WO2011060349A1

WO2011060349A1 - Methods of modulating mesenchymal stem cells

Info

Publication number: WO2011060349A1
Application number: PCT/US2010/056684
Authority: WO
Inventors: Arnold R. Brody; Kenneth B. Adler; Indu Parikh
Original assignee: North Carolina State University; Biomarck Pharmaceuticals Ltd
Current assignee: North Carolina State University; Biomarck Pharmaceuticals Ltd
Priority date: 2009-11-13
Filing date: 2010-11-15
Publication date: 2011-05-19
Anticipated expiration: 2012-05-13

Abstract

The present invention is related to methods of modulating migration of a mesenchymal stem cell (MSC) comprising contacting or admixing a migration-inhibiting amount of a migration-modulating peptide with the MSC to form a migration-inhibited MSC and administering the migration-inhibited MSC to a subject in need of treatment.

Description

METHODS OF MODULATING MESENCHYMAL STEM CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

61/261,132, filed on November 13, 2009, and entitled "METHODS OF MODULATING MESENCHYMAL STEM CELLs," the contents of which is herein incorporated by reference in its entirety and for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: BMRK_005_01WO_SeqList.txt, date recorded: November 15, 2010, file size 74 kilobytes).

FIELD OF THE INVENTION

[0003] This invention relates to compositions and methods useful to modulate the migration of mesenchymal stem cells. In a preferred embodiment, this invention relates to compositions and methods useful to modulate mesenchymal stem cells in the presence of chemotactic agents, and particularly to modulate the migration of mesenchymal stem cells.

BACKGROUND OF THE INVENTION

[0004] Mesenchymal stem cells, herein also referred to as MSCs, are multi-potential stem cells capable of differentiating into a number of cell types, which types can include osteoblasts, chondrocytes, chondroblasts, myocytes, adipocytes, adipoblasts, fibrocytes, fibroblasts, myofibroblasts, tenoblasts, neuroblasts, endothelial and epithelial cells and beta-pancreatic islet cells. Mesenchymal stem cells can be found in diverse tissues such as bone marrow, cord blood, and dental pulp; for example, see Peister et al. Blood.

103(5): 1662-1668, (2004). Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential.

[0005] Mesenchymal stem cells have been characterized; for example, see Prockop et al., Cytotherapy 3:393-396, (2001), and Baksh et al, Stem Cells 25: 1384-1392, (2007).

[0006] MSCs appear to act as progenitors in replacing and possibly repairing injured tissues in the lung: see, for example Albera et al., Tissue Eng. 11 :1115-1 121, (2005); and in other organs, for example, see Porada et al., Curr Stem Cell Res Ther. 1 :365-369, (2006).

[0007] Certain growth factors and cytoskeletal components may be essential or may be significant to one or more mechanisms of MSC migration and to biological responses engendered by or involving MSCs; for example, see Discher et al., Science 324: 1673-1677, (2009).

[0008] Mesenchymal stem cells have been used or administered in animal model systems, for example, by intravascular injection such as in Moodley et al., Am J Pathol 175:303-313, (2009); and, for example, by bone marrow transplantation such as in Spees et al, Am J Respir Crit Care Med 176:385-394, (2007).

[0009] Mesenchymal stem cells can alleviate lung injury from bleomycin exposure by paracrine responses, and mesenchymal stem cells can interfere with fibroblast conversion to myofibroblasts that produce extracellular matrix. Administration of MSCs may ameliorate fibrogenesis caused by bleomycin; see, for example, Ortiz et al., Proc Natl Acad Sci USA 104:1 1002-11007, (2007). MSCs block production of the inflammatory cytokines TNFa and IL1 that apparently mediate bleomycin-induced lung injury. MSC or stem-like cells residing at sites of injury may play a role in protecting the lung from additional consequences of such injury. MSC migrate to sites of tissue injury and ameliorate damage from multiple etiologies. For example, intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice: see Gupta et al., J Immunol. 179(3): 1855- 1863, (2007); and endotoxin-induced systemic response is prevented by bone marrow- derived mesenchymal stem cells in mice: see Xu et al, Am J Physiol Lung Cell Mol Physiol. 293(1):L131-141, (2007). [0010] Administration of MSCs can ameliorate fibrogenesis caused by radiation; see, for example, Abe et al., Am J Respir Crit Care Med 170: 1158-1 163, (2004); and administration, such as by intrapulmonary administration, of MSCs can ameliorate fibrogenesis in acute lung injury; see, for example, Gupta et al.(2007).

[0011] In certain cases of chronic injury, MSC may exacerbate damage and contribute to fibrosis: see Li et al., J Hepatol. 50(6): 1 174-1183, (2009).

[0012] Whatever role MSC might be playing at sites of injury, it appears that the cells can exhibit a homing capacity, and can migrate from the bone marrow, transit through the vasculature, and arrive at the affected tissues: see, for example, Sordi V. Transplantation. 87(9S):S42-45, (2009). Discovery of a method to modulate

mesenchymal stem cell migration to a site of injury or disease in a tissue or fluid in a subject, thereby modulating the beneficial and/or deleterious effects such stem cells can exhibit can therefore useful in methods of treatment of such disease or injury.

[0013] Reduction of airway remodeling may occur if Stem Cell Factor (SCF) is blocked and bone marrow-derived mesenchymal cells consequently are inhibited from reaching the sites of injury, thus impugning bone marrow-derived fibroblasts in airway remodeling; see, for example, Dolgachev et al., Am J Pathol 174:390-400, (2009). Both circulating fibroblasts (i.e. fibrocytes), which are progenitor cells with multiple functions and which appear to be an indicator of poor prognosis in idiopathic pulmonary fibrosis (see Moeller et al., Am J Respir Crit Care Med 179:588-594, (2009)) and MSCs can accumulate preferentially at sites of lung injury.

[0014] The molecular mechanism (or mechanisms) through which transferred (or administered) MSCs migrate from the vasculature and move among other cell types and along interstitial pathways in the lung or elsewhere is currently unresolved. It is known that the actin cytoskeleton is required for successful cell migration. Also unresolved is the role or mechanism by which inflammatory cytokines and mediators, which the MSCs synthesize and secrete at sites of lung injury, may induce responses in surrounding cells in developing lesions.

[0015] Elucidation of one or more molecular mechanisms that mediate MSC migration could be central to controlling the arrival and departure of these cells (MSCs) from developing pulmonary lesions. [0016] Directed cellular chemotaxis is a complex process. In one aspect, an attractant (or chemokine) can bind to a specific membrane receptor, and signal transduction pathways become activated; see, for example, Stephens et al., Curr Biol. 18(1 l):R485-494, (2008). These activated pathways induce actin polymerization at the leading edge of the polarized cell, with the ultimate result of production of movement of the cell towards increasing gradient concentrations of chemokines (see, for example, Pollard Cell. 1 12(4):453-465, (2003); Rowlands et al, Am J Physiol Cell Physiol.

295(4):C1037-1044, (2008); and DeFea, Annu Rev Physiol. 69:535-560, (2007)).

[0017] Fibrocytes are circulating mesenchymal cell progenitors that are involved in tissue repair and fibrosis. Circulating fibrocytes are progenitors for fibroblasts and may participate in the pathogenesis of lung fibrosis (Moeller, 2009). Application of the compositions and methods of this invention as described below directed to fibrocytes may find useful application in the treatment of lung fibrosis.

BRIEF SUMMARY OF THE INVENTION

[0018] Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous multi-functional protein that is activated after being phosphorylated by Protein Kinase C (PKC); see, for example, Sundaram et al, Biochem Cell Biol. 82(l): 191-200, (2004). MARCKS can bind F-actin filaments (Hartwig et al, Nature. 356:618-622, (1992)), and has been identified at the leading edge of polarized cells (Sundaram, 2004).

Overexpression of MARCKS can inhibit cell adhesion to extracellular matrix

components (Spizz et al., J Biol Chem. 276(34):32264-73, (2001)), and studies have indicated a potential involvement in cell movement in vitro (Thelen et al., Nature.

351 :320-322, (1991)).

[0019] The present inventors have discovered that MARCKS is involved in directed chemotaxis of MSCs. Further, it also has been found that a synthetic peptide identical to the N-terminus of MARCKS, named the MANS (myristoylated N-terminal sequence) peptide (SEQ ID No: 1), which is myristoylated at the N-terminus of the peptide, as well as active fragments of MANS peptide, can affect MARCKS function, resulting in disruption of vesicular transport within cells as well as cell migration. [0020] That MARCKS protein is required for MSCs to migrate in vitro is illustrated in Figure 6 A and 6B. Pre-treatment of MSCs with a MARCKS-specific peptide such as the MANS peptide, which blocks mucus hypersecretion and biological activity of MARCKS in a mouse model of asthma (see Singer et al., Nat Med 10: 193- 196, (2004)), modulates or attenuates the migration of MSCs.

[0021] The present inventors propose that MARCKS is a key molecule for MSC migration in vivo, and when the cells arrive at sites of lung injury in a subject suffering from a disease of involving the lung, they release cytokines and influence surrounding cells that modulate the development of disease. Controlling the migration of

mesenchymal stem cells, which is an object of this invention, with, for example, the MANS peptide, will be crucial when these mesenchymal stem cells are used in therapeutic approaches.

[0022] MSCs apparently are dependant on MARCKS protein for migration.

MARCKS is widespread in eukaryotic cells and has multiple functions. The protein contains three separate evolutionarily-conserved domains: the myristoylated N-terminus can function to attach MARCKS protein to cell membranes; the Multiple Homology 2 domain (MH2) has an unknown function; and the phosphorylation site domain (PSD) is the phosphorylation target of protein kinase C (PKC), as well as the site that binds and cross-links actin filaments and binds calcium/calmodulin (see Yarmola et al., The Journal of biological chemistry 276:22351-22358, 2001).

[0023] MARCKS appears to be a central molecule regulating degranulation in several cell types, including airway epithelium (see Park et al., American journal of respiratory cell and molecular biology 39:68-76, 2008.) and leukocytes (see Takashi et al., American journal of respiratory cell and molecular biology 34:647-652, 2006).

[0024] Mesenchymal stem cells (MSCs) migrate to sites of lung injury in several animal models and in human lungs. Mesenchymal stem cells are mobile, and synthesize and secrete a number of potent cytokines. Thus, in one aspect, modulation of migration of MSCs to sites of lung injury in a subject suffering from lung injury or disease may ameliorate lung injury.

[0025] One of the many proteins that play a pivotal role in cytoskeletal formation is myristoylated alanine-rich C kinase substrate (known as MARCKS as disclosed in U.S. Patent No. 7,265,088 and in cited publications, see below), a linear protein found in numerous cell types. The nucleotide and amino acid sequences of human MARCKS cDNA and protein, as reported by Harlan et al., J. Biol. Chem. 266: 14399 (1991) (GenBank Accession No. M68956), are provided as in U.S. Patent No. 7,265,088 . The nucleotide and amino acid sequences of human MARCKS cDNA and protein as reported by Sakai et al., Genomics 14: 175 (1992) are also provided in U.S. Patent No. 7,265,088. An additional publication (Harlan et al., J Biol. Chem. 266(22):14399 (1991)) provides a nucleotide sequence for human MARCKS that differs from that of Sakai et al. at nucleotides 619 and 724; in this sequence, G is substituted for T at position 619 and C is substituted for G at position 724. Additional allelic variants of human and other

MARCKS proteins would be expected. MARCKS performs a number of roles within cells, including PIP₂ binding, calmodulin binding, vesicular transport, and F-actin binding. When MARCKS is inhibited in polymorphonuclear leukocytes (PMNs), cell motility is impaired.

[0026] In this invention, MSCs, such as primary bone marrow MSCs (BM-

MSCs), express MARCKS protein. This protein is key to organization of the cytoskeleton that mediates cell migration. Mouse BM-MSC migration can be inhibited by exposure to MANS peptide (SEQ ID No: 1), implying MARCKS plays a significant role in migration of these cells.

[0027] In this invention, it is shown that mouse primary BM-MSCs exhibit non- random migration towards chemoattractants, and that BM-MSCs move toward both stem cell-specific and general inflammatory chemokines.

[0028] Primary mouse BM-MSCs are shown to be capable of non-random movement (or migration) toward a chemokine gradient concentration established in vitro.

[0029] MANS peptide and activated fragments of MANS peptide such as N- terminal myristoylated fragments of MANS peptide can block or inhibit or reduce the rate of BM-MSC migration in a dose-dependent manner toward chemokine gradient concentrations. Mouse BM-MSC migration can be inhibited by exposure to MANS peptide.

[0030] MARCKS protein plays a significant role in migration of these

mesenchymal stem cells in vitro. [0031] Exposure of mouse BM-MSC to MANS peptide disrupts mouse BM-MSC motility.

[0032] In one aspect, the present invention comprises a method of modulating a chemotactic migration of a mesenchymal stem cell (MSC) moving toward an increasing gradient concentration of a chemokine, the method comprising administration of a migration-inhibiting amount of a migration-modulating peptide to the MSC to form a migration-inhibited MSC, followed by exposure of said migration-inhibited MSC to an increasing gradient concentration of a chemokine.

[0033] In another aspect of the present invention, the migration-modulating peptide is a MANS peptide, N-myristoyl-GAQFSKTAA GEAAAERPGEAAVA (SEQ ID NO: 1).

[0034] In another aspect of the present invention, the migration-modulating peptide can be an active fragment of the MANS peptide, which active fragment can be selected from the group consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGE AAV (SEQ ID No: 2); N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID No: 4); N- myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl- GAQFSKTAAKGEAAAERPGE (SEQ ID No: 1 1); N-myristoyl- GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl- GAQFSKTAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-

GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFS TAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl- GAQFSKTAAKGEAA (SEQ ID No: 56); N-myristoyl-GAQFSKTAAKGEA (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl- GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFSKTAA (SEQ ID No: 121 ); N-myristoyl-GAQFSKTA (SEQ ID No: 137); N-myristoyl-GAQFSKT (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), N-myristoyl-GAQF (SEQ ID No: 211), and a combination thereof. Each of these fragments contains the contiguous amino acid sequence GAQF, which is the order of the N-terminal amino acid sequence found in MANS peptide, and each of these fragments is myristoylated at the N-terminal glycine, which is the site of myristoylation found in MANS peptide. A preferred embodiment comprises N-myristoyl-GAQFSKTAAK (SEQ ID No: 106).

[0035] In another aspect of the present invention, the migration-modulating peptide can be an N-terminal- and/or C-terminal-chemically modified peptide, which peptide consists of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence of from at least 4 to no more than 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1); and

(b) an amino acid sequence substantially identical to the sequence defined in (a); wherein

(i) the C-terminal amino acid of said chemically modified peptide is chemically modified and the N-terminal amino acid of the peptide is optionally chemically modified; or

(ii) the C-terminal amino acid of said chemically modified peptide is not chemically modified and the N-terminal amino acid of the peptide is chemically modified and is or is not myristoylated;

with the proviso that said chemically modified peptide contains no more than 23 amino acids; and

wherein administration of a migration-inhibiting amount of said chemically modified peptide to an MSC forms a migration-inhibited MSC. Such chemically modified peptides are described, for example, in U.S. Patent 7,524,926, in

WO2006/078899, the disclosure of each of which is incorporated herein in its entirety by reference.

[0036] In another aspect of the invention, the amino acid sequence of the migration-modulating peptide can comprise L-enantiomers of amino acids in a MANS peptide sequence.

[0037] In another aspect of the invention, the amino acid sequence of the migration-modulating peptide can comprise D-enantiomers of amino acids in a MANS peptide sequence.

[0038] In another aspect of the invention, the migration-modulating peptide can be an active fragment of MARCKS protein, wherein the active fragment can be selected from the group consisting of peptides 10 to 50 amino acids in length having amino acid sequence found in the N-terminal region of the MARCKS protein, and wherein such fragment may be myristoylated or non-myristoylated, with the proviso that such fragments demonstrate in vitro a migration-modulating effect on MSCs. Examples of such a group of MARCKS fragment peptides are described in European Patent

EP1154786, the disclosure of which is incorporated herein in its entirety by reference.

[0039] The present inventors have found that certain peptides such as the MANS peptide and an active fragment thereof are useful to modulate or block or inhibit the migration of mesenchymal stem cells (MSCs). These are cells that can differentiate into multiple cell types and are being used with increasing frequency as a potential therapeutic tool for diseases such as asthma, fibrosis and cancer such as lung cancer. The ability to control MSC function and migration will be a major part of being able to use these cells in new therapeutic approaches. The present inventors have used MANS to block the migration of MSCs (both human and mouse) in vitro, and our data show an effect on MSC function in vivo in a mouse model of asthma. The present inventors further have found that the chemotactic influence of several well known chemoattractants is blocked by MANS peptide. Peptides which have amino acid sequences identical to amino acid sequences in fragments of MANS peptide (referred to herein as peptide fragments of MANS), and peptides which are analogs of MANS and analogs of peptide fragments of MANS, which are modified at least at the N-terminal and/or the C-terminal end of the peptide, that are disclosed herein, can also find utility to modulate the chemotactic influence of several well known chemoattractants on MSCs.

[0040] This invention may find use in treatment of acute and chronic lung diseases, which afflict millions of individuals world wide.

[0041] This invention relates to the emerging field of therapy comprising the application (i.e., administration) of adult mesenchymal stem cells (MSCs) into the lung, such as by way of the trachea, vasculature or through whole bone marrow

transplantation, which routes of administration are preferred routes for administration of compounds and of pharmaceutical compositions of this invention, which can comprise a migration-modulating peptide and/or a migration-inhibited MSC. Addition of unmodified exogenous MSCs or transplantation is known to result in these cells homing to lesions and differentiating into a variety of cell types, ameliorating the lesion and providing a beneficial outcome. The present inventors provide data demonstrating that myristoylated alanine rich C kinase substrate (MARCKS) is required for MSCs to migrate inasmuch as the MANS peptide and fragments and analogs thereof, block directed chemotactic migration of the MSCs in vitro. In addition, the present inventors show that pre-treatment of MSCs with MANS blocks MARCKS function and alters the accumulation of inflammatory cells in a mouse model of allergic airway remodeling. The ability to control MSC migration with MANS and with active fragments of MANS peptide and/or of MARCKS protein as described herein will be an important component in using MSCs as a therapeutic tool.

[0042] Pharmaceutical compositions of this invention can comprise MANS peptide and/or an active fragment thereof. Pharmaceutical compositions of this invention can further comprise MANS peptide and/or an active fragment thereof together with a carrier such as a pharmaceutically acceptable carrier such a sterile aqueous saline solution which can be isotonic in the presence of MCS. Pharmaceutical compositions of this invention can further comprise an MCS which has been treated with or exposed to MANS peptide or an active fragment thereof.

[0043] As used herein, the term "modulation of migration" means a change in rate of migration over a given period of time relative to the rate of migration observed in a control. In one embodiment, the change can be a decrease in rate of migration. In another embodiment, the change can be an increase in the rate of migration. In a preferred embodiment, the change can be referred to as a decrease, or as an attenuation, or as a reduction, or as an inhibition, or as a slowing, of rate of migration of

mesenchymal stem cells (MSCs) subsequent to treatment (or pretreatment) of unmodified MSCs with an active peptide of this invention prior to measurement of the number of migrated treated MSCs in a field of measurement after a period of time in comparison to a similar measurement of number of cells in the same field in a control or reference migration of untreated MSCs for the same period of time. Attenuation of migration obtains when fewer cells of peptide-treated MSCS are observed per field of measurement compared to the number of cells per field observed in a control of untreated MSCs. The term "modulated" preferably means "attenuated" or "reduced" or "inhibited" and the term "modulating" preferably means "attenuating" or "reducing" or "inhibiting."

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 illustrates that mouse primary bone marrow mesenchymal stem cells (BM-MSCs) migrate in response to the presence of at least chemokines MCP-1, C5a, and SDF-1 in vitro.

[0045] Figure 2 A illustrates a determination of an optimum chemokine SDF-1 concentration for mouse BM-MSC migration in vitro.

[0046] Figure 2B illustrates a determination of an optimum chemokine C5a concentration for mouse BM-MSC migration in vitro.

[0047] Figure 2C illustrates a determination of an optimum chemokine SCF concentration for mouse BM-MSCs migration in vitro.

[0048] Figure 3 illustrates a Western blot of MARC S protein in mouse primary

BM-MSCs.

[0049] Figure 4 illustrates that mouse primary BM-MSC migration in vitro can be modulated or disrupted by MANS peptide (N-terminally myristoylated SEQ ID No: 1).

[0050] Figure 5 illustrates that MANS peptide (N-terminally myristoylated SEQ

ID No: 1) modulation or attenuation or inhibition of mouse BM-MSC movement is concentration-dependent.

[0051] Figure 6A illustrates the effect of MANS peptide (N-terminally myristoylated SEQ ID No: 1) in modulating or attenuating or reducing BM-MSC migration relative to the effect of RNS peptide (N-terminally myristoylated SEQ ID No: 232), which RNS peptide contains the same amino acids as found in MANS but in a randomized sequence order, in the presence of C5a and of SDF-1 a.

[0052] Figure 6B illustrates the effect of MANS peptide (N-terminally myristoylated SEQ ID No: 1) in modulating or attenuating or reducing BM-MSC migration relative to the effect of RNS peptide (N-terminally myristoylated SEQ ID No: 232), in the presence of C5a at increasing concentrations of C5a. [0053] Figure 7 A illustrates that mice sensitized to, and challenged with, OVA

(OVA-Sham cells) demonstrate in vivo a significant increase in lavage eosinophils compared to alum-treated mice (Alum Sham cells).

[0054] Figure 7B illustrates in vivo PAS semi-quantitative scoring in OVA- treated mice for mucus cell metaplasia which was enhanced in mice that received MANS-incubated MSCs.

[0055] Figure 8 illustrates a proposed mechanism for MARCKS protein- dependent mesenchymal stem cell migration in the presence of a chemokine gradient.

[0056] Figure 9 illustrates intensity per mm (mm squared) as a function of time and sample in blots designated Pi-MARCKS and GAPDH.

[0057] Figure 10 illustrates the modulating or attenuating effect of incubation of

MCSs with an N-terminal myristoylated peptide, N-myristoyl-GAQFSKTAAK (SEQ ID No: 106) on inhibition of migration of mouse bone marrow derived stem cells in response to SDF-la.

DETAILED DESCRIPTION OF THE INVENTION

[0058] MARCKS, a protein of approximately 82 kD, has three evolutionarily- conserved regions (see Aderem et al., Nature 332:362-364 (1988); Thelen et al., Nature 351 :320-322 (1991); Hartwig et al., Nature 356:618-622 (1992); Seykora et al, J Biol Chem 271 : 18797-18802 (1996)): an N-terminus, a phosphorylation site domain (PSD), and a multiple homology 2 (MH2) domain.

[0059] The N-terminus of MARCKS protein, a 24 amino acid sequence with a myristic acid moiety attached to a terminal glycine residue is involved in binding of MARCKS to membranes (Seykora 1996) and possibly to calmodulin (Matsubara et al., J Biol Chem 278:48898-48902 (2003)). This N-terminal myristoylated 24 amino acid sequence is known as the MANS peptide and has the N-terminal myristoylated amino acid structure: N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA SEQ ID No: 1).

[0060] The MANS peptide and active fragments thereof, can compete with native

MARCKS in cells for membrane binding. Involvement of MARCKS protein in release of inflammatory mediators from the granules of infiltrating leukocytes is relevant to inflammation in diseases in all tissues and organs, including lung diseases characterized by airway inflammation, such as asthma, COPD and cystic fibrosis. However, inflammation and mucus secretion in the airways are two separate and independent processes (Li et al., J Biol Chem 276:40982-40990 (2001); Singer et al., Nat Med 2004; 10:193-196 (2004)).

[0061] In one aspect, the present invention comprises a method of modulating

(inhibiting or reducing) migration of a mesenchymal stem cell (MSC) in a subject, the method comprising admixing a migration-inhibiting amount of a migration-modulating peptide with the MSC to form a migration-inhibited MSC and administering the migration-inhibited MSC to a subject in need of treatment.

[0062] In the embodiments described herein, a useful migration-inhibiting amount of a migration-modulating (migration-inhibiting) peptide can be in the range from about 1 nanomolar to about 1000 micromolar, more preferably from about 10 nanomolar to about 100 micromolar, and even more preferably from about 500 nanomolar to about 100 micromolar of an isotonic aqueous solution or suspension of the peptide, which solution or suspension is compatible with stem cell viability. A useful amount of peptide is about 50 micromolar. A useful pH is in the range of 6.6 to 8, preferably in the range of 7.2 to 7.6. Useful preincubation time for exposure of the peptide to the MCS prior to cell migration assay or use in tissue or fluid (e.g., blood, serum, lymph, urine, mucus, saline, lacrimate, perspiration, and ascites fluid) of a mammal is in the range of about 1 minute to about 1 hour, preferably from about 10 minutes to about 30 minutes, and more preferably from 15 minutes to 20 minutes.

[0063] In a further aspect, the present invention comprises method of modulating a chemotactic migration of a mesenchymal stem cell (MSC), the method comprising admixing a migration-inhibiting amount of a migration-modulating peptide with the MSC to form a migration-inhibited MSC, followed by exposure of said migration-inhibited MSC to a chemokine gradient, wherein the migration-inhibited MSC exhibits modulated chemotactic migration properties. In one aspect, this method further includes where the exposure comprises an in vivo administration to a human or animal tissue. In another aspect, the tissue is a lung tissue. For an in vitro use, this method further comprises wherein the admixing is followed by exposure of said migration-inhibited MSC to an increasing gradient concentration of a chemokine.

[0064] In one aspect, the present invention comprises a method of modulating a chemotactic migration of a mesenchymal stem cell (MSC) moving toward an increasing gradient concentration of a chemokine, the method comprising administration of a migration-inhibiting amount of a migration-modulating peptide to the MSC to form a migration-inhibited MSC, followed by exposure of said migration-inhibited MSC to an increasing gradient concentration of a chemokine.

[0065] The method (methods) described above further includes a migration- modulating peptide that comprises a MANS peptide, N-myristoyl- GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). Further, the migration- modulating peptide comprises an active fragment of the MANS peptide. More specifically, the active fragment of the MANS peptide is selected from the group consisting of N-myristoyl-GAQFSKTAAKGE AAAERPGEAAV (SEQ ID No: 2); N- myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID No: 4); N-myristoyl- GAQFSKTAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl- GAQFS TAAKGEAAAERPGE (SEQ ID No: 1 1); N-myristoyl- GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl- GAQFSKTAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-

GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl- GAQFSKTAAKGEAA (SEQ ID No: 56); N-myri stoyl-G AQF SKT AAKGE A (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl- GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFSKTAA (SEQ ID No: 121); N-myristoyl-GAQFSKTA (SEQ ID No: 137); N-myristoyl-GAQFSKT (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), N-myristoyl-GAQF (SEQ ID No: 211), and a combination thereof.

[0066] In a preferred embodiment, MANS peptide and active fragments thereof are myristoylated. [0067] Further, in another aspect, the migration-modulating peptide comprises an

N-terminal- and/or C-terminal-chemically modified peptide, which peptide consists of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence of from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1), wherein the amino acid sequence of the peptide begins at the N-terminal amino acid of the reference sequence, or an amino acid sequence substantially identical thereto (wherein

"substantially identical" as used herein means an amino acid substitution variant with respect to the MANS peptide sequence, which substitution variant is at least 75% identical to the amino acid sequence of the MANS peptide, and preferably differing from the MANS amino acid sequence by no more than one amino acid in a tetramer (4-mer) through to a heptamer (7-mer); preferably differing from the MANS amino acid sequence by no more than two amino acids (i.e., by 1 or by 2 amino acids) in an octamer (8-mer) through to an 11-mer; preferably differing from the MANS amino acid sequence by no more than three amino acids (i.e., by 1 or by 2 or by 3 amino acids) in a 12-mer through to a 15-mer; preferably differing from the MANS amino acid sequence by no more than four amino acids (i.e., by 1 or by 2 or by 3 or by 4 amino acids) in a 16-mer through to a 19-mer; preferably differing from the MANS amino acid sequence by no more than five amino acids (i.e., by 1 or by 2 or by 3 or by 4 or by 5 amino acids) in a 20-mer through to a 23-mer; and preferably differing from the MANS amino acid sequence by no more than six amino acids (i.e., by 1 or by 2 or by 3 or by 4 or by 5 or by 6 amino acids) in a 24- mer, which 24-mer is MANS); and wherein alanine (A) may be substituted with lysine (K), valine (V), leucine (L), or isoleucine (I); glutamic acid (E) may be substituted with aspartic acid (D); glycine (G) may be substituted with proline (P); lysine ( ) may be substituted with arginine (R), glutamine (Q), or asparagine (N); phenylalanine (F) may be substituted with leucine (L), valine (V), isoleucine (I), or alanine (A); proline (P) may be substituted with glycine (G); glutamine (Q) may be substituted with glutamic acid (E) or asparagine (N); arginine (R ) may be substituted with lysine ( ), glutamine (Q), or asparagine (N); serine (S) may be substituted with threonine; threonine (T) may be substituted with serine (S); and valine (V) may be substituted with leucine (L), isoleucine (I), methionine (M), phenylalanine (F), alanine (A), or norleucine (Nle) and (b) an amino acid sequence having from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1) or an amino acid sequence substantially identical thereto as defined above, which begins at the amino acid at position 2 through the amino acid at position 21 of the reference sequence; and wherein

wherein administration of a migration-inhibiting amount of said chemically modified peptide to an MSC forms a migration-inhibited MSC. In a preferred embodiment, the peptide is myristoylated at the N-terminal amine in the form of an amide of myristic acid.

[0068] The present invention further comprises a pharmaceutical composition comprising a migration-modulating peptide selected from the group consisting of MANS peptide, an active fragment of MANS peptide, an N-terminal chemically modified active fragment of MANS peptide, a C-terminal chemically modified active fragment of MANS peptide, a C-terminal chemically modified MANS peptide and a N-terminal and C- terminal chemically modified active fragment of MANS peptide, a fragment, as described herein, wherein the migration-modulating peptide is formulated in an aqueous medium suitable for inhalation, injection, or transplantation use.

[0069] The present invention further comprises a pharmaceutical composition comprising a migration-modulating peptide in the presence of a mesenchymal stem cell, together in an aqueous medium suitable for inhalation, injection, or transplantation use.

[0070] The present invention further comprises a pharmaceutical composition comprising a migration-modulating peptide selected from the group consisting of an active N-terminal MARCKS protein segment-peptide from 4 to 50 amino acids in length, an N-terminal chemically modified version of said MARCKS protein segment- peptide (such as an N-terminally myristoylated fragment of the MARCKS protein), a C- terminal chemically modified version of said MARCKS protein segment-peptide, and a C-terminal and N-terminal chemically modified version of said MARCKS protein segment-peptide, as described herein, wherein the migration-modulating peptide is formulated in an aqueous medium suitable for inhalation, injection, or transplantation use.

[0071] In another aspect of the present invention, the migration-modulating peptide is a MANS peptide, N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1).

[0072] In another aspect of the present invention, the migration-modulating peptide can be an active fragment of the MANS peptide.

[0073] An active fragment of a MANS peptide can be an N-terminal

myristoylated fragment of a MANS peptide selected from the group consisting of N- myristoyl-GAQFSKTAAKGEAAAERPGEAAV (SEQ ID No: 2); N-myristoyl- GAQFSKTAAKGEAAAERPGEAA (SEQ ID No: 4); N-myristoyl- GAQFSKTAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl- GAQFSKTAAKGEAAAERPGE (SEQ ID No: 1 1); N-myristoyl- GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl- GAQFSKTAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-

GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl- GAQFSKTAAKGEAA (SEQ ID No: 56); N-myristoyl-GAQFSKTAAKGEA (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl- GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFSKTAA (SEQ ID No: 121); N-myristoyl-GAQFSKTA (SEQ ID No: 137); N-myristoyl-GAQFSKT (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), and N-myristoyl-GAQF (SEQ ID No: 211),, which peptides are described in U.S. Patent 7,544,772, the disclosure of which is incorporated herein by reference.

[0074] The presence of the hydrophobic N-terminal myristoyl moiety in these peptides can enhance their compatibility with and presumably their permeability to plasma membranes, as well as potentially enable the peptides to be taken up by cells. The hydrophobic insertion of myristate into a bilayer can provide a partition coefficient or apparent association constant with lipids of up to 10⁴ M^"1 or a unitary Gibbs free binding energy of about 8 kcal/mol (see, for example, Peitzsch, R.M., and McLaughlin, S. 1993, Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry. 32: 10436-10443) which is sufficient, at least in part, to permit a partitioning of the MANS peptide and of myristoylated MANS peptide fragments as described herein into the plasma membrane of a cell while additional functional groups and their interactions within the MANS peptide (which is

myristoylated) and within myristoylated MANS peptide fragments can potentiate their relative membrane permeabilities. The fragments can each exhibit partition coefficients and membrane affinities that are representative of their respective structure. The fragments can be prepared by methods of peptide synthesis known in the art, such as by solid phase peptide synthesis (see, for example, the methods described in Chan, Weng C. and White, Peter D.Eds., Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, New York, New York (2000); and Lloyd- Williams, P. et al. Chemical Approaches to the Synthesis of Peptides and Proteins (1997)) and purified by methods known in the art, such as by high pressure liquid chromatography. Molecular weight of each peptide can be confirmed by mass spectroscopy with each showing a peak with an appropriate molecular mass. Efficacy of the individual peptides and of combinations of individual peptides (for example, combinations of 2 of the peptides, combinations of 3 of the peptides, combinations of 4 of the peptides) in the methods of this disclosure can be readily determined without undue experimentation using the procedures described in the examples disclosed herein. A preferred combination will comprise two of the peptides; a preferred molar ratio of the peptides can be from 50:50 to 99.99 to 0.01 , which ratio can be readily determined using the procedures described in the examples disclosed herein.

[0075] An active fragment of a MARCKS protein, or of a MANS peptide, is at least four amino acids in length in one aspect. In another aspect, an active fragment of a MARCKS protein, or of a MANS peptide, is at least six amino acids in length. As used herein, an "active fragment" of a MARCKS protein is one that affects (modulates, inhibits or enhances) the MARCKS protein-mediated migration of a stem cell.

[0076] In one aspect of the present invention, the migration-modulating peptide can be an N-terminal- and/or C-terminal-chemically modified peptide, which modified peptide consists of an active fragment amino acid sequence of MANS peptide, which peptide consists of an amino acid sequence selected from the group consisting of:

(b) an amino acid sequence substantially identical to the sequence defined in (a), wherein "substantially identical" is as explained above;

wherein

with the proviso that said chemically modified peptide contains no more than 23 amino acids; and wherein administration of a migration-inhibiting amount of said chemically modified peptide to an MSC forms a migration-inhibited MSC. Such chemically modified peptides are described in U.S. Patent 7,524,926, the disclosure of which is incorporated herein in its entirety by reference.

[0077] In a further aspect of the present invention, the migration-modulating peptide can be an N-terminal- and/or C-terminal-chemically modified peptide, which peptide consists of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence having from at least 4 to no more than 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1) or an amino acid sequence substantially identical thereto, wherein the amino acid sequence of the peptide begins at the N-terminal amino acid of the reference sequence, and wherein the N-terminal amino acid of the peptide may be myristoylated, and wherein "substantially identical" is as explained above;

(b) an amino acid sequence having from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1) or an amino acid sequence substantially identical thereto, which begins at begins at the amino acid at position 2 through the amino acid at position 21 of the reference sequence and wherein "substantially identical" is as explained above; and wherein at least one of the N- terminal amino acid or the C-terminal amino acid of the peptide selected from group (a) or group (b) is independently chemically modified,

[0078] Peptides having at least 4 to no more than 23 amino acids and which have amino acid sequences identical to amino acid sequences found in the MANS peptide as the reference peptide are listed in Table 1 , which table also includes the amino acid sequence of MANS peptide as Peptide No. 1 as well as the random sequence (RNS) peptide No. 232, which is used as a control to demonstrate that amino acid sequence order is relevant to efficacy in this invention., as well as random sequence control peptide 233.

[0079] TABLE 1

peptide 8 AQFSKTAAKGEAAAERPGEAA SEQ ID NO: 8 peptide 9 QFSKTAAKGEAAAERPGEAAV SEQ ID NO: 9 peptide 10 FSKTAAKGEAAAERPGEAAVA SEQ ID NO: 10 peptide 11 GAQFSKTAAKGEAAAERPGE SEQ ID NO: 11 peptide 12 AQFSKTAAKGEAAAERPGEA SEQ ID NO: 12 peptide 13 QFSKTAAKGEAAAERPGEAA SEQ ID NO: 13 peptide 14 FSKTAAKGEAAAERPGEAAV SEQ ID NO: 14 peptide 15 SKTAAKGEAAAERPGEAAVA SEQ ID NO: 15 peptide 16 GAQFSKTAAKGEAAAERPG SEQ ID NO: 16 peptide 17 AQFSKTAAKGEAAAERPGE SEQ ID NO: 17 peptide 18 QFSKTAAKGEAAAERPGEA SEQ ID NO: 18 peptide 19 FSKTAAKGEAAAERPGEAA SEQ ID NO: 19 peptide 20 SKTAAKGEAAAERPGEAAV SEQ ID NO: 20 peptide 21 KTAAKGEAAAERPGEAAVA SEQ ID NO: 21 peptide 22 GAQFSKTAAKGEAAAERP SEQ ID NO: 22 peptide 23 AQFSKTAAKGEAAAERPG SEQ ID NO: 23 peptide 24 QFSKTAAKGEAAAERPGE SEQ ID NO: 24 peptide 25 FSKTAAKGEAAAERPGEA SEQ ID NO: 25 peptide 26 SKTAAKGEAAAERPGEAA SEQ ID NO: 26 peptide 27 KTAAKGEAAAERPGEAAV SEQ ID NO: 27 peptide 28 TAAKGEAAAERPGEAAVA SEQ ID NO: 28 peptide 29 GAQFSKTAAKGEAAAER SEQ ID NO: 29 peptide 30 AQFSKTAAKGEAAAERP SEQ ID NO: 30 peptide 31 QFSKTAAKGEAAAERPG SEQ ID NO: 31 peptide 32 FSKTAAKGEAAAERPGE SEQ ID NO: 32 peptide 33 SKTAAKGEAAAERPGEA SEQ ID NO: 33 peptide 34 KTAAKGEAAAERPGEAA SEQ ID NO: 34 peptide 35 TAAKGEAAAERPGEAAV SEQ ID NO: 35 peptide 36 AAKGEAAAERPGEAAVA SEQ ID NO: 36 peptide 37 GAQFSKTAAKGEAAAE SEQ ID NO: 37 peptide 38 AQFSKTAAKGEAAAER SEQ ID NO: 38 peptide 39 QFSKTAAKGEAAAERP SEQ ID NO: 39 peptide 40 FSKTAAKGEAAAERPG SEQ ID NO: 40 peptide 41 SKTAAKGEAAAERPGE SEQ ID NO: 41 peptide 42 KTAAKGEAAAERPGEA SEQ ID NO: 42 peptide 43 TAAKGEAAAERPGEAA SEQ ID NO: 43 peptide 44 AAKGEAAAERPGEAAV SEQ ID NO: 44 peptide 45 AKGEAAAERPGEAAVA SEQ ID NO: 45 peptide 46 GAQFSKTAAKGEAAA SEQ ID NO: 46 peptide 47 AQFSKTAAKGEAAAE SEQ ID NO: 47 peptide 48 QFSKTAAKGEAAAER SEQ ID NO: 48 peptide 49 FSKTAAKGEAAAERP SEQ ID NO: 49 peptide 50 SKTAAKGEAAAERPG SEQ ID NO: 50 peptide 51 KTAAKGEAAAERPGE SEQ ID NO: 51 peptide 52 TAAKGEAAAERPGEA SEQ ID NO: 52 peptide 53 AAKGEAAAERPGEAA SEQ ID NO: 53 peptide 54 AKGEAAAERPGEAAV SEQ ID NO: 54 peptide 55 KGEAAAERPGEAAVA SEQ ID NO: 55 peptide 56 GAQFSKTAAKGEAA SEQ ID NO: 56 peptide 57 AQFSKTAAKGEAAA SEQ ID NO: 57 peptide 58 QFSKTAAKGEAAAE SEQ ID NO: 58 peptide 59 FSKTAAKGEAAAER SEQ ID NO: 59 peptide 60 SKTAAKGEAAAERP SEQ ID NO: 60 peptide 61 KTAAKGEAAAERPG SEQ ID NO: 61 peptide 62 TAAKGEAAAERPGE SEQ ID NO: 62 peptide 63 AAKGEAAAERPGEA SEQ ID NO: 63 peptide 64 AKGEAAAERPGEAA SEQ ID NO: 64 peptide 65 KGEAAAERPGEAAV SEQ ID NO: 65 peptide 66 GEAAAERPGEAAVA SEQ ID NO: 66 peptide 67 GAQFSKTAAKGEA SEQ ID NO: 67 peptide 68 AQFSKTAAKGEAA SEQ ID NO: 68 peptide 69 QFSKTAAKGEAAA SEQ ID NO: 69 peptide 70 FSKTAAKGEAAAE SEQ ID NO: 70 peptide 71 SKTAAKGEAAAER SEQ ID NO: 71 peptide 72 KTAAKGEAAAERP SEQ ID NO: 72 peptide 73 TAAKGEAAAERPG SEQ ID NO: 73 peptide 74 AAKGEAAAERPGE SEQ ID NO: 74 peptide 75 AKGEAAAERPGEA SEQ ID NO: 75 peptide 76 KGEAAAERPGEAA SEQ ID NO: 76 peptide 77 GEAAAERPGEAAV SEQ ID NO: 77 peptide 78 EAAAERPGEAAVA SEQ ID NO: 78 peptide 79 GAQFSKTAAKGE SEQ ID NO: 79 peptide 80 AQFSKTAAKGEA SEQ ID NO: 80 peptide 81 QFSKTAAKGEAA SEQIDNO: 81 peptide 82 FSKTAAKGEAAA SEQ ID NO: 82 peptide 83 SKTAAKGEAAAE SEQ ID NO: 83 peptide 84 KTAAKGEAAAER SEQ ID NO: 84 peptide 85 TAAKGEAAAERP SEQ ID NO: 85 peptide 86 AAKGEAAAERPG SEQ ID NO: 86 peptide 87 AKGEAAAERPGE SEQ ID NO: 87 peptide 88 KGEAAAERPGEA SEQ ID NO: 88 peptide 89 GEAAAERPGEAA SEQ ID NO: 89 peptide 90 EAAAERPGEAAV SEQ ID NO: 90 peptide 91 AAAERPGEAAVA SEQIDNO: 91 peptide 92 GAQFSKTAAKG SEQ ID NO: 92 peptide 93 AQFSKTAAKGE SEQ ID NO: 93 peptide 94 QFSKTAAKGEA SEQ ID NO: 94 peptide 95 FSKTAAKGEAA SEQ ID NO: 95 peptide 96 SKTAAKGEAAA SEQ ID NO: 96 peptide 97 KTAAKGEAAAE SEQ ID NO: 97 peptide 98 TAAKGEAAAER SEQ ID NO: 98 peptide 99 AAKGEAAAERP SEQ ID NO: 99 peptide 100 AKGEAAAERPG SEQ ID NO: 100 peptide 101 KGEAAAERPGE SEQ ID NO: 101 peptide 102 GEAAAERPGEA SEQIDNO: 102 peptide 103 EAAAERPGEAA SEQIDNO: 103 peptide 104 AAAERPGEAAV SEQIDNO: 104 peptide 105 AAERPGEAAVA SEQIDNO: 105 peptide 106 GAQFSKTAAK SEQ ID NO: 106 peptide 107 AQFSKTAAKG SEQIDNO: 107 peptide 108 QFSKTAAKGE SEQIDNO: 108 peptide 109 FSKTAAKGEA SEQIDNO: 109 peptide 110 SKTAAKGEAA SEQIDNO: 110 peptide 111 KTAAKGEAAA SEQIDNO: 111 peptide 112 TAAKGEAAAE SEQIDNO: 112 peptide 113 AAKGEAAAER SEQIDNO: 113 peptide 114 AKGEAAAERP SEQIDNO: 114 peptide 115 KGEAAAERPG SEQID O: 115 peptide 116 GEAAAERPGE SEQ ID NO: 116 peptide 117 EAAAERPGEA SEQ ID NO: 117 peptide 118 AAAERPGEAA SEQ ID NO: 118 peptide 119 AAERPGEAAV SEQ ID NO: 119 peptide 120 AERPGEAAVA SEQ ID NO: 120 peptide 121 GAQFSKTAA SEQ ID NO: 121 peptide 122 AQFSKTAAK SEQ ID NO: 122 peptide 123 QFSKTAAKG SEQ ID NO: 123 peptide 124 FSKTAAKGE SEQ ID NO: 124 peptide 125 SKTAAKGEA SEQ ID NO: 125 peptide 126 KTAAKGEAA SEQ ID NO: 126 peptide 127 TAAKGEAAA SEQ ID NO: 127 peptide 128 AAKGEAAAE SEQ ID NO: 128 peptide 129 AKGEAAAER SEQ ID NO: 129 peptide 130 KGEAAAERP SEQ ID NO: 130 peptide 131 GEAAAERPG SEQ ID NO: 131 peptide 132 EAAAERPGE SEQ ID NO: 132 peptide 133 AAAERPGEA SEQ ID NO: 133 peptide 134 AAERPGEAA SEQ ID NO: 134 peptide 135 AERPGEAAV SEQ ID NO: 135 peptide 136 ERPGEAAVA SEQ ID NO: 136 peptide 137 GAQFSKTA SEQ ID NO: 137 peptide 138 AQFSKTAA SEQ ID NO: 138 peptide 139 QFSKTAAK SEQ ID NO: 139 peptide 140 FSKTAAKG SEQ ID NO: 140 peptide 141 S TAAKGE SEQ ID NO: 141 peptide 142 KTAAKGEA SEQ ID NO: 142 peptide 143 TAAKGEAA SEQ ID NO: 143 peptide 144 AA GEAAA SEQ ID NO: 144 peptide 145 AKGEAAAE SEQ ID NO: 145 peptide 146 KGEAAAER SEQ ID NO: 146 peptide 147 GEAAAERP SEQ ID NO: 147 peptide 148 EAAAERPG SEQ ID NO: 148 peptide 149 AAAERPGE SEQ ID NO: 149 peptide 150 AAERPGEA SEQ ID NO: 150 peptide 151 AERPGEAA SEQ ID NO: 151 peptide 152 ERPGEAAV SEQ ID NO: 152 peptide 153 RPGEAAVA SEQ ID NO: 153 peptide 154 GAQFSKT SEQ ID NO: 154 peptide 155 AQFSKTA SEQ ID NO: 155 peptide 156 QFSKTAA SEQ ID NO: 156 peptide 157 FSKTAAK SEQ ID NO: 157 peptide 158 SKTAAKG SEQ ID NO: 158 peptide 159 KTAAKGE SEQ ID NO: 159 peptide 160 TAAKGEA SEQ ID NO: 160 peptide 161 AAKGEAA SEQ ID NO: 161 peptide 162 A GEAAA SEQ ID NO: 162 peptide 163 KGEAAAE SEQ ID NO: 163 peptide 164 GEAAAER SEQ ID NO: 164 peptide 165 EAAAERP SEQ ID NO: 165 peptide 166 AAAERPG SEQ ID NO: 166 peptide 167 AAERPGE SEQ ID NO: 167 peptide 168 AERPGEA SEQ ID NO: 168 peptide 169 ERPGEAA SEQ ID NO: 169 peptide 170 RPGEAAV SEQ ID NO: 170 peptide 171 PGEAAVA SEQ ID NO: 171 peptide 172 GAQFSK SEQ ID NO: 172 peptide 173 AQFSKT SEQ ID NO: 173 peptide 174 QFSKTA SEQ ID NO: 174 peptide 175 FSKTAA SEQ ID NO: 175 peptide 176 S TAAK SEQ ID NO: 176 peptide 177 KTAAKG SEQ ID NO: 177 peptide 178 TAAKGE SEQ ID NO: 178 peptide 179 AAKGEA SEQ ID NO: 179 peptide 180 A GEAA SEQ ID NO: 180 peptide 181 GEAAA SEQ ID NO: 181 peptide 182 GEAAAE SEQ ID NO: 182 peptide 183 EAAAER SEQ ID NO: 183 peptide 184 AAAERP SEQ ID NO: 184 peptide 185 AAERPG SEQ ID NO: 185 peptide 186 AERPGE SEQ ID NO: 186 peptide 187 ERPGEA SEQ ID NO: 187 peptide 188 RPGEAA SEQIDNO: 188 peptide 189 PGEAAV SEQIDNO: 189 peptide 190 GEAAVA SEQIDNO: 190 peptide 191 GAQFS SEQIDNO: 191 peptide 192 AQFSK SEQIDNO: 192 peptide 193 QFSKT SEQIDNO: 193 peptide 194 FSKTA SEQIDNO: 194 peptide 195 S TAA SEQIDNO: 195 peptide 196 KTAAK SEQIDNO: 196 peptide 197 TAAKG SEQIDNO: 197 peptide 198 AAKGE SEQIDNO: 198 peptide 199 A GEA SEQIDNO: 199 peptide 200 GEAA SEQ ID NO: 200 peptide 201 GEAAA SEQIDNO: 201 peptide 202 EAAAE SEQ ID NO: 202 peptide 203 AAAER SEQ ID NO: 203 peptide 204 AAERP SEQ ID NO: 204 peptide 205 AERPG SEQ ID NO: 205 peptide 206 ERPGE SEQ ID NO: 206 peptide 207 RPGEA SEQ ID NO: 207 peptide 208 PGEAA SEQ ID NO: 208 peptide 209 GEAAV SEQ ID NO: 209 peptide 210 EAAVA SEQ ID NO: 210 peptide 211 GAQF SEQIDNO: 211 peptide 212 AQFS SEQ ID NO: 212 peptide 213 QFSK SEQIDNO: 213 peptide 214 FSKT SEQIDNO: 214 peptide 215 SKTA SEQ ID NO: 215 peptide 216 KTAA SEQID O: 216 peptide 217 TAA SEQIDNO: 217 peptide 218 AAKG SEQIDNO: 218 peptide 219 AKGE SEQ ID NO: 219 peptide 220 KGEA SEQ ID NO: 220 peptide 221 GEAA SEQ ID NO: 221 peptide 222 EAAA SEQ ID NO: 222 peptide 223 AAAE SEQ ID NO: 223 peptide 224 AAER SEQ ID NO: 224 peptide 225 AERP SEQ ID NO: 225

peptide 226 ERPG SEQ ID NO: 226

peptide 227 RPGE SEQ ID NO: 227

peptide 228 PGEA SEQ ID NO: 228

peptide 229 GEAA SEQ ID NO: 229

peptide 230 EAAV SEQ ID NO: 230

peptide 231 AAVA SEQ ID NO: 231

Peptide 232 GTAPAAEGAGAEV RASAEAKQAF SEQ ID NO: 232

Peptide 233 GKASQFAKTA SEQ ID NO: 233

[0080] Peptides which can be useful in the current invention can be selected from the group consisting of isolated peptides having amino acid sequences listed in Table 1 from SEQ ID NO: 1 to SEQ ID NO: 231, where peptides of amino acid sequences SEQ ID NO: 1 through 231 as listed in Table 1 which are optionally N-terminal- and/or C- terminal-chemically modified.

[0081] Preferred independent N-terminal chemical modifications of the peptides from SEQ ID NO: 1 to SEQ ID NO: 231 listed in Table 1 include N-terminal amine group modification by acylation of the N-terminal amino acid of the peptide in the form of an amide selected from the group consisting of:

an amide of a C₂ (acetyl) to C₂₄ aliphatic carboxylic acid which may be linear, branched, saturated, or unsaturated,

an amide of trifluoroacetic acid,

an amide of benzoic acid, and

an amide of a C_\ to C₂₄ aliphatic alkyl sulfonic acid; or

the N-terminal amine group of the N-terminal amino acid of the peptide can be alkylated with a group selected from the group consisting of:

a Cj to C₂₄ aliphatic alkyl group,

a linear 2-(C] to C₂₄ aliphatic alkyl)oxyethyl group,

an omega-methoxy-poly( ethyl eneoxy)_n-ethyl group, where n is from 0 to 10.

[0082] Preferred independent C-terminal chemical modifications of the peptides from SEQ ID NO: 1 to SEQ ID NO: 231 listed in Table 1 include amide formation at the C- terminal carboxylic acid group of the C-terminal amino acid of the peptide in the form of an amide selected from the group consisting of:

an amide of ammonia, an amide of a Ci to C₂₄ aliphatic alkyl amine,

an amide of a hydroxyl-substituted C₂ to C₂₄ aliphatic alkyl amine,

an amide of a linear 2-(Cl to C24 aliphatic alkyl)oxyethylamine group, and an amide of an omega-methoxy-poly(ethyleneoxy)_n-ethylamine group, where n is from 0 to 10.

[0083] In addition, the C-terminal carboxylic acid group of the C-terminal amino acid of the peptide is optionally in the form of an ester selected from the group consisting of: an ester of a Ci to C₂₄ aliphatic alkyl alcohol,

an ester of a 2-(omega-methoxy-poly(ethyleneoxy)_n)-ethanol group, where n is from 0 to 10

an ester of a linear PEG-amine, the PEG component of molecular weight from 1 ,000 to 25,000 Daltons.

[0084] In one embodiment, aliphatic portions of carboxylic acid groups and sulfonic acid groups and alcohol and amino groups can comprise a ring of at least C3 (i.e., at least a cyclopropyl ring).

[0085] In one embodiment, the peptide can be N-terminally modified, for example, by an acetyl group or a myristoyl group, as an N-terminal amide, such as Acetyl-GAQFSKTAAK (N-terminal acetyl SEQ ID No: 106) and myristoyl- GAQFSKTAAK (N-terminal myristoyl SEQ ID No: 106), respectfully. In another embodiment, the peptide can be C-terminally modified (for example by an amide with ammonia) such as GAQFSKTAAK-NH₂ (SEQ ID No: 106 C-terminal amide). In another embodiment, the peptide can be N-terminally modified and C-terminally modified, for example as N-acetyl-peptide-C-amide (with ammonia) such as Acetyl-GAQFSKTAAK- NH₂. (N-terminal acetyl SEQ ID No: 106 C-terminal amide) and Myristoyl- GAQFS TAAK-NH₂ (N-terminal myristoyl SEQ ID No: 106 C-terminal amide). These peptides can be used in the methods of this invention to determine their ability to form migration-inhibited MSCs.

[0086] In one embodiment, a peptide which may find use in this invention can be selected from the group of peptides which contain the amino acid sequence AKGE (SEQ ID No: 219). Such peptides include SEQ ID No: 1 through SEQ ID No: 54, SEQ ID No: 56 through SEQ ID No: 64, SEQ ID No: 67 through SEQ ID No: 75, SEQ ID No: 79 through SEQ ID No: 87, SEQ ID No: 93 through SEQ ID No: 100, SEQ ID No: 108 through SEQ ID No: 1 14, SEQ ID No: 124 through SEQ ID No: 129, SEQ ID No: 141 through SEQ ID No: 145, SEQ ID No: 159 through SEQ ID No: 162, SEQ ID No: 178 through SEQ ID No: 180, SEQ ID No: 198, SEQ ID No: 199, and SEQ ID No: 219. In one currently preferred embodiment, these peptides are myristoylated at the N-terminal amino group.

[0087] In one embodiment, this invention discloses a method of modulating (i.e., of attenuating) the MARC S -related migration of a mesenchymal stem cell toward an increasing concentration gradient of a chemotactic agent in a fluid or tissue in a subject, the method comprising treatment of said mesenchymal stem cell with a migration- inhibiting amount of a migration-modulating peptide and incubation of said cell with said peptide to form a migration-inhibited mesenchymal stem cell, and administering the migration-inhibited mesenchymal stem cell to said fluid or tissue.

[0088] In one aspect, the migration-modulating peptide is selected from the group consisting of MANS peptide (N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID No: 1), and an active fragment peptide thereof having from 4 to 23 amino acids in the sequence of amino acids found in MANS peptide.

[0089] In another aspect, the active peptide fragment comprises the amino acid sequence GAQFSKTAAK (SEQ ID No: 106).

[0090] In another aspect, the migration-modulating peptide is selected from the group consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID No: 1) N-myristoyl-GAQFSKTAAKGEAAAERPGEAAV (SEQ ID No: 2); N-myristoyl- GAQFSKTAAKGEAAAERPGEAA (SEQ ID No: 4); N-myristoyl- GAQFSKTAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl- GAQFSKTAAKGEAAAERPGE (SEQ ID No: 1 1); N-myristoyl- GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl- GAQFSKTAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-

GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl- GAQFSKTAAKGEAA (SEQ ID No: 56); N-myristoyl-GAQFSKTAAKGEA (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl- GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFS TAA (SEQ ID No: 121); N-myristoyl-GAQFS TA (SEQ ID No: 137); N-myristoyl-GAQFS T (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), N-myristoyl-GAQF (SEQ ID No: 211), and a combination thereof.

[0091] In another aspect, the chemotactic agent is a chemokine or cytokine.

Further the chemotatic agent is selected from the group consisting of MCP-1 (Monocyte chemotactic protein-1), SDF-1 (Stromal cell-derived factor 1), SDF-Ια (Stromal cell- derived factor-1 alpha), SCF(stem cell factor) and C5a (Complement factor 5a).

[0092] In a preferred aspect, the concentration gradient of the chemotactic agent is established with a concentration in the range from 0.5 to 200 ng/mL. In a preferred aspect, the migration-inhibiting amount of the migration-modulating peptide is in the range of 100 nanoM to 100 microM. In a preferred aspect, the incubation is for a period of 10 to 30 minutes. In a preferred aspect, the tissue is injured or diseased such as injured or diseased lung tissue. In a preferred aspect, the fluid is selected from the group consisting of blood, serum, lymph, urine, mucus, saline, lacrimate, perspiration, and ascites fluid.

[0093] In another aspect, the active fragment peptide is an N- terminal- and/or C- terminal-chemically modified peptide, which peptide consists of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence of from 4 to 23 contiguous amino acids of a reference sequence, GAQFS TAA GEAAAERPGEAAVA (SEQ ID NO: 1), wherein the amino acid sequence of the peptide begins at the N-terminal amino acid of the reference sequence, or an amino acid sequence substantially identical thereto; and

(b) an amino acid sequence having from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1) or an amino acid sequence substantially identical thereto, which begins at the amino acid at position 2 through the amino acid at position 21 of the reference sequence; and wherein (i) the C-terminal amino acid of said chemically modified peptide is chemically modified and the N-terminal amino acid of the peptide is optionally chemically modified; or

(ii) the C-terminal amino acid of said chemically modified peptide is not chemically modified and the N-terminal amino acid of the peptide is chemically modified;

wherein administration of a migration-inhibiting amount of said chemically modified peptide to a mesenchymal stem cell forms a migration-inhibited mesenchymal stem cell. In a preferred aspect, the peptide is amidated with a linear, branched, saturated, or unsaturated C2 to C24 aliphatic carboxylic acid at the N-terminal amino acid of the peptide. In a more preferred aspect, the peptide is myristoylated at the N-terminal amino acid of the peptide. In another aspect, the peptide comprises the amino acid sequence AKGE (SEQ ID NO: 219).

[0094] Peptides and chemically modified peptides described herein can find utility in the methods of this invention to form migration-inhibited MSCs.

[0095] The peptides which find utility in this invention can be in the form of pharmaceutically acceptable salts.

[0096] The peptides which find utility in this invention can be in the form of a pharmaceutical composition comprising a peptide of the invention and a

pharmaceutically acceptable carrier. Such a composition is preferably sterile, and/or sterilized, and/or sterilizable. Such a composition is preferably isotonic to avoid rupture or lysis of the MSCs.

[0097] Figure 1 illustrates that mouse primary bone marrow mesenchymal stem cells (BM-MSCs) migrate in response to the presence of at least chemokines MCP-1, C5a, and SDF-1 in vitro. The height of the bars represent the mean number of migrated BM-MSCs ± SEM observed within 45 randomly chosen microscopic fields

(magnification = 400x) under the conditions associated with the bars and which are identified as follows: bar number 10 shows a mean number of 0 cells per field related to use of SF-IMDM (0) = serum-free Iscove's Modified Dulbecco's Medium as a negative control; bar number 1 1 shows a mean number of 2.5 cells per field related to use of SF- IMDM (4) = serum-free Iscove's Modified Dulbecco's Medium as a random control; bar number 12 shows a mean number of 43 cells per field related to use of cIMDM = IMDM containing 20% serum; bar number 13 shows a mean number of 25 cells per field related to use of MCP-1 = 50 ng/ml MCP-1 in SF-IMDM; bar number 14 shows a mean number of 23 cells per field related to use of C5a = 50ng/ml C5a in SF-IMDM; and bar number 15 shows a mean number of 39 cells per field related to use of SDF-1 = 50 ng/ml SDF-1 in SF-IMDM.

[0098] Figure 2 A illustrates a determination of an optimum chemokine SDF-1 concentration for mouse BM-MSC migration in vitro. Thus, after 1.5 x 10⁵ vital mouse BM-MSCs are loaded into 5 mm pore transwell inserts with lower chambers containing SF-IMDM supplemented with increasing concentrations of the chemokine SDF-1 , the following results are obtained. Bar number 23 shows a mean number of 24 cells per field related to use of 25 ng/mL SDF-1 ; bar number 24 shows a mean number of 14 cells per field related to use of 50 ng/mL SDF-1 ; and bar number 25 shows a mean number of 6 cells per field related to use of 100 ng/mL SDF-1. Bar 20 (which shows a mean number of 0 cells per field) and bar 21 (which shows a mean number of 4 cells per field) are related to SF-IMDM and represent negative or random movement controls. Bar 22, related to cIMDM, is a positive migration control. Each bar represents the mean number of migrated BM-MSCs ± SEM within 45 randomly chosen microscopic fields

(magnification = 400x). Starred bars represent statistically significant decrease in migrated BM-MSCs, determined by unpaired two-tailed t tests.

[0099] Figure 2B illustrates a determination of an optimum chemokine C5a concentration for mouse BM-MSC migration in vitro. Thus, after 1.5 x 10⁵ vital mouse BM-MSCs are loaded into 5 mm pore transwell inserts with lower chambers containing SF-IMDM supplemented with increasing concentrations of the chemokine C5a, the following results are obtained. Bar number 33 shows a mean number of 9 cells per field related to use of 25 ng/mL C5a; bar number 34 shows a mean number of 19 cells per field related to use of 50 ng/mL C5a; bar number 35 shows a mean number of 15 cells per field related to use of 100 ng/mL C5a; and bar number 36 shows a mean number of 10 cells per field related to use of 200 ng/mL C5a. Bar 30 (mean ~1 cell) and bar 31 (mean ~4 cells) are related to SF-IMDM and represent negative or random movement controls. Bar 32 (mean ~59 cells), related to cIMDM, is a positive migration control. Each bar represents the mean number of migrated BM-MSCs ± SEM within 45 randomly chosen microscopic fields (magnification = 400x). Starred bars represent statistically significant decrease in migrated BM-MSCs, determined by unpaired two-tailed t tests.

[00100] Figure 2C illustrates a determination of an optimum chemokine SCF concentration for mouse BM-MSCs migration in vitro. Thus, after 1.5 x 10⁵ vital mouse BM-MSCs are loaded into 5 mm pore transwell inserts with lower chambers containing SF-IMDM supplemented with increasing concentrations of the chemokine SCF, the following results are obtained. Bar number 43 shows a mean number of 38 cells per field related to use of 0.5 ng/mL SCF; bar number 44 shows a mean number of 30 cells per field related to use of 5 ng/mL SCF; bar number 45 shows a mean number of 34 cells per field related to use of 25 ng/mL SCF; bar number 46 shows a mean number of 16 cells per field related to use of 50 ng/mL SCF; and bar number 47 shows a mean number of 6 cells per field related to use of 100 ng/mL SCF. Bar 40 (mean ~0 cells) and bar 41 (mean ~3 cells) are related to SF-IMDM and represent negative or random movement controls. Bar 42 (mean -62 cells), related to cIMDM, is a positive migration control. Each bar represents the mean number of migrated BM-MSCs ± SEM within 45 randomly chosen microscopic fields (magnification = 400x). Starred bars represent statistically significant decrease in migrated BM-MSCs, determined by unpaired two-tailed t tests.

[00101] Figure 3 illustrates a Western blot of MARCKS protein in mouse primary BM-MSCs. Each lane contains 20 mg per well of resolved whole cell lysate. The lane labels and their representations are as follows: MW = Dual color molecular weight ladder; BSA = bovine serum albumin fraction V; J774A.1 = monocyte-macrophage J774A.1 cell line; NHLF = native human lung fibroblasts; Mouse BM-MSC = primary mouse bone marrow mesenchymal stem cells; and Human CB-MSC = human cord blood mesenchymal stem cells. MARCKS protein resolves at 80kDa.

[00102] Figure 4 illustrates that mouse primary BM-MSC migration in vitro can be disrupted by MANS peptide. Thus, 1.5 x 10⁵ BM-MSCs were incubated at 37 °C with 100 μΜ MANS peptide or separately incubated with 100 μΜ RNS peptide for 15 minutes prior to migration experiments. Bar number 51 shows a mean number of 10 migrating cells per field related to use of BSA, and represents a carrier negative control; bar number 52 shows a mean number of 55 migrating cells per field related to use of cIMDM, and represents a positive control; bar number 53 shows a mean number of 26 migrating cells per field related to use of C5a and represents cells moving towards 50 ng/ml C5a; bar number 54 shows a mean number of 11 migrating cells per field related to use of C5a with cells which are preincubated with MANS peptide as an inhibitor of stem cell migration, and represents migrating BM-MSCs which have been incubated with MANS peptide and which are moving towards 50 ng/ml C5a, and demonstrates that MANS peptide can inhibit (1 1/26 = 42 or a 58% reduction in migration of MCSs) in vitro mesenchymal stem cell migration relative to the number of MSCs migrating in the absence of MANS peptide; bar number 55 shows a mean number of 31 migrating cells per field related to use of C5a with cells which are preincubated with RNS peptide, and represents BM-MSCs which have been incubated with RNS peptide moving towards 50 ng/ml C5a; bar number 56 shows a mean number of 21 migrating cells per field related to use of SDF-1 and represents cells moving towards 25 ng/ml SDF-1 ; bar number 57 shows a mean number of 8 migrating cells per field related to use of SDF-1 with cells which are preincubated with MANS peptide as an inhibitor of stem cell migration, and represents migrating BM-MSCs which have been incubated with MANS peptide and which are moving towards 25 ng/ml SDF-1, and demonstrates that MANS peptide can inhibit in vitro mesenchymal stem cell migration relative to the number of MSCs migrating in the absence of MANS peptide, the ratio of 8/21 being 38 representing a reduction of 62%; and bar number 58 shows a mean number of 49 migrating cells per field related to use of SDF-1 with cells which are preincubated with RNS peptide, and represents BM-MSCs which have been incubated with RNS peptide moving towards 25 ng/ml SDF-1. Each bar represents the mean number of migrated BM-MSCs ± SEM within 30 randomly chosen microscopic fields (magnification = 400x). Starred bars represent statistically significant decrease in migrated BM-MSCs, determined by unpaired two-tailed t tests.

[00103] Figure 5 illustrates that MANS peptide inhibition of mouse BM-MSC movement is concentration-dependent. Thus, after 1.5 x 10⁵ mouse BM-MSCs were incubated at 37 °C with increasing concentrations of MANS peptide (500 nM (nanoM) to 50 μΜ (microM); bars 64, 65, and 66) or 50 μΜ (microM) RNS peptide (bar 67) for 15 minutes prior to exposure to 50 ng/ml C5a. Each bar represents the mean number of migrated BM-MSCs ± SEM within 30 randomly chosen microscopic fields (magnification = 400x). Starred bars represent statistically significant decrease in migrated BM-MSCs, determined by unpaired two-tailed t tests.) Bar 60 (mean ~1 cell) represents a negative control with SF-IMDM; bar 61 shows a mean number of 6 cells per field related to use of SF-IMDM as random movement control. Bar 62 shows a mean number of 50 cells per field related to use of cIMDM and is a positive control. Bar 63 shows a mean number of 24 cells per field related to use of C5a alone, and represents migration of cells not exposed to MANS peptide and exposed only to 50 ng/ml C5a. Bar 64 shows a mean number of 41 migrating cells per field related to use of C5a with BM- MSCs which are preincubated with 500 nM of MANS peptide, which peptide the inventors have discovered is a concentration dependent inhibitor of stem cell migration, and which bar 64 represents the number of migrating BM-MSCs which have been incubated with 500 nM of MANS peptide and which are moving towards 50 ng/ml C5a. Bar 65 shows a mean number of 22 migrating cells per field related to use of C5a with BM-MSCs which are preincubated with 5 μΜ of MANS peptide, which peptide the inventors have discovered is a concentration dependent inhibitor of stem cell migration, and which bar 65 represents the number of migrating BM-MSCs which have been incubated with 5 μΜ of MANS peptide and which are moving towards 50 ng/ml C5a. Bar 66 shows a mean number of 2.5 migrating cells per field related to use of C5a with BM-MSCs which are preincubated with 50 μΜ of MANS peptide, which peptide the inventors have discovered is a concentration dependent inhibitor of stem cell migration, and which bar 66 represents the number of migrating BM-MSCs which have been incubated with 50 μΜ of MANS peptide and which are moving towards 50 ng/ml C5a. Bar 67 shows a mean number of 43 migrating cells per field related to use of C5a with BM-MSCs which are preincubated with 50 μΜ of RNS peptide, a peptide consisting of the same type and number of amino acids found in MANS peptide but arranged in a randomized order relative to MANS peptide. This experiment suggests that the amino acid sequence of MANS peptide is a significant factor in the ability of MANS peptide to act as an inhibitor of stem cell migration, and is consistent with inhibition of a

MARC S-related mechanism of stem cell migration.

[00104] There is compelling evidence to suggest that MARCKS regulates cytoskeletal rearrangement as well as binding of the actin cytoskeleton to the leading edge of the plasma membrane during migration. MARCKS has been shown to bind and cross-link actin in a variety of cell types: see Thelen et al., Nature 351 :320-322 (1991); and see Vergeres et al., The Journal of biological chemistry 270:19879-19887 (1995); and see Hartwig et al., Nature 356:618-622 (1992). The inventors propose that a close relationship exists between MARCKS protein and the cytoskeletal machinery responsible for directed migration in mesenchymal stem cells. MANS peptide has been shown to inhibit MARCKS protein binding to granule membranes within bronchial epithelial cells, while not affecting phosphorylation levels (see Park et al., Am J Pathol 171 : 1822-1830 (2007)). MANS peptide occupation of MARCKS protein binding sites on a cell plasma membrane may prevent endogenous MARCKS protein from reassociating with the membrane after phosphorylation. By potentially forcing MARCKS protein to a modified concentration distribution in the cytosol, MANS peptide may also be inhibiting migration by displacing MARCKS protein to an inappropriate subcellular compartment, disrupting MARCKS function in migration and perhaps disrupting MARCKS function in adhesion. This is shown schematically in Figure 8, described below.

[00105] Figure 6A illustrates the effect of MANS peptide on BM-MSC migration in gradients of C5a and of SDF-Ια. Thus, 1.5 x 10⁵ Mouse BM-MSC were mock- exposed, or exposed to 100 μΜ MANS peptide, or 100 μΜ RNS peptide at 37°C, then loaded into 5 μιη pore Transwell™ chambers. The lower well contained: no chemokine (bar 70, height ~1 cells/field), 0.1% BSA (bar 71 , height -9 cells/field), 20% serum (bar 72, height -55 cells/field), 50 ng/ml of C5a (bar 73, height -25 cells/field), or 25 ng/ml of SDF-Ια in SF-IMDM. All samples were maintained for 3 hours at 37°C in humidified 5% C0₂ atmosphere, except To (time zero) samples SF-IMDM (0). Each bar represents the mean number of cells per field of 45 randomly selected fields at 400x magnification. Error is SEM. Stars [***] indicate mean number of cells per field (bar height) is significantly different (p < .05.). While experiments employing 50 ng mL of C5a alone produced a bar graph height of 25 cells/field (bar 73, exposure to MANS peptide reduced the migration of MSCs by more than 50% as shown by a bar height of 10 cells/field (bar 74). Use of RNS peptide under identical conditions produced a bar height of 31 cells cells/field (bar 75), indicating no decrease in MCS migration as a result of exposure to RNS peptide. In addition, in experiments employing 25 ng/mL of SDF-Ια alone, a bar graph height of 21 cells/field was produced (bar 76), while exposure to MANS peptide reduced the migration of MSCs by more than 50% as shown by a bar height of 7 cells/field (bar 77). Use of RNS peptide under identical conditions produced a bar height of 49 cells cells/field (bar 78), indicating no decrease in MCS migration (and possible increase) as a result of exposure to RNS peptide.

[00106] Figure 6B illustrates the effect of MANS peptide on BM-MSC migration in gradients of C5a after treatment with MANS peptide. Thus, 1.5 x 10⁵ Mouse BM-MSC were mock-exposed, separately exposed to MANS peptide at concentrations of 500 nM, 5 μΜ, and 50 μΜ, or exposed to 50 μΜ RNS peptide, each at 37°C, then loaded into 5 μιη Transwell™ chambers. The lower well contained: no chemokine (bar 80, height = ~0 cells/field), or 20% serum (bar 82, height 50 cells/field), or 50 ng/ml of C5a in SF-IMDM (bar 83, height = 24 cells/field. All samples were maintained for 3 hours at 37°C in humidified 5% C0₂ atmosphere, except T₀ (time zero) samples SF-IMDM (0). Each bar represents the mean number of cells per field of 45 randomly selected fields at 400x magnification. Error is SEM. Stars [***] indicate mean number of cells per field (bar height) is significantly different (p < .05.) Bar 84 with height of 41 cells/field was produced in experiments using C5a and exposure to 500 nM MANS peptide. This bar height was reduced by about 50% to 22 cells/field in experiments using C5a and exposure to 5 μΜ (5 microM) MANS peptide (bar 85). This bar height was further significantly reduced to 3 cells/field in experiments using C5a and exposure to 50 μΜ (50 microM) MANS peptide (bar 86), reflecting the migration-inhibiting effect of MANS peptide on MSCs. Interestingly, in experiments using C5a and exposure to 50 μΜ (50 microM) random sequence RNS peptide, the bar height of 43 cells/field (bar 87) indicated no significant migration inhibiting effect was induced by the RNS peptide.

[00107] Figure 7A illustrates that mice sensitized to, and challenged in vivo with, OVA aerosol (OVA-sham cells, bar 91) demonstrate a significant increase in lavage eosinophils compared to alum-treated mice (Alum-sham cells, bar 90). Adoptive transfer of MSCs pre-incubated with the MANS peptide, was associated with reduced lung eosinophil accumulation (OVA-MSC-MANS; bar 93, height of 18 x 10³ lavage eosinophils/mL) relative to mice that received MSCs pre-incubated with a control peptide (OVA-MSC-RNS; bar 92, height of 43 x 10³ lavage eosinophils/mL). [00108] Figure 7B illustrates that mice sensitized to, and challenged in vivo with, OVA aerosol (OVA-sham cells, bar 94) demonstrate an increase in mucus cell metaplasia as assessed by PAS semi-quantitative scoring compared to alum-treated mice (Alum- sham cells). Similar trends were found for mucus cell metaplasia as assessed by PAS semi-quantitative scoring. PAS score was zero in alum-treated mice (not shown). OVA- treatment resulted in mucus cell metaplasia which was enhanced in mice that received MANS-incubated MSCs (PAS score of 2.0, bar 96) compared to RNS-incubated MSCs (PAS score of 2.5, bar 95).

[00109] From these in vivo experiments, it appears that the delivery of MSCs prior to OVA aerosol exacerbates eosinophilic inflammation and mucus cell metaplasia, but inhibition of MSC MARCKS function with MANS reduces the infiltration of lung eosinophils and PAS score. Airway responsiveness was unaffected by MSC

administration in this model. These two components of the experiment are consistent with a fundamental role for MARCKS, and therefore MANS, in controlling cell migration.

[00110] The inventors have previously demonstrated the non-cytotoxic nature of the MANS peptides (see, for example, Singer et al, 2004; Takashi et al, 2006). Thus, the above results are not related to potential toxicity of MANS peptide. Moreover, for the studies herein, after incubation of MSCs with MANS peptide, the MSCs were thoroughly washed before i.v. injection into mice. Thus, this lack of toxicity is true for MSCs as well.

[00111] Figure 8 illustrates MARCKS protein-dependent mesenchymal stem cell

(MSC) migration. With respect to chemotaxis of an MSC toward a chemokine gradient, MSC migration can obtain with the leading edge of the MSC proximal to and directed toward a monotonic increasing chemokine gradient field while the trailing edge of the MSC resides distal with respect to the direction of the chemokine gradient field. In one aspect, the inventors propose that MARCKS protein binds to the leading edge of the MSC membrane via the N-terminal region of MARCKS and simultaneously with the actin cytoskeleton (represented by straight lines in the figure) via the effector domain in response to chemokine stimulation. This interaction drives migration of the MSC along or towards the increasing chemokine gradient. According to this hypothesis, the MANS peptide or an active fragment thereof can inhibit MSC migration by preventing

MARCKS protein binding to the leading edge of the mesenchymal stem cell membrane.

[00112] Figure 10 illustrates the effect (see bar 103) of an N-terminal

myristoylated peptide N-myristoyl-GAQFSKTAAK (SEQ ID No: 106) as an attenuator or inhibitor of migration of mouse bone marrow derived stem cells (BM-MSC) in comparison with the effects (see bar 100) of serum-free IMDM as a negative control (relative bar height - -16 cells per field); of IMDM plus 20% serum as a positive control (see bar 101 ; relative bar height = -48 cells per field); of SDF-Ια (i.e., Stromal Cell Derived Factor-l a) at 25 ng/mL) (see bar 102; relative bar height = -42 cells per field); and of peptide 233 (see bar 104), which was employed in the same fashion as peptide N- myristoyl-GAQFSKTAAK (SEQ ID No: 106) and which contains the same amino acids found in N-myristoyl-peptide 106 (which is N-myristoyl-GAQFSKTAAK (SEQ ID No: 106)) but in a randomized sequence and with N-terminal acetylation (i.e., N-terminal acetyl-GKASQFAKTA (SEQ ID NO: 233)) and which produced a bar height = -46 cells per field, which is indicative of having no effect on stem cell migration under these experimental conditions. N-myristoyl peptide 106 (N-myristoyl-GAQFSKTAAK (SEQ ID No: 106)) was given to cells 15 minutes prior to exposure to SDF-Ι , and migration was measured after 3 hours. N-myristoyl-GAQFSKTAAK (SEQ ID No: 106) produced a relative bar height of -26 cells per field under these conditions, indicative of at least 60% reduction or inhibition or attenuation of migration of stem cells in this in vitro

experiment..

[00113] Relevant to Figure 10, to study the effect of a peptide of this invention on BM-MCS migration in vitro, 1.5x10⁵ live primary mouse BM-MSCs were exposed to 100 μΜ of a peptide such as N-myristoyl-GAQFSKTAAK (SEQ ID No: 106) to form a migration-inhibited stem cell, or controls, at 37°C for 15 min, then loaded into 5 μηι pore transwell chambers. The lower well contained either SF-IMDM alone or supplemented with 25ng/ml SDF-Ια. Each bar represents the mean of 45 randomly selected fields at 400x magnification ± 1 SEM. N-myristoyl-GAQFSKTAAK (SEQ ID No: 106) attenuated BM-MSC migration in response to SDF-la.

[00114] The results indicate that primary mouse bone marrow mesenchymal stem cells (BM-MSC) migrate towards chemotactic agents known to be involved in inflammation or stem cell recruitment. Western blotting identified MARC S protein in MSC cell lysates, and MARCKS phosphorylation was induced by exposure to the chemotactic agents. Treatment of these cells with a MANS peptide (N-myristoyl SEQ ID No: 1) and with a N-terminal-myristoylated peptide fragment of MANS peptide, i.e., with N-myristoyl-GAQFSKTAAK (SEQ ID No: 106), but not with a missense (RNS peptide, SEQ ID No: 232) control, attenuated, in a concentration-dependent manner, directional chemotaxis of BM-MSC. As a predictive model to anticipate clinical trial use of MSCs, that migration of MSCs can be attenuated helps to understand how these cells migrate to sites of injury (i.e., sites of injured or diseased tissue) under chemotaxis and ultimately whether or not their presence is beneficial to the repair process.

[00115] Methods: Bone marrow-derived mesenchymal stem cells (BM-MSC) from C57B1/6J mice (1) (Institute for Regenerative Medicine, College Station, TX) were propagated in complete Iscove's Modified Dulbecco's Medium (cIMDM) as described (3). All cells were incubated at 37°C in a humidified 5% C0₂ atmosphere.

[00116] Chemotaxis assays: Following 12hr incubation in serum-free (SF)- IMDM, 1.5xl0⁵ BM-MSCs were introduced to the upper chamber of 24- well 5μιη pore transwell inserts coated with rat-tail collagen. Lower chambers contained SF-IMDM with either: nothing added (negative control); 20% serum (positive control); or the chemotactic agent 25ng ml Stromal Cell Derived Factor- 1 alpha (SDF-Ια). Cells were incubated with or 50μΜ of test peptide for 15 min at 37°C prior to the migration assay. Cells treated with peptides were exposed only to the 25ng/ml SDF-Ια. After 3 hr incubation, inserts were removed, and the inner surface rinsed and wiped to remove non- migrated cells. Cells on the outside of the inserts were formalin-fixed and stained, and the inserts were attached to glass slides. Cells were counted in 15 random fields at 400x magnification per insert. The mean number of cells from 45 fields per experimental condition was calculated. As indicated in Figure 10, the BM-MSC's migrated in response to SDF- l a significantly above control levels, reaching approximately 50% cells migrating. Preincubation of the cells, as with MANS peptide (N-myristoyl-SEQ ID No: 1), or with N-myristoyl-GAQFSKTAAK (SEQ ID No: 106), appeared to significantly inhibit the migration of the BM-MSC to a gradient of SDF- la. Preincubation of the cells with randomized amino acid sequence peptide N-acetyl-GKASQFAKTA (SEQ ID No: 233) did not produce a reduction or inhibition of migration of BM-MSC to a gradient of SDF-la.

[00117] The inventors have established that MARCKS binds to both actin and myosin and to cell membranes. Using the MANS peptides (MANS and active fragments thereof) we now anticipate that MARCKS will bind to both actin and the plasma membrane on the leading edge of the migrating cells, thereby transferring the contractile force to the cell membrane, allowing movement of the cell. At the optimal concentration of chemotactic stimuli and time following exposure as determined above MARCKS binding characteristics can be assessed via biochemical and morphological approaches:

a) Western blot showing MARCKS association with actin and cell membrane, including quantitative (densitometric) analysis of enhanced binding following exposure to chemotactic stimuli.

b) Ultrastructural immunohistochemical analysis of MARCKS binding to cytoskeleton and to the inner face of the leading edge of the plasma membrane in stimulated and migrating MSC's, utilizing gold-labeling of MARCKS and actin.

c) Determination of additional proteins that associate with MARCKS constitutively and after stimulation with chemotactic agent exposure. The involvement of chaperones, such as Heat Shock Proteins; other cytoskeletal proteins, such as myosin isoforms; and adhesion-type proteins, such as integrins, is anticipated. Confirmation of involvement of these entities can be performed using co-immunoprecipitation and immunoblotting as described previously.

d) Laser-scanning confocal microscopy can be utilized to follow the progression of MARCKS protein immunolabeled with fluorescent probes. With a heated microscope stage, observations in real time can be made to determine where MARCKS concentrates in the presence and absence of chemotactic stimulation over the time course described in experiments above. Further co-immunolabeling of the actin cytoskeleton, proteins within the leading edge of polarized cell membrane, and MANS peptides and active fragments thereof can be used to examine MARCKS function in greater detail. Proteins detected by co-immunoprecipitation experiments described above can be included in these studies. [00118] Alternatively, advances in fluorescence resonance energy transfer (FRET) reagents have made it possible to monitor protein interactions within a living cell. Using EnSpire Alpha labeling systems (Perkin Elmer), determination can be made of the kinetics of MARCKS interaction with actin, integrins, Heat Shock Proteins, and co- immunoprecipitated proteins and chaperones identified in experiments described previously. Monitoring the binding frequency of MARCKS and labeled target proteins in cells mock-exposed or exposed to chemotactic agents (SDF-la, C5a, MCP-1) over time, as well as when treated with MANS and RNS peptides, can provide a further clarification of the protein interactions with MARCKS during cell chemotaxis.

[00119] The MANS peptide (which has an amino acid sequence identical to the N- terminus of MARCKS protein) can disrupt binding of MARCKS to membranes. The inventors propose that MANS attenuates (inhibits, reduces) MSC migration by blocking binding of MARCKS to the inner face of the plasma membrane of the leading edge of the migrating cell (Figure 8). Thus, MARCKS may still bind to actin but, after treatment with MANS of an active fragment thereof, may no longer transfer the contractile force to the cell membrane, attenuating migration.

[00120] The protocols involve pre-incubating the MSCs with a range of concentrations of MANS peptide or an active fragment thereof (for example,

concentrations from 10 to 100 μΜ (microM) of MANS peptide or an active fragment thereof) or a missense control peptide, such as RNS (N-terminally myristoylated SEQ ID NO: 232), which is a random sequence control peptide, for 30 minutes, then exposing the cells to the chemotactic stimuli and in essence repeating the experiments described above. MANS peptide and active fragments thereof, but not random or scrambled or missense sequence peptides such as RNS, can inhibit binding of MARCKS to the inner face of the plasma membrane in the leading edge of the migrating MSCs.

[00121] Treatment of the MSCs with MANS Peptide or an active fragment thereof is provided. Once the effective timing and dose of MSC treatment is elucidated, for example, in an asthma model (see, for example, Walker et al., J Clin Invest 112:566-574 (2003)), experiments can be repeated with MSCs treated with MANS peptide or an active fragment thereof (or a control random sequence peptide such as RNS). The MSCs can be incubated for 15 min with peptide just prior to infusion. In 150 μΐ. of media, 500,000 cells can be administered through the retrobulbar sinus plexus or into the airways by oropharyngeal aspiration. These experiments can determine the in vivo role for

MARCKS and MANS or an active fragment thereof in regulating the effect of MSC migration and ultimately how the cells influence airway remodeling.

[00122] The inventors have developed evidence that MARCKS protein is a key molecular regulator of MSC migration in response to numerous chemotactic stimuli. Such MSC migration which is regulated by MARCKS protein can be referred to herein as MARCKS-related MSC migration. Using the MANS peptide or an active fragment thereof it can be established the mechanism through which MARCKS serves as a molecular bridge between the actin cytoskeleton and the inner face of the plasma membrane in the leading edge of migrating cells in vitro and in vivo. In addition, the inventors have shown that MSC migration can be modulated (attenuated, inhibited, or reduced) with MANS peptide or an active fragment thereof. Efficacy of other MARCKS- related peptides such as, for example, active fragments of MARCKS peptide having an amino acid sequence from 4 to 50 amino acids from the N- terminal of MARCKS peptide, or from 10 to 50 or from 10 to 100 amino acids from the N-terminal of MARCKS peptide, which can be myristoylated at the N-terminal glycine of each peptide in preferred embodiments, can also be demonstrated. MSCs clearly migrate to sites of lung injury, and the MANS peptide and active fragments thereof and other MARCKS-related peptides can be used to control MSC migration and function of inflammatory cells.

[00123] RNS peptide is a missense control peptide having the same amino acids and number of amino acids as found in MANS peptide but ordered in a random sequence with respect to the amino acid sequence in MANS peptide. RNS peptide, where RNS = Random N-terminal Sequence, has the 24 amino acid sequence: N-myristoyl

GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 232). RNS peptide is described, for example, in U.S. Patent 7,544,772, the disclosure of which is incorporated herein by reference.

METHODS

[00124] Cell Culture Methods The monocyte macrophage cell line J774A.1 was purchased from the American Type Cell Culture (ATCC; Manassas, VA) and propagated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with: 10% Fetal Bovine Serum (FBS), lOOU/ml penicillin, lOOU/ml streptomycin, and 2mML-glutamine (Invitrogen, Carlsbad, CA). Primary mouse bone marrow mesenchymal stem cells (BM-MSC) were harvested from C57B1/6J wt mice (Institute for Regenerative Medicine; Texas A&M University, College Station, TX). Mouse BM-MSC lines were propagated from passage 4 to 8 in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with: 2mML- glutamine, lOOU/ml penicillin, lOOU/ml streptomycin, 10% FBS, 10% horse serum, and 0.25mg/ml amphotericinB (Invitrogen). Cells were cultured at 37°C in a humidified 5% C0₂ atmosphere.

[00125] Transwell™ Migration Assay

Mouse BM-MSCs were grown to 70% surface coverage in T-150 cm flasks (Corning), then incubated for 12 hours in serum-free (SF-) IMDM media. Once the incubation period was complete, cells were treated with Trypsin-EDTA (Invitrogen), pelleted, and enumerated using 0.4% trypan blue stain in conjunction with a

hemacytometer. 1.5 x 10⁵ live BM-MSCs were introduced to the upper chamber of each collagen-treated transwell insert with 5 mm pores. The lower well was filled with 0.6 ml SF-IMDM medium supplemented with a chemokine to establish a concentration gradient [MCP-1 was used at concentrations of 25, 50, 100, and 200 ng/ml MCP-1 ; SDF-1 was used at concentrations of 25, 50, and 100 ng ml SDF-1 ; C5a was used at concentrations of 25, 50, 100, and 200 ng/ml C5a; and SCF was used at concentrations of 0.5, 5, 25, 50, and 100 ng/ml of SCF (R&D Systems, Minneapolis, MN)], (other chemotactic cytokines can be employed in the methods of this invention at similar concentrations), 0.001%) bovine serum albumin (BSA), 20% serum-supplemented medium (positive control), or SF-IMDM (negative/random movement control). Exposure to or pre-incubation with a peptide described in this invention such as myristoylated N-terminal sequence peptide (MANS) or the control random N-terminal sequence peptide (RNS) was performed by incubating 1.5 x 10⁵ BM-MSC per ml with 0.5, 5, 50, or 100 mM MANS peptide or with 50, or 100 mM RNS peptide (Genemed Synthesis, San Francisco, CA) in a 37 °C water bath for 20 minutes, then introduced into the transwell system. Experiments using vital dye identified no toxicity to BM-MSCs due to MANS peptide exposure at the concentrations detailed here (data not shown). Once the optimum time for incubation, which was determined empirically, had elapsed, cells that had not migrated were dislodged and washed away, the filters were fixed in 4% formalin, and the inserts were stained in filtered hematoxylin and 0.8% malachite green oxylate. Inserts were removed with a scalpel, fixed to microscope slides, and examined by light microscope. Cells from 15 randomly chosen fields per insert were counted at 400x magnification, and the mean ± SEM calculated per each experimental condition using Graphpad Prism v.4.

[00126] Western Blot

Twenty mg per well of whole cell lysates (J774A.1, native human lung fibroblast (NHLF), mouse BM-MSC, human cord blood MSC) were resolved on 10% NuPAGE Bis-Tris cassettes and transferred to nitrocellulose following manufacturer's protocols (Invitrogen). Nitrocellulose membranes were probed with mouse monoclonal anti-MARCKS antibodies (Millipore) and visualized using Supersignal West Pico Chemiluminescent Substrate kit (Pierce). Chemiluminescent images were captured on HyblotCL film (Denville Scientific) and scanned to a digital file.

[00127] Statistics

Results were analyzed using unpaired two-tailed t tests in conjunction with F tests to determine statistical significance (Graphpad Prism v.4; Graphpad Software). A p value <0.05 was considered to be statistically significant. All values are expressed as mean ± SEM where n = 30 or 45 fields counted at 400x magnification.

[00128] The presence of MARCKS was identified in MSCs by growing mouse bone marrow MSCs (Tulane University Center for Gene Therapy), protein extraction by the Laemmli Method, and identification on a Western Blot using mouse anti-MARCKS monoclonal antibody (Millipore). A positive control was J774A.1 monocyte cell lysate, and a negative control was bovine serum albumin (BSA). A characteristic band on the Western blot at 80 kDa identified MARCKS for the first time in the MSC cell lysate. Human cord blood MSCs are currently being analyzed, and studies to determine additional influence and role of MARCKS in MSC migration are under way. Peptides described in this invention can control certain functions of MARCKS in MSC migration. Targeting MARCKS protein using peptides described in the current invention can limit, inhibit, or reduce the migration of MSCs in the tissue such as lung tissue.

[00129] Additional background references are provided below. The disclosures of each of the journal references and patent documents cited herein are hereby incorporated by reference in their entirety.

Kolf, CM., E.Cho, R.S.Tuan. 2007. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: Regulation of niche, self-renewal and differentiation. Arthritis Res Ther. 9(1):204.

Kim, J., J. Chu, X. Shen, J. Wang, S.H. Orkin. 2008. An extended transcriptional network for pluripotency of embryonic stem cells. Cell. 132(6) : 1049- 1061.

Ortiz, L.A., M. Dutreil, C. Fattman, A.C. Pandey, G. Torres, K. Go, D.G. Phinney. 2007.

Interleukin 1 receptor antagonist mediates the anti-inflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci USA. 104(26): 11002-

1 1007.

Warshamana, G.S., M. Corti, A.R.Brody. 2001. TNF-alpha, PDGF, and TGF-beta(l) expression by primary mouse bronchiolar-alveolar epithelial and mesenchymal cells: TNF-alpha induces TGF-beta(l). Exp Mol Pathol. 71(l):13-33.

Spees, J.L., D.A. Pociask, D.E. Sullivan, M.J.Whitney, J.A. Lasky, D.J. Prockop, A.R.Brody. 2007. Engraftment of bone marrow progenitor cells in a rat model of asbestos-induced pulmonary fibrosis. Am J Respir Crit Care Med. 176(4):385-394.

Singer,M., L.D. Martin, B.B. Vargaftig, J.Park, A.D.Gruber, Y.Li, K.B.Adler. 2004. A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med. 10(2): 193-196.

Claims

1. A method of modulating the MARCKS -related migration of a mesenchymal stem cell toward an increasing concentration gradient of a chemotactic agent in a fluid or tissue, the method comprising treatment of said mesenchymal stem cell with a migration- inhibiting amount of a migration-modulating peptide and incubation of said cell with said peptide to form a migration-inhibited mesenchymal stem cell, and administering the migration-inhibited mesenchymal stem cell to said fluid or tissue.

2. The method of claim 1 , wherein the modulating comprises attenuating the MARCKS-related migration of a mesenchymal stem cell.

3. The method of claim 1 or claim 2, wherein the active peptide fragment comprises the amino acid sequence GAQFSKTAAK (SEQ ID No: 106).

4. The method of claim 1 or claim 2, wherein the migration-modulating peptide is selected from the group consisting of MANS peptide (N-myristoyl- GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID No: 1), and an active fragment peptide having from 4 to 23 amino acids in the amino acid sequence found in MANS peptide.

5. The method of claim 1 or claim 2, wherein the migration-modulating peptide is selected from the group consisting of N-myristoyl- GAQFS TAAKGEAAAERPGEAAVA (SEQ ID No: 1) N-myristoyl-

G AQFS KTAA GE AAAERPGE AA V (SEQ ID No: 2); N-myristoyl- GAQFS TAAKGEAAAERPGEAA (SEQ ID No: 4); N-myristoyl- GAQFS TAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl- G AQF SKT AAKGE AAAERP GE (SEQ ID No: 11); N-myristoyl- GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl- GAQFS TAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-

GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl- GAQFSKTAAKGEAA (SEQ ID No: 56); N-myristoyl-GAQFSKTAAKGEA (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl- GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFSKTAA (SEQ ID No: 121); N-myristoyl-GAQFSKTA (SEQ ID No: 137); N-myristoyl-GAQFSKT (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), N-myristoyl-GAQF (SEQ ID No: 211), and a combination thereof.

6. The method of claim 1 or claim 2, wherein the chemotactic agent is selected from the group consisting of MCP-1 , SDF-1 , SDF-Ια, SCF and C5a.

7. The method of claim 1 or claim 2, wherein the concentration gradient of the chemotactic agent is established with a concentration in the range from 0.5 to 200 ng/mL.

8. The method of claim 1 or claim 2, wherein the migration-inhibiting amount of the migration-modulating peptide is in the range of 100 nanoM to 100 microM.

9. The method of claim 1 or claim 2, wherein the incubation is for a period of 10 to 30 minutes.

10. The method of claim 1 or claim 2, wherein the tissue is injured or diseased.

11. The method of claim 1 or claim 2, wherein the tissue is lung tissue.

12. The method of claim 1 or claim 2, wherein the fluid is selected from the group consisting of blood, serum, lymph, urine, mucus, saline, lacrimate, perspiration, and ascites fluid.

13. The method of claim 1 or claim 2, wherein the fluid or tissue is present in a subject.

14. The method of claim 1 or claim 2, wherein the active fragment peptide is an N- terminal- and/or C-terminal-chemically modified peptide, which peptide consists of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence of from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1), wherein the amino acid sequence of the peptide begins at the N-terminal amino acid of the reference sequence, or an amino acid sequence substantially identical thereto; and

(b) an amino acid sequence having from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1) or an amino acid sequence substantially identical thereto, which begins at the amino acid at position 2 through the amino acid at position 21 of the reference sequence; and wherein

wherein administration of a migration-inhibiting amount of said chemically modified peptide to a mesenchymal stem cell forms a migration-inhibited mesenchymal stem cell.

15. The method of claim 14, wherein the peptide is amidated with a linear, branched, saturated, or unsaturated C2 to C24 aliphatic carboxylic acid at the N-terminal amino acid of the peptide.

16. The method of claim 14, wherein the peptide is myristoylated at the N-terminal amino acid of the peptide.

17. The method of claim 14, wherein the peptide comprises the amino acid sequence AKGE (SEQ ID NO: 219).