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
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Bone marrow contains at least two types of stem cells, hematopoietic stem cells and stem cells of non-hematopoietic tissues variously referred to as mesenchymal stem cells or mesenchymal stromal cells (MSCs) or bone marrow stromal cells (BMSCs). These terms are used synonymously throughout herein. MSCs are of interest because they are easily isolated from a small aspirate of bone marrow, or other mesenchymal stem cell sources, and they readily generate single-cell derived colonies. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib, knee or other mesenchymal tissues.
• MSCs mesenchymal stromal cells
• BMSCs bone marrow stromal cells
• MSCs include embryonic yolk sac, placenta, umbilical cord, skin, fat, synovial tissue from joints, and blood.
• the presence of MSCs in culture colonies may be verified by specific cell surface markers which are identified with monoclonal antibodies. See U.S. Patent Nos. 5,486,359 and 7, 153,500.
• the single-cell derived colonies can be expanded through as many as 50 population doublings in about 10 weeks, and can differentiate into osteoblasts, adipocytes, chondrocytes (Friedenstein et al., 1970 Cell Tissue inet. 3:393-403; Castro-Malaspina, et al., 1980 Blood 56:289-301 ; Beresford et al., 1992 J.
• MSCs serve as progenitors for multiple mesenchymal cell lineages including bone, cartilage, ligament, tendon, adipose, muscle, cardiac tissue, stroma, dermis, and other connective tissues. See U.S. Patent Nos. 6,387,369 and 7.101 ,704. For these reasons, MSCs currently are being tested for their potential use in cell and gene therapy of a number of human diseases (Horwitz et al.irri 1999 Nat. Med. 5:309-31 3; Caplan, et al. 2000 Clin. Orthoped. 379:567-570).
• MSCs constitute an alternative source of pluripotent stem cells. Under physiological conditions they maintain the architecture of bone marrow and regulate hematopoiesis with the help of different cell adhesion molecules and the secretion of cytokines, respectively (Clark and Keating, 1995 Ann NY Acad Sci 770:70-78). MSCs grown out of bone marrow by their selective attachment to tissue culture plastic can be efficiently expanded (Azizi et al, 1 98 Proc Natl Acad Sci USA 95:3908-3913; Colter et al, 2000 Proc Natl Acad Sci USA 97:32 13-21 8) and genetically manipulated (Schwarz et al. 1999 Hum Gene Ther 10:2539-2549).
• MSCs also are referred to as mesenchymal stem cells because they are capable of differentiating into multiple mesodermal tissues, including bone (Beresford el al., 1992 J Cell Sci 102:341 -35 1 ), cartilage (Lermon et al., 1995 Exp Cell Res 219:21 1-222), fat (Beresfordet al., 1992 1 Cell Sci. 102:341 -351 ) and muscle (Wakitani et al., 1995 Muscle Nerve 18: 1417- 1426).
• hBMSCs human bone marrow stromal cells
• the present invention provides the necessary data to establish that MSCs have added therapeutic benefit in treating diseases.
• Figure 1 is a series of images demonstrating the methodology for the generation of h SC spheroids and the acquisition of spheroid-derived cells that maintain mesenchymal surface features; however, they can be distinguished from MSCs prepared in standard monolayer cultures by decreased expression of the surface marker PODXL and increased expression of the surface marker CD49b.
• Figure 2 is a series of images demonstrating that MSCs derived from cultured spheroids proliferate slowly but remain highly viable.
• Figure 3 is a series of images demonstrating the MSC spheroid-derived cells are significantly smaller than MSCs cultured as monolayers.
• Figure 4. is a series of images demonstrating that MSCs cultured as spheroids in hanging drops express higher levels than MSCs from monolayers of a series of therapeutic genes: TNF-a stimulated gene protein 6, or TSG-6, an anti -inflammatory protein; stanniocalcin I , or STC-l , an anti-apoptotic protein; leukemia inhibitory factor; or LIF, a protein that regulates cell growth and development; IL- 1 1 , a protein that regulates hematopoiesis; TNF-a related apoptosis inducing ligand, or TNFSF 10 (also known as TRAIL), a protein that kills some cancer cells and regulates immune response; IL-24, a protein that kills some cancer cells; CXC chemokine receptor 4, or CXCR4, a protein that regulates homing of cells; JTGA2 (also known as integrin a2), a protein involved in cell adhesion and cell signaling; and IL-8.
• MSC spheroid-derived cells express much higher levels of the same genes that three other kinds of cells grown as spheroids, i.e. human dermal fibroblasts (hDF), a lung epithelial cancer cell line (A549), and human neural progenitor cells (hNPC).
• hDF human dermal fibroblasts
• A549 a lung epithelial cancer cell line
• hNPC human neural progenitor cells
• Figure 5 is a series of images demonstrating that production of the antiinflammatory protein TSG-6, the anti-apoptotic protein STC-l , and the cell regulatory protein LIF is enhanced notably in MSCs derived from hanging drop cultures.
• Figure 6 is a series of images demonstrating that TNFa levels produced from LPS stimulated macrophages were decreased markedly by MSC spheroid-derived cells. The effects of the MSCs spheroid-derived cells are much greater than those of MSCs cultured as monolayers.
• FIG. 7 is an image demonstrating that MSC spheroid-derived cells exhibit antiinflammatory effects in vivo.
• peritonitis was induced in mice by injection of the irritant Zymosan, and then MSC spheroid-derived cells injected into the peritoneum.
• the MSC spheroid-derived cells decreases inflammation as indicated by the decrease in serum plasmin activity, a biomarker for inflammation.
• Figure 8. is a series of images demonstrating that hMSCs aggregated rapidly into three dimensional spheroids when grown in hanging drops or on a nonadherent surface.
• Figure 9. is a series of images demonstrating that hMSCs in smaller spheroids show high viability.
• Figure 10 is a series of images demonstrating that hMSC spheroids express high levels of anti-inflammatory molecule TSG-6.
• Figure 1 is an image developed from analyses by micro-arrays demonstrating that hMSC spheroids express high levels of a series of cytokines and cell adhesion molecules while the expression of cell cycle and cytoskeletal genes is down-regulated. The data demonstrated that the profile of expressed genes in hMSC spheroids is different markedly from the profile of expressed gene in hMSCs cultured as monolayers.
• Figure 12 is an image demonstrating that hMSC spheroids express high levels of molecules having therapeutic effects: TSG-6. an anti-inflammatory protein; STC 1 , an anti- apoptotic protein; LIF, a protein that regulates cell growth and development; 1L-24, a protein that kills some cancer cells, and TRAIL, a protein that kills some cancer cells and also modulates the immune system.
• TSG-6 an anti-inflammatory protein
• STC 1 an anti- apoptotic protein
• LIF a protein that regulates cell growth and development
• 1L-24 a protein that kills some cancer cells
• TRAIL a protein that kills some cancer cells and also modulates the immune system.
• Figure 13 is a series of images demonstrating that hMSC spheroids secrete large amounts of the proteins TSG-6, STC-1, and LIF.
• Figure 14 is a series of images demonstrating that LPS stimulated macrophages secrete less TNF-a when co-cultured with hMSC spheroids. MSCs cultured as monolayers were less effective.
• Figure 1 5 is an image demonstrating that hMSC spheroids show the same antiinflammatory effects as hMSC spheroid-derived cells ( Figure 6) in a mouse model of peritonitis.
• FIG. 16 The expression of TSG-6 was increased as hMSCs aggregated into spheroids in hanging drops.
• A Phase contrast microscopy showing the time course of the aggregation of 25,000 hMSCs into a spheroid in a hanging drop.
• B H&E staining of hMSC spheroid sections from 3 day hanging drop cultures. Surface (Top), and center (Middle and Bottom) of a spheroid.
• Scale bar 50 ⁇ .
• C Real-time RT PCR measurements of TSG-6 expression in hMSCs shown as relative to Adh Low sample (n - 3).
• Adh Low hMSCs plated at 100 cells/cm 2 for 7-8 days unlil about 70% confluent
• Adh High hMSCs harvested from same Adh Low cultures, plated at 5,000 cells/cm 2 and incubated for 3 days
• Sph 10k -250k hMSCs harvested from same Adh Low cultures and incubated for 3 days in hanging drops at 10,000-250,000 cells/drop.
• FIG. 17 Viability of hMSCs in spheroids.
• FIG. 18 Size analysis and i.v. infusion of spheroid hMSCs.
• B Cell size assayed by microscopy.
• C Relative tissue distribution of i.v. infused hMSCs. NOD/scid mice were infused i.v. with 10 6 monolayer or spheroid derived hMSCs.
• FIG. 1 Spheroid hMSCs retain the properties of hMSCs from adherent cultures.
• A Differentiation of hMSCs in osteogenic medium (Osteo Dif) and control medium (Osteo Con). Cultures were stained with Alizarin Red after 14 days.
• B Differentiation of hMSCs in adipogenic medium (Adipo Dif) and control medium (Adipo Con). Cultures were stained with Oil Red O after 14 days. (Scale bar, 200 ⁇ )
• FIG. 20 Microarray assays of hMSCs from two donors.
• A Hierarchical clustering of differentially expressed genes. Genes that were either up- (236 genes) or down- regulated (230 genes) in spheroids (Sph 25k) at least twofold compared with their adherent culture counterparts (Adh Low and Adh High), were used in hierarchical clustering. The most significant Gene Ontology terms for up-regulated genes and down-regulated genes are shown next to the heat map.
• FIG. 21 Spheroid hMSCs express high levels of anti-inflammatory and anti- tumorigenic molecules.
• A Real-time RT PCR measurements for anti-inflammatory genes (TSG-6, STC- 1 , and LIF), anti-tumorigenic genes (IL-24 and TRAIL), gene for an MSC homing receptor (CXCR4), and gene for the Wnt signaling inhibitor (D K I ) for two donors. Values are mean RQ ⁇ 95% confidence interval from triplicate assays compared with Adh Low sample.
• FIG 22 hMSC spheroids exhibit enhanced anti-inflammatory effects in vitro and in vivo.
• A Schematic of the mouse macrophage (mMO) assay. mM ⁇ l>s were seeded in the upper chamber of a transwell, stimulated with LPS for 90 min, the LPS was removed, and the chamber transferred to a six- well dish plated with monolayer (Adh), spheroid (Sph), or spheroid-derived hMSCs (Sph DC) at the same cell density. M ⁇ D:hMSC (2: 1 ). After 5 hours, medium was collected for ELISAs.
• B ELISA for mT Fa in medium from cocultures (n ; 3).
• FIG. 23 Spheroid hMSCs maintain high viability with culture in animal product- free media and after freezing. Spheroids were cultured in the described commercially available Xeno-free medium. After 3 days, the spheroids were dissociated using trypsin/ED I A and then frozen in DMSO. The cells were thawed and the viability determined by flow cytometry measuring PI uptake and annexin V-FITC cell surface labeling. Unlabeled cells were considered viable.
• CCM Complete culture medium (aMEM wilh 17% FBS); F luSA, Human serum albumin (Baxter Healthcare); Stpro, StemPro Xeno-free medium (Gibco); Mes, Mesencult Xeno-free medium (Stem Cell Technologies).
• FS forward scatter
• the present invention relates to the discovery that mesenchymal stromal cells (MSCs) can be manipulated in culture to possess novel therapeutic characteristics and therefore can be useful in therapy of a desired disease.
• MSCs mesenchymal stromal cells
• mesenchymal stem cells in a spheroidal aggregate or mesenchymal stem cells obtained from a spheroidal aggregate wherein the mesenchymal stem cells express increased amounts of at least one therapeutic protein compared to mesenchymal stem cells cultured as a monolayer.
• the at least one therapeutic protein is expressed by the mesenchymal stem cells in a spheroidal aggregate or by the mesenchymal stem cells obtained from a spheroidal aggregate in an amount at least 20% greater than the amount expressed by mesenchymal stem cells cultured as a monolayer.
• the at least one therapeutic protein is expressed by the mesenchymal stem cells in a spheroidal aggregate or by the mesenchymal stem cells obtained from a spheroidal aggregate in an amount of at least 3-fold greater than the amount expressed by mesenchymal stem cells cultured as a monolayer.
• the at least one therapeutic protein is expressed by the mesenchymal stem cells in a spheroidal aggregate or the mesenchymal stem cells obtained from a spheroidal aggregate in an amount of at least 10-fold greater than the amount expressed by mesenchymal stem cells cultured as a monolayer.
• the at least one therapeutic protein is expressed by the mesenchymal stem cells in a spheroidal aggregate or by the mesenchymal stem cells obtained from a spheroidal aggregate in an amount which is at least 50-fold greater than the amount expressed by mesenchymal stem cells cultured as a monolayer.
• the at least one therapeutic protein is expressed by the mesenchymal stem cells in a spheroidal aggregate or by the mesenchymal stem cells obtained from a spheroidal aggregate in an amount which is at least 500-fold greater than the amount expressed by mesenchymal stem cells cultured as a monolayer. In yet another non-limiting embodiment, the at least one therapeutic protein is expressed by the mesenchymal stem ceils in a spheroidal aggregate or by the mesenchymal stem cells obtained from a spheroidal aggregate in an amount which is at least 1 ,000-fold greater than the amount expressed by the mesenchymal stem cells cultured as a monolayer.
• the at least one therapeutic protein is selected from the group consisting of anti-inflammatory proteins, anti-apoptotic proteins, proteins that regulate cell growth and development, proteins that regulate an immune response, proteins that regulate hematopoiesis, proteins which inhibit, prevent, or destroy the growth of tumors, proteins that regulate the homing of cells, proteins that are involved in cell adhesion and/or cell signaling, proteins that enhance angiogenesis, and combinations thereof.
• the MSCs can be used to treat diseases associated with including but not limited to, inflammation, apoptosis of cells, immune dysregulation, including autoimmune disea'ses, tumors, including cancer, and the like.
• diseases associated with including but not limited to, inflammation, apoptosis of cells, immune dysregulation, including autoimmune disea'ses, tumors, including cancer, and the like.
• the MSCs of the invention can be manipulated to possess therapeutic characteristics as a result of expressing increased amounts of one or more therapeutic proteins, including but not limited to an anti -inflammatory protein, an anti-apoptotic protein, a protein that regulates hematopoiesis, a protein that kills tumor cells and/or cancer cells, a protein that regulates immune response, a protein that regulates homing of cells, a protein involved in cell adhesion and/or cell signaling, a protein that enhances angiogenesis, and the like, as well as combinations thereof.
• the invention provides the use of MSCs as an antiinflammatory therapy.
• the invention provides the use of MSCs to decrease the programmed cell death (apopiosis) that is associated with oxygen deprivation (ischemia and hypoxia) and with multiple kinds of injury to cells and tissues.
• the invention provides the use of MSCs as an anti-tumor therapy, i.e., which inhibits, prevents, or destroys the growth of tumors, including cancerous tumors.
• the invention provides the use of MSCs for immune regulation, including, but not limited to, the treatment of autoimmune diseases.
• the invention is related to the discovery that MSCs aggregate rapidly into spheroids when grown in hanging drops or on non-adherent dishes.
• MSCs are referred to herein as a "spheroidal aggregate" of MSCs or "MSC spheroids".
• MSC spheroids exhibit high viability and express high levels of one or more of a series of genes for therapeutic proteins.
• TNF-a stimulated gene protein 6, or TSG-6 an anti-inflammatory protein
• growth differentiation factor-15 or GDF- 15, an anti-inflammatory protein
• stanniocalcin-l or STC-1
• an anli- apoptotic and anti-inflammatory protein an anli- apoptotic and anti-inflammatory protein
• leukemia inhibitory factor, or LIF a protein that regulates cell growth and development
• IL-l l a protein that regulates hematopoiesis
• TNF-a related apoptosis inducing ligand, or TNFSF10 also known as TRAIL
• IL-24 a protein that kills some cancer cells
• CD82 another protein that kills cancer cells and suppresses metastases
• CXC chemokine receptor 4, or CXCR4 a protein that regulates homing of cells
• ITGA2 also known as integrin a2 or CD49b
• ITGA2 also known as integrin a2 or CD49b
• the present invention provides methods for pre-programming MSCs to express one or more therapeutically beneficial proteins, including but not limited to, anti-inflammatory, anti- apoptotic, immune modulatory, anti-apoptotic, and anti-tumorigenic proteins prior to the administration thereof to an animal, including human and non-human animals. Definitions
• an element means one element or more than one element.
• autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
• biocompatible lattice is meant to refer to a substrate that can facilitate formation into three-dimensional structures conducive for tissue development.
• cells can be cultured or seeded onto such a biocompatible lattice, such as one that includes extracellular matrix material, synthetic polymers, cytokines, growth factors, etc.
• the lattice can he molded into desired shapes for facilitating the development of tissue types.
• the medium and/or substrate is supplemented with factors (e.g., growth factors, cytokines, extracellular matrix material, etc.) that facilitate the development of appropriate tissue types and structures.
• bone marrow stromal cells As used herein, the term “bone marrow stromal cells,” “stromal cells,” “mesenchymal stem cells,” “mesenchymal stromal cells” or “MSCs” arc used interchangeably and refer to a cell derived from bone marrow (reviewed in Prockop, 1997), peripheral blood (Kuznetsov et al, 2001 ), adipose tissue (Guilak et al., 2004), umbilical cord blood (Rosada et al., 2003), synovial membranes (De Bari et al., 2001 ), and periodontal ligament (Seo et al., 2005), embryonic yolk sac, placenta, umbilical cord, skin, and blood (U.S.
• MSCs are characterized by their ability to adhere to plastic tissue culture surfaces (Friedenstein et al.; reviewed in Owen & Fricdenstein, 1988), and by being an effective feeder layers for hematopoietic stem cells (Eaves et al interchange 2001 ).
• MSCs can be differentiated both in culture and in vivo into osteoblasts and chondrocytes, into adipocytes, muscle cells (Wakitani et al., 1995) and cardiomyocytes (Fukuda and Yuasa, 2006).
• MSCs Mesenchymal stem cells
• MSCs Mesenchymal stem cells
• “Graft” refers to a cell, tissue, organ or otherwise any biological compatible lattice for transplantation.
• Allogeneic refers to a graft derived from a different animal of the same species.
• Xenogeneic refers to a graft derived from an animal of a different species.
• Transplant refers to a biocompatible lattice or a donor tissue, organ or cell, to be transplanted.
• An example of a transplant may include, but is not limited to, skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung and liver.
• the transplant is a human neural stem cell.
• BMSC bone marrow stromal cell
• Donor antigen refers to an antigen expressed by the donor tissue to be transplanted into the recipient.
• Alloantigen is an antigen that differs from an antigen expressed by the recipient.
• an "effector cell” refers to a cell which mediates an immune response against an antigen.
• the effector cells can be the recipient's own cells that elicit an immune response against an antigen present in the donor transplant.
• the effector cell can be part of the transplant, whereby the introduction of the transplant into a recipient results in the effector cells which are present in the transplant eliciting an immune response against the recipient of the transplant.
• a “therapeutically effective amount” is the amount of BMSCs which is sufficient to provide a beneficial effect to the subject to which the BMSCs are administered.
• endogenous refers to any material from or produced inside an organism, cell, or system.
• Exogenous refers to any material introduced from or produced outside an organism, cell, or system.
• Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
• a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
• Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cD A.
• nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
• An 'isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which normally are adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
• the term also applies to nucleic acids which have been purified substantially from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which accompany it naturally in the cell.
• the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
• nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “LP refers to uridine.
• a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
• vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphophilic compounds, plasmids, and viruses.
• the term “vector” includes an autonomously replicating plasmid or a virus.
• the term also should be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
• viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
• “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
• An expression vector comprises sufficient cis ⁇ acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
• Expression vectors include all those known in the art, such as cosmids, plasmids (e.g.. naked or contained in liposomes), and viruses that incorporate the recombinant polynucleotide.
• the present invention relates to the discovery that when MSCs and in particular human MSCs (hMSCs) aggregate into spheroids, the hMSC spheroids and cells obtained from hMSC spheroids express increased levels of one or more genes encoding therapeutic proteins.
• therapeutic proteins include, but are not limited to anti-inflammatory agents, anli-apoptotic agents, proteins that regulate an immune response, proteins that regulate hematopoiesis, agents that inhibit, prevent, or destroy the growth of tumors, proteins that regulate the homing of cells, proteins that are involved in cell adhesion and cell signaling, and proteins that enhance angiogenesis, and combinations thereof.
• Such proteins include, but are not limited to, TSG-6, an anti-inflammatory protein; growth differentiation factor- 15, or GDF-15. an antiinflammatory protein; STC-I, an anti-apoptotic and anti-inflammatory protein; L1F, a protein that regulates cell growth and development; IL-1 1 , a protein that regulates hematopoiesis; TNF- ⁇ related apoptosis inducing ligand, or TNFSF 10 (also known as TRAIL), a protein that kills some cancer cells and regulates immune response; 1L-24, a protein that kills some cancer cells; C 82, another protein that kills cancer cells and suppresses metastases, CXC chemokine receptor 4, or CXCR4, a protein that regulates homing of cells; ITGA2 (also known as integrin a2 or CD49b), a protein involved in cell adhesion and cell signaling; and IL-8, a protein that enhances angiogenesis.
• ITGA2 also known as integrin a
• hMSC spheroids and hMSCs obtained from hMSC spheroids exhibit anti-inflammatory effects in an in vitro inflammation assay and are and -inflammatory in an in vivo mouse model of peritonitis. Accordingly, hMSC spheroids and hMSCs obtained from hMSC spheroids are useful as antiinflammatory therapy in many diseases. In some instances, hMSC spheroids and hMSCs obtained from hMSC spheroids are useful as anti-tumor therapy in many cancers.
• the present invention encompasses methods and compositions for reducing and/or eliminating an inflammatory response in a mammal by treating the mammal with an amount of MSC spheroids, or cells obtained from hMSC spheroids, effective to reduce or inhibit inflammation in the mammal.
• the present invention provides methods of pre-programming MSCs to express increased amounts of therapeutic proteins, including but not limited to, therapeutically beneficial antiinflammatory, anti-apoptotic, immune regulatory, and anti-tumori genie proteins prior to the administration or transplantation thereof.
• the method includes activating MSCs and administer MSCs so that anti-inflammatory, anti-apoptotic. immune modulatory. and/or anti-tumorigenic proteins are at maximal expression when administered to a patient. Therapy to inhibit inflammation and/or anti-tumor therapy
• the present invention includes a method of using MSC spheroids or cells obtained from hMSC spheroids as a therapy to inhibit or modulate inflammation.
• the invention is based on the discovery that MSCs when aggregated rapidly form spheroids.
• the hMSC spheroids exhibit high viability and express high levels of anti-inflammatory, anti-apoptotic, immune modulatory, and/or anti-tumorigenic molecules.
• the hMSC spheroids or cells obtained from hMSC spheroids secrete high levels of the anti-inflammatory proteins.
• the hMSC spheroids or cells obtained from hMSC spheroids also exhibited anti-inflammatory effects in an in vitro inflammation assay and in a mouse model of peritonitis.
• hMSC spheroids or cells obtained from hMSC spheroids to suppress inflammation provides a means for an anti-inflammatory therapy.
• MSCs can be obtained from any source.
• the MSCs may be autologous with respect to the recipient (obtained from the same host) or allogeneic with respect to the recipient.
• the MSCs may be xenogeneic to the recipient (obtained from an animal of a different species); for example, rat MSCs may be used to suppress inflammation in a human.
• MSCs used in the present invention can be isolated, from the bone marrow of any species of mammal, including but not limited to, human, mouse, rat, ape, gibbon, bovine.
• the MSCs are isolated from a human, a mouse, or a rat.
• the MSCs are isolated from a human.
• MSCs can be isolated and expanded in culture in vitro to obtain sufficient numbers of cells for use in the methods described herein provided that the MSCs are cultured in a manner that promotes aggregation and formation of spheroids.
• MSCs can be isolated from human bone marrow and cultured in complete medium (DMEM low glucose containing 4 mM L-glutamine, 10% PBS, and 1% penicillin streptomycin) in hanging drops or on non-adherent dishes; however, the invention should in no way be construed to be limited to any one method of isolating or to any culturing medium. Rather, any method of isolating and any culturing medium should be construed to be included in the present invention provided that the MSCs are cultured in a manner that promotes aggregation and formation of spheroids.
• Any medium capable of supporting MSCs in vitro may be used to culture the MSCs.
• Media formulations that can support the growth of MSCs include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium (exMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like.
• DMEM Dulbecco's Modified Eagle's Medium
• exMEM alpha modified Minimal Essential Medium
• RPMI Media 1640 Roswell Park Memorial Institute Media 1640
• up to 20% fetal bovine serum (PBS) or 1 -20% horse serum is added to the above medium in order to support the growth of MSCs.
• PBS fetal bovine serum
• a defined medium can also be used if the growth factors, cytokines, and hormones necessary for culturing MSCs are provided at appropriate concentrations in the medium.
• Media useful in the methods of the invention may contain one or more compounds of interest, including but not limited to antibiotics, milogenic or differentiation compounds useful for the culturing of MSCs.
• the cells may be grown , in one non-limiting embodiment, at temperatures between 27°C to 40°C, in another non-limiting embodiment, at 31 °C to 37°C, and in another non-limiting embodiment, in a humidified incubator.
• the carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1 % and 22%.
• the invention should in no way be construed to be limited to any one method of isolating and culturing MSCs. Rather, any method of isolating and culturing MSCs should be construed to be included in the present invention.
• Antibiotics which can be added into the medium include, but are not limited to, penicillin and streptomycin.
• concentration of penicillin in the culture medium is about 10 to about 200 units per ml.
• concentration of streptomycin in the culture medium is about 10 to about 200 ⁇ g/ml.
• the mesenchymal stem cells are cultured under conditions which, as noted hereinabove, provide for the aggregation of the mesenchymal stem cells into a spheroidal aggregate, and provide for optimal expression of the therapeutic protein(s).
• the mesenchymal stem cells are cultured in a medium, such as complete culture medium (CCM), for example, which includes serum in an amount effective to upregulate one or more of the hereinabove noted therapeutic proteins.
• a medium such as complete culture medium (CCM)
• the medium may include fetal bovine serum in an amount of up to 20%.
• the fetal bovine serum is present in an amount of about 17%.
• the mesenchymal stem cells are cultured under conditions and for a period of time (for example, 7 or 8 days) sufficient to provide a sufficient number of cells for further culturing.
• the culture medium may include growth factors other than or in addition to serum to upregulate one or more of the hereinabove noted therapeutic proteins.
• the cells then are cultured under conditions which promote the formation of spheroidal aggregates of the cells, in one non-limiting embodiment, the cells are cultured as hanging drops.
• Each drop of cells contains mesenchymal stem cells in an amount which provides for optimal expression of the at least one therapeutic protein.
• the hanging drops of the cells are cultured in a medium, such as complete culture medium, containing fetal bovine serum in an amount of up to 20%. In a non-limiting embodiment, the fetal bovine serum is present in an amount of about 17%.
• each hanging drop of mesenchymal stem cells that is cultured contains from about 10,000 to about 500,000 cells/drop. In another non-limiting embodiment, each hanging drop of mesenchymal slem cells that is cultured contains from about 10,000 to about 250,000 cells/drop. In a further non-limiting embodiment, each hanging drop of cells contains from about 10,000 to about 25,000 cells/drop. In yet another non-limiting embodiment, each hanging drop of cells contains about 25,000/drop.
• the hanging drops of mesenchymal stem cells are cultured for a period of time sufficient for forming spheroidal aggregates of the mesenchymal stem cells.
• the drops of cells are cultured for a period of time of up to 4 days.
• the mesenchymal stem cells may, if desired, be dissociated from the spheroids by incubating the spheroids in the presence of a dissociation agent, such as trypsin and/or EDTA, for example.
• a dissociation agent such as trypsin and/or EDTA
• the spheroidal aggregates of mesenchymal stem cells, or mesenchymal stem cells derived from the spheroidal aggregates may be administered to an animal to provide a desired therapeutic effect.
• the animal may be a mammal, including but not limited to, human and non- human primates.
• MSCs can be administered by a route which is suitable for the placement of the transplant, i.e. a biocompatible lattice or a donor tissue, organ or cell, to be transplanted.
• MSCs can be administered systemically, i.e., parenterally, by intravenous injection or can be targeted to a particular tissue or organ, such as bone marrow.
• MSCs can be administered via a subcutaneous implantation of cells or by injection of the cells into connective tissue, for example, muscle.
• MSCs can be suspended in an appropriate pharmaceutical carrier or diluent.
• Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the MSCs and with the recipient, such as buffered saline solution or other suitable excipients.
• the composition for administration can be formulated, produced and stored according to standard methods complying with proper sterility and stability.
• the dosage of the MSCs varies within wide limits and may be adjusted to the individual requirements in each particular case.
• the number of cells used depends on the age. weight, sex, and condition of the recipient, the number and/or frequency of administrations, the disease or disorder being treated, and the extent or severity thereof, and other variables known to those of skill in the art.
• the MSCs of the present invention can be used in conjunction with current modes, for example the use of anti- inflammatory therapy, for the treatment diseases, disorders, or conditions associated with inflammation.
• An advantage of using MSCs in place of or in conjunction with anti-inflammatory agents is that by using the methods of the present invention to ameliorate the severity of inflammation in the recipient, the amount of anti-inflammatory agents used and/or the frequency of administration of anti-inflammatory agents can be reduced.
• a benefit of reducing the use of anti-inflammatory agents is the alleviation of unwanted side effects associated with anti-inflammatory agents.
• the cells of the present invention may be administered into a recipient as a "one-lime" therapy for the treatment of inflammation.
• a one-time administration of MSCs into the recipient eliminates the need for chronic anti-inflammatory therapy. If desired, however, multiple administrations of MSCs may also be employed.
• the invention described herein also encompasses a method of preventing or treating inflammation by administering MSCs in a prophylactic or therapeutically effective amount for the prevention, treatment or amelioration of inflammation.
• An effective amount of MSCs can be determined by comparing the level of inflammation in a recipient prior to the administration of MSCs thereto, with the level of inflammation present in the recipient following the administration of MSCs thereto. A decrease, or the absence of an increase, in the level of inflammation in the recipient with the administration of MSCs thereto, indicates that the number of MSCs administered is a therapeutic effective amount of MSCs.
• the MSCs of the present invention can be used in conjunction with current modes, for example the use of anti-tumor therapy, for the treatment of cancer.
• Cancers which may be treated by the mesenchymal stem cells of the present invention include, but are not limited to, lung cancer, Kaposi's sarcoma, colorectal cancer, glioma, breast cancer, including breast metastases, melanoma, including melanoma metastases, hepatomas, pancreatic cancer, and osteosarcomas.
• An advantage of using MSCs in place of or in conjunction with anti-tumor agents is that by using the methods of the present invention to ameliorate the severity of cancer in the recipient, the amount of anti-tumor agents used and/or the frequency of administration of anti-tumor agents can be reduced.
• a benefit of reducing the use of anti-tumor agents is the alleviation of unwanted side effects associated with anti-tumor agents.
• the cells of the present invention may be administered into a recipient as a "one-time" therapy for the treatment of cancer.
• a one-time administration of MSCs into the recipient eliminates the need for chronic anti-tumor therapy. If desired, however, multiple administrations of MSCs may also be employed.
• the invention described herein also encompasses a method of preventing or treating cancer by administering MSCs in a prophylactic or therapeutically effective amount for the prevention, treatment or amelioration of cancer.
• An effective amount of MSCs can be determined by comparing the level of cancer in a recipient prior to the administration of MSCs thereto, with the level of cancer present in the recipient following the administration of MSCs thereto. A decrease, or the absence of an increase, in the level of cancer in the recipient with the administration of MSCs thereto, indicates that the number of MSCs administered is a therapeutic effective amount of MSCs.
• the MSCs of the present invention can be used in conjunction with current modes, for example the use of immune modulation therapy for graft versus host disease following transplants of bone marrow or organs or to treat or prevent transplant rejection, or for autoimmune diseases such as lupus and autoimmune related diseases such as Type I diabetes, rheumatoid arthritis, thyroiditis, and psoriasis, and auioproliferative diseases- Based upon the disclosure herein, it is envisioned that the MSCs of the present invention can be used in conjunction with current modes, for example the use of therapies to limit programmed cell death as occurs following injury to tissues from lack of oxygen (ischemia or hypoxia) or in diseases such as Alzheimer's disease, parkinsonism, and other neurodegenerative diseases, as well as stroke, brain trauma, or con
• the mesenchymal stem cells in a spheroidal aggregate or the mesenchymal stem cells obtained from a spheroidal aggregate may, in addition to the treatments described hereinabove, be used in other therapies employing mesenchymal stem cells, with the added advantage that the mesenchymal stem cells of the present invention express increased amounts of one or more of the therapeutic proteins hereinabove described.
• the mesenchymal stem cells of the present invention may be administered to an animal, whereby such mesenchymal stem cells differentiate into a desired cell type and/or generate or regenerate a desired tissue in an animal.
• the mesenchymal stem cells of the present invention may be administered to an animal, whereby such mesenchymal stem cells may differentiate into ceils such as osleocytes. adipocytes, chondrocytes, myocytes, astrocytes, oligodendrocytes, neurons, and/or may generate bone, cartilate. ligaments, tendons, adipose tissue, muscle, cardiac tissue, stroma, dermal tissue, and/or other connective tissues in the animal.
• ceils such as osleocytes.
• mesenchymal stem cells of the present invention may be used to provide an animal with any of a plurality of desired cell types, and/or generate or regenerate any of a plurality of desired tissues in an animal, while providing the animal with the therapeutic proteins hereinabove described.
• the mesenchymal stem cells in a spheroidal aggregate, or the mesenchymal stem cells obtained from a spheroidal aggregate may be administered to an animal to repair or regenerate bone, tendons, and or cartilage, including chondrogenesis/knee and joint repair, or may be used as an adjunct therapy through protein production and immune mediation.
• the mesenchymal stem cells in a spheroidal aggregate, or the mesenchymal stem cells obtained from a spheroidal aggregate may be used for the in vivo production of cytokines for the support of cotransplanted hematopoietic stem cells, and for producing enzymes that are deficient in animal models of lysosomal storage disorders.
• mesenchymal stem cells in a spheroidal aggregate may be used to regenerate cardiac tissue and/or effect revascularization of cardiac tissue following myocardial infarction, as well as for repair of intervertebral disc defects and spine therapy, repair of tissue or blood vessel damage caused by stroke, therapy for epilepsy, and skeletal tissue repair.
• the mesenchymal stem cells in a spheroidal aggregate, or mesenchymal stem cells obtained from a spheroidal aggregate also may be employed in treating wounds.
• the mesenchymal stem cells in a spheroidal aggregate, or mesenchymal stem cells obtained from a spheroidal aggregate may be administered syslemically, or may be applied to the wound topically.
• the mesenchymal stem cells in a spheroidal aggregate, or mesenchymal stem cells obtained from a spheroidal aggregate interact with other wound cells through paracrine mechanisms, and interaction with vascular endothelial cells and immuno- modulation accelerate wound healing and reduce scar formation upon completion of the healing process.
• the mesenchymal stem cells in spheroidal aggregates, or mesenchymal stem cells obtained from spheroidal aggregates may be employed in treating diseases or disorders of the lung.
• mesenchymal stem cells in spheroidal aggregates may be employed in treating diseases or disorders of the lung.
• mesenchymal stem cells “home” to areas of diseased or damaged lung tissue, whereby the mesenchymal stem cells may replace or repair the damaged or diseased lung tissue.
• the mesenchymal stem cells in a spheroidal aggregate, or mesenchymal stem cells obtained from a spheroidal aggregate include, but are not limited to, lung cancer, cystic fibrosis, ⁇ I -antitrypsin deficiency, and idiopathic pulmonary fibrosis, or IPF.
• the mesenchymal stem cells in a spheroidal aggregate, or mesenchymal stem cells obtained from a spheroidal aggregate may be employed in treating brain injuries or disorders, and in repairing and/or regenerating brain tissue, as well as repairing and/or regenerating blood vessels in the brain, or promoting angiogenesis in the brain.
• the mesenchymal stem cells in a spheroidal aggregate may be used in repairing and/or regenerating brain tissues, and/or repairing and/or regenerating blood vessels in the brain which have been damaged as a result of a stroke or a concussion.
• mesenchymal stem cells in a spheroidal aggregate or mesenchymal stem cells obtained from a spheroidal aggregate, when administered, escape trapping in the lung, thereby providing an advantage with respect to treating diseases in distal organs, including cancer and other diseases or disorders hereinabove described.
• the mesenchymal stem cells become "pre-activated," i.e., the mesenchymal stem cells in spheroidal aggregates, or mesenchymal stem cells obtained from spheroidal aggregates, express in vivo the increased amounts of the one or more therapeutic proteins described herein immediately upon administration to an animal, as opposed to delayed expression of the therapeutic protein(s) in the animal, e.g., at about 10 to 24 hours after administration of mesenchymal stem cells which are not in the form of spheroidal aggregates or not obtained from spheroidal aggregates.
• mesenchymal stem cells in spheroidal aggregates, or mesenchymal stem cells obtained from spheroidal aggregates, in a non-limiting embodiment may be administered alone, or in combination with drugs or other pharmaceutical agents known to be employed in the treatments hereinabove described.
• the mesenchymal stem cells in spheroidal aggregates, or mesenchymal stem cells obtained from a spheroidal aggregate may be genetically engineered with an exogenous polynucleotide encoding a therapeutic agent.
• polynucleotide may be contained in an appropriate expression vector such as those hereinabove described, and such vector may be introduced into the mesenchymal stem cells of the present invention by means known to those skilled in the art.
• mesenchymal stem cells may be employed in gene therapy treatments, with the advantage that such genetically engineered mesenchymal stem cells express increased amounts of one or more therapeutic agents.
• Applicants also have discovered that media in which spheroidal aggregates have been cultured may provide a therapeutic effect as hereinabove described.
• a method of providing a therapeutic effect in an animal comprising administering to the animal a composition comprising a medium in which there have been cultured previously spheroidal aggregates of mesenchymal stem cells.
• the composition includes the medium in an amount effective to provide a therapeutic effect in said animal.
• the therapeutic effects may be those hereinabove described, including, but not limited to, an anti-inflammatory effect, an anti-tumor effect, or the regulation of an immune response.
• Such medium in a non-limiting embodiment, may be prepared by culturing mesenchymal stem cells in a medium, such as those hereinabove described, which promotes the aggregation and formation of spheroids of mesenchymal stem cells, and under conditions and for a period of time, such as hereinabove described, which also promote the formation of spheroidal aggregates of mesenchymal stem cells.
• a medium such as those hereinabove described, which promotes the aggregation and formation of spheroids of mesenchymal stem cells, and under conditions and for a period of time, such as hereinabove described, which also promote the formation of spheroidal aggregates of mesenchymal stem cells.
• the medium also referred to as a conditioned medium
• a conditioned medium then may be administered to an animal in an amount effective to provide a desired therapeutic effect in the animal.
• the conditioned medium is administered in conjunction with an appropriate pharmaceutical carrier or diluent, such as, for example, buffered saline solution or other excipients as hereinabove described.
• an appropriate pharmaceutical carrier or diluent such as, for example, buffered saline solution or other excipients as hereinabove described.
• the composition can be formulated, produced, and stored according to standard methods complying with proper sterility and stability.
• the dosage of the conditioned medium varies within wide limits and may be adjusted to the individual requirements in each particular case.
• the amount of media to be administered depends on the age, weight, sex. and condition of the recipient, the number and/or frequency of administrations, the disease or disorder being treated, and the extent or severity thereof, and other variables known to those skilled in the art.
• Example 1 Unique characteristics of human mesenchymal stromal cells derived from multicellular spheroid cultures Human mesenchymal stromal cells (hMSCs) show great promise for the repair of damaged and dysfunctional tissues. Our preliminary studies showed that hMSCs cultured as hanging drops aggregated into spheroids with anti-inflammatory propensities. In the current work we sought to define the unique characteristics of the spheroid derived cells (SDCs) in comparison to hMSC monolayer cultures and spheroids derived from other cell types. To obtain SDCs for further studies, spheroids were dissociated with trypsin/EDTA and the cells were harvested by centrifugation.
• SDCs spheroid derived cells
• SDCs By microscopy the measured diameter of SDCs was 1 /2 and the volume approximately 1/4 of the monolayer hMSCs. Interestingly, the SDC size was independent of the initial cell number within the hanging drop, and therefore, spheroid size. High SDC viability also was confirmed by microscopy as well as flow cytometry utilizing trypan blue and Annexin/Pl. Moreover, SDCs maintained their mesenchymal characteristics as determined by flow cytometry (CD73+, CD90+, CD105+) and CFU-F assays; however, SDC expression of CD49b, was increased, while PODXL, CD49d, and CD49f were decreased.
• hMSC spheroids did not grow, nor did the cells divide significantly (less than 3% in S-phase) within these aggregates similar to fibroblasts, but in contrast to neurospheres and cancer spheres. Also in contrast to other sphere types, anti-tumorigenic (TRAIL and IL-24) and anti-inflammatory (TSG-6, STC-1, and LIF) genes were up-regulated in hMSC spheroids. The elevated secretion of these anti-inflammatory factors in hMSC SDCs was measured with Kl.ISAs and the anti-inflammatory properties were demonstrated using a co-culture system with LPS activated macrophages and in a mouse model of peritonitis. In conclusion, we determined distinct features of hMSC SDCs in terms of morphology, phenotype, growth, viability, and antiinflammatory characteristics. These results suggest that SDCs are unique pre-activated small cells that could show beneficial effect in regenerative therapies.
• nucleated cells were isolated from bone marrow aspirates by density gradient centrifugation and resuspended in complete culture medium (CCM): a-MK , 17% FBS, penicillin/streptomycin, L-glutamine. Nucleated cells were plated and after 24 hours nonadherent cells were discarded. Adherent cells were expanded until approximately 70% confluent, harvested with trypsin/KDTA, and replated at 50 cells/cm 2 in cell factories. The cells were expanded until 70% confluent then frozen as passage 1 ceils. Frozen vials of passage I MSCs were thawed, plated at 100 cells/cm 2 , and further expanded for 7 days prior to freezing.
• CCM complete culture medium
• MSCs derived from cultured spheroids proliferate slowly but remain highly viable
• MSCs were plated as monolayers at 100 cells/cm 2 for 6 days (Adh 6), at 5,000 cells s/cm 2 for 3 days (Adh), and as hanging drops at 25,000 cells/drop for 3 days.
• Adherent MSCs were harvested with trypsin/EDTA.
• SDCs spheroids were collected and dissociated with trypsin/EDTA.
• Figure 2A For cell cycle analysis, MSCs were fixed with 70% Ethanol, washed in PBS, incubated with RNase, then stained with Prodium iodide (PI ) overnight.
• PI Prodium iodide
• MSC spheroid-derived cells are significantly smaller than MSCs cultured as monolayers
• MSCs cultured in hanging drops display a unique expression profile of anti-inflammatorv. anti-apoptotic. immune modulatory, and anti-tumorigenic genes
• Multi-cellular spheroids generated by suspending MSCs in hanging drops (25,000 cells/drop), were dissociated with trypsin/EDTA.
• the cells acquired (SDCs) were plated in 6- well plates at 200,000 cells/well in 1 .5 ml of CCM. After 24h, conditioned medium was harvested and utilized for KLISAs. The results obtained were compared to MSCs derived from adherent monolayer cultures (Adh) seeded at equal density/volume and also cultured for 24h. Mean values are shown as secreted TSG-6, STC- 1 , or LIF (pg/ml). Error bars represent standard deviations. Experiments were performed in triplicate. ( Figure 5).
• TNFq levels produced from LPS stimulated macrophages were decreased
• Mouse macrophages (mM ⁇ J>), seeded in the upper chamber of a transwell (4.67cm ? growth area, 0.4 ⁇ m pores) at 400,000 cells/well, were stimulated with 0.1 ⁇ /ml of LPS for 90min. After LPS was removed, MSCs, derived from adherent monolayer cultures (Adh) and from spheroids generated by hanging drop technique (SDC), were plated beneath the transwell chamber at 200,000 cells/well. After 5 hours, medium conditioned by mM l was collected for mT F-a ELISA ( Figure 6A).
• hMSCs suspended at high cell densities in hanging drops aggregate to form compact multi-cellular spheroids mesenchymal surface features and growth characteristics are maintained and or reacquired in the majority of hMSCs derived from multi-cellular spheroids; hMSCs cultured as multi-cellular spheroids in hanging drops show low proliferation rates but remain highly viable; hMSC spheroid-derived cells are significantly smaller than MSCs cultured on adherent dishes suggesting that SDCs may have enhanced mobility in the vasculature following infusion.
• hMSC spheroids In contrast to aggregates of human fibroblasts, cancer cells and neural progenitors, it was observed that hMSC spheroids exhibited a robust upregulation in expression of numerous anti-inflammatory (e.g., TSG-6, STC- l, LIF) and anti-tumorigenic (e.g., IL- l , TNFSF10, 1L-24) genes. Significant upregulation of the homing receptor CXCR4 and the pro-angiogenic factor IL-8 also was exclusive to MSC spheroids.
• TSG-6 TSG-6, STC- l, LIF
• anti-tumorigenic e.g., IL- l , TNFSF10, 1L-24
• hMSC spheroid-derived cells reduced TNF-a secreted by mouse macrophages and attenuated inflammatory response elicited by zymosan in a mouse model of peritonitis.
• hanging drop technique is useful in preprogramming MSCs to express therapeutically beneficial anti-inflammatory, anti-apoptotic, immune modulatory, and anti-tumorigenic proteins prior to transplantation.
• Example 2 Aggregation of human mesenchymal stromal cells into three dimensional multicellular spheroids enhances their anti-inflammatory properties
• TSG-6 After 72-96 hours in hanging drops the expression of TSG-6 peaked, more than 1000-fold over their monolayer counterparts, but the viability of the cells derived from spheroids declined significantly after 72 hours.
• hMSC spheroids but not monolayer hMSCs, significantly suppressed the mTNF-a secretion by LPS stimulated macrophages in a co-culture system, verifying the anti-inflammatory capacity of the spheroids.
• hMSC spheroids were anti-inflammatory in a mouse model of peritonitis.
• hMSCs can be activated to secrete high amounts of anti-inflammatory cytokines, without chemical induction, by culturing them in hanging drops or on low adherent dishes as spheroids.
• Human MSCs were isolated from 1 -4 ml bone marrow aspirates taken from the iliac crest of normal adult donors. Nucleated cells were isolated with density gradient and resuspended in complete culture medium (CCM): a-ME , 17% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. Nucleated cells were plated on a 175 cm 2 culture flask and incubated at 37°C with 5% C0 2 . After 24 hours, nonadherent cells were discarded.
• CCM complete culture medium
• Adherent cells were incubated 4- 1 1 days until approximately 70% confluent, harvested with 0.25% trypsin and 1 mM EDTA for 5 mm at 37°C, and replated at 50 cells/cm 2 in an intercommunicating system of culture flasks. The cells were incubated 7- 12 days until approximately 70% confluent, harvested with trypsin/EDTA, and frozen in 5% DMSO and 30% FBS as passage I cells. A frozen vial of passage 1 MSCs (donor 1 or 2) was thawed and the cells were plated in a 145 cm 2 culture dish in CCM.
• MSCs were plated either as hanging drops on the lid of a culture dish in 35 ⁇ of CCM (25,000 cells/drop), or on non-adherent surface at 200,000 cells/ml for up to 3 days. Scale bar 100 ⁇ . It was observed that hMSCs aggregated rapidly into three dimensional spheroids when grown in hanging drops or on a nonadherent surface ( Figure 8). hMSCs in smaller spheroids show high viability
• MSCs from 2 donors were plated at 100 cells cm 2 and grown as monolayers for 7-8 days before harvesting (Adh-St) for viability assay by FACS using Annexin (Anx) and Propidium iodide (PI) staining.
• Harvested cells were also plated on adherent dishes at 5000 cells/cm 2 (Adh), on nonadherent dishes at 200,000 cells/ml (Non-adh), and as hanging drops (Drop) at different cell densities; 10.000 ( 10k), 25,000 (25k), 100,000 ( 100k), and 250,000 (250k). Cells from all conditions were harvested for viability assay at day 3.
• Viability assay also was performed on cells from monolayer (Adh) and 25k spheroid cultures (25k Drop) at day 1 ( Id), day 2 (2d), day 3 (3d), and day 4 (4d). It was observed that hMSCs in smaller spheroids show high viability ( Figure 9). hMSC spheroids express high levels of anti-inflammatory molecule TSG-6
• MSCs from 2 donors were plated at 100 cells/cm 2 and grown as monolayers for 7-8 days before harvesting for RNA (Adh-St) and subsequent cultures.
• Harvested cells were plated on adherent dishes at 5000 cells/cm 2 (Adh), on non-adherent dishes at 200,000 cells/ml (Non-adh), and as hanging drops (Drop) at different cell densities; 10,000 ( 10k), 25,000 (25k), 100,000 ( 100k), and 250,000 (250k).
• Cells were harvested for RNA at day 1 ( Id), day 2 (2d), day 3 (3d), and day 4 (d4).
• MSCs from 2 donors were plated at 100 cells/ cm 2 and grown as monolayers for 7 days before harvesting for RNA (Adh-St) and subsequent cultures.
• Harvested cells were plated on adherent dishes at 5000 cells/cm 2 (Adh), on non-adherent dishes at 200,000 cells/ml (Non-adh), and as hanging drops at 25.000 cells/drop (25k-Drop).
• Cells were harvested for RNA at day 3 (3d) and prepared for microarray using Whole Transcript Sense Target Labeling Assay. Labeled and fragmented samples were hybridized on Human Exon 1.0 ST arrays and gene level analysis was performed with Partek Genomics Suite 6.4.
• Microarray results were confirmed with real-time RT-PCR for several anti-inflammatory (TSG-6, STC- 1 , and LIF) and anti-tumorigenic (1L-24 and TRAIL) molecules, and a Wnt signaling inhibitor (DK 1); Figure 12.
• Results are shown as relative quantity (RQ) compared to MSCs plated at 100 cells/cm 2 on adherent dishes and grown for 7 days (Adh St). MSCs were grown for 3 days on adherent dishes at 5000 cells/cm 2 (Adh 3d), on non-adherent dishes at 200.000 cells/ml (Non-adh 3d), and as hanging drops at 25,000 cells/drop (25k-Drop 3d). 18S was used as an endogenous control. Error bars are 95% confidence intervals from triplicate reactions. hMSC spheroids secrete large amounts of anti-inflammatory cytokines
• MSCs were plated at 100 cells/cm 2 and grown as monolayers for 7 days. Cells were harvested and plated at 5000 cells/cm 2 on adherent dishes and as hanging drops at 25,000 cells/drop. After 3 days, monolayer cultures were harvested and plated on adherent dishes (Adh) at 200,000 cells/well in 1.5 ml of CC in triplicate. Spheroids grown for 3 days were plated either on adherent (25k-Adh) or non-adherent (25k-Non adh) dishes at 8 spheroids/well in 1 .5 ml of CCM in triplicate. Conditioned medium was collected after 24 hours, cleared from cellular material, and used for Kl.lSAs.
• Mouse macrophages were plated in the upper chamber of the transweli (0.4 ⁇ ) at 400,000 cells/ well. Cells were stimulated with 0.1 g/ml of LPS in DM EM supplemented with 10% FBS and penicillin streptomycin. After 90 min, LPS was removed and replaced with fresh medium and MSCs were plated in the bottom chamber of the transweli at 200,000 cells/well (Adh), or 8 spheres/well (25k). After 5 hours, ⁇ were harvested for RNA and subsequent real-time RT-PCR for mTNF-a. in addition, conditioned medium was harvested for T F- ⁇ EL1SA ( Figure 14). Error bars are standard deviations from triplicate experiments. ANOVA was used to determine the significance levels.
• C57BL/6 mice were injected IP with 1% zymosan in I ml of HBSS. Total of 1 .5 x I0 6 monolayer MSCs (Adh) or 60 spheres (25k) were injected IP 15 min. later in 160 ⁇ 1 of HBSS. A ter 24 hours, blood was collected from the right ventricle and serum was isolated. Plasmin activity was measured from the serum with Chromozym PL cleavage reaction ( Figure 15). Ftrror bars are standard deviations for 4-6 animals per group. ANOVA was used to determine the signi icance levels.
• hMSCs aggregate rapidly into spheroids when grown in hanging drops or on non-adherent dishes; hMSC spheroids show high viability and TSG-6 expression when grown as small spheroids (25k) for 3 days; hMSC spheroids express high levels of anti-inflammatory (TSG-6, STC- 1 , and LIF) and anti- tumorigenic (IL-24 and TRAIL) molecules and secrete high levels of the anti-inflammalory proteins; hMSC spheroids show ami -inflammatory effects in an in vitro inflammation assay and are anti-inflammatory in a mouse model of peritonitis. The results suggest that hMSC spheroids are useful as an anti-inflammatory therapy in many diseases.
• hMSC Cell Culture Frozen vials of passage 1 hMSCs from bone marrow were obtained from the Center for the Preparation and Distribution of Adult Stem Cells (http://'Medicine.tamhsc.edu/irm/msc-distrtbution.html). After 24-hours recovery, hMSCs were seeded at low density ( 100 cells/cm 2 ), and incubated in complete culture medium (CCM) containing 17% FBS for 7-8 days until approximately 70% confluent. hMSCs were passed under the same conditions through no more than three passages before being used for assays.
• CCM complete culture medium
• hMSCs Spheroid Generation and Dissociation.
• hMSCs were plated in hanging drops in 35 ⁇ of CCM containing 10,000-250,000 cells/drop for up to 4 days.
• spheroids were incubated with trypsin/EDTA for 5-30 min (depending on the size of the spheroid) while pipetting every 2-3 min.
• hMSC spheroids were dependent on the number of hMSCs suspended in the hanging drops (Fig. 16E).
• hMSC spheroids of all sizes expressed and secreted very high levels of the anti-inflammatory molecule TSG-6 compared with either low or high density monolayer cultures, but spheroids of 25,000 cells (Sph 25k) showed the highest expression and secretion of TSG-6 (Fig. 16C and D).
• TSG-6 expression increased in a time dependent manner with spheroids of 25,000 hMSCs and consistently was much higher than in standard cultures of adherent hMSCs (Fig. 16F).
• hMSCs in Spheroids may have less access to nutrients, it was of interest to establish whether the cells remained viable.
• 3 day cultures of spheroids of 10,000 or 25.000 hMSCs. almost 90% of the harvested cells were viable as assayed by propidium iodide (PI) uptake and labeling with annexin V-FITC (Fig. 17A).
• PI propidium iodide
• Fig. 17A The number of apoptotic or necrotic cells was greater in spheroids prepared with 100,000 or 250,000 hMSCs (Fig. 17A).
• the number of apoptotic or necrotic cells increased slightly when the incubation period was extended from 3 days to 4 days (Fig. 17B).
• hMSCs in spheroids appeared smaller than hMSCs from standard monolayer cultures.
• the cells released from spheroids by tripsinization were nearly half the diameter and approximately one- fourth the volume of hMSCs derived from adherent monolayers as shown by flow cytometry (Fig. I 8A) and microscopy (Fig. 18B).
• both monolayer and spheroid hMSCs were injected i.v. into the tail vein of NOD/scid mice.
• Real-time PCR for human Alu sequences in the lungs collected 15 min after hMSC infusion suggested that the number of trapped cells decreased by about 25% with spheroid-derived hMSCs compared with monolayer hMSCs.
• a larger fraction of infused spheroid hMSCs were recovered in the liver, spleen, kidney, and heart (Fig. 18C).
• hMSCs Dissociated from Spheroids Retain the Properties of Adherent hMSCs.
• hMSCs dissociated from spheroids retained the ability to differentiate into mineralizing cells and adipocytes (Fig. 1 A and B).
• the dissociated cells expanded more slowly during an initial passage and then more rapidly than adherent hMSCs through four passages before reaching senescence at about the same number of population doublings (Fig. 19C).
• the dissociated cells readily generated colonies (CFUs) when plated at clonal densities ( Fig. 19D). Consistent with the data on rates of propagation (Fig.
• the number of CFUs from spheroid cells was initially less than the number of CFUs from adherent cultures but was greater in later passages (Fig. 19D).
• the surface epitopes of the hMSCs dissociated from spheroids were similar to the surface epitopes of hMSCs from adherent monolayers when dissociated under the same conditions with trypsin ( 10 mm at 37°C): the dissociated cells were negative for hematopoietic markers, and they were slightly less positive for CD73, CD90, and CD 105, apparently because of the smaller size of the cells (Fig. 1 E).
• Fig. 21 A Real time RT PCR assays demonstrated marked increases in the expression of TSG-6; stanniocalcin-1 (STC-I), an anti-inflammatory/ anti-apoptotic protein; leukemia inhibitory factor (LIF), a cytokine for growth and development; IL-24, a tumor suppressor protein; TNF--a related apoptosis inducing ligand (TRAIL), a protein with selectivity for killing certain cancer cells: and CXC chemokine receptor 4 (CXCR4), a receptor involved in SC homing.
• STC-I stanniocalcin-1
• LIF leukemia inhibitory factor
• TRAIL TNF--a related apoptosis inducing ligand
• CXCR4 CXC chemokine receptor 4
• Spheroid hMSCs Secrete Anti-inflammatory Proteins.
• Spheroids of hMSCs plated on adherent culture surfaces gradually generated spindle-shaped cells that migrated away from the spheroids (Fig. 2 I B). No migration was seen with spheroids plated on non-adherenl surfaces (Fig. 21 B).
• ELlSAs demonstrated that hMSCs either in spheroids or dissociated from spheroids continued to secrete TSG-6, STC-I , and LIF when plated on culture dishes for 24 hours (Fig. 2I C-E). The levels of all three factors were much higher than with adherent monolayer hMSCs.
• Spheroid hMSCs Decrease Activation of Macrophages in Vitro and Inflammation in Vivo.
• the increased secretion of anti-inflammatory molecules TSG-6 and STC- 1 by the spheroid hMSCs suggested that the cells would be more effective than adherent monolayer cultures of hMSCs in reducing inflammatory responses.
• mouse macrophages were pre-activated with LPS in the upper chamber of a transwell, followed by a transfer of the chamber to a test well (Fig. 22A). Under the conditions of the experiment, the presence in the lest well of hMSCs from adherent monolayers had no significant effect on the expression or secretion of TNFa by the stimulated macrophages (Fig, 22B).
• STC-1 is important because STC- I also reduces reactive oxygen species, or ROS. ROS are an early trigger for inflammation, and apoptotic at high levels. Thus, STC-I is anti-inflammatory and anti-apoptotic.
• hMSC spheroids significantly decreased the protein content of the lavage fluid and the volume , neutrophil activity, as assayed by secreted myeloperoxidase (MPO) (Fig. 22D), and levels of the pro-inflammatory molecules TNFa (Fig.
• hMSC spheroids serum levels of plasmin activity, an inflammation associated protease that is inhibited by TSG-6 (Wisniewski, et al., Cytokine Growth Factor, Vol. 15, pgs. 129-146 (2004)), were decreased significantly by hMSC spheroids (Fig. 22F). Serum plasmin activity was reduced approximately to the levels of non- inflammatory control animals 24 hours after spheroid injection (Fig. 22F). Spheroid-derived hMSCs also substantially decreased levels of the inflammatory markers assayed, although to a lesser extent than intact spheroids (Fig. 22 C-F). Moreover. hMSC spheroids were significantly more effective than adherent monolayer hMSC in suppressing inflammation (Fig. 22 C-F).
• hMSCs cultured as spheroids depend critically on the experimental conditions.
• the cells In hanging drops, the cells first formed a network and then most of the cells coalesced into a single spheroid.
• Optimal levels of TSG-6 expression were observed with spheroids approximately 500 ⁇ in diameter and incubated for 3 days. Expression levels remained high but were lower in larger spheroids, and more of the cells became apoptotic or necrotic in the larger spheroids. Also, more of the cells became apoptotic or necrotic with longer times of incubation.
• the cells in spheroids retained most of the surface epitopes of hMSCs from adherent cultures.
• hMSCs dissociated from spheroids retained the potential to differentiate into mineralizing cells and adipocytes. They also expanded at a similar rate as hMSCs from adherent monolayer cultures after a delay through one passage. In addition, spheroid-dissociated hMSCs remained highly clonogenic.
• both spheroids and spheroid derived hMSCs may be more effective than hMSCs from adherent cultures in modulating inflammator reactions.
• the suggestion was confirmed by the demonstration that the spheroids and spheroid derived hMSCs were more effective in suppressing TNFa production by LPS stimulated macrophages in culture. In addition, they were more effective in suppressing inflammation in an in vivo model for zymosan induced peritonitis.
• the spheroid hMSCs expressed high levels of transcripts for the tumor suppressor protein IL-24, an observation made previously with 3D cultures of hMSCs prepared using spinner flasks and a rotating wall vessel bioreactor (Frith, 2010).
• the spheroid hMSCs prepared under the conditions optimized to express TSG-6 also expressed high levels of transcripts for TRAIL that is selective for killing certain cancer cells (Mahmood, et al., Exp. Cell Res., Vol. 316, pgs. 887-899 (2010); Mellier, et al, Mol. Aspects Med., Vol. 31, pgs.
• hMSC spheroids and spheroid derived hMSCs may be particularly effective as an adjunct therapy for some types of cancers, particularly for therapy of cancers sensitive to anti -inflammatory agents such as aspirin or steroids (Grivennikov, et al., Cell, Vol. 140, pgs.
• a further advantage of the spheroid hMSCs was that they were less than one-fourth the volume of hMSCs from adherent cultures. Therefore a significantly smaller number was trapped in the lung after i.v. infusion and thus larger numbers were found in many tissues (Lee Cell Stem Cell, 2009; Lee, Blood, 2009).
• hMSCs can be activated non-chemically in hanging drops to secrete substantial quantities of potent anti-inflammatory proteins and express anti-tumorigenic molecules. Therefore spheroid hMSCs may have advantages for many therapeutic applications. In addition, hMSCs dissociated from spheroids provide extremely small activated cells that could have major advantages for i.v. administration.
• EXAMPLE 4 Spheroids of human mesenchymal stem cells were cultured in complete culture medium (CCM) containing a-MEM with 17% FBS, human serum albumin (HuSA) (Baxter Healthcare) Stem Pro Xeno-free Medium (Stpro) (Gibco), or Mesencult Xeno-free Medium (Stem Cell Technologies), or Mes. After 3 days, the spheroids were dissociated using trypsin/EDTA, and then were frozen in dimethylsulfoxide (DMSO). The cells then were thawed and the viability of the cells was determined by flow cytometry, measuring PI uptake and annexin V-FITC cell surface labeling. Unlabeled cells were considered viable. As shown in Figure 23, the culturing of spheroids of mesenchymal stem cells in different media does not affect their viability.
• CCM complete culture medium
• HuSA human serum albumin
• Stpro Stem Pro Xeno-free Medium
• Spheroids of human mesenchymal stem cells were cultured for 3 days in CCM or in various commercially available Xeno-free media as described in Fxample 4. The spheroids then were dissociated using trypsin EDTA, and then were frozen in DMSO. After a minimum of 1 week, the cells were thawed and then labeled with the viability dyes calcein AM and 7AAD to exclude dead cells from the analysis. Representative linear scatter plots determined through flow cytometric determination of the viable population are shown in Figure 24. Size was quantified by comparing forward scatter (FS) properties of the cells with beads of known diameter (3, 7, 15, and 25 ⁇ ). Brackets were applied to the scatter plot at locations corresponding to the appropriate bead size.
• FS forward scatter
• Figure 24 shows that the mesenchymal stem cells dissociated from spheroids are larger, as shown by greater forward scattering of light (FS Lin) when the spheroids are cultured in media that do not contain fetal calf serum (FCS) that is found in the complete culture medium (CCM).
• FCS fetal calf serum
• the therapeutic proteins TSG-6. STC-1 , and GDF- 1 5 are not expressed when the spheroids are cultured in media that does not contain fetal calf serum; however, the addition of human serum albumin (HuSA) increases expression of such proteins.
• Human serum albumin Human serum albumin
• Spheroids of human mesenchymal stem cells were cultured for 3 days in the commercially available Xeno-free media described in Example 4.
• the media conditioned by the spheroids (conditioned medium, or CM) were collected, diluted 1 :50, and added to mouse macrophages in the presence of 100 ng/ml LPS.
• Macrophages cultured with LPS (sMO) or without LPS (MO), and with non-conditioned media in the presence of LPS, served as controls. After 18 hours, the macrophage media were harvested and assayed for mTNFet by KLISA. Values are mean ⁇ SD (n 3).
• CM conditioned media
• CCM fetal calf serum

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