WO2008097828A2 - Method for isolating mesenchymal stromal cells - Google Patents

Abstract

The present invention relates to an isolated population of non-cancerous cells which have been enriched for ganglioside GD2 mesenchymal stromal cells (MSCs). Methods for isolating GD2+ MSCs and using the same in therapeutic applications are also provided.

Description

METHOD FOR ISOLATING MESENCHYMAL STROMAL CELLS

Introduction This application claims benefit of priority to U.S. Provisional Patent Application Serial No. 60/887,844, filed February 2, 2007, the content of which is incorporated herein by reference in its entirety.

This invention was made in the course of research sponsored by the National Institutes of Health (Grant Nos . ROl HL077643 and T32 -CA070089) . The government has certain rights in the invention.

Background of the Invention Human mesenchymal stromal cells (MSCs) , also commonly referred to as mesenchymal stem cells, are multipotent progenitors that can differentiate to bone, fat, cartilage and other mesenchymal tissues (Horwitz, et al . (2005) Cytotherapy 7:393-395). MSCs can be isolated from a variety of tissues, but those from bone marrow are the most widely studied and best characterized. Interest in MSCs for diverse applications has grown and certain criteria have been suggested for defining MSCs including plastic-adherence when maintained in standard culture conditions; expression of CD105, CD73 and CD90, and lack of CD45, CD34, CD14 or CDlIb, CD79alpha or CD19 and HLA-DR surface molecule expression; and the ability to differentiate to osteoblasts, adipocytes and chondroblasts in vitro (Dominici, et al . (2006) Cytotherapy 8:315-317) . Methods for isolating MSCs have been suggested. For example, U.S. Patent No. 5,486,359 discloses a method of preparing human marrow mesenchymal stromal cell cultures using special culture medium and adherence to a plastic or glass culture dish. The first monoclonal antibodies used to characterize

MSCs were SH2 and SH3 (Caplan (1991) J. Orthop. Res. 9:641-

650) , which later were shown to recognize epitopes on CD105

(Barry, et al . (1999) Biochem. Biophys . Res. Cornmun. 265:134-139) and CD73 (Barry et al . (2001) Biochem. Biophys.

Res. Comm. 289:519-524), respectively. While these antigens have been suggested as the cornerstone of human MSC identification (Dominici, et al . (2006) supra) , they are also expressed on hematopoietic and endothelial cells. Neural antigen expression on MSCs has also been suggested

(Deng, et al . (2006) Stem Cells 24:1054-1064; Padovan, et al. (2003) Cell Transplant. 12:839-848).

Ganglioside GD2 is a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids (i.e., n- acetylneuraminic acid) linked on the sugar chain, which is a marker commonly found on cells of the nervous system, tumor cells and tumor side populations (Hirschmann-Jax, et al .

(2004) Proc. Natl. Acad. Sci . USA 101:14228-14233). Given its presence on the surface of neural cells and tumor cells, anti-GD2 antibodies and GD2 ligands have been suggested for use in the detection of tumor cells (see U.S. Patent Application No. 20060159652; Modak, et al . (2002) Med. Pediatr. Oncol. 39 (6) : 547-51 ; Warzynski , et al . (2002) Cytometry 50 (6) : 298-304 ; Heiner, et al . (1987) Cancer Res. 47(20) : 5377-81) and in clearing tissue such as bone marrow of tumor cells (see, U.S. Patent No. 4,675,287; WO 86/00909; Frost, et al . (1997) Cancer 80:317-33; Ozkaynak, et al . (2000) J. Clin. Oncol. 18:4077-85; Ifversen, et al . (2000) J. Hematother. Stem Cell Res. 9(6):867-75; Osenga, et al . (2006) Clin. Cancer Res. 12 (6) : 1750-9) as well as in isolating neural stem cells (see U.S. Patent Application No. 20030040023) . Moreover, therapeutics which target GD2 have been suggested for treating cancers of neuroectodermal origin (U.S. Patent Nos . 20030147808 and 20050202021) . Summary of the Invention

The present invention is a method for isolating an enriched population of ganglioside GD2 mesenchymal stromal cells by selecting from a population of non-cancerous cells a population of cells which have ganglioside GD2 on their surface. In certain embodiments, the method further involves expanding the enriched population of ganglioside GD2 mesenchymal stromal cells. The present invention also provides an isolated population of non-cancerous cells which have been enriched for ganglioside GD2 mesenchymal stromal cells. In particular embodiments, the isolated population of cells have been transfected with exogenous genetic material encoding a protein to be expressed.

The present invention is also a method for treating a subject in need of mesenchymal stromal cells by administering mesenchymal stromal cells which are GD2+ to the subject. In some embodiments, the mesenchymal stromal cells are administered to generate bone formation, whereas in other embodiments, the mesenchymal stem cells are administered to treat or repair a connective tissue defect in the patient .

Detailed Description of the Invention

It has now been found that ganglioside GD2 , or simply GD2, is expressed on the surface of MSCs, but not other cells within the bone marrow. Therefore, GD2 is useful as a single definitive marker of bone marrow-derived MSCs. Advantageously, GD2 is consistently expressed at a high level on all cells of this population, whether investigated after ex vivo expansion, or using freshly harvested bone marrow. Its expression clearly distinguishes MSCs from other spindle-shaped adherent cells, such as skin fibroblasts, and from other elements of the bone marrow microenvironment .

Accordingly, the present invention provides an isolated population of cells which has been enriched for ganglioside

GD2 MSCs and a method for obtaining the same. Moreover, having identified GD2 as a marker for MSCs, GD2 ligands

(e.g., anti-GD2 antibodies) can be used as effective probes for identifying, quantifying, and purifying GD2+ MSCs, regardless of their source in the body. GD2+ MSCs of the present invention can be used in therapeutic applications as well as in studying the full differentiation potential of this population of cells.

As used herein, the term mesenchymal stromal cells (MSCs), or mesenchymal stem cells, refers to multipotent cells naturally found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into more than one specific type of mesenchymal or connective tissue (i.e., the tissues of the body that support the specialized elements; e.g., adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines. Moreover, MSCs of the invention adhere to plastic when maintained in standard culture conditions; express one or more of CD105, CD73 or CD90; and lack expression of one or more of CD45, CD34, CD14, CDlIb, CD79alpha, CD19 or HLA-DR.

A population of cells enriched for ganglioside GD2+ MSCs is intended to mean that 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in a population of cells have ganglioside GD2 present on the cell surface. "Isolated" as used herein signifies that the cells are placed into conditions other than their natural environment; however, the term "isolated" does not preclude the later use of these cells thereafter in combinations or mixtures with other cells. To obtain an enriched population of GD2+ MSCs, the present invention also provides a method for isolating said a population of cells. The method involves obtaining a tissue or cell sample from a subject and selecting GD2+ cells from the sample to obtain an enriched population of GD2+ MSCs. MSCs of the present invention can be selected and isolated from any non-cancerous tissue or cell population that includes GD2+ MSCs, such as peripheral blood (Villaron, et al . (2004) Haematologica 89:1421-1427), placenta (Fukuchi, et al . (2004) Stern Cells 22:649-658), bone marrow, or skin. In one embodiment, the tissue or cells is of an adult, i.e., not embryonic tissue or cells) . In another embodiment, the tissue or cells is non-cancerous, i.e., the tissue or cell does not contain a benign or malignant tumor or cell. In another embodiment, the adult tissue is non- neuronal , i.e., a tissue or cell which is not of the peripheral or central nervous system. In still a further embodiment, the tissue or sample is from a mammal including a human, as well as a zoological {e.g., zebra, lion, elephant and the like), veterinary (e.g., horse, cow and the like), or companion animal (e.g., cat, dog and the like) .

The enriched population of GD2+ MSCs of the invention can be isolated from a heterogenous population of cells (e.g., a tissue with a mixed population of mesenchymal and hematopoietic cells such as bone marrow) or a population of mesenchymal cells. Several techniques are known to those of skill in the art for obtaining mesenchymal cells. For example, approaches to mesenchymal cell preparation include leucopheresis , density gradient fractionation, immunoselection and differential adhesion separation.

The present invention is not limited to a specific method for selecting GD2+ MSCs. For example, the step of selecting GD2+ MSCs can be achieved using antibodies which specifically recognize and bind GD2. Such antibodies can be produced de novo by immunizing a host, including a goat, rabbit, chicken, rat, mouse, human, etc., by injection with synthetic or natural GD2. Methods for producing antibodies are well-known in the art. See, e.g., Kohler and Milstein ((1975) Nature 256:495-497) and Harlow and Lane (Antibodies:

A Laboratory Manual (Cold Spring Harbor Laboratory, New York

(1988)). Alternatively, anti-GD2 antibodies such as 14.G2A,

4G12, chl4.18, 3F8, VIN-IS-56 and HB-8568™ can be obtained from commercial sources including BD Biosciences Pharmingen, Chemicon, Novus Biologicals, or American Tissue Culture Collection.

Generally, the use of antibodies to isolate cells involves providing a cell suspension containing MSCs; contacting the cell suspension with one or a combination of antibodies (e.g., monoclonal or polyclonal) which recognize an epitope on the MSCs; and separating and recovering from the cell suspension the cells bound by the antibodies. The antibodies can be linked to a solid-phase (e.g., a microbead) and utilized to capture MSCs from tissue or cell samples. The bound cells can then be separated from the solid phase by known methods depending on the nature of the antibody and solid phase.

Antibody-based systems appropriate for preparing the desired cell population include magnetic bead/paramagnetic particle columns utilizing antibodies for either positive or negative selection; separation based on biotin or streptavidin affinity; or high speed flow cytometric sorting of immunofluorescent-stained MSCs (e.g., fluorescence- activated cell sorting (FACS) ) mixed in a suspension of other cells.

In one embodiment, the isolation of the cell population of the present invention can include the use of a combination of one or more antibodies that recognize a known marker on MSCs as well as an antibody which recognizes GD2. One method for such preparation of the cells of the present invention is to first select a population of cells expressing a marker identifying MSCs, e.g., SH3 or SH2 , by immunomagnetic selection of a bone marrow cell sample, and subsequently selecting for GD2+ MSCs using an anti-GD2 antibody.

Alternatively, GD2+ MSCs can be isolated by procedures which do not use antibodies. For example, GD2+ MSCs can be isolated using GD2 ligands (see U.S. Patent Application No. 20060159652) .

In certain embodiments, the GD2+ MSC population of the present invention is capable of differentiation into most, if not all, of the mesenchymal cell lineages including bone, cartilage, fat, tendon and muscle tissues (Prockop, et al . (1997) Science 276:71-74), and can also be used to obtain other types of tissue-forming cells such as hepatic (Petersen, et al . (1999) Science 284:1168-1170), airway epithelial (Wang, et al . (2005) Proc . Natl. Acad. Sci . USA 102:186-191), renal (Poulsom, et al . (2003) J. Am. Soc. Nephrol. 14:48S-54S), cardiac (Orlic, et al . (2001) Nature 410:701-705), and neural cells (Mezey, et al . (2000) Science 290:1779-1782; Brazelton, et al . (2000) Science 290:1775- 1779; Croft and Przyborski (2004) Stem Cells Dev. 13:409- 420) . Markers commonly used to characterize cells which have differentiated into various tissue types are well-known in the art. For example, bone cells can be identified by the presence of markers including, but not limited to cbfal, bone-specific alkaline phosphatase (BAP) , hydroxyapatite, osteocalcin (OC) and osteopontin. Identification of adipocytes can be achieved using markers such as adipocyte lipid-binding protein (ALBP) , Fatty acid transporter (FAT) , adipocyte lipid-binding protein (ALBP) and lipoprotein lipase, whereas the identification cartilage can be achieved using collagen types II and IV, keratin, and sulfated proteoglycan as markers .

GD2+ MSCs of the present invention can be maintained in culture media which can be a chemically defined serum- free medium or can be a "complete medium", such as Dulbecco ' s Modified Eagles Medium Supplemented with 10% serum (DMEM) . Suitable chemically defined serum- free media are described in U.S. Patent No. 5,908,782 and WO 96/39487, and "complete media" are described in U.S. Patent No. 5,486,359. Chemically Defined Medium comprises a minimum essential medium such as Iscove's Modified Dulbecco ' s Medium (IMDM)

(GIBCO) , supplemented with human serum albumin, human EX-

CYTE lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine and a mitogen. These media stimulate mesenchymal stem cell growth without differentiation.

The MSCs of the present invention can be frozen for storage, culture-expanded using a media described herein, and/or differentiated using established methods. See, e.g., Rickard, et al . ((1994) Dev. Biol. 161:218-228) for the differentiation of MSCs to osteoblasts; Nuttall, et al .

((1998) J^". Bone Miner. Res. 13:371-382) and U.S. Patent No.

6,709,864 for the differentiation of MSCs to adipocytes; and

Johnstone, et al . ((1998) Exp. Cell Res. 238:265-272) for the differentiation of MSCs to chondroblasts . Moreover, MSCs of the present invention can be further purified. In this regard, "purified" indicates that the cell population contains less than 5% impurities, impurities being for example, cells that are not GD2+. The purified cell population can later be used in combinations or mixtures as is appropriate.

The present invention also relates to various methods of utilizing the population of cells enriched for GD2+ MSCs for therapeutic and/or diagnostic purposes. In accordance with therapeutic methods of the invention, a subject in need of mesenchymal stromal cells is identified and administered an effective amount of the GD2+ MSCs disclosed herein. For purposes of this invention, administration of an effective amount of a GD2+ MSC disclosed herein (undifferentiated or differentiated) results in a beneficial or desired clinical result including, but are not limited to, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.

Subjects in need of treatment include those in need of regenerating mesenchymal tissues which have been damaged through acute injury, abnormal genetic expression, or acquired disease and those in need of cell replacement therapy. Specifically, the MSCs can be infused alone or added to bone marrow cells for bone marrow transplant procedures. Other applications also particularly contemplated are orthopedic, such as augmentation of bone formation. Other applications include, for example, the treatment of osteoarthritis, osteoporosis, traumatic or pathological conditions involving any of the connective tissues, such as a bone defects, connective tissue defects, skeletal defects or cartilage defects. Additional treatments using MSCs are described in U.S. Patent No. 6,875,430. Moreover, given their immunosuppressive effects and their ability to trigger the release of cytokines, MSCs can be used to treat immune disorders such as GVHD, or rheumatologic (autoimmune) disorders (Djouad, et al . (2005) Arthritis & Rheumatism 52:1595-1603). MSCs can also be coadministered with hematopoietic stem cells (HSCs) in the context of hematopoietic stem cell transplants (HSCT) (Fibbe and Noort (2003) Ann. NY Acad. Sci . 996:235-244). MSCs are expected to promote engraftment of HSCs by triggering cytokine release.

Treating a subject having damaged mesenchymal tissue can be achieved, e.g., by removal of small aliquots of bone marrow, isolation of their GD2+ MSCs and treatment of damaged tissue with an enriched population of their own GD2+

MSCs combined with a suitable biocompatible carrier material

(e.g., a hydrogel) for delivering the MSCs to the damaged tissue sites.

The GD2+ MSCs of the invention can also be used for detecting and evaluating growth factors or inhibitory factors relevant to MSC self-regeneration and differentiation into committed mesenchymal lineages; and developing mesenchymal cell lineages and assaying for factors with mesenchymal tissue development.

In particular embodiments, the present invention also embraces ex vivo introduction of exogenous genetic material into GD2+ MSCs such that the cells produce exogenous proteins in vitro or in vivo. Genetic modification of mesenchymal stromal cells is discussed more fully in U.S. Patent No. 5,591,625; Marx, et al . (1999) Hum. Gene Ther. 10:1163-1173; Zhang, et al . (2004) J. Virol. 78:1219-1229; and Wang, et al . (2005) supra. Generally, introduction of exogenous genetic material into MSCs involves inserting the gene of interest into a vector, e.g., an adenoviral vector or a lentivirus vector pseudotyped with modified RD114 envelope glycoproteins (Zhang, et al . (2004) supra), transducing the MSCs with high-titer viral supernatant and selecting transduced GD2+ MSCs based upon the presence of a selectable or expression fo the gene of interest .

Exogenous genetic material which can be transfected into GD2+ MSCs includes, by way of illustration, nucleic acid molecules encoding wild type cystic fibrosis transmembrane conductance regulator [CFTR) for treating cystic fibrosis (Wang, et al . (2005) supra), BMP2 to facilitate bone formation (Tsuda, et al . (2003) MoI. Ther. 7 (3) :354-365) , and BDNF to ameliorate functional deficits after stroke (Kurozumi, et al . (2004) MoI. Ther. 9(2):189- 97) . Other applications of transduced MSCs are well-known in the art.

The invention is described in greater detail by the following non- limiting examples.

Example 1: Materials and Methods

Isolation of Human MSCs. Bone marrow MSCs were isolated according to a conventional protocol approved by the Institutional Review Board (Marx, et al . (1999) supra) . Adipose-derived MSCs were obtained from tissue according to a standard protocol (Mitchell, et al . (2006) Stem Cells 24:376-385). Cord blood MSCs were isolated by placing mononuclear cells (ALLCELLS, Berkeley, CA) in tissue culture at a density of 8 x 10⁵ cells/cm² and then processing the cells as described for marrow MSCs.

Immunocytochemistry. Immunocytochemical studies were performed on formalin-fixed cells which were culture expanded on a sterile chamber slide, using a murine monoclonal antibody against GD2 (clone 14.G2A, BD Biosciences, San Jose, CA) . The reaction was visualized with a biotinylated rabbit anti -mouse secondary antibody with ABC substrate and Nova Red as the chromogen (Vector Labs, Burlingame, CA) . All slides were lightly counterstained with hematoxylin. Reverse Transcription PCR. RNA was extracted with

TRIZOL Reagent according to the manufacturer's instructions

(INVITROGEN, Carlsbad, CA) . Primers for amplification of GD2 synthase included forward primer 5'-CCA ACT CAA CAG GCA ACT

AC-3' (SEQ ID N0:l) and reverse primer 5'-GAT CAT AAC GGA GGA AGG TC-3' (SEQ ID NO : 2 ) , which yielded a 230-bp product, and forward primer 5'-GAC AAG CCA GAG CGC GTT A-3' (SEQ ID

NO: 3) and reverse primer 5'-TAC TTG AGA CAC GGC CAG GTT-3'

(SEQ ID N0:4) , which yielded a 99-bp product. Primers for amplification of β2 -microglobulin were forward primer 5'-CTC

GCG CTA CTC TCT CTT TCT TGG-3' (SEQ ID NO: 5) and reverse primer 5'-GCT TAC ATG TCT CGA TCC ACT TAA-3' (SEQ ID NO: 6), which yielded a 333 bp-product. PCR conditions were denaturation at 95°C for 12 minutes; amplification for 35 cycles of 95°C for 1 minute, 59°C for 1 minute, and 72⁰C for

I minute; and one cycle at 72⁰C for 10 minute.

MSC Differentiation. Using conventional methods, culture-expanded MSCs were differentiated in vitro to osteoblasts (Rickard, et al . (1994) supra), adipocytes (Nuttall, et al . (1998) supra), and chondroblasts (Johnstone, et al . (1998) supra) . The differentiated osteoblasts were stained with Alizarin Red S (Bodine, et al . (1996) J. Bone Miner. Res. 11:806-819), the adipocytes with Oil Red 0 (Sekiya, et al . (2004) J. Bone Miner. Res. 19:256- 264), and the chondroblasts with Alcian Blue (Wang, et al . (2005) J^". Bone Min. Res. 20:1624-1636) according to published protocols.

Flow Cytometry. All analyses were performed on a BD LSR

II flow cytometer, with antibodies from BD Biosciences, except that the monoclonal antibody against human fibroblasts, clone D7-FIB, was from SEROTEC (Raleigh, NC) . The data was analyzed with CELLQUEST Pro Software Version 5.2.1 (BD Biosciences).

Example 2 : Neural Ganglioside GD2 Expressed by MSCs

Human MSCs isolated from bone marrow showed the characteristic features of spindle shape and plastic adherence. Flow cytometric analysis demonstrated the expression of distinguishing MSC antigens (CD105, CD73 and CD90), and the absence of hematopoietic and endothelial antigens (CD45, CD34, CD19, CD3 , CDlIb, and HLA DR) . The culture-expanded cells were capable of in vitro differentiation to osteoblasts as demonstrated by Alizarin Red staining; adipocytes, as demonstrated by Oil Red 0 staining; and chondroblasts as demonstrated by Alcian Blue staining. Thus, the isolated cells met the essential criteria used to define MSCs (Dominici, et al . (2006) supra) . Immunocytochemical staining of the marrow-derived MSCs revealed striking expression of the neural ganglioside GD2. Comparable staining was found on all MSCs, indicating a pancellular expression of GD2 among these bone marrow elements. Staining of MSCs without the primary antibody yielded negative results demonstrating the lack of nonspecific staining. To assess GD2 expression by an alternative method, culture-expanded cells were analyzed by flow cytometry, wherein high levels of GD2 surface expression was observed. Reverse-transcription PCR analysis using two different primer pairs, with a neuroblastoma cell line and blood mononuclear cells serving as positive and negative controls, respectively, showed that MSCs expressed the mRNA for GD2 synthase, an essential enzyme for GD2 biosynthesis. These results provide supportive evidence of genuine ganglioside expression on MSCs.

Cells previously analyzed were subsequently expanded in culture (passage 2) . Since GD2 can play a role in cellular adhesion (Jabbar, et al . (2006) Pediatr. Blood Cancer 46:292-299), it was determined whether plastic adherence of MSCs in vitro might induce or alter the expression of GD2 in a manner different from the normal regulation of the ganglioside. Newly harvested bone marrow mononuclear cells

(MNCs) were analyzed by four-color flow cytometry, gating first on marrow MNCs that lacked CD45 expression. These gated cells were then analyzed for CD105 and CD73 expression. About 95% of the CD45- CD105+ CD73+ cells expressed GD2. Further, newly harvested marrow MNCs that expressed either CD271 or D7Fib, both of which have been suggested to represent MSCs (Jones, et al . (2002) Arthritis Rheum. 46:3349-3360; Jones, et al . (2004) Arthritis Rheum. 50:817-827), were found to co-express GD2. The intensity of GD2 expression exceeded that of either CD271, which is lost during culture, or D7Fib, which is also expressed on skin fibroblasts. Notably, MSCs continued to express GD2 at similar levels though 8 culture passages.

To determine if GD2 expression was unique to marrow- derived MSCs, cells from adipose tissue and umbilical cord blood were analyzed. MSCs from both of these sources also expressed GD2. The adipose-derived cells seemed to express GD2 at approximately the same level as marrow-derived MSCs; however, substantially lower GD2 expression levels were found in cells from umbilical cord blood, consistent with recognized differences between adult and fetal MSCs (Campagnoli, et al . (2001) Blood 98:2396-2402; Gotherstrom, et al . (2005) Haematologica 90:1017-1026). Foreskin fibroblasts, by contrast, lacked GD2 expression altogether.

Furthermore, immunohistochemical staining of normal bone marrow biopsy specimens and flow cytometric analysis of normal marrow aspirates did not detect other GD2 -expressing cells, consistent with prior reports (Hoon, et al . (2001) Am. J. Pathol. 159:493-500; Lo Piccolo, et al . (2001) Cancer 92:924-931; Cheung and Cheung (2001) Clin. Cancer Res. 7 = 1698-1705) .

Claims

What is claimed is :

1. A method for isolating an enriched population of ganglioside GD2 mesenchymal stromal cells comprising selecting from a population of non-cancerous cells a population of cells which have ganglioside GD2 on their surface thereby isolating an enriched population of ganglioside GD2 mesenchymal stromal cells.

2. The method of claim 1, further comprising expanding the enriched population of ganglioside GD2 mesenchymal stromal cells.

3. An isolated population of cells comprising a population of non-cancerous cells which have been enriched for ganglioside GD2 mesenchymal stromal cells.

4. The isolated population of cells of claim 3, wherein said cells are transfected with exogenous genetic material encoding a protein to be expressed.

5. A method for treating a subject in need of mesenchymal stromal cells comprising administering mesenchymal stromal cells which are GD2+ to a subject in need of treatment .

6. The method of claim 5, wherein the mesenchymal stromal cells are administered to generate bone formation.

7. The method of claim 5, wherein the mesenchymal stromal cells are administered to treat or repair a connective tissue defect in the patient.