1. Products under consideration LentiGlo TM – ready to use lentiviruses co-expressing a novel luciferase along with the flourescent protein Bioluminescent stem cells – adult human mesenchymal stem cells engineered to express a novel luciferase along with the fluorescent protein Applications: Study stem cell growth, survival, and differentiation in vitro and in vivo

2. Overview of presentation  Novel luciferase reporters  Lenti-luciferases (LentiGlo TM )  Stable co-expression of different luciferases and fluorescent proteins in human adult stem cells using ready to use lentivirus (LentiGlo TM ) vectors  In vivo imaging to track stem cell growth and survival  Profect protein delivery technology to effect directed differentiation of stem cells into specific lineages  Targeting Systems IP review

3. Luciferase activities & emission max of different luciferase reporters

4. Substrate specificities of different luciferases

5. Kinetics of luciferase activity of different luciferase reporters

6. Dual luciferase assays

7. A Triple Luciferase assay based on Cyridina, Green Renilla, and Red Luciola luciferases

8.  Secreted luciferase reporters enable non-invasive analysis of cell growth, survival and in vivo imaging by sample blood or urine  LentiGlo TM vectors contain novel, bright luciferases increasing sensitivity  Available of several Lenti-luciferase products expressing luciferases emitting at different wavelengths and utilizing different luciferin substrates allows for efficient multiplexing options such as tracking different types of stem cells in the same animal Why LentiGlo TM ?

9. LentiGlo TM vectors that enable tracking stem cells, assessing stem cell growth and survival in vivo and also facilitate studies on stem cell differentiation or gene silencing in vivo. Lenti-UBC-RedFluc-GFP Lenti-UBC-GrFluc-RFP Lenti-UBC-GrRenLuc-RFP Lenti-UBC-GLuc-RFP Lenti-UBC-CypLuc-RFP Lenti-NFkB-GrRenLuc-RFP Lenti-NFkB-GLuc-RFP LentiGlo TM Stem Track Collection

10. LentiGlo TM Lentiviral Vectors –LentiGlo TM Gluc-GFP: A lentiviral vector expressing gaussia luciferase under control of the CMV promoter and a green fluorescent protein under an IRES –Choice of CMV or UbC promoter –Choice of Gluc, RenLuc, GrFLuc, RedFLuc or VargLuc

11. LentiGlo TM : The power of lentiviral delivery with novel luciferases Panel of novel luciferase reporters – The brightest and the best Human mesenchymal stem cells transduced with a LentiGlo TM vector expressing Gluc and GFP

12. StemTrack TM - Stem cells transduced to co-express a novel luciferase along with a fluorescent protein StemTrack TM Human stem cells expressing bioluminescent genes Primary human bone marrow-derived mesenchymal stem cells transduced with LentiGlo TM Gluc-GFP

13. Gaussia luciferase as an in vivo reporter to monitor cell survival or gene expression in vivo 1 day Post- 4 days Pos-implantation implantation Data using primary Ad HMSCs transduced with LentiGlo vector expressing Gluc- GFP and subcutaneously implanted into mice

14. Use of LentiGlo TM (expressing red firefly luciferase) for imaging human stem cells One million such cells expressing a different luciferase, the red Italica (firefly) luciferase were implanted into the transverse processes in nude mice. After 2 weeks, mice were injected with luciferin intraperitoneally and imaged using a CCD camera. Even though only 500,000 stem cells were implanted a robust signal was observed

15. Non-invasive, quantitative measurement of stem cell growth in vivo by measurement of Gluc activity in blood One millions stem cells expressing Gluc and GFP or PBS control were injected intravenously in nude mice. Prior to injection, the Gluc activity was monitored. The Gluc level in blood indicated that a significant number of cells survived the injection and did not proliferate

16. Gluc level in blood is linear with respect to implanted cell number

17. Applications of LentiGlo TM technology in tumor imaging

18. Efficient protein delivery into stem cells enables novel approaches for stem cell differentiation:  Targeted nuclear delivery of transcription factors to mediate differentiation of adult human stem cells into specific lineages  Protein delivery to mediate expansion of human adult stem cells (Tanaka et al, Stem Cells paper) Protein Delivery

19. Proprietary technology for highly efficient delivery of functionally active proteins and peptides into human and animal stem cells  Does not result in permanent genetic modification of cells  Successful ex-vivo expansion of CD34 positive hematopoietic stem cells has been recently been demonstrated  Does not require covalent modification of protein to be delivered  Shown to be applicable for expansion of hematopoietic stem cells (see stem cell paper in list of 11 citations using Profect). This technology does not involve covalent modification of the protein being delivered and has been used to deliver one or several proteins, peptides, transcription factors, enzymes, antibodies etc to the cytoplasm or nucleus of the cell Proprietary technology

20. Profect-P1: A lipid reagent that forms a non-covalent complex with proteins Profect-P2: A non-lipid reagent that forms non-covalent complexes with proteins and enables protein transport across both the cell membrane and the nuclear membrane. Protein delivery reagents

21. An important tool in stem cell research and therapeutics Applications:  Delivery of transcription factors to mediate differentiation of adult stem cells into specific lineages  Expansion of stem cells  Delivery of bioluminescent (chemiluminescent proteins) to track implanted stem cells Directed Protein Delivery

22. Profect TM Optimized for protein delivery into stem cells Mesenchymal Stem Cells transfected with Pluriport showing Histone and Dapi imaged at 20X magnification Efficient protein delivery into adult human stem cells

23. Antibody delivery into the nucleus of Cos-7 cells using Profect P-2 Antibody Delivery Cos-7 cells were transfected with Alexa 594 conjugated IgG and Alexa 488 conjugated histone using the Profect P-2 reagent.

24. LentiGlo TM Stem Cells Myogenic pics of pCMV-Gluc-GFP transduced adipose derivered mesenchymal stem cells treated with myogenic treatment Cardiomyocyte-like pics of pCMV-Gluc-GFP transduced adipose derivered mesenchymal stem cells treated with myogenic treatment Control – untreated. Ad- HMSCs transduced with pCMV-Gluc-GFP lenti without further treatment Osteogenic Towards osteocytes?

25. Differentiation of Ad-HMScs into islet cells Differentiation of adipose tissue -derived mesenchymal stem cells into pancreatic Beta cells (top panel) Bottom panel (negative control). Staining for insulin C peptide, an islet sp marker

26. Differentiation of Ad-HMSCs into skeletal muscle cells Cells were stained with antibodies to skeletal muscle troponin T, a marker for skeletal muscle cells

27. Differentiation of AD-HMScs into osteocytic lineage Cells stained for BMP-2 (Bone morphogenetic protein) after treatment (controls were clearly negative hence not shown)