstrated use in HTS and HCA assay systems April 26, 2011 PROPRIETARY AND CONFIDENTIAL
  2. 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. 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. 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. 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. 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. 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. 8. Effect of Antimycin A on hN2™ neurite outgrowth 0 µM 1 µM 10 µM 100 µM PROPRIETARY AND CONFIDENTIAL 8
  9. 9. Compound effects on hN2™ neurite outgrowth PROPRIETARY AND CONFIDENTIAL 9
  10. 10. Differentiation of hNP1™ for 14 days in 96-well format PROPRIETARY AND CONFIDENTIAL 10
  11. 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. 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

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