US20080025957A1 - Stem Cells Suitable for Transplantation, their Preparation and Pharmaceutical Compositions Comprising Them - Google Patents

Stem Cells Suitable for Transplantation, their Preparation and Pharmaceutical Compositions Comprising Them Download PDF

Info

Publication number
US20080025957A1
US20080025957A1 US10/578,291 US57829104A US2008025957A1 US 20080025957 A1 US20080025957 A1 US 20080025957A1 US 57829104 A US57829104 A US 57829104A US 2008025957 A1 US2008025957 A1 US 2008025957A1
Authority
US
United States
Prior art keywords
cells
cxcr4
stem cells
population
sdf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/578,291
Other languages
English (en)
Inventor
Tsvee Lapidot
Joy Kahn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yeda Research and Development Co Ltd
Original Assignee
Yeda Research and Development Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yeda Research and Development Co Ltd filed Critical Yeda Research and Development Co Ltd
Assigned to YEDA RESEARCH AND DEVLOPMENT CO. LTD. reassignment YEDA RESEARCH AND DEVLOPMENT CO. LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KAHN, JOY, LAPIDOT, TSVEE
Publication of US20080025957A1 publication Critical patent/US20080025957A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to stem cells suitable for transplantation and to methods for their preparation.
  • HSC transplantation procedures mimic the physiological process of HSC migration from the circulation into the bone marrow (BM) occurring during late embryonic development and steady state hematopoiesis in adults throughout life 1-3 .
  • Gene transfer into human HSC may serve as a promising tool in the correction of a wide variety of hematopoietic and genetic disorders.
  • HSC transplantation can be used to durably deliver these genetically modified cells to the BM, which in turn will release mature cells with the corrected gene into the circulation throughout life.
  • Enhanced efficacy of stem cell engraftment could improve the outcome of clinical transplantations as well as gene therapy protocols, and might be achieved by modulating the ability of stem cells to home to and repopulate the recipient BM. For this purpose, a better understanding of the mechanisms, which regulate these processes, is necessary.
  • CXCR4 expression is a dynamic process, which is regulated by environmental factors such as cytokines, chemokines, stromal cells, adhesion molecules, and proteolytic enzymes 18 .
  • CXCR4 can be upregulated from intracellular pools by short term ( ⁇ 40 hr) in vitro cytokine culture 13,19 or stimulation of cord blood (CB) CD34 + with proteolytic enzymes such as MMP-2 and MMP-9 20 . This subsequently enhances their in vitro migration towards an SDF-1 gradient 13 as well as their in vivo homing and repopulation capacities in transplanted NOD/SCID and serially transplanted ⁇ 2 mnull NOD/SCID mice 12,13 , linking stem cell self renewal and development with motility.
  • SDF-1/CXCR4 interactions are also involved in retention of stem and progenitor cells in the BM 10,32,33 .
  • This hypothesis has also been confirmed by other studies which demonstrated the involvement of SDF-1/CXCR4 interactions in the anchorage of human HSC injected directly into the murine BM cavity 34,35 . Interference of these interactions induces release/mobilization of both human and murine progenitors from the BM into the circulation 36-41 .
  • Lentiviral vectors have been used to introduce transgenes into SCID repopulating cells (SRCs) 43-46 , due to their unique ability to transduce non-dividing cells 47 . Furthermore, a significant clinical breakthrough in gene therapy was made in patients with human severe combined immunodeficiency (SCID)-X1 resulting in full correction of disease phenotype 48,49 , proving that gene therapy can work in practice. However, emerging evidence exists for impaired homing 8 and low engraftment 9 of retrovirally-transduced human CD34 + cells.
  • LTCIC long-term culture initiating cells
  • LTC-IC Long-term culture-initiating cells
  • CFU colony-forming unit-cells
  • BM bone marrow
  • the invention relates to a method for preparing a population of cells comprising stem cells with a high amount of immature primitive progenitors, the method comprising collecting stem cells and introducing into the cells a DNA fragment comprising the sequence of CXCR4.
  • the population of cells exhibits improved CXCR4 signaling in response to low concentration of SDF-1.
  • the population of cells exhibits improved CXCR4 signaling in response to high concentration of SDF-1.
  • the stem cells are hematopoietic stem cells, preferably, CD34+ enriched.
  • the immature primitive progenitors are of the CD34+/CD38 ⁇ /low lineage.
  • the collection of the stem cells is effected after a stem cell mobilization procedure; and/or after a surgical procedure.
  • stem cells having CXCR4 levels above a predetermined threshold are isolated e.g. by FACS.
  • the stem cells of the invention are capable of differentiating towards the myeloid and erythroid lineages.
  • the amount of the immature primitive progenitors of the CD34+/CD38 ⁇ /low lineage are about 1-5% of the population.
  • the amount of immature primitive progenitors of the CD34+/CD38 ⁇ /low lineage are equal to or higher than about 3% of the stem cells.
  • the low concentration of SDF-1 is equal to or lower than about 50 ng/ml.
  • improved signaling is manifested by enhancement of cell migration mediated by low concentrations of SDF-1.
  • improved signaling is manifested by enhancement of cell proliferation mediated by low concentrations of SDF-1.
  • the high concentration of SDF-1 is equal to or higher than about 1 microgram/ml.
  • improved signaling is manifested by a reduction in desensitization by SDF-1.
  • the invention provides a population of cells comprising stem cells comprising a high amount of immature primitive progenitors, and exhibiting improved CXCR4 signaling capability in response to low and/or high concentrations of SDF-1, prepared by introducing to the stem cells a DNA fragment comprising the CXCR4 sequence.
  • the stem cells are hematopoietic stem cells.
  • the population of cells is capable of differentiating towards the myeloid and erythroid lineages.
  • the hematopoietic stem cells are CD34+ enriched stem cells.
  • the immature primitive progenitors are of the CD34+/CD38 ⁇ /low lineage.
  • the amount of CD34+/CD38 ⁇ /low is about 1-5% of the population.
  • the amount of CD34+/CD38 ⁇ /low is about and above 3% of the population.
  • the low concentration of SDF-1 is about and below 50 ng/ml.
  • the high concentration of SDF-1 is equal to or higher than about 1 microgram/ml.
  • the invention provides the use of the population of cells of the invention in the manufacture of a medicament for increasing homing of stem cells to a target tissue in a subject in need.
  • the invention provides the use of the population of cells of the invention in the manufacture of a medicament for increasing repopulation of cells to a target tissue in a subject in need.
  • the target tissue is selected from the group consisting of bone marrow, blood vessel, heart, lung, liver, pancreas, kidney, nervous system, skin, bone and skeletal muscle.
  • the population of cells of the invention is used to facilitate transplantation.
  • transplantation follows chemotherapy protocols.
  • transplantation is autologous. In another further embodiment of the invention, the transplantation involves mobilization of autologous cells.
  • transplantation is heterologous.
  • transplantation is carried out with mobilized stem cells.
  • the invention provides a method of treating a disorder in a subject, requiring cell or tissue replacement, the method comprising providing to a subject in need thereof a therapeutically effective amount of the population of cells according to the invention.
  • the invention provides a method for preparing a population of cells comprising stem cells exhibiting CXCR4 with intact 6H8 epitope, the method comprises collecting stem cells and introducing to the stem cells a DNA fragment comprising the sequence of CXCR4.
  • the invention provides a population of cells comprising stem cells comprising intact CXCR4 6H8 epitope prepared by introducing to the stem cells a DNA fragment comprising the sequence of CXCR4, and use of the population of cells in the manufacture of a medicament for transplantation in a subject in need.
  • An additional aspect of the invention relates to a method for treating a disorder requiring cell or tissue replacement, the method comprises providing to a subject in need thereof a therapeutically effective amount of a population of cells according to the invention.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a population of cells comprising stem cells exhibiting intact CXCR4 6H8 epitope prepared by introducing to stem cells a DNA fragment comprising the sequence of CXCR4.
  • FIG. 1 shows a schematic representation of the lentiviral vector constructs. Only the relevant portions of the integrated provirus are depicted.
  • the EF1- ⁇ promoter is used to drive expression of either green fluorescent protein (GFP) cDNA in the control vector (upper panel) or the CXCR4-IRES-GFP bicistronic cassette of the experimental vector (lower panel).
  • GFP green fluorescent protein
  • SA splice acceptor
  • pA polyadenylation signal
  • SIN self-inactivating vector.
  • FIG. 2 shows cell surface CXCR4 expression on lentiviral-transduced human CD34+ cells.
  • CB cord blood
  • MPB mobilized peripheral blood
  • CD34 + cells were analyzed by flow cytometry for either GFP expression alone or GFP together with CXCR4 expression using an anti-hCXCR4-PE antibody.
  • A. Data show a representative FACS analysis from CB CD34 + cells. Numbers indicate percent of total CD34 + cells. Forward scattered (FSC) indicative of the cell size.
  • B. Results indicate percentage of CB and MPB CD34 + cells expressing CXCR4 and represent mean ⁇ SE of 7 independent experiments. * p ⁇ 001 compared to control GFP-infected cells
  • C. Immunofluorescence detection of cell surface (upper panel) and intracellular (lower panel) CXCR4 expression of GFP transduced (GFP), CXCR4 transduced (CXCR4) or isotype control cells.
  • FIG. 3 shows clonogenic progenitor content of transduced CB CD34+ cells.
  • Lentiviral transduced CB CD34 + cells were seeded in semisolid methylcellulose culture.
  • Upper panel indicates BFU-E (a) and CFU-GM (b) colonies from control vector transduced cells.
  • Lower panel indicates BFU-E (c) and CFU-GM (d) from CXCR4 transduced cells.
  • a representative experiment is shown.
  • B. Data indicate total number of CFU-GM and BFU-E colonies. Numbers above bars indicate percentage of GFP+ colonies out of total colonies. Bars represent mean ⁇ SE of three independent experiments.
  • CB and MPB CD34+ cells were labeled with human anti-CD34 and anti-CD38 mAbs. Numbers indicate percent positive cells from entire population. A representative experiment out of 3 is shown.
  • FIG.4 shows functionality of CXCR4 expressed on transduced CD34+ cells.
  • A. CXCR4-transduced CB CD34 + cells were stimulated with SDF-1 (300 ng/ml) for the indicated times and intracellular F-actin content was measured by FACS. Data indicates fold increase in F-actin content following stimulation with SDF-1 compared to unstimulated cells. Data represent mean ⁇ SE of three independent experiments.
  • B. CXCR4-transduced CB and MPB CD34+ cells were tested in a transwell migration assay for their migration towards different SDF-1 concentrations as indicated. Data indicate percent migrating cells to SDF-1. Bars represent mean ⁇ SE of five independent experiments.
  • CB *p ⁇ 0.04
  • MPB *p ⁇ 0.05
  • MPB *p ⁇ 0.05
  • FIG. 5 shows response of CXCR4-overexpressing CB CD34+ cells to different SDF-1 concentrations.
  • A Lentiviral transduced CB CD34 + cells were incubated for seven days in serum free conditions with SDF-1 (50 ng/ml) in combination with SCF (50 ng/ml), FLT-3L (50 ng/ml) and IL-6 (50 ng/ml). Results are shown as fold increase in number of viable cells compared to cells incubated in the absence of SDF-1. Results represent mean ⁇ SE of three independent experiments performed in duplicate.
  • B Lentiviral transduced CB CD34 + cells were incubated for seven days in serum free conditions with SDF-1 (50 ng/ml) in combination with SCF (50 ng/ml), FLT-3L (50 ng/ml) and IL-6 (50 ng/ml). Results are shown as fold increase in number of viable cells compared to cells incubated in the absence of SDF-1. Results represent mean ⁇ SE of three independent
  • Lentiviral transduced CB CD34 + cells were incubated overnight with 1 ⁇ g/ml SDF-1 and examined for (i) CXCR4 expression by immunostaining and (ii) SDF-1 (125 ng/ml) induced in vitro migration using a transwell migration system. Bars represent mean ⁇ SE of two independent experiments performed in duplicate. *p ⁇ 0.05 compared to untreated cells (black bars).
  • FIG. 6 shows in vivo multilineage reconstitution and GFP expression of CXCR4 overexpressing SCID repopulating cells (SRC).
  • CXCR4-transduced CB CD34+ cells were injected into sublethally irradiated NOD/SCID mice. Five weeks post transplantation mice were examined for the presence of human repopulating cells when compared to control GFP-transduced cells.
  • A. Murine BM (i), spleen (ii) and peripheral blood (PB) (iii) were analyzed for human cell engraftment by FACS analysis detecting % of human CD45+ cells. Bars represent mean ⁇ SE of nine independent experiments performed in duplicate or triplicate. *p ⁇ 0.005 compared to control GFP-transduced cells.
  • B. Lymphoid and myeloid differentiation of human SRC in a representative NOD/SCID transplant recipient is shown by CD19 and CD33 antibody staining respectively. Numbers indicate percent of positive cells out of total live population.
  • FIG. 7 shows a schematic illustration of CXCR4.
  • FIG. 8 shows that CXCR4 overexpression on CB CD34+ cells prevents deterioration of the 6H8 epitope in vivo.
  • B Representative histogram FACS analysis of the mean CXCR4 fluorescence as in A following staining of GFP (control) and CXCR4 overexpressing cells with 12G5 and 6H8 mAbs.
  • the present invention relates to stem cells enriched with immature primitive progenitors, and having improved CXCR4 signaling in response to low and high concentration of SDF-1, and to methods of generating and using the same. More specifically, the cells of the invention are capable of responding to low concentrations of SDF-1 and are less desensitized by high concentrations of SDF-1. Specifically, the present invention allows treatment of disorders requiring cell or tissue replacement. The present invention is based on results demonstrating that transgenic stem cells overexpressing CXCR4 exhibit enhanced levels of CD34+/CD38 ⁇ /low cell population and/or exhibit improved CXCR4 signaling capability in response to low and high concentration of SDF-1 and exhibit intact CXCR4 6H8 epitope.
  • HSC Hematopoietic stem cell
  • chondrocyte therapy for cartilage defects
  • neuronal cell therapy for neurodegenerative diseases
  • stem cell therapy for numerous applications [Forbes (2002) Clinical Science 103:355-369].
  • One of the problems associated with stem cell therapy is the difficulty of achieving long-term successful engraftment of cells at the target tissue.
  • patients who were successfully transplanted exhibit very low levels of stem cells and immature progenitors, which generate cells with the desired phenotype.
  • CD34 + progenitor cells Treatment of CD34 + progenitor cells with cytokines such as HGF and MMPs has been previously shown to upregulate expression of CXCR4 and exhibit better response to SDF-1. However, under increasing concentrations of SDF-1, CXCR4 is internalized and the cells are less responsive to SDF-1. Also, increasing the levels of SDF-1 induces desensitization and quiescence of proliferating human long term culture initiating cells (LTCIC) and primitive human fetal liver CD34 + stem cells capable of serial repopulation of transplanted NOD/SCID mice 30,31 .
  • LTCIC long term culture initiating cells
  • stem cells capable of serial repopulation of transplanted NOD/SCID mice 30,31 .
  • One of the problems of BM transplantation is the reduced levels of long lasting immature progenitors (1 log reduction) in the BM of transplanted patients compared to healthy individuals 4-7 .
  • transgenic hematopoietic stem cells overexpressing CXCR4 show unexpected high levels of the CD34+/CD38 ⁇ /low cell population.
  • transgenic stem cells such as CB and MBP CD34+ enriched cells
  • transduced cells are 87 ⁇ 2.7% (CB) and 80 ⁇ 4% (MPB) positive for cell surface CXCR4 expression whereas only 28 ⁇ 3.1% of both CB and MPB CD34 + cells infected with the GFP vector expressed endogenous CXCR4.
  • transgenic cells overexpressing CXCR4 are not affected in their ability to differentiate towards the myeloid and erythroid lineages.
  • transduced CD34 + cells both control and CXCR4
  • BFU-E burst-forming unit-erythroid
  • CFU-GM colony-forming unit-granulocyte, macrophage
  • MPB CD34+ CXCR4 transduced cells demonstrated a higher percentage (2.9%) of CD34+/CD38 ⁇ /low population compared to 0.5% in control cells. This effect was not observed in CB CD34+ cells ( FIG. 3C ). CXCR4 transduction therefore support grow and/or preservation of the CD34+/CD38 ⁇ /low primitive population.
  • CXCR4 transduced stem cells show more intact CXCR4 6H8 epitope in the surface of the stem cells.
  • CXCR4-transduced CB CD34+ cells had almost doubled their seeded number, while control cell number, had decreased to below the original amount seeded.
  • a representative FACS staining with CD19 and CD33 monoclonal antibodies demonstrates that multilineage hematopoiesis into lymphoid and myeloid populations respectively was maintained in transduced cells ( FIG. 6B ) with a trend to more B-cell lymphopoiesis in mice transplanted with CXCR4-transduced cells ( FIG. 6C ), most probably since SDF-1 is also a Pre B cell growth factor.
  • an average of 36% ⁇ 19% (range 7.5% to 77%) of the CD45 cells were found to express GFP ( FIG.
  • the present findings enable the generation of stem cells, which can be efficiently recruited to a target tissue and repopulate it, and as such can be used in numerous clinical applications, such as in repair of liver injury and in liver or bone marrow transplantation.
  • a method to get “extended long-term culture-initiating cells” or ELTC-IC or of increasing the primitive CD34 + /CD38 ⁇ /low cell population According to another aspect of the present invention there is provided a method to get ELTC-IC cells capable to respond to SDF-1 even when exposed to increased SDF-1 concentrations.
  • stem cells refers to cells, which are capable of differentiating into other cell types having a particular, specialized function (i.e., “fully differentiated” cells).
  • transgenic cell is a cell carrying an introduced gene or segment.
  • Non-limiting examples of stem cells which can be used according to this aspect of the present invention, are hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) obtained from bone marrow tissue of an individual at any age or from cord blood of a newborn individual, embryonic stem (ES) cells obtained from the embryonic tissue formed after gestation (e.g., blastocyst), or embryonic germ (EG) cells obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation.
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • ES embryonic stem
  • EG embryonic germ
  • HSCs Hematopoietic stem cells
  • fetal liver fetal liver
  • umbilical cord blood fetal cord blood
  • bone marrow and peripheral blood which are capable of differentiating into any of the specific types of hematopoietic or blood cells, such as erythrocytes, lymphocytes, macrophages and megakaryocytes.
  • HSCs typically reside in niches that support all the requisite factors and adhesive properties to maintain their ability and produce an appropriate balanced output of mature progeny over the life time of the organism [Whetton (1999) Trends Cell Biol 9:233-238; Weissman (2000) Cell 100:157-168; Jankowska-Wieczorek (2001) Stem Cells 19:99-107; Chan (2001) Br. J. Haematol. 112:541-557].
  • HSCs according to this aspect of the present invention are preferably CD34+ cells and more preferably CD34+/CD38 ⁇ /low cells, which are a more primitive stem cell population and are therefore less lineage-restricted, and were shown to be the major long-term BM repopulating cells.
  • MSCs Mesenchymal stem cells are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into more than one specific type of mesenchymal or connective tissue (i.e. the tissues of the body that support the specialized elements; e.g. adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines.
  • connective tissue i.e. the tissues of the body that support the specialized elements; e.g. adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues
  • MSCs Approximately, 30% of human marrow aspirate cells adhering to plastic are considered as MSCs. These cells can be expanded in vitro and then induced to differentiate. The fact that adult MSCs can be expanded in vitro and stimulated to form bone, cartilage, tendon, muscle or fat cells render them attractive for tissue engineering and gene therapy strategies. In vivo assays have been developed to assay MSC function. MSCs injected into the circulation can integrate into a number of tissues described hereinabove.
  • skeletal and cardiac muscle can be induced by exposure to 5-azacytidine and neuronal differentiation of rat and human MSCs in culture can be induced by exposure to ⁇ -mercaptoethanol, DMSO or butylated hydroxyanisole [Tomita (1999) 100:11247-11256; Woodbury (2000) J. Neurosci. Res. 61:364-370].
  • MSC-derived cells are seen to integrate deep into brain after peripheral injection as well as after direct injection of human MSCs into rat brain; they migrate along pathways used during migration of neural stem cells developmentally, become distributed widely and start lose markers of HSC specialization [Azizi (1998) Proc. Natl. Acad. Sci. USA 95:3908-3913].
  • Methods for promoting mesenchymal stem and lineage-specific cell proliferation are disclosed in U.S. Pat. No. 6,248,587.
  • Epitopes on the surface of the human mesenchymal stem cells such as SH2, SH3 and SH4 described in U.S. Pat. No. 5,486,359 can be used as reagents to screen and capture mesenchymal stem cell population from a heterogeneous cell population, such as exists, for example, in bone marrow.
  • Precursor mesenchymal stem cells which are positive for CD45, are preferably used according to this aspect of the present invention, since these precursor mesenchymal stem cells can differentiate into the various mesenchymal lineages.
  • Preferred stem cells according to this aspect of the present invention are human stem cells.
  • Table 1 below provides examples of adult stem cells, which can be used to obtain the indicated phenotype in a target tissue of interest, according to this aspect of the present invention.
  • stem cells according to this aspect of the present invention are successfully transduced with lentiviral vectors with high expression of the CXCR4 transgene.
  • polypeptide and protein in the present specification are interchangeable.
  • the present invention also concerns muteins of the above CXCR4 protein of the invention, which muteins retain essentially the same biological activity of the CXCR4 protein having essentially only the naturally occurring sequences of the CXCR4.
  • Such “muteins” may be ones in which up to about 20, preferably no more than 10 amino acid residues may be deleted, added or substituted by others in the CXCR4 protein respectively, such that modifications of this kind do not substantially change the biological activity of the protein mutein with respect to the protein itself.
  • muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable thereof.
  • Any such mutein preferably has a sequence of amino acids sufficiently duplicative of that of the basic the CXCR4 such as to have substantially similar activity thereto.
  • Muteins of the CXCR4 protein which can be used in accordance with the present invention, or nucleic acid coding thereof, include a finite set of substantially the CXCR4 corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • Preferred changes for muteins in accordance with the present invention are what are known as “conservative” substitutions.
  • Conservative amino acid substitutions of those in the protein having essentially the naturally-occurring CXCR4 sequences may include synonymous amino acids within a group, which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule, see Grantham, Science, Vol. 185, pp. 862-864 (1974).
  • insertions and deletions of amino acids may also be made in the above-defined sequence without altering its function, particularly if the insertions or deletions only involve a few amino acids, e.g., under 50, and preferably under 20 CXCR4 and do not remove or displace amino acids which are critical to a functional conformation, e.g., cysteine residues, Anfinsen, “Principles That Govern The Folding of Protein Chains”, Science, Vol. 181, pp. 223-230 (1973). Muteins produced by such deletions and/or insertions come within the purview of the present invention.
  • the synonymous amino acid groups are those defined in Table A. More preferably, the synonymous amino acid groups are those defined in Table B; and most preferably the synonymous amino acid groups are those defined in Table C.
  • Amino Acid Synonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met Pro Ala, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met, Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser His His, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp Trp
  • Examples of production of amino acid substitutions in proteins which can be used for obtaining muteins of the protein for use in the present invention include any known method steps, such as presented in US patents RE 33,653, U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Mark et al; U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No. 4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to Chong et al; and U.S. Pat. No. 5,017,691 to Lee et al; and lysine substituted proteins presented in U.S. Pat. No. 4,904,584 (Straw et al).
  • any mutein of the CXCR4 protein for use in the present invention has an amino acid sequence essentially corresponding to that of the above noted CXCR4 protein of the invention.
  • the term “essentially corresponding to” is intended to comprehend muteins with minor changes to the sequence of the basic protein which do not affect the basic characteristics thereof, particularly insofar as its ability to the CXCR4 is concerned.
  • the type of changes which are generally considered to fall within the “essentially corresponding to” language are those which would result from conventional mutagenesis techniques of the DNA encoding the CXCR4 protein of the invention, resulting in a few minor modifications, and screening for the desired activity for example increasing the sensitivity of stem cells to a chemoattractant.
  • the present invention also encompasses CXCR4 variants.
  • a preferred CXCR4 variant are the ones having at least 80% amino acid identity, a more preferred the CXCR4 variant is one having at least 90% identity and a most preferred variant is one having at least 95% identity to CXCR4 amino acid sequence.
  • sequence identity means that the amino acid sequences are compared by alignment according to Hanks and Quinn (1991) with a refinement of low homology regions using the Clustal-X program, which is the Windows interface for the ClustalW multiple sequence alignment program (Thompson et al., 1994).
  • the Clustal-X program is available over the internet at ftp://ftp-igbmc.u-strasbg.fr/pub/clustalx/.
  • this link becomes inactive, those of ordinary skill in the art could find versions of this program at other links using standard internet search techniques without undue experimentation.
  • the most recent version of any program referred herein, as of the effective filing date of the present application is the one, which is used in order to practice the present invention.
  • sequence identity Another method for determining “sequence identity” is the following.
  • the sequences are aligned using Version 9 of the Genetic Computing Group's GDAP (global alignment program), using the default (BLOSUM62) matrix (values ⁇ 4 to +11) with a gap open penalty of ⁇ 12 (for the first null of a gap) and a gap extension penalty of ⁇ 4 (per each additional consecutive null in the gap).
  • percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the claimed sequence.
  • Muteins in accordance with the present invention include those encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA under stringent conditions and which encodes a the CXCR4 protein in accordance with the present invention, comprising essentially all of the naturally-occurring sequences encoding the CXCR4 and sequences which may differ in its nucleotide sequence from the naturally-derived nucleotide sequence by virtue of the degeneracy of the genetic code, i.e., a somewhat different nucleic acid sequence may still code for the same amino acid sequence, due to this degeneracy.
  • hybridization shall include any process by which a strand of nucleic acid joins with complementary strand through a base pairing (Coombs J, 1994, Dictionary of Biotechnology, stokton Press, New York N.Y.). “Amplification” is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach and Dveksler, 1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.).
  • “Stringency” typically occurs in a range from about Tm ⁇ 5° C. 5° C. below the melting temperature of the probe) to about 20° C. to 25° C. below Tm.
  • stringent conditions refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, N.Y., 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
  • stringency conditions are a function of the temperature used in the hybridization experiment, the molarity of the monovalent cations and the percentage of formamide in the hybridization solution.
  • Tm melting temperature
  • Tm 81.5 C+ 16.6(LogM)+0.41(% GC) ⁇ 0.61(% form) ⁇ 500/L
  • % GC is the percentage of G and C nucleotides in the DNA
  • % form is the percentage of formamide in the hybridization solution
  • L is the length of the hybrid in base pairs.
  • highly stringent conditions are those which provide a Tm which is not more than 10 C below the Tm that would exist for a perfect duplex with the target sequence, either as calculated by the above formula or as actually measured.
  • Modely stringent conditions are those, which provide a Tm, which is not more than 20 C below the Tm that would exist for a perfect duplex with the target sequence, either as calculated by the above formula or as actually measured.
  • examples of highly stringent (5-10 C below the calculated or measured Tm of the hybrid) and moderately stringent (15-20 C below the calculated or measured Tm of the hybrid) conditions use a wash solution of 2 ⁇ SSC (standard saline citrate) and 0.5% SDS (sodium dodecyl sulfate) at the appropriate temperature below the calculated Tm of the hybrid.
  • SSC standard saline citrate
  • SDS sodium dodecyl sulfate
  • a common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above is hybridization in a solution of 6 ⁇ SSC (or 6 ⁇ SSPE (standard saline-phosphate-EDTA), 5 ⁇ Denhardt's reagent, 0.5% SDS, 100 & micro; g/ml denatured, fragmented salmon sperm DNA at a temperature approximately 20 to 25 C below the Tm. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel, 1987, 1999).
  • TMAC tetramethyl ammonium chloride
  • Adult stem cells can be obtained using a surgical procedure such as bone marrow aspiration or can be harvested using commercial systems such as those available from Nexell Therapeutics Inc. Irvine, Calif., USA.
  • Stem cells utilized by the present invention are preferably collected (i.e., harvested) using a stem cell mobilization procedure, which utilizes chemotherapy or cytokine stimulation to release of HSCs into circulation of subjects. Stem cells are preferably retrieved using this procedure since mobilization is known to yield more HSCs and progenitor cells than bone marrow surgery.
  • Stem cell mobilization can be induced by a number of molecules.
  • examples include but are not limited to cytokines such as, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-7, IL-3, IL-12, stem cell factor (SCF), and flt-3 ligand; chemokines like IL-8, Mip-1 ⁇ , Gro ⁇ , or SDF-1; and the chemotherapeutic agents cyclophosphamide (Cy) and paclitaxel.
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-7 interleukin
  • IL-3 interleukin-7
  • IL-12 interleukin-12
  • SCF stem cell factor
  • flt-3 ligand chemokines like IL-8, Mip-1
  • G-CSF is preferably used alone or in combination such as with cyclophosphamide to mobilize the stem cells.
  • G-CSF is administered daily at a dose of 5-10 ⁇ g/kg for 5-10 days.
  • Methods of mobilizing stem cells are disclosed in U.S. Pat. Nos. 6,447,766 and 6,162,427.
  • Human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage.
  • the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting.
  • the ICM is then plated in a tissue culture flask containing the appropriate medium, which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated.
  • Human ES cells can be purchased from the NIH human embryonic stem cells registry ( ⁇ http://escr.nih.gov>).
  • Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, and TE32.
  • Human EG cells can be retrieved from the primordial germ cells obtained from human fetuses of about 8-11 weeks of gestation using laboratory techniques known to anyone skilled in the arts. The genital ridges are dissociated and cut into small chunks, which are thereafter disaggregated into cells by mechanical dissociation.
  • the EG cells are then grown in tissue culture flasks with the appropriate medium.
  • the cells are cultured with daily replacement of medium until cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.
  • Shamblott et al. [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.
  • CD34+ stem cells can be concentrated using affinity columns or FACS as further described hereinunder.
  • Culturing of stem cells under proliferative conditions may also be effected in cases where stem cell numbers are too low for use in treatment. Culturing of stem cells is described in U.S. Pat. Nos. 6,511,958, 6,436,704, 6,280,718, 6,258,597, 6,184,035, 6,132,708 and 5,837,5739.
  • stem cells are obtained, they are transfected with DNA comprising the sequence encoding CXCR4 or an active portion thereof.
  • the “transformed, transfected or transgenic” cells are cultured under suitable conditions, which allow the expression of the protein encoded by the polynucleotide or DNA.
  • the stem cells can be transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector comprising the CXCR4 coding sequence; lentiviruses systems are preferably used to express the CXCR4 or the active portion thereof.
  • transformed cells are cultured under effective conditions, which allow for the expression of CXCR4.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective medium refers to any medium in which a cell is cultured to produce the recombinant modified polypeptide of the present invention.
  • Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • the expression construct includes a cis-acting regulatory element active in mammalin cells (examples above), which may be inducible, growth specific or tissue specific conditions.
  • cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., ( 1989 ) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.
  • the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
  • the inducible cis-acting regulatory element is regulatable by changes in the environment of the stem cells during the homing-implantation process.
  • the nucleic acid construct of the present invention can further include an IRES element, preferably EMCV IRES, which can be in between the CXCR4 gene and a marker or selection gene sequence and can function in up regulating the translation of the marker or selection gene.
  • IRES element preferably EMCV IRES
  • Identification and isolation of such cells overexpressing CXCR4 according to this aspect of the present invention can be effected using a number of cytological, biochemical and molecular methods, which are well known in the art.
  • Analysis of receptor level can be effected by flow cytometry.
  • This approach employs instrumentation that scans single cells flowing past excitation sources in a liquid medium.
  • the technology can provide rapid, quantitative, multiparameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission.
  • This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules.
  • To isolate cell populations using fluorescence activated cell sorter stem cells of the present invention are contacted with anti CXCR4 commercially available from R&D, 614 McKinley Place NE Minneapolis, Minn.
  • cytological or biochemical methods for quantitatively assessing the level of the chemotactic receptor expression include but are not limited to binding analysis using a labeled (e.g., radioactively labeled) chemokine, western blot analysis, cell-surface biotinylation and immunofluorescent staining. It will be appreciated that the receptor expression levels can also be determined at the mRNA level. For example, CXCR4 mRNA may be detected in cells by hybridization to a specific probe. Such probes may be cloned DNAs or fragments thereof, RNA, typically made by in-vitro transcription, or oligonucleotide probes, usually generated by solid phase synthesis.
  • Quantification of mRNA levels can be also effected using an amplification reaction [e.g., PCR, “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990)], employing primers, which hybridize specifically to the mRNA of a chemotactic receptor of interest.
  • an amplification reaction e.g., PCR, “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990)
  • samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
  • chemotactic receptor expression can also be used to determine the chemotactic receptor expression.
  • a chemotaxis assay which employs a gradient of the chemotactic agent (e.g., SDF-1) and follows stem cell migration through a membrane towards the chemotactic agent can be utilized to identify and isolate stem cells exhibiting increased chemotaxis. If the cells do not express enough levels of the chemotactic receptor (e.g., CXCR4), then the majority of the cells will remain on the membrane.
  • the chemotactic receptor e.g., CXCR4
  • Stem cells exhibiting improved CXCR4 signaling capability in response to low or high SDF-1 concentrations and/or stem cell exhibiting enhanced levels of immature primitive progenitors, such CD34+/CD38 ⁇ /low population, can be used in a wide range of clinical applications.
  • a method of treating a disorder requiring cell or tissue replacement is effected by providing to a subject in need thereof a therapeutically effective amount of transgenic stem cells overexpressing CXCR4 or an active portion thereof and thereby providing increasing levels of immature primitive progenitors such as CD34+/CD38 ⁇ /low population with better response to SDF-1 even in an environment with increased concentrations of SDF-1, to thereby treat the disorder requiring the cell or tissue replacement in the subject.
  • Disorders requiring cell or tissue replacement include but are not limited to various immunodeficiencies such as in T and/or B-lymphocytes, or immune disorders, such as rheumatoid arthritis.
  • Such immunodeficiencies may be the result of viral infections, HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment; Hematological deficiencies including but not limited to leukemias, such as acute lymphoblastic leukemia (ALL), acute nonlymphoblastic leukemia (ANLL), acute myelocytic leukemia (AML) or chronic myelocytic leukemia (CML).
  • ALL acute lymphoblastic leukemia
  • ANLL acute nonlymphoblastic leukemia
  • AML acute myelocytic leukemia
  • CML chronic myelocytic leukemia
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • XSCID X-linked SCID
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • XSCID X-linked SCID
  • Other disorders requiring cell or tissue replacement include those associated with liver failure, pancretic failure, neurological disorders, those disorders requiring augmented bone formation such as osteoartbritis, osteoporosis, traumatic or pathological conditions involving any of the connective tissues, such as a bone defects, connective tissue defects, skeletal defects or cartilage defects.
  • Preferred individual subjects according to the present invention are mammals such as canines, felines, ovines, porcines, equines, bovines and preferably humans.
  • stem cells according to this aspect of the present invention are preferably obtained from the subject to be treated.
  • stem cells may also be obtained from a syngeneic, allogeneic and less preferably from a xenogeneic donor.
  • the stem cells of the present invention can be genetically modified to express any therapeutic gene such as an antiviral agent against hepatitis further described in U.S. Pat. No. 5,928,638.
  • the stem cells are transplanted into the recipient subject. This is generally effected using methods well known in the art, and usually involves injecting or introducing the treated stem cells into the subject using clinical tools well known by those skilled in the art (U.S. Pat. No. 6,447,765, 6,383,481, 6,143,292, and 6,326,198).
  • introduction of the stem cells of the present invention can be effected locally or systematically via intravascular administration, such as intravenous or intraarterial administration, intraperitoneal administration, and the like.
  • Intravascular administration such as intravenous or intraarterial administration, intraperitoneal administration, and the like.
  • Cells can be injected into a 50 mol Fenwall infusion bag using sterile syringes or other sterile transfer mechanisms.
  • the cells can then be immediately infused via IV administration over a period of time, such as 15 minutes, into a free flow IV line into the patient.
  • additional reagents such as buffers or salts may be added as well.
  • the composition for administration must be formulated, produced and stored according to standard methods complying with proper sterility and stability.
  • Stem cell dosages can be determined according to the prescribed use. In general, in the case of parenteral administration, it is customary to administer from about 0.01 to about 5 million cells per kilogram of recipient body weight. The number of cells used will depend on the weight and condition of the recipient, the number of or frequency of administrations, and other variables known to those of skill in the art.
  • CXCR4 overexpressing system may serve as an effective tool to improve the compromised homing and engraftment following gene therapy protocols 8,9 .
  • CXCR4 overexpresion on human CD34 + cells may facilitate improved clinical CB CD34 + transplantation as well as autologous mobilized PBL CD34 + transplantation following chemotherapy treatment, which can both be limited due to low cell yield 6 .
  • other cell types such as mesenchymal stem cells possess the ability to migrate to various organs, however their levels of engraftment are particularly low.
  • the effect of the treatment may be evaluated, if desired, as known in the art.
  • the treatment may be repeated as needed or required.
  • CXCR4-transduced human CD34 + cells have increased surface CXCR4 expression.
  • CXCR4 was overexpressed on human CB and MPB CD34 + enriched cells using a HIV-derived lentiviral gene transfer system. Transduced cells were analyzed for CXCR4 surface expression.
  • CB Cored blood
  • MPB peripheral blood
  • CD34 + cell enrichment was effected using magnetic bead separation as previously described [Kollet (2001) Blood 97:3283-3291].
  • CXCR4 expression was determined by flow cytometry using purified anti human CXCR4 (clone 12G5, R&D, Minneapolis, Minn.) and secondary F (ab′)2 fragment of goat anti mouse IgG FITC (Jackson, West Grove, Pa.).
  • the human CXCR4 gene lentiviral expression vector was constructed by isolating a 1.2 kb CXCR4 cDNA from human CB cells and linking it to the enhanced green fluorescent protein (GFP) gene via an internal ribosome entry site (IRES).
  • the fragment containing the CXCR4-IRES-GFP was ligated with the EF-1 ⁇ promoter to generate a self-inactivating (SIN) vector where the fragment containing the EF-1 ⁇ -CXCR4-IRES-GFP bicistronic cassette was inserted into a pHR′-SIN vector backbone (Woods et al. Blood. 2000 December 1;96(12):3725-33.) kindly provided by Dr.
  • Replication-defective, self-inactivating HIV-derived lentiviral vector was generated by transient transfection of the 293T packaging cell line by means of FuGENE 6 transfection reagent (Roche Diagnostics, Mannheim Germany), utilizing a three-plasmid system: transfer vector pHR′-EF1 ⁇ -GFP-SIN (control vector) or pHR′-EF1 ⁇ -CXCR4-IRES-GFP-SIN for CD34+ cells or pHR′-CMV-GFP (control vector) or pHR′-CMV-CXCR4-IRES-GFP (experimental vector) for hMSCs, the envelope coding plasmid pMD.G and the packaging construct pCMVR8.91 (Zufferey R, i Nat.
  • CD34 + cells Transduction of CD34 + cells was performed using a double transduction protocol in HSC 50 .
  • CD34 + cells (up to 4 ⁇ 10 5 per well) were pre-stimulated with SCF (50 ng/ml) in 400 ⁇ l serum free medium for 24 hours in a 12-well plate.
  • Viral supernatant (1.6 ml/35 mm well) supplemented with SCF (50 ng/ml), FLT-3L (50 ng/ml) both from R&D Systems (Minneapolis, Minn.), and IL-6 (50 ng/ml; Interpharm Laboratories, Ares-Serono Group, Ness Ziona, Israel) was added to the cells with a viral load as much as 2 ⁇ 10 7 TU/ml (first infection). Transduction was repeated 24 hours later (second infection).
  • Infection efficiency was determined according to cell expression of CXCR4 using specific antihuman CXCR4-PE (12G5, B D Pharmingen, San Diego, Calif.) and GFP (FL1 channel) by flow cytometric analysis (Kollet 2002 blood vol 100 page 2778) (FACSCalibur, Becton Dickinson (BD), San Jose, Calif.) 72 hours post transduction. Mock cells were cultured in the same conditions as transduced cells, without exposure to lentiviral vectors.
  • CB and MPB CD34 + cells showed high transduction efficencies as scored by flow cytometry, reaching 70% GFP positive cells in GFP vector (control) transduced cells and 50% in CXCR4-transduced cells ( FIG. 2A-upper panel). Furthermore, CXCR4-infected CD34 + cells were 87 ⁇ 2.7% (CB) and 80 ⁇ 4% (MPB) positive for cell surface CXCR4 expression whereas only 28 ⁇ 3.1% of both CB and MPB CD34 + cells infected with the GFP vector expressed endogenous CXCR4, resembling levels of non-transduced cells (mock cells) ( FIG. 2A-lower panel (representative) and B).
  • CXCR4 transduced cells showed a higher mean fluorescence intensity (MFI) of 89.4 compared to 10.2 of GFP vector transduced cells ( FIG. 2C ).
  • MFI mean fluorescence intensity
  • FIG. 2C their intracellular CXCR4 expression was lower than their control (GFP) counterparts ( FIG. 2C ).
  • cells transduced with the CXCR4 vector had less GFP+ cells, consistent with earlier reports showing reduced expression levels of genes which are placed downstream from an IRES51.
  • CXCR4-overexpressing human CD34 + cells maintain their in vitro differentiation potential and transgene expression.
  • Stem and progenitor cells are capable in vitro of multilineage differentiation into both myeloid and erythroid lineages when provided with the appropriate cytokine cocktail (Metcalf d recent results in cancer research 1977 vol 61 page 1). We therefore assessed the effect of transgene expression on the ability of transduced cells to differentiate in vitro, in the differentiated lineages.
  • CFU assay In order to detect the levels of human progenitors as well as maintenance of transgene expression following transduction in ex vivo cultures, semisolid cultures were performed as previously described 52 .
  • CB CD34 + transduced cells (3 ⁇ 10 3 cells/ml) were plated in 0.9% methylcellulose (Sigma), 30% FCS, 5 ⁇ 10 -5 M 2 ME, 50 ⁇ ng/ml SCF, 5 ng/ml IL-3, 5 ng/ml GM-CSF (R&D), and 2 u/ml erythropoietin (Orto Bio Tech, Don Mills, Canada). The cultures were incubated at 37° C. in a humidified atmosphere containing 5% CO 2 and scored 14 days later by phase contrast microscopy for GFP+ as well as myeloid or erythroid colonies by morphologic criteria.
  • Transduced CD34 + cells both control and CXCR4
  • BFU-E burst-forming unit-erythroid
  • CFU-GM colony-forming unit-granulocyte, macrophage
  • Chemokines induce cell motility by activating a cascade of intracellular events leading to cytoskeletal arrangements and particularly actin polymerization 54 .
  • As a means for determining functionality of the inserted receptor we examined the effect of CXCR4 overexpression on SDF-1 induced activation of the motility machinery.
  • Actin polymerization assay Transduced cells were stimulated with SDF-1 a (300 ng/ml, Peprotech, Rocky Hill, N.J.) in serum-free RPMI at 37° C. for indicated times. Reaction was stopped by adding 3 volumes of 3.7% paraformaldehyde at RT for 10 min, followed by washing with PBS and permeabilization on ice for 2 min with 0.1% Triton-Hepes (20 mM Hepes, 300 mM sucrose, 50 mM NaCl, 3 mM MgC12, 0.1% Triton). Cells were then stained with FITC-Phalloidin (2 mg/ml, Sigma) for 30 min at RT, washed and analyzed by flow cytometry.
  • SDF-1 a 300 ng/ml, Peprotech, Rocky Hill, N.J.
  • RPMI RPMI (600 ⁇ l) supplemented with 10% FCS containing 125 ng/ml SDF-1 ⁇ were added to the lower chamber of a Costar 24-wells transwell (Corning (pore size 5 ⁇ m), N.Y.). 1 ⁇ 10 5 transduced CD34 cells in 100 ⁇ l medium were loaded to the upper chamber and were allowed to migrate for 4 hours at 37° C. Migrating cells were collected from the lower chamber and counted for 30 seconds using a FACSCalibur. Control spontaneous migration was performed without SDF-1 ⁇ in the lower chamber.
  • CB CD34 + cells exhibited a peak of actin polymerization after 30 s stimulation with SDF-1 ( FIG. 4A ).
  • CXCR4 transduced CD34 + cells are more responsive to low SDF-1 concentrations.
  • RPMI 600 ⁇ l
  • FCS 10% FCS containing either 10 ng/ml or 125 ng/ml SDF-1 ⁇
  • 1 ⁇ 10 5 transduced CD34 cells in 100 ⁇ l medium were loaded to the upper chamber and were allowed to migrate for 4 hours at 37° C.
  • Migrating cells were collected from the lower chamber and counted for 30 seconds using a FACSCalibur. Control spontaneous migration was performed without SDF-1 ⁇ in the lower chamber.
  • Proliferation assay Fluorescence Activated Cell Sorting assay—Following a 96 hour transduction protocol, CB CD34 + cells were cultured for 7 days in duplicates in serum free medium supplemented with SCF 50 ng/ml, FLT-3L 50 ng/ml and IL-6 50 ng/ml in the presence or absence of SDF-1 50 ng/mil. Cells were counted daily and viability was evaluated by trypan blue exclusion.
  • Control cells however, only increased their cell number on day five by up to only 1.2 ⁇ 0.05 fold, and by day 7 their number had decreased to below the original amount seeded ( FIG. 5A ). Notably, this enhanced proliferative effect of CXCR4 overexpressing cells was not detected at higher (100 ng/ml) SDF-1 concentrations (data not shown). Longer time points in culture led to cell differentiation, ruling out the possibility of cellular transformation following lentiviral transduction. Taken together, our results indicate that overexpression of CXCR4 on CD34 + cells, enhances their response to low SDF-1 concentrations, increasing both their motility and proliferation/survival, with a lesser response to high concentrations when compared to control cells.
  • CXCR4 overexpressing cells are less responsive to SDF-1 induced desensitization.
  • CXCR4 cell surface expression CXCR4-transduced CB CD34 + cells were incubated overnight with 1 ⁇ g/ml SDF-1.
  • Cell surface CXCR4 expression was determined by labeling of cells with antihuman CXCR4-PE (12G5, BD Pharmingen, San Diego, Calif.) and analyzed by flow cytometry (FACSCalibur, Becton Dickinson (BD), San Jose, Calif.).
  • RPMI RPMI (600 ⁇ l) supplemented with 10% FCS containing 125 ng/ml SDF-1 ⁇ were added to the lower chamber of a Costar 24-wells transwell (Corning (pore size 5 ⁇ m), N.Y.). 1 ⁇ 10 5 transduced CD34 + cells in 100 ⁇ l medium were loaded to the upper chamber and were allowed to migrate for 4 hours at 37° C. Migrating cells were collected from the lower chamber and counted for 30 seconds using a FACSCalibur. Control spontaneous migration was performed without SDF-1 ⁇ in the lower chamber.
  • CXCR4-transduced CB CD34 + cells were incubated overnight with 1 ⁇ g/ml SDF-1 and analyzed for CXCR4 cell surface expression. Unexpectedly, there was only a 40% (p ⁇ 0.05) decrease in cell surface receptor expression in CXCR4 overexpressing cells, whereas in control cells there was up to 90% (p ⁇ 0.05) receptor internalization (FIG. 5 Bi). Following this desensitization, cells were also assayed in vitro for directional migration towards an SDF-1 (125 ng/ml) gradient. Control cells showed a significant (p ⁇ 0.05) decrease in SDF-1 mediated migration, while the migration of CXCR4-transduced cells was hardly affected (FIG. 5 Bii). This suggests that the internalization of CXCR4 is compensated for by constant overexpression of the receptor, which continuously remains functional.
  • CXCR4 overexpression improves SRC engraftment of NOD/SCID mice.
  • transduced progenitors were transplanted in NOD/SCID mice.
  • mice NOD/LtSz-Prkdcscid mice were bred and maintained as previously described 18 . All the experiments were approved by the animal care committee of the Weizmann Institute. Eight—ten week old mice were sublethally irradiated (375 cGy, from a 60 Co source) and transplanted with human cells as indicated [2 ⁇ 10 5 cells/mouse (engraftment) and 5 ⁇ 10 5 cells/mouse (homing)] 24 hours post irradiation.
  • Human cell engraftment and homing Human cell engraftment and homing—Mice were sacrificed around five weeks post transplantation and bone marrow and spleen cells were harvested and resuspended into single cell suspension. Human cell engraftment was assayed by flow cytometry (FACSCalibur, BD) using specific antihuman CD45-APC mAb (BD Pharmingen). Lineage analysis was performed by staining with anti-CD19-PE (BD Pharmingen), or anti-CD33-PE (BD). The more primitive cell population was analysed using CD34-APC mAb (BD Pharmingen) together with anti-CD38-PE (BD). Human cells were also analysed for GFP expression (FL1 channel). Human plasma and mouse IgG were used to block Fc receptors. Isotype control antibodies and cells obtained from mice that did not undergo transplantation were used as negative controls and human CB CD34 + cells were used as positive controls.
  • FIG. 6B a representative FACS staining with CD19 and CD33 monoclonal antibodies demonstrates that multilineage hematopoiesis into lymphoid and myeloid populations respectively was maintained in the murine BM ( FIG. 6B ) with a trend to more B-cell lymphopoiesis in mice transplanted with CXCR4-transduced cells ( FIG. 6C ), most probably since SDF-1 is also a Pre B cell growth factor. Furthermore, an average of 36% ⁇ 19% (range 7.5% to 77%) of the CD45 + cells were found to express GFP ( FIG. 6B ). Transgene expression was also detected in both myeloid and lymphoid populations ( FIG. 6B ).
  • mice transplanted with CXCR4 overexpressing cells showed a four fold increase in the primitive CD34 + /CD38 ⁇ /low cell population compared to mice injected with control vector-transduced cells ( FIG. 6D ), suggesting that the higher engraftment levels of CXCR4-overexpressing cells are due to increased repopulation of the more primitive cell population.
  • Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood. 2000;95:756-768

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
  • Peptides Or Proteins (AREA)
US10/578,291 2003-11-13 2004-11-08 Stem Cells Suitable for Transplantation, their Preparation and Pharmaceutical Compositions Comprising Them Abandoned US20080025957A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IL15886803A IL158868A0 (en) 2003-11-13 2003-11-13 Methods of generating and using stem cells enriched with immature primitive progenitor
IL158868 2003-11-13
PCT/IL2004/001018 WO2005047494A2 (en) 2003-11-13 2004-11-08 Haematopoietic stem cells suitable for transplantation, their preparation and pharmaceutical compositions comprising them

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2004/001018 A-371-Of-International WO2005047494A2 (en) 2003-11-13 2004-11-08 Haematopoietic stem cells suitable for transplantation, their preparation and pharmaceutical compositions comprising them

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/031,466 Continuation US8367057B2 (en) 2003-11-13 2011-02-21 Stem cells suitable for transplantation, their preparation and pharmaceutical compositions comprising them

Publications (1)

Publication Number Publication Date
US20080025957A1 true US20080025957A1 (en) 2008-01-31

Family

ID=34044273

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/578,291 Abandoned US20080025957A1 (en) 2003-11-13 2004-11-08 Stem Cells Suitable for Transplantation, their Preparation and Pharmaceutical Compositions Comprising Them
US13/031,466 Expired - Fee Related US8367057B2 (en) 2003-11-13 2011-02-21 Stem cells suitable for transplantation, their preparation and pharmaceutical compositions comprising them

Family Applications After (1)

Application Number Title Priority Date Filing Date
US13/031,466 Expired - Fee Related US8367057B2 (en) 2003-11-13 2011-02-21 Stem cells suitable for transplantation, their preparation and pharmaceutical compositions comprising them

Country Status (9)

Country Link
US (2) US20080025957A1 (enExample)
EP (1) EP1682653B1 (enExample)
JP (2) JP2007511219A (enExample)
AU (1) AU2004288879B2 (enExample)
CA (1) CA2545409A1 (enExample)
ES (1) ES2534724T3 (enExample)
IL (2) IL158868A0 (enExample)
NO (1) NO335515B1 (enExample)
WO (1) WO2005047494A2 (enExample)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8198083B1 (en) 2007-10-31 2012-06-12 William Gunter Loudon Organotypic slices of the central nervous system
US8834928B1 (en) 2011-05-16 2014-09-16 Musculoskeletal Transplant Foundation Tissue-derived tissugenic implants, and methods of fabricating and using same
US8883210B1 (en) 2010-05-14 2014-11-11 Musculoskeletal Transplant Foundation Tissue-derived tissuegenic implants, and methods of fabricating and using same
US9352003B1 (en) 2010-05-14 2016-05-31 Musculoskeletal Transplant Foundation Tissue-derived tissuegenic implants, and methods of fabricating and using same
US10130736B1 (en) 2010-05-14 2018-11-20 Musculoskeletal Transplant Foundation Tissue-derived tissuegenic implants, and methods of fabricating and using same
US10531957B2 (en) 2015-05-21 2020-01-14 Musculoskeletal Transplant Foundation Modified demineralized cortical bone fibers
WO2023010068A3 (en) * 2021-07-28 2023-03-02 Cartesian Therapeutics, Inc. Multiprotein-engineered cells secreting a multispecific antibody

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050271639A1 (en) * 2002-08-22 2005-12-08 Penn Marc S Genetically engineered cells for therapeutic applications
CN101495623B (zh) 2006-03-24 2013-09-11 儿童医疗中心有限公司 调节造血干细胞生长的方法
EP2679221B1 (en) 2006-10-20 2020-09-23 Children's Medical Center Corporation Method to enhance tissue regeneration
WO2008073748A1 (en) 2006-12-08 2008-06-19 University Of Rochester Expansion of hematopoietic stem cells
CA2682469A1 (en) 2007-03-30 2008-10-09 The Cleveland Clinic Foundation Method of treating ischemic disorders
WO2009079451A2 (en) 2007-12-14 2009-06-25 The Cleveland Clinic Foundation Compositions and methods of promoting wound healing
EP3031907B1 (en) 2008-11-06 2021-01-06 Indiana University Research and Technology Corporation Materials and methods to enhance hematopoietic stem cells engraftment procedures
US20120283315A1 (en) 2009-08-28 2012-11-08 Penn Marc S Sdf-1 delivery for treating ischemic tissue
MX359398B (es) 2011-09-30 2018-09-27 Bluebird Bio Inc Compuestos para transduccion viral mejorada.
WO2013082243A1 (en) * 2011-12-02 2013-06-06 Fate Therapeutics, Inc. Enhanced stem cell composition
EP3413896B1 (en) 2016-02-12 2021-03-17 Bluebird Bio, Inc. Vcn enhancer compositions and methods of using the same
CA3014078A1 (en) 2016-02-12 2017-08-17 Bluebird Bio, Inc. Vcn enhancer compositions and methods of using the same
GB201807944D0 (en) * 2018-05-16 2018-06-27 Ospedale San Raffaele Srl Compositions and methods for haematopoietic stem cell transplantation
WO2022054999A1 (ko) * 2020-09-14 2022-03-17 주식회사 제너로스 생착능이 증가된 줄기세포의 제조 방법

Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4588585A (en) * 1982-10-19 1986-05-13 Cetus Corporation Human recombinant cysteine depleted interferon-β muteins
US4737462A (en) * 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4873316A (en) * 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US4879111A (en) * 1986-04-17 1989-11-07 Cetus Corporation Treatment of infections with lymphokines
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US4959314A (en) * 1984-11-09 1990-09-25 Cetus Corporation Cysteine-depleted muteins of biologically active proteins
US4965195A (en) * 1987-10-26 1990-10-23 Immunex Corp. Interleukin-7
US5017691A (en) * 1986-07-03 1991-05-21 Schering Corporation Mammalian interleukin-4
USRE33653E (en) * 1983-04-15 1991-07-30 Cetus Corporation Human recombinant interleukin-2 muteins
US5116743A (en) * 1989-02-06 1992-05-26 Mitsubishi Petrochemical Co., Ltd. L-alanine production with two microorganisms having fumarase inactivity in a single reaction tank
US5486359A (en) * 1990-11-16 1996-01-23 Osiris Therapeutics, Inc. Human mesenchymal stem cells
US5837539A (en) * 1990-11-16 1998-11-17 Osiris Therapeutics, Inc. Monoclonal antibodies for human mesenchymal stem cells
US5843780A (en) * 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US5928638A (en) * 1996-06-17 1999-07-27 Systemix, Inc. Methods for gene transfer
US6090622A (en) * 1997-03-31 2000-07-18 The Johns Hopkins School Of Medicine Human embryonic pluripotent germ cells
US6132708A (en) * 1997-10-10 2000-10-17 Oregon Health Sciences University Liver regeneration using pancreas cells
US6143292A (en) * 1995-05-25 2000-11-07 Baxter International Inc. Allogeneic cell therapy for cancer following allogeneic stem cell transplantation
US6184035B1 (en) * 1998-11-18 2001-02-06 California Institute Of Technology Methods for isolation and activation of, and control of differentiation from, skeletal muscle stem or progenitor cells
US6248587B1 (en) * 1997-11-26 2001-06-19 University Of Southern Cailfornia Method for promoting mesenchymal stem and lineage-specific cell proliferation
US6258597B1 (en) * 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6280718B1 (en) * 1999-11-08 2001-08-28 Wisconsin Alumni Reasearch Foundation Hematopoietic differentiation of human pluripotent embryonic stem cells
US6326198B1 (en) * 1990-06-14 2001-12-04 Regents Of The University Of Michigan Methods and compositions for the ex vivo replication of stem cells, for the optimization of hematopoietic progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion and/or IL-6 secretion of human stromal cells
US6383481B1 (en) * 1998-03-30 2002-05-07 Japan Immunoresearch Laboratories Co., Ltd. Method for transplantation of hemopoietic stem cells
US6436704B1 (en) * 2000-04-10 2002-08-20 Raven Biotechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
US6447766B1 (en) * 1993-06-08 2002-09-10 Smithkline Beecham Corporation Method of mobilizing hematopoietic stem cells
US6447765B1 (en) * 1998-03-03 2002-09-10 University Of Southern California Use of cytokines and mitogens to inhibit graft versus host disease
US6511958B1 (en) * 1997-08-14 2003-01-28 Sulzer Biologics, Inc. Compositions for regeneration and repair of cartilage lesions
US20050163760A1 (en) * 2001-12-06 2005-07-28 Nathalie Cartier-Lacave Use of cd34+ hematopoietic progenitor cells for the treatment of cns disorders

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2874751B2 (ja) 1986-04-09 1999-03-24 ジェンザイム・コーポレーション 希望する蛋白質をミルク中へ分泌する遺伝子移植動物
DK2371361T3 (da) * 2001-07-31 2019-08-19 Genzyme Corp Fremgangsmåder til mobilisering af progenitor-/stamceller
IL146970A0 (en) * 2001-12-06 2002-08-14 Yeda Res & Dev Migration of haematopoietic stem cells and progenitor cells to the liver

Patent Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4588585A (en) * 1982-10-19 1986-05-13 Cetus Corporation Human recombinant cysteine depleted interferon-β muteins
US4737462A (en) * 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
USRE33653E (en) * 1983-04-15 1991-07-30 Cetus Corporation Human recombinant interleukin-2 muteins
US4959314A (en) * 1984-11-09 1990-09-25 Cetus Corporation Cysteine-depleted muteins of biologically active proteins
US4879111A (en) * 1986-04-17 1989-11-07 Cetus Corporation Treatment of infections with lymphokines
US5017691A (en) * 1986-07-03 1991-05-21 Schering Corporation Mammalian interleukin-4
US4873316A (en) * 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US4965195A (en) * 1987-10-26 1990-10-23 Immunex Corp. Interleukin-7
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US5116743A (en) * 1989-02-06 1992-05-26 Mitsubishi Petrochemical Co., Ltd. L-alanine production with two microorganisms having fumarase inactivity in a single reaction tank
US6326198B1 (en) * 1990-06-14 2001-12-04 Regents Of The University Of Michigan Methods and compositions for the ex vivo replication of stem cells, for the optimization of hematopoietic progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion and/or IL-6 secretion of human stromal cells
US5486359A (en) * 1990-11-16 1996-01-23 Osiris Therapeutics, Inc. Human mesenchymal stem cells
US5837539A (en) * 1990-11-16 1998-11-17 Osiris Therapeutics, Inc. Monoclonal antibodies for human mesenchymal stem cells
US6447766B1 (en) * 1993-06-08 2002-09-10 Smithkline Beecham Corporation Method of mobilizing hematopoietic stem cells
US5843780A (en) * 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US6143292A (en) * 1995-05-25 2000-11-07 Baxter International Inc. Allogeneic cell therapy for cancer following allogeneic stem cell transplantation
US5928638A (en) * 1996-06-17 1999-07-27 Systemix, Inc. Methods for gene transfer
US6090622A (en) * 1997-03-31 2000-07-18 The Johns Hopkins School Of Medicine Human embryonic pluripotent germ cells
US6511958B1 (en) * 1997-08-14 2003-01-28 Sulzer Biologics, Inc. Compositions for regeneration and repair of cartilage lesions
US6258597B1 (en) * 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6132708A (en) * 1997-10-10 2000-10-17 Oregon Health Sciences University Liver regeneration using pancreas cells
US6248587B1 (en) * 1997-11-26 2001-06-19 University Of Southern Cailfornia Method for promoting mesenchymal stem and lineage-specific cell proliferation
US6447765B1 (en) * 1998-03-03 2002-09-10 University Of Southern California Use of cytokines and mitogens to inhibit graft versus host disease
US6383481B1 (en) * 1998-03-30 2002-05-07 Japan Immunoresearch Laboratories Co., Ltd. Method for transplantation of hemopoietic stem cells
US6184035B1 (en) * 1998-11-18 2001-02-06 California Institute Of Technology Methods for isolation and activation of, and control of differentiation from, skeletal muscle stem or progenitor cells
US6280718B1 (en) * 1999-11-08 2001-08-28 Wisconsin Alumni Reasearch Foundation Hematopoietic differentiation of human pluripotent embryonic stem cells
US6436704B1 (en) * 2000-04-10 2002-08-20 Raven Biotechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
US20050163760A1 (en) * 2001-12-06 2005-07-28 Nathalie Cartier-Lacave Use of cd34+ hematopoietic progenitor cells for the treatment of cns disorders

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8198083B1 (en) 2007-10-31 2012-06-12 William Gunter Loudon Organotypic slices of the central nervous system
US8883210B1 (en) 2010-05-14 2014-11-11 Musculoskeletal Transplant Foundation Tissue-derived tissuegenic implants, and methods of fabricating and using same
US9352003B1 (en) 2010-05-14 2016-05-31 Musculoskeletal Transplant Foundation Tissue-derived tissuegenic implants, and methods of fabricating and using same
US10130736B1 (en) 2010-05-14 2018-11-20 Musculoskeletal Transplant Foundation Tissue-derived tissuegenic implants, and methods of fabricating and using same
US11305035B2 (en) 2010-05-14 2022-04-19 Musculoskeletal Transplant Foundatiaon Tissue-derived tissuegenic implants, and methods of fabricating and using same
US8834928B1 (en) 2011-05-16 2014-09-16 Musculoskeletal Transplant Foundation Tissue-derived tissugenic implants, and methods of fabricating and using same
US10531957B2 (en) 2015-05-21 2020-01-14 Musculoskeletal Transplant Foundation Modified demineralized cortical bone fibers
US11596517B2 (en) 2015-05-21 2023-03-07 Musculoskeletal Transplant Foundation Modified demineralized cortical bone fibers
US12295848B2 (en) 2015-05-21 2025-05-13 Musculoskeletal Transplant Foundation Implants including modified demineralized cortical bone fibers and methods of making same
WO2023010068A3 (en) * 2021-07-28 2023-03-02 Cartesian Therapeutics, Inc. Multiprotein-engineered cells secreting a multispecific antibody

Also Published As

Publication number Publication date
IL158868A0 (en) 2004-05-12
AU2004288879B2 (en) 2011-06-16
IL175430A0 (en) 2006-09-05
WO2005047494A2 (en) 2005-05-26
AU2004288879A1 (en) 2005-05-26
ES2534724T3 (es) 2015-04-27
CA2545409A1 (en) 2005-05-26
EP1682653B1 (en) 2015-03-25
US8367057B2 (en) 2013-02-05
JP2012125249A (ja) 2012-07-05
JP2007511219A (ja) 2007-05-10
NO335515B1 (no) 2014-12-22
NO20062709L (no) 2006-08-11
US20110268712A1 (en) 2011-11-03
EP1682653A2 (en) 2006-07-26
IL175430A (en) 2015-01-29
WO2005047494A3 (en) 2005-06-23
JP5547221B2 (ja) 2014-07-09

Similar Documents

Publication Publication Date Title
US8367057B2 (en) Stem cells suitable for transplantation, their preparation and pharmaceutical compositions comprising them
Kahn et al. Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation
JP5362659B2 (ja) 化学誘引物質に対する増大させた感受性を有する幹細胞およびそれを産生および使用する方法
US8846393B2 (en) Methods of improving stem cell homing and engraftment
AU2006321172B2 (en) Methods of improving stem cell homing and engraftment
US8252588B2 (en) Stem cells having increased sensitivity to SDF-1 and methods of generating and using same
Kauser et al. Bone marrow-derived progenitors
Duckles et al. Handbook of Experimental Pharmacology
HK1164367A (en) Methods of improving stem cell homing and engraftment

Legal Events

Date Code Title Description
AS Assignment

Owner name: YEDA RESEARCH AND DEVLOPMENT CO. LTD., ISRAEL

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAPIDOT, TSVEE;KAHN, JOY;REEL/FRAME:018829/0694;SIGNING DATES FROM 20060719 TO 20060724

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION