WO2019200244A1 - Methods and compositions for differentiation and maturation of stem cells and tissue explants prepared thereby - Google Patents

Abstract

This invention relates to methods for promoting the differentiation and maturation mammalian stem cells using CXCL12 and fragments or derivatives thereof. This invention further relates to methods for producing tissue explants suitable for transplantation and tissue explants produced by the methods.

Description

METHODS AND COMPOSITIONS FOR DIFFERENTIATION AND MATURATION OF STEM CELLS AND TISSUE EXPLANTS PREPARED THEREBY

RELATED APPLICATIONS

[0001] This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 62/656,629, filed April 12, 2018, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to methods for promoting the differentiation and maturation mammalian stem cells using CXCL12 and fragments or derivatives thereof. This invention further relates to methods for producing tissue explants suitable for transplantation and tissue explants produced by the methods.

BACKGROUND OF THE INVENTION

[0003] The concept of implanting cells and tissues in a subject to replace failing or nonfunctional tissue has been around for many years. Sources of explant material include tissues and cells from animals (e.g., pigs) and cells from humans that have been cultured and expanded. Ideally, the explant material includes stem cells and/or progenitor cells that can mature and differentiate into desirable cell types that have the biological functions of the tissues being replaced (e.g., glucose-stimulated insulin production in pancreatic islet cells). In practice, however, the stems cells and progenitor cells often fail to differentiate or fail to develop or maintain the desired biological functions.

[0004] There is a long felt need in the art for methods of preparing explant materials that develop and maintain desirable biological functions.

SUMMARY OF THE INVENTION

[0005] The present invention is based in part on the development of techniques for improving the differentiation and/or maturation of mammalian stem cells and/or progenitor cells and providing tissue explants with improved biological properties and improved suitability for tissue transplantation. In particular, methods for isolating stem cells and/or progenitor cells and preparing explants in which the cells are contacted with a CXCR4 and/or CXCR7 binding agent have been developed.

[0006] Thus, one aspect of the invention relates to a method for promoting the

differentiation and/or maturation of mammalian stem cells and/or progenitor cells, the method comprising contacting the stem cells and/or progenitor cells with an effective amount of a CXCR4 and/or CXCR7 binding agent, thereby promoting the differentiation and/or maturation of the cells.

[0007] Another aspect of the invention relates to a method of preparing a tissue explant, comprising:

a) obtaining a mammalian tissue sample comprising stem cells and/or progenitor cells from fetal, newborn, neonatal, juvenile, or adult tissue;

b) culturing the tissue sample for a period of time; and

c) encapsulating cells from the tissue sample in a matrix comprising a CXCR4 and/or CXCR7 binding agent.

[0008] A further aspect of the invention relates to a tissue explant prepared by the methods of the invention.

[0009] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows the effect of CXCL12 on encapsulated differentiation of neonatal porcine islets cell clusters (NPICC).

[0011] FIG. 2 shows the effect of CXCL12 on insulin production of NPICC.

[0012] FIG. 3 shows the effect of CXCL12 on insulin production of NPICC.

[0013] FIG. 4 shows the effect of CXCL12 on insulin production of NPICC.

[0014] FIG. 5 shows the effect of CXCL12 on viability of NPICC.

DETAILED DESCRIPTION

[0015] After reading this description, it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, not all embodiments of the present invention are described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below.

[0016] Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

Definitions

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

[0019] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0020] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0021] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0022] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0023] As used herein, the singular forms“a”,“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] Also as used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of

combinations when interpreted in the alternative (“or”). [0025] All numerical designations, e.g., pH, temperature, time, concentration, amounts, and molecular weight, including ranges, are approximations which are varied (+) or (-) by 10%, 1%, or 0.1%, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term“about.” It is also to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

[0026] “Optional” or“optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0027] The term“comprising” or“comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of ⁵ shall mean excluding more than trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention.

[0028] The terms“patient,”“subject,”“individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In one embodiment, the patient, subject, or individual is a mammal. In some embodiments, the mammal is a mouse, a rat, a guinea pig, a non-human primate, a dog, a cat, or a domesticated animal (e.g., horse, cow, pig, goat, sheep). In certain embodiments, the patient, subject or individual is a human.

[0029] The term“modulate,”“modulates,” or“modulation” refers to enhancement (e.g. , an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0030] The term“enhance” or“increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold.

[0031] The term“inhibit” or“reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible level or activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0032] The term“contact” or grammatical variations thereof as used with respect to a polypeptide and a receptor, refers to bringing the polypeptide and the receptor in sufficiently close proximity to each other for one to exert a biological effect on the other. In some embodiments, the term contact means binding of the polypeptide to the receptor.

[0033] The term“treat,”“treating,” or“treatment” (and grammatical variations thereof) covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e. , causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.

[0034] The terms“prevent,”“preventing,” and“prevention” (and grammatical variations thereof) refer to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g. , the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present invention.

[0035] The term“administering” or“administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, by inhalation, or parenterally (intravenously, intramuscularly, intraperitoneally, subdermally, or subcutaneously). Administration includes self-administration and the administration by another.

[0036] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean“substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. [0037] The term“therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0038] The term“therapeutically effective amount” or“effective amount” refers to an amount of the agent that, when administered, is sufficient to cause the desired effect. For example, an effective amount of CXCL12 may be an amount sufficient to have a

differentiation effect on a stem cell or progenitor cell. The therapeutically effective amount of the agent will vary depending on the disease or condition being treated and its severity as well as the age, weight, etc., of the subject to be treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder.

[0039] A“prevention effective” amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0040] The term“CXCR4 and/or CXCR7 binding agent” refers to a compound or molecule that binds to CXCR4 and/or CXCR7 and activates a CXCL12/CXCR4 and/or

CXCL12/CXCR7 pathway thereby causing a differentiation and/or maturation effect on a cell.

[0041] The term“fragment,” as applied to a peptide, will be understood to mean an amino acid sequence of reduced length relative to a reference peptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical to the reference peptide or amino acid sequence. Such a peptide fiagment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 5, 10, 15, 20, 25, 30, 35, 46. 50, 55, or 60 or more consecutive amino acids of a peptide or amino acid sequence according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of less than about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 or less consecutive amino acids of a peptide or amino acid sequence according to the invention.

[0042] As used herein, the terms“protein” and“polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0043] A“fusion protein” or“fusion polypeptide” refers to a hybrid polypeptide which comprises polypeptide portions from at least two different polypeptides. A“fusion protein” as defined herein, is a fusion of a first amino acid sequence (protein) comprising, for example a CXCL12 polypeptide of the invention, joined to a second amino acid sequence comprising a targeting polypeptide.

[0044] The portions may be from proteins of the same organism, in which case the fusion protein is said to be“interspecies,”“intergenic,” etc. In various embodiments, the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide. In the case where more than one amino acid sequence is fused to a first polypeptide, the fusion sequences may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences. A first polypeptide may be fused to the N-terminus, the C-terminus, or the N- and C-terminus of a second polypeptide. Furthermore, a first polypeptide may be inserted within the sequence of a second polypeptide.

[0045] As used herein, a“functional” polypeptide or“functional fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g. , binding to or activating CXCR4 and/or CXCR7). In particular embodiments, the “functional” polypeptide or“functional fragment” substantially retains all of the activities possessed by the unmodified polypeptide. By“substantially retains” biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A“non-functional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as receptor binding and activation can be measured using assays that are well known in the art and as described herein. [0046] As used herein, the term“homolog” is used to refer to a molecule which differs from a naturally occurring polypeptide by minor modifications to the naturally occurring polypeptide, but which significantly retains a biological activity of the naturally occurring polypeptide.

Minor modifications include, without limitation, changes in one or a few amino acid side chains, changes to one or a few amino acids (including deletions, insertions, and/or substitutions), changes in stereochemistry of one or a few atoms, and minor derivatizations, including, without limitation, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and addition of glycosylphosphatidyl inositol. The term

“substantially retains,” as used herein, refers to a fragment, homolog, or other variant of a polypeptide that retains at least about 50% of the activity of the naturally occurring polypeptide (e.g., binding to or inhibiting a calcium channel), e.g., about 70%, 80%, 90% or more. Other biological activities, depending on the polypeptide, may include pH sensitivity, enzyme activity, receptor binding, ligand binding, induction of a growth factor, a cell signal transduction event, etc.

[0047] In certain embodiments, the polypeptide of the invention comprises at least one modified terminus, e.g., to protect the polypeptide against degradation. In some embodiments, the N-terminus is acetylated and/or the C-terminus is amidated. In some embodiments, the polypeptide comprises one or two D-alanines at the amino- and/or carboxyl-terminal ends.

[0048] In certain embodiments, the polypeptide of the invention comprises at least one non natural amino acid (e.g. , 1, 2, 3, or more) or at least one terminal modification (e.g., 1 or 2). In some embodiments, the peptide comprises at least one non-natural amino acid and at least one terminal modification.

[0049] A“pharmaceutically-acceptable carrier” as used herein means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0050] A“pharmaceutically-acceptable salt” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds.

[0051] The term“antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, IgE and IgY. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. The term “antibody” also includes an antibody fragment as defined herein.

[0052] The term“antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody’s specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)₂, scFv, Fv, dsFv diabody, and Fd fragments.

The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids. [0053] The term“Fab fragment” refers to a fragment of an antibody comprising an antigen- binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule.

[0054] The term“F(ab’)₂ fragment” refers to a fragment of an antibody containing two antigen-binding sites, generated by cleavage of the antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0055] The term“Fc fragment” refers to the fragment of an antibody comprising the constant domain of its heavy chain.

[0056] The term“Fv fragment” refers to the fragment of an antibody comprising the variable domains of its heavy chain and light chain.

[0057] The term“engineered antibody” refers to a recombinant molecule that comprises at least an antibody fragment comprising an antigen binding site derived from the variable domain of the heavy chain and/or light chain of an antibody and may optionally comprise the entire or part of the variable and/or constant domains of an antibody from any of the Ig classes (for example IgA, IgD, IgE, IgG, IgM and IgY). Examples of engineered antibodies include enhanced single chain monoclonal antibodies and enhanced monoclonal antibodies. Examples of engineered antibodies are further described in PCT/US2007/061554, the entire contents of which are incorporated herein by reference. An“engineered antibody” includes an engineered antibody fragment, according to the method of the invention, and as defined herein.

[0058] The term“single chain variable fragment or scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

[0059] The term“multivalent antibody” refers to an antibody or engineered antibody comprising more than one antigen recognition site. For example, a“bivalent” antibody has two antigen recognition sites, whereas a“tetravalenf’ antibody has four antigen recognition sites.

The terms“monospecific,”“bispecific,”“trispecific,”“tetraspecific,” etc., refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody. For example, a“monospecific” antibody’s antigen recognition sites all bind the same epitope. A“bispecific” antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope. A“multivalent monospecific” antibody has multiple antigen recognition sites that all bind the same epitope. A“multivalent bispecific” antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.

[0060] The term“epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0061] An“antigen” refers to a target of an immune response induced by a composition described herein. An antigen may be a protein antigen and is understood to include an entire protein, fragment of the protein exhibited on the surface of a virus or an infected, foreign, or tumor cell of a subject, as well as a peptide displayed by an infected, foreign, or tumor cell as a result of processing and presentation of the protein, for example, through the typical MHC class 1 or II pathways. Examples of such foreign cells include bacteria, fungi, and protozoa.

Examples of bacterial antigens include Protein A (PrA), Protein G (PrG), and Protein L (PrL).

[0062] The term“antigen binding site” refers to a region of an antibody or fragment thereof, that specifically binds an epitope on an antigen.

[0063] The term“linker” is art-recognized and refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or group of molecules (for example, 2 or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and at least one spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0064] A“spacer molecule” includes any amino acid segment that is not related to the two protein segments it separates. For example, in a fusion consisting of a CXCL12 polypeptide and a targeting polypeptide, a spacer molecule would consist of a stretch of amino acids that is unrelated to the polypeptides comprising the fusion protein. A“spacer molecule” useful according to the invention includes neutral amino acids such as glycine, leucine, valine, alanine, rather than acidic or basic amino acids like aspartate, or arginine respectively. Methods for promoting differentiation and maturation of stem and progenitor cells

[0065] The present invention is based in part on the development of techniques for improving the differentiation and/or maturation of mammalian stem cells and/or progenitor cells and providing tissue explants with improved biological properties and improved suitability for tissue transplantation. In particular, methods for isolating stem cells and/or progenitor cells and preparing explants in which the cells are contacted with a CXCR4 and/or CXCR7 binding agent have been developed.

[0066] Thus, one aspect of the invention relates to a method for promoting the

differentiation and/or maturation of mammalian stem cells and/or progenitor cells, the method comprising contacting the stem cells and/or progenitor cells with an effective amount of a CXCR4 and/or CXCR7 binding agent, thereby promoting the differentiation and/or maturation of the cells.

[0067] The cells may be obtained from any suitable mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is one that can provide cells and tissues suitable for transplantation into a human, e.g., a pig or a primate.

[0068] The stem cells and/or progenitor cells may obtained from fetal, newborn, neonatal, juvenile, or adult tissue of a mammal. In some embodiments, the cells may be obtained from a neonate, e.g., between day 1 and day 10 after birth, e.g., between day 3 and day 8 after birth.

[0069] The stem cells and/or progenitor cells may be obtained from any tissue known to contain suitable cells, including but not limited to pancreatic, heart, liver, neural, skin, lung, muscle, bone, bone marrow, adipose, or dental pulp tissue. In some embodiments, the stem cells and/or progenitor cells may be obtained from a hormone-producing tissue, e.g., endocrine glands such as the pineal gland, hypothalamus, pituitary gland, parathyroid gland, thyroid gland, thymus gland, adrenal gland, pancreas, ovaries, or testes, or other hormone producing tissues such as heart, stomach, duodenum, liver, kidney, skin, or adipose tissue.

[0070] In some embodiments, at least some of the stem cells and/or progenitor cells express CXCR4 and/or CXCR7 (e.g, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,

45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more). The methods of the present invention provide advantageous benefits in that the differentiation and/or viability of the stem cells and/or progenitor cells is maintained or increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent. In some

embodiments, the level of differentiation of the cells is increased compared to controls, e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 100%, or more. In some embodiments, the viability of the cells is increased compared to controls, e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 100%, or more.

[0071] In one particular advantage of the present invention, at least one biological function of the stem cells and/or progenitor cells is increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent, e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 100%, or more. In some embodiments, the stem cells and/or progenitor cells are obtained from hormone-producing tissue and the level of hormone secretion is increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent, e.g, by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 100%, or more. In one nonlimiting example, the stem cells and/or progenitor cells are obtained from pancreatic islet tissue and the level of glucose- stimulated insulin secretion is increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent, e.g, by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 100%, or more. The increase in at least one biological function {e.g, glucose-stimulated insulin secretion) may be a long-lasting effect, e.g, lasting at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 days or more.

[0072] In some embodiments, the step of contacting the stem cells and/or progenitor cells with a CXCR4 and/or CXCR7 binding agent comprises encapsulating the stem cells and/or progenitor cells in a matrix comprising the CXCR4 and/or CXCR7 binding agent. The matrix may be any matrix suitable for encapsulating cells while maintaining the viability of the cells. In some embodiments, the matrix is a polymer matrix, e.g, an alginate matrix. In some embodiments, the matrix is not an alginate matrix.

[0073] The encapsulated cells or explant may be a composition suitable for implantation or injection into a patient, wherein the explant comprises an effective amount of a CXCR4 and/or CXCR7 binding agent. In some embodiments, the explant is in the form of particles, e.g., biodegradable particles, wherein the particles are loaded with the CXCR4 and/or CXCR7 binding agent. In some embodiments, the particles encapsulate or are coated with the CXCR4 and/or CXCR7 binding agent. In some embodiments, the particles have an average diameter of between about 1 micron to about 20 microns, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns or any range therein.

[0074] The matrix may be selected such that the CXCR4 and/or CXCR7 binding agent elutes from the matrix and continuously contacts the encapsulated cells. In some

embodiments, the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a steady rate, e.g., at a rate that provides a concentration of the agent that is effective to promote the differentiation and/or maturation of the cells. In some embodiments, the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a rate of about 0.1 to about 10 pmol/hour, e.g., about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 pmol/hour or any range therein, e.g., 0.1-2, 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 0.1-4, 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 0.1-5, 1-6, 2-7, 3-8, 4-9, 5-10, 0.1-6, 1-7, 2-8, 3-9, 4-10, 0.1-6, 1-7, 2-

8, 3-9, or 4-10 pmol/hour. In some embodiments, the CXCR4 and/or CXCR7 binding agent elutes from the matrix for a period of at least one month after implantation or injection, e.g., at least 2, 3, 4, 5, or 6 months.

[0075] In one aspect, the explant is in a composition which is in the form of an injectable formulation, a sprayable formulation for use in subdermal applications such as a surgical field, or an inhalable formulation. The type of formulation is dependent, at least in part, on the intended use of the formulation.

[0076] The matrix encapsulating the cells may comprise a biocompatible material. The type of biocompatible material depends on the intended use. For example, the biocompatible material may be biodegradable or non-biodegradable. Without being bound by theory, it is believed that a biodegradable particle is preferred, for example, where the CXCR4 and/or CXCR7 binding agent is required at the site of implantation for a short period of time (e.g., hours to days or a week); where the particle cannot easily be removed from the implantation site; and/or where particles may cause damage or injury if left in place for a long period of time. In contrast, and without being bound by theory, it is believed that a non-biodegradable particle is preferred, for example, where the CXCR4 and/or CXCR7 binding agent is required at the site of implantation for a long period of time (e.g. , weeks to months or longer), and/or the particle can be easily removed from the implantation site.

[0077] In one embodiment, the biocompatible material is a biocompatible polymer. The biocompatible polymer can be carbohydrate-based, protein-based, and/or synthetic, e.g.,

PLA. Biocompatable materials suitable for use in matrices include, but are not limited to, poly-dimethyl-siloxane (PDMS), poly-glycerolsebacate (PGS), polylactic acid (PLA), poly- L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid- polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly( alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyesterspolyacrylates,

polymethacrylate, acyl substituted cellulose acetates, nondegradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulphonated polyolefins, polyethylene oxide, polyvinyl alcohol, nylon silicon, poly(styrene-block- butadiene), polynorbomene, and hydrogels. Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N.Y., 1989). Combinations of these polymers may also be used. In one embodiment, the biocompatible polymer is alginate. In one embodiment, the biocompatible polymer is not alginate.

[0078] In one embodiment, the particle is retrievable. For example, the particle may comprise a metal that allows for magnetic retrieval of the particle(s) from the subject.

[0079] In one embodiment, the particle is visualizable. That is, a clinician can visualize the particle(s) within the subject in a non-invasive manner. For example, the particle may comprise a metal or other element that can be visualized non-invasively (e.g., by X-ray, MRI, CAT-scan, etc.). In some embodiments, the particle comprises a fluorescent marker or radioactive label that can be visualized non-invasively.

[0080] In some embodiments of the method, the stem cells and/or progenitor cells are cultured for a period of time prior to encapsulation. The cells may be cultured, for example, for 1 to 12 days prior to encapsulation, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or any range therein, e.g, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 2-6, 3-7, 4-8, 5-9, 6-10, or 7-11 days. In some embodiments, the stem cells and/or progenitor cells are cultured in the absence of serum or growth hormones/growth factors. In some embodiments, the stem cells and/or progenitor cells are cultured in the absence of a CXCR4 and/or CXCR7 binding agent.

[0081] The CXCR4 and/or CXCR7 binding agent is a CXCR4-binding molecule or a CXCR7-binding molecule that activates the CXCL12/CXCR4 and/or CXCL12/CXCR7 pathway. CXCR4 and/or CXCR7 binding agents are described, for example, in U.S. Patent No. 6,448,054, which is incorporated herein by reference in its entirety. CXCR4-binding molecules include, but are not limited to, CXCL12 or active fragments or derivatives, homologs, or analogs thereof, including protease-resistant derivatives, an agonistic antibody or antibody fragment or derivative to CXCR4, or any other ligand for CXCR4. CXCR4 agonists are disclosed, for example, in US Publication Nos. 2017/0079971, 2016/0228413, 2015/0157630, 2015/0038509, 2013/0324552, 2013/0210709, 2013/0079292, 2013/0035347, 2013/0005944, and 2012/0301427, each incorporated by reference herein in its entirety. CXCR7-binding molecules include, but are not limited to, CXCL12 or active fragments or derivatives, homologs, or analogs thereof, including protease-resistant derivatives, an agonistic antibody or antibody fragment or derivative to CXCR7, or any other ligand for CXCR7. CXCR7 agonists are disclosed, for example, in US Publication Nos. 2016/0107997, 2015/0307556, 2013/0345199, 2013/0225506, 2013/0023483, 2009/0098091, and

2007/0160574, each incorporated by reference herein in its entirety.

[0082] In some embodiments, the CXCR4-binding molecule is CXCL12 (CXCL12 polypeptide). CXCL12 or CXCL12 polypeptide refers to a protein or fragment thereof that binds a CXCL12 specific antibody and that has chemorepellant or differentiation/maturation activity. Chemorepellant activity is determined by assaying the direction of T cell migration (e.g., toward or away from an agent of interest). See, e.g., Poznansky et ah, Nature Medicine 2000, 6:543-8.

[0083] CXCL12 polypeptides are known in the art. See, e.g., Poznansky et ah, Nature Medicine 2000, 6:543-8, which is incorporated herein in its entirety. In one embodiment, a CXCL12 polypeptide has at least about 85%, 90%, 95%, or 100% amino acid sequence identity to NP_00l029058 and has chemokine activity. Examples of SDF-l (CXCL12) isoforms can be found in PCT Publication No. WO 2015/069256, which is incorporated herein by reference in its entirety. These include SDF-l alpha (Accession No. NP_954637), SDF-l beta (Accession No. P48061), SDF-l gamma (Accession No. NP_001029058), SDF-l delta

(MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNNLISAAPAGKRVIAGARALHPSPPR ACPTARALCEIRLWPPPEWSWPSPGDV (SEQ ID NO:l)), SDF-l epsilon

(MNAKVYVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIV ARLKNNNRQ V CIDPKLK WIQE YLEKALNN C (SEQ ID NO:2)), and SDF-l phi (MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIV ARLKNNNRQ V CIDPKLKWIQEYLEK ALNKIWL Y GN AETSR (SEQ ID NO:3)).

In another embodiment, the sequence of the CXCL12/SDF-1 polypeptide is

MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIV ARLKNNNRQ V CIDPKLKWIQEYLEKALNKGRREEKV GKKEKIGKKKRQKKRK AAQKRKN (SEQ ID NO:4). In one embodiment, a CXCL12 polypeptide has at least about 85%, 90%, 95%, or 100% amino acid sequence identity to a sequence described herein and has chemokine or differentiation/maturation activity.

[0084] The CXCL12 polypeptide may be a variant such as the ones disclosed in US Application No. 15/887,467, incorporated by reference herein in its entirety. In some embodiments, 1 to 5 of the first consecutive amino acid residues of the CXCL12 polypeptide sequence are deleted relative to the wild-type CXCL12 sequence. Reference to the first five consecutive amino acid residues of the CXCL12 polypeptide sequence refers to the sequence KPVSL (SEQ ID NO:5) in mature human CXCL12 and the corresponding residues from CXCL12 of other species. In some embodiments, 1, 2, 3, 4, or 5 of the first consecutive amino acid residues of the CXCL12 polypeptide sequence are deleted. In some

embodiments, the recombinant CXCL12 polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

MNAKVVVVLVLVLTALCLSDGSYRCPCRFFESHVARANVKHLKILNTPNCALQIVA RLKNNNRQV CIDPKLKWIQE YLEKALNK (SEQ ID NO:6)

[0085] In some embodiments, the sixth amino acid residue of the CXCL12 polypeptide sequence is substituted relative to the wild-type CXCL12 sequence. The sixth amino acid residue in human CXCL12 is a serine. The residue may be substituted with a conservative substitution. In some embodiments, the residue is substituted with alanine. In some embodiments, the recombinant CXCL12 polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:7.

MNAKVVVVLVLVLTALCLSDGKPVSLAYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQY CIDPKLKWIQEYLEKALNK (SEQ ID NO:7)

[0086] In some embodiments, the deletion of 1 to 5 of the first consecutive amino acid residues of the CXCL12 polypeptide sequence is combined with the substitution of the sixth residue. Thus, the modified CXCL12 polypeptide may have 1, 2, 3, 4, or 5 of the first consecutive amino acid residues deleted in combination with substitution of the sixth residue, e.g., to alanine. In some embodiments, the CXCL12 polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:8.

MNAKVVVVLYLVLTALCLSDGAYRCPCRFFESHVARANVKHLKILNTPNCALQIVA RLKNNNRQ V CIDPKLKWIQEYLEKALNK (SEQ ID NO:8)

[0087] In one embodiment, the CXCL12 polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:9.

SYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYL EKALNK (SEQ ID NO:9)

[0088] In some embodiments, the CXCL12 polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10.

AYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYL EKALNK (SEQ ID NO: 10) [0089] In some embodiments, the CXCL12 polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:ll.

KPV SLAYRCPCRFFESHV ARANVKHLKILNTPNC ALQIVARLKNNNRQ V CIDPKLKW IQEYLEKALNK (SEQ ID NO: 11)

[0090] The CXCL12 polypeptide that is part of the present invention may be a CXCL12 polypeptide variant, homolog, or derivative that activates the CXCL12/CXCR4 and/or CXCL12/CXCR7 pathway. Activators of CXCR4 useful with the invention may include, but are not limited to, CXCL12 (SDF-l) mutants, fusion proteins/genes, truncations and/or analogues. Non-limiting examples of CXCL12 mutants that may be useful as activators of CXCR4 include S-SDF-l(S4V) (Segers et al. Circulation·.! 16(5): 1683-1692 (2007) and Segers et al. Circulation 123:1306-1315 (2011)), CXCL12-GL (CXCL12 fused to Gaussia luciferase) (Luker et al. Biotechniques 47(l):625-632 (2009)) and AAV-[S4V]-SDF-la and V-[S4V]-SDF-la (Baumann et al. J. Controlled Release 162:68-75 (2012)). Non-limiting examples of CXCL12 fusion proteins/genes that may be useful as activators of CXCL4 include SDF1-GPVI protein (SDF-l -glycoprotein VI)(Ziegler et al. Circulation l25(5):685- 696 (2012)), SDF-l /HOXB4 (Chen et al. Am. J. Transl. Res.6(6): 691-702 (2014)), S1FG (SDF-1/CXCL12 fused to fractalkine mucin stalk and GPI anchor from LFA-3) (Stachel et al. Stem Cells 31 :1795-1805 (2013)), and affinity tagged CXCL12 (e.g., CXCLl2-Strep) (Picciocchi et al. PLoS One 9(l):e87394 (2014)). Non-limiting examples of CXCL12 truncation variants that can activate CXCR4 include CXCLl2[22-89] and CXCLl2[22-88] (Richter et al. Stem Cells Dev. 23(16): 1959-1974 (2014)). Non-limiting examples of

CXCL12 analogues that may be useful as activators of CXCR4 include lactam analogues of CXCL12, CTCE 0021 and CTCE 0214 (Patrussi et al. Curr. Med. Chem. 18:497-512 (2011)), HSEFFR-CPC-RFFESH (SDF-l {H-H}) (Palladino et al. FEBS Letters 579:5293-5298 (2005)), and SDF-l analogue (N33A) (Ueda et al. J. Biol. Chem. 272(4):24966-24970 (1997)).

[0091] The CXCL12 polypeptide variant, homolog, or derivative may have up to 20 amino acid additions, deletions and/or substitutions {e.g., conservative substitutions) relative to the wild-type sequence, e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions and/or substitutions. Conservative amino acid substitutions in the CXCL12 polypeptides of the invention may be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0092] In identifying amino acid sequences encoding polypeptides other than those specifically disclosed herein, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (see, Kyte and Doolittle, J. Mol. Biol. 157'.105 (1982); incorporated herein by reference in its entirety). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0093] Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, id.), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);

methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);

aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0094] Accordingly, the hydropathic index of the amino acid (or amino acid sequence) may be considered when modifying the peptides specifically disclosed herein.

[0095] It is also understood in the art that the substitution of amino acids can be made on the basis of hydrophilicity. U.S. Patent No. 4,554,101 (incorporated herein by reference in its entirety) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0096] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (±3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);

threonine (-0.4); proline (-0.5 ± I); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). [0097] Thus, the hydrophilicity of the amino acid (or amino acid sequence) may be considered when identifying additional peptides beyond those specifically disclosed herein.

[0098] In certain embodiments, the CXCL12 polypeptide is capable of dimerizing with another protein. In some embodiments, the CXCL12 polypeptide is capable of forming a homodimer, e.g., through the natural formation of CXCL12 dimers. Wild-type CXCL12 is present as monomers and homodimers under physiological conditions (Ray et al. , Biochem.

J. 442:433 (2012)). In some embodiments, the CXCL12 polypeptide is capable of forming a heterodimer, e.g., through the binding of the CXCL12 polypeptide to wild-type CXCL12 or a CXCL12 fragment or variant.

[0099] In some embodiments, the CXCL12 polypeptide is a locked monomer polypeptide, e.g., wherein at least one cysteine is substituted relative to the wild-type CXCL12 sequence, such that the polypeptide is unable to form a disulfide bond with another CXCL12 monomer. In some embodiments, two cysteines are substituted relative to the wild-type CXCL12 sequence, e.g., with a conservative substitution, e.g, alanine. In certain embodiments, residues at positions 55 and 58 (numbering with respect to human CXCL12) are substituted with cysteine to resist peptide-induced dimerization by maintaining steric repulsion of the chemokine helix. Locked monomers are disclosed, for example, in US Patent No. 9,908,923, incorporated by reference herein in its entirety. As used herein, a“locked monomer polypeptide” is a CXCL12 polypeptide that preferentially does not form a dimer when present in a liquid. In some embodiments, when a locked monomer polypeptide is present in a liquid, less than 10% of the polypeptide is in the form of a dimer, e.g, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0100] In some embodiments, the CXCL12 polypeptide is a locked dimer polypeptide, wherein the dimer comprises two monomers locked together. Locked dimers are disclosed, for example, in US Patent No. 7,923,016, incorporated by reference herein in its entirety. As used herein, a“locked dimer polypeptide” is a CXCL12 polypeptide that preferentially is in the form of a dimer when present in a liquid. In some embodiments, when a locked dimer polypeptide is present in a liquid, less than 10% of the polypeptide is in the form of a monomer, e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. [0101] The CXCL12 locked dimer polypeptide of the invention may be locked by substituting one or more amino acid residues in the monomers with cysteine. In some embodiments, two residues are substituted with cysteine. In certain embodiments, residues at positions 36 and 65 (numbering with respect to human CXCL12) are substituted with cysteine.

[0102] In some embodiments, the CXCL12 may comprise a leader sequence on the N- terminus, e.g. , to allow for secretion of the protein from the host cell in which it is synthesized. In one embodiment, the leader sequence is a wild-type CXCL12 leader sequence. The human CXCL12 leader sequence has the amino acid sequence of SEQ ID

NO: 12.

MNAKVVVVLVLVLTALCLSDG (SEQ ID NO:12)

In one embodiment, the leader sequence is a heterologous leader sequence, e.g., one that is functional in the host cell in which the protein will be expressed. The leader sequence may be a sequence for a prokaryotic or eukaryotic protein, e.g., one suitable for expression in bacteria, yeast, human cells, plant cells, insect cells, etc. In one embodiment, the leader sequence is a plant leader sequence, e.g., from Arabidopsis extensin, Nicotiana extensin, barley alpha amylase, or PR1 A. In certain embodiments, the leader sequence comprises the amino acid sequence of any one of SEQ ID NOS:13-16.

Arabidopsis extensin MGSPMASLVATLLYLTISLTFYSQSTA (SEQ ID NO: 13)

Nicotiana extensin MGKMASLFATFLVVLVSLSLASESSA (SEQ ID NO:14)

barley alpha amylase MANKHMSLSLFIVLLGLSCSLASG (SEQ ID NO: 15)

PR1A MGFVLFSQLPSFLLVSTLLLFLVISHSCRA (SEQ ID NO:16)

[0103] In certain embodiments, the CXCL12 has at least 80% identity to any one of the sequences disclosed herein, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 98%, or 99% identity.

[0104] In some embodiments, CXCR4 and/or CXCR7 binding agent is a fusion protein comprising a CXCL12 polypeptide fused in frame to a targeting polypeptide. As used herein, a“CXCL12 polypeptide” refers to the mature polypeptide after the leader sequence is cleaved. Such fusion proteins are disclosed, for example, in US Application No. 62/536,203, incorporated by reference herein in its entirety.

[0105] The targeting polypeptide may be any polypeptide capable of delivering the fusion protein to a specific target, e.g., a stem cell or progenitor cell. In some embodiments, the targeting polypeptide is an antigen binding domain. An antigen binding domain is any peptide sequence that specifically binds to an antigen and can function as part of a fusion protein. The antigen binding domain may be a natural sequence, e.g. , an antibody or a fragment thereof, a ficolin, a collectin, etc. The antigen binding domain may be a synthetic sequence, e.g., an engineered antibody, an antibody-like peptide, an antibody mimetic, an aptamer, etc. The antigen binding domain may comprise, for example, at least one scFv, at least one Fab fragment, at least one Fv fragment, etc. It may be monovalent or it may be multivalent. In embodiments wherein the engineered antibody is multivalent, it may be bivalent, trivalent, tetravalent, etc. The multivalent antibodies may be monospecific or multispecific, e.g., bispecific, trispecific, tetraspecific, etc. The multivalent antibodies may be in any form, such as a diabody, triabody, tetrabody, etc. In certain embodiments, the engineered antibody is a Tandab.

[0106] In some embodiments, the targeting polypeptide targets the fusion protein to a specific cell type, e.g., a stem cell or progenitor cell. The targeting polypeptide may specifically bind to a stem cell or progenitor cell marker, e.g., a cell surface marker.

[0107] The CXCL12 polypeptide and targeting polypeptide may be present in the fusion protein in any arrangement. In some embodiments, the CXCL12 polypeptide is directly or indirectly fused to the N-terminal side of the targeting polypeptide. In some embodiments, the CXCL12 polypeptide is directly or indirectly fused to the C-terminal side of the targeting polypeptide. In some embodiments, the fusion protein comprises more than one CXCL12 polypeptide which may be the same or different. In some embodiments, the fusion protein comprises more than one targeting polypeptide which may be the same or different. When there are three or more polypeptides in the fusion protein they may be arranged in any order, e.g., CXCL12 polypeptide-targeting polypeptide-CXCLl2 polypeptide or targeting polypeptide-CXCLl2 polypeptide- targeting polypeptide. [0108] In certain embodiments, the fusion protein further comprises a linker between the CXCL12 polypeptide and the targeting polypeptide. If there are multiple CXCL12 polypeptides and/or targeting polypeptides present in the fusion protein, there may be a linker present between some or all of the polypeptides. The linker may be any amino acid sequence that provides a suitable length and/or flexibility. In one embodiment, the linker sequence is a repeating sequence, e.g., a repeat of G₄S. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of: GGSSRSS (SEQ ID NO:17), (GGGSGGG)₄ (SEQ ID NO:18), GGGGS GGGGS GGGGS (SEQ ID NO:19),

GGS SRS S S S GGGGSGGGG (SEQ ID NO:20), and GGSSESSSSGGGGSGGGG (SEQ ID NO:21).

[0109] In certain embodiments, the CXCL12 protein of the invention can be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the CXCL12 protein in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. For example, one or more non-naturally occurring amino acids, such as D-alanine, can be added to the termini. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Additionally, the peptide terminus can be modified, e.g., by acetylation of the N-terminus and/or amidation of the C-terminus.

Likewise, the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable“carrier” proteins prior to administration.

[0110] In some embodiments, the CXCL12 protein may comprise an additional domain that provides a desired characteristic. For example, the CXCL12 protein may comprise a domain that increase the stability of the CXCL12 protein, e.g., by inhibiting degradation of the CXCL12 protein. In some embodiments, the CXCL12 protein comprises an isolated Fc domain of an antibody, e.g., from IgGl or IgG2. In some embodiments, the CXCL12 protein does not comprise an isolated Fc domain of an antibody. Tissue Explants and methods for making them

[0111] Another aspect of the invention relates to methods for preparing tissue explants using the methods described above. Some embodiments include a method of preparing a tissue explant, comprising:

a) obtaining a mammalian tissue sample comprising stem cells and/or progenitor cells from fetal, newborn, neonatal, juvenile, or adult tissue;

b) culturing the tissue sample for a period of time; and

c) encapsulating cells from the tissue sample in a matrix comprising a CXCR4 and/or CXCR7 binding agent.

[0112] The stem cells and/or progenitor cells may be any of the cells described above and may be isolated as described above.

[0113] The matrix and encapsulation process may be any of the ones described above.

[0114] The cells may be cultured, for example, for 1 to 12 days prior to encapsulation, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or any range therein, e.g., 1-3, 1-4, 1-5, 1-6, 1-7, 1- 8, 1-9, 1-10, 1-11, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 2-6, 3-7, 4-8, 5-9, 6- 10, or 7-11 days. In some embodiments, the stem cells and/or progenitor cells are cultured in the absence of serum or growth hormones/growth factors. In some embodiments, the stem cells and/or progenitor cells are cultured in the absence of a CXCR4 and/or CXCR7 binding agent.

[0115] A further aspect of the invention relates to tissue explants prepared by the methods of the invention. In some embodiments, the tissue explant comprising mammalian stem cells and/or progenitor cells encapsulated in a matrix comprising a CXCR4 and/or CXCR7 binding agent.

[0116] The cells may be from any suitable mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is one that can provide cells and tissues suitable for transplantation into a human, e.g., a pig or a primate.

[0117] The stem cells and/or progenitor cells may be from fetal, newborn, neonatal, juvenile, or adult tissue of a mammal. In some embodiments, the cells may be from a neonate, e.g., between day 1 and day 10 after birth, e.g., between day 3 and day 8 after birth.

[0118] The stem cells and/or progenitor cells may be from any tissue known to contain suitable cells, inducing but not limited to pancreatic, heart, liver, neural, skin, lung, muscle, bone, bone marrow, adipose, or dental pulp tissue. In some embodiments, the stem cells and/or progenitor cells may be from a hormone-producing tissue, e.g., endocrine glands such as the pineal gland, hypothalamus, pituitary gland, parathyroid gland, thyroid gland, thymus gland, adrenal gland, pancreas, ovaries, or testes, or other hormone producing tissues such as heart, stomach, duodenum, liver, kidney, skin or adipose tissue.

[0119] In some embodiments, at least some of the stem cells and/or progenitor cells express CXCR4 and/or CXCR7.

[0120] The matrix may be any matrix suitable for encapsulating cells while maintaining the viability of the cells. In some embodiments, the matrix is a polymer matrix, e.g., an alginate matrix. The matrix may be selected such that the CXCR4 and/or CXCR7 binding agent elutes from the matrix and continuously contacts the encapsulated cells. In some

embodiments, the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a steady rate, e.g., at a rate that provides a concentration of the agent that is effective to promote the differentiation and/or maturation of the cells. In some embodiments, the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a rate of about 0.1 to about 10 pmol/hour, e.g, about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 pmol/hour or any range therein, e.g., 0.1-2, 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 0.1-4, 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 0.1-5, 1-6, 2-7, 3-8, 4-9, 5-10, 0.1-6, 1-7, 2-8, 3-9, 4-10, 0.1-6, 1-7, 2-

8, 3-9, or 4-10 pmol/hour.

[0121] In some embodiments, the stem cells and/or progenitor cells are cultured for a period of time prior to encapsulation. The cells may be cultured, for example, for 1 to 12 days prior to encapsulation, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or any range therein, e.g., 1-3,

1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12,

2-6, 3-7, 4-8, 5-9, 6-10, or 7-11 days. In some embodiments, the stem cells and/or progenitor cells are cultured in the absence of serum or growth hormones/growth factors. In some embodiments, the stem cells and/or progenitor cells are cultured in the absence of a CXCR4 and/or CXCR7 binding agent.

[0122] The present invention further relates to compositions comprising the tissue explants of the invention. The compositions may be pharmaceutical compositions further comprising a pharmaceutically acceptable carrier. [0123] By“pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.

[0124] The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0125] The explants of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (2 I^th Ed. 2005). In the manufacture of a pharmaceutical formulation according to the invention, the explant is typically admixed with, inter alia, an acceptable carrier. [0066] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, or bacteriostatic water.

[0126] Various modifications and variations can be made to the compounds, compositions and methods described herein. Other aspects of the compounds, compositions and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions and methods disclosed herein. It is intended that the specification and examples be considered as embodiments, and are not intended to be limiting.

[0127] The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art, which would similarly permit one to successfully perform the intended invention.

Example 1

Effective of CXCL12 on differentiation and maturation of neonatal porcine islet cell clusters

[0128] The effect of CXCL12 on encapsulated differentiation of NPICC was tested.

NPICC were isolated from three-day old piglets and cultured for six days in serum free media. On day six, cells were encapsulated in 1.6% BP 1410-28 alginate with or without 1 pg/mL CXCL12 and cultured for 28 days with or without 10 ng/mL CXCL12 supplemented in the media. On day 28 post encapsulation, capsules were dissolved and NPICC were dissociated with 0.05% trypsin EDTA and attached to a MATRIGEL™ coated slide. Cells were fixed with 4% paraformaldehyde and stained for insulin, glucagon, somatostatin, and DAPI. Slides were imaged and the number of positive cells was counted and expressed as a percentage of DAPI⁺ cells. The results indicated that cells that were encapsulated with CXCL12 and had CXCL12 supplemented in cell culture media had higher percentage of positive cells for all the markers analyzed (FIG. 1). This suggests that CXCL12 increases the differentiation of pancreatic progenitor cells into mature endocrine cell subsets.

[0129] To test the ability of CXCLl2-treated NPICC to secrete insulin in response to glucose, NPICC isolated from three-day old piglets were cultured for 6 days and then encapsulated either with or without 1 pg/mL of CXCL12. After encapsulations, capsules were cultured in the media supplemented with or without 10 ng/mL of CXCL12. Twenty days post-encapsulation, a static glucose-stimulated insulin secretion (GSIS) assay was performed. Capsules were washed in Kreb’s buffer and then one set of capsules was exposed to low glucose (2.8 mM) while the other set was exposed to high glucose (20 mM) solution. Following a 2-hr incubation, solutions were collected and insulin levels were quantitated using an insulin ELISA kit (Mercodia, Sweden). The insulin values were normalized to the number of IEQs used in the assay. The data show that capsules prepared and cultured with hCXCLl2 (++) secreted higher amounts of insulin than control (— ) capsules (FIG. 2). The results suggest that the presence of CXCL12 in the alginate and culture media might be stimulating insulin production and/or beta-cell differentiation and maturation.

[0130] To study the persistence of glucose-stimulated insulin secretion, NPICC isolated from three-day old piglets were cultured for 6 days and then encapsulated either with or without 1 pg/mL of CXCL12. After encapsulations, capsules were cultured in the media supplemented with or without 10 ng/mL of CXCL12. GSIS assays were performed at day-6, day- 13 and day-29 post-encapsulation in a perifusion device. Briefly, capsules were washed in Krebs buffer, and then exposed to low glucose (3.3 mM) and high glucose (16.7 mM) for 90 mins, and lastly to KCL (25 mM) solution for 45 mins. Perfusates were collected every 15 mins and insulin levels were quantitated using an insulin ELISA kit (Mercodia, Sweden). The insulin levels were normalized to the number of IEQs. The results shows that higher levels of insulin were secreted from capsules encapsulated and cultured in CXCL12 (FIG. 3). Additionally, increasingly higher levels of insulin were secreted from capsules at later time points post-encapsulation. [0131] To test the effect of the age of the piglet on NPICC function, NPICC isolated from eight-day old piglets were cultured for 5 days and then encapsulated either with or without 1 mg/mL of CXCL12. After encapsulations, capsules were cultured in the media supplemented with or without 10 ng/mL of CXCL12. GSIS assays were performed at day-6, day-l3 and day-29 post-encapsulation in a perifusion device. Briefly, capsules were washed in Krebs buffer, and then exposed to low glucose (3.3 mM) and high glucose (16.7 mM) for 90 mins, and lastly to KCL (25 mM) solution for 45 mins. Perfusates were collected every 15 mins and insulin levels were quantitated using an insulin ELISA kit (Mercodia, Sweden). The insulin levels were normalized to the number of IEQs. The results shows that higher levels of insulin were secreted from capsules encapsulated and cultured in CXCL12 (FIG. 4).

Furthermore, the levels of insulin secreted were higher than the levels secreted from NPICC isolated from three-day old piglets.

[0132] The effect of human recombinant CXCL12 on the viability of NPICC was tested. NPICCs from three-day old piglets were encapsulated in 1.6% w/v alginate six days after isolation. The encapsulations were performed either in the presence or absence of 1 pg/mL CXCL12. These microcapsule cultures were maintained in vitro for 28 days in media supplemented with or without 10 ng/ml CXCL12. The viability of these encapsulated NPICCs was investigated via a live/dead assay using fluorescein diacetate and propidium iodide (FDA/PI). In these conditions CXCL12 had no effect on the viability of the encapsulated NPICC.

[0133] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

WHAT IS CLAIMED IS:

1. A method for promoting the differentiation and/or maturation of mammalian stem cells and/or progenitor cells, the method comprising contacting the stem cells and/or progenitor cells with an effective amount of a CXCR4 and/or CXCR7 binding agent, thereby promoting the differentiation and/or maturation of the cells.

2. The method of claim 1, wherein the stem cells and/or progenitor cells are obtained from fetal, newborn, neonatal, juvenile, or adult tissue.

3. The method of claim 2, wherein the stem cells and/or progenitor cells are obtained from pancreatic, heart, liver, neural, skin, lung, muscle, bone, bone marrow, adipose, or dental pulp tissue.

4. The method of claim 2, wherein the stem cells and/or progenitor cells are obtained from a hormone-producing tissue.

5. The method of any one of claims 1-4, wherein at least some of the stem cells and/or progenitor cells express CXCR4 and/or CXCR7.

6. The method of any one of claims 1-5, wherein the differentiation and/or viability of the stem cells and/or progenitor cells is maintained or increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent.

7. The method of any one of claims 1-6, wherein a biological function of the stem cells and/or progenitor cells is increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent.

8. The method of claim 7, wherein the stem cells and/or progenitor cells are obtained from hormone-producing tissue and the level of hormone secretion is increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent.

9. The method of claim 8, wherein the stem cells and/or progenitor cells are obtained from pancreatic islet tissue and the level of insulin secretion is increased relative to control cells that have not been contacted with a CXCR4 and/or CXCR7 binding agent.

10. The method of claim 9, wherein the increase in insulin secretion lasts for at least 60 days.

11. The method of any one of claims 1-10, wherein the contacting comprises

encapsulating the stem cells and/or progenitor cells in a matrix comprising the CXCR4 and/or CXCR7 binding agent.

12. The method of claim 11 , wherein the matrix is a polymer matrix.

13. The method of claim 12, wherein the matrix is an alginate matrix.

14. The method of any one of claims 11-13, wherein the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a steady rate.

15. The method of any one of claims 11-14, wherein the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a rate of about 0.1 to about 10 pmol/hour.

16. The method of any one of claims 1-15, wherein the stem cells and/or progenitor cells are obtained from neonatal tissue between day 1 and day 10 after birth.

17. The method claims 16, wherein the stem cells and/or progenitor cells are obtained from neonatal tissue between day 3 and day 8 after birth.

18. The method of any one of claims 11-17, wherein the stem cells and/or progenitor cells are cultured for a period of time prior to encapsulation.

19. The method of claim 18, wherein the stem cells and/or progenitor cells are cultured for 1 to 12 days prior to encapsulation.

20. The method of claim 18 or 19, wherein the stem cells and/or progenitor cells are cultured in the absence of serum or growth hormones/growth factors.

21. The method of any one of claims 18-20, wherein the stem cells and/or progenitor cells are cultured in the absence of a CXCR4 and/or CXCR7 binding agent.

22. A method of preparing a tissue explant, comprising:

a) obtaining a mammalian tissue sample comprising stem cells and/or progenitor cells from fetal, newborn, neonatal, juvenile, or adult tissue;

b) culturing the tissue sample for a period of time; and

c) encapsulating cells from the tissue sample in a matrix comprising a CXCR4 and/or CXCR7 binding agent.

23. The method of claim 22, wherein the stem cells and/or progenitor cells are obtained from pancreatic, heart, liver, neural, skin, lung, muscle, bone, bone marrow, adipose, or dental pulp tissue.

24. The method of claim 22, wherein the stem cells and/or progenitor cells are obtained from a hormone-producing tissue.

25. The method of any one of claims 22-24, wherein at least some of the stem cells and/or progenitor cells express CXCR4 or CXCR7.

26. The method of any one of claims 22-25, wherein the matrix is a polymer matrix.

27. The method of claim 26, wherein the matrix is an alginate matrix.

28. The method of any one of claims 22-27, wherein the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a steady rate.

29. The method of claim 28, wherein the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a rate of about 5 pmol/hour.

30. The method of any one of claims 22-29, wherein the stem cells and/or progenitor cells are obtained from neonatal tissue between day 1 and day 10 after birth.

31. The method claims 30, wherein the stem cells and/or progenitor cells are obtained from neonatal tissue between day 3 and day 8 after birth.

32. The method of any one of claims 22-31 , wherein the stem cells and/or progenitor cells are cultured for a period of time prior to encapsulation.

33. The method of claim 32, wherein the stem cells and/or progenitor cells are cultured for 1 to 12 days prior to encapsulation.

34. The method of claim 32 or 33, wherein the stem cells and/or progenitor cells are cultured in the absence of serum or growth hormones/growth factors.

35. The method of any one of claims 32-34, wherein the stem cells and/or progenitor cells are cultured in the absence of a CXCR4 and/or CXCR7 binding agent.

36. A tissue explant prepared by the method of any one of claims 22-35.

37. A tissue explant comprising mammalian stem cells and/or progenitor cells obtained from fetal or neonatal tissue encapsulated in a matrix comprising a CXCR4 and/or CXCR7 binding agent.

38. The tissue explant of claim 37, wherein the stem cells and/or progenitor cells are obtained from pancreatic, heart, liver, neural, skin, lung, muscle, bone, bone marrow, adipose, or dental pulp tissue.

39. The tissue explant of claim 37, wherein the stem cells and/or progenitor cells are obtained from a hormone-producing tissue.

40. The tissue explant of any one of claims 37-39, wherein at least some of the stem cells and/or progenitor cells express CXCR4 or CXCR7.

41. The tissue explant of any one of claims 37-40, wherein the matrix is a polymer matrix.

42. The tissue explant of claim 41, wherein the matrix is an alginate matrix.

43. The tissue explant of any one of claims 37-42, wherein the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a steady rate.

44. The tissue explant of claim 43, wherein the CXCR4 and/or CXCR7 binding agent elutes from the matrix at a rate of about 5 pmol/hour.

45. The tissue explant of any one of claims 37-44, wherein the stem cells and/or progenitor cells are obtained from neonatal tissue between day 1 and day 10 after birth.

46. The tissue explant claims 45, wherein the stem cells and/or progenitor cells are obtained from neonatal tissue between day 3 and day 8 after birth.

47. The tissue explant of any one of claims 37-46, wherein the stem cells and/or progenitor cells are cultured for a period of time prior to encapsulation.

48. The tissue explant of claim 47, wherein the stem cells and/or progenitor cells are cultured for 1 to 12 days prior to encapsulation.

49. The tissue explant of claim 47 or 48, wherein the stem cells and/or progenitor cells are cultured in the absence of serum or growth hormones.

50. The tissue explant of any one of claims 47-49, wherein the stem cells and/or progenitor cells are cultured in the absence of a CXCR4 and/or CXCR7 binding agent.