Oligonucleotides stimulatory of the mesenchymal stem cell proliferation and uses thereof

This invention relates to the use of oligonucleotides for the manufacture of pharmaceutical compositions which induce "in vitro" and "in vivo" proliferation of MSCs. More in details it refers to oligonucleotides having about 14 to 100 nucleotides that have the ability to greatly stimulate the proliferation of MCSs in animals, including human.

These oligonucleotides have the ability to greatly stimulate the proliferation of pluripotent mesenchymal stem cell "in vitro" and "in vivo" of animals, including humans are disclosed. They can be used in a wide range of clinical procedures such as (1) regeneration of mesenchymal tissues which have been damaged through acute injury, abnormal genetic expression or acquired disease by inoculation of the ODNs of this invention; (2) treatment of a host with damaged mesenchymal tissue by removal of small aliquots of bone marrow, isolation of their mesenchymal stem cells and treatment of the damaged tissue with MSCs culture-expanded by incubation with one or more of the ODNs of this invention combined with a biocompatible carrier suitable for delivering the MSCs to the damaged body site(s); (3) production "in vitro" of various mesenchymal tissues by directed differentiation of the MSCs culture- expanded by incubation with one or more of the ODNs of this invention, to replace and restore tissue damage or defects with say "in vitro" obtained mesenchymal tissues combined with a biocompatible carrier suitable for delivering the "in vitro" produced tissues to the damaged body site(s); and (4) treatment of a host with abnormal genetic expression with MSCs culture- expanded by incubation with one or more of the ODNs of this invention and transformed by genetic engineering procedures to express a protein able to replace the genetic deffect.

Methods for generation of mesenchymal tissues in an area of an animal where say tissues are deficient are provided. They consist in the local or systemic administration of a composition comprising one or more of the oligonucleotides to the animal, in a pharmaceutically acceptable carrier. The composition is administered in an amount effective to induce repair at the damaged site.

Methods for extensive proliferation of MSCs of an animal "in vitro" are also provided. They consist in incubation of small aliquot of bone marrow extracted from the animal, "in vitro" incubation of the cells present in say aliquot of bone marrow in a culture media containing an effective concentration of one or more of the ODNs of this invention for a time period sufficient to obtain the amount of cells necessary for a successful medical treatment. BACKGROUND OF THE INVENTION

Mesenchymal stem cells (MSC) also known as marrow stromal cells or mesenchymal progenitor cells, are blast cells found, among other body sites, in bone marrow, blood, dermis and periosteum that are capable of differentiating "in vitro" into bone, cartilage, fat, tendon, muscle, hepatic, renal, cardiac, and neural cells depending upon influences from various bioactive factors (Alhadlaq, A and Mao, JJ. Stem Cells Dev. 13:436, 2004). Also, cultured MSC have the ability to differentiate into different cell specific lineages when placed "in vivo" at the site of a given damaged tissue. Therefore, MSC are a valuable material for medical applications such as systemic transplantation for systemic diseases, local implantation for local tissue defects, gene therapy protocols or generation of transplantable tissues and organs in tissue engineering protocols. In order to isolate MSC, it is necessary to separate them from other cells in the bone marrow or other MSC source. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullar spaces. Other sources of MSC include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood and other mesenchymal stem cell tissues. A number of processes have been developed for isolation, purification and replication of MSC in cultures (Alhadlaq, A and Mao, JJ. Stem Cells Dev. 13:436, 2004). However, limited proliferative potential of MSC in addition to a very low number in the bone marrow or other sources have so far limited their clinical applications. Immortalization of MSC using viral transforming proteins has been described. However, transformed cells are unlikely to be accepted for standard medical treatments own to its possible tumor inducing potential. DESCRIPTION OF THE INVENTION

We now disclose that oligonucleotides having about 14 to 100 nucleotides, preferably from 14 to 40, even more preferably from 14 to 26, are compounds with potent activity to stimulate the proliferation of pluripotent mesenchymal stem cell of animals, including humans, "in vitro" and "in vivo". According to preferred embodiments, the oligonucleotides to be used in accordance to the present invention consists of 25 or less nucleotides and are not antisense. According to one embodiment of the invention, a preferred oligonucleotide has at least one subsequence with the following composition: PyNTTTTNT, wherein Py is C or T and wherein N is any deoxyribonucleotide. Suitable oligonucleotides may be selected from the group consisting of:

TCATCATTTTGTCATTTTGTCATT (SEQ ID N°2); TCCTCCTTTTGTCCTTTTGTCCTT (SEQ ID N°3); TCTTCTTTTTGTCTTTTTGTCTTT (SEQ ID N°4); TTGTTGTTTTGTTGTTTTGTTGTT (SEQ ID N°7); TCATTTTTTTGTTTTTTTGTCATT (SEQ ID N ⁰ IO); TCATTGTTTTGTTGTTTTGTCATT (SEQ ID N ⁰ I l); TCATTCTTTTGTTCTTTTGTCATT (SEQ ID N°12); TCATTATTTTGTTATTTTGTCATT (SEQ ID N°15); TCATCCTTTTGTCCTTTTGTCATT (SEQ ID N°17); TCATCTTTTTGTCTTTTTGTCATT (SEQ ID N°18); CATTTTGTTTTTTTTTTTTTTTTT (SEQ ID N° 20); TTCATTTTGTTTTTTTTTTTTTTT (SEQ ID N°21); TTTTCATTTTGTTTTTTTTTTTTT (SEQ ID N°22); TTTTTTCATTTTGTTTTTTTTTTT (SEQ ID N°23); TTTTTTTTCATTTTGTTTTTTTTT (SEQ ID N°24);

TTTTTTTTTTCATTTTGTTTTTTT (SEQ ID N°25); _{TTTTTTTTTTTTCATTTTGTTTTT (} - _SE Q _{ID NO2} φ.

_{TTTTTTTTTTTTTTCATTTTGTTT} ^ _SE Q _{ID NO27} ^ _{TTTTTTTTTTTTTTTTCATTTTGT} ^ _{SEQ ID NO 28} ^.

TTTTCATTTTGTCATTTTGTTTTT (SEQ ID N°29); TCATCAATTTGTCAATTTGTCATT (SEQ ID N°30);

ACATCATTTTGTCATTTTGTCATT (SEQ ID N°46); CCATCATTTTGTCATTTTGTCATT (SEQ ID N°47); GCATCATTTTGTCATTTTGTCATT (SEQ ID N°48); TAATCATTTTGTCATTTTGTCATT (SEQ ID N°49); TTATCATTTTGTCATTTTGTCATT (SEQ ID N°50); TGATCATTTTGTCATTTTGTCATT (SEQ ID N°51); TCCTCATTTTGTCATTTTGTCATT (SEQ ID N°52); TCTTCATTTTGTCATTTTGTCATT (SEQ ID N°53); TCAACATTTTGTCATTTTGTCATT (SEQ ID N°55); TCACCATTTTGTCATTTTGTCATT (SEQ ID N°56); TCAGCATTTTGTCATTTTGTCATT (SEQ ID N°57); TCATCATTTTGTCATTTTGTAATT (SEQ ID N°58); TCATCATTTTGTCATTTTGTTATT (SEQ ID N°59); TCATCATTTTGTCATTTTGTGATT (SEQ ID N°60); TCATCATTTTGTCATTTTGTCCTT (SEQ ID N°61); TCATCATTTTGTCATTTTGTCTTT (SEQ ID N°62); TCATCATTTTGTCATTTTGTCAAT (SEQ ID N°64); TCATCATTTTGTCATTTTGTCACT (SEQ ID N°65); TCATCATTTTGTCATTTTGTCAGT (SEQ ID N°66); TCATCATTTTGTCATTTTGTCATA (SEQ ID N°67); TCATCATTTTGTCATTTTGTCATC (SEQ ID N°68); TCATCATTTTGTCATTTTGTCATG (SEQ ID N°69); TCATCAATTGGTCAATTGGTCATT (SEQ ID N°75); TCATCAACTGGTCAACTGGTCATT (SEQ ID N°77); TCATCATTGTGTCATTGTGTCATT (SEQ ID N°84); TCATCATTTGGTCATTTGGTCATT (SEQ ID N°87); TGCTGCTTTTGTGCTTTTGTGCTT (SEQ ID N°91); TCATCATCTTGTCATCTTGTCATT (SEQ ID N°95); TCATCATGTTGTCATGTTGTCATT (SEQ ID N°96);

GGGGGTCTTTTTTTCTTTTTTTTT (SEQ ID N°107); _{TTTTTTCTTTTTTTCTTTTTTGGG} ^ _{SEQ ID N} o ₁₀₈ ^.

AAAAATCTTTTTTTCTTTTTTTTT (SEQ ID N°109);

TGCTGCTTTTATGCTTTTATGCTT (SEQ ID N ⁰ I lO); TCATCATTCTGTCATTCTGTCATT (SEQ ID N ⁰ I I l); XTXTXTCTTTTTTTCTTTTTTTTT (SEQ ID N°112); TGCTGCTTTTCTGCTTTTCTGCTT (SEQ ID N°113); XXTTTTCTTTTCTTTTTTTTTTTT (SEQ ID N°114); TTTTTCCTTTTTTCCTTTTTTTTT (SEQ ID N°115);

CCCCCTCTTTTTTTCTTTTTTTTT (SEQ ID N°116); _{ττττττcτττττcτττττττττττ} ^ _{SEQ ro N} o _{1 17} ^. _{ττττττcτττττττcττττττccc} ^ _{SEQ ID N} o _{1 1 8} ). _{ττττττcτττττcτcτττττcτcτ (} - _SE Q _{ro J} ^ ₁₁₉ J.

TTTTTGCTTTTTTGCTTTTTTTTT (SEQ ID N°120); TTTTTACTTTTTTACTTTTTTTTT (SEQ ID N°121);

TCATAATTTTGTAATTTTGTCATT (SEQ ID N°122); _{TTTTTTCTTTTTTTCTTTTTTAAA} ^ _{SEQ ro N} o ₁₂₃ ). _{ττττττcττττττcττττττττττ} ^ _{SEQ ID N} o ₁₂₄ ^. _{TTTTTTTTTTTTCATTTTGTGGGG} ^ _{SEQ ro N} o ₁₂₅ ^. _{TTTTTTTTTTTTCATTTTGTTTTG} ^ _{SEQ ID N} o ₁₂₆ ^.

GGGTTTTTTTTTCATTTTGTTTTT (SEQ ID N°127); _{GTTTTTTTTTTTCATTTTGTTTTG (} - _SE Q _{ID No128} ).

_{TTTTTTTTTTTTCATTTTGTTTGG} ^ _SE Q _{ro J} ^ ₁₂₉ J. _{TTTTTTTTTTTTCATTTTGTTTTA} ^ _SE Q _ro JJ ₀ J ₃₀ ^ _{TTTTTTTTTTTTCATTTTGTTTGA} ( _{SEQ ID} J^ ₁₃₁ ). _{TTTTTTTTTTTTCATTTTGTTTAG} ^ _{SEQ ID} N _O132 ). _{TTTTTTTTTTTTCATTTTGTTTAA} ^ _{SEQ ID} N _O1 33)

It is an object of the present invention to provide one or more of the oligonucleotides of this invention to an animal with a tissue deficiency to produce normal new normal tissue where it is needed. Another object of the present invention is to provide a method to enhance the replication of pluripotent mesenchymal stem cell "in vitro" so that an adequate number of these cells can be obtained for successful medical treatments. These objects will become apparent to those skilled in the art.

The first of the above objects is achieved by providing a method for tissue regeneration at a site of an animal where the tissue is deficient and which consists in the administration of an effective amount of a composition comprising one or more of the oligonucleotides of this invention to the animal, locally at the site or systemically as needed in each case, in a pharmaceutically acceptable carrier, the composition being administered in an amount effective to induce tissue regeneration at the site.

This aspect of the invention enables the generation of normal tissue in general or locally as required. Pre-clinical results using as example some of the osteogenic ODNs of this invention described below show tissue formation in tissue defects in rats.

The second of the above objects is achieved by providing a method to enhance the replication of pluripotent mesenchymal stem cell "in vitro" which consists in obtention of bone marrow cells from a suitable source like iliac crest, femora, tibiae, spine, rib or other medullar spaces, culturing these cells "in vitro" using a culture medium to the art supplemented with one or more of the oligonucleotides of this invention for a time period sufficient to obtain the amount of cells necessary for a successful medical treatment.

In particular, the oligonucleotides according to the present invention can be used for the manufacture of a medicament for the treatment of the tissue damage own to a heart stroke, for the treatment of skeletal deficiency or alteration, for the treatment of the tissue damage own to a cartilage deficiency or alteration, for the treatment of the tissue damage own to a nervous system deficiency or alteration, for the treatment of the tissue damage own to a adipose tissue deficiency or alteration, for the treatment of the tissue damage own to a liver deficiency or alteration, for the treatment of the tissue damage own to a muscle deficiency or alteration, for the treatment of the tissue damage own to a skin deficiency or alteration, for the treatment of the tissue damage own to a burn accident, for the treatment of the tissue damage own to a pancreas deficiency or alteration, for the treatment of the tissue damage own to a mucosal deficiency or alteration, for the treatment of the tissue damage own to a blood vessel deficiency or alteration, for

the treatment of the tissue damage own to a blood deficiency or alteration and/or for improvement of bone marrow engraftment.

The oligonucleotides for use in the present invention may have any kind of modification of the natural (phosphodiester) phosphate backbone, as for instance a phosphate backbone modification on the 5' inter-nucleotide linkages or a phosphate backbone modification on the 3' inter-nucleotide linkages, or they may have at least one of the internucleotide linkages as a phosphorotioate linkage.

The medicament containing at least one of the above discussed oliconucleotides is preferably administered to a human, the oligonucleotide being preferably present in amounts of 1 μg to lOOmg per dose. The medicament may be administered by intradermic, intramuscular or intravenous injection or by oral, intranasal, anal, vaginal, or trans-dermal route; the medicament may be selected from the group consisting of liquid, gel and lyophilized formulations. The oligonucleotide may be included in a pharmaceutically acceptable carrier and/or may be encapsulated in a slow release delivery vehicle.

The oligonucleotide may also be administered to a subject combined with a device aimed to aid in the treatment of a tissue or organ deficiency or alteration.

According to an embodiment, the mesenchymal stem cells are obtained from bone marrow or from periosteum. According to a further embodiment, the mesenchymal stem cells differentiate into cells of more than one tissue cell type, said tissue being preferably selected from bone, cartilage, adipose, tendon, ligament, dermis, epidermis, neuronal, cardiac, renal, glandular and hepatic tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. Ia shows the replication of MSCs extracted from the femoral bone marrow of rats stimulated or not in the presence of ODN 504 (SEQ ID N°2).

Fig Ib shows the MSC morphology of cells in controls and ODN IMT504 treated cultures.

Fig 2a shows adipocytes, fibroblasts and macrophages in LTBM culture supplemented with ODN IMT504.

Fig 2b shows calcium deposits in osteoblasts obtained from rat bone marrow cells in osteogenic medium supplemented with ODN IMT504.

Fig 3 shows the MSCs replication capacity in cultures supplemented with CpG

ODNs prototypes ODN 2006 and ODN 2216 as compared with the MSCs replication capacity in a culture supplemented with ODN 504.

Fig 4 shows MSCs colony forming units obtained by incubation of bone marrow cells of rats inoculated or not with ODN 504.

Fig 5a shows the radiographic evolution of a defect experimentally provoked in the tibial bone of a rat injected with the ODN IMT504 as compared with a rat injected with placebo after 4 weeks of treatment.

Fig 5b shows the histological analysis of a defect experimentally provoked in the tibial bone of a rat injected with the ODN IMT504 as compared with a rat injected with placebo after 4 weeks of treatment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Repairing a tissue means promoting the formation of morphologically normal, mature tissue only at a site where there is a tissue deficiency that needs to be replaced.

By "animal" is meant any animal having a vertebrate structure, preferably a mammal, and most preferably a human.

A "subject" refers to an animal of the order primate, including humans.

As used herein, the term "oligonucleotide" or "oligo" shall mean multiple nucleotides (i.e. molecules comprising a sugar, e.g. ribose or deoxyribose, linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term "oligonucleotide" as used herein refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). The term "oligonucleotide" shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).

An "oligonucleotide" refers to multiple nucleotides linked by phosphodiester bonds.

An "immunostimulatory oligonucleotide" refers to an oligonucleotide which stimulates (i.e. has a mitogenic effect on, induces, increases or decreases cytokine expression by) a cell of the immune system (i.e. a lymphocyte or a macrophage) in a statistically significant manner.

A "CpG" refers to a cytosine-guanine dinucleotide.

A "CpG oligonucleotide" refers to an oligonucleotide which stimulates a cell of the immune system, and whose immunostimulatory activity critically depends on the presence of at least one CpG in its sequence.

A "non-CpG oligonucleotide" refers to an oligonucleotide that stimulates a cell of the immune system, and whose immunostimulatory activity does not critically depend on the presence of a CpG in its sequence.

Modes for Carrying Out the Invention EXAMPLE 1 Materials and Methods

The following materials and methods were generally used throughout the examples.

1) Oligonucleotides

Oligonucleotides having phosphorothioate internucleotide linkages were purchased, purified by high-pressure liquid chromatography (HPLC), from Operon Technologies (Alameda, California) or Annovis (Aston, Pennsylvania) or Oligos Etc (Bethel, Maine). ODNs were suspended in depyrogenated water, assayed for LPS contamination using the Limulus test and kept at -20 ⁰ C until used. Purity was assessed by HPLC and PAGE assays. ODN preparations were used if LPS levels were undetectable.

2) Animal Experiments

2a- Bone marrow (BM) extraction: Rat BM-derived MSCs were harvested from 8 to 12 weeks old (about 350 g) male Spragϋe Dawley rats. Animals were anesthetized by intraperitoneal ( i. p.) injection of a mixture of ketamine (50 mg/kg) and xilacine (5 mg/kg). After removing epiphyses and gaining access to the marrow cavities, whole BM plugs were flushed out from femoral bones using a 1 ml syringe with α-MEM medium supplemented with 100 IU/ml gentamicin and 25 μg/ml amphotericin.

2b- "In vivo" MSCs replication stimulation by oligonucleotide treatment: Young (8-12 weeks old) adult male Spragϋe Dawley rats weighing about 350 g were subcutaneously injected with 300 μl of PBS containing 250 μg of the oligonucleotide IMT504 (Seq. ID N°2) (treated animals) or 300 μl of PBS (control animals) once a day for five days. Two days after this, the animals were sacrificed and the BM-derived MSCs extracted as described above. 2c- "In vivo" osteogenesis stimulation by oligonucleotide treatment: Young (8-12 weeks old) adult male Spragϋe Dawley rats weighing about 350 g were used. Animals were anesthetized by intraperitoneal ( i. p.) injection of a mixture of ketamine (50 mg/kg) and xilacine (5 mg/kg). After skin overlying the hind limb was shaved and sterilized. A 1.5 cm longitudinal incision was performed in the tibia forefront zone. To generate a defect in the bone, an osteotomy was made using a low-speed dental drill attached to a round diamond saw under saline irrigation. For the osteotomy, a site free of muscular insertions was selected 15 mm below the ankle. The wound was deep enough to reach the bone marrow. A single dose (as stated in each experiment) of ODN in 3 μl of methylcellulose 1% was introduced into the defect made in the right tibia. As a control, the same volume (3 μl) of vehicle was introduced into the defect made in the left tibia. Fracture callus formation was evaluated radiographically on days 0, 21 and 28. On day 28, animals were euthanized under ether atmosphere, and the tibias removed and photographed. After this, the tibias were fixed in 10% formol solution, decalcified in 10% EDTA solution, and embedded in paraffin. A longitudinal section of each tibia was cut, stained with the Masson's trichromic technique to visualize calcified zones and examined under a light microscope. 3) Tissue culture

3a- MSCs colony forming units determination ( Castro-Malaspina H., Gay R. E., Resnick G., Kapoor N., Meyers P., Chiarieri D., McKenzie S., Broxmeyer H.E., Moore M.A. Characterization of human bone marrow fibroblast colony- forming cells (CFU-F) and their progeny Blood. 1980; 56(2): 289-301): 2 x 10 ⁶ cells extracted from femoral bones as described above were cultured in 25 cm ² culture dish containing 10 ml of α-MEM medium supplemented with 100 IU/ml gentamicin, 25 μg/ml amphotericin, 2 mM L-glutamine and 20% fetal calf serum.

Incubation was in 5% CO2 at 37°C.After seven days unattached cells were wash out and fresh medium added. ODN treated cultures contained lμM of the indicated ODN during the first seven days of culture. Incubation proceeded until day 14. After this, attached cells were washed twice with PBS, fixed with methanol and stained using the May-Grungwald-Giemsa technique. Colonies were observed and counted using an optical microscope.

3b- Long Term Bone Marrow (LTBM) culture: This culture technique was used in order to asses the differentiation capacity of the cultured MSCs (Gartner S., Kaplan H. S. Long-term culture of human bone marrow cells. Proc Natl Acad Sci U S A. 1980; 77(8): 4756-4759). 10 x 10 ⁶ extracted from femoral bones as described above were cultured in 25 cm ² culture dish containing 10 ml of α- MEM medium supplemented with 100 IU/ml gentamicin, 25 μg/ml amphotericin, 2 mM L-glutamine, 12,5 % fetal calf serum, 12,5% horse serum, and 10 ^"8 M hydrocortisone. Incubation was in 5% CO2 at 37°C for 28 days. Unattached cells were eliminated every seven days by centrifugation of the cell supernatant. After this, half of the supernatant was replaced by fresh medium and returned to the culture dish. ODN treated cultures contained 1 μM of the indicated ODN during the first seven days of culture. After this, the stimulus was progressively diluted with the medium replacement. After incubation, attached cells were washed twice with PBS, fixed with methanol and stained using the May-Grunwald-Giemsa technique. Cells were observed using an optical microscope. 3c- Osteogenic differentiation of the MSCs "in vitro" (Bruder S. P, Jaiswal N., Haynesworth S.E. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997; vol. 64, p. 278): 2 x 10 ⁶ cells extracted from femoral bones as described above were cultured in 25 cm ² culture dish containing 10 ml of D-MEM medium supplemented with 100 IU/ml gentamicin, 2.5 μg/ml amphotericin, 2 mM L-glutamine and 10% fetal calf serum. Cultures also contained lμM of the indicated ODN. After three days culture unattached cells were wash out and fresh medium and ODN were added. After seven days more of culture, cells were trypsinized and seeded in a new culture dish at 3000 cells/cm ² containing the differentiating medium. This medium

consisted in D-MEM supplemented with 10-8 dexametazone, 0.2 mM ascorbic acid and 1OmM β-glicerophosphate. The cell supernatant was replaced every three days with fresh medium. After 21 days attached cells were washed twice with

PBS, fixed with methanol and stained using the Alizarin Red technique to visualize calcium deposits.

EXAMPLE 2

ODN stimulation of the MSCs replication "in vitro"

The oligonucleotide used in these experiments was IMT504 (SEQ ID N°2). This oligonucleotide is 24 nucleotides long; its nucleotide sequence is 5'-

TCATCATTTTGTCATTTTGTCATT-3', and all the DNA (natural) phosphodiester bonds have been replaced with phosphorothioate bonds to protect it from enzymatic degradation.Figure Ia shows that the replication of MSCs extracted from the femoral bone marrow of rats are greatly stimulated in the presence of ODN 504. Fig Ib shows the typical MSC morphology of cells in controls and treated cultures. Many other oligonucleotides (e.g.: SEQ ID N°3,

SEQ ID N°4, SEQ ID N°7 and SEQ ID N ⁰ IO) were assayed with similar results.

EXAMPLE 3

MSCs replicated "in vitro" under ODN stimulation conserve the multipotent differentiation capacity

Fig 2 a shows that MSCs replicated "in vitro" under ODN stimulation could differentiate in LTBM culture to adipocytes, fibroblasts and macrophages as expected for normal MSCs (Gartner S., Kaplan H.S. Long-term culture of human bone marrow cells. Proc Natl Acad Sci U S A. 1980; 77(8): 4756-4759). On the other hand, Fig 2 b shows that in osteogenic medium (Bruder S.P, Jaiswal N.,

Haynesworth S.E. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997; vol. 64, p. 278) MSCs replicated "in vitro" under ODN stimulation could differentiate into osteoblasts that form calcium deposits.

EXAMPLE 4

CpG ODNs are poor stimulators of the MSCs replication "in vitro"

1. Fig 3 shows that CpG ODNs prototypes ODN 2006 and ODN 2216 (Krieg, A.M. GpG motifs in bacterial DNA and their immune effects. 2002. Annu. Rev. Immunol. 20, 709-760) are poor stimulators of the MSCs replication as compared with ODN 504. Many other CpG ODNs were assayed with identical results. EXAMPLE 5

ODN stimulation of the MSCs replication "in vivo"

Fig 4 shows that bone marrow of rats inoculated with ODN 504 have a very high level of MSCs colony forming cells as compared with rats inoculated with placebo. This result indicates that the stimulation of the MSCs replication by ODNs also occurs "in vivo". EXAMPLE 6

ODN stimulation of the osteogenesis "in vivo"

Since stimulation of the replication of MSCs could be induced by the ODN and since MSCs are osteogenic when injected in an animal (Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem. 1994 Nov;56(3):283-94. Review), the osteogenic potential of the MSCs over-replicated "in vivo" by ODN injection was assayed. Fig 5a and 5b shows that an experimental defect provoked in the tibial bone of rats is rapidly repaired in animals treated with the ODN IMT504 as compared with rats injected with placebo.

RELEVANT REFERENCES U.S. Patent Documents General application of MSCs

United States Patent 6,387,367 Human mesenchymal stem cells United States Patent 5,827,735 Pluripotent mesenchymal stem cells and methods of use thereof

United States Patent 5,486,359 Human mesenchymal stem cells US Patent (appl.) 20040258669 Mesenchymal stem cells and methods of use thereof

US Patent (appl.) 20040048375 Application of pluripotential stem cells for body organ tissue repair and venous capillary expansion

MSCs isolation methods

US Patent (appl.) 20030211602 Isolation of mesenchymal stem cells and use thereof

Use of MSCs in bone regeneration

United States Patent 6,863,900 Regeneration and augmentation of bone using mesenchymal stem cells.

United States Patent 6,541,024 Regeneration and augmentation of bone using mesenchymal stem cells

US Patent (appl.) 20030031695 Regeneration and augmentation of bone using mesenchymal stem cells

Use of MSCs in regeneration of adipose tissue

United States Patent 6,709,864 Adipogenic differentiation of human mesenchymal stem cells United States Patent 6,322,784 Adipogenic differentiation of human mesenchymal stem cells

United States Patent 5,827,740 Adipogenic differentiation of human mesenchymal stem cells

Use of MSCs in regeneration of liver tissue

United States Patent 6,458,589 Hepatocyte lineage cells derived from pluripotent stem cells

Use of MSCs in regeneration of cardiac tissue

United States Patent 6,387,369 Cardiac muscle regeneration using mesenchymal stem cells

US Patent (appl.) 20040180043 Cardiac transplantation of stem cells for the treatment of heart failure

US Patent (appl.) 20030103951 Cardiac muscle regeneration using mesenchymal stem cells

Use of MSCs cartilage repair

United States Patent 6,214,369 Mesenchymal stem cells for cartilage repair

United States Patent 5,908,784 In vitro chondrogenic induction of human mesenchymal stem cells

United States Patent 5,906,934 Mesenchymal stem cells for cartilage repair

US Patent (appl.) 20030026786 Chondrogenic differentiation of human mesenchymal stem cells

US Patent (appl.) 20020005205 Joint repair using mesenchymal stem cells

Use of MSCs in regeneration of pancreatic tissue

US Patent (appl.) 20050054102 Method for differentiating stem cells into insulin-producing cells

Use of MSCs in regeneration of neural tissue

US Patent (appl.) 20040151701 Method for differentiating mesenchymal stem cells into neural cells

US Patent (appl.) 20040115806 Method of generating neurons from stem cells and medium for culturing stem cells

Use of MSCs in prevention of the immune response in transplantation

US Patent (appl.) 20020085996 Mesenchymal stem cells for prevention and treatment of immune responses in transplantation

Use of MSCs in skin regeneration

AUOOOOOl 832601a Skin Regeneration Using Mesenchymal

Stem Cells

Use of MSCs in muscle regeneration

AU000000706026b Myogenic Differentiation Of Human

Mesenchymal Stem Cells