Compositions and methods for treatment of human diseases with glycocalyx-modified mesenchymal stem cells

Abstract

The present disclosure provides, inter alia, compositions and methods for treating diseases and conditions (such as skeletal diseases) using glycocalyx-modified mesenchymal stem cells. According to some aspects, provided herein are compositions and methods to improve tissue colonization of intravascularly administered human MSCs and, thereby, optimize the intended biologic benefit(s) for any clinical indication where MSCs are utilized.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF HUMAN DISEASES WITH GLYCOCALYX-MODIFIED MESENCHYMAL STEM CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent ApplicationSerial No. 63 / 666,458, filed on July 1, 2024, which is incorporated by reference herein in its entirety. FIELD OF INVENTION

[0002] The present disclosure relates to the field of regenerative medicine, in particular, tothe tissue reparative / restorative effects of human mesenchymal stem cells derived from older individuals that have been administered following glycocalyx modification. In one embodiment, a method for treating bone diseases or skeletal conditions using glycocalyx-modified human mesenchymal stem cells is disclosed. BACKGROUND OF THE INVENTION

[0003] Abundant preclinical studies (encompassing both in vitro studies and animalstudies) have provided compelling evidence for existence of multipotent mammalian adult stem cells defined by their capacity to (1) adhere to plastic, (2) expand in tissue culture (i.e., undergo culture-expansion), (3) express cell surface structures CD44, CD73, CD90, and CD105, without expression of CD45, CD11b, CD14, and CD19, and, most critically, (4) develop into various characteristic progeny cells including fibroblasts, osteoblasts, chondroblasts, and adipocytes under standard in vitro differentiation conditions. This multipotent adult stem cell type is generally called a “mesenchymal stem cell” and is denoted by the acronym “MSC”, but is also referred to by various names including, but not limited to, “multipotent stromal cells” or “mesenchymal stromal cells” or “mesenchymal stem / stromal cells” or “medicinal signaling cells” or USA.617191053.7 / 45S“mesenchymal progenitor cells” or “mesenchymal precursor cells”. As used herein, the acronym “MSC” incorporates any cell type with the characteristics described above.

[0004] In the case of intended MSC-based medical therapies in human beings, there canexist a gap between the promise of clinical applicability as proposed based on results of animal models and the reality of clinical applicability of human MSCs to improve outcomes for patients in need. In large part, this gap can exist because MSCs from various mammals can diverge in biologic properties from human MSCs (e.g., due to species-specific differences in the secretome and the paracrine properties of MSCs), and, thus, an animal’s MSC operational capacity in an animal model of a human disease may not mirror that of a human MSC. Moreover, the animal “model equivalent” of the human disease may not fully recapitulate the pathobiology of the human disease. Lastly, in the context of use of an allogeneic MSC as opposed to an autologous MSC (i.e., a non-self-sourced versus a self-sourced MSC, respectively), an additional confounder is introduced due to the inherent variations of the immunobiology of alloreactivity / host defense between that of the animal and that of a human being, which can further limit the extrapolation of results of an MSC-based animal study to elucidate the clinical applicability of human MSCs in human diseases. Thus, it is ideal to perform clinical trials to unveil the true breadth and scope of human MSC tissue reparative / restorative properties in human hosts.

[0005] Beyond heterogeneity in MSC biology in mammals, abundant in vitro studiesindicate that MSC biology varies as a function of the age of the donor from which the cells are obtained. In particular, it has been extensively reported that MSCs derived from younger mammals display much greater tissue reparative functions, have greater differentiation capacity, and higher proliferative ability than MSCs from older mammals. Functional alterations and replicative exhaustion of human MSCs each rise precipitously as a function of age, with humanMSCs become increasingly senescent after age 30. For this reason, to date, all human MSC preparations that are in commercial development for therapeutic applications consist of cells harvested from healthy donors below age 30 years, indeed, most favorably from umbilical cord, umbilical cord blood, or placental sources. Importantly, clinical trials to evaluate the potency of “off-the-shelf” human MSCs for various clinical indications / medical conditions exclusively utilize MSCs obtained from younger, healthy individuals. Beyond age-related changes in MSC biology, a variety of diseases have ascribed a pathobiologic role to MSCs themselves (e.g., osteoporosis, osteoarthritis, and autoimmune diseases, among many other medical conditions), mandating that MSCs for clinical applications be obtained from healthy donors and precluding clinical use of MSCs obtained from patients with any acute or chronic disease(s), especially avoiding MSCs obtained from any inflamed / diseased tissue itself. However, while there can exist species-specific and age-specific variations in MSC biology, all mammalian MSCs share a fundamental biologic deficit: they all lack molecular effectors of cell migration that can guide the trafficking of intravascularly administered cells to sites of tissue injury. While there may be some localization of intravascularly administered MSCs within a given tissue due to vascular “entrapment” (a random process whereby these large cells (MSC diameter ranges from 20-40 microns) lodge within small bore (<10 micron diameter) microvessels), this process is random and does not result in efficient MSC colonization within an intended anatomic area. As such, the vast majority of animal-based studies – and clinical (human) studies – enforce MSC colonization of a target tissue by the direct injection of the MSCs into the target diseased / inflamed tissue.

[0006] From a technical standpoint, though direct injection ensures that MSCs aredeposited within the intended tissue of interest (i.e., the inflamed / injured / diseased tissue itself),direct injection is an invasive procedure that requires use of needles (or other administrative devices, e.g., catheters) with subsequent instillation of MSCs suspended in a crystalloid solution into the affected site as a bolus under hydrostatic pressure; altogether, this approach disrupts the native tissue microenvironment, and this technique can thus result in worsening the tissue injury within the affected site as well as collateral tissue damage in unaffected sites consequent to the invasive procedure itself. Of equal fundamental importance, direct tissue injection is only possible for tissues with strict anatomic boundaries, with tissue planes / histologic structure permitting placement of MSCs at defined affected sites, and with sufficient structural integrity / consistency capable to withstand the invasive injection (i.e., this approach is feasible for the heart, but not feasible for the vast majority of tissues (e.g., the lungs, GI tract, pancreas, or the central nervous system)). Furthermore, direct tissue injection is not feasible for diseases and / or conditions that are multi-focal or generalized in nature (e.g., multiple sclerosis, muscular dystrophies, osteoporosis, etc.). For all these reasons, there is a pressing need to develop methods to optimize tissue delivery of intravascularly administered MSCs in order to fully achieve their intended therapeutic benefit(s).

[0007] Migration of intravascular (circulating) cells to tissue sites is critically dependenton adhesive interactions between the blood-borne cells and endothelial cells within the target tissue. This process depends on the capacity of circulating cells to bind to target tissue endothelial cells with sufficient potency to withstand the hemodynamic shear forces of the prevailing blood flow. The preeminent mediator of this process is a tetrasaccharide glycan expressed on the surface of the circulating cell which is called “sialylated Lewis X” (“sLeX”; CD15s). sLeX is the principal binding determinant (ligand) for E-selectin, an endothelial lectin: adhesive interactions between sLeX displayed on the surface of blood-borne cells and E-selectin expressed on endothelial cellspromotes trafficking to the target tissue with subsequent colonization / engraftment of the sLeX- bearing circulating cell at the pertinent site. E-selectin is constitutively displayed on microvessels of marrow and skin, and, notably, its expression is induced / upregulated at all endothelial beds at inflamed / diseased tissues. As will be described in further detail below, the constitutive expression of E-selectin on marrow microvascular endothelial cells provides a “gateway” for highly efficient marrow colonization of sLeX-bearing blood-borne cells.

[0008] There are a variety of skeletal conditions that affect bone integrity and / or bonehealing that result in disabling and life-threatening consequences. Some of these conditions result from genetic deficits (e.g., osteogenesis imperfecta), some are due to trauma (e.g., fractures, and, in particular, delayed-healing or non-healing fractures), some are due to infections (e.g., osteomyelitis), some are due to metabolic derangements (e.g., renal osteodystrophy), some are due to drugs (e.g., steroids), some are due to endocrinopathies (e.g., hyperparathyroidism), some are due to malnutrition (e.g., inadequate calcium absorption or intake), and some are due to degenerative diseases (e.g., osteoporosis). Of all the skeletal conditions affecting human welfare, osteoporosis is the most prevalent and the most insidious, leading to fragility fractures and associated disability. This generalized skeletal disease is most commonly caused by the loss of estrogen in menopause (“postmenopausal osteoporosis”) and it affects hundreds of millions of women world-wide. Importantly, with the steadily increasing aging of the population world-wide, the incidence of osteoporosis is continuing to rise, especially among aging men.

[0009] In normal bone, there is a continuous bone “turn-over” whereby bone destructionis matched by bone destruction and the bone structure is maintained in steady-state. However, in osteoporosis (literally, “porous bone”), bone production (mediated by osteoblasts, cells derived from mesenchymal stem cells (MSCs)) is outpaced by bone destruction (mediated by osteoclasts,cells derived from hematopoietic stem cells). The combat zone of this tug-of-war battle between osteoblasts and osteoclasts is within the marrow of the affected bones, at the bony interfaces within the marrow known as the “endosteal” surfaces. This process leads to bone erosion, most prominently within “trabecular bone” that forms a meshwork of stress struts that crisscross the cavity within bone marrow (thereby forming a shock-absorbing skeletal lattice within the “Medullary Cavity”). Osteoblasts create bone but are terminally differentiated cells (i.e., cells that have limited ability to multiply / proliferate), and they are generated / replenished by MSCs located within the marrow. Thus, to maximally induce bone regeneration (“osteorestoration”) and, thereby tilt the balance of the osteoporosis tug-of-war within the marrow towards bone creation, one needs to increase the colonization of MSCs within the marrow itself.

[0010] Osteoporosis is “generalized” (i.e., a systemic disease) in that all skeletal sites areaffected. As such, direct injection of reparative / regenerative cells – either the osteoblast (OB) itself or the mesenchymal stem cell (MSC) – into the marrow at all skeletal sites where osteoblast replenishment is needed is impossible. Both MSCs and OBs can be readily culture-expanded in numbers sufficient to treat a patient suffering from any skeletal condition. Accordingly, to achieve intended osteorestoration, the proximate hurdle is to develop the means to deliver the culture- expanded MSCs or the OBs to all sites that are affected so that they can exert their beneficial effect(s) where needed, an imperative that is best achieved by exploiting vascular delivery (i.e., introducing the osteorestorative / osteoreparative cells into the blood and then having the cells migrate to all the marrow located throughout the body). But, even in cases where the bone disease may not be generalized (e.g., at a given fracture site or at sites of several fractures, such as might occur with trauma) or in cases where there may be introduction of a scaffold to enable bonereconstruction (e.g., a bone graft or any pertinent scaffold / implant), it may be beneficial to seed the affected bone area with MSCs and / or with osteoblasts, using the vascular route of delivery.

[0011] The migration of blood-borne cells to marrow is called “osteotropism”, a processoccurring at specialized medullary microvascular endothelial cells that constitutively express E- selectin, a lectin binding a glycan determinant called “sialylated Lewis X” (sLeX; CD15s). Hematopoietic stem / progenitor cells (HSPCs) display sLeX, and E-selectin-sLeX interactions empower HSPC osteotropism requisite for successful hematopoietic stem cell transplantation. sLeX is a tetrasaccharide, a “type 2” lactosamine (i.e., galactose (Gal) β(1,4)-linked to N- acetylglucosamine (GlcNAc)), bearing terminal sialic acid (NeuAc) and fucose (Fuc) substitutions: NeuAc-α(2,3)-Gal-β(1,4)-[Fuc-α(1,3)]-GlcNAc-R. Natively, MSCs do not display sLeX yet uniformly express a CD44 glycovariant that bears terminal α(2,3)-sialylated type 2 lactosamines (i.e., NeuAc-α(2,3)-Gal-β(1,4)-GlcNAc-R) missing only Fuc α(1,3)-linked to GlcNAc to complete the sLeX motif. The missing Fuc can be placed stereospecifically using ^(1,3)-fucosyltransferases, thereby engendering sLeX decorations on the MSC surface; when injected intravascularly, these “glycoengineered” MSCs are then capable of osteotropism. This process is called, without limitation, a variety of terms including “cell surface glycoengineering” or “glycocalyx editing” or “glycosyltransferase-programmed stereosubstitution” (GPS), and, more generally, the glycoengineered MSCs are called “glycocalyx-modified MSCs”.

[0012] MSCs can fuel osteorestoration via direct differentiation into osteoblasts and / or viaMSC-mediated trophic effects to improve resident osteoblast biology and / or via MSC-mediated dampening of inflammation, thereby effectively treating bone conditions / diseases such as osteoporosis. MSCs are located within many tissues of the body (e.g., bone marrow, fat, periodontal ligament, umbilical cord, amniotic membrane, etc.), and can also be found in blood(especially in umbilical cord blood). However, there are abundant studies indicating that the MSC extracted from bone marrow has the most potent capacity to make osteoblasts and, commensurately, the greatest capacity to make new bone (this is known as “osteogenic capacity”). Still, there is no doubt that MSCs extracted from any tissue site have osteogenic capacity.

[0013] For all studies of tissue regeneration / repair using tissue stem / progenitor cells, noprior study has ever sought to extract and then culture-expand the reparative stem / progenitor cell for restoration / regeneration of the “affected tissue” (i.e., the diseased tissue) from the diseased tissue itself. Indeed, primary clinical principles would dictate that this approach would not be prudent as stem / progenitor cells extracted from an inflammatory / injured tissue milieu could be physiologically compromised by the antagonistic conditions of the pertinent diseased tissue microenvironment, and, furthermore, deficits in the reparative abilities of the tissue-derived stem / progenitor cell could perhaps be etiologic in the disease process itself. Indeed, experiments in animal models indicate that MSCs extracted from sites of tissue injury / inflammation are compromised in terms of tissue repair / restoration. In the case of osteoporosis, it has been reported that MSCs extracted from marrow of osteoporosis patients show deficits in osteogenic capacity in in vitro assays, indicating that disease associated functional deficiencies of MSCs could contribute to the imbalance between bone destruction and bone production. As such, one might consider extraction of the MSC from native (“healthy”) marrow of another person. However, in the case of skeletal conditions, the precursor cell (the MSC) creates a highly immunoreactive progeny cell (the osteoblast). Hence, a non-self (i.e., “allogeneic”) source of MSCs would engender “foreign” osteoblasts that would be immunologically rejected. To create a universal (“off-the-shelf”) MSC product that would then differentiate into immune-evasive osteoblasts, one could undertake gene editing of MSCs to remove all the relevant molecular effectors inducing immunorecognition ofosteoblasts (e.g., removal of major histocompatibility antigens (“tissue antigens”)) and / or could genetically introduce immunoregulatory molecules to hinder / avoid immune recognition (e.g., check-point inhibitors), but such efforts would encompass genetic manipulations that pose myriad practical challenges and could in themselves trigger adverse biologic processes that could markedly alter the properties and function of the progeny (gene-edited) osteoblast. Nonetheless, should an allogeneic source of MSCs and / or an MSC gene-edited approach become feasible, the platform glycoengineering approach described herein would be applicable to the requisite distribution of the osteoregenerative cells throughout the entire skeleton. Similarly, one could deliver the pertinent progeny of MSCs – the osteoblast (including gene-edited osteoblasts) derived from differentiation of MSCs in culture - by modifying the osteoblast surface using the glycoengineering approach described herein and then intravascularly administering the cells (thereby achieving increased osteoblast content within the affected skeletal sites).

[0014] Regardless of whether the MSC is obtained autologously or from a “non-self”source, there exists a need for efficient delivery of the human MSCs to human marrow, thereby improving MSC-mediated osteorestorative capacity within affected bones in situ. As a fundamental prerequisite, it is critical to assess whether culture-expanded MSCs obtained autologously from the marrow of a person suffering from osteoporosis, then glycoengineered and administered intravascularly, would yield any adverse effects. Beyond safety and tolerability, it is imperative that this approach would engender a beneficial clinical response, measured by the reduction in incidence of fragility fractures following the administration of the autologous culture- expanded MCSs. The inventions disclosed herein are directed to meeting these and other needs.SUMMARY OF THE INVENTION

[0015] According to some aspects, provided herein are compositions and methods toimprove tissue colonization of intravascularly administered human MSCs and, thereby, optimize the intended biologic benefit(s) for any clinical indication where MSCs are utilized.

[0016] Some embodiments provided herein are based, in part, on the surprising findingthat a single intravascular infusion of human MSCs that have undergone cell surface glycoengineering (glycocalyx editing; glycosyltransferase-programmed stereosubstitution (GPS)) to enforce display of the cell surface glycan sLeX results in a profound beneficial reparative / restorative effect within a human tissue whose endothelial beds express E-selectin. The embodiments provided herein are also based in part on the surprising finding that human MSCs obtained from “aged” donors (in particular, human beings older than age 30 years) are capable of exerting significant tissue reparative / restorative effects. Moreover, the embodiments provided herein are based in part on the surprising finding that culture-expanded human MSCs harvested from a tissue location that harbors a disease / inflammatory process retain the capacity to exert significant tissue reparative / restorative effects, i.e., contrary to numerous reports, MSCs sourced from a site of pathology are not compromised with regards to tissue regenerative properties.

[0017] In accordance with the above, provided herein are methods and compositions foraugmenting the functional capabilities and therapeutic impact of intravascularly administered human MSCs for use in any human disease / inflammatory process whereby the endothelial beds of the affected tissue(s) express E-selectin.

[0018] In certain embodiments provided herein, human MSCs can be treated in vitro withglycosyltransferases and appropriate donor nucleotide donors under conditions sufficient to installdisplay of sLeX on the MSC surface. Glycosyltranferases can include a fucosyltransferase, a sialyltransferase, a galactosyltransferase, or an N-acetylglucosaminotransferase, either singly or in combination. These glycosyltransferases may be used following treatment of the MSCs with sialidases, with subsequent resialylation of the MSC surface followed by fucosylation. The fucosyltransferase may be an ^(1,3)-fucosyltransferase (for example, but not limited to, Fucosyltransferase VI (FTVI, FT6) or Fucosyltransferase VII (FTVII, FT7) or Fucosyltransferase III (FTIII, FT3) or Fucosyltransferase V (FTV or FT3)), and / or an ^(2,3)-sialyltransferase (for example, but not limited to, ST3GalIII, ST3GalIV, or ST3GalVI). Confirmation of the enforced expression of sLeX on the cell surface can be verified by staining using antibodies directed to sLeX (e.g., the rat IgM mAb HECA-452 or the mouse IgG mAb CSLEX-1) and / or by staining E- selectin-Fc chimera using fluorochrome-tagged antibodies / reagents and standard flow cytometry techniques.

[0019] As illustrative, without limitation thereto or thereof for the therapeutic utility ofglycoengineered human MSC for human diseases / conditions, the application of glycocalyx- modified (cell surface glycoengineered) human MSCs derived from bone marrow of patientssuffering from osteoporosis to repair / restore damaged bone is disclosed herein. Postmenopausalosteoporosis affects older women (i.e., women after the age of menopause). No prior study has ever investigated the ability of human MSCs harvested exclusively from older individuals to perform their biologic function. Indeed, there is significant evidence that MSCs from older persons have deficits in a variety of MSC properties, including, but not limited to, their osteogenic / osteorestorative capacity. Of equal importance, there is concern that MSCs obtained from diseased tissue sites may have deficits in biologic activity, including a possible contributory role to the tissue pathobiology. In particular, there is evidence that derangements in MSC biologyfuel degenerative diseases: in the case of osteoporosis, there is evidence that MSCs harvested from marrow of affected patients have deficiencies in osteogenic capacity. Moreover, stem / progenitor cells obtained from older persons, and then culture-expanded, could harbor age-associated mutations that could result in aberrant cell proliferation and / or differentiation leading to cancer; in the case of MSCs, the possibility of highly aggressive cancers derived from “older” MSCs called “sarcomas” (including osteosarcomas).

[0020] Here, it has been surprisingly discovered that culture-expanded MSCs obtainedfrom the marrow of osteoporotic bone (marrow) of older persons, when glycocalyx-modified and then intravascularly administered into a patient with osteoporosis, can improve osteogenic / osteoreparative capacity and thereby achieve osteorestoration sufficient to decrease fragility fractures. There are no serious adverse effects, notably, no evidence whatsoever of cancer development. As disclosed herein, the colonization of intravascularly administered culture- expanded human MSCs at all skeletal sites within the recipient was accomplished by modifying the composition of the cell “glycocalyx” (i.e., the sugar-coat displayed on the cell surface), via a process that is generically called “cell surface glycan engineering”, “glycoengineering”, “glycocalyx editing”, “glycosyltransferase-programmed stereosubstitution” (GPS), and / or “glycocalyx modification”. In particular, all MSCs possess a membrane glycoprotein called “CD44” whose sugars (glycans) can be engineered to display sLeX. This sugar (glycan) engineering of the human MSC surface CD44 involves the glycosyltransferase-mediated installation of fucose onto α(2,3)-sialylated type 2 lactosaminyl glycans of CD44 using an α(1,3)- fucosyltransferase together with GDP-fucose donor (“exofucosylation”), thereby enforcing expression of the sLeX-laden glycoform of CD44 called “Hematopoietic Cell E- / L-selectinLigand” (HCELL), the most potent E-selectin ligand expressed on any human cell. HCELL is the “bone marrow homing receptor” – the cell surface molecule that mediates osteotropism.

[0021] According to some aspects, the present disclosure provides a method of treating orameliorating a skeletal / bone disease or condition in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the skeletal / bone disease or condition; modifying the glycocalyx of the MSCs, ex vivo, using one or more glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce “glycocalyx-modified MSCs”; introducing the glycocalyx- modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for bone regeneration.

[0022] In some embodiments, the present disclosure provides a method of treating orameliorating a disease or condition (e.g., a bone disease) in a subject in need thereof. In some embodiments of, e.g., a bone disease, the present disclosure provides the steps comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the disease or condition, and then differentiating the MSCs in vitro into osteoblasts, which may themselves be further culture-expanded; modifying the glycocalyx of the osteoblasts, ex vivo, using one or more glycosyltransferases to enforce cell surface expression of sLeX on the osteoblasts to produce “glycocalyx-modified osteoblasts”; introducing the glycocalyx-modified osteoblasts into the subject, wherein the glycocalyx-modified osteoblasts are effective for bone regeneration.

[0023] In some embodiments, the glycosyltransferase-mediated reactions enforce sLeXdisplay on the intrinsic cell surface glycoprotein CD44, thereby engendering the expression of the distinct CD44 glycoform known as HCELL. on the surface of the glycocalyx-modified MSCs orglycocalyx-modified osteoblasts. In some embodiments, the population of culture-expanded MSCs is obtained from the bone marrow of the subject suffering from the skeletal / bone disease or condition. In other embodiments, the MSCs may be obtained from non-marrow sources, including, but not limited to, adipose tissue, dental pulp, periodontal ligament, umbilical cord, umbilical cord blood, menstrual blood, placenta, and amniotic membrane

[0024] In some embodiments, the skeletal / bone disease or condition is caused by one ormore of congenital / genetic diseases, trauma, degenerative diseases, metabolic diseases, endocrinopathies, neoplastic diseases, and iatrogenic (drug) effects. In some embodiments, the bone disease or condition is caused by one or more of dietary deficiencies / malnutrition, endocrinologic conditions, gastrointestinal conditions / malabsorption states, pregnancy / lactation- induced bone loss, or decreased bone density due to cancer or due to medications. In some embodiments, the bone disease or condition is osteoporosis caused by one or more of an autoimmune disorder, digestive / gastrointestinal disorder, medical procedure, cancer, hematologic / blood disorder, neurological / nervous system disorder, blood / bone marrow disorder, endocrine / hormonal disorder, and pregnancy / lactation. In some embodiments, the bone condition is one or more of fracture and trauma-related injuries (including crush injuries and bone destruction requiring repair with scaffolds / implants).

[0025] In some embodiments, the MSCs or osteoblasts are glycocalyx-modified ex vivoby exofucosylation. In some embodiments, the MSCs or osteoblasts are glycocalyx-modified ex vivo by transfection with a nucleic acid encoding a fucosyltransferase. In some embodiments, the glycocalyx-modified MSCs or glycocalyx-modified osteoblasts are introduced into the subject one time per year. In some embodiments, the glycocalyx-modified MSCs or glycocalyx-modified osteoblasts are introduced into the subject multiple times per year (e.g., a plurality of times / year).

[0026] In some embodiments, the subject suffers from the skeletal / bone diseaseosteoporosis. In some embodiments, the subject suffers from the bone disease postmenopausal osteoporosis (also called “age-related osteoporosis” because this entity also occurs in elderly men). In some embodiments, the subject is greater than 1 month old. In some embodiments, the subject suffers from a congenital / genetic condition whereby production of bone is compromised, such as occurs in osteogenesis imperfecta, hypophosphatasia, and idiopathic juvenile osteoporosis. In some embodiments, the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has evidence of bone loss by imaging studies (e.g., dual- energy X-ray absorptiometry scan (DXA scan) that can identify reduced bone density. In some embodiments, the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject suffers from one or more bone fractures. In some embodiments, the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has suffered a traumatic fracture of a bone. In some embodiments, the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has suffered an atraumatic fracture of a bone. In some embodiments, the glycocalyx-modified MSC or glycocalyx-modified osteoblast is introduced to the subject after the subject has suffered an atraumatic fracture of one or more of vertebra, hip, distal forearm / carpal joint, and pelvis. In some embodiments, the MSC is obtained from the bone marrow (i.e., bone marrow-derived MSCs (“BM-MSCs”) of the subject (i.e., “autologous” BM-MSCs (“autoBM-MSCs”) suffering from the bone disease and the subject is greater than 1 month old. In some embodiments, the MSC is obtained from osteoporotic bone marrow of the subject. In some embodiments, the MSC is obtained from osteoporotic bone marrow of the subject and the subject is greater than 30 years old.

[0027] In some embodiments, the MSC surface is glycocalyx-modified ex vivo byexofucosylation with a fucosyltransferase VI or fucosyltransferase VII (“exofucosylated” autoBM (“Fuc-autoBM-MSCs”)). In some embodiments, the MSC is glycocalyx-modified ex vivo by transfection or transduction with a nucleic acid encoding a fucosyltransferase VI or a fucosyltransferase VII.

[0028] In some embodiments, the subject is undergoing one or more osteoporosistreatments. In some embodiments, the subject is undergoing one or more osteoporosis treatments including, but not limited to, calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents / analogs.

[0029] In some embodiments, the glycocalyx-modified MSCs are effective to increaseplasma levels of bone neoformation marker N-terminal propeptide of type I procollagen (P1NP).

[0030] In some embodiments, 0.5x106 to 10x106 glycocalyx-modified MCSs / kg ofrecipient body weight is introduced to the subject. In some embodiments, the MSC is introduced to the subject via intravascular administration.

[0031] According to some aspects, the present disclosure provides: a method of treating adisease in a subject in need thereof, wherein the endothelial beds of the diseased tissue express E- selectin, comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the diseased tissue of the subject; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject.

[0032] In some embodiments, the disease is a skeletal / bone disease or condition. In someembodiments, the skeletal / bone disease or condition is osteoporosis. In some embodiments,modifying the glycocalyx of the MSCs comprises exofucosylation using one or more fucosyltransferases. In some embodiments, the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.

[0033] In some embodiments, introducing the glycocalyx-modified MSCs comprisesintravascular administration. In some embodiments, 0.5x106to 50x106glycocalyx-modified MSCs / kg of recipient body weight are introduced to the subject. In some embodiments, the subject is undergoing one or more treatments for the disease in addition to the introduction of glycocalyx- modified MSCs.

[0034] According to some aspects, the present disclosure provides a method of treating askeletal / bone disease in a subject in need thereof comprising: obtaining a population of culture- expanded mesenchymal stem cells (MSCs) from diseased tissue of the subject, wherein the subject suffers from the skeletal / bone disease; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for skeletal regeneration.

[0035] In some embodiments, the skeletal / bone disease is osteoporosis. In someembodiments, the glycocalyx of the MSCs comprises exofucosylation using one or more of fucosyltransferases. In some embodiments, the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs. In some embodiments, introducing the glycocalyx-modified MSCs comprises intravascular administration.

[0036] In some embodiments, 0.5x106 to 50x106 glycocalyx-modified MSCs / kg ofrecipient body weight are introduced to the subject. In some embodiments, the subject is undergoing one or more treatments for the skeletal / bone disease in addition to the introduction of glycocalyx-modified MSCs. In some embodiments, the one or more treatments include at least one of calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents.

[0037] According to some aspects, the present disclosure provides a method of treating adisease in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from a subject older than 30 years; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject.

[0038] In some embodiments, the subject is older than 50 years. In some embodiments,modifying the glycocalyx of the MSCs comprises exofucosylation using one or more fucosyltransferases. In some embodiments, the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.

[0039] In some embodiments, introducing the glycocalyx-modified MSCs comprisesintravascular administration. In some embodiments, 0.5x106to 50x106glycocalyx-modified MSCs / kg of recipient body weight are introduced to the subject. In some embodiments, the disease is a skeletal disease or condition. In some embodiments, the skeletal disease or condition is osteoporosis.

[0040] According to some aspects, the present disclosure provides a method of treatingosteoporosis in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from a subject older than 30 years; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on CD44 molecules of the MSCs to produce glycocalyx-modified MSCs expressing HCELL; and introducing the glycocalyx-modified MSCs into the subject via intravascular administration.

[0041] In some embodiments, the glycosyltransferases comprise one or morefucosyltransferases. In some embodiments, modifying the glycocalyx of the MSCs comprises exofucosylation using the one or more of fucosyltransferase VI and fucosyltransferase VII.

[0042] In some embodiments, 0.5x106 to 50x106 glycocalyx-modified MSCs / kg ofrecipient body weight are introduced to the subject. In some embodiments, the subject is undergoing one or more additional treatments for osteoporosis. In some embodiments, the one or more additional treatments include at least one of calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents.

[0043] According to some aspects, the present disclosure provides a method of makingimmunomodulatory / restorative / reparative mesenchymal stem cells (MSCs) comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or wherein the subject is greater than 30 years old.

[0044] According to some aspects, the present disclosure provides a method of preparinga secretome comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affectedtissue; and / or wherein the subject is greater than 30 years old; and harvesting the secretome (e.g. extracellular vesicles) from the MSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The patent or application file contains at least one drawing executed in color.Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0046] The following drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0047] Fig. 1A: Temporal distribution of patient fragility (i.e., spontaneous) fracturesbefore and after Fuc-autoBM-MSC infusion. Timeline of years / months are shown at bottom of figure, and each patient is presented as a separate row, in sequence by date of accrual to study. For each patient, the date of Fuc-autoBM-MSC administration is represented by solid purple circle, and date of completion of the protocol-mandated 2-year evaluation period is shown by blue vertical bar; the date and number of fragility fractures (if >1, so listed as “x#”) are shown in red cross- marks on horizontal bars depicting the patient evaluation interval before (pink bars) and after (blue bars) Fuc-autoBM-MSC infusion. Horizontal bars colored orange indicate the treatment period for those patients who received PTH analog (teriparatide), in each case administered as per discretion of primary physician. At time of infusion (see Table 1), only one patient (#1) was on bisphosphonates, two patients were receiving vitamin D with (#3) or without calcium (#7), four patients (#2, #4, #6, #8) were receiving denosumab / vitamin D / calcium, and two patients (#9, #10)were receiving teriparatide / vitamin D. One patient (#5) was receiving no treatment but had been receiving denosumab with final dose 6 months before Fuc-autoBM-MSC infusion; this was the only patient that manifested a fragility fracture within the protocol-mandated evaluation period, with fracture occurring at 9 months following denosumab withdrawal. The incidence of fragility fractures decreased sharply following Fuc-autoBM-MSC administration: comparing the fracture incidence within the 2-year protocol-mandated evaluation period to that of 2 years prior to infusion, fracture incidence was reduced by 93% (from 7 events / year to 0·5 events / year (p=0·006)).

[0048] Fig. 1B: Incidence of fragility fractures for 2 years pre-infusion and within the 2-year post-infusion period; fragility fracture incidence decreased sharply following Fuc-autoBM- MSC administration (93% reduction (from 7 events / year to 0.5 events / year) (p=0.006, paired t- test). (Right): Incidence of fragility fractures pre-infusion and post-infusion for all years of observation for all patients (p=0.005, Welch´s t-test).

[0049] Fig 2A-B: Changes in plasma levels of bone neoformation markers after Fuc-autoBM-MSC infusion. Fig. 2A shows levels of each biomarker at designated point of measurement. Red solid line (patient #4) indicates initiation of teriparatide (PTH analog) treatment; red dashed line (patients #9 and #10) indicates discontinuation of teriparatide. Fig.2B top table shows mean of aggregated delta percent change from baseline levels for each biomarker per patient for the first 24 months post-infusion. Bottom left table shows Mean ± SEM of aggregated delta percent change from baseline levels of each biomarker for all patients; Bottom right table shows Mean ± SEM of aggregated delta percent change from baseline levels for each biomarker excluding values from teriparatide-treated patients (#4, #9, and #10). Abbreviations:P1NP: N-terminal propeptide of type I procollagen; BGLAP: osteocalcin; Bone ALP: bone alkaline phosphatase.

[0050] Fig.3. Changes in plasma levels of bone resorption biomarkers after Fuc-autoBM-MSC infusion. Top two panels show levels of each biomarker at designated point of measurement. Red solid line (patient #4) indicates initiation of teriparatide (PTH analog) treatment; red dashed line (patients #9 and #10) indicates discontinuation of teriparatide. Top table shows mean of aggregated delta percent change from baseline levels of each biomarker per patient for the first 24 months post-infusion. Bottom left table shows Mean ± SEM of aggregated delta percent change from baseline levels of each biomarker for all patients; Bottom right table shows Mean ± SEM of aggregated delta percent change from baseline levels of each biomarker excluding values from teriparatide-treated patients (#4, #9, and #10). Abbreviations: b-CTx: C-terminal telopeptide of type I collagen; NTX: N-terminal telopeptide of type I collagen.

[0051] Fig. 4A: Histomorphometrical analysis of bone tissue area in subjects before (day0) and 120 days after systemic administration of fucosylated autologous bone marrow-derived MSC. Bone Tissue Area (BTA) was measured within bone biopsies (obtained at the same anatomic area for each subject) before (basal, day +0) and 120 days following MSC infusion (day +120). Each high-resolution analysis per histological slice is shown as a circle. Panel at left shows data for each tissue plane (mm2) for each subject· Mean + SD for BTA values are shown, and differences between basal and day 120 values are as noted: significantly increased in 7 patients (*p<0·05, ***p<0·001), significantly decreased in patient #6 (3·5 mm2 to 2·5 mm2) and patient #7 (2·2 mm2 to 0·8 mm2)(^ΔΔΔp<0·001), or unchanged in patient #9 (2·4 mm2 to 2·3 mm2; ns) (Welch’s t-test).

[0052] Fig. 4B: Bone Tissue Area (BTA, mm2) and Volumetric Bone Mineral Density(QTS Score). Accumulated (all data points from all subjects) BTA and QTS scores are shown, with dashed and solid lines in each panel representing respective Mean + SEM. Left Panel: BTA was measured within iliac crest bone biopsies (obtained at the same anatomic area for each subject) at baseline (day 0) and 120 days after Fuc-autoBM-MSC infusion (day +120). BTA increased significantly post-infusion from Mean + SEM of 1·42 ± 0·03 mm2to 2·41 ± 0·05 mm2(***p<0·001, Welch’s t-test). Right Panel: QTS scores for all patients at baseline (day 0) and month 24 after Fuc-autoBM-MSC infusion. The QTS score Mean + SEM at month 24 (6·49 ± 0·39) was significantly increased from baseline (5·3 ± 0·15) (*p<0·05 (Welch’s t-test)). DETAILED DESCRIPTION

[0053] According to some aspects, the present disclosure provides a method of treating orameliorating a disease or condition in a subject in need thereof using glycocalyx-modified MSCs, wherein the glycocalyx-modified MSCs are obtained from a diseased / affected tissue. According to some aspects, the present disclosure provides a method of treating or ameliorating a disease or condition in a subject in need thereof using glycocalyx-modified MSCs, wherein the MSCs are obtained from the subject and the subject is of advanced age (e.g. greater than: 30 years old, 40 years old, 50 years old, 60 years old, 70 years old). According to some aspects, the present disclosure provides a method of treating or ameliorating a bone disease or condition in a subject in need thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the bone disease or condition; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effectivefor bone regeneration. As disclosed herein, it was surprisingly discovered that osteorestoration can be achieved using the population of culture-expanded MSCs from the bone tissue (marrow) of a subject suffering from a bone disease, such as osteoporosis. In some embodiments, the population of culture-expanded MSCs are obtained from elderly subjects. In some such embodiments, the culture-expanded MSCs can be used alone or in a composition to treat bone disease. As disclosed herein, it has also been surprisingly discovered that a single infusion of exofucosylated MSCs would be sufficient to drive forward a beneficial clinical effect.

[0054] As used herein, the terms "treat," "treating," "treatment" and grammatical variationsthereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because not every treated subject may respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.

[0055] As used herein, the terms “ameliorate”, "ameliorating" and grammatical variationsthereof mean to decrease the severity of the symptoms of a disease in a subject.

[0056] As used herein, the term “substantially free of divalent metal cation co-factors” or“substantially free of stabilizer compounds” means that the pertinent solution / buffer may have a concentration of a pertinent divalent cation and / or of a cryoprotective agent / cell stabilizer, respectively, at a molar level that does not cause cell death or cytotoxicity.

[0057] In the present disclosure, an "effective amount" of a therapeutic is an amount ofsuch therapeutic that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine. In general, a suitable dose of a therapeutic according to the disclosure will be that amount of the agent, which is the lowest dose effective to produce the desired effect with no or minimal side effects. The effective dose of a therapeutic according to the present disclosure may be administered as a single dose, or two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the treatment course (e.g., not necessarily on the same day).

[0058] Administration of compositions, pharmaceutical compositions, including cellpopulations disclosed herein for therapeutic indications can be achieved in a variety of ways, in each case as clinically warranted, using a variety of anatomic access devices, a variety of administration devices, and a variety of anatomic approaches, with or without support of anatomic imaging modalities (e.g., radiologic, MRI, ultrasound, etc.) or mapping technologies (e.g., epiphysiologic mapping procedures, electromyographic procedures, electrodiagnostic procedures, etc.). The compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered systemically, via either peripheral vascular access (e.g., intravenous placement, peripheral venous access devices, etc.) or central vascular access (e.g., central venous catheter / devices, arterial access devices / approaches, etc.). The compositions, pharmaceuticalcompositions and cell populations of the present disclosure can be delivered intravascularly into anatomic feeder vessels of an intended tissue site using catheter-based approaches or other vascular access devices (e.g., arterial catheterization, etc.) that will deliver a vascular bolus of cells to the intended site. The compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered directly into body cavities or anatomic compartments by either catheter-based approaches or direct injection into a pertinent anatomic site (e.g., intrabone / intramedullary (i.e., within the marrow itself) or within scaffolds / implants. The compositions, pharmaceutical compositions and cell populations of the present disclosure can be introduced by direct local tissue injection, using either intravascular approaches, or percutaneous approaches, or via surgical exposure / approaches to the tissue, or via arthroscopic approaches, or directly into the accessible tissue sites and / or guided by imaging techniques. The compositions, pharmaceutical compositions and cell populations of the present disclosure can also administered into tissue or structural support devices (e.g., tissue scaffold devices and / or embedded within scaffolds placed into tissues, etc.), and / or administered in gels, and / or administered together with enhancing agents (e.g., admixed with supportive cells, cytokines, growth factors, resolvins, anti- inflammatory agents, etc.).

[0059] According to some embodiments, the compositions, pharmaceutical compositionsand cell populations of the present disclosure are administered to the subject with an enforced expression of glycosylation. According to some embodiments, the enforced glycosylation on the surface of administered cells will aid in tissue repair / regeneration. Moreover, whether cells are administered systemically, intravascularly, by direct local tissue injection, or by placement onto relevant tissue surfaces / sites or implants / scaffolds, the enforced expression of sLeX on administered cells promotes lodgment of cells within the affected tissue milieu, in apposition tocells bearing E-selectin (i.e., endothelial cells) and / or L-selectin (i.e., leukocytes), respectively, within the target site. Thus, the spatial distribution and localization of administered cells within the target tissue is modulated by the enforced glycosylation of the surface of the administered cells. Exofucosylation

[0060] According to some embodiments, cells of the present disclosure are contacted witha glycosyltransferase to enforce a glycan on the cell surface. In some embodiments, the glycosylstransferase is a human glycosyltransferase. In some embodiments, the glycosyltransferase is a non-human glycosyltransferase. According to some embodiments, fucosylated lactosaminyl glycans are enforced by a member of the α(1,3)-fucosyltransferase family. The human α(1,3)-fucosyltransferase family includes Fucosyltransferase III (also called FTIII, FT3, FUTIII, or FUT3), Fucosyltransferase IV (also called FTIV, FT4, FUTIV, or FUT4), Fucosyltransferase V (also called FTV, FT5, FUTV, or FUT5), Fucosyltransferase VI (also called FTVI, FT6, FUTVI, or FUT6), Fucosyltransferase VII (also called FTVII, FT7, FUTVII, or FUT7), Fucosyltransferase IX (also called FTIX, FT9, FUTIX, or FUT9), and variants thereof. The cDNA / protein sequences for the α(1,3)-fucosyltransferase family are as follows Name GenBank Acc. No. F lt f III

[0061] As used herein, the notation for a fucosyltransferase should not be construed aslimiting to the nucleotide sequence or the amino acid sequence. For example, the notation of Fucosyltransferase VII, FTVII, FT7, FUTVII or FUT7 are used interchangeably as meaning the nucleotide, amino acid sequence, or both, of Fucosyltransferase VII. According to some embodiments, cells are contacted by one or more of the α(1,3)-fucosyltransferase family members to enforce fucosylated lactosaminyl glycans.

[0062] In some embodiments, fragments of α(1,3)-fucosyltransferase family members arecontacted with a cell. For example, a peptide / nucleotide having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an α(1,3)-fucosyltransferase family member is contacted with a cell. As used herein, the term “identity” and grammatical versions thereof means the extent to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.

[0063] In some embodiments, the cells may be contacted with the desiredfucosyltransferase via exofucosyltation using, for example, the methods disclosed herein. U.S. Pat. Nos.7,875,585 and 8,084,236, (which disclosures are expressly incorporated by reference as if recited in full herein) provide non-limiting examples of compositions and methods for ex vivo modification of cell surface glycans on a viable cell, which may be used to enforce expression of fucosylated lactosaminyl glycans on a cell according to the present disclosure. In some embodiments, the cells may be contacted with a purified glycosyltransferase polypeptide and a physiologically acceptable solution, for use together with appropriate donor nucleotide sugars in reaction buffers and reaction conditions specifically formulated to retain cell viability. In someembodiments, the physiologically acceptable solution may be free or substantially free of divalent metal co-factors, to such extent that cell viability is not compromised. In these and other embodiments, the cells may be contacted with a solution that is also free or substantially free of stabilizer compounds such as for example, glycerol, again, to such extent that cell viability is not compromised. Glycosyltransferases of the present disclosure include for example, one or more fucosyltransferase. In certain embodiments, the fucosyltransferase is an α(1,3)-fucosyltransferase such as an α(1,3)-fucosyltransferase III, α(1,3)-fucosyltransferase IV, an α(1,3)-fucosyltransferase V, an α(1,3)-fucosyltransferase VI, an α(1,3)-fucosyltransferase VII, or an α(1,3)- fucosyltransferase IX. However, it should be understood that fucosyltransferases other than these, for example the ^(1,3)-fucosyltransferase from H. Pylori, could possess requisite capacity to install fucose in ^(1,3)-linkage to GlcNAc and thus convert a Type 2-α(2,3)-sialylated type 2 lactosamine into sLeX; as such, it should not be viewed that the exofucosylation reaction is limited to use of those ^(1,3)-fucosyltransferase described herein.

[0064] In some embodiments, glycans are glycocalyx-modified on the surface of a cell bycontacting a population of cells with one or more glycosyltransferase compositions described above. In some embodiments, the cells are contacted with the glycosyltransferase composition together with an appropriate nucleotide sugar donor (e.g., GDP-fucose) under conditions in which the glycosyltransferase has enzymatic activity. For example, cells may be incubated for 60 min at 37ºC in fucosyltransferase reaction buffer composed of Hank’s Balanced Salt Solution (HBSS) (without Ca2+and Mg2+) (Lonza) containing 20 mM HEPES (Lonza), 0.1% human serum albumin (HSA) (Grifols, Barcelona, Spain), 30 µg / ml fucosyltransferase, and 1 mM GDP-fucose. Glycan modification according to this method results in cells according to the present disclosure that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more viability at 24 hours ormore after treatment. In one embodiment, for example, the cells of the present disclosure have at least 70% viability at 48 hours after treatment. In one such embodiment, for example, the cells of the present disclosure have at least 75% viability at 48 hours after treatment. In one embodiment, for example, the cells of the present disclosure have at least 80% viability at 48 hours after treatment. In addition, the phenotype of the cells of the present disclosure (other than the glycan modification) is preferably preserved after treatment. By preserved phenotype, it is meant the cell of the present disclosure maintains its native function and / or activity. For example, if the cell of the present disclosure is an HSPC, its relevant multipotency or oligopotency or unipotency is maintained, as would be characteristic of that particular stem cell type.

[0065] According to some embodiments, glycosyltransferases are contacted with cells ofthe present disclosure in reaction buffer that is free of or substantially free of) divalent metal co- factors (e.g. divalent cations such as manganese, magnesium, calcium, zinc, cobalt or nickel) and stabilizers such as glycerol. In some embodiments, a purified glycosyltransferase polypeptide and a physiologically acceptable solution free of or substantially free of divalent metal co-factors is used to enforce a desired glycosylation pattern. Such a composition is free of or substantially free of cryoprotective agents / stabilizer compounds (such as for example, glycerol), in any case such that the composition contains such agents / stabilizers at levels that do not affect cell viability. The glycosyltransferases used with solutions that are free or substantially free of divalent metal cofactors, and free of or substantially free of cryoprotective agents / stabilizer compounds, include for example, α(1,3)-fucosyltransferases such as an α(1,3)-fucosyltransferase III, α(1,3)- fucosyltransferase IV, an α(1,3)- fucosyltransferase VI, an α(1,3)- fucosyltransferase VII, or an α(1,3)-fucosyltransferase IX. According to some embodiments, the glycosyltransferase is biologically active. As used herein “biologically active” means that the glycosyltransferase iscapable of transferring a sugar molecule from a donor to acceptor. For example, a glycosyltransferase according to the present disclosure is capable of transferring 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 5, 10 or more µmoles of sugar per minute at pH 6.5 at 37° C. In some embodiments, the contacting of a glycosyltransferase with a cell occurs in a physiologically acceptable solution, which is any solution that does not cause cell damage, e.g. death. For example, the viability of the cell is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more after treatment with the compositions of the invention. Suitable physiologically acceptable solutions include, for example, Hank's Balanced Salt Solution (HBSS), Dulbecco's Modified Eagle Medium (DMEM), a Good's buffer such as a HEPES buffer, a 2-Morpholinoethanesulfonic acid (MES) buffer, or phosphate buffered saline (PBS).

[0066] According to some embodiments the human or mammalian cells of the presentdisclosure may be contacted with a desired fucosyltransferase by transfecting a DNA or RNA nucleotide sequence encoding the desired fucosyltransferase into the cell. According to some embodiments, modified RNA (modRNA) encoding the relevant α(1,3)-FT transcripts is used to enforce the desired pattern of fucosylated lactosaminyl glycans. In some embodiments, the transfected nucleotide sequence encodes a full length or partial peptide sequence of the desired fucosyltransferase. In some embodiments, the nucleotide sequence encodes a naturally existing isoform of a fucosyltransferase. (See, e.g., Mondal N. et al. Distinct human α(1,3) – fucosyltransferases drive Lewis-X / sialyl Lewis-X assembly in human cells. J Biol Chem. 2018; 293(19):7300-7314.)

[0067] According to some embodiments, the cells may be contacted with the desiredfucosyltransferase by transfecting the cells with a recombinant DNA or RNA molecule. (Levy 2013, Warren 2010) As used herein, the term “recombinant DNA or RNA” means a DNA orRNA molecule formed through recombination methods to splice fragments of DNA or RNA from a different source or from different parts of the same source. In some embodiments the recombinant DNA may comprise a plasmid vector, which controls expression of the DNA in the cell. Proteins, such as the enzymes disclosed herein, which are encoded by recombinant DNA or RNA are recombinant proteins. In some embodiments, the nucleic acid may be in the form of modified RNA (“modRNA”).

[0068] In some embodiments, the MSC or the osteoblast may have undergone in vitro pre-treatment with agents which stimulate osteogenic activity (agents having “osteoinductive effects”) prior to intravascular administration of the cells. Such agents include, but are not limited to, growth factors (e.g., bone morphogenetic proteins (BMPs, in particular BMP-2), TGF-^^^among others), and chemicals (e.g., fucoidan, dexamethasone, ascorbic acid, ^-glycerophosphate, among others). In some embodiments, the patient will receive agents that stimulate new bone formation (osteoanabolic agents) prior to and / or coincident with and / or following the intravascular administration of glycocalyx-modified MSCs or glycocalyx-modified osteoblasts; such osteoanabolic agents include, but are not limited to, hormones (e.g., parathyroid hormone, analogs of parathyroid hormone (e.g., teriparatide, abaloparatide), among others) and romosozumab. In some embodiments, the patient will receive agents that inhibit bone resorption (antiresorptive agents) prior to and / or coincident with and / or following the intravascular administration of glycocalyx-modified MSCs or glycocalyx-modified osteoblasts; such agents include, but are not limited to, bisphosphonates, denosumab, and estrogens / estrogen analogs. In some embodiments, the culture-expanded MSCs or culture-expanded osteoblasts may have undergone transfection or transduction to introduce nucleic acid sequences encoding the production of proteins that promoteosteogenesis (e.g., transcription factors such as osterix, and RUNX2, BMPs and other growth factors, among others). Administration of cells

[0069] Administration of compositions, pharmaceutical compositions, including cellpopulations disclosed herein for therapeutic indications can be achieved in a variety of ways, in each case as clinically warranted, using a variety of anatomic access devices, a variety of administration devices, and a variety of anatomic approaches, with or without support of anatomic imaging modalities (e.g., radiologic, MRI, ultrasound, etc.) or mapping technologies (e.g., epiphysiologic mapping procedures, electromyographic procedures, electrodiagnostic procedures, etc.). The compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered systemically, via syringe-based injection, or either peripheral vascular access (e.g., intravenous placement, peripheral venous access devices, etc.) or central vascular access (e.g., central venous catheter / devices, arterial access devices / approaches, etc.). The compositions, pharmaceutical compositions and cell populations of the present disclosure can be delivered intravascularly into anatomic feeder vessels of an intended tissue site using catheter- based approaches or other vascular access devices (e.g., cardiac catheterization, etc.) that will deliver a vascular bolus of cells to the intended site. The compositions, pharmaceutical compositions and cell populations of the present disclosure can be introduced into the spinal canal and / or intraventricularly intrathecally, into the subarachnoid space to distribute within cerebrospinal fluid and / or within the ventricles). The compositions, pharmaceutical compositions and cell populations of the present disclosure can be administered directly into body cavities or anatomic compartments by either catheter-based approaches or direct injection (e.g., intraperitoneal, intrapleural, intrapericardial, intravesicularly (e.g., into bladder, into gall bladder,into bone marrow, into biliary system (including biliary duct and pancreatic duct network), intraurethrally, via renal pelvis / intraureteral approaches, intravaginally, etc.)). The compositions, pharmaceutical compositions and cell populations of the present disclosure can be introduced by direct local tissue injection, using either intravascular approaches (e.g., endomyocardial injection), or percutaneous approaches, or via surgical exposure / approaches to the tissue, or via laparoscopic / thoracoscopic / endoscopic / colonoscopic approaches, or directly into anatomically accessible tissue sites and / or guided by imaging techniques (e.g., intra-articular, intra-ocular, into spinal discs and other cartilage, into bones, into muscles, into skin, into connective tissues, and into relevant tissues / organs such as central nervous system, peripheral nervous system, heart, liver, kidneys, spleen, joints, eye, etc.). The compositions, pharmaceutical compositions and cell populations of the present disclosure can also be placed directly onto relevant tissue surfaces / sites (e.g., placement onto tissue directly, onto ulcers, onto burn surfaces, onto serosal or mucosal surfaces, onto epicardium, etc.). The compositions, pharmaceutical compositions and cell populations of the present disclosure can also administered into tissue or structural support devices (e.g., tissue scaffold devices and / or embedded within scaffolds placed into tissues, etc.), and / or administered in gels, and / or administered together with enhancing agents (e.g., admixed with supportive cells, cytokines, growth factors, resolvins, anti-inflammatory agents, etc.).

[0070] According to some embodiments, the compositions, pharmaceutical compositionsand cell populations of the present disclosure are administered to the subject following enforced glycosylation of the cell surface (i.e., following glycocalyx modification). According to some embodiments, the enforced glycosylation on the surface of administered cells will aid in revascularization, in host defense (e.g., against infection or cancer) and / or in tissue repair / regeneration and / or mediate immunomodulatory processes that will dampen inflammationand / or prevent inflammation. According to some embodiments, the enforced glycosylation pattern guides delivery of intravascularly administered cells to the marrow, to the skin, or to sites of inflammation / tissue injury by mediating binding of blood-borne cells to vascular E-selectin expressed on endothelial cells at sites of inflammation. Moreover, whether cells are administered systemically, intravascularly, into the spinal canal and / or intraventricularly intrathecally, into the subarachnoid space to distribute within cerebrospinal fluid), directly into body cavities or compartments, by direct local tissue injection, or by placement onto relevant tissue surfaces / sites, the enforced expression of ligands for E-selectin and / or L-selectin on administered cells promotes lodgment of cells within the affected tissue milieu, in apposition to cells bearing E-selectin (i.e., endothelial cells) and / or L-selectin (i.e., leukocytes), respectively, within the target site. Thus, the spatial distribution and localization of administered cells within the target tissue is modulated by the enforced glycosylation on administered cells.

[0071] Particularly, the colonization of a desired cell type at a site of inflammation occursas a result of the enforced glycosylation on the administered cells, such that the administered cells have augmented binding to E-selectin, thereby promoting the systemic delivery of the desired cells and / or the lodgement of cells when injected directly into the affected site. For example, the enforced glycosylation of E-selectin ligands (e.g., HCELL) is advantageously capable of anchoring directly injected cells within E-selectin-expressing vessels within the marrow, within the skin, and at sites of inflammation, tissue injury, or cancer. Thus, the present methods augment efficiency in the delivery of relevant cells at or to a site of inflammation, tissue injury, or cancer, including, for example, the capacity to deliver immunomodulatory cells (e.g., mesenchymal stem cells).

[0072] In some embodiments the disease, disorder, or medical condition having associatedinflammation can be treated using the instant methods even in the absence of differentiation of the cell population in the subject. That is, there are trophic effects of administered cells at the site of inflammation without persistent engraftment and / or repopulation of the administered cells, irrespective of the type of tissue involved. These trophic effects include release of cytokines / growth factors that promote revascularization (e.g., VEGF), that promote tissue repair (e.g., TGF-β), that are immunomodulatory (e.g., IL-10), that stimulate growth / proliferation of tissue-resident progenitors (e.g., SCF, LIF, etc) and many other tissue-reparative processes (e.g., mitochondria delivery to cells). In addition, administered cells (e.g., MSCs) may have potent immunomodulatory properties, including direct suppression of activated lymphocytes (e.g., via expression of PDL-1).

[0073] According to some aspects, the present disclosure provides a pharmaceuticalcomposition comprising conditioned media obtained from a population of CD44+cells that have been modified ex vivo via treatment with a CD44 ligand for a period of time sufficient to prime the cells to produce elevated levels of one or more anti-inflammatory or immunomodulatory molecules relative to a native populations of CD44+cells. According to some aspects, the present disclosure provides a pharmaceutical composition comprising conditioned media obtained from a population of CD44+cells that have been (1) glycocalyx-modified ex vivo via exofucosylation to enforce hematopoietic cell E-Selectin / L-Selectin Ligand (HCELL) expression and treated with E- selectin or L-selectin for a period of time sufficient to prime the cells to produce interleukin-10 (IL-10) and at least one additional anti-inflammatory cytokine; or (2) modified ex vivo via treatment with hyaluronic acid (HA) for a period of time sufficient to prime the cells to produce interleukin-10 (IL-10) and at least one additional anti-inflammatory cytokine, wherein themodified cells of (1) or (2) produce elevated levels of IL-10 and at least one additional anti- inflammatory cytokine relative to a native population of CD44+cells.

[0074] In some embodiments, the conditioned media may be administered to a subject byany of the methods and routes of administration disclosed herein. In some embodiments, the conditioned media may be administered to a subject in aerosolized form. Indications

[0075] The present disclosure is directed to the treatment of a disease, disorder, or medicalcondition wherein E-selectin is expressed in endothelial beds of the affected tissue(s) and / or L- selectin-expressing leukocytes have infiltrated / accumulated in the affected tissue(s). As discussed above, E-selectin and L-selectin each bind to sialylated, fucosylated carbohydrates, and enforced expression of these sialofucosylated glycan structures on the cell surface serves to program binding to these selectins. Accordingly, the disclosure describes methods to enhance homing to target tissue(s) by augmenting the expression of E-selectin ligands on administered cells; additionally, in describing methods to enhance expression of potent E-selectin and L-selectin ligands (such as HCELL) on administered cells to promote adherence to E-selectin on vascular endothelial cells and / or adherence to L-selectin expressed on tissue-infiltrating leukocytes within affected tissue(s), the disclosure provides a means to augment colonization / lodgement of the cells within relevant tissue microenvironments where biologic effects are intended. Pertinent salutary clinical effects may be engendered by the biologic activity of the infiltrating cell itself (e.g., immunologic effects mediated by a leukocyte, a CAR-T / NK cell, a regulatory T cell or B cell, etc.), and / or by the release of tissue-reparative / anti-inflammatory (trophic) factors, and / or by release of (desired) cytotoxic agents by the infiltrating cell, and / or or by differentiation of the infiltrating cell into a given cell type within the tissue milieu. In general, the methods described herein have utility in improvingthe outcome of any cell-based therapeutic approach, be it in host defense (e.g., infusion of any type of leukocyte), immunotherapy applications (e.g. , administration of culture-expanded antigen- specific T cells and / or culture expanded NK cells for cancer or infectious disease applications, administration of culture-expanded chimeric antigen receptor (CAR) T or NK cells, administration of antigen-pulsed dendritic cells, etc.), immunomodulatory / immunosuppressive therapeutic applications (e.g. , administration of culture-expanded regulatory T cells (Tregs) or B cells (Bregs), administration of antigen-pulsed immunomodulatory dendritic cells, administration of mesenchymal stem cells, administration of culture-expanded NKT cells, etc.), or tissue repair / regenerative medicine applications (e.g. , use of stem and / or progenitor cells or other tissue- reparative cells for tissue regeneration / restoration; use of culture-expanded stem cells and / or culture-expanded progenitor cells for tissue regeneration / restoration). Within utility in regenerative medicine applications, it is understood that administered cells may themselves contribute to regenerate the target tissue by way of long-term engraftment (with attendant proliferation / differentiation) yielding tissue-specific cells (e.g. , such as in transplantation of hematopoietic stem cells for blood cell production) and / or may deliver a tissue restorative / reparative effect without long-term engraftment or differentiation into tissue-resident cells (e.g. , via delivery of trophic effects that stimulate resident stem / progenitors to repair the injured tissue(s) and / or by dampening inflammatory processes that promote injury and impede repair). All applications for all indications described herein can be used alone or in combination with enhancing agents (e.g. , growth factors, tissue scaffolds, etc.).

[0076] Any and all diseases, disorders, or medical conditions having associatedinflammation (e.g., acute and / or chronic), tissue injury / damage or neoplastic conditions may be treated in accordance with the methods described herein, including, but not limited to thoseinitiated by direct tissue injury (e.g., burns, trauma, bone fracture, bone deformities, decubitus ulcers, etc.), ischemic / vascular events (e.g., myocardial infarct, stroke, shock, hemorrhage, coagulopathy, etc.), infections (e.g. , cellulitis, pneumonia, meningitis, cystitis, sepsis, SIRS, etc.), neoplasia (e.g. , breast cancer, lung cancer, prostate cancer, renal cell cancer, lymphoma, leukemia, etc.), immunologic / autoimmune conditions (e.g. , acute or chronic GVHD, multiple sclerosis, diabetes, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), rheumatoid arthritis, psoriasis, etc.), degenerative diseases (e.g., osteoporosis, osteoarthritis, spinal disc degeneration, Alzheimer's disease, atherosclerosis, etc.), congenital / genetic diseases (e.g. , epidermolysis bullosa, osteogenesis imperfecta, muscular dystrophies, lysosomal storage diseases, Huntington's disease, etc.), adverse drug effects (e.g., chemotherapy-induced tissue / organ toxicity, radiotherapy toxicity, drug-induced hepatitis, drug-induced cardiac injury, etc.), toxic injuries (e.g. , radiation exposure(s), chemical exposure(s), alcoholic hepatitis, alcoholic pancreatitis, alcoholic cardiomyopathy, cocaine cardiomyopathy, etc.), metabolic derangements (e.g. , uremic pericarditis, metabolic acidosis, etc.), iatrogenic conditions (e.g. , radiation-induced tissue injury, surgery-related complications, etc.), and / or idiopathic processes (e.g. , amyotrophic lateral sclerosis, Parsonnage-Turner Syndrome, etc.).

[0077] Other general and specific diseases, disorders, or medical condition that may betreated in accordance with the methods described herein include, but are not limited to:

[0078] Acute Leukemias, e.g., Acute Biphenotypic Leukemia, Acute LymphocyticLeukemia (ALL), Acute Myelogenous Leukemia (AML), and Acute Undifferentiated Leukemia;

[0079] Myelodysplastic Syndromes, Amyloidosis (of any type), Chronic MyelomonocyticLeukemia (CMML), Refractory Anemia (RA), Refractory Anemia with Excess Blasts (RAEB),Refractory Anemia with Excess Blasts in Transformation (RAEB-T), and Refractory Anemia with Ringed Sideroblasts (RARS);

[0080] Myeloproliferative Disorders, e.g., Acute Myelofibrosis, Agnogenic MyeloidMetaplasia (Myelofibrosis), Essential Thrombocythemia, chronic myelogenous leukemia, and Polycythemia Vera;

[0081] Phagocyte Disorders, e.g., Chediak-Higashi Syndrome, Chronic GranulomatousDisease, Leukocyte adhesion deficiencies, myeloperoxidase deficiency, Neutrophil Actin Deficiency, and Reticular Dysgenesis;

[0082] Lysosomal Storage Diseases, e.g. , Adrenoleukodystrophy, Alpha Mannosidosis,Gaucher's Disease, Hunter's Syndrome (MPS-II), Hurler's Syndrome (MPS-IH), Krabbe Disease, Maroteaux-Lamy Syndrome (MPS-VI), Metachromatic Leukodystrophy, Morquio Syndrome (MPS-IV), Mucolipidosis II (l-cell Disease), Mucopolysaccharidoses (MPS), Niemann-Pick Disease, Sanfilippo Syndrome (MPS-III), Scheie Syndrome (MPS-IS), Sly Syndrome, Beta- Glucuronidase Deficiency (MPS-VII), and Wolman Disease;

[0083] Inherited Erythrocyte Abnormalities, e.g.,_Beta Thalassemia, Blackfan-DiamondAnemia, Pure Red Cell Aplasia, and Sickle Cell Disease;

[0084] Inherited Platelet Abnormalities, e.g., Amegakaryocytosis / CongenitalThrombocytopenia, Gray platelet syndrome;

[0085] Solid organ malignancies, e.g., Brain Tumors, Ewing Sarcoma, Neuroblastoma,Ovarian Cancer, Renal Cell Carcinoma, Lung Cancers, Breast cancers, Gastric cancers, Esophageal cancers, Skin cancers, Oral cancers, Endocrine cancers, Liver cancers, Biliary system cancers, Pancreatic cancer, Prostate Cancer, and Testicular Cancer;

[0086] Other Applications, e.g.,_Bone Marrow Transplants, Heart Disease (myocardialinfarction), Liver Disease, Muscular Dystrophy, Alzheimer's Disease, Parkinson's Disease, Spinal Cord Injury, Spinal disc disease / degeneration, Bone disease, Bone fracture, Stroke, Peripheral Vascular Disease, Head trauma, Bullous diseases, Mitochondrial diseases, Ex vivo and In vivo expanded stem and progenitor cell populations, In vitro fertilization application and enhancement, Hematopoietic Rescue Situations (following Chemotherapy / Radiation therapy), Stem cells and progenitor cells derived from various tissues sources, Application in humans and animals, and Limb regeneration, reconstructive surgical procedures / indications, alone or in combination with enhancing agents;

[0087] Chronic Leukemias, e.g., Chronic Lymphocytic Leukemia (CLL), ChronicMyelogenous Leukemia (CML), Juvenile Chronic Myelogenous Leukemia (JCML), and Juvenile Myelomonocytic Leukemia (JMML);

[0088] Stem Cell Disorders, e.g., Aplastic Anemia (Severe), Congenital Cytopenia,Dyskeratosis Congenita, Fanconi Anemia, and Paroxysmal Nocturnal Hemoglobinuria (PNH);

[0089] Lymphoproliferative Disorders, e.g., Hodgkin's Disease, Non-Hodgkin'sLymphomas, and Prolymphocytic Leukemia;

[0090] Histiocytic Disorders, e.g., Familial Erythrophagocytic Lymphohistiocytosis,Hemophagocytosis, Hemophagocytic Lymphohistiocytosis, Histiocytosis-X, and Langerhans' Cell Histiocytosis;

[0091] Congenital (Inherited) Immune System Disorders, e.g., Absence of T and B Cells,Absence of T Cells, Normal B Cell SCID, Ataxia-Telangiectasia, Bare Lymphocyte Syndrome, Common Variable Immunodeficiency, DiGeorge Syndrome, Kostmann Syndrome, Leukocyte Adhesion Deficiency, Omenn's Syndrome, Severe Combined Immunodeficiency (SCID), SCIDwith Adenosine Deaminase Deficiency, Wiskott-Aldrich Syndrome, and X-Linked Lymphoproliferative Disorder;

[0092] Other Inherited Disorders, e.g., Cartilage-Hair Hypoplasia, Ceroid Lipofuscinosis,Congenital Erythropoietic Porphyria, Familial Mediterranean Fever, Glanzmann Thrombasthenia, Lesch-Nyhan Syndrome, Osteopetrosis, and Sandhoff Disease;

[0093] Plasma Cell Disorders, e.g., Multiple Myeloma, Plasma Cell Leukemia, andWaldenstrom's Macroglobulinemia; and

[0094] Autoimmune Diseases, including but not limited to, Myasthenia Gravis,amyotrophic lateral sclerosis, Multiple Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus, Scleroderma, Ankylosing spondylitis, Diabetes Mellitus, and Inflammatory Bowel Diseases.

[0095] Articular and skeletal diseases / conditions, e.g., disc degeneration, synovial disease,cartilage degeneration, cartilage trauma, cartilage tears, arthritis, bone fractures, bone deformities, bone reconstruction, osteogenesis imperfecta, congenital bone diseases / conditions, genetic bone diseases / conditions, osteoporosis, osteopetrosis, hypophosphatasia, metabolic bone disease, etc.

[0096] Skin / soft tissue diseases and conditions such as bullous diseases, psoriasis, eczema,epidermolysis bullosa, ulcerative skin conditions, soft tissue deformities (including post-surgical skin and soft tissue deformities), plastic surgery / reconstructive surgery indications, etc.

[0097] In general, associated inflammation symptoms that include, without limitation,fever, pain, edema, hyperemia, erythema, bruising, tenderness, stiffness, swollenness, chills, respiratory distress, hypotension, hypertension, stuffy nose, stuffy head, breathing problems, fluid retention, blood clots, loss of appetite, weight loss, polyuria, nocturia, anuria, dyspnea, dyspnea on exertion, muscle weakness, sensory changes, increased heart rate, decreased heart rate,arrythmias, polydipsia, formation of granulomas, fibrinous, pus, non-viscous serous fluid, or ulcers. The actual symptoms associated with an acute and / or chronic inflammation are well known and can be determined by a person of ordinary skill in the art by taking into account factors, including, without limitation, the location of the inflammation, the cause of the inflammation, the severity of the inflammation, the tissue or organ affected, and the associated disorder.

[0098] Specific patterns of acute and / or chronic inflammation are seen during particularsituations that arise in the body, such as when inflammation occurs on an epithelial surface, or pyogenic bacteria are involved. For example, granulomatous inflammation is an inflammation resulting from the formation of granulomas arising from a limited but diverse number of diseases, include, without limitation, tuberculosis, leprosy, sarcoidosis, and syphilis. Purulent inflammation is an inflammation resulting in large amount of pus, which consists of neutrophils, dead cells, and fluid. Infection by pyogenic bacteria such as staphylococci is characteristic of this kind of inflammation. Serous inflammation is an inflammation resulting from copious effusion of non- viscous serous fluid, commonly produced by mesothelial cells of serous membranes, but may be derived from blood plasma. Skin blisters exemplify this pattern of inflammation. Ulcerative inflammation is an inflammation resulting from the necrotic loss of tissue from the epithelial surface, exposing lower layers and forming an ulcer.

[0099] An acute and / or chronic inflammation symptom can be associated with a large,unrelated group of disorders which underlay a variety of diseases and disorders. The immune system is often involved with acute and / or chronic inflammatory disorders, demonstrated in both allergic reactions, arthritic conditions, and some myopathies, with many immune system disorders resulting in abnormal inflammation. Non-immune diseases with etiological origins in acute and / or chronic inflammatory processes include amyloidosis, cancer, atherosclerosis, and ischaemic heartdisease. Non-limiting examples of disorders exhibiting acute and / or chronic inflammation as a symptom include, without limitation, acne, acid reflux / heartburn, age related macular degeneration (AMD), allergy, allergic rhinitis, Alzheimer's disease, amyotrophic lateral sclerosis, anemia, appendicitis, arteritis, arthritis, asthma, atherosclerosis, autoimmune disorders, balanitis, blepharitis, bronchiolitis, bronchitis, a bullous pemphigoid, burn, bursitis, cancer, cardiac arrest, carditis, celiac disease, cellulitis, cervicitis, cholangitis, cholecystitis, chorioamnionitis, chronic obstructive pulmonary disease (COPD) (and / or acute exacerbations thereof), cirrhosis, colitis, congestive heart failure, conjunctivitis, drug-induced tissue injury (e.g., cyclophosphamide- induced cystitis), cystic fibrosis, cystitis, common cold, dacryoadenitis, decubitus ulcers, dementia, dermatitis, dermatomyositis, diabetes, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic ulcer, digestive system disease, eczema, emphysema, encephalitis, endocarditis, endocrinopathies, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibromyalgia, fibrosis, fibrositis, gastritis, gastroenteritis, gingivitis, glomerulonephritis, glossitis, heart disease, heart valve dysfunction, hepatitis, hidradenitis suppurativa, Huntington's disease, hyperlipidemic pancreatitis, hypertension, ileitis, infection, inflammatory bowel disease, inflammatory cardiomegaly, inflammatory neuropathy, insulin resistance, interstitial cystitis, interstitial nephritis, iritis, ischemia, ischemic heart disease, keratitis, keratoconjunctivitis, laryngitis, lupus nephritis, macular degeneration, mastitis, mastoiditis, meningitis, metabolic syndrome (syndrome X), a migraine, mucositis, multiple sclerosis, myelitis, myocarditis, myositis, nephritis, neuronitis, non-alcoholic steatohepatitis, obesity, omphalitis, oophoritis, orchitis, osteochondritis, osteopenia, osteomyelitis, osteoporosis, osteitis, otitis, pancreatitis, Parkinson's disease, parotitis, pelvic inflammatory disease, pemphigus vularis, pericarditis, peritonitis, pharyngitis, phlebitis, pleuritis, pneumonitis, polycystic nephritis, proctitis, prostatitis, psoriasis,pulpitis, pyelonephritis, pylephlebitis, radiation-induced injury, renal failure, reperfusion injury, retinitis, rheumatic fever, rhinitis, salpingitis, sarcoidosis, sialadenitis, sinusitis, spastic colon, stasis dermatitis, stenosis, stomatitis, stroke, surgical complication, synovitis, tendonitis, tendinosis, tenosynovitis, thrombophlebitis, thyroiditis, tonsillitis, trauma, traumatic brain injury, transplant rejection, trigonitis, tuberculosis, tumor, ulcers, urethritis, ursitis, uveitis, vaginitis, vasculitis, and vulvitis.

[0100] General categories of diseases, disorders, and trauma that can result in or otherwisecause acute and / or chronic inflammation include, but are not limited to genetic diseases, neoplasias, direct tissue injury, autoimmune diseases, infectious diseases, vascular diseases / complications (e.g., ischemia / reperfusion injury), iatrogenic causes (e.g. drug adverse effects, radiation injury, etc.), and allergic manifestations.

[0101] In one embodiment, an acute and / or chronic inflammation comprises a tissueinflammation. In general, tissue inflammation is an acute and / or chronic inflammation that is confined to a particular tissue or organ. Thus, for example, a tissue inflammation may comprise a skin inflammation, a muscle inflammation, a tendon inflammation, a ligament inflammation, a bone inflammation, a cartilage / joint inflammation, a lung inflammation, a heart inflammation, a liver inflammation, a gall bladder inflammation, a pancreatic inflammation, a kidney inflammation, a bladder inflammation, an gum inflammation, an esophageal inflammation, a stomach inflammation, an intestinal inflammation, an anal inflammation, a rectal inflammation, a vessel inflammation, a vaginal inflammation, a uterine inflammation, a testicular inflammation, a penile inflammation, a vulvar inflammation, a neuron inflammation, an oral inflammation, an ocular inflammation, an aural inflammation, a brain inflammation, a ventricular / meningial inflammation and / or inflammation involving central or peripheral nervous system cells / elements.

[0102] In another embodiment, an acute and / or chronic inflammation comprises a systemicinflammation. Although the processes involved are similar if not identical to tissue inflammation, systemic inflammation is not confined to a particular tissue but rather involves multiple sites within the body, involving the epithelium, endothelium, nervous tissues, serosal surfaces and organ systems. When it is due to infection, the term sepsis can be used, with bacteremia being applied specifically for bacterial sepsis and viremia specifically to viral sepsis. Vasodilation and organ dysfunction are serious problems associated with widespread infection that may lead to septic shock and death.

[0103] In another embodiment, an acute and / or chronic inflammation is induced by anarthritis. Arthritis includes a group of conditions involving damage to the joints of the body due to the inflammation of the synovium including, for example, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies like ankylosing spondylitis, reactive arthritis (Reiter's syndrome), psoriatic arthritis, enteropathic arthritis associated with inflammatory bowel disease, Whipple disease and Behcet disease, septic arthritis, gout (also commonly referred to as gouty arthritis, crystal synovitis, metabolic arthritis), pseudogout (calcium pyrophosphate deposition disease), and Still's disease. Arthritis can affect a single joint (monoarthritis), two to four joints (oligoarthritis) or five or more joints (polyarthritis) and can be either an autoimmune disease or a non-autoimmune disease.

[0104] In another embodiment, an acute and / or chronic inflammation is induced by anautoimmune disorder. Autoimmune diseases can be broadly divided into systemic and organ- specific autoimmune disorders, depending on the principal clinico-pathologic features of each disease. Systemic autoimmune diseases include, for example, systemic lupus erythematosus (SLE), Sjogren's syndrome, Scleroderma, rheumatoid arthritis and polymyositis. Localautoimmune diseases may be endocrinologic (Diabetes Mellitus Type 1 , Hashimoto's thyroiditis, Addison's disease, etc.), dermatologic (pemphigus vulgaris), hematologic (autoimmune haemolytic anemia), neural (multiple sclerosis) or can involve virtually any circumscribed mass of body tissue. Types of autoimmune disorders include, without limitation, acute disseminated encephalomyelitis (ADEM), Addison's disease, an allergy or sensitivity, amyotrophic lateral sclerosis (ALS), anti-phospholipid antibody syndrome (APS), arthritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune pancreatitis, bullous pemphigoid, celiac disease, Chagas disease, chronic obstructive pulmonary disease (COPD) (including acute exacerbations thereof), diabetes mellitus type 1 (IDDM), endometriosis, fibromyalgia, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD), interstitial cystitis, lupus (including discoid lupus erythematosus, drug-induced lupus erythematosus, lupus nephritis, neonatal lupus, subacute cutaneous lupus erythematosus and systemic lupus erythematosus), morphea, multiple sclerosis (MS), myasthenia gravis, myopathies, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia, primary biliary cirrhosis, recurrent disseminated encephalomyelitis (multiphasic disseminated encephalomyelitis), rheumatic fever, schizophrenia, scleroderma, Sjogren's syndrome, tenosynovitis, vasculitis, and vitiligo. In one particular embodiment, the acute and / or chronic inflammation results from or is otherwise caused by diabetes in the subject. In another particular embodiment, the acute and / or chronic inflammation results from or is otherwise caused by multiple sclerosis in the subject.

[0105] In another embodiment, an acute and / or chronic inflammation is induced by amyopathy. In general, myopathies are caused when the immune system inappropriately attackscomponents of the muscle, leading to inflammation in the muscle. A myopathy includes, for example, an inflammatory myopathy and an autoimmune myopathy. Myopathies include, for example, dermatomyositis, inclusion body myositis, and polymyositis.

[0106] In another embodiment, an acute and / or chronic inflammation is induced by avasculitis. Vasculitis is a varied group of disorders featuring inflammation of a vessel wall including lymphatic vessels and blood vessels like veins (phlebitis), arteries (arteritis) and capillaries due to leukocyte migration and resultant damage. The inflammation may affect any size blood vessel, anywhere in the body. It may affect either arteries and / or veins. The inflammation may be focal, meaning that it affects a single location within a vessel, or it may be widespread, with areas of inflammation scattered throughout a particular organ or tissue, or even affecting more than one organ system in the body. Vasculitis include, without limitation, Buerger's disease (thromboangiitis obliterans), cerebral vasculitis (central nervous system vasculitis), ANCA- associated vasculitis, Churg-Strauss arteritis, cryoglobulinemia, essential cryoglobulinemic vasculitis, giant cell (temporal) arteritis, Golfer's vasculitis, Henoch-Schonlein purpura, hypersensitivity vasculitis (allergic vasculitis), Kawasaki disease, microscopic polyarteritis / polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoid vasculitis, Takayasu arteritis, Wegener's granulomatosis, and vasculitis secondary to connective tissue disorders like systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), relapsing polychondritis, Behcet's disease, or other connective tissue disorders, vasculitis secondary to viral infection.

[0107] In another embodiment, an acute and / or chronic inflammation is induced by a skindisorder. Skin disorders include, for example, an acne, including acne vulgaris, a bullous phemigoid, a dermatitis, including atopic dermatitis and acute and / or chronic actinic dermatitis, aneczema-like atopic eczema, contact eczema, xerotic eczema, seborrhoeic dermatitis, dyshidrosis, discoid eczema, venous eczema, dermatitis, dermatitis herpetiformis, neurodermatitis, and autoeczematization, and stasis dermatitis, diabetic skin complications, hidradenitis suppurativa, lichen planus, psoriasis including plaqure psoriasis, nail psoriasis, guttate psoriasis, scalp psoriasis, inverse psoriasis, pustular psoriasis, erythrodermis psoriasis, and psoriatic arthritis, rosacea and scleroderma including morphea, ulcers.

[0108] In another embodiment, an acute and / or chronic inflammation is induced by agastrointestinal disorder. A gastrointestinal disorder includes, for example, irritable bowel disease (IBD), an inflammatory bowel disease including Crohn's disease and an ulcerative colitis like ulcerative proctitis, left-sided colitis, pancolitis, and fulminant colitis.

[0109] In another embodiment, an acute and / or chronic inflammation is induced by acardiovascular disease. When LDL cholesterol becomes embedded in arterial walls, it can invoke an immune response. Acute and / or chronic inflammation eventually can damage the arteries, which can cause them to burst. In general, cardiovascular disease is any of a number of specific diseases that affect the heart itself and / or the blood vessel system, especially the veins and arteries leading to and from the heart. There are over 60 types of cardiovascular disorders including, for example, a hypertension, endocarditis, myocarditis, heart valve dysfunction, congestive heart failure, myocardial infarction, a diabetic cardiac conditions, blood vessel inflammation like arteritis, phlebitis, vasculitis; arterial occlusive disease like arteriosclerosis and stenosis, inflammatory cardiomegaly, a peripheral arterial disease; an aneurysm; an embolism; a dissection; a pseudoaneurysm; a vascular malformation; a vascular nevus; a thrombosis; a thrombophlebitis; a varicose veins; a stroke. Symptoms of a cardiovascular disorder affecting the heart include, without limitation, chest pain or chest discomfort (angina), pain in one or both arms, the leftshoulder, neck, jaw, or back, shortness of breath, dizziness, faster heartbeats, nausea, abnormal heartbeats, feeling fatigued. Symptoms of a cardiovascular disorder affecting the brain include, without limitation, sudden numbness or weakness of the face, arm, or leg, especially on one side of the body, sudden confusion or trouble speaking or understanding speech, sudden trouble seeing in one or both eyes, sudden dizziness, difficulty walking, or loss of balance or coordination, sudden severe headache with no known cause. Symptoms of a cardiovascular disorder affecting the legs, pelvis and / or arm include, without limitation, claudication, which is a pain, ache, or cramp in the muscles, and cold or numb feeling in the feet or toes, especially at night.

[0110] In another embodiment, an acute and / or chronic inflammation is induced by acancer. In general, inflammation orchestrates the microenvironment around tumors, contributing to proliferation, survival and migration. For example, fibrinous inflammation results from a large increase in vascular permeability which allows fibrin to pass through the blood vessels. If an appropriate procoagulative stimulus is present, such as cancer cells, a fibrinous exudate is deposited. This is commonly seen in serous cavities, where the conversion of fibrinous exudate into a scar can occur between serous membranes, limiting their function. In another example, a cancer is an inflammatory cancer like a NF-KB-driven inflammatory cancer.

[0111] In another embodiment, an acute and / or chronic inflammation is apharmacologically induced inflammation. Certain drugs or exogenic chemical compounds, including deficiencies in key vitamins and minerals, are known to effect inflammation. For example, Vitamin A deficiency causes an increase in an inflammatory response, Vitamin C deficiency causes connective tissue disease, and Vitamin D deficiency leads to osteoporosis. Certain pharmacologic agents can induce inflammatory complications, e.g., drug-induced hepatitis. Certain illicit drugs such as cocaine and ecstasy may exert some of their detrimentaleffects by activating transcription factors intimately involved with inflammation (e.g., NF-κΒ). Radiation therapy can induce pulmonary toxicity, burns, myocarditis, mucositis, and other tissue injuries depending on site of exposure and dose.

[0112] In another embodiment, an acute and / or chronic inflammation is induced by aninfection. An infectious organism can escape the confines of the immediate tissue via the circulatory system or lymphatic system, where it may spread to other parts of the body. If an organism is not contained by the actions of acute inflammation it may gain access to the lymphatic system via nearby lymph vessels. An infection of the lymph vessels is known as lymphangitis, and infection of a lymph node is known as lymphadenitis. A pathogen can gain access to the bloodstream through lymphatic drainage into the circulatory system. Infections include, without limitation, bacterial cystitis, bacterial encephalitis, pandemic influenza, viral encephalitis, and viral hepatitis (A, B and C).

[0113] In another embodiment, an acute and / or chronic inflammation is induced by atissue or organ injury. Tissue or organ injuries include, without limitation, a burn, a laceration, a wound, a puncture, or a trauma.

[0114] In another embodiment, an acute and / or chronic inflammation is induced by atransplant rejection. Transplant rejection occurs when a transplanted organ or tissue is not accepted by the body of the transplant recipient because the immune system of the recipient attacks the transplanted organ or tissue. An adaptive immune response, transplant rejection is mediated through both T-cell-mediated and humoral immune (antibodies) mechanisms. A transplant rejection can be classified as a hyperacute rejection, an acute rejection, or a chronic rejection. Acute and / or chronic rejection of a transplanted organ or tissue is where the rejection is due to a poorly understood acute and / or chronic inflammatory and immune response against thetransplanted tissue. Also included as transplant rejection is graft-versus-host disease (GVHD), either acute or chronic GVHD. GVHD is a common complication of allogeneic bone marrow transplantation in which functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount an immunologic attack. It can also take place in a blood transfusion under certain circumstances. GVHD is divided into acute and chronic forms. Acute and chronic GVHD appear to involve different immune cell subsets, different cytokine profiles, somewhat different host targets, and respond differently to treatment. In another embodiment, an acute and / or chronic inflammation is induced by a Th1 -mediated inflammatory disease. In a well- functioning immune system, an immune response should result in a well-balanced pro- inflammatory Th1 response and anti-inflammatory Th2 response that is suited to address the immune challenge. Generally speaking, once a pro-inflammatory Th1 response is initiated, the body relies on the anti-inflammatory response invoked by a Th2 response to counteract this Th1 response. This counteractive response includes the release of Th2 type cytokines such as, e.g., IL- 4, IL-5, and IL-13 which are associated with the promotion of IgE and eosinophilic responses in atopy, and also IL-10, which has an anti-inflammatory response. A Th1 -mediated inflammatory disease involves an excessive proinflammatory response produced by Th1 cells that leads to acute and / or chronic inflammation. The Th1 -mediated disease may be virally, bacterially or chemically (e.g., environmentally) induced. For example, a virus causing the Th1 -mediated disease may cause a chronic or acute infection, which may cause a respiratory disorder or influenza.

[0115] In another embodiment, an acute and / or chronic inflammation comprises an acuteand / or chronic neurogenic inflammation. Acute and / or chronic neurogenic inflammation refers to an inflammatory response initiated and / or maintained through the release of inflammatory molecules like Substance P (SP) or calcitonin gene-related peptide (CGRP) which released fromperipheral sensory nerve terminals (i.e., an efferent function, in contrast to the normal afferent signaling to the spinal cord in these nerves). Acute and / or chronic neurogenic inflammation includes both primary inflammation and secondary neurogenic inflammation. Primary neurogenic inflammation refers to tissue inflammation (inflammatory symptoms) that is initiated by, or results from, the release of substances from primary sensory nerve terminals (such as C and A-delta fibers). Secondary neurogenic inflammation refers to tissue inflammation initiated by non- neuronal sources (e.g., extravasation from vascular bed or tissue interstitium-derived, such as from mast cells or immune cells) of inflammatory mediators, such as peptides or cytokines, stimulating sensory nerve terminals and causing a release of inflammatory mediators from the nerves. The net effect of both forms (primary and secondary) of acute and / or chronic neurogenic inflammation is to have an inflammatory state that is maintained by the sensitization of the peripheral sensory nerve fibers. The physiological consequence of the resulting acute and / or chronic neurogenic inflammation depends on the tissue in question, producing, such as, e.g., cutaneous pain (allodynia, hyperalgesia), joint pain and / or arthritis, visceral pain and dysfunction, pulmonary dysfunction (asthma, COPD), and bladder dysfunction (pain, overactive bladder). Single Administration / Low Cell Dose

[0116] As disclosed herein, it has been surprisingly discovered that that MSCs obtainedfrom diseased tissue and / or tissue of subjects of advanced age retain significant (if not complete) biological activity. Moreover, it has been surprisingly discovered that the retained biological activity makes it possible to achieve a clinical benefit from administering MSCs obtained from diseased tissue and / or tissue of subjects of advanced age in a single administration and / or low cell dose. Given the surprising potency of the glycocalyx-modified cells disclosed herein, it is possibleto treat subjects with autologous cells in a single administration and / or low cell dose, even when MSCs are obtained from diseased tissue and / or tissue of subjects of advanced age.

[0117] In some embodiments, the low dose compositions, including glycocalyx-modifiedcell populations, pharmaceutical compositions and use of such compositions in the methods disclosed herein comprises at least about 50,000 cells / kg (based on the weight of the subject), at least about 200,000 cells / kg (based on the weight of the subject), at least about 400,000 cells / kg (based on the weight of the subject), at least about 500,000 cells / kg (based on the weight of the subject), at least about 600,000 cells / kg (based on the weight of the subject), at least about 700,000 cells / kg (based on the weight of the subject), at least about 800,000 cells / kg (based on the weight of the subject), or at least about 900,000 cells / kg (based on the weight of the subject). In some embodiments, the low dose compositions, including cell populations, pharmaceutical compositions and use of such compositions in the methods disclosed herein comprises about 50,000 glycocalyx-modified cells, about 200,000 glycocalyx-modified cells, about 400,000 glycocalyx-modified cells, about 500,000 glycocalyx-modified cells, about 600,000 glycocalyx- modified cells, about 700,000 glycocalyx-modified cells, about 800,000 glycocalyx-modified cells, or about 900,000 glycocalyx-modified cells. In some embodiments, the low dose compositions, including cell populations, pharmaceutical compositions and use of such compositions in the methods disclosed herein comprise less than 200,000 glycocalyx-modified cells. Priming MSCs

[0118] As disclosed herein, it has been surprisingly discovered that that MSCs obtainedfrom diseased tissue and / or tissue of subjects of advanced age retain significant (if not complete)biological activity. Thus, according to some embodiments, the MSCs disclosed herein may be primed to produce anti-inflammatory molecules.

[0119] According to some aspects, the present disclosure provides compositions andmethods directed to one or more MSCs that have been modified ex vivo to increase production of one or more anti-inflammatory or immunomodulatory molecules via binding of CD44 with a ligand. As used herein, the term “ligand” and grammatical variation thereof means a natural or artificial molecule(s) which bind to CD44 directly or indirectly, and that is effective to promote production of anti-inflammatory or immunomodulatory molecules when ligated to the CD44 present on a cell.

[0120] In some embodiments, the CD44 is ligated ex vivo with a molecule that is effectiveto promote production of anti-inflammatory or immunomodulatory molecules by a cell. In some embodiments, CD44 ligands include naturally occurring ligands (such as an extracellular matrix component) or artificial ligands that are effective to promote production of anti-inflammatory or immunomodulatory molecules by a cell. In some embodiments, CD44 ligands that are effective to promote production of anti-inflammatory or immunomodulatory molecules by a cell include, but are not limited to, hyaluronic acid (HA), osteopontin (OPN), bone morphometric proteins (BMPs), TGF (and other tissue reperative cytokines), collagens (e.g., Type I and VI), serglycins, galectins (e.g. gal-8), Siglecs, matrix metalloproteinases (MMPs), ARHGEF1, Ezrin (via PIP2), epidermal growth factor receptor (Hyaluronan-dependent), fibrin and fibrinogen, fibronectin, FYN, Lck, selectins, and Src. In some embodiments, the CD44 ligands that are effective to promote production of anti-inflammatory and / or immunomodulatory molecules by a cell are selectins. In some embodiments, the CD44 ligands that are effective to promote production of anti-inflammatory and / or immunomodulatory molecules by a cell include, but are not limited to, E- selectin, L-selectin, and P-selectin.

[0121] In some embodiments, the CD44 ligand that is effective to promote production ofanti-inflammatory and / or immunomodulatory molecules by a cell is hyaluronic acid (HA). In some embodiments, the HA is a high molecular weight HA (HW HA) that exhibits pro-inflammatory effects in vivo, such as HA with a molecular weight of at least 100,000 daltons.

[0122] In some embodiments, the CD44 is ligated with a molecule with a specific bindingaffinity for CD44, such as for example, an antibody or antigen binding fragment thereof derived from any animal source, including, but not limited to monoclonal antibodies, polyclonal antibodies, phagemids, aptamers, Camel Ig (a camelid antibody (VHH)), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody ("scFv"), bis- scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, Nanobody). In some embodiments, the CD44 is ligated with a monoclonal or polyclonal antibody effective to initiate or enhance production of anti- inflammatory cytokines in the CD44+ cells.

[0123] In some embodiments, the CD44 is ligated with a ligand that is effective to enhancethe binding of HA to the CD44. In some embodiments, the CD44 is ligated with a monoclonal or polyclonal antibody that is effective to enhance the binding of HA to the CD44. Examples of antibodies that enhance the binding of HA to CD44 include, but are not limited to, IRAWB14 antibody (see, e.g, Zheng, Z., et al., Monoclonal Antibodies to CD44 and Their Influence on Hyaluronan Recognition, The Journal of Cell Biology, Vol. 130, No. 2, 485-495). In some embodiments, the cell bearing CD44 is activated with a small molecule that is effective in enhancing the capacity of CD44 to bind to HA. Examples of small molecules that enhance CD44binding to HA include, but are not limited to, phorbol myristate acetate (PMA) (see, e.g., Sionov, R.V., et al., Cell Adhes Commun.1998;6(6):503-23).

[0124] In some embodiments, the CD44 ligand may be a modified ligand. In someembodiments, the CD44 ligand may be a hybrid of, or conjugated to, one or more other molecules. For example, in some embodiments, an E-selectin may be an E-selectin or L-selectin chimera with IgG (i.e., an E-Ig chimera or L-Ig chimera). Such chimeras are readily synthesized by those of skill in the art (or may be obtained from commercial sources (e.g., Bio-Techne / R&D Systems).

[0125] In some embodiments, the cells are treated with the CD44 ligand in amounts andfor durations effective to promote production of anti-inflammatory and / or immunomodulatory molecules in the CD44+ cells. In some embodiments, the cells are treated for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, or at least 48 hours. In some embodiments, the cells are treated with the CD44 ligand for between about 30 minutes and about 48 hours, such as between about 30 minutes and about 2 hours, between about 30 minutes and 90 minutes, and between about 30 minutes and one hour. In some embodiments, the CD44 is ligated ex vivo with HA for a period of time sufficient to prime the CD44+ cells to initiate or enhance production of anti-inflammatory cytokines.

[0126] In some embodiments, the CD44 is altered prior to, or concurrent with, ligation topromote interaction with a ligand. For example, the interactions of CD44 with ligands may be regulated via certain naturally occurring modifications to the intracellular or extracellular regions of CD44. Those naturally occurring modifications may block or reduce the ability of CD44 to bind to a ligand. In some embodiments, the naturally occurring modifications of CD44 are altered ex vivo to permit and / or promote binding of a ligand to CD44. For example, in some embodiments,terminal sialic acids on CD44 O-glycans or N-glycans, which are known to block HA binding, are removed by treatment with one or more sialidases, prior to or concurrent with ligation of HA.

[0127] In some embodiments, the glycans decorating CD44 are altered to promote bindingof one or more ligands, such as selectins. In some embodiments, CD44 expressing cells are treated ex vivo with one or more glycosidases and / or glycosyltransferases to construct on the CD44 a glycan structure that is effective for ligand binding. In some embodiments, CD44 expressing cells are treated ex vivo with one or more fucosyltransferases, for example, to enforce expression of HCELL, which binds to E-selectin and L-selectin.

[0128] In some embodiments, a modification of a cell’s glycocalyx is achieved by use ofa glycosyltransferase to install a chemically reactive group, orthogonal functional group, or molecular tag that is attached to the donor nucleotide sugar. For example, in some embodiments, fucosyltransferase-mediated installation of a modified-fucose by use of a donor GDP-fucose wherein the fucose has been modified by methods known in the art with a chemically reactive group, or orthogonal functional group, or molecular tag (e.g., biotinylated GDP-fucose, azido- GDP-fucose, etc.) thereby allowing for subsequent linkage of other molecules onto the installed fucose within cell surface lactosaminyl glycans (examples of this approach include, but are not limited to, use of biotinylated GDP-fucose with subsequent complexing using streptavidin- conjugated molecules and / or use of “click chemistry” wherein the azido-containing fucose molecule is then complexed to an alkyne-containing molecule). In other embodiments, molecules covalently linked to the donor nucleotide fucose (i.e., GDP-fucose with covalent attachment of additional molecule(s)) can be stereospecifically added in a distinct pattern onto cell surface lactosaminyl glycans to endow CD44 with the ability to bind to desired ligand.

[0129] In some embodiments, the CD44 is modified ex vivo to provide structures receptiveto ligands. In some embodiments, the CD44 is modified ex vivo via exofucosylation to enforce hematopoietic cell E-Selectin / L-Selectin Ligand (HCELL) expression. In some such embodiments, the modified CD44 may be ligated with one or more of a selectin (e.g., E-selectin and L-selectin) and monoclonal antibodies (mAbs) (e.g., the mAbs “CSLEX-1” and “HECA452”) effective to increase production of anti-inflammatory or immunomodulatory molecules in the CD44+cells.

[0130] In some embodiments, the CD44 modifying enzymes, e.g., glycosidases,glycosyltransferases and fucosyltransferases are obtained from any convenient source, e.g., purified from eukaryotic or prokaryotic cells or obtained from commercial sources, including R&D Systems, SigmaAldrich, SCHsciences, and CarbExplore Research.

[0131] In some embodiments, ligation of the CD44 is effective to produce elevated levelsof at least one anti-inflammatory or immunomodulatory molecule in the CD44+cells. As used herein, the term “anti-inflammatory molecule” and grammatical variation thereof means any molecule produced by a cell that acts to dampen inflammation, for example, by suppressing or restraining the action(s) of inflammatory effectors in a cell or tissue. As used herein, the term “immunomodulatory molecule” and grammatical variation thereof means any molecule produced by a cell that modulates an innate or adaptive immune response, excluding deleterious molecules that exacerbate an inflammatory disease or condition (e.g., pro-inflammatory molecules).

[0132] In some embodiments, the anti-inflammatory molecule is an anti-inflammatorycytokine. As used herein, the term “cytokine” includes, but is not limited to, leukocyte-generated peptides (e.g., lymphocyte-generated lymphokines, monocyte-produced monokines), chemokines, interferons, interleukins, adipocyte-secreted adipokines and muscle-generated myokines. In someembodiments, the anti-inflammatory molecules include, but are not limited to, Interleukin-10 (IL- 10), TGF-β, IDO, nitric oxide (NO) metabolites, PGE2 and combinations thereof. In some embodiments, ligation of the CD44 is effective to elevate production of at least one anti- inflammatory molecule at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to native population of cells, e.g., an untreated population of cells, such as an untreated population of CD44+cells. In some embodiments, ligation of the CD44 is effective to elevate production of at least one immunomodulatory molecule at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to a native population of cells, e.g., an untreated population of cells, such as an untreated population of CD44+cells.

[0133] In some embodiments, the anti-inflammatory and / or immunomodulatory moleculeis released from the CD44+cells via secretome. In some embodiments, the anti-inflammatory and / or immunomodulatory molecule is released from the CD44+cells via extracellular vesicle (e.g., exosome). In some embodiments, the anti-inflammatory and / or immunomodulatory molecule is released from the CD44+cells in soluble form. In some embodiments, anti-inflammatory and / or immunomodulatory molecules are produced by the CD44+cells via secretome / release of trophic agents.without priming.

[0134] In some embodiments, the anti-inflammatory or immunomodulatory moleculesproduced by the CD44+cells are effective to promote tissue repair in a subject. As used herein, the term “subject” and grammatical variation thereof, includes, but is not limited to, any mammal, such as a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In some embodiments,the production of elevated level of anti-inflammatory cytokines (such as IL-10) by the CD44+cells are effective to facilitate regenerative healing of damaged tissue in a subject. In some embodiments, the production of elevated level of anti-inflammatory cytokines (such as IL-10) by the CD44+cells are effective to facilitate regenerative healing via one or more of promoting the production and deposition of extracellular matrix components in damaged tissue of a subject, modulating fibroblast function, modulating myofibroblast differentiation, and modulating endothelial progenitor cell survival and function. In some embodiments, ligation of the CD44 is effective to produce elevated levels of at least one anti-inflammatory or immunomodulatory molecules in the CD44+cells that is effective to promote tissue repair in a subject.

[0135] The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0136] The following examples are provided to further illustrate the compositions andmethods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way. EXAMPLES

[0137] Background / Summary: Osteoporosis results from bone destruction outpacingbone production. Mesenchymal stem / stromal cells (MSCs), the precursors of osteoblasts, can regenerate bone. However, the efficacy of vascularly-administered MSCs for treatment of osteoporosis is impeded by inadequate homing to bone marrow, a deficit that is correctable byMSC surface fucosylation (“exofucosylation”). Mesenchymal stem / stromal cells (MSCs) are osteoregenerative, however, their therapeutic efficacy for skeletal conditions is hampered by poor bone-homing (“osteotropism”). In preclinical models this deficit is correctable by glycocalyx editing to enforce sLeX expression, programming MSC trafficking to marrow microvessels that are portals for osteotropism. We conducted a first-in-human clinical trial (ClinicalTrials.gov NCT02566655) involving a single intravenous infusion of glycocalyx-edited autologous bone marrow-derived MSCs in ten women with advanced-stage osteoporosis. The protocol-mandated evaluation spanned two years, including clinical assessments, radiographic studies, biopsies to quantify bone tissue area (BTA), and bone mineral density (BMD) measurements. Thereafter, fracture and safety monitoring continued for >3 additional years for each patient. No serious adverse events occurred. Fragility fractures were markedly and durably reduced, with improved BTA and volumetric BMD. These findings indicate that glycocalyx editing effectuates MSC-based osteoporosis therapy and also refute notions that MSCs derived from older persons and / or diseased-tissue sites are biologically compromised.

[0138] Methods: We conducted an open-label clinical Phase I trial of intravenouslyadministered exofucosylated autologous bone marrow-derived MSCs (Fuc-autoBM-MSCs) for treatment of ten women with postmenopausal osteoporosis. The first four patients received a single infusion of 2x106Fuc-autoBM-MSCs / Kg, and subsequent six received 5x106Fuc-autoBM- MSCs / Kg. At time of Fuc-autoBM-MSC infusion, patients continued their usual antiosteoporosis pharmacotherapy, with medication modifications permitted post-infusion as clinically indicated. The protocol-mandated safety evaluation period was two years, including eleven clinical visits, yearly radiographic studies, and interval assessments of pain / disability / QOL, bone mineral density (BMD, both areal (via DXA) and volumetric (via quantitative computed tomography)), andbiomarkers of bone turnover. Moreover, iliac crest biopsy was performed pre-infusion and at day- 120 post-infusion for histomorphometric analysis of bone tissue area (BTA). Following the initial protocol-mandated 2-year evaluation, surveillance for adverse events and fractures was continued for a minimum of 3 additional years in all patients.

[0139] Findings: There were no serious treatment-related adverse events in any patient,with a median observation period exceeding 6 years. Surprisingly, fragility fracture incidence was strikingly reduced, with statistically significant improvements in bone density / quality / structure as assessed by both BTA and volumetric BMD.

[0140] Interpretation: The safety profile was excellent, and, thus, the Phase 1 studyendpoint was met. Fuc-autoBM-MSC infusion yields improved trabecular bone quality / density, an osteoregenerative effect that decreases fragility fracture risk. This remarkable, unanticipated efficacy outcome following a single infusion of Fuc-autoBM-MSC indicates that culture-expanded BM-MSCs, using source MSCs harnessed from older individuals, retain significant osteoanabolic activity. Equally important, these MSCs have been collected from a diseased tissue, i.e., osteoporotic bone, indicating that resident BM-MSCs from osteoporotic patients retain sufficient biologic potency to engender osteorestoration and reverse osteoporosis. Collectively, these data unveil a highly promising cell-based regenerative therapeutics approach for treating osteoporosis.

[0141] Introduction

[0142] Osteoporosis is a progressive generalized skeletal disorder, primarily affectingwomen due to menopause-associated estrogen loss, whereby osteoclast-mediated bone resorption outpaces osteoblast-mediated bone formation. This process develops within the bone medullary cavity (“marrow cavity”) and predominantly erodes trabecular bone, markedly disrupting skeletal integrity / architecture, resulting in bone weakness and fragility (i.e., spontaneous and / or low-impact) fractures. Globally, one-in-three women over age 50 will suffer an osteoporosis-related fracture mounting to ~10 million fractures / year, yielding a world-wide rate of a fracture every 3 seconds. This chronic, debilitating and life-threatening condition reduces quality of life and lifespan, with immense global health, societal, and economic impacts.

[0143] Currently, osteoporosis is incurable. The principal goal of all osteoporosis therapyis to prevent new fragility fractures. Presently, this is achieved primarily by slowing bone loss, either by decreasing osteoclast-mediated bone resorption (“antiresorptives”: e.g., bisphosphonates; denosumab (a monoclonal antibody that inhibits osteoclast development by targeting the cytokine “receptor activator of nuclear factor-^b ligand” (RANKL) thereby blunting its binding to the osteoclast receptor RANK), and / or stimulating bone formation (“osteoanabolics”: e.g., parathyroid hormone (PTH) analogs; romosozumab (a mAb that targets sclerostin and thereby neutralizes its inhibition of Wnt signaling, leading to both osteoanabolic and antiresorptive effects)). All current medications require life-long adherence to pharmacologic or biologic agents, with significant side- effects that are compounded by cumulative dosing. These pharmacotherapeutic issues result in poor adherence: ~30%-35% of women who should be on antiosteoporotic agents avoid treatment, a clinical challenge known as the osteoporosis “treatment gap”. Ideally, therapeutic strategies will be developed capable of achieving cure of osteoporosis.

[0144] Mesenchymal stem / stromal cells (MSCs) are multipotent adult stem cellsthat are the precursors of osteoblasts, cells that create and repair bone. MSCs are readily harvested from marrow, rapidly expand in tissue culture, and, in particular, bone marrow-derived MSCs (“BM-MSCs”) are distinctly specialized for osteogenesis and osteoregeneration. Importantly, osteoblasts are post-mitotic cells whose replenishment depends on BM-MSC differentiation into osteoblasts. MSCs also secrete bioactive molecules that dampen inflammation and support tissuerepair. The combination of MSC osteogenic and paracrine-mediated anti-inflammatory properties raises hope that marrow delivery of culture-expanded BM-MSCs at affected skeletal sites could drive bone regeneration, preventing and / or reversing osteoporosis. Because osteoporosis is a “generalized” disease (i.e., not anatomically localized), intravascular (IV) administration is required to achieve necessary systemic biodistribution of therapeutic cells. BM-MSC autografts are preferable to allografts to avoid immunorejection of osteolineage progeny, but, at odds with this clinical benefit are safety concerns: MSCs originating from older persons or from a diseased tissue source could pose a potential heightened risk of malignancy and other undesired tissue formation, these cells are also more likely to be senescent and / or biologically compromised, and, more intrinsically, functional derangements in BM-MSCs of osteoporotic patients might be contributory to the pathobiology. These theoretic concerns mandate rigorous clinical evaluation of any potential adverse effect(s) of intravascularly administered autologous BM-MSCs (autoBM- MSCs). Importantly, to mitigate any additional risks arising from cellular manipulation(s) intended to optimize marrow migration / colonization (“osteotropism”) of infused autoBM-MSCs, it would be ideal to avoid technical approaches involving intracellular modifications / alterations. This goal can be achieved via cell surface glycan engineering.

[0145] All eukaryotic cells, and most prokaryotic cells, are blanketed by a dense matrix ofcarbohydrates collectively called the “glycocalyx”. Distinct glycan motifs within the glycocalyx, comprised of defined oligosaccharide clusters containing specific monosaccharides arranged in a characteristic spatial arrangement / configuration, license specific cellular properties impacting multiple biologic events (including cell migration, immune responses, pathogen invasiveness, cell / tissue development, and carcinogenesis). These oligosaccharides are assembled within the Golgi apparatus by action of glycosyltransferases that covalently link, in step-wise fashion, thecomponent monosaccharides in a regio- and stereospecific manner upon a pertinent “acceptor” glycan structure. This chemo-enzymatic reaction can be performed directly on a living cell’s surface (i.e., exogenously) using pertinent glycosyltransferases together with their respective donor nucleotide sugars under reaction conditions that are non-toxic and preserve native cell biology. This technique is called “glycosyltransferase-programmed stereosubstitution” (GPS) and it engenders exceptionally specific and definitive glycocalyx motif-editing.

[0146] One well-characterized glycocalyx motif is the structure known as “sialylatedLewis X” (sLeX; CD15s), a tetrasaccharide comprised of sialic acid (“NeuAc”), galactose (“Gal”), fucose (“Fuc”), and N-acetylglucosamine (“GlcNAc”) covalently assembled as follows: NeuAc- α(2,3)-Gal-β(1,4)-[Fuc-α(1,3)]-GlcNAc-β1-R). sLeX is the canonical binding determinant for E- selectin (CD62E), a Ca2+-dependent lectin constitutively expressed on specialized bone marrow microvessels that serve as gateways for entry of circulating cells into the marrow. Hematopoietic stem / progenitor cells (HSPCs) characteristically display sLeX, and sLeX-E-selectin interactions mediate HSPC osteotropism, a prerequisite for successful hematopoietic stem cell transplantation. Natively, MSCs do not express sLeX, and, accordingly, they lack osteotropic capacity. However, the MSC glycocalyx contains terminal α(2,3)-sialylated type 2 lactosamines (i.e., NeuAc-α(2,3)- Gal-β(1,4)-GlcNAc-R) missing only fucose α(1,3)-linked to GlcNAc to compose sLeX. Conspicuously, on MSCs, this trisaccharide acceptor is displayed uniquely on a glycovariant of the cell membrane glycoprotein CD44. Glycoengineering of human MSC surface “standard” CD44 via α(1,3)-fucosyltransferase-mediated installation of Fuc results in temporary expression (~48 hours) of sLeX motifs on CD44. This glycocalyx editing process is known as “exofucosylation”, and it generates the operationally specialized CD44 glycoform called “Hematopoietic Cell E- / L-selectin Ligand” (HCELL).

[0147] HCELL is prominently expressed on human HSPCs. This glycoprotein is anextremely potent E-selectin ligand and a principal molecular effector of osteotropism. Preclinical human-murine xenotransplant studies employing intravital microscopy in combination with long- term engraftment analysis of human HSPCs and of exofucosylated (i.e., HCELL+) human MSCs have shown that HCELL engagement of E-selectin on marrow microvessels engenders chemokine- independent trans-endothelial migration, with subsequent highly efficient colonization of these cells within the marrow parenchyma, followed by engraftment. In particular, results of these in vivo studies reveal that glycocalyx editing to engender transient HCELL expression on intravenously (IV)-infused human MSCs is sufficient to empower efficient endosteal lodgment and in situ human osteoid production within murine bone. Based on this compelling evidence, we hypothesized that IV administration of glycocalyx-edited HCELL+ autoBM-MSCs could have an osteorestorative effect in osteoporosis.

[0148] As a fundamental necessary first step toward testing this hypothesis, we performeda “safety study”, a Phase 1 clinical trial focusing on potential risks / adverse events of two dose levels of a single IV administration of exofucosylated (glycocalyx-edited) culture-expanded autoBM-MSCs (“Fuc-autoBM-MSCs”) in patients with advanced osteoporosis (ClinicalTrials.gov ID# NCT02566655). The results show that this treatment approach has an excellent safety profile and is feasible. However, strikingly and unexpectedly, beyond providing key insights on these primary issues, the results also reveal a profound and remarkably durable reduction in the incidence of fragility fractures accompanied by radiographic and histomorphometric evidence of an osteorestorative effect within trabecular bone. These findings indicate that BM-MSCs derived from osteoporotic marrow, and from older donors, retain significant salutary clinical effects. Thus, precision glycocalyx editing effectuates MSC-based therapy to reverse osteoporosis, shifting thetherapeutic approach in treatment of this disease from pharmacologic approaches to regenerative medicine.

[0149] Methods

[0150] Study Design

[0151] This open-label, single arm, single center, Phase 1 clinical trial was conductedaccording to Declaration of Helsinki principles to assess the safety / tolerability of a single intravenous infusion of Fuc-autoBM-MSCs in women with postmenopausal osteoporosis and fragility-related fractures. Patients provided informed consent under a protocol approved by the University Hospital Virgen de la Arrixaca (Murcia, Spain) and the Spanish Agency of Medicine and Medical Devices. This trial was registered with European Clinical Trials Database (EudraCT 2012-005814-20) and ClinicalTrials.gov (NCT02566655).

[0152] Accrual was limited to 10 patients. In escalating-dose sequence, the first fourpatients received 2x106Fuc-autoBM-MSCs / Kg, with subsequent six receiving 5x106Fuc-autoBM- MSCs / Kg. Primary endpoint was safety: serious and non-serious adverse events were monitored at the time of infusion and within a protocol-mandated 2-year evaluation period; this evaluation included eleven in-person clinical assessments, with particular vigilance regarding skeletal integrity and signs / symptoms of neoplastic disorders. Secondary endpoints included fracture incidence, biomarkers of bone turnover, quantification of bone mineral density (BMD) by both DXA (areal BMD) and computed tomography (volumetric BMD), histomorphometric analysis of bone biopsy specimens, and patient pain, functional status, and quality-of-life (QOL) scores.

[0153] The study formally concluded when the last patient accrued had completed theprotocol-mandated 2-year evaluation (which occurred in May 2020). Notably, in March 2020, Spain declared a state emergency pursuant to the COVID-19 pandemic, and, given theuncertainties regarding COVID-19-related pathobiology, health status and fracture incidence monitoring via primary physician visits and phone interviews was continued (“extended surveillance period” for all patients following the initial 2-year protocol-mandated evaluation) until median follow-up for the entire cohort was 6 years (i.e., three-times the protocol-mandated period).

[0154] Patients

[0155] Postmenopausal osteoporotic women aged 50-75 years with one or more fragilityfractures were recruited according to prespecified protocol criteria. Key exclusion criteria were immune arthritides, concomitant severe systemic diseases, cancer, genetic conditions associated with secondary osteoporosis, positive serologies for bloodborne transmissible viral diseases, or treatment with steroids or with drugs interfering with cell proliferation. Osteoporosis was defined via central DXA scan at the lumbar spine (LS) and at the femoral neck (FN) meeting the criteria of a T-score of -2·5 or lower, or T-score <-1·0 with history of at least one hip, vertebral or forearm fragility fracture. Detailed eligibility criteria are provided in the protocol.

[0156] AutoBM-MSC Culture and Exofucosylation

[0157] Bone marrow (~100 mL) was obtained by needle aspirations of the iliac crest understerile conditions, and autoBM-MSCs were processed and cultured under GMP conditions as previously reported. Culture-expanded autoBM-MSCs were then exofucosylated under sterile conditions followed by flow cytometry to confirm enforced expression of sLeX, as described. Fuc- autoBM-MSC products were released only if meeting technical specifications that included characteristic MSC immunophenotypic profile, >90% cell viability, 100% sLeX expression, sterility (assessed by aerobic and anaerobic microbiologic assays, and endotoxin-negativity), and genetic stability as evaluated by G-band karyotyping and / or CGH-arrays.

[0158] Fuc-autoBM-MSCs Administration

[0159] Patients were hospitalized for infusion. Following premedication (50 mghydrocortisone / 5 mg dexchlorpheniramine IV), Fuc-autoBM-MSCs (5x106 / mL in saline) were administered via peripheral venous catheter at 4-6 mL / min. The first four patients received 2x106cells / Kg, and subsequent six patients received 5x106cells / Kg. Vital signs were monitored during infusion and for 24 hours before discharge.

[0160] Radiologic Studies

[0161] All patients underwent X-ray studies of dorsal and lumbar spine (AP and lateral),and of pelvis and hips (AP), at baseline, and at 12 months and 24 months post-Fuc-autoBM-MSC infusion.

[0162] Assessment of Bone Mineral Density by Dual-energy X-ray Absorptiometry(DXA)

[0163] DXA was performed (Hologic Discovery Wi densitometer) at 6, 12, and 24 monthspost-infusion to assess areal bone mineral density (BMD, expressed as grams / cm2) at the lumbar spine (L1 to L4, with operator exclusion of up to two vertebral bodies from analysis when confounding structural abnormalities were present), and at femoral neck. T-score was then calculated according to World Health Organization recommendations.

[0164] Volumetric BMD by Quantitative Computed Tomography (QCT) and Quality ofTrabecular Structure (QTS) Assessment

[0165] High resolution peripheral QCT (General Electric 64-MDCT) was performed atright distal radius at baseline and at months 4, 12 and 24 post-infusion. Trabecular bone microstructure was quantitatively analyzed by using Quibim Precision Software tool, as previously described15,16. This tool provides bone volume percentage, trabecular thickness, separation, andirregularity analysis, and combines these values into a single score (the “QTS”). Higher QTS values indicate better bone quality and lower values indicate a higher fracture risk.

[0166] Measurement of Bone Tissue Area (BTA)

[0167] BTA was measured via histomorphometry of sequentially-cut non-decalcified 5-^m sections of iliac crest bone biopsies obtained under sterile conditions at baseline and at month 4 post-Fuc-autoBM-MSC administration. Sections were prepared and stained with von Kossa / Nuclear Fast Red as described; following proper color deconvolution, high resolution images were obtained for BTA scoring (analyzed using ImageJ v.153p).

[0168] Patient-reported Outcomes and Measurement of Serum Biomarkers of BoneTurnover

[0169] At multiple time-points post-infusion, patient pain, functional status, and QOLwere assessed by Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), and EuroQoL 5-D questionnaire, respectively, and biomarkers of bone turnover (both bone formation and resorption) were measured in serum samples: N-terminal propeptide of type I procollagen (P1NP), osteocalcin (BGLAP), and C-terminal telopeptide of type I collagen (^-CTx;^^^Cross Laps”) by electrochemiluminescent immunoassay (Roche Diagnostics, Switzerland); N-terminal telopeptide of type I collagen (NTX) by ELISA; and bone alkaline phosphatase (ALP) by chemiluminescense (DiaSorin, Italy).

[0170] Statistical Analysis

[0171] Bone biomarker levels were evaluated as percent change from baseline levels(“delta % change”), with values summarized as mean + SEM. For BTA and QTS score analyses, variance homogeneity was assessed by Levene test followed by Welch's t-test or Student's t-test for non-homogeneous or homogeneous variances, respectively. Kolmogorov-Smirnov test wasundertaken to analyze data distribution (non-parametric, p<0.05). For assessing changes in areal BMD and volumetric BMD (QTS), delta % change was calculated and Student 2-tailed t-test was employed. For VAS, ODI, and EuroQol measurements, delta % change was measured and Wilcoxon test was employed. P values <0.05 were considered statistically significant.

[0172] Results

[0173] Patient Characteristics

[0174] Thirteen patients were recruited, with three excluded because the autoBM-MSCproduct did not meet technical specifications. Characteristics of the ten enrolled patients are listed in Table 1. TABLE 1

[0175] *“V / P” indicates the number of atraumatic fractures within the vertebra (V) versusthose within the hip, distal forearm / carpal joint and / or pelvis (peripheral, P) for each patient at time of recruitment. Osteoporosis treatment at time of Fuc-autoBM-MSC infusion (Day 0) is shown for each patient. Five patients (#1, #3, #6, #7, #8) had no medication changes within the 2- year post-infusion evaluation period. For patients #2, #4, #5, #9, and #10, medication changes were as follows: Patient #2 received final dose of Denosumab at 12 months post-infusion and was continued on Vitamin D / calcium; Patient #4 received Denosumab 2 months prior to infusion, but patient refused Denosumab post-infusion and was started on Vitamin D / calcium at 6 months with change to teriparatide / Vitamin D at 9 months post-infusion; Patient #5 (who discontinued Denosumab 6 months prior to infusion) had a fragility fracture at 3 months post-infusion (**denotes that this patient was the only patient to have a fracture within the 2-year post-infusion period) and was then started on Denosumab / Vitamin D / calcium; Patient #9 terminated teriparatide / vitamin D at 21·5 months post-infusion and was then started on Denosumab / Vitamin D / calcium; Patient #10 terminated teriparatide / Vitamin D at 5·5 months post-infusion and was then started on Denosumab / Vitamin D / calcium.

[0176] All patients had at least one fragility fracture within 5 years prior to receiving Fuc-autoBM-MSC infusion, and, with exception of patient #5, all were deemed “high risk” _ENREF_3for recurrent fragility fractures. Median age was 60·7 years (range 51-72 years); two patients had only peripheral fractures (#1 hip; #5 distal radius), and eight patients had history of vertebral fractures, of which only one (#4) had both vertebral and peripheral fractures (at pelvis and both radii). All treatment modalities were dictated by each patient’s primary treating physician (Table 1). Notably, two patients (#9, #10) were receiving teriparatide at time of Fuc-autoBM-MSC infusion, and three patients (#3, #5, #7) were not on any prescription antiosteoporotic medications.

[0177] Primary Endpoint: Safety

[0178] All patients completed the protocol-mandated 2-year evaluation. Four patientsreported transient pain and tissue edema at marrow harvest sites. There were no Fuc-autoBM-MSC infusion-related adverse events, and all patients were discharged 24 hours post-infusion in usual health. Within 24 months post-infusion, two patients had SAEs (atypical chest pain; ileus), in each case many months post-infusion: both required hospitalization, with neither event deemed related to Fuc-autoBM-MSC treatment (Table 2). TABLE 2: The absolute number of serious adverse events (SAE) are shown, with percentage of patients affected in parenthesis.

[0179] Post-infusion evaluation included cancer screening given conceptual concerns thatmutations ensuant to cell aging and / or ex vivo MSC proliferation could be tumorigenic. Moreover, BM-MSCs from osteoporotic patients could harbor osteogenic deficiencies, raising vigilanceregarding bone integrity. Within 2 years post-infusion, and throughout the entire patient assessment period (median of ~6 years (72·7 months); average being 6·14 years (range 5·32-7·27 years); minimum follow-up >5·5 years), all patients were free of neoplastic disease, with no manifestations of non-osteoporotic skeletal pathologies, and without new diseases / conditions requiring medical interventions.

[0180] Secondary Endpoints: Fragility Fracture Incidence; Biomarkers of BoneTurnover; Areal and Volumetric BMD Measurements; Histomorphometric analysis of Bone; Patient Pain / Disability / QOL Assessments

[0181] Fragility fractures: Figure 1A is a swimmer’s plot depicting fragility fractures pre-and post-Fuc-AutoBM-MSC infusion. Only one patient (#5) sustained a fragility fracture within 2-years post-infusion. This patient received denosumab / vitamin D / calcium for 2-years pre- infusion, but refused medication at intended 4thdose (6 months pre-infusion): fracture occurred 9 months after discontinuation (3 months post-infusion); denosumab / vitamin D / calcium were then re-initiated and maintained, and another fragility fracture occurred 4.5 years post-infusion. Another patient (#6) sustained fragility fractures within the extended surveillance period: patient received denosumab / vitamin D / calcium pre- and post-infusion, with 2 fragility fractures occurring at 28 months post-infusion; denosumab was then discontinued and teriparatide / vitamin D / calcium was initiated, with another fragility fracture at 7 months of this treatment. Figure 1B shows incidence of fragility fractures for 2 years pre-infusion and within the 2-year post-infusion period; fragility fracture incidence decreased sharply following Fuc-autoBM-MSC administration (93% reduction (from 7 events / year to 0.5 events / year) (p=0.006, paired t-test). (Right): Incidence of fragility fractures pre-infusion and post-infusion for all years of observation for all patients (p=0.005, Welch´s t-test).

[0182] Biomarkers of bone turnover: At baseline, and 4, 6, 9, 12, 18 and 24 months post-infusion, serum levels were measured for bone neoformation biomarkers P1NP, osteocalcin (BGLAP), and bone ALP (Fig.2A, B), and for bone resorption biomarkers ^-CTx and NTX (Fig. 3). For each patient, the percent change from baseline values was assessed for all biomarkers and mean + SEM was calculated (Fig. 2B; Fig 3). There was a trend toward increases in bone formation markers P1NP and BGLAP, even when excluding patients (#4, #9, #10) who received teriparatide at any time post-infusion (Fig.2B).

[0183] Histomorphometric analysis of bone tissue biopsies: Iliac crest biopsies wereobtained at baseline and at day +120 post-Fuc-autoBM-MSC administration. Baseline and post- infusion analysis performed per patient (Fig.4A) showed statistically significant increases in mean BTA values in seven patients (#1, #2, #3, #4, #5, #8, #10), decreases in two (#6, #7), and no change in one patient (#9).

[0184] Histomorphometric analysis showed a significant global increase in the mean bonetissue area (BTA) per slice, from 1·42 + 0·03 mm2to 2·41 + 0·05 mm2(mean + SEM; ***p<0·001) (Fig.4B (left)).

[0185] Bone mineral density (BMD) as assessed by DXA (Areal BMD) and by QCT(Volumetric BMD; QTS score): DXA was performed at LS and FN at baseline and at 6,12 and 24 months post-infusion. There were no significant differences in mean delta % values from baseline at months 6, 12 or 24 in areal BMD, nor for T-scores, for LS or FN (Table 3). However, for QTS score, the mean delta % values trended higher at 4 and 12 months, and a statistically significant increase in QTS score was observed from baseline (5·3 + 0·15) to month 24 post-infusion (6·5 +0·4 (mean + SEM); *p<0·05) (Figure 4B (right); Table 3).TABLE 3: Mean of Delta Percent Change (mean % change) in Areal (DXA) and Volumetric (QTS) BMD(A) Mean of Delta Percent Change (mean % change) in Areal BMD: DXA and T-score at Lumbar SpineMean BMD % change Mean T-score % change(B) Mean of Delta Percent Change (mean % change) in Areal BMD: DXA and T-score at Femoral NeckMean BMD % change Mean T-score % change(C) Mean of Delta Percent Change (mean % change) in Volumetric BMD: Radial QTS Month 4 Month 12 Month 24Abbreviations: BMD (Bone mineral density, in g / cm ); DXA (Dual energy X-ray absorptiometry); QTS (Quality of trabecular structure score) Normality has been assumed as per Kolmogorov-Smirnov test (p > 0·05)*p-value: calculated using Student 2-tailed T-test (Baseline Areal BMD, T-score, or QTS as reference); significance in bold

[0186] Pain / Functional Status / QOL Assessments: Most patients described painimprovement post-Fuc-autoBM-MSC infusion, reaching statistical significance in mean delta % within 15-days (p=0·01), and then at months 9, 18 and 24 post-infusion (-34% at month 24, p=0·02) (Table 4). TABLE 4: Mean of Delta Percent Change (mean % change) in VASNormality has been assumed using Kolmogorov-Smirnov test (p>0·05) *p-value: calculated using Student 2-tailed T-test (Baseline VAS as reference). Significance: p<0·05

[0187] There were also statistically significant changes in mean delta % values of disabilityscores at months 12 and 24 (-30% at month 24, p=0·02; Table 5). TABLE 5: Mean of Delta Percent Change (mean % change) in ODIODI: Oswestry disability index data (0-100) Normality has been assumed using Kolmogorov-Smirnov test (p>0·05) *p-value: calculated using Student 2-tailed T-test (Baseline ODI as reference). Significance accepted with p<0·05

[0188] Although improved QOL scores were evident post-Fuc-autoBM-MSC infusion,these metrics did not reach statistical significance (Table 6). TABLE 6: Mean of Delta Percent Change (mean % change) in EuroQol EuroQol: Quality of life questionnaire results (0-100) Normality has been assumed using Kolmogorov-Smirnov test (p>0·05)*p-value: calculated using Student 2-tailed T-test (Baseline EuroQol as reference). Significance: p<0·05

[0189] Discussion

[0190] This Phase I clinical trial was undertaken to evaluate the safety / tolerability andfeasibility of the intravenous administration of exofucosylated, culture-expanded, autologous BM- derived MSCs in patients with osteoporosis and history of low impact fractures. BM-MSCs were utilized as they have been reported to possess higher osteogenic potential compared to MSCs derived from other tissues (e.g., fat). Autologous BM-MSCs were used because osteoblasts are highly immunogenic, potently stimulating T cell alloreactivity. Exofucosylation of auto-BM- MSCs was performed prior to intravascular MSC administration to enforce expression of HCELL, thereby programming marrow delivery (osteotropism) so that the cells could colonize the affected tissue, i.e., the entire skeleton.

[0191] Safety assessment was the primary endpoint. Over a median observation period of~6 years (72·7 months), no immediate nor long-term adverse events were associated with Fuc- autoBM-MSC infusion in any patient. Though possible that MSCs derived from older patients could harbor age-associated “clonal mutations” prompting malignant transformation, and such mutations could also theoretically arise during culture-expansion, no neoplasms of any type arose. Notably, though age-related deficits in human MSC functionality have been reported (including age-associated decline in BM-MSC osteogenic capacity), there were no signs nor symptoms of bone abnormalities / atypical fractures, no ectopic bone formation, nor accentuation of adverse events characteristically associated with the use of antiosteoporotic agents. Importantly, to our knowledge, this trial is the first to assess the potential for adverse events following the intravascular administration of MSCs obtained exclusively from older individuals (i.e., donors >50 years old), and, more specifically, obtained from within the diseased tissue itself. Due to the aforementionedconcerns of age-related alterations in stem cell biology, all prior clinical trials, especially those employing third-party MSCs, prefer administering cells obtained from younger donors (<30 years old).

[0192] Following a single infusion of Fuc-autoBM-MSCs, the incidence of fragilityfractures dramatically decreased. Across all patients, the amount of fragility fractures within the time-span of 2-years pre-infusion compared to 2 years post-infusion (the protocol-mandated post- infusion evaluation period) dropped precipitously, from 14 total fractures pre-infusion to 1 fracture post-infusion (a 93% reduction in fragility fractures, an incidence change from 7 events / year to 0.5 events / year (p=0·006)) (Figs. 1A and 1B). In the 4 patients that received the lower dose of Fuc-autoBM-MSCs (2x106cells / Kg), no fragility fractures occurred over the entire post-infusion evaluation period (median of 7.5 years). Even more noteworthy, no fragility fractures occurred at any point post-Fuc-autoBM-MSC infusion among those patients >60 years old (#2, #4, #7, #10), suggesting that BM-MSCs that have been harvested from older individuals retain antiosteoporotic properties. Consistent with patient-reported outcomes showing statistically significant decreases in pain and disability scores following Fuc-autoBM-MSC infusion (Tables 4-5), only one patient (#5) sustained a fragility fracture within the protocol-mandated 2-year evaluation period. This fracture occurred at 9-months following denosumab withdrawal, timing commensurate with post- denosumab “rebound” fractures due to osteoclast activation. Within the extended surveillance period (i.e., beyond the protocol-mandated 2-year evaluation period), fragility fractures occurred in just two patients (#5 and #6; see Fig.1A).

[0193] The observed profound and sustained decrease in the incidence of fragility fracturesafter a single infusion of Fuc-autoBM-MSCs merits attention, especially since fragility fracture reduction is the key metric for FDA / EMA approval of any antiosteoporotic therapy. Though thispilot study was focused on safety, some mechanistic insights into this finding may be gleaned. First, medication modifications do not account for the decreased fracture incidence, since no fractures occurred in patients #1, #3, #7 and #8, each of whom had no medication changes (and patients #3 and #7 were not on antiosteoporosis prescription medications). Secondly, there was a statistically significant improvement in trabecular bone microarchitecture (BTA) at 4 months post- infusion (p<0·001), and in volumetric BMD (QTS score) at 24 months (p<0·05) (Fig.4B); these trends persisted upon excluding patients (#4, #9, #10) that received teriparatide post-infusion, indeed, with increased statistical significance of the 24-month QTS scores (p=0·0156 (all patients) versus p=0·0028 (teriparatide-excluded patients)). Regarding bone turnover, the most sensitive and specific bone neoformation biomarker is P1NP, and the mean level of this biomarker increased prominently in 8 / 10 patients, with one patient (#10) having a sharp drop from baseline P1NP levels solely due to discontinuation of teriparatide at 5.5 months post-Fuc-autoBM-MSC infusion (Figure 2A, B). Notably, there was also a pronounced trend of increased ^-CTx levels indicative of enhanced bone resorption (Fig. 3), suggesting that MSCs do not inhibit osteoclast-mediated remodeling as is characteristic of antiresorptive medications. This pattern of increased levels in both bone neoformation and resorption biomarkers, with concomitant augmented trabecular bonecontent as measured by BTA and QTS scores (Fig. 4B), is similar to the osteoanabolic effect ofPTH whereby bone turnover increases but trabecular bone structure is enhanced. Increases in areal BMD (DXA) and improved T-scores were also evident within the lumbar spine following Fuc- autoBM-MSC infusion, with less effect in the femoral neck (Table 3); this pattern likewise resembles DXA / T-score changes observed with PTH / PTH-analog administration. Conspicuously, the observed trends in biomarkers also persist with exclusion of data from patients that received teriparatide (Fig.2B; Fig.3). Collectively, these results suggest that administered MSCs mediatean osteorestorative / osteoanabolic effect predominantly within trabecular bone. This effect could be secondary to direct differentiation of administered MSCs into osteoblasts (as was observed in preclinical human-mouse MSC xenotransplantation studies), and / or an MSC-mediated paracrine / trophic influence supporting osteoblast integrity / bioactivity and / or an MSC anti- inflammatory effect dampening bone injury and thereby sustaining bone neoformation. Moreover, since the MSCs used herein were harvested from a disease-laden tissue site (osteoporotic marrow), the finding of the osteoanabolic effect post-Fuc-autoBM-MSC infusion suggests that the osteoporotic inflammatory milieu of the marrow does not abolish the intrinsic MSC osteogenic activity.

[0194] Osteoporosis is treatable, yet ~30% of patients that suffer from an osteoporosis-related fracture in the US avoid antiosteoporosis medications, and treatment rates worsen with age. This so-called “osteoporosis treatment gap” highlights the critical unmet medical needs of patients suffering from this disease_ENREF_2: new osteoporosis treatments are desired that are safe, readily adherable / tolerable, and,could engender durable bone regeneration without necessitating recurrent-dosing, life-long therapy. To date, >1500 clinical trials have been undertaken to assess the impact of MSCs for treatment of various medical conditions, but no clinical studies have examined the utility of MSCs in treatment of osteoporosis, nor have examined use of MSCs derived exclusively from older patients for any medical condition. Moreover, no prior clinical study has evaluated MSCs that have undergone exofucosylation to enforce expression of E-selectin binding so as to enhance colonization of vascularly-administered MSCs at tissue(s) whose endothelial beds express E-selectin. The finding that administration of exofucosylated human MSCs is feasible and safe, together with the promising clinical responses observed following just a single infusion of Fuc-autoBM-MSCs, provides rationale for conducting largertrials to evaluate the antiosteoporosis efficacy of this treatment approach. Collectively, the results of this Phase 1 clinical trial raise optimism that a cell-based regenerative therapeutic strategy will be developed that will address the unmet medical needs of the hundreds of millions of women that are suffering from postmenopausal osteoporosis. Conclusion

[0195] Though cellular gene editing, and commensurately, cellular protein editing, arenow well-established, the field of cellular glycan editing remains relatively underappreciated and underexplored. This difference is due in large part to the fact that, unlike nucleic acids and proteins, glycan biosynthesis is not template-instructed. Nevertheless, glycan assembly is a highly ordered process, executed by exceptionally precise glycosyltransferase-mediated step-wise addition of relevant monosaccharides onto specific precursors, the “acceptor glycans”. Thus, a living cell’s glycocalyx can be custom-modified to display a desired glycan motif via glycosyltransferase- programmed stereo- and regio-specific installation of the relevant monosaccharide(s) on a pertinent acceptor glycan. This compositional editing critically depends on creation of glycosyltransferases that can function at the cell surface (i.e., exogenously) utilizing reaction conditions that are non-toxic to the target cell and preserve the cell’s innate biologic phenotype. However, before this glycocalyx editing technique can be applied to optimize outcomes of cell- based therapeutics, it is paramount to establish the safety and feasibility of its use on the intended cell type and for the pertinent clinical indication.

[0196] This study (a first-in-human “Phase 1” clinical trial) was undertaken with primaryend-point to evaluate the safety / tolerability and feasibility of the intravenous administration of glycocalyx-edited culture-expanded autoBM-MSCs in patients with advanced osteoporosis. Because of the generalized nature of osteoporosis, primary clinical principles dictate use of thevascular route of MSC administration to enable MSC biodistribution to all affected skeletal tissue. Glycocalyx editing of human MSCs was performed via ^(1,3)-fucosyltransferase-mediated exofucosylation to install sLeX motifs on the MSC membrane glycoprotein CD44, thereby generating HCELL, a potent molecular effector of osteotropism. The resultant enforced HCELL expression is transient (duration ~48 hours) due to natural cell surface CD44 turn-over, and, thereafter, the MSC glycocalyx reverts to its usual composition. Importantly, exofucosylation does not alter MSC osteogenic capacity as evaluated by in vitro assays, and, also, by in vivo studies of intravascularly administered HCELL+ human MSCs into immunodeficient mice.

[0197] BM-MSCs were utilized in this study as they have been reported to possesshigher osteogenic potential compared to MSCs derived from other tissues (e.g., fat). In design of this trial, ethical considerations mandated that the MSC infusion be an “add-on” to existing therapies in each patient. Autologous, as opposed to allogeneic, BM-MSCs were used to avoid potential immunorejection because osteoblasts are highly immunogenic, potently stimulating T cell alloreactivity. Consistent with this notion, efforts to treat osteogenesis imperfecta (a congenital skeletal disease) using intravascularly administered allogeneic MSCs (without glycocalyx editing) attained only a brief, and relatively modest, osteogenic effect. Uniformly, the data indicate that infusion of exofucosylated autologous BM-MSCs (Fuc-autoBM-MSCs) in patients suffering from osteoporosis is both well-tolerated and has an excellent safety profile: over a median observation period of ~6 years (72.7 months), no immediate nor long-term adverse events were associated with Fuc-autoBM-MSC infusion in any patient. Thus, this trial’s primary end-point was met.

[0198] At present, ClinicalTrials.gov (“CT.gov”) lists >1500 clinical trials (searchedvia terms “mesenchymal stem cell” or “mesenchymal stromal cell” within the prompt “Other Terms”) that have been undertaken using MSCs for a variety of medical conditions. Yet,conspicuously, though MSCs are the precursors of osteoblasts, there is a paucity of studies related to MSC-based therapy for osteoporosis. Using the search terms “osteoporosis” and “mesenchymal stem cell” (or “mesenchymal stromal cell” or “MSC”), apart from our study, only one other trial has been registered in CT.gov to assess the applicability of intravenously administered MSCs for osteoporosis treatment: that Phase 1 trial utilized (allogeneic) umbilical cord-derived (UC-derived) MSCs and the trial was suspended (without associated explanation; NCT04501354). To date, no prior study has evaluated use of autoBM-MSCs in treatment of osteoporosis, no prior study has undertaken long-term follow-up (i.e., >1-year post-infusion) of patients that have received MSCs intravascularly for treatment of osteoporosis, and no prior clinical trial has utilized MSCs that have been expressly modified to empower marrow infiltration / colonization upon IV administration.

[0199] Importantly, in all clinical trials to date investigating MSC-based therapy forany medical condition, no study has utilized MSCs derived exclusively from older donors or from a diseased tissue site: this trial is the first to assess adverse events following the IV administration of MSCs obtained entirely from older individuals (i.e., donors >50 years old), and, more specifically, MSCs obtained from a pathological locus (i.e., osteoporotic marrow). Indeed, clinical trials of MSC-based therapeutic indications generally mandate MSCs obtained from healthy, young donors (typically, donors <30 years old). Thus, vigilance was heightened regarding the possibilities of age-associated or disease-related MSC biologic deficits, with highest attention to neoplasia development. Regarding the former, though age-related deficits in human MSC functionality have been reported (including age-associated decline in BM-MSC osteogenic capacity), no patient manifested signs or symptoms of bone abnormalities / atypical fractures, nor ectopic bone formation, nor accentuation of adverse events characteristically associated with the use of antiosteoporotic agents. Regarding the latter, MSCs derived from older patients could harborage-associated “clonal mutations” prompting malignant transformation, and such mutations could also theoretically arise during culture-expansion. Furthermore, MSCs can abet tumorigenicity by contributing to elaboration of the “tumor microenvironment” (TME) that supports tumor growth, tumor immune-evasiveness, and therapy resistance. Importantly, in this trial of MSCs harvested from, and administered to, patients all of whom are >50 years old at enrollment, no patient developed any neoplastic condition; this detail is especially noteworthy because age >45 years is itself the greatest risk factor for neoplasia and the post-infusion clinical observation period exceeded 5 years in all patients.

[0200] Though theoretic concerns exist regarding use of autologous MSCs for treatmentof osteoporosis due to potential age-associated and / or disease-related deficits in their osteoregenerative biology or in their osteogenic progeny, the observed salutary clinical effect following a single infusion of Fuc-autoBM-MSCs refutes this concern. Notably, across all patients, the incidence of fragility fractures dropped precipitously in comparing the time-span of 2-years pre-infusion to 2 years post-infusion (i.e., the protocol-mandated post-infusion evaluation period): specifically, a change in incidence from 7 events / year to 0.5 events / year, reflecting a 93% reduction in fragility fractures (p=0.006)) (Figure 1A). Though larger clinical trials are warranted to more fully assess the effect(s) of Fuc-autoBM-MSC administration on fragility fracture incidence, as compared to all available clinical trial data of current agents used to treat osteoporosis, the observed level of reduction in fragility fracture incidence in this trial is unprecedented. Consistent with patient-reported outcomes showing statistically significant decreases in pain and disability scores following Fuc-autoBM-MSC infusion (Tables 4-5), only one patient (#5) sustained a fragility fracture within the protocol-mandated 2-year evaluation period; notably, this fracture occurred 9-months after denosumab withdrawal, timing consistentwith the well-documented denosumab-withdrawal (“rebound”) fracture occurrence due to osteoclast activation.

[0201] Within the extended surveillance period (i.e., after protocol-mandated 2-yearevaluation period), fragility fractures occurred in just two patients (#5 and #6; see Figure 1A). Thus, the observed reduction in the incidence of fragility fractures was remarkably durable, with a reduction in total fragility fracture rate from 0.52 fractures / year pre-infusion to 0.08 fractures / year post-infusion (p=0.0047; Figure 1B). Importantly, over the entire post-infusion evaluation period (median of >6 years), the two patients (#3 and #7) that never received antiosteoporosis medications did not manifest any fragility fractures, and no fragility fractures occurred in the four patients (#1-4) that received the lower dose of Fuc-autoBM-MSCs (2x106cells / kg). Even more noteworthy, no fragility fractures occurred at any point post-Fuc-autoBM- MSC infusion among those patients >60 years old (#2, #4, #7, #10), offering further evidence that BM-MSCs harvested from older individuals, and from osteoporotic marrow, retain osteoregenerative capability.

[0202] The observed profound and sustained decrease in the incidence of fragility fracturesafter a single infusion of Fuc-autoBM-MSCs merits attention (Figure 1A and 1B), especially since fragility fracture reduction is the foremost goal of all antiosteoporosis therapies. Moreover, reduction in fragility fractures is the most objective measurement of effectiveness for any osteoporosis treatment regimen, and, most importantly, it is the principal metric / end-point for FDA / EMA approval of any proposed new antiosteoporotic therapy. Though this study was focused on safety, some mechanistic insights into this finding may be gleaned. First, the sharp decrease in fracture incidence cannot be attributed to medication modifications (see Table 1 legend) since no fractures occurred within 2 years post-infusion in patients #1, #3, #6, #7 and #8, each of whomhad no medication changes (and patients #3 and #7 never received antiosteoporosis prescription medications post-Fuc-autoBM-MSC infusion). Secondly, there was a statistically significant improvement in trabecular bone microarchitecture (BTA) at 4 months post-infusion (p<0.001), and in volumetric BMD (QTS score) at 24 months (p<0.05). This osteoregenerative profile post- infusion is independent of teriparatide administration: these trends persist upon excluding patients (#4, #9, #10) that received teriparatide, indeed, with increased statistical significance of the 24- month QTS scores (p=0.016 (all patients) versus p=0.004 (teriparatide-excluded patients)). Notably, the QCT (volumetric BMD) is more specific for trabecular bone integrity, whereas DXA is more specific for cortical bone integrity. Though not reaching statistical significance, increases in areal BMD (DXA) and improved T-scores were evident within the lumbar spine following Fuc- autoBM-MSC infusion, with less effect in the femoral neck (Table 3). Regarding bone turnover, the most sensitive and specific bone neoformation biomarker is P1NP, and the mean level of this biomarker increased prominently in 8 / 10 patients, with one patient (#10) having a sharp drop from baseline P1NP levels solely due teriparatide discontinuation at 5.5 months post-Fuc-autoBM-MSC infusion (Figures 2A and 2B).

[0203] Collectively, the results suggest that the administered Fuc-AutoBM-MSCs mediatean osteoregenerative effect predominantly within trabecular bone. This is a critical issue given that trabecular bone is far more metabolically active than cortical bone, and, accordingly, osteoporosis predominantly disintegrates trabecular bone. This beneficial effect could be secondary to direct differentiation of administered MSCs into osteoblasts (as observed in preclinical human-mouse MSC xenotransplantation studies), and / or an MSC-mediated paracrine influence supporting osteoblast integrity / bioactivity and / or an MSC-based anti-osteoclastic effect and / or an MSC- induced anti-inflammatory effect dampening bone injury and thereby sustaining boneneoformation. Though future studies are needed to elucidate the extent to which these putative etiologies contribute to the observed reduction in fragility fractures, the results of this study establish that BM-MSCs from osteoporotic patients are not osteogenically inert.

[0204] Osteoporosis is treatable, yet ~30% of patients that suffer from an osteoporosis-related fracture in the US avoid antiosteoporosis medications, and treatment compliance rates worsen with age. This so-called “osteoporosis treatment gap” highlights the critical unmet medical needs of patients suffering from this disease:_ENREF_2new osteoporosis treatments are desired that are safe, readily adherable / tolerable, and, optimally, could engender durable bone regeneration without necessitating recurrent-dosing, life-long therapy. To date, no clinical studies have examined use of culture-expanded MSCs derived exclusively from older patients for any medical condition, no prior clinical study has utilized culture-expanded MSCs derived from any tissue site harboring any disease, and no prior clinical study has evaluated culture-expanded MSCs that have undergone glycocalyx editing to operationally program E-selectin binding so as to enhance colonization of vascularly administered MSCs at tissue(s) whose endothelial beds express E- selectin. Specifically, the capability to perform precision glycocalyx motif-editing to install sLeX display (i.e., enforce HCELL expression) on MSCs has expressly enabled a heretofore unattainable analysis of the biology of culture-expanded human bone marrow-derived MSCs within human beings suffering from osteoporosis, thereby providing a more accurate and complete understanding, as well as a greater appreciation, of how these cells may transform patient care. The finding that administration of glycocalyx motif-edited human MSCs is feasible and safe, together with the promising clinical responses observed following just a single infusion of Fuc- autoBM-MSCs, provides rationale for conducting larger trials to further evaluate the antiosteoporosis efficacy of this treatment approach. Collectively, the results of this clinical trialraise optimism that a cell-based regenerative therapeutic strategy will be developed that will address the unmet medical needs of the hundreds of millions of women -- and the markedly increasing number of aging men -- that are suffering from osteoporosis.

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[0206] All documents cited in this application are hereby incorporated by reference as ifrecited in full herein. In the event of a conflict between the teachings of this application and those of the incorporated documents, the teachings of this application control.

[0207] The embodiments described in this disclosure can be combined in various ways.Any aspect or feature that is described for one embodiment can be incorporated into any other embodiment mentioned in this disclosure. While various novel features of the inventive principles have been shown, described and pointed out as applied to particular embodiments thereof, it should be understood that various omissions and substitutions and changes can be made by those skilled in the art without departing from the spirit of this disclosure. Those skilled in the art will appreciate that the inventive principles can be practiced in other than the described embodiments, which are presented for purposes of illustration and not limitation.Various Embodiments:

[0208] A method of treating or ameliorating a disease in a subject in need thereofcomprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases together with the pertinent requisite donor nucleotide sugars to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; introducing the glycocalyx-modified MSCs into the subject.

[0209] A method of treating or ameliorating a bone disease or condition in a subject inneed thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from the bone disease or condition; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases together with the pertinent requisite donor nucleotide sugars to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx-modified MSCs are effective for bone regeneration.

[0210] A method making immunomodulatory / restorative / reparative mesenchymal stemcells (MSCs) comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or wherein the subject is greater than 30 years old.

[0211] An immunomodulatory / restorative / reparative mesenchymal stem cell (MSC)produced by the process of: obtaining a population of MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or wherein the subject is greater than 30 years old. USA.617191053.7 / 45S

[0212] A method of preparing a secretome (containing all extracellular vesicles releasedfrom MSCs as well as all trophic / bioactive factors released by the cell) comprising the steps of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or wherein the subject is greater than 30 years old; and harvesting the secretome (e.g., exosomes and other extracellular vesicles) from the MSCs.

[0213] A secretome comprising immunomodulatory / regenerative / reparative / restorativevesicles and bioactive factors produced by the process of: obtaining a population MSCs from a subject; wherein the subject suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or wherein the subject is greater than 30 years old; and harvesting the secretome (e.g., extracellular vesicles) from the MSCs.

[0214] A pharmaceutical composition comprising a population of MSCs produced by theprocess of: (a) obtaining a population of culture-expanded MSCs originating from the subject (i.e., autologously-derived MSCs); (b) modifying the glycocalyx of the culture-expanded MSCs, ex vivo, using glycosyltransferase(s) and attendant nucleotide sugar donor(s) to enforce cell surface expression of sLeX on the MSC surface to produce glycocalyx-modified MSCs; (c) Introducing the glycocalyx-modified MSCs into the subject intravascularly so as to induce a tissue reparative or tissue restorative effect in the affected tissue(s), wherein one or both of the following apply:

[0215] (i) The MSCs are obtained from one of the disease-affected tissues of thesubject

[0216] (ii) The subject is over 30 years of age.

[0217] The method or composition of any preceding embodiment wherein theglycolsyltransferases enforce sLeX on the cell surface molecule CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.

[0218] The method or composition of any preceding embodiment wherein the populationof culture-expanded MSCs is obtained from the bone marrow of the subject suffering from a skeletal disease or condition.

[0219] The method or composition of any preceding embodiment wherein the skeletaldisease or condition is caused by one or more of congenital / genetic diseases, trauma, infection, degenerative diseases, metabolic diseases, neoplastic diseases, medications, iatrogenic effects, dietary deficiencies / malnutrition, endocrinologic conditions, gastrointestinal conditions / malabsorption states, and pregnancy / lactation-induced bone loss.

[0220] The method or composition of any preceding embodiment wherein the skeletaldisease or condition is osteoporosis caused by one or more of an autoimmune disorder, a genetic disorder, digestive / gastrointestinal disorder, metabolic condition, kidney disease, malnutrition, Vitamin D deficiency, medical procedure, medications, cancer, hematologic / blood disorder, neurological / nervous system disorder, bone marrow disorder, endocrine / hormonal disorder, menopause, and pregnancy / lactation.

[0221] The method or composition of any preceding embodiment wherein the skeletalcondition is one or more of fracture and trauma.

[0222] The method or composition of any preceding embodiment wherein the MSCs areglycocalyx-modified ex vivo by exofucosylation.

[0223] The method or composition of any preceding embodiment wherein the MSCs areglycocalyx-modified ex vivo by transfection or transduction with a nucleic acid encoding a fucosyltransferase.

[0224] The method or composition of any preceding embodiment wherein the glycocalyx-modified MSCs are introduced into the subject 1 - 365 times per year.

[0225] The method or composition of any preceding embodiment wherein the subjectsuffers from the bone disease osteoporosis.

[0226] The method or composition of any preceding embodiment wherein the subjectsuffers from osteogenesis imperfecta.

[0227] The method or composition of any preceding embodiment wherein the subjectsuffers from hypophosphatasia.

[0228] The method or composition of any preceding embodiment wherein the subject isgreater than 1 month old.

[0229] The method or composition of any preceding embodiment wherein the glycocalyx-modified MSC is introduced to the subject after the subject suffers from one or more bone fractures or skeletal deformities.

[0230] The method or composition of any preceding embodiment wherein the glycocalyx-modified MSC is introduced to the subject after the subject has suffered an atraumatic fracture of a bone.

[0231] The method or composition of any preceding embodiment where the glycocalyx-modified MSC is introduced to the subject after to subject has suffered an atraumatic fracture of one or more of vertebra, hip, distal forearm / carpal joint, and pelvis.

[0232] The method or composition of any preceding embodiment where the MSC isobtained from the bone marrow of the subject suffering from the skeletal disease and the subject is greater than 1 month old.

[0233] The method or composition of any preceding embodiment wherein the MSC isobtained from osteoporotic bone marrow of the subject.

[0234] The method or composition of any preceding embodiment wherein the MSC isobtained from osteoporotic bone marrow of the subject and the subject is greater than 30 years old.

[0235] The method or composition of any preceding embodiment wherein the MSC isglycocalyx-modified ex vivo by exofucosylation with one or more of a fucosyltransferase VI and fucosyltransferase VII.

[0236] The method or composition of any preceding embodiment, wherein the MSC isglycocalyx-modified ex vivo by transfection with a nucleic acid encoding one or more of a fucosyltransferase VI and fucosyltransferase VII.

[0237] The method or composition of any preceding embodiment, wherein the subject isundergoing one or more osteoporosis treatments.

[0238] The method or composition of any preceding embodiment, wherein the subject isundergoing one or more osteoporosis treatments including calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents / analogs.

[0239] The method or composition of any preceding embodiment, wherein the glycocalyx-modified MSCs are effective to increase plasma levels of bone neoformation marker N-terminal propeptide of type I procollagen (P1NP).

[0240] The method or composition of any preceding embodiment, wherein 0.5x106 to50x106glycocalyx-modified MCSs / kg of recipient body weight is introduced to the subject.

[0241] The method or composition of any preceding embodiment wherein the glycocalyx-modified MSC is introduced to the subject via intravascular administration.

[0242] A method of treating or ameliorating a bone disease or condition in a subject inneed thereof comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the subject, wherein the subject suffers from a skeletal disease or condition, and then differentiating the MSCs in vitro into osteoblasts; modifying the glycocalyx of the osteoblasts, ex vivo, using one or more glycosyltransferases to enforce cell surface expression of sLeX on the osteoblasts to produce glycocalyx-modified osteoblasts; and introducing the glycocalyx-modified osteoblasts into the subject, wherein the glycocalyx-modified osteoblasts are effective for bone regeneration.

[0243] A method for treating a disease in a human subject wherein the endothelial beds ofthe tissue(s) affected by the disease express E-selectin, comprising the steps of: (a) obtaining a population of culture-expanded MSCs originating from the subject (i.e.,autologously-derived MSCs); (b) modifying the glycocalyx of the culture-expanded MSCs, ex vivo, usingglycosyltransferase(s) and attendant nucleotide sugar donor(s) to enforce cell surface expression of sLeX on the MSC surface to produce glycocalyx-modified MSCs; (c) Introducing the glycocalyx-modified MSCs into the subject intravascularly so as toinduce a tissue reparative or tissue restorative effect in the affected tissue(s), wherein one or both of the following apply: (i) The MSCs are obtained from one of the disease-affected tissues of the subject(ii) The subject is over 30 years of age

[0244] The method or composition of any preceding embodiment wherein the subject isgreater than 40 years old.

[0245] The method or composition of any preceding embodiment wherein the subject isgreater than 50 years old.

[0246] The method or composition of any preceding embodiment wherein the subject isgreater than 60 years old.

[0247] The method or composition of any preceding embodiment wherein the subject isgreater than 70 years old.

[0248] The method or composition of any preceding embodiment wherein the subject isgreater than 80 years old.

[0249] The method or composition of any preceding embodiment wherein the subject isgreater than 90 years old.

[0250] The method or composition of any preceding embodiment wherein the MSCs havethe functional immunomodulatory / restorative / regenerative / reparative attributes of MSCs from younger subjects (i.e. less than 30 years old) and / or healthy subjects.

Claims

What is claimed is:

1. A method of treating a disease in a subject in need thereof, wherein the endothelial beds of the diseased tissue express E-selectin, comprising the steps of: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from the diseased tissue of the subject; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject.

2. The method of claim 1, wherein the disease is a skeletal disease or condition.

3. The method of claim 2, wherein the skeletal disease or condition is osteoporosis.

4. The method of claim 1, wherein modifying the glycocalyx of the MSCs comprises exofucosylation using one or more fucosyltransferases.

5. The method of claim 4, wherein the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.

6. The method of claim 1, wherein introducing the glycocalyx-modified MSCs comprises intravascular administration.

7. The method of claim 6, wherein 0.5x106to 50x106glycocalyx-modified MSCs / kg of recipient body weight are introduced to the subject.

8. The method of claim 1, wherein the subject is undergoing one or more treatments for the disease in addition to the introduction of glycocalyx-modified MSCs.

9. A method of treating a skeletal disease in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from diseased tissue of the subject, wherein the subject suffers from the skeletal disease; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject, wherein the glycocalyx- modified MSCs are effective for skeletal regeneration.

10. The method of claim 9, wherein the skeletal disease is osteoporosis.

11. The method of claim 9, wherein modifying the glycocalyx of the MSCs comprises exofucosylation using one or more of fucosyltransferases.

12. The method of claim 11, wherein the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.

13. The method of claim 9, wherein introducing the glycocalyx-modified MSCs comprises intravascular administration.

14. The method of claim 13, wherein 0.5x106to 50x106glycocalyx-modified MSCs / kg of recipient body weight are introduced to the subject.

15. The method of claim 9, wherein the subject is undergoing one or more treatments for the skeletal disease in addition to the introduction of glycocalyx-modified MSCs.

16. The method of claim 15, wherein the one or more treatments include at least one of calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents.

17. A method of treating a disease in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from a subject older than 30 years; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on the MSCs to produce glycocalyx-modified MSCs; and introducing the glycocalyx-modified MSCs into the subject.

18. The method of claim 17, wherein the subject is older than 50 years.

19. The method of claim 17, wherein modifying the glycocalyx of the MSCs comprises exofucosylation using one or more fucosyltransferases.

20. The method of claim 19, wherein the exofucosylation enforces sLeX expression on CD44 to produce Hematopoietic Cell E- / L-selectin Ligand (HCELL) on the glycocalyx-modified MSCs.

21. The method of claim 17, wherein introducing the glycocalyx-modified MSCs comprises intravascular administration.

22. The method of claim 21, wherein 0.5x106to 50x106glycocalyx-modified MSCs / kg of recipient body weight are introduced to the subject.

23. The method of claim 17, wherein the disease is a skeletal disease or condition.

24. The method of claim 23, wherein the skeletal disease or condition is osteoporosis.

25. A method of treating osteoporosis in a subject in need thereof comprising: obtaining a population of culture-expanded mesenchymal stem cells (MSCs) from a subject older than 30 years; modifying the glycocalyx of the MSCs, ex vivo, using glycosyltransferases to enforce cell surface expression of sLeX on CD44 molecules of the MSCs to produce glycocalyx- modified MSCs expressing HCELL; andintroducing the glycocalyx-modified MSCs into the subject via intravascular administration.

26. The method of claim 25, wherein the glycosyltransferases comprise one or more fucosyltransferases.

27. The method of claim 26, wherein modifying the glycocalyx of the MSCs comprises exofucosylation using the one or more of fucosyltransferase VI and fucosyltransferase VII.

28. The method of claim 25, wherein 0.5x106to 50x106glycocalyx-modified MSCs / kg of recipient body weight are introduced to the subject.

29. The method of claim 25, wherein the subject is undergoing one or more additional treatments for osteoporosis.

30. The method of claim 29, wherein the one or more additional treatments include at least one of calcium supplementation, Vitamin D supplementation, bisphosphonates, denosumab, romosozumab, and hormonal agents.

31. A method making immunomodulatory / restorative / reparative mesenchymal stem cells (MSCs) comprising the steps of:obtaining a population of MSCs from a subject; wherein the subject: (1) suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or (2) wherein the subject is greater than 30 years old.

32. A method of preparing a secretome comprising the steps of: obtaining a population of MSCs from a subject; wherein the subject: (1) suffers from a disease or condition and the MSCs are obtained from the diseased or affected tissue; and / or (2) wherein the subject is greater than 30 years old; and harvesting the secretome (e.g. extracellular vesicles) from the MSCs.

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