WO2008144052A2 - Bioluminescent imaging of stem cells - Google Patents
Bioluminescent imaging of stem cells Download PDFInfo
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- WO2008144052A2 WO2008144052A2 PCT/US2008/006429 US2008006429W WO2008144052A2 WO 2008144052 A2 WO2008144052 A2 WO 2008144052A2 US 2008006429 W US2008006429 W US 2008006429W WO 2008144052 A2 WO2008144052 A2 WO 2008144052A2
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- A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
Definitions
- the present invention relates to methods and compositions for detecting, localizing and tracking light-emitting stem cells in vitro and in vivo, and to methods and compositions to differentiate stem cells into specific lineages by intracellular delivery of functionally active proteins.
- HSC Hematopoietic stem cells
- the present invention provides methods for advantageously monitoring stem cells in vivo or in vitro, as well as methods for directing cell differentiation and/or expansion.
- the monitoring method generally involve the use of a secreted luciferase, especially a Gaussia luciferase.
- the present methods enable tracking of the location, survival, and/or expansion of stem cells by detecting and/or locating and/or quantitating luciferase activity.
- the invention further provides methods allowing transient detection or monitoring of cells without permanent or even long term alteration of the cells, including stem cells, using the transfer of luciferase protein into cells.
- a first aspect of the invention concerns a method for monitoring stem cells in vivo in an animal (e.g., a human) by detecting luminescence from a luciferase expressed in stem cells or progenitor cells transfected with a vector expressing a secreted luciferase.
- the secreted luciferase is Gaussia, Metridia, Pleuromama, or Cypridina luciferase or a derivative thereof;
- the detecting involves detecting teh luminescence from luciferase in blood;
- the stem cells are human mesenchymal stem cells, CD34 positive hematopoietic stem cells, neural stem cells, or mammalian stem cells;
- the cells are progenitor cells; expression of the luciferase is under the control of a CMV promoter, an HIV promoter, or an SV40 promoter.
- the detecting involves quantifying light emitted by reaction of the luciferase; quantification involves use of a charge coupled device (CCD) detector.
- CCD charge coupled device
- the vector also includes an expressible fluorescent protein; the fluorescent protein is under the control of an internal ribosome entry site (IRES); the fluorescent protein comprises a green fluorescent protein, or a red fluorescent protein; the method also includes detecting fluorescence from the fluorescent protein; detecting fluorescence from the fluorescent protein includes quantifying light emitted by reaction of the fluorescent protein; the detecting is performed in a mammal; the cells are also transfected with a vector expressing one or more proteins regulating or mediating stem cell expansion or differentiation; the vector expressing the luciferase and the vector expressing the proteins regulating or mediating stem cell expansion or differentiation are the same vector; the vector expressing the luciferase and the vector expressing the proteins regulating or mediating stem cell expansion or differentiation are the same vector.
- IVS internal ribosome entry site
- the cell is transfected using a cationic transfection reagent, e.g., including a cationic lipid, a cationic polyamine, or a dendrimer; transfection is enhanced using a replication deficient adenovirus; the cell is also transfected with a second plasmid expressing one or more proteins that regulate stem cell expansion or one or more proteins mediating differentiation of stem cells into a specific phenotype; the cell is transfected with a construct expressing both Gaussia luciferase under control of the CMV promoter or other strong promoter and a target gene coding sequence subcloned after the stop codon of the Gaussia luciferase gene, and an siRNA directed against a target gene; the vector is a lentiviral vector or a plasmid vector.
- a cationic transfection reagent e.g., including a cationic lipid, a cationic polyamine, or a dendrimer
- transfection is
- a second aspect concerns a non-invasive method for detecting expression of a heterologous gene in a selected cell type by transfecting the selected cell type with a vector (e.g., a plasmid vector) which includes an expressable secreted luciferase, where delivery of the plasmid vector is effected by complexing the vector with a cationic transfection agent and a replication- deficient adenovirus; and detecting the expression of the luciferase, e.g., from an extracellular medium.
- a vector e.g., a plasmid vector
- a vector e.g., a plasmid vector
- delivery of the plasmid vector is effected by complexing the vector with a cationic transfection agent and a replication- deficient adenovirus
- detecting the expression of the luciferase e.g., from an extracellular medium.
- the method is carried out as described for the preceding aspect; the cell is transfected using a cationic transfection reagent, such as one including a cationic lipid, a cationic polyamine, or a dendrimer; the cell is also transfected with a second plasmid expressing one or more proteins that regulate stem cell expansion or one or more proteins mediating differentiation of stem cells into a specific phenotype.
- a related aspect of the invention concerns a method for transient monitoring of a cell by transfecting the cell with a luciferase protein and detecting the presence of the luciferase.
- the luciferase protein is a Gaussia luciferase protein or other luciferase as specified for one of the above aspects; the luciferase is detected within the cell; the luciferase is detected outside the cell; the luciferase is detected in the blood of an animal; the transfecting is performed ex vivo, the cells are implanted in an animal, and the detecting is performed following said implantation; the cell is transfected using a cationic transfection reagent, e.g., as described for an above aspect or otherwise described herein.
- Another aspect concerns a method for visualization of stem cells by transfecting stem cells with a lentiviral vector comprising a sequence encoding Gaussia luciferase (or other secreted luciferase) as a reporter gene in a under control of the CMV promoter; and detecting the presence of the luciferase.
- the invention provides a method for determining survival of transplanted stem cells in vivo in an animal by measuring luciferase activity in the blood of said animal from a secreted luciferase expressed from the stem cells.
- the luciferase activity is from a Gaussia luciferase or other secreted luciferase as described herein.
- a related aspect concerns a method for quantitatively detecting expansion of transplanted stem cells in vivo in an animal (e.g., a human) by measuring luciferase activity in the blood of the animal, where the luciferase activity is from a secreted luciferase expressed in the stem cells.
- luciferase activity is from a Gaussia luciferase or other secreted luciferase as described herein.
- Another related aspect concerns a method of detecting migration and localization of transplanted stem cells by detecting luciferase activity from a Gaussia luciferase or other secreted luciferase in the cells.
- the cells are transfected with a vector expressing the luciferase; the cells are transfected with the luciferase protein, e.g., Gaussia luciferase protein.
- An aspect of the invention provides a method for studying gene regulation in stem cells by determining the expression of Gaussia luciferase (or other secreted luciferase) from or in cells comprising a Gaussia luciferase coding sequence under the control of a promoter for a gene of interest.
- the promoter regulates expression of a transcription factor or regulatory protein involved in stem cell differentiation.
- a further aspect concerns a method for monitoring gene silencing in vivo by determining luciferase activity from cells expressing a secreted luciferase, preferably a Gaussia luciferase, co-regulated with a target gene, wherein the cells are also transfected with a vector expressing a siRNA targeting that target gene.
- the invention also concerns a method for causing stem cell differentiation or expansion by transfecting stem cells with a regulatory protein, e.g., a transcription factor.
- a regulatory protein e.g., a transcription factor
- the transfection results in detectable transient persistence of the regulatory protein in the cells for 1 to 21 days, 2 to 21, 4 to 21 days, 7 to 21 days, 4 to 14 days, or 4 to 10 days;
- the stem cells are mesenchymal stem cells and the regulatory protein is selected from the group consisting of the proteins listed in Table 1.
- the cell is co-transfected with a secreted luciferase as described herein.
- Another aspect concerns a chimeric polypeptide comprising a first domain having a bioluminescent polypeptide (GFP or any fluorescent protein), a second domain having a secreted chemiluminescent peptide, and an endogenous protease cleavage motif positioned between the first and second domains.
- GFP bioluminescent polypeptide
- second domain having a secreted chemiluminescent peptide
- endogenous protease cleavage motif positioned between the first and second domains.
- the secreted chemiluminescent peptide is a secreted luciferase, e.g., a Gaussia luciferase, Metridia luciferase, Pleuromama luciferase, and Cypridina luciferease.
- a secreted luciferase e.g., a Gaussia luciferase, Metridia luciferase, Pleuromama luciferase, and Cypridina luciferease.
- the invention provides a purified polypeptide characterized as having Gaussia luciferase activity and a recognition site specifically cleavable by a protease, wherein cleavage results in a increase in luciferase activity and wherein said recognition site is a peptide sequence selected from the group consisting of DEVD, LEHD, IETD, VEHD, LETD, IEPD, DETD, WEHD, YVAD , VEID, and any combination thereof.
- the protease is a caspase-family protease, a matrix metalloproteinase, or a serine protease; the caspase-family protease is selected from the group consisting of a Caspase-3, a Caspase-6, a Caspase-8, and a Caspase-9; the endogenous protease cleavage motif is specifically cleaved by an endogenous cellular protease; the endogenous protease cleavage recognition motif comprises a PACE/furin cleavage recognition motif.
- Yet another aspect concerns a method for detecting intracellular protease activity by detecting extracellular chemiluminescent activity from a secreted chemiluminescent peptide from cells expressing a polypeptide of any of the preceding two aspects.
- the polypeptide is a GFP-protease cleavage site-GLUC polypeptide; the protease activity is detected from in vivo cells; the protease activity is indicative of apoptosis.
- stem cell means totipotent or pluripotent cells, such as embryonic or non-embryonic stem cells of any origin, e.g., bone marrow, fetus, periphereal blood, amniotic fluid from any mammal. It includes but is not limited to CD 34 positive cells, mesenchymal and stromal cells and multipotent stromal cells. It also includes stem cells derived from other tissues such as muscle, adipocyte tissue, and neural stem cells. Unless expressly indicated to the contrary, as used herein the term “stem cell” includes progenitor cells.
- Progenitor cell As used herein , the term “progenitor cell” refers to a partially differentiated, multipotent cell that can different to give rise to distinct cell lines.
- vector refers to a replicon, such as virus, plasmid, phage, or cosmid, to which another nucleic acid (usually DNA) segment may be attached so as to bring about the replication of the attached segment.
- the vector is genetically engineered to include nucleic acid sequences that are adapted for insertion of desired nucleic acid sequences.
- plasmid vector refers to a circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA, and is adapted for insertion of one or more desired DNA sequences.
- a "viral vector” is a vector that includes nucleic acid sequences obtained from or derived from a virus. Examples include “lentiviral vector” and “adenoviral vector”, derived from lentiviruses and adenoviruses respectively.
- transfection refers to the transfer of exogenous biopolymer, such as nucleic acid (usually DNA) or protein, into a cell.
- transduction refers to the transfer of genetic material (e.g., DNA) into a cell using a viral vector.
- the transducing DNA may (as in the case of lentiviral vectors) or may not be integrated (covalently linked) into the genome of the cell.
- promoter refers to a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 1 direction) coding sequence.
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5 1 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
- a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
- Various promoters may be used to drive transcription from vectors.
- IRES internal ribosome entry site
- Fig. 1 Infection of human mesenchymal stem cells with a lentiviral vector expressing both Gaussia luciferase and a Fluorescent protein.
- Fig. 2 Delivery of Alexa-488-conjugated histone into human Mesenchymal stem cells
- Fig. 3A Visualization of implanted human mesenchymal stem cells (transduced with a lentiviral vector to express Gaussia luciferase) into nude mice using biuoluminescent imaging techniques (a CCD camera) at day 21 and day 4 following implantation.
- Fig. 3B Quantitation of the bioluminescent signal intensity at days 1 and 4 following implantation
- Fig. 4 Plasmid map of the psiScreen vector used in gene silencing:
- Fig. 5 A schematic representation of the use of Gaussia luciferase to screen different siRNAs for their effectiveness in silencing a target gene
- Fig. 6 Screening of different siRNAs (small interfering siRNAs) against p53 using the psiScreen system
- Fig. 7 Dose dependence of siRNA on silencing of the human tumor suppressor p53 gene in HEK-293 cells :
- Fig. 8 A, 8B Screening of different siRNAs (small interfering siRNAs) against p53 using the psiScreen system in supernatants (Fig. 8A) and cell lysates (Fig. 8B) of human mesenchymal stem cells.
- Fig. 9 Differentiation of Ad-HMSCs into pancreatic Beta cells two weeks after transfection with PDX-1.
- Panels A and B Ad-HMSCs were transfected with recombinant PDX-1 using the Profect P- 1 reagent and then two weeks later examined for differentiation into pancreatic beta cells by immunostaining for insulin C peptide (Panels A and B).
- Panel C Control cells transfected with histone using the Profect P1 reagents and stained for insulin C peptide.
- Panel D Control cells stained fro PDX-1.
- Panel E Ad-HMSCs transfected with recombinant PDX-1 protein using Profect P1 and immunostained for PDX-1 using an anti PDX-1 antibody
- Fig. 10 Differentiation of Ad-HSCs into skeletal myobalsts two weeks following transfection with MyoD: Panels A-B: Ad-HMCs (genetically transduced with a lentivirus expressing Gaussia luciferase and GFP ) and then transfected with MyoD using Profect P-1. The cells start differentiating within 2 weeks following transfection with MyoD. The morphology of the cells (visualized by fluorescence microscopy because of their ability to express GFP) clearly indicates a myogenic phenotype which is further confirmed by staining for skeletal muscle troponin. Note that the control cells (transfected with histone using Profect P1) maintain the normal morphology of Ad-HMSCs (Panel C-Control Panel)
- the present invention provides methods for tracking and imaging implanted stem cells in vivo and in vitro using bioluminescent imaging techniques based on a distinctive luciferase, Gaussia luciferase (26, 12) which is over 1000 times brighter than the firefly and Renilla luciferases presently used in bioluminescent imaging. Luciferase based bioluminescent imaging techniques have been previously used for imaging tumors (10,20) or studying the fate of the transplanted cells (16).
- Gaussia luciferase being over a 1000-fold brighter than firefly and renilla luciferase
- the use of Gaussia luciferase enables imaging of small numbers of implanted cells in vivo using non-invasive bioluminescent imaging techniques.
- the system and methods described in the present invention satisfy many of the criteria for an Ideal Imaging Technology for Stem Cell Tracking During Clinical Trials, such as low toxicity, quantification of cell number and cell survival, minimal or no dilution with cell division and non-invasive imaging in the living subject over a prolonged period.
- the present invention provides methods and compositions that provide a non-invasive approach to detect, localize and track stem cells, as well as other entities, in a living host, such as a mammal, as well as in w ⁇ ro.using a novel secreted luciferase.
- the methods and compositions described provide noninvasive real time quantitation of both stem cell survival and quantification of stem cell multiplication both in vitro and in vivo following transplantation into animals.
- Gaussia luciferase as a reporter gene to monitor stem cell survival and growth of implanted stem cells in vivo (see data in provisional patent.
- a useful composition of the assay reagent for assaying Gaussia luciferase activity in the blood or urine is disclosed herein.
- Gaussia luciferase activity as an ultrasensitive method to quantitate cell number in vitro has also been shown in a very recent study.
- Fig 2 in the Badr et al, 2007 reference below shows that secreted Glue activity is a very sensitive indicator of cell number and is directly proportional to the number of cells over a very wide range. This corroborates the statement in the prior prior provisional application that Glue can be used to assess cell growth in vitro.
- the data in the Wurdinger et al, 2007 reference shows that Glue can be used to monitor cell growth and survival in vivo by quantitating Gaussia luciferase acitivty in the blood.
- Gaussia luciferase has also been shown to be very useful in monitoring survival of implanted tumor cells in vivo.
- the Glue blood assay complements in vivo bioluminescence imaging, which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in experimental disease and therapy models.
- a later section in the present disclosure describes exemplarly methods and composition of reagents for assaying Gaussia luciferase activity in the blood.
- Embodiments of the present invention also allow for studying regulation of gene expression and gene silencing in stem cells in vitro and in vivo, as well as being applicable to studying the mechanisms underlying expansion of stem cells and stem cell differentiation This can be accomplished, for example, by expression of key regulatory proteins into stem cells using either a gene delivery approach or a protein delivery approach. Controlled expression of specific regulatory proteins at varying stages of development is a critical step in differentiation of stem cells into a specific phenotype. Of key importance in this process are transcription factors which act at the nuclear level to activate key regulatory genes involved in stem cell differentiation.
- a major advantage of the proposed methods is that they enable simultaneous imaging of transplanted stem cells by expression of a bioluminescent gene and simultaneous differentiation or expansion of the transplanted stem cells in vivo or in vitro by co-delivery of a second gene or protein involved in stem cell differentiation, expansion, and/or regulation.
- Another embodiment of the current invention allows studying the effect of gene silencing in transplanted stem cells in vivo simply by genetically transducing stem cells infected with a vector, preferably a lentiviral vector, expressing Gaussia luciferase, with an siRNA directed against a target gene.
- a vector preferably a lentiviral vector, expressing Gaussia luciferase
- siRNA directed against a target gene.
- Gaussia luciferase can also be used for quantitative assessment of gene silencing in vivo.
- One approach is to transduce stem cells with an expression vector in which the gene of interest is subcloned after the stop codon of a luciferase gene under control of a strong promoter. Co-transfection of this vector with an siRNA directed against the target gene results in a decrease in luciferase expression that is directly proportional to the decline in Gaussia luciferase activity.
- the level of gene silencing can be quantitatively evaluated by assaying the Gaussia luciferase activity in the blood.
- An implementation of this approach is provided by the siScreen system as discussed herein, and it's applicability to study gene silencing in stem cells (also applicable to other cells) is discussed herein.
- Bioluminescent Imaging has been shown to be a useful tool for tumor, immune, and hematopoietic cell tracking studies (12,19-25).
- transplanted cells stably expressing luciferase can be detected in vivo through the tissue of live animals, e.g., using ultrasensitive cooled charged-coupled device (CCD) cameras, after administration of the appropriate luciferin, the luciferase substrate.
- CCD ultrasensitive cooled charged-coupled device
- long-term tracking of primary cultured cells has been limited by the relative difficultly in obtaining a high level of sustained reporter gene expression, including luciferase.
- a lentiviral vector preferably based on a viral sequence modified to prevent pathogenic infection, e.g., a third-generation self- inactivating lentiviral vector system based on human immunodeficiency virus type 1 (reference 12, Tannous et al, Molecular Therapy), can be used.
- This viral vector offers safe, high titer transduction and efficient integration into cells irrespective of their state of division.
- This type of vector can be used to achieve efficient delivery and stable expression of luciferase and/or green fluorescent protein (GFP) (or other second reporter protein) in order to monitor transplanted cells for in vivo and/or in vitro studies.
- GFP green fluorescent protein
- a novel gene delivery system in which an adenovirus is complexed with a cationic transfection reagent (e.g., lipid, polyamine or dendrimer) and a plasmid encoding the luciferase gene has been used to achieve high levels of transient gene expression lasting several weeks without permanent modification of the transfected cells with the bioluminescent luciferase.
- a cationic transfection reagent e.g., lipid, polyamine or dendrimer
- a plasmid encoding the luciferase gene has been used to achieve high levels of transient gene expression lasting several weeks without permanent modification of the transfected cells with the bioluminescent luciferase.
- References 7 and 8 discuss the details of enhancement of cationic liposome-mediated gene transfer using replication - deficient adenovirus.
- References 9, 10 and 11 also discuss applications of this technology.
- the present methods and materials are useful in detecting and/or monitoring many different types of cells. As indicated above, these methods and materials are particularly advantageous for use with stem cells and/or other progenitor cells.
- Stem cells are attractive as therapeutic candidates because of their pluripotent or even totipotent potential. As they multiply in culture they tend to lose their plasticity. Transduction of stem cells with lentiviral vectors expressing Gaussia luciferase results in integration of the Gaussia luciferase gene into the genome of the host cell thus circumventing the need for making a stable cell line.
- the Targefect plus Virofect gene delivery system and the Profect protein delivery systems described in the present study have the advantage of efficient transient delivery into stem cells without the need for making a recombinant virus. Gene expression from transient transfection persists for at least a week and can be exploited for differentiation or expansion of stem cells by transfecting appropriate genes and/or proteins.
- stem cells expressing Gaussia luciferase can be very useful in stem cell research because they can now be transduced with a second gene causing either cell expansion or differentiation into a specific phenotype, and the success of the technology can be studied by non-invasive bioluminescent imaging of the target organ.
- This approach is generally applicable to any type of stem cells originating from any animal (e.g., human) and any organ/blood/amniotic fluid source. It is also applicable to progenitor cells from any source. Examples of cell expansion factors that can be transfected either as gene or proteins include trancription factors such as the Hox protein or peptides (31). This approach can also be used to study the fate of normal cells such as hepatocytes, muscle cells or endothelial cells that have been genetically transduced ex vivo and then transplanted back into the recipient.
- Transduction of stem cells with genes expressing specific transcription factors or the corresponding protein or peptide factors can also be useful in mediating differentiation of human stem cells into specific cell lineages such as pancreatic cells, muscle cells or neural cells. Such differentiation can, if desired, be monitored or tracked using a Gaussia luciferase or other luciferase as described herein.
- Adipose- tissue-derived mesenchymal stem cells can be directed towards a myogenic phenotype in vitro by the addition of specific inductive media. Conversion of adipose-tissue-derived cells to a myogenic phenotype is enhanced by co-culture with primary myoblasts in the absence of cell contact and is maximal when the two cell types are co-cultured in the same plate. Conversely, in vitro expanded adipose-tissue derived mesenchymal stem cells required direct contact with muscle cells to generate skeletal myotubes.
- RT-PCR and immunohistochemistry confirmed the increased expression of characteristic downstream molecule myogenin and mysion heavy chains during myogenic differentiation.
- Ecoptic myogenesis was found and remained the stable phenotype when the transfected cells were seeded in vivo.
- MyoD is a determining factor of myogenic lineages, and it may play an important role in the cell therapy and cell-mediated gene therapy of the skeletal muscle.
- the present invention describes a simple way of differentiating adipose tissue-derived mesenchymal stem cells into skeletal muscle cells by transfection of Ad-HMSCs with recombinant MyoD, e.g., using the Protect P1 reagent.
- Ad- HMScs at passage 2 were transfected with Myo D and cultured for 2 weeks in mesenchymal stem cell culture media (containging approx 3% serum). After two weeks the cells were observed to be developing a skeletal muscle cell-type morphology.
- Ad-HMSCs assumed the typical elongated multinucleated skeletal muscle-type morphology and stained positive for the skeletal muscle markers (skeletal muscle troponin, myogenin and myosin heavy chain. Images of results of MyoD transfection are shown in Fig. 10 for differentiation of Ad-HSCs into skeletal myobalsts two weeks following transfection with MyoD. Panels A-B: Ad-HMCs (genetically transduced with a lentivirus expressing Gaussia luciferase and GFP) and then transfected with MyoD using Profect P-1. The cells start differentiating within 2 weeks following transfection with MyoD.
- the morphology of the cells clearly indicates a myogenic phenotype which is further confirmed by staining for skeletal muscle troponin as shown below.
- Control cells transfected with histone using Profect P1 maintained the normal morphology of Ad-HMSCs (Panel C).
- pancreatic Beta cells differentiation into pancreatic Beta cells is associated with the expression of specific transcription factors.
- Five different genes have been identified including those encoding the tissue-specific transcription factors expressed in pancreatic ⁇ -cells, i.e. HNF-4alpha (MODY1), HNF-1 alpha (MODY3), IPF-1 (also known as IDX-1 and PDX-1) (MODY4), and HNF-1 ⁇ (MODY5).
- HNF-4alpha MODY1
- HNF-1 alpha MODY3
- IPF-1 also known as IDX-1 and PDX-1)
- HNF-1 ⁇ MODY5
- IDX-1 Homozygosity for an inactivating mutation in IDX-1 results in pancreatic agenesis in a child and ldx-1-null mice also have pancreatic agenesis.
- IDX-1 transactivates several genes essential for the differentiated ⁇ -cell phenotype, and is important for glucose sensing and metabolism in the ⁇ -cell, including insulin, glucose-transporter-2 (GLUT-2), and glucokinase. More recently, the elegant studies of Yoshida et al have shown that IDX-1 can induce ⁇ -cell- specific gene expressions in some non- ⁇ -cells such as intestinal epithelial cells and may therefore be useful for future diabetes gene/cell therapy.
- Ad-HMSCs adult mesenchymal stem cells
- IDX-1 also known as PDX-1
- PDX-1 transcription factor 1
- Protein delivery was performed using the Profect P-1 reagent (Targeting Systems/Pluristem Innovations) according to the manufacturer's protocols. Efficient intracellular delivery of PDX-1 into Ad-HMSCs was ascertained by immunostaining for PDX-1. PDX-1 was found to persist in the transfected cells for at least one week after transfection. It is believed that the transcription factor PDX-1 mediates differentiation into pancreatic Beta cells by initiating a cascade of events activating other factors that eventually results in commitment of the cells into the pancreatic Beta cell type. Once the cells are committed they will not revert back to the undifferentiated state. Results of the test are shown in Fig.
- Ad-HMSCs were transfected with recombinant PDX-1 using the Profect P-1 reagent and then two weeks later examined for differentiation into pancreatic beta cells by immunostaining for staining for insuin
- Panel C shows control cells transfected with histone using the Protect P1 reagents and stained for insulin C peptide.
- Panel D shows control cells stained for PDX-1.
- Ad-HMSCs were transfected with recombinant PDX-1 protein using Profect P1 and immunostained for PDX-1 using an anti PDX-1 antibody
- transfection of stem cells with either an expression vector encoding IDX-1 or the IDX-1 protein itself can mediate differentiation of stem cells into pancreatic Beta cells, with or without Gaussia luciferase-expression.
- Protein delivery is thus a very powerful and safe tool for directed differentiation of stem cells into a specific lineage as it does not cause any permanent alteration to the cells in other ways, unlike gene transfer methods that involve recombinant viruses.
- Using the Profect reagents it is also possible to control the level of intracellular protein delivery by manipulating the concentration of protein used during complex formation, thereby providing an added advantage of the system.
- transfection of recombinant HOX B4 protein into hematopoietic stem cells should result in marked increase in the growth rate of stem cells as determined by the secreted Gaussia luciferase assay.
- the HLH transcription factors NeuroD and Neurogenin can mediate differentiation of mesenchymal stem cells into neuronal cells.
- the transcription factors neuronal helix-loop-helix protein (NEX)/mammalian atonal homolog 2 (Math-2), BETA2/neuronal determination factor (NeuroD), and NeuroD-related factor (NDRF)/NeuroD2 comprise a family of Drosophila atonal-related basic helix- loop-helix (bHLH) proteins with highly overlapping expression in the developing forebrain.
- Ngn1 the bHLH transcription factor neurogenin
- Ngn1 inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis.
- thse studies indicate that regulated expression of NeuroD and Neurogenin in mesenchymal stem cells would direct differentiation in neurons, and can be used in the present invention.
- the transcription factor Gata4 can mediate differentiation of mesenchymal cells into cardia myocytes.
- the transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart.
- a recent study by Oka et al, 2006, ref 73) showed that deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium.
- the results of this study showed that cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood.
- GAT A4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability, and can be used in the present invention.
- the present invention also applies to transfection of active small peptides (or sequences encoding such small peptides) into stem cells. Certain such peptides can mediate differentiation of stem cells into specific phenotypes, and thus can also be performed using the present compositions and methods.
- Glucagon-like peptide-1 GLP-1 an incretin hormone derived from proglucagon gene, has been identified as an inducer of IDX-1 expression.
- GLP-1 administered to diabetic mice was found to stimulate insulin secretion and effectively lower their blood sugar levels. GLP-1 also enhances ⁇ -cell neogenesis and islet size.
- GLP-1 The increase on insulin secretion by GLP-1 has been shown to be mediated by an I DX-1 -dependant transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis.
- GLP-1 is also capable of regulation the transcription of three genes that determine the pancreatic ⁇ -cell- specific phenotype: insulin, GLUT-2, and glucokinase.
- transfection of either the gene expressing GLIP-1 or transfection of the GLIP-1 peptide itself can mediate differentiation of human stem cells into pancreatic Beta cells.
- this can also be applied to other peptides possessing differentiation-mediating activity.
- the present invention can be used in cell expansion.
- the homeobox transcription factor HOX B4 plays a key role in the expansion and self renewal of hematopoietic stem cells.
- HSCs expanded by HOXB4 treatment reconstituted all hematopoietic lineages in mice that received transplants mice without causing leukemia, indicating that HSCs expressing HOXB4 were regulated by the hematopoietic system [48).
- Krosl et al. tried to expand murine HSCs by delivering HOXB4 protein [49].
- TAT-HOXB4 cell membrane-permeable, recombinant TAT-HOXB4 protein was added to the culture medium, inducing a fivefold net expansion of HSCs.
- HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was comparable to that induced by the human HOXB4 retrovirus during a similar period of observation.
- TAT- HOXB4 was supposed to be delivered with high efficiency, its half-life was estimated to be only 1 hour.
- the TAT protein has many undesirable side effect such as activating transcription of host cell genes such as the gene for transforming growth factor alpha and epidermal growth factor (reference 86).
- Amsellem et al. tried to expand human CB HSCs using HOXB4 protein [52]. They used HOXB4 protein secreted into the culture supernatant from cocultured MS-5 murine stromal cells, and this approach increased NOD/SCID mouse repopulating cells (SRCs) 2.5-fold. However, the efficiency of protein delivery was not very high, and the coculture system may not be practical for clinical applications. In contrast, the paper by Tanaka et al (31) showed that the peptide decHOX could be delivered into more than 70% of CB CD34 + hHSC/HPCs and was detected in these cells even after 4 days.
- HOXB4 influences the expression of genes involved in pivotal cell-intrinsic pathways such as regulation of cell cycle, differentiation, and apoptosis. It also modulates the response to multiple conserved extrinsic signals provided by the microenvironment (see Figure 2 in the Schiedlmeier paper). HOXB4 mediates expansion not only of bone marrow -derived hematopoietic stem cells but also embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically (53).
- HSCs embryo-derived hematopoietic stem cells
- recombinant HOX B4 has advantages over gene delivery approaches as it does not cause any permanent genetic modification of the cells. Also delivery of recombinant HX protein using protein delivery reagents such as Profect P1 or Profect P-2 (Targeting Systems, Santee, CA), does not require any modification of the HOX protein and proteins delivery using these reagents causes minimal toxicity to the cells. In fact the study by Tanaka et al (31) used the Profect reagent to deliver HOX peptides into hematopoietic stem cells.
- Gaussia princeps luciferase is the smallest known luciferase and is over 1000 times brighter than the existing firefly and renilla luciferases. Gaussia luciferase uses coelenterazine as the substrate. In the presence of coelenterazine and oxygen a bright blue bioluminescence is observed. In the present invention several approaches have been taken for bioluminescent imaging of stem cells (as well as other cells) using this novel luciferase.
- Gaussia luciferase Since Gaussia luciferase is efficiently expressed intracellular ⁇ as well as secreted into the medium, it is now possible to quantitate luciferase activity in the blood at different time intervals without killing the animal. Since factors such as serum in blood can affect Gaussia luciferase activity, specialized reagents are used for measuring Gaussia luciferase activity in the blood samples. The Gaussia luciferase assay reagents described in the patent application can be used for quantitaive measurement of Gaussia luciferase activity in blood samples. Since the luciferase activity is directly proportional to the number of stem cells, it provides an indication of cell growth after transplantation. Stem cells expressing Gaussia luciferase can be very useful in high throughput assays to screen for different compounds that induce stem cell expansion.
- This technology also enables one to use Gaussia luciferase as an in vivo reporter to study real time regulation of gene expression in stem cells in vivo by transfecting constructs that express Gaussia luciferase under control of the promoter of interest into stem cells. This would not be possible using the firefly and renilla luciferases as they are expressed intracellular ⁇ .
- All embodiments of the present invention are also applicable to Metridia luciferase, another secreted luciferase that has a high degree of homology (approx 90%) to Gaussia luciferase and shows almost identical sequence at the active site.
- Gaussia luciferase is approximately 1000/times brighter than Metridia luciferase (Metridia luciferase is only about 5 times brighter than firefly and Renilla luciferase (reference 17, Markova et al)
- a second reporter molecule can be encoded in a vector used to transfect (or co-transfect) cells, and especially stem cells.
- such second reporter will be a fluorescent protein such as green fluorescent protein or red fluorescent protein.
- Such second reporter can encoded in a separate vector molecule (which may be the same or different from the type of vector in which the luciferase is encoded), or may be encoded in the same vector molecule.
- the second reported may be encoded in the same or different open reading frames, e.g., using separate promoters in the case of different open reading frames or one promoter and an IRES in the case of a single open reading frame.
- Second reporter molecules could be useful to study effects of turning on certain genes on the expansion of stem cells in vivo.
- Second reporter plasmids expressing siRNA directed against specific cellular genes can also be co-transfected with vectors expressing Gaussia luciferase to study effects of silencing of specific target genes on stem cell expansion, cell survival and/or cell differentiation in vivo
- human stem cells have been infected with a lentiviral vector expressing Gaussia luciferase alone under control of the CMV promoter or expressing both Gaussia luciferase (e.g., under control of the CMV promoter) and a second reporter protein such as GFP (e.g., under control of an IRES).
- a lentiviral vector expressing Gaussia luciferase alone under control of the CMV promoter or expressing both Gaussia luciferase (e.g., under control of the CMV promoter) and a second reporter protein such as GFP (e.g., under control of an IRES).
- GFP green fluorescent protein
- the test was carried out by transduction of mesenchymal stem cells with a lentiviral vector encoding a green fluorescent protein (under control of an IRES) and Gaussia luciferase (under control of the CMV promoter).
- the cells were transduced using 50 lentiviral particles per cell.
- the genetically transduced stem cells upon subcutaneous injection into mice can be visualized using coelenterazine (see, e.g., Fig. 3 and Fig. 4), the substrate for Gaussia luciferase.
- the present invention thus provides an approach for non-invasive bioluminescent imaging of stem cells using a novel secreted reporter Gaussia luciferase.
- a plasmid vector expressing Gaussia luciferase e.g., under control of the CMV promoter, can also be delivered into stem cells.
- such plasmid vector can be efficiently delivered into mesenchymal stem cells using an appropriate transfection system, e.g., the Targefect F-1 or Targefect F-2 transfection reagent in combination with the Virofect enhancer, all from Targeting Systems, Santee, CA.
- an appropriate transfection system e.g., the Targefect F-1 or Targefect F-2 transfection reagent in combination with the Virofect enhancer, all from Targeting Systems, Santee, CA.
- Transfection with such a vector and transfection system provides transient expression of the luciferase, allowing tracking and/or other monitoring of the cells without permanent modification of the cells.
- While delivery of vectors encoding one or more reporter molecules provides highly advantageous methods for detecting and monitoring cells, in some cases it is beneficial to deliver protein reporter molecules directly, e.g., for transient detection.
- protein reporter molecule delivery can be accomplished using commercially available protein delivery reagents.
- the Profect P-1 and Profect P2 reagents from Targeting Systems have been used to successfully deliver proteins into mesenchymal and hematopoietic human stem cells (see Fig. 2 on histone delivery into mesenchymal stem cells and paper on delivery of HOX peptides into hematopoietic stem cells, reference 31 )
- transfection of the Gaussia luciferase protein into stem cells has been accomplished using the Profect P1 and Profect P2 reagents from Targeting Systems.
- the delivered protein is functionally active for about 3-4 days after transfection making this technique useful for those situations where transient visualization of implanted cells is desirable without any genetic modification of the cells.
- Gaussia luciferase as a reporter to study gene silencing in human stem cells:
- the psiScreen vector has a multiple cloning site after the stop codon of the Gaussia luciferase gene. (See Fig. 4)
- the psiScreen Vectors are designed to provide a rapid, quantitative approach for evaluation and optimization RNA interference (RNAi). These vectors enable monitoring of changes in expression of a target gene fused to a novel luciferase reporter gene.
- RNAi RNA interference
- Gaussia luciferase is used as a primary reporter gene, and the target gene of interest can be cloned into multiple cloning sites located downstream of the translational stop codon of the luciferase gene.
- Transfection of cells with the psiScreen vectors results in the production of an mRNA in which the mRNA encoding Gaussia luciferase is expressed as a fusion with the mRNA encoding the target gene.
- Initiation of the gene silencing occurs when co-transfection of si RNA towards the target gene of interest results in cleavage and subsequent degradation of fusion mRNA.
- Measurement of decreased Gaussia luciferase activity serves as an indicator of RNA interference. Since the Target gene is sub-cloned after the stop codon of Gaussia luciferase, the activity of Gaussia luciferase is unaffected by the fusion partner.
- the psiScreen system provides an easy method to screen a wide variety of siRNAs for gene silencing.
- the effectiveness of different siRNAs to silence the target gene of interest is evaluated quantitatively simply by measuring the luciferase activity.Gaussia luciferase is a secreted into the media. It is therefore necessary to only assay cell supernatants for luciferase activity without the need for lysing the cells. Considerable time is saved since time course experiments can be performed using the same group of transfected cells without lysing at each time point.
- Gaussia luciferase a thermostable enzyme, is 1000 times brighter than Renilla and firefly Luciferase thus increasing sensitivity of the assay.
- Gaussia Luciferase as a sensor for detection intracellular protease activity.
- Protein cleavage is a central event in many regulated biological processes.
- a system for detecting intracellular proteolysis using a positive readout assay based on increased secretion of a reporter polypeptide Gaussia luciferase (GLUC) following enzymatic cleavage of a protease cleavage site placed between a secretion inhibitor polypeptide (preferably a second reporter molecule such as a green fluorescent protein (GFP)) and GLUC fused to the C terminal of the secretion inhibitor polypeptide (e.g., GFP).
- a secretion inhibitor polypeptide preferably a second reporter molecule such as a green fluorescent protein (GFP)
- GFP green fluorescent protein
- the assay is based on the principle that N-terminal modification of a secreted reporter such as Gaussia luciferase with a polypeptide such as green fluorescent protein (GFP) results in marked reduction in secreted Gaussia luciferase activity.
- a secreted reporter such as Gaussia luciferase
- a polypeptide such as green fluorescent protein (GFP)
- Gaussia luciferase and GFP we have observed that active Gaussia luciferase is released in amounts proportional to the intracellular proteolytic activity thus generating a positive readout of intracellular protease activity without killing the cells.
- the sensitivity of the assay was further enhanced when constructs containing two (or more) protease cleavage sites flanked by amino acid spacers (e.g., 5 amino acid spacers) were placed between the GFP and the Gaussia luciferase sequence.
- a construct expressing a GFP -DEVD- GLUC fusion protein was transfected into HEK-293 cells as a sensor for measuring intracellular caspase activity (induced by addition of doxyrubicin, an inducer of apoptosis to the cells).
- the GFP used in this construct was the Ptilosarcus GFP from Nanolight Technologies, AZ.
- GFP green fluorescent protein
- SEAP secreted alkaline phosphatase
- Photinus luciferase secreted luciferases offer many advantages over cellular reporter enzymes as they can be non-destructively harvested from cellular supernatants over time.
- secreted luciferases have been reported, from the marine copepods Gaussia phnceps[76], and Metridia longa[77], the ostracod Vargula hilgendorfii[78]].
- intracellular luciferases such as from the sea pansy Renilla reniformis can be engineered to be secreted and stable in the extracellular milieu[81].
- Gaussia Luciferase offers a luminous output much higher than traditional bioluminescent proteins allowing for an ultra sensitive reporter of cell death through caspase secretion.
- the Gaussia activity is greatly reduced. Then when apoptosis occurs, the DEVD site is cleaved and the GLUC is reactivated allowing for a quantitative measure of cell death over time.
- Example 1 Transduction of cultured human mesenchymal stem cells with a recombinant lentiviral vector expressing Gaussia luciferase
- Human mesenchymal stem cells were isolated from the bone marrow and grown in mesenchymal stem cell culture media provided by Cell Applications Inc, San Diego. The human mesenchymal stem cells were transduced with a lentiviral vector encoding a green fluorescent protein (under control of an IRES) and Gaussia luciferase (under control of the CMV promoter). The cells were transduced using 50 lentiviral particles per cell. As shown in Fig. 1 , the transfection efficiency was almost 100%.
- a variety of proteins can be efficiently delivered into human stem cells using Profect reagents (reference 31).
- Alexa 488-conjugated histone which contains a nuclear localizaition signal was used aa a reporter protein to demonstrate the efficancy of intracellular protein delivery using Porfect P-1 and Profect P-2 reagents. Both commercially available from Targetign Systems, Santee, CA.
- Fluorescent histone is a good reporter for assessing efficient delivery of functionally active proteins because if the protein is functionally intact then it translocates to the nucleus because of the nuclear localization signal.
- the nuclear location of the fluorescent histone can be assessed by fluorescence microscopy after staining cells with the DAPI stain for nucleaur visualization.
- HMSC cells were infected with a lentivirus vector carrying the expression cassette for Glue under the control of CMV promoter. 4 days after infection, 2 million of these cells were implanted subcutaneously with Matrigel in nude mice. Mice were injected i.v. with 100 ⁇ g of coelenterazine, prepared by diluting it in methanol to a concentration of 5 ⁇ g/ ⁇ l, than 20 ⁇ l_ was added to 130 ⁇ l_ of PBS and all was i.v. injected. Mice were imaged at different time points using a CCD camera and photon counts were acquired over five minutes. The sum of photon counts/min is plotted in Fig. 3B.
- Images are displayed as a pseudo-color photon count image, superimposed on a grayscale anatomic white light image, allowing assessment of both bioluminescent intensity and its anatomical source.
- the implanted stem cells can be easily visualized and cell growth tracked in real time using a CCD camera without killing the animal. Further it is possible to quantitate the Gaussia luciferase activity in the blood, e.g., using the GAR B2 reagents from Targeting Systems.
- Fig. 6 shows ability of different siRNAs for silencing of the target gene (human tumor suppressor p53 gene).
- siRNA 1 and siRNA 2 represent 23 bp synthetic siRNAs with different sequences designed to silence the human tumor suppressor p53 gene.
- HEK-293 cells (1 million) were co-transfected with the psiScreen vector (1 ⁇ g) containing the p53 gene cloned into the multiple cloning site after the stop codon of the luciferase gene and 100 pmols of an unrelated siRNA (control, green bar) or 100 pmols of a mixed siRNA pool against the p53 gene purchased from New England Biolabs (NEB) or 100 pmols of synthetic siRNAs (oligo 1 and oligo 2)synthesized by Dharmacon, USA.
- the co-transfection experiments were performed using the Targefect F-2 reagent from Targeting Systems according to the manufacturer's recommended protocol. The data has been normalized for transfection efficiency using a firefly luciferase expression vector as a control plasmid. Unrelated siRNA was shown to be ineffective in gene silencing (data not shown)
- Fig. 7 shows dose response of siRNA for gene silencing of the target gene (human tumor suppressor p53 gene) in HEL-293 cells.
- the data has been normalized for transfection efficiency using a firefly luciferase expression vector as a control plasmid. Unrelated siRNA was shown to be ineffective in gene silencing (data not shown)
- Example 5 Use of Gaussia luciferase as a reporter gene to study gene silencing in human mesenchymal stem cells:
- FIG. 8 A, 8B show the results of screening of different siRNAs (small interfering siRNAs) against p53 using the psiScreen system in supernatants (Fig. 8A) and cell lysates (Fig. 8B) of human mesenchymal stem cells.
- the results of this experiment show the strong potential of the Gaussia luciferase-based siScreen system to study real time gene silencing in stem cells in vitro.
- the easiest way to perform such experiments is to co-transfect a small number of stem cells genetically transduced to express Gaussia luciferase with siRNA against a target gene and then implant them into an animal in vivo. Implantation of as few as 2 million stem cells genetically modified with Gaussia luciferase can be visualized using an appropriate CCD camera. Transfection of Gaussia lcuiferase- expressing stem cells with siRNA against a given target gene enables one to track the effects of silencing the target gene on the fate of the implanted stem cells using in vivo bioluminescent imaging techniques.
- Gaussia luciferase activity in the blood.
- the GAR B2 reagent enables one to quantitatively measure Gaussia luciferase in the blood.
- differentiation and homing of stem cells to a particular location following gene silencing can be assessed by BLI (bioluminescent imaging techniques).
- BLI bioluminescent imaging techniques
- Gaussia luciferase as a reporter for high throughput screening (HTS) of siRNA libraries targeted against genes whose silencing may lead to stem cell expansion :
- Stem cells transduced with lentiviral vectors to express Gaussia luciferase would be very useful in screening siRNA libraries for their ability to mediate stem cell expansion by silencing key target genes.
- stem cells transfected with siRNAs that silence target genes to mediate stem cell growth would show increased secreted Gaussia luciferase activity compared to control cells.
- the level of luciferase activity would directly co-relate to the effectiveness of a given siRNA to mediate stem cell expansion for instance Ex vivo targeting of p21 gene has been shown to permit relative expansion of human hematopoietic stem cells. ( reference 30).
- Gaussia luciferase activity in the blood is Gaussia luciferase activity in the blood:
- compositions with EDTA give lower activity but improved stability of the bioluminescent signal
- HNF-4alpha gene connects the transcription factors involved in maturity-onset diabetes of the young.
- PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.
- HLH helix loop helix
- Root DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat Methods 2006, 3:715- 719.
- telomere confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic linea ⁇ e.Stem Cells. 2005 Apr;23(4):516-29.
- a second Construct was made identical to the one above except with the addition of Glycine spacers and a second DEVD site.
- the sequence of the spacers and DEVD is PCMV-GFP-(GLYHSER-DEVD-[GLYHSER-DEVD-[GLYHSER-GIUC
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Abstract
Methods and compositions for detecting and localizing light originating from cultured stem cells or stem cells injected into tissue or an animal, especially a mammal, are described. Also disclosed are methods for localization of stem cells in selected regions, as well as for tracking stem cells within the mammal, and for causing stem cell differentiation.
Description
BIOLUiViIiSiESCEMT IMAGING OF STEM CELLS
RELATED APPLICATIONS
[0001] This application claims the benefit of WaNa, U.S. Provisional Appl. 60/939,046, filed May 18, 2007, entitled Bioluminescent Imaging of Stem Cells, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions for detecting, localizing and tracking light-emitting stem cells in vitro and in vivo, and to methods and compositions to differentiate stem cells into specific lineages by intracellular delivery of functionally active proteins.
BACKGROUND OF THE INVENTION
[0003] The following discussion is provided solely to assist the understanding of the reader, and does not constitute an admission that any of the information discussed or references cited constitute prior art to the present invention.
[0004] Stem cells hold tremendous potential for cancer therapy, tissue engineering, and cellular therapeutics. Understanding stem cell behavior and biology in the context of the living body, where the influences of tissue and organ systems remain intact, is essential for successful development of stem cell-based therapies . Hematopoietic stem cells (HSC) are well characterized, self-renewing, multipotent cells that can produce lifelong, complete hematopoietic reconstitution (16,). HSC function has been studied from a variety of experimental approaches, but HSC function typically must be assayed once tissues are removed from the body, often weeks or months after transplantation. Yet the critical early events of homing to the spleen and bone marrow (BM), seeding in the stromal microenvironment, and initial expansion of short- and long-term reconstituting cells occur within the first hours to days after transplantation (23-28) and are therefore inaccessible to investigation in most studies.
[0005] Several recent studies on differentiation of stem cells have identified key regulatory proteins that play very important roles in differentiation of stem cells
into specific lineages. In most instances these regulatory proteins are transcription factors which act by initiating a cascade of other factors whose coordinate expression results in differentiation into a specific lineage. Notably regulated expression of MyoD is believed to play an important role for differentiation of stem cells into myoblasts and expression of IDX-1 (also known as PDX-1) has been shown to play an important role in differentiation into pancreatic beta cells. Another group of transcription factors the Hox proteins are believed to play an important role in the self-renewal and expansion of adult and embryo-derived hematopoietic cells.
[0006] In vivo bioluminescence imaging (BLI) methods have been used to elucidate the spatiotemporal trafficking patterns of malignant cells, lymphocytes, and other mature immune cells within living animal models of human biology and disease (18-22). This technology utilizes low-light imaging systems based on charge-coupled device (CCD) cameras to detect luminescent signals that emanate from within the body from cells expressing the enzyme luciferase. For BLI detection of luciferase activity in living animals, the substrate for luciferase, , luciferin, is administered intravenously or intraperitoneally and rapidly diffuses throughout all tissues and enters many cell types. The level of photon emission and the spectrum of emitted light from luciferase-expressing mammalian cells is adequate to penetrate the tissues of small research animals, such as mice and rats, and thus can be detected externally with low-light imaging cameras (18-22).
SUMMARY OF THE INVENTION
[0007] The present invention provides methods for advantageously monitoring stem cells in vivo or in vitro, as well as methods for directing cell differentiation and/or expansion. The monitoring method generally involve the use of a secreted luciferase, especially a Gaussia luciferase. Advantageously, the present methods enable tracking of the location, survival, and/or expansion of stem cells by detecting and/or locating and/or quantitating luciferase activity. The invention further provides methods allowing transient detection or monitoring of cells without permanent or even long term alteration of the cells, including stem cells, using the transfer of luciferase protein into cells.
[0008] Thus, a first aspect of the invention concerns a method for monitoring stem cells in vivo in an animal (e.g., a human) by detecting luminescence from a luciferase expressed in stem cells or progenitor cells transfected with a vector expressing a secreted luciferase.
[0009] In particular embodiments, the secreted luciferase is Gaussia, Metridia, Pleuromama, or Cypridina luciferase or a derivative thereof; the detecting involves detecting teh luminescence from luciferase in blood; the stem cells are human mesenchymal stem cells, CD34 positive hematopoietic stem cells, neural stem cells, or mammalian stem cells; the cells are progenitor cells; expression of the luciferase is under the control of a CMV promoter, an HIV promoter, or an SV40 promoter.
[0010] In certain embodiments, the detecting involves quantifying light emitted by reaction of the luciferase; quantification involves use of a charge coupled device (CCD) detector.
[0011] In particular embodiments, the vector also includes an expressible fluorescent protein; the fluorescent protein is under the control of an internal ribosome entry site (IRES); the fluorescent protein comprises a green fluorescent protein, or a red fluorescent protein; the method also includes detecting fluorescence from the fluorescent protein; detecting fluorescence from the fluorescent protein includes quantifying light emitted by reaction of the fluorescent
protein; the detecting is performed in a mammal; the cells are also transfected with a vector expressing one or more proteins regulating or mediating stem cell expansion or differentiation; the vector expressing the luciferase and the vector expressing the proteins regulating or mediating stem cell expansion or differentiation are the same vector; the vector expressing the luciferase and the vector expressing the proteins regulating or mediating stem cell expansion or differentiation are the same vector.
[0012] For particular embodiments, the cell is transfected using a cationic transfection reagent, e.g., including a cationic lipid, a cationic polyamine, or a dendrimer; transfection is enhanced using a replication deficient adenovirus; the cell is also transfected with a second plasmid expressing one or more proteins that regulate stem cell expansion or one or more proteins mediating differentiation of stem cells into a specific phenotype; the cell is transfected with a construct expressing both Gaussia luciferase under control of the CMV promoter or other strong promoter and a target gene coding sequence subcloned after the stop codon of the Gaussia luciferase gene, and an siRNA directed against a target gene; the vector is a lentiviral vector or a plasmid vector.
[0013] A second aspect concerns a non-invasive method for detecting expression of a heterologous gene in a selected cell type by transfecting the selected cell type with a vector (e.g., a plasmid vector) which includes an expressable secreted luciferase, where delivery of the plasmid vector is effected by complexing the vector with a cationic transfection agent and a replication- deficient adenovirus; and detecting the expression of the luciferase, e.g., from an extracellular medium.
[0014] In certain embodiments, the method is carried out as described for the preceding aspect; the cell is transfected using a cationic transfection reagent, such as one including a cationic lipid, a cationic polyamine, or a dendrimer; the cell is also transfected with a second plasmid expressing one or more proteins that regulate stem cell expansion or one or more proteins mediating differentiation of stem cells into a specific phenotype.
[0015] Likewise, a related aspect of the invention concerns a method for transient monitoring of a cell by transfecting the cell with a luciferase protein and detecting the presence of the luciferase.
[0016] In certain embodiments, the luciferase protein is a Gaussia luciferase protein or other luciferase as specified for one of the above aspects; the luciferase is detected within the cell; the luciferase is detected outside the cell; the luciferase is detected in the blood of an animal; the transfecting is performed ex vivo, the cells are implanted in an animal, and the detecting is performed following said implantation; the cell is transfected using a cationic transfection reagent, e.g., as described for an above aspect or otherwise described herein.
[0017] Another aspect concerns a method for visualization of stem cells by transfecting stem cells with a lentiviral vector comprising a sequence encoding Gaussia luciferase (or other secreted luciferase) as a reporter gene in a under control of the CMV promoter; and detecting the presence of the luciferase.
[0018] In particular embodiments, the detection is performed in vitro; the detection is performed in vivo or from in vivo cells; the transfection is performed using a transfection complex comprising a plasmid vector expressing Gaussia luciferase as a reporter gene under control of the CMV promoter, a lipid or nonlipid cationic transfection reagent, and a replication-deficient adenovirus; the transfection is performed using a transfection complex comprising a plasmid vector expressing Gaussia luciferase as a reporter gene under control of the CMV promoter, a lipid or non-lipid cationic transfection reagent, and a lentivirus; the
[0019] Likewise, the invention provides a method for determining survival of transplanted stem cells in vivo in an animal by measuring luciferase activity in the blood of said animal from a secreted luciferase expressed from the stem cells.
[0020] In certain embodiments, the luciferase activity is from a Gaussia luciferase or other secreted luciferase as described herein.
[0021] A related aspect concerns a method for quantitatively detecting expansion of transplanted stem cells in vivo in an animal (e.g., a human) by
measuring luciferase activity in the blood of the animal, where the luciferase activity is from a secreted luciferase expressed in the stem cells.
[0022] The method of claim 54, wherein said luciferase activity is from a Gaussia luciferase or other secreted luciferase as described herein.
[0023] Another related aspect concerns a method of detecting migration and localization of transplanted stem cells by detecting luciferase activity from a Gaussia luciferase or other secreted luciferase in the cells.
[0024] In particular embodiments, the cells are transfected with a vector expressing the luciferase; the cells are transfected with the luciferase protein, e.g., Gaussia luciferase protein.
[0025] An aspect of the invention provides a method for studying gene regulation in stem cells by determining the expression of Gaussia luciferase (or other secreted luciferase) from or in cells comprising a Gaussia luciferase coding sequence under the control of a promoter for a gene of interest.
[0026] In particular embodiments, the promoter regulates expression of a transcription factor or regulatory protein involved in stem cell differentiation.
[0027] A further aspect concerns a method for monitoring gene silencing in vivo by determining luciferase activity from cells expressing a secreted luciferase, preferably a Gaussia luciferase, co-regulated with a target gene, wherein the cells are also transfected with a vector expressing a siRNA targeting that target gene.
[0028] The invention also concerns a method for causing stem cell differentiation or expansion by transfecting stem cells with a regulatory protein, e.g., a transcription factor.
[0029] In particular embodiments, the transfection results in detectable transient persistence of the regulatory protein in the cells for 1 to 21 days, 2 to 21, 4 to 21 days, 7 to 21 days, 4 to 14 days, or 4 to 10 days; the stem cells are mesenchymal stem cells and the regulatory protein is selected from the group consisting of the proteins listed in Table 1.
[0030] In particular embodiments, the cell is co-transfected with a secreted luciferase as described herein.
[0031] Another aspect concerns a chimeric polypeptide comprising a first domain having a bioluminescent polypeptide (GFP or any fluorescent protein), a second domain having a secreted chemiluminescent peptide, and an endogenous protease cleavage motif positioned between the first and second domains.
[0032] In particular embodiments, the secreted chemiluminescent peptide is a secreted luciferase, e.g., a Gaussia luciferase, Metridia luciferase, Pleuromama luciferase, and Cypridina luciferease.
[0033] Likewise in a related aspect, the invention provides a purified polypeptide characterized as having Gaussia luciferase activity and a recognition site specifically cleavable by a protease, wherein cleavage results in a increase in luciferase activity and wherein said recognition site is a peptide sequence selected from the group consisting of DEVD, LEHD, IETD, VEHD, LETD, IEPD, DETD, WEHD, YVAD , VEID, and any combination thereof.
[0034] In certain embodiments, the protease is a caspase-family protease, a matrix metalloproteinase, or a serine protease; the the caspase-family protease is selected from the group consisting of a Caspase-3, a Caspase-6, a Caspase-8, and a Caspase-9; the endogenous protease cleavage motif is specifically cleaved by an endogenous cellular protease; the endogenous protease cleavage recognition motif comprises a PACE/furin cleavage recognition motif.
[0035] Yet another aspect concerns a method for detecting intracellular protease activity by detecting extracellular chemiluminescent activity from a secreted chemiluminescent peptide from cells expressing a polypeptide of any of the preceding two aspects.
[0036] In certain embodiments, the polypeptide is a GFP-protease cleavage site-GLUC polypeptide; the protease activity is detected from in vivo cells; the protease activity is indicative of apoptosis.
[0037] "Stem cell" As used herein, the term "stem cell" means totipotent or pluripotent cells, such as embryonic or non-embryonic stem cells of any origin, e.g., bone marrow, fetus, periphereal blood, amniotic fluid from any mammal. It includes but is not limited to CD 34 positive cells, mesenchymal and stromal cells and multipotent stromal cells. It also includes stem cells derived from other tissues such as muscle, adipocyte tissue, and neural stem cells. Unless expressly indicated to the contrary, as used herein the term "stem cell" includes progenitor cells.
[0038] "Progenitor cell" As used herein , the term "progenitor cell" refers to a partially differentiated, multipotent cell that can different to give rise to distinct cell lines.
[0039] The term "vector" refers to a replicon, such as virus, plasmid, phage, or cosmid, to which another nucleic acid (usually DNA) segment may be attached so as to bring about the replication of the attached segment. In most cases, the vector is genetically engineered to include nucleic acid sequences that are adapted for insertion of desired nucleic acid sequences. Thus, a "plasmid vector" refers to a circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA, and is adapted for insertion of one or more desired DNA sequences. Similarly, a "viral vector" is a vector that includes nucleic acid sequences obtained from or derived from a virus. Examples include "lentiviral vector" and "adenoviral vector", derived from lentiviruses and adenoviruses respectively.
[0040] In the present context, the term "transfection" refers to the transfer of exogenous biopolymer, such as nucleic acid (usually DNA) or protein, into a cell.
[0041] As used herein, the term "transduction" refers to the transfer of genetic material (e.g., DNA) into a cell using a viral vector. The transducing DNA may (as in the case of lentiviral vectors) or may not be integrated (covalently linked) into the genome of the cell.
[0042] The terms "promoter", "promoter sequence", and the like, refer to a DNA regulatory region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (31 direction) coding sequence. The promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (51 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters may be used to drive transcription from vectors.
[0043] The terms "IRES", "internal ribosome entry site" and the like refer to a DNA sequence that allows for translation initiation in the middle of the mRNA.
[0044] Additional embodiments will be apparent from the Detailed Description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Fig. 1 : Infection of human mesenchymal stem cells with a lentiviral vector expressing both Gaussia luciferase and a Fluorescent protein.
[0046] Fig. 2:Delivery of Alexa-488-conjugated histone into human Mesenchymal stem cells
[0047] Fig. 3A: Visualization of implanted human mesenchymal stem cells (transduced with a lentiviral vector to express Gaussia luciferase) into nude mice using biuoluminescent imaging techniques (a CCD camera) at day 21 and day 4 following implantation.
[0048] Fig. 3B: Quantitation of the bioluminescent signal intensity at days 1 and 4 following implantation
[0049] Fig. 4: Plasmid map of the psiScreen vector used in gene silencing:
[0050] Fig. 5: A schematic representation of the use of Gaussia luciferase to screen different siRNAs for their effectiveness in silencing a target gene
[0051] Fig. 6: Screening of different siRNAs (small interfering siRNAs) against p53 using the psiScreen system
[0052] Fig. 7: Dose dependence of siRNA on silencing of the human tumor suppressor p53 gene in HEK-293 cells :
[0053] Fig. 8 A, 8B: Screening of different siRNAs (small interfering siRNAs) against p53 using the psiScreen system in supernatants (Fig. 8A) and cell lysates (Fig. 8B) of human mesenchymal stem cells.
[0054] Fig. 9: Differentiation of Ad-HMSCs into pancreatic Beta cells two weeks after transfection with PDX-1. Panels A and B : Ad-HMSCs were transfected with recombinant PDX-1 using the Profect P- 1 reagent and then two weeks later examined for differentiation into pancreatic beta cells by immunostaining for insulin C peptide (Panels A and B). Panel C: Control cells transfected with histone using the Profect P1 reagents and stained for insulin C
peptide. Panel D: Control cells stained fro PDX-1. Panel E: Ad-HMSCs transfected with recombinant PDX-1 protein using Profect P1 and immunostained for PDX-1 using an anti PDX-1 antibody
[0055] Fig. 10: Differentiation of Ad-HSCs into skeletal myobalsts two weeks following transfection with MyoD: Panels A-B: Ad-HMCs (genetically transduced with a lentivirus expressing Gaussia luciferase and GFP ) and then transfected with MyoD using Profect P-1. The cells start differentiating within 2 weeks following transfection with MyoD. The morphology of the cells (visualized by fluorescence microscopy because of their ability to express GFP) clearly indicates a myogenic phenotype which is further confirmed by staining for skeletal muscle troponin. Note that the control cells (transfected with histone using Profect P1) maintain the normal morphology of Ad-HMSCs (Panel C-Control Panel)
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] The present invention provides methods for tracking and imaging implanted stem cells in vivo and in vitro using bioluminescent imaging techniques based on a distinctive luciferase, Gaussia luciferase (26, 12) which is over 1000 times brighter than the firefly and Renilla luciferases presently used in bioluminescent imaging. Luciferase based bioluminescent imaging techniques have been previously used for imaging tumors (10,20) or studying the fate of the transplanted cells (16). However, a major limitation in tracking small numbers of stem cells to study their survival, expansion or differentiation potential in vivo is that the sensitivity of the firefly and Renilla luciferase may not be sufficient to give a strong bioluminescent signal, especially when working with small numbers of implanted cells. Also since the firefly and renilla genes are both expressed intracellular^, quantitative assessment of stem cell survival and multiplication would not be possible. However, because Gaussia luciferase is a secreted protein, the survival and multiplication of stem cells can be quantitatively determined by measuring the Gaussia luciferase activity in the bloodstream. In addition, due to Gaussia luciferase being over a 1000-fold brighter than firefly and renilla luciferase, the use of Gaussia luciferase enables imaging of small numbers of implanted cells in vivo using non-invasive bioluminescent imaging techniques.
[0057] The system and methods described in the present invention satisfy many of the criteria for an Ideal Imaging Technology for Stem Cell Tracking During Clinical Trials, such as low toxicity, quantification of cell number and cell survival, minimal or no dilution with cell division and non-invasive imaging in the living subject over a prolonged period. Thus, the present invention provides methods and compositions that provide a non-invasive approach to detect, localize and track stem cells, as well as other entities, in a living host, such as a mammal, as well as in wϊro.using a novel secreted luciferase.
[0058] In addition, the methods and compositions described provide noninvasive real time quantitation of both stem cell survival and quantification of stem cell multiplication both in vitro and in vivo following transplantation into animals. We have successfully used Gaussia luciferase as a reporter gene to monitor stem
cell survival and growth of implanted stem cells in vivo (see data in provisional patent. A useful composition of the assay reagent for assaying Gaussia luciferase activity in the blood or urine is disclosed herein.
[0059] The measurement of Gaussia luciferase activity as an ultrasensitive method to quantitate cell number in vitro has also been shown in a very recent study. For example, Fig 2 in the Badr et al, 2007 reference below shows that secreted Glue activity is a very sensitive indicator of cell number and is directly proportional to the number of cells over a very wide range. This corroborates the statement in the prior prior provisional application that Glue can be used to assess cell growth in vitro. The data in the Wurdinger et al, 2007 reference shows that Glue can be used to monitor cell growth and survival in vivo by quantitating Gaussia luciferase acitivty in the blood. In fact Gaussia luciferase has also been shown to be very useful in monitoring survival of implanted tumor cells in vivo. The Glue blood assay complements in vivo bioluminescence imaging, which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in experimental disease and therapy models. A later section in the present disclosure describes exemplarly methods and composition of reagents for assaying Gaussia luciferase activity in the blood.
[0060] Embodiments of the present invention also allow for studying regulation of gene expression and gene silencing in stem cells in vitro and in vivo, as well as being applicable to studying the mechanisms underlying expansion of stem cells and stem cell differentiation This can be accomplished, for example, by expression of key regulatory proteins into stem cells using either a gene delivery approach or a protein delivery approach. Controlled expression of specific regulatory proteins at varying stages of development is a critical step in differentiation of stem cells into a specific phenotype. Of key importance in this process are transcription factors which act at the nuclear level to activate key regulatory genes involved in stem cell differentiation. A major advantage of the proposed methods is that they enable simultaneous imaging of transplanted stem cells by expression of a bioluminescent gene and simultaneous differentiation or expansion of the transplanted stem cells in vivo or in vitro by co-delivery of a
second gene or protein involved in stem cell differentiation, expansion, and/or regulation.
[0061] Another embodiment of the current invention allows studying the effect of gene silencing in transplanted stem cells in vivo simply by genetically transducing stem cells infected with a vector, preferably a lentiviral vector, expressing Gaussia luciferase, with an siRNA directed against a target gene. The consequences of silencing of the target gene, e.g., on cell expansion and differentiation can then be studied by in vivo bioluminescent imaging techniques which track the implanted cells.
[0062] Gaussia luciferase can also be used for quantitative assessment of gene silencing in vivo. One approach is to transduce stem cells with an expression vector in which the gene of interest is subcloned after the stop codon of a luciferase gene under control of a strong promoter. Co-transfection of this vector with an siRNA directed against the target gene results in a decrease in luciferase expression that is directly proportional to the decline in Gaussia luciferase activity. The level of gene silencing can be quantitatively evaluated by assaying the Gaussia luciferase activity in the blood. An implementation of this approach is provided by the siScreen system as discussed herein, and it's applicability to study gene silencing in stem cells (also applicable to other cells) is discussed herein.
[0063] Bioluminescent Imaging (BLI) has been shown to be a useful tool for tumor, immune, and hematopoietic cell tracking studies (12,19-25). In this method, transplanted cells stably expressing luciferase can be detected in vivo through the tissue of live animals, e.g., using ultrasensitive cooled charged-coupled device (CCD) cameras, after administration of the appropriate luciferin, the luciferase substrate. However, long-term tracking of primary cultured cells has been limited by the relative difficultly in obtaining a high level of sustained reporter gene expression, including luciferase.
[0064] To address this limitation, a lentiviral vector, preferably based on a viral sequence modified to prevent pathogenic infection, e.g., a third-generation self- inactivating lentiviral vector system based on human immunodeficiency virus type
1 (reference 12, Tannous et al, Molecular Therapy), can be used. This viral vector offers safe, high titer transduction and efficient integration into cells irrespective of their state of division. This type of vector can be used to achieve efficient delivery and stable expression of luciferase and/or green fluorescent protein (GFP) (or other second reporter protein) in order to monitor transplanted cells for in vivo and/or in vitro studies.
[0065] In another embodiment of the present invention, a novel gene delivery system in which an adenovirus is complexed with a cationic transfection reagent (e.g., lipid, polyamine or dendrimer) and a plasmid encoding the luciferase gene has been used to achieve high levels of transient gene expression lasting several weeks without permanent modification of the transfected cells with the bioluminescent luciferase. References 7 and 8 discuss the details of enhancement of cationic liposome-mediated gene transfer using replication - deficient adenovirus. References 9, 10 and 11 also discuss applications of this technology.
Cells for Detection and Stem Cell Differentiation
[0066] The present methods and materials are useful in detecting and/or monitoring many different types of cells. As indicated above, these methods and materials are particularly advantageous for use with stem cells and/or other progenitor cells.
[0067] Stem cells are attractive as therapeutic candidates because of their pluripotent or even totipotent potential. As they multiply in culture they tend to lose their plasticity. Transduction of stem cells with lentiviral vectors expressing Gaussia luciferase results in integration of the Gaussia luciferase gene into the genome of the host cell thus circumventing the need for making a stable cell line. The Targefect plus Virofect gene delivery system and the Profect protein delivery systems described in the present study have the advantage of efficient transient delivery into stem cells without the need for making a recombinant virus. Gene expression from transient transfection persists for at least a week and can be exploited for differentiation or expansion of stem cells by transfecting appropriate genes and/or proteins.
[0068] Thus, in addition to a variety of other applications, stem cells expressing Gaussia luciferase can be very useful in stem cell research because they can now be transduced with a second gene causing either cell expansion or differentiation into a specific phenotype, and the success of the technology can be studied by non-invasive bioluminescent imaging of the target organ.
[0069] This approach is generally applicable to any type of stem cells originating from any animal (e.g., human) and any organ/blood/amniotic fluid source. It is also applicable to progenitor cells from any source. Examples of cell expansion factors that can be transfected either as gene or proteins include trancription factors such as the Hox protein or peptides (31). This approach can also be used to study the fate of normal cells such as hepatocytes, muscle cells or endothelial cells that have been genetically transduced ex vivo and then transplanted back into the recipient. Transduction of stem cells with genes expressing specific transcription factors or the corresponding protein or peptide factors can also be useful in mediating differentiation of human stem cells into specific cell lineages such as pancreatic cells, muscle cells or neural cells. Such differentiation can, if desired, be monitored or tracked using a Gaussia luciferase or other luciferase as described herein.
[0070] Additional embodiments of the present application clearly show that direct intracellular delivery of functionally active key transcription factors can be used as a powerful tool enabling directed differentiation of multipotent stem cells into a specific lineage, with or without the use of luciferase for tracking.
[0071] For example, transfection of adult mesenchymal stem cells transfected with the transcription factor, MyoD, was found to result in differentiation of stem cells into myoblasts 2 weeks following transfection. MyoD is a transcription factor that has been documented in several studies to play an important role in the differentiation of stem cells into skeletal muscle cells.
[0072] Recent studies (e.g., Di Rocco et al, 2006) have shown that Adipose- tissue-derived mesenchymal stem cells can be directed towards a myogenic phenotype in vitro by the addition of specific inductive media. Conversion of adipose-tissue-derived cells to a myogenic phenotype is enhanced by co-culture
with primary myoblasts in the absence of cell contact and is maximal when the two cell types are co-cultured in the same plate. Conversely, in vitro expanded adipose-tissue derived mesenchymal stem cells required direct contact with muscle cells to generate skeletal myotubes. The latter study also showed that uncultured adipose-tissue-associated cells have a high regenerative capacity in vivo since they can be incorporated into muscle fibers following ischemia and can restore significantly dystrophin expression in mdx mice. More recently, studies by Sun et al, 2007 showed that the transcription factor MyoD plays an important role in the differentiation of mesenchymal stem cells into skeletal muscle cells. This study showed that in vitro, the rat MSCs could be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs could be increased, with a higher degree of the differentiation into the skeletal muscle.
[0073] Other evidence supporting an important role of Myo D comes from the studies of Xiu et al, 2007. In this study, stably transfected NIH3T3-derived cell lines were established, in which exogenous MyoD was expressed at high levels. Transcriptional activation of endogenous muscle regulatory gene and induction towards the skeletal muscle lineages were observed with phase-contrast microscopy when continuously cultured in vitro. Moreover, to determine their myogenic formation ability in vivo, the transfected cells were implanted in nude mice subcutaneously for up to 10 weeks. Myogenesis of fibroblasts incubated in the medium was activated by overexpression of MyoD, and the cells were accumulated and fused into multinucleated myotubes. Correlatively, RT-PCR and immunohistochemistry confirmed the increased expression of characteristic downstream molecule myogenin and mysion heavy chains during myogenic differentiation. Ecoptic myogenesis was found and remained the stable phenotype when the transfected cells were seeded in vivo. Our results indicate that MyoD is a determining factor of myogenic lineages, and it may play an important role in the cell therapy and cell-mediated gene therapy of the skeletal muscle.
[0074] The present invention describes a simple way of differentiating adipose tissue-derived mesenchymal stem cells into skeletal muscle cells by transfection
of Ad-HMSCs with recombinant MyoD, e.g., using the Protect P1 reagent. Ad- HMScs at passage 2 were transfected with Myo D and cultured for 2 weeks in mesenchymal stem cell culture media (containging approx 3% serum). After two weeks the cells were observed to be developing a skeletal muscle cell-type morphology. At the end of the third week the Ad-HMSCs assumed the typical elongated multinucleated skeletal muscle-type morphology and stained positive for the skeletal muscle markers (skeletal muscle troponin, myogenin and myosin heavy chain. Images of results of MyoD transfection are shown in Fig. 10 for differentiation of Ad-HSCs into skeletal myobalsts two weeks following transfection with MyoD. Panels A-B: Ad-HMCs (genetically transduced with a lentivirus expressing Gaussia luciferase and GFP) and then transfected with MyoD using Profect P-1. The cells start differentiating within 2 weeks following transfection with MyoD. The morphology of the cells (visualized by fluorescence microscopy because of their ability to express GFP) clearly indicates a myogenic phenotype which is further confirmed by staining for skeletal muscle troponin as shown below. Control cells (transfected with histone using Profect P1) maintained the normal morphology of Ad-HMSCs (Panel C).
[0075] As another example, differentiation into pancreatic Beta cells is associated with the expression of specific transcription factors. Five different genes have been identified including those encoding the tissue-specific transcription factors expressed in pancreatic β-cells, i.e. HNF-4alpha (MODY1), HNF-1 alpha (MODY3), IPF-1 (also known as IDX-1 and PDX-1) (MODY4), and HNF-1β (MODY5). (B). Of these, the homeodomain transcription factor IDX-1 has been the most well characterized, both in its crucial role in pancreatic development and in the development of the diabetic state. IDX-1 is critical for the development of the pancreas. Homozygosity for an inactivating mutation in IDX-1 results in pancreatic agenesis in a child and ldx-1-null mice also have pancreatic agenesis. IDX-1 transactivates several genes essential for the differentiated β-cell phenotype, and is important for glucose sensing and metabolism in the β-cell, including insulin, glucose-transporter-2 (GLUT-2), and glucokinase. More recently, the elegant studies of Yoshida et al have shown that IDX-1 can induce β-cell- specific gene expressions in some non-β-cells such as intestinal epithelial cells
and may therefore be useful for future diabetes gene/cell therapy. Further evidence linking the potency of IDX-1 in driving β-cell like differentiation in non β- cells comes from the studies of Yoshida et al. These investigators showed that transfection of epithelial cells with IDX-1 induces the expression of β-cell specific genes such as Nkx6.1 , amylin, glucokinase and insulin.
[0076] Thus, in initial experiments, transfection of adult mesenchymal stem cells (derived from adipose tissue (Ad-HMSCs) with the transcription factor IDX-1 (also known as PDX-1) was found to result in differentiation of Ad-HMSCs into pancreatic beta cells (based in part on positive staining for insulin C peptide). Insulin C peptide which results from cleavage of proinsulin by pancreatic Beta cells is the most distinctive marker for identification of pancreatic beta cells. The differentiation of Ad-HMSCs into pancreatic beta cells was observed at approximately 2 weeks after transfection with the IDX-1 (PDX-1) recombinant protein. The results demonstrated that a large fraction (at least 70%) of Ad- HMSCs differentiate into the pancreatic Beta cell type approximately two weeks after transfection with the PDX-1 (IDX-1) protein. Further, transfection of Ad- HMSCs with the PDX-1 transcription factor was also found to result in a 4-fold increase in growth rate of Ad-HMSCs 3 days following transfection with PDX-1 protein.
[0077] Protein delivery was performed using the Profect P-1 reagent (Targeting Systems/Pluristem Innovations) according to the manufacturer's protocols. Efficient intracellular delivery of PDX-1 into Ad-HMSCs was ascertained by immunostaining for PDX-1. PDX-1 was found to persist in the transfected cells for at least one week after transfection. It is believed that the transcription factor PDX-1 mediates differentiation into pancreatic Beta cells by initiating a cascade of events activating other factors that eventually results in commitment of the cells into the pancreatic Beta cell type. Once the cells are committed they will not revert back to the undifferentiated state. Results of the test are shown in Fig. 9, where for Panels A and B, Ad-HMSCs were transfected with recombinant PDX-1 using the Profect P-1 reagent and then two weeks later examined for differentiation into pancreatic beta cells by immunostaining for staining for insuin
C peptide (Panels A and B). Panel C shows control cells transfected with histone
using the Protect P1 reagents and stained for insulin C peptide. Panel D shows control cells stained for PDX-1. For Panel E, Ad-HMSCs were transfected with recombinant PDX-1 protein using Profect P1 and immunostained for PDX-1 using an anti PDX-1 antibody
[0078] Thus transfection of stem cells with either an expression vector encoding IDX-1 or the IDX-1 protein itself can mediate differentiation of stem cells into pancreatic Beta cells, with or without Gaussia luciferase-expression. Protein delivery is thus a very powerful and safe tool for directed differentiation of stem cells into a specific lineage as it does not cause any permanent alteration to the cells in other ways, unlike gene transfer methods that involve recombinant viruses. Using the Profect reagents, it is also possible to control the level of intracellular protein delivery by manipulating the concentration of protein used during complex formation, thereby providing an added advantage of the system.
[0079] Further, transfection of recombinant HOX B4 protein into hematopoietic stem cells should result in marked increase in the growth rate of stem cells as determined by the secreted Gaussia luciferase assay.
[0080] Still further, our initial results also indicate that transfection of Ad-HMSCs with telomerase (as recombinant protein) results in differentiation of Ad-HMSCs into osteoblasts as identified by immunostaining for BMP-2 (Bone morphogenic protein 2), a bone specific marker.
[0081] Similarly transfection of Ad-HMSCs with recombinant Neuro D or Neurogenein 3 should result in differentiation into neuron-like cells 2-3 weeks after transfection.
[0082] Such results and consideration of prior work demonstrates that transfection of adult or fetal mesenchymal stem cells with other transcription factors such as those listed in TABLE 1 can result in directed differentiation of adult or fetal mesenchymal stem cells into several lineages.
[0083] The HLH transcription factors NeuroD and Neurogenin can mediate differentiation of mesenchymal stem cells into neuronal cells. The transcription
factors neuronal helix-loop-helix protein (NEX)/mammalian atonal homolog 2 (Math-2), BETA2/neuronal determination factor (NeuroD), and NeuroD-related factor (NDRF)/NeuroD2 comprise a family of Drosophila atonal-related basic helix- loop-helix (bHLH) proteins with highly overlapping expression in the developing forebrain. Recent studies (72) have shown that injection of mRNA encoding BETA2/NeuroD and NDRF into Xenopus oocytes was found to convert ectodermal cells into neurons suggesting that they play an important role in specifying neuronal cell fate. The recent studies of Schwab et al, 2000 (72) showed that mice lacking BETA2/NeuroD, and in NEX*BETA2/NeuroD had developmental defects in the brain demonstrating that bHLH proteins are required in vivo for terminal neuronal differentiation. The primary developmental arrest appears to be restricted to granule cells in which an autoregulatory system involving all three neuronal bHLH genes has failed. More recently studies by Sun et al (74) showed that in addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis.. Taken together thse studies indicate that regulated expression of NeuroD and Neurogenin in mesenchymal stem cells would direct differentiation in neurons, and can be used in the present invention.
[0084] Likewise, the transcription factor Gata4 can mediate differentiation of mesenchymal cells into cardia myocytes. The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. A recent study by Oka et al, 2006, ref 73) showed that deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. The results of this study showed that cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Thus, GAT A4 is a necessary regulator of
cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability, and can be used in the present invention.
[0085] The present invention also applies to transfection of active small peptides (or sequences encoding such small peptides) into stem cells. Certain such peptides can mediate differentiation of stem cells into specific phenotypes, and thus can also be performed using the present compositions and methods. Recently, Glucagon-like peptide-1 (GLP-1) an incretin hormone derived from proglucagon gene, has been identified as an inducer of IDX-1 expression. Also, GLP-1 administered to diabetic mice was found to stimulate insulin secretion and effectively lower their blood sugar levels. GLP-1 also enhances β-cell neogenesis and islet size. The increase on insulin secretion by GLP-1 has been shown to be mediated by an I DX-1 -dependant transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. GLP-1 is also capable of regulation the transcription of three genes that determine the pancreatic β-cell- specific phenotype: insulin, GLUT-2, and glucokinase. Thus transfection of either the gene expressing GLIP-1 or transfection of the GLIP-1 peptide itself can mediate differentiation of human stem cells into pancreatic Beta cells. Of course, this can also be applied to other peptides possessing differentiation-mediating activity.
Expansion of hematopoietic stem cells by Intracellular delivery of functionally active key regulatory transcription factors
[0086] In addition to cell differention, the present invention can be used in cell expansion. As described in references 48, 49, 50, 52. 53, the homeobox transcription factor HOX B4 plays a key role in the expansion and self renewal of hematopoietic stem cells. These results are further supported by a published study which shows that transfection of CD 34 positive hematopoietic stem cells with HOX decoy peptides also results in expansion of CD34 positive stem cells (reference 31)
[0087] Our observation on the effect of direct protein delivery on expansion of hematopoietic stem cells is supported by several earlier studies cited below:
[0088] Recent studies (48) suggest that homeobox-containing transcription factors, particularly HOXB4, and Notch signaling provide 2 important regulatory mechanisms that control proliferation and self-renewal of hematopoietic stem cells. These pathways may be manipulated to develop protocols for expansion of stem cells HOXB4 has attracted particular interest during the last few years because its gene transfer induced -40-fold murine and -30-fold human HSC expansion ex vivo, suggesting possible clinical applications [ 48,50]. Regarding clinical use, there was an initial concern that constitutive expression of HOXB4 in HSCs might cause leukemia. This is because deregulated expression of HOXB8 was found in myeloid leukemia, and HOX family genes are sometimes involved in leukemogenic chromosomal translocations, yielding NUP98-HOXA9 [51) . However, HSCs expanded by HOXB4 treatment reconstituted all hematopoietic lineages in mice that received transplants mice without causing leukemia, indicating that HSCs expressing HOXB4 were regulated by the hematopoietic system [48). To eliminate any deleterious effects caused by stable HOXB4 gene transfer, Krosl et al. tried to expand murine HSCs by delivering HOXB4 protein [49]. In the latter study, cell membrane-permeable, recombinant TAT-HOXB4 protein was added to the culture medium, inducing a fivefold net expansion of HSCs. HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was comparable to that induced by the human HOXB4 retrovirus during a similar period of observation. Although TAT- HOXB4 was supposed to be delivered with high efficiency, its half-life was estimated to be only 1 hour. Also the TAT protein has many undesirable side effect such as activating transcription of host cell genes such as the gene for transforming growth factor alpha and epidermal growth factor (reference 86).
[0089] Amsellem et al. tried to expand human CB HSCs using HOXB4 protein [52]. They used HOXB4 protein secreted into the culture supernatant from cocultured MS-5 murine stromal cells, and this approach increased NOD/SCID mouse repopulating cells (SRCs) 2.5-fold. However, the efficiency of protein delivery was not very high, and the coculture system may not be practical for clinical applications. In contrast, the paper by Tanaka et al (31) showed that the
peptide decHOX could be delivered into more than 70% of CB CD34+ hHSC/HPCs and was detected in these cells even after 4 days. After 7 days of culture in serum-free medium containing a cytokine cocktail, cultures treated with decHOX (HOX decoy peptides) had approximately twofold-increased numbers of CD34+ cells and primitive multipotent progenitor cells compared with control cells. Furthermore, decHOX-treated cells reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficiency mice more rapidly and more effectively (more than twofold greater efficiency, as determined by a limiting dilution method) than control cells. DecHOX-treated cells were also able to repopulate secondary recipients. The studies by Tanaka et al there showed that in combination with growth factors and/or other approaches, decHOX might be a useful new tool for the ex vivo expansion of hematopoietic stem/progenitor cells.
[0090] A recent study (Schiedlmeier et al, 2007, reference 53) identified a set of overlapping genes that likely represent "universal" targets of HOXB4. HOXB4 influences the expression of genes involved in pivotal cell-intrinsic pathways such as regulation of cell cycle, differentiation, and apoptosis. It also modulates the response to multiple conserved extrinsic signals provided by the microenvironment (see Figure 2 in the Schiedlmeier paper). HOXB4 mediates expansion not only of bone marrow -derived hematopoietic stem cells but also embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically (53).
[0091] In summary, the studies cited above (48-50) have shown a 30-fold expansion in human hematopoietic following gene transfer of the HOX B4 protein.. HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was somewhat comparable to that induced by the human HOXB4 retrovirus during a similar period of observation. Based on these earlier studies, transfection of CD34 positive hematopoietic stem cells with recombinant HOX-4 protein using Profect P1 will also result in an approximately 20-30 fold expansion of hematopoietic stem cells over a period of 10 days
[0092] Delivery of recombinant HOX B4 has advantages over gene delivery approaches as it does not cause any permanent genetic modification of the cells. Also delivery of recombinant HX protein using protein delivery reagents such as Profect P1 or Profect P-2 (Targeting Systems, Santee, CA), does not require any modification of the HOX protein and proteins delivery using these reagents causes minimal toxicity to the cells. In fact the study by Tanaka et al (31) used the Profect reagent to deliver HOX peptides into hematopoietic stem cells.
[0093] In keeping with the examples described above, the following table lists a number of functionally active proteins and peptides which can be used with the present invention. In most cases, this table summarizes the role of key regulatory proteins in directing differentiation of stem cells into a specific lineage.
TABLE-1 : Differentiation of fetal or adult mesenchymal/embryonic stem cells into specific lineages by intracellular delivery of functionally active proteins:
Luciferase
[0094] Gaussia princeps luciferase is the smallest known luciferase and is over 1000 times brighter than the existing firefly and renilla luciferases. Gaussia luciferase uses coelenterazine as the substrate. In the presence of coelenterazine and oxygen a bright blue bioluminescence is observed. In the present invention several approaches have been taken for bioluminescent imaging of stem cells (as well as other cells) using this novel luciferase.
[0095] Since Gaussia luciferase is efficiently expressed intracellular^ as well as secreted into the medium, it is now possible to quantitate luciferase activity in the blood at different time intervals without killing the animal. Since factors such as serum in blood can affect Gaussia luciferase activity, specialized reagents are used for measuring Gaussia luciferase activity in the blood samples. The Gaussia luciferase assay reagents described in the patent application can be used for quantitaive measurement of Gaussia luciferase activity in blood samples. Since the luciferase activity is directly proportional to the number of stem cells, it provides an indication of cell growth after transplantation. Stem cells expressing Gaussia luciferase can be very useful in high throughput assays to screen for different compounds that induce stem cell expansion.
[0096] This technology also enables one to use Gaussia luciferase as an in vivo reporter to study real time regulation of gene expression in stem cells in vivo by transfecting constructs that express Gaussia luciferase under control of the promoter of interest into stem cells. This would not be possible using the firefly and renilla luciferases as they are expressed intracellular^.
10097] All embodiments of the present invention are also applicable to Metridia luciferase, another secreted luciferase that has a high degree of homology (approx 90%) to Gaussia luciferase and shows almost identical sequence at the active site. However Gaussia luciferase is approximately 1000/times brighter than Metridia luciferase (Metridia luciferase is only about 5 times brighter than firefly and Renilla luciferase (reference 17, Markova et al)
Second Reporter Molecules
[0098] In addition to the inclusion of an appropriate luciferase, a second reporter molecule can be encoded in a vector used to transfect (or co-transfect) cells, and especially stem cells. In many cases, such second reporter will be a fluorescent protein such as green fluorescent protein or red fluorescent protein. Such second reporter can encoded in a separate vector molecule (which may be the same or different from the type of vector in which the luciferase is encoded), or may be encoded in the same vector molecule. When encoded in the same vector molecule, the second reported may be encoded in the same or different open reading frames, e.g., using separate promoters in the case of different open reading frames or one promoter and an IRES in the case of a single open reading frame. Second reporter molecules could be useful to study effects of turning on certain genes on the expansion of stem cells in vivo. Thus one can simultaneously study the consequences of upregulation or downregualtion of the promoter of the gene of interest on stem cell survival, differentiation or expansion using the fluorescence intensity as a measure of promoter activity and Gaussia luciferase based chemiluminescence as an indicator of cell location and division. Second reporter plasmids expressing siRNA directed against specific cellular genes can also be co-transfected with vectors expressing Gaussia luciferase to study effects of silencing of specific target genes on stem cell expansion, cell survival and/or cell differentiation in vivo
Transduction of stem cells with lentiviral vectors expressing Gaussia luciferase alone or expressing both Gaussia luciferase and a fluorescent protein or other second reporter:
[0099] In one embodiment of the present invention human stem cells have been infected with a lentiviral vector expressing Gaussia luciferase alone under
control of the CMV promoter or expressing both Gaussia luciferase (e.g., under control of the CMV promoter) and a second reporter protein such as GFP (e.g., under control of an IRES). In a test, and as shown in Fig. 1 almost 100% of the mesenchymal stem cells were transduced with the lentiviral vector and expressed both GFP (green fluorescent protein) and Gaussia luciferase. The test was carried out by transduction of mesenchymal stem cells with a lentiviral vector encoding a green fluorescent protein (under control of an IRES) and Gaussia luciferase (under control of the CMV promoter).The cells were transduced using 50 lentiviral particles per cell. The genetically transduced stem cells upon subcutaneous injection into mice can be visualized using coelenterazine (see, e.g., Fig. 3 and Fig. 4), the substrate for Gaussia luciferase. The present invention thus provides an approach for non-invasive bioluminescent imaging of stem cells using a novel secreted reporter Gaussia luciferase.
Transient transfection of stem cells
[00100] A plasmid vector expressing Gaussia luciferase, e.g., under control of the CMV promoter, can also be delivered into stem cells.
[00101] Thus, for example, such plasmid vector can be efficiently delivered into mesenchymal stem cells using an appropriate transfection system, e.g., the Targefect F-1 or Targefect F-2 transfection reagent in combination with the Virofect enhancer, all from Targeting Systems, Santee, CA.
[00102] Transfection with such a vector and transfection system provides transient expression of the luciferase, allowing tracking and/or other monitoring of the cells without permanent modification of the cells.
Protein Delivery of Reporter Molecules
[00103] While delivery of vectors encoding one or more reporter molecules provides highly advantageous methods for detecting and monitoring cells, in some cases it is beneficial to deliver protein reporter molecules directly, e.g., for transient detection.
[00104] Generally, such protein reporter molecule delivery can be accomplished using commercially available protein delivery reagents. The Profect P-1 and Profect P2 reagents from Targeting Systems have been used to successfully deliver proteins into mesenchymal and hematopoietic human stem cells (see Fig. 2 on histone delivery into mesenchymal stem cells and paper on delivery of HOX peptides into hematopoietic stem cells, reference 31 )
[00105] For example, transfection of the Gaussia luciferase protein into stem cells has been accomplished using the Profect P1 and Profect P2 reagents from Targeting Systems. The delivered protein is functionally active for about 3-4 days after transfection making this technique useful for those situations where transient visualization of implanted cells is desirable without any genetic modification of the cells. ( reference 31)
[00106] The experiments described in this application have been performed using either human cord blood and bone marrow derived mescenchymal stem cells and CD34 positive stem cells and should be applicable to any stem cell or other cell type.
Application of Gaussia luciferase as a reporter to study gene silencing in human stem cells:
[00107] The psiScreen vector has a multiple cloning site after the stop codon of the Gaussia luciferase gene. (See Fig. 4)
[00108] The psiScreen Vectors are designed to provide a rapid, quantitative approach for evaluation and optimization RNA interference (RNAi). These vectors enable monitoring of changes in expression of a target gene fused to a novel luciferase reporter gene. In both vectors, Gaussia luciferase is used as a primary reporter gene, and the target gene of interest can be cloned into multiple cloning sites located downstream of the translational stop codon of the luciferase gene. Transfection of cells with the psiScreen vectors results in the production of an mRNA in which the mRNA encoding Gaussia luciferase is expressed as a fusion with the mRNA encoding the target gene. Initiation of the gene silencing occurs when co-transfection of si RNA towards the target gene of interest results in
cleavage and subsequent degradation of fusion mRNA. Measurement of decreased Gaussia luciferase activity serves as an indicator of RNA interference. Since the Target gene is sub-cloned after the stop codon of Gaussia luciferase, the activity of Gaussia luciferase is unaffected by the fusion partner.
Advantages of the psiScreen system:
[00109] The psiScreen system provides an easy method to screen a wide variety of siRNAs for gene silencing. The effectiveness of different siRNAs to silence the target gene of interest is evaluated quantitatively simply by measuring the luciferase activity.Gaussia luciferase is a secreted into the media. It is therefore necessary to only assay cell supernatants for luciferase activity without the need for lysing the cells. Considerable time is saved since time course experiments can be performed using the same group of transfected cells without lysing at each time point.
[00110] Gaussia luciferase, a thermostable enzyme, is 1000 times brighter than Renilla and firefly Luciferase thus increasing sensitivity of the assay.
Use of Gaussia Luciferase as a sensor for detection intracellular protease activity.
[00111] Protein cleavage is a central event in many regulated biological processes. As another aspect of the invention, a system is provided for detecting intracellular proteolysis using a positive readout assay based on increased secretion of a reporter polypeptide Gaussia luciferase (GLUC) following enzymatic cleavage of a protease cleavage site placed between a secretion inhibitor polypeptide (preferably a second reporter molecule such as a green fluorescent protein (GFP)) and GLUC fused to the C terminal of the secretion inhibitor polypeptide (e.g., GFP). The assay is based on the principle that N-terminal modification of a secreted reporter such as Gaussia luciferase with a polypeptide such as green fluorescent protein (GFP) results in marked reduction in secreted Gaussia luciferase activity.
[00112] By engineering constructs which contain a DEVD sequence between the
Gaussia luciferase and GFP, we have observed that active Gaussia luciferase is
released in amounts proportional to the intracellular proteolytic activity thus generating a positive readout of intracellular protease activity without killing the cells. The sensitivity of the assay was further enhanced when constructs containing two (or more) protease cleavage sites flanked by amino acid spacers (e.g., 5 amino acid spacers) were placed between the GFP and the Gaussia luciferase sequence. As an example, a construct expressing a GFP -DEVD- GLUC fusion protein was transfected into HEK-293 cells as a sensor for measuring intracellular caspase activity (induced by addition of doxyrubicin, an inducer of apoptosis to the cells). The GFP used in this construct was the Ptilosarcus GFP from Nanolight Technologies, AZ.
[00113] While many assays for proteolysis use fluorescent or luminescent reporters such as green fluorescent protein (GFP), secreted alkaline phosphatase (SEAP) or Photinus luciferase, secreted luciferases offer many advantages over cellular reporter enzymes as they can be non-destructively harvested from cellular supernatants over time. Several secreted luciferases have been reported, from the marine copepods Gaussia phnceps[76], and Metridia longa[77], the ostracod Vargula hilgendorfii[78]]. In addition, intracellular luciferases such as from the sea pansy Renilla reniformis can be engineered to be secreted and stable in the extracellular milieu[81]. Of these reporters Gaussia Luciferase offers a luminous output much higher than traditional bioluminescent proteins allowing for an ultra sensitive reporter of cell death through caspase secretion. By fusing the end terminal of the Gaussia gene with GFP and DEVD the Gaussia activity is greatly reduced. Then when apoptosis occurs, the DEVD site is cleaved and the GLUC is reactivated allowing for a quantitative measure of cell death over time.
[00114] The sequence of an exemplary plasmid useful for expressing a chimeric construct useful for this approach is shown in Table 2.
EXAMPLES
Example 1 - Transduction of cultured human mesenchymal stem cells with a recombinant lentiviral vector expressing Gaussia luciferase
[00115] Human mesenchymal stem cells were isolated from the bone marrow and grown in mesenchymal stem cell culture media provided by Cell Applications Inc, San Diego. The human mesenchymal stem cells were transduced with a lentiviral vector encoding a green fluorescent protein (under control of an IRES) and Gaussia luciferase (under control of the CMV promoter). The cells were transduced using 50 lentiviral particles per cell. As shown in Fig. 1 , the transfection efficiency was almost 100%.
Example 2 - Delivery of functionally active proteins into human stem cells using Profect
[00116] A variety of proteins can be efficiently delivered into human stem cells using Profect reagents (reference 31). Alexa 488-conjugated histone which contains a nuclear localizaition signal was used aa a reporter protein to demonstrate the efficancy of intracellular protein delivery using Porfect P-1 and Profect P-2 reagents. Both commercially available from Targetign Systems, Santee, CA. Fluorescent histone is a good reporter for assessing efficient delivery of functionally active proteins because if the protein is functionally intact then it translocates to the nucleus because of the nuclear localization signal. The nuclear location of the fluorescent histone can be assessed by fluorescence microscopy after staining cells with the DAPI stain for nucleaur visualization. 5 μg of Alexa-488-conjugated histone was complexed with 5 μl of the Profect transfection reagent in 0.5 ml of high glucose medium, incubated for 20 minutes at room temperature to form transfection complexes and then added to the cells according to the manufacturer's protocols. The cell culture medium was aspirated from the cells prior to addition of transfection complexes. The cells were incubated with the histone-Profect transfection complexes at 37 0C overnight and then washed three times with serum free DMEM (Dulbecco's modified Eagles's medium, stained with DAPI (a fluorescent stain that stains the nuclei deep blue and then visualized using fluorescence microscopy. As shown in Fig. 2, the transfection efficiency approximates 75% . Cells transfected with functionally active Alexa-499 histone appear bright blue due to co-localization of the Alexa488- Hisotne (green fluorescence) with the DAPI nuclear stain (deep blure fluorescence.
Example 3 - Visualization of implanted stem cells in a mice using Bioluminescent Imaging
[00117] HMSC cells were infected with a lentivirus vector carrying the expression cassette for Glue under the control of CMV promoter. 4 days after infection, 2 million of these cells were implanted subcutaneously with Matrigel in nude mice. Mice were injected i.v. with 100 μg of coelenterazine, prepared by diluting it in methanol to a concentration of 5 μg/μl, than 20 μl_ was added to 130 μl_ of PBS and all was i.v. injected. Mice were imaged at different time points using a CCD camera and photon counts were acquired over five minutes. The sum of photon counts/min is plotted in Fig. 3B. Images are displayed as a pseudo-color photon count image, superimposed on a grayscale anatomic white light image, allowing assessment of both bioluminescent intensity and its anatomical source. As shown in Fig. 3A the implanted stem cells can be easily visualized and cell growth tracked in real time using a CCD camera without killing the animal. Further it is possible to quantitate the Gaussia luciferase activity in the blood, e.g., using the GAR B2 reagents from Targeting Systems.
Example 4 - Evaluation of the psiScreen system for screening si RNAs against the human tumor suppressor p53 gene:
[00118] In order to evaluate Guassia luciferase as a reporter for studying gene silencing in vitro an experiment was designed to evaluate several siRNAs against the human tumor suppressor p53 gene. Different siRNAs against human p53 gene were synthesized and evaluated for their ability to silence the p53 gene by co-transfection with a siScreen vector in which the p53 coding sequence was subcloned after the stop codon. As expected siRNA sequences with a high score showed much more effective gene silencing. The data plotted shows results after normalization for transfection efficiency using a firefly luciferase expression vector pCMV-Fluc as a denominator plasmid.
[00119] In connection with the screening of different siRNAs (small interfering RNAs) against p53 using the psiScreen system, Fig. 6 shows ability of different siRNAs for silencing of the target gene (human tumor suppressor p53 gene). siRNA 1 and siRNA 2 represent 23 bp synthetic siRNAs with different sequences designed to silence the human tumor suppressor p53 gene. HEK-293 cells (1
million) were co-transfected with the psiScreen vector (1 μg) containing the p53 gene cloned into the multiple cloning site after the stop codon of the luciferase gene and 100 pmols of an unrelated siRNA (control, green bar) or 100 pmols of a mixed siRNA pool against the p53 gene purchased from New England Biolabs (NEB) or 100 pmols of synthetic siRNAs (oligo 1 and oligo 2)synthesized by Dharmacon, USA. The co-transfection experiments were performed using the Targefect F-2 reagent from Targeting Systems according to the manufacturer's recommended protocol. The data has been normalized for transfection efficiency using a firefly luciferase expression vector as a control plasmid. Unrelated siRNA was shown to be ineffective in gene silencing (data not shown)
[00120] Fig. 7 shows dose response of siRNA for gene silencing of the target gene (human tumor suppressor p53 gene) in HEL-293 cells. The data has been normalized for transfection efficiency using a firefly luciferase expression vector as a control plasmid. Unrelated siRNA was shown to be ineffective in gene silencing (data not shown)
Example 5 - Use of Gaussia luciferase as a reporter gene to study gene silencing in human mesenchymal stem cells:
[00121] Primary human mesenchymal stem cells were co-transfected with a psiScreen plasmid vector and siRNA directed against p53 or against Gaussia luciferase using the Targefect F-2 reagent according to the manufacturer's protocol. The cell supernatants and lysates were assayed for Gaussia luciferase activity 48 hrs post transfection to assess the efficiency of gene silencing. Oligo 5 is siRNA against Gaussia luciferase; Oligo 7 is siRNA against p53; Cy3 is Cy3- labeled negative control siRNA. The figures described below show the effect of these different siRNAs on luciferase activity in supernatants and cell lysates from the transfected cells. Gaussia luciferase activity in supernatants and cell lysates was measured using the GAR-1 reagent from Targeting Systems.
[00122] FIG. 8 A, 8B show the results of screening of different siRNAs (small interfering siRNAs) against p53 using the psiScreen system in supernatants (Fig. 8A) and cell lysates (Fig. 8B) of human mesenchymal stem cells.
[00123] The results of this experiment show the strong potential of the Gaussia luciferase-based siScreen system to study real time gene silencing in stem cells in vitro. These results in combination with the in vivo experiments for visualization of a small number of implanted stem cells using Gaussia luciferase also suggest the applicability of Gaussia luciferase to study gene silencing in vivo in real time. The easiest way to perform such experiments is to co-transfect a small number of stem cells genetically transduced to express Gaussia luciferase with siRNA against a target gene and then implant them into an animal in vivo. Implantation of as few as 2 million stem cells genetically modified with Gaussia luciferase can be visualized using an appropriate CCD camera. Transfection of Gaussia lcuiferase- expressing stem cells with siRNA against a given target gene enables one to track the effects of silencing the target gene on the fate of the implanted stem cells using in vivo bioluminescent imaging techniques. For instance if silencing a particular gene leads to expansion of stem cells in vivo then this can be quantitatively assessed by assaying Gaussia luciferase activity in the blood. The GAR B2 reagent enables one to quantitatively measure Gaussia luciferase in the blood. Also differentiation and homing of stem cells to a particular location following gene silencing can be assessed by BLI (bioluminescent imaging techniques). The brightness of Gaussia luciferase provides significant advantages over firefly and renilla luciferase for such applications
[00124] Use of Gaussia luciferase as a reporter for high throughput screening (HTS) of siRNA libraries targeted against genes whose silencing may lead to stem cell expansion :
[00125] Stem cells transduced with lentiviral vectors to express Gaussia luciferase would be very useful in screening siRNA libraries for their ability to mediate stem cell expansion by silencing key target genes. In such screening assays stem cells transfected with siRNAs that silence target genes to mediate stem cell growth would show increased secreted Gaussia luciferase activity compared to control cells. The level of luciferase activity would directly co-relate to the effectiveness of a given siRNA to mediate stem cell expansion for instance Ex vivo targeting of p21 gene has been shown to permit relative expansion of human hematopoietic stem cells. ( reference 30).
Example 6 - Composition of exemplary assay reagent (GAR-BL2) for measuring Gaussia luciferase activity in the blood:
[00126] Many substances in the blood can interfere with the Gaussia luciferase assay. The following assay buffers have been specially formulated to measure
Gaussia luciferase activity in the blood:
4OuM -10OuM coelenterazine, 0.02 % NP40, 1X PBS
40 uM-100 uM coelenterazine, 0.02% NP40, 1X PBS1 1% EDTA
[00127] The above formulations with inclusion of 2% NP40 instead of 0.02 % NP40 result in a marked improvement in stability of the Bioluminescent signal. Increased coelenterazine concentration results in an increase in the sensitivity of the bioluminescent signal.
[00128] The compositions with EDTA give lower activity but improved stability of the bioluminescent signal
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References for differentiation into skeletal muscle cells
69. Giuliana Di Rocco1'*, Maria Grazia lachininoto2, Alessandra Tritarelli2, Stefania Straino2, Antonella Zacheo2, Antonia Germani1, Filippo Crea3 and Maurizio C. Capogrossi2'*' (2006) Myogenic potential of adipose-tissue-derived cells . Journal of Cell Science 119, 2945-2952 (2006)
70. In vitro differentiation of rat mesenchymal stem cells into skeletal muscle cells induced by myoblast differentiation factor and 5-azacytidine]
Z Sun, Z Chen, and B Wang. Chinese Journal of reparative and reconstructive surgery. 2007 Dec;21(12):1371-5
71. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, December 1, 2007; 21(12): 1371-5 Regulation of skeletal muscle differentiation in fibroblasts by exogenous MyoD gene in vitro and in vivo.RF Qin, TQ Mao, XM Gu, KJ Hu, YP Liu, JW Chen, and X Nie MoI Cell Biochem, August 1, 2007; 302(1-2): 233-9.
References citing role of neuroD and neurogenin in directing differentiationinto neuronal cells
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Differentiation of mesenchymal cells into cardia myocytes:
73. Toru Oka, Marjorie Maillet, Alistair J. Watt, Robert J. Schwartz, Bruce J. Aronow, Stephen A. Duncan, Jeffery D. Molkentin Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability Circulation Research. 2006;98:837. Integrative Physiology
74. Sun Y, Nadal-Vicens M, Misono S1 Lin MZ, Zubiaga A, Hua X, Fan G, Greenberg ME. Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms.Cell. 2001 Feb 9;104(3):365-76.
75λ. Root DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM: Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat Methods 2006, 3:715- 719.
76. Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO: Codon- optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. MoI Ther 2005, 11:435-443.
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80. Nakajima Y, Kobayashi K, Yamagishi K, Enomoto T, Ohmiya Y: cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci Biotechnol Biochem 2004, 68:565-570.
81. Liu J, O'Kane DJ, Escher A: Secretion of functional Renilla reniformis luciferase by mammalian cells. Gene 1997, 203:141-148.
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83. Simonsen JL, Rosada C, Serakinci N, Justesen J, Stenderup K, Rattan Sl, Jensen TG, Kassem M. (2002) Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells.. Nat Biotechnol. 2002 Jun;20(6):592-6.
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85. Jun ES, Lee TH, Cho HH, Suh SY, Jung JS.(2004) Expression of telomerase extends longevity and enhances differentiation in human adipose tissue-derived stromal cells. Cell Physiol Biochem. 2004; 14(4-6):261-8
Table 2: Plasmid map
Molecule: pCMV-hptGFP-DEVD-Gluc, 6486 bps DNA Circular Description: Clone ID 7030612
Molecule Features:
Type Start End Name Description
REGION 209 863 CMVP CMV Promoter
REGION 864 882 T7P T7 Promoter
REGION 889 901 MCS Polylinker
GENE 900 1630 hpt-GFP Humanized pt GFP ORF
GENE 1642 2199 Glue Gaussia Luciferase Gene
REGION 2219 2263 pAl Synthetic pA site
REGION 2235 2252 SP6 P SP6 promoter
REGION 2804 3139 SV40P
REGION 2918 3003 SV40 Ori SV40 Origin or replication
GENE 3175 3969 Neo Neomycin ORF
REGION 4024 4396 SV40 pA
REGION 4656 5329 CoIEl
GENE 5474 6334 Amp Ampicillin resistance gene
1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
901 atgaaccgca acgtgctgaa gaacaccggc ctgaaggaga tcatgagcgc caaggccagc
961 gtggagggca tcgttaacaa ccacgtgttc agcatggagg gcttcggcaa gggcaacgtg
1021 ctgttcggca accaattgat gcagatccgc gtgaccaagg gcggccccct gcccttcgcc
1081 ttcgacatcg tgagcatcgc cttccagtac ggcaaccgta cgttcaccaa gtaccccgac
1141 gacatcgccg actacttcgt gcagagcttc cccgccggct tcttctacga gcgcaacctg
1201 cgcttcgagg acggcgccat cgtggacatc cgcagcgaca tcagcctgga ggacgacaag
1261 ttccactaca aggtggagta ccgcggcaac ggcttcccca gcaacgggcc cgtgatgcag
1321 aaggccatcc tgggcatgga gcccagcttc gaggtggtgt acatgaacag cggcgtgctg
1381 gtgggcgagg tggacctggt gtacaagctt gagagcggca actactacag ctgccacatg
1441 aagaccttct accgttcgaa gggcggcgtg aaggagttcc ccgagtacca cttcatccac
1501 caccgcctcg agaagaccta cgtggaggag ggcagcttcg tggagcagca cgaggccgcc
1561 atcgcccagc tgaccaccat cggcaagccc ctgggatccc tgcacgagtg ggtggacgaa
1621 gtggacgcag atccagccac catgggagtc aaagttctgt ttgccctgat ctgcatcgct
1681 gtggccgagg ccaagcccac cgagaacaac gaagacttca acatcgtggc cgtggccagc
1741 aacttcgcga ccacggatct cgatgctgac cgcgggaagt tgcccggcaa gaagctgccg 1801 ctggaggtgc tcaaagagat ggaagccaat gcccggaaag ctggctgcac caggggctgt 1861 ctgatctgcc tgtcccacat caagtgcacg cccaagatga agaagttcat cccaggacgc 1921 tgccacacct acgaaggcga caaagagtcc gcacagggcg gcataggcga ggcgatcgtc 1981 gacattcctg agattcctgg gttcaaggac ttggagccca tggagcagtt catcgcacag 2041 gtcgatctgt gtgtggactg cacaactggc tgcctcaaag ggcttgccaa cgtgcagtgt 2101 tctgacctgc tcaagaagtg gctgccgcaa cgctgtgcga cctttgccag caagatccag 2161 ggccaggtgg acaagatcaa gggggccggt ggtgactaag cggccgctcg agcatgcatc 2221 tagaaataat tcttactgtc atgccaagta agatgctttt ctgtgctgca atagcaggca 2281 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 2341 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2401 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 2461 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg 2521 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2581 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2641 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 2701 ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta 2761 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 2821 ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 2881 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 2941 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3001 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3061 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 3121 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg 3181 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga 3241 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt 3301 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct 3361 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg 3421 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt 3481 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc 3541 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc 3601 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga 3661 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg 3721 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat 3781 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg 3841 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc 3901 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta 3961 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg 4021 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 4081 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 4141 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4201 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 4261 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 4321 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 4381 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 4441 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 4501 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 4561 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 4621 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 4681 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 4741 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 4801 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 4861 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 4921 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 4981 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 5041 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5101 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5161 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 5221 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 5281 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
5341 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 5401 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 5461 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 5521 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 5581 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 5641 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5701 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 5761 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 5821 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 5881 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 5941 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 6001 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 6061 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 6121 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 6181 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 6241 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 6301 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 6361 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 6421 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 6481 gacgtc
A second Construct was made identical to the one above except with the addition of Glycine spacers and a second DEVD site. The sequence of the spacers and DEVD is PCMV-GFP-(GLYHSER-DEVD-[GLYHSER-DEVD-[GLYHSER-GIUC
[00129] All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.
[00130] One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.
[00131] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein
without departing from the scope and spirit of the invention. For example, variations can be made to by using other types of stem cells or other cells, by using Metridia luciferase instead of Gaussia luciferase, and/or the detection method may be varied. Thus, such additional embodiments are within the scope of the present invention and the following claims.
[00132] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
[00133] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
[00134] Also, unless indicated to the contrary, where various numerical values or value range endpoints are provided for embodiments, additional embodiments are described by taking any 2 different values as the endpoints of a range or by taking two different range endpoints from specified ranges as the endpoints of an additional range. Such ranges are also within the scope of the described invention. Further, specification of a numerical range including values greater than one includes specific description of each integer value within that range.
[00135] Thus, additional embodiments are within the scope of the invention and within the following claims.
Claims
1. A method for monitoring stem cells in vivo in an animal, comprising detecting luminescence from a luciferase expressed in stem cells or progenitor cells transfected with a vector expressing a secreted luciferase.
2. The method of claim 1 , wherein said secreted luciferase is Gaussia luciferase or a derivative thereof.
3. The method of claim 1 , wherein said secreted luciferase is a Metridia luciferase or a derivative thereof.
4. The method of claim 1 , wherein said detecting comprises detecting said luminescence from luciferase in blood.
5. The method of claim 1 , wherein said stem cells are human mesenchymal stem cells.
6. The method of claim 1 , wherein said stem cells are CD34 positive hematopoietic stem cells.
7. The method of claim 1 , wherein said stem cells are neural stem cells.
8. The method of claim 1 , wherein said stem cells are mammalian stem cells.
9. The method of claim 1 , wherein said cells are progenitor cells.
10. The method of claim 1 , wherein expression of said luciferase is under the control of a CMV promoter.
11. The method of claim 1 , wherein expression of said luciferase is under the control of an HIV promoter.
12. The method of claim 1 , wherein expression of said luciferase is under the control of an SV40 promoter.
13. The method of claim 1 , wherein said detecting comprises quantifying light emitted by reaction of said luciferase.
14. The method of claim 13, wherein said quantification comprises use of a charge coupled device (CCD) detector.
15. The method of claim 1 , wherein said vector further comprises an expressible fluorescent protein.
16. The method of claim 15, wherein said fluorescent protein is under the control of an internal ribosome entry site (IRES).
17. The method of claim 15, wherein said fluorescent protein comprises a green fluorescent protein.
18. The method of claim 15, wherein said fluorescent protein comprises a red fluorescent protein.
19. The method of claim 15, further comprising detecting fluorescence from said fluorescent protein.
20. The method of claim 19, wherein detecting fluorescence from said fluorescent protein comprises quantifying light emitted by reaction of said fluorescent protein.
21. The method of claim 1 , wherein said detecting is performed in a mammal.
22. The method of claim 1 , wherein said cells are also transfected with a vector expressing one or more proteins regulating or mediating stem cell expansion or differentiation.
23. The method of claim 22, wherein said vector expressing said luciferase and said vector expressing said proteins regulating or mediating stem cell expansion or differentiation are the same vector.
24. The method of claim 22, wherein said vector expressing said luciferase and said vector expressing said proteins regulating or mediating stem cell expansion or differentiation are the same vector.
25. The method of claim 1 , wherein said cell is transfected using a cationic transfection reagent.
26. The method of claim 25, wherein said cationic transfection reagent comprises a cationic lipid, a cationic polyamine, or a dendrimer.
27. The method of claim 26, wherein said transfection is enhanced using a replication deficient adenovirus.
28. The method of claim 1, wherein said cell is also transfected with a second plasmid expressing one or more proteins that regulate stem cell expansion or one or more proteins mediating differentiation of stem cells into a specific phenotype.
29. The method of claim 1 , wherein the cell is transfected with a construct expressing both Gaussia luciferase under control of the CMV promoter or other strong promoter and a target gene coding sequence subcloned after the stop codon of the Gaussia luciferase gene; and an siRNA directed against said target gene.
30. The method of claim 1 , wherein said vector is a lentiviral vector.
31. The method of claim 1 , wherein said vector is a plasmid vector.
32. A non-invasive method for detecting expression of a heterologous gene in a selected cell type, comprising transfecting said selected cell type with a vector comprising an expressable secreted luciferase, wherein delivery of said plasmid vector is effected by complexing said plasmid vector with a cationic transfection agent and a replication-deficient adenovirus; and detecting the expression of said luciferase.
33. The method of claim 32, wherein said cell is transfected using a cationic transfection reagent.
34. The method of claim 33, wherein said cationic transfection reagent comprises a cationic lipid, a cationic polyamine, or a dendrimer.
35. The method of claim 32, wherein said cell is also transfected with a second plasmid expressing one or more proteins that regulate stem cell expansion or one or more proteins mediating differentiation of stem cells into a specific phenotype.
36. A method for transient monitoring of a cell, comprising transfecting said cell with a luciferase protein; and detecting the presence of said luciferase.
37. The method of claim 36, wherein said luciferase protein is a Gaussia luciferase protein.
38. The method of claim 36, wherein said luciferase protein is a Metridia luciferase protein.
39. The method of claim 36, wherein said luciferase is detected within said cell.
40. The method of claim 36, wherein said luciferase is detected outside said cell.
41. The method of claim 36, wherein said luciferase is detected in the blood of an animal.
42. The method of claim 36, wherein said transfecting is performed ex vivo, said cells are implanted in an animal, and said detecting is performed following said implantation.
43. The method of claim 36, wherein said cell is transfected using a cationic transfection reagent.
44. A method for visualization of stem cells, comprising transfecting stem cells with a lentiviral vector comprising a sequence encoding Gaussia luciferase as a reporter gene in a under control of the CMV promoter; and detecting the presence of said luciferase.
45. The method of claim 44, wherein said detection is performed in vitro.
46. The method of claim 45, wherein said transfection is performed using a transfection complex comprising a plasmid vector expressing Gaussia luciferase as a reporter gene under control of the CMV promoter, a lipid or non-lipid cationic transfection reagent, and a replication-deficient adenovirus.
47. The method of claim 45, wherein said transfection is performed using a transfection complex comprising a plasmid vector expressing Gaussia luciferase as a reporter gene under control of the CMV promoter, a lipid or non-lipid cationic transfection reagent, and a lentivirus.
48. The method of claim 44, wherein said detection is performed in vivo.
49. The method of claim 48, wherein said transfection is performed using a transfection complex comprising a plasmid vector expressing Gaussia luciferase as a reporter gene under control of the CMV promoter, a lipid or non-lipid cationic transfection reagent, and a replication-deficient adenovirus.
50. The method of claim 48, wherein said transfection is performed using a transfection complex comprising a plasmid vector expressing Gaussia luciferase as a reporter gene under control of the CMV promoter, a lipid or non-lipid cationic transfection reagent, and a lentivirus.
51. A method for determining survival of transplanted stem cells in vivo in an animal, comprising measuring luciferase activity in the blood of said animal from a secreted luciferase expressed from said stem cells.
52. The method of claim 51 , wherein said luciferase activity is from a Gaussia luciferase.
53. The method of claim 51 , wherein said luciferase activity is from a Metridia luciferase.
54. A method for quantitatively detecting expansion of transplanted stem cells in vivo in an animal, comprising measuring luciferase activity in the blood of said animal, wherein said luciferase activity is from a secreted luciferase expressed in said stem cells.
55. The method of claim 54, wherein said luciferase activity is from a Gaussia luciferase.
56. The method of claim 54, wherein said luciferase activity is from a Metridia luciferase.
57. A method of detecting migration and localization of transplanted stem cells, comprising detecting luciferase activity from a Gaussia luciferase in said cells.
58. The method of claim 57, wherein said cells are transfected with a vector expressing said luciferase.
59. The method of claim 57, wherein said cells are transfected with Gaussia luciferase protein.
60. A method for studying gene regulation in stem cells, comprising determining the expression of Gaussia luciferase in cells comprising a Gaussia luciferase coding sequence under the control of a promoter for a gene of interest.
61. The method of claim 60, wherein said promoter regulates expression of a transcription factor or regulatory protein involved in stem cell differentiation.
62. A method for monitoring gene silencing in vivo, comprising determining luciferase activity from cells expressing a Gaussia luciferase co-regulated with a target gene, wherein said cells are also transfected with a vector expressing a siRNA targeting said target gene.
63. A method for causing stem cell differentiation or expansion, comprising transfecting stem cells with a regulatory protein.
64. The method of claim 63, wherein said transfection results in detectable transient persistence of said regulatory protein in said cells for 4 to 14 days.
65. The method of claim 63, wherein stem cells are mesenchymal stem cells and said regulatory protein is selected from the group consisting of
66. A chimeric polypeptide comprising a first domain comprising a bioluminescent polypeptide (GFP or any fluorescent protein); a second domain comprising a secreted chemiluminescent peptide; and an endogenous protease cleavage motif positioned between said first and second domains.
67. The chimeric polypeptide of claim 66, wherein said secreted chemiluminescent peptide is a secreted luciferase.
68. The chimeric polypeptide of claim 67, wherein said secreted luciferase is selected from the group consisting of Gaussia luciferase, Metridia luciferase, Pleuromama luciferase, and Cypridina luciferease.
69. A purified polypeptide characterized as having Gaussia luciferase activity and a recognition site specifically cleavable by a protease, wherein cleavage results in a increase in luciferase activity and wherein said recognition site is a peptide sequence selected from the group consisting of DEVD, LEHD , IETD, VEHD, LETD, IEPD, DETD, WEHD, YVAD , VEID, and any combination thereof.
70. The purified polypeptide of claim 69, wherein the protease is a caspase- family protease, a matrix metalloproteinase, or a serine protease.
71. The purified polypeptide of claim 70, wherein the caspase-family protease is selected from the group consisting of a Caspase-3, a Caspase-6, a Caspase- 8, and a Caspase-9.
72. The chimeric polypeptide of claim 69, wherein the endogenous protease cleavage motif is specifically cleaved by an endogenous cellular protease.
73. The chimeric polypeptide of claim69, wherein the endogenous protease cleavage recognition motif comprises a PACE/furin cleavage recognition motif.
74. A method for detecting intracellular protease activity, comprising detecting extracellular chemiluminescent activity from a secreted chemiluminescent peptide from cells expressing a polypeptide of any of claims 69-73.
75. The method of claim 74, wherein said polypeptide is a GFP-protease cleavage site-GLUC polypeptide.
76. The method of claim 74, wherein said protease activity is detected from in vivo cells.
77. The method of claim 74, wherein detection of said protease activity is indicative of apoptosis.
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