Products under consideration

• LentiGloTM – 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

Overview of presentation

Novel luciferase reporters

Lenti-luciferases (LentiGloTM)

Stable co-expression of different luciferases and fluorescent proteins in human adult stem cells using ready to use lentivirus (LentiGloTM) 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

Luciferase activities & emission max of different luciferase reporters

LUCIFERASE SUBSTRATE

Gaussia luciferase CoelenterazineRenilla luciferase CoelenterazineRed Luciola luciferase Firefly luciferinCypridina luciferase Cypridina luciferin

(Vargulin )

Substrate specificities of different luciferases

Kinetics of luciferase activity of different luciferase reporters

Dual luciferase assays

Vargula (463nm) and Red Italica (613nm)

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Red Italica(613nm)

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

Vargula (463nm), Green Renilla (527nm) and Red Italica (613nm)

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Green Renilla (527nm)

Red Italica (613nm)

Vargula (463nm)

Secreted luciferase reporters enable non-invasive

analysis of cell growth, survival and in vivo imaging by

sample blood or urine

LentiGloTM

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 LentiGloTM?

LentiGloTM 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-GFPLenti-UBC-GrFluc-RFPLenti-UBC-GrRenLuc-RFPLenti-UBC-GLuc-RFPLenti-UBC-CypLuc-RFPLenti-NFkB-GrRenLuc-RFPLenti-NFkB-GLuc-RFP

LentiGloTM Stem Track

Collection

LentiGloTM Lentiviral Vectors

– LentiGloTM 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

LentiGloTM: 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 LentiGloTM vector expressing Gluc and GFP

StemTrackTM- Stem cells transduced to co-express a novel luciferase along with a fluorescent protein

StemTrackTM

Human stem cells expressing bioluminescent genes

Primary human bone marrow-derived mesenchymal stem cells transduced

with LentiGloTM Gluc-GFP

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

Use of LentiGloTM (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

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

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

Applications of LentiGloTM technology in tumor imaging

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

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 cellsSuccessful ex-vivo expansion of CD34 positive hematopoietic stem cells has been recently been demonstratedDoes 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

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

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

ProfectTM

 

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

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.

LentiGloTM 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

OsteogenicTowards osteocytes?

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

Differentiation of Ad-HMSCs into skeletal muscle cells

Cells were stained with antibodies to skeletal muscle troponin T, a marker for skeletal muscle cells

Differentiation of AD-HMScs into osteocytic lineage

Cells stained for BMP-2 (Bone morphogenetic protein) after treatment (controls were clearly negative hence not shown)