WO2015004609A2

WO2015004609A2 - Adherent cells from placenta and use thereof in treatment of injured tendons

Info

Publication number: WO2015004609A2
Application number: PCT/IB2014/062963
Authority: WO
Inventors: Zami Aberman; Rachel Ofir; Nirit Drori-Carmi
Original assignee: Pluristem Ltd.
Priority date: 2013-07-09
Filing date: 2014-07-09
Publication date: 2015-01-15
Also published as: WO2015004609A3

Abstract

A method of treating injured tendons in a subject in need thereof is disclosed. The method comprises administering to the subject a therapeutically effective amount of placenta- derived adherent stromal cells.

Description

ADHERENT CELLS FROM PLACENTA AND USE

THEREOF IN TREATMENT OF INJURED TENDONS

FIELD

Disclosed herein are methods of treating diseases using adherent cells from placenta, more specifically treating tendon injuries and sequelae thereof, using the adherent cells.

BACKGROUND

In the developing medical world, a growing need exists for large amounts of adult stem cells for the purpose of cell engraftment and tissue engineering. In addition, adult stem cell therapy is continuously developing for treating and curing various conditions such as hematopoietic disorders, heart disease, Parkinson's disease, Alzheimer's disease, stroke, burns, muscular dystrophy, autoimmune disorders, diabetes and arthritis.

Mesenchymal stem cells (MSCs), also referred to as mesenchymal stromal cells or marrow stromal cells, have been from many adult tissues, such as placenta, bone marrow and adipose tissue. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed minimal criteria to define human MSCs (Dominici et al, 2006): 1. Plastic adherence when maintained in standard culture conditions (a minimal essential medium plus 20% fetal bovine serum (FBS)). 2. Expression of CD105, CD73 and CD90, and lack of expression of CD45, CD34, CD14 or CDl lb, CD79a or CD19 and HLA- DR surface molecules. 3. Differentiation into osteoblasts, adipocytes and chondroblasts in vitro.

In recent years, considerable activity has focused on the therapeutic potential of MSCs for various medical applications, including tissue repair of damaged organs such as the brain, heart, bone and liver, and in support of bone marrow transplantations. MSCs may be obtained from sources such as bone marrow, adipose tissue, placenta, and blood. Certain populations of MSC are capable of differentiating into different types of mesenchymal mature cells (e.g. reticular endothelial cells, fibroblasts, adipocytes, osteogenic precursor cells), depending upon environmental cues. Accordingly, MSCs have been widely studied in regenerative medicine for generation of tissues such as bone, cartilage and fat, for replacing injured or pathologic tissues, and as treatments for genetic and acquired diseases [Fibbe and Noort, Ann N Y Acad Sci (2003) 996: 235-44; Horwitz et al., Cytotherapy (2005) 7(5): 393-5; Zimmet and Hare, Basic Res Cardiol (2005) 100(6): 471-81].

The Applicant has previously devised three-dimensional (3D) culturing conditions suitable for expansion of placental-derived MSCs, which are described in PCT Publ. No. WO 2007/108003, which is fully incorporated herein by reference in its entirety.

Orthopedic applications of MSCs

Various conditions and pathologies require connective tissue (e.g., bone, tendon and ligament) regeneration and/or repair. These include, for example, bone fractures, burns, burn wound, deep wound, degenerated bone, various cancers associated with connective tissue loss (e.g., bone cancer, osteosarcoma, bone metastases), and articular cartilage defects.

The use of autologous BM-MSCs to enhance bone healing has been described for veterinary and human orthopedic applications and include percutaneous injection of bone marrow for ligament healing (Carstanjen et al., 2006), treatment of bone defects by autografts or allografts of bone marrow in orthopedic clinic (Horwitz et al., 1999, Horwitz et al., 2002), regeneration of critical-sized bone defect in dogs using allogeneic [Arinzeh TL, et al., J Bone Joint Surg Am. 2003, 85-A(10): 1927-35] or autologous [Bruder SP, et al., J Bone Joint Surg Am. 1998 Jul;80(7):985-96] bone marrow-MSCs loaded onto ceramic cylinder consisting of hydroxyapatite-tricalcium phosphate, or in rabbit using allogeneic peripheral blood derived MSCs (Chao et al., 2006.), and extensive bone formation using MSCs implantation in baboon (Livingston et al, 2003).

Rabbit models for injured tendons showed that stromal cell-treated tissues were stronger and stiffer than natural repaired tissues (Gordon et al., 2005). In addition, seeding of cultured MSCs into a tendon gap resulted in significantly improved repair biomechanics (Young et al., 1998, Osiris Therapeutics, www.osiris.com).

PCT Pub. No. WO 2009/037690 discloses treatment of medical conditions requiring connective tissue repair by administration of MSC that are capable of differentiating into osteocytes and osteoblasts.

SUMMARY

It has been discovered that certain populations of adherent cells derived from placenta are capable of increasing proteoglycan content, increasing collagen content, and decreasing inflammation in injured tendons. In certain embodiments, the cells are mesenchymal-like adherent stromal cells, which exhibit a marker pattern similar to mesenchymal stromal cells, but do not differentiate into osteocytes, under conditions where "classical" mesenchymal stem cells would differentiate into osteocytes. In other embodiments, the cells are mesenchymal- like adherent stromal cells, which exhibit a marker pattern similar to mesenchymal stromal cells, but do not differentiate into adipocytes, under conditions where mesenchymal stem cells would differentiate into adipocytes. In still other embodiments, the cells are mesenchymal- like adherent stromal cells, which exhibit a marker pattern similar to mesenchymal stromal cells, but do not differentiate into osteocytes or adipocytes, under conditions where mesenchymal stem cells would differentiate into osteocytes or adipocytes, respectively. Alternatively or in addition, the cells have been cultured in 2-dimensional (2D) culture, followed by 3-dimensional culture.

According to some embodiments, the tendon injury or disorder is selected from tendon degeneration, tendon strain, tendinitis, tendinosis, and tenosynovitis, each of which is considered a separate embodiment. In certain embodiments, the tendon degeneration is a result of tendon strain.

Unless otherwise indicated, all ranges mentioned herein are inclusive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the embodiments of the invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a diagram of a bioreactor that can be used to prepare the cells. FIG. 2 depicts gross rat patellar tendon appearance following collagenase injury, treatment with adherent stromal cells, and tendon harvest at 1, 2, and 4 weeks after cell treatment. In each of panels A-C, a stromal cell-treated (hereinafter "treated") tendon is shown at left, and a tendon treated with vehicle (saline) alone (hereinafter "untreated") is shown at right.

FIG. 3 shows fluorescent microscopy images of CFSE-labeled adherent cells in rat patellar tendons 4 days (panel A), 2 weeks (panel B), and 4 weeks (panel C) after administration of the cells following collagenase injury. Clusters of fluorescent cell-like signals are visible up to four weeks after administration of the cells. At each harvest time- point, values for treated and untreated tendons are shown by the left and right bars, respectively.

FIG. 4 is a graph of load-to-failure values (measured in N) of rat patellar tendons injured by collagenase treatment, treated with stromal cells, and then harvested one week, two weeks, and four weeks after administration of the cells. At each harvest time-point, values for treated and untreated tendons are shown by the left and right bars, respectively.

FIG. 5 is a graph of stiffness over time (measured in Newtons/millimeter [N/mm]) of rat patellar tendons injured by collagenase treatment, treated with stromal cells, and then harvested 1, 2, and 4 weeks after administration of the cells. At each harvest time-point, values for treated and untreated tendons are shown by the left and right bars, respectively.

FIG. 6 depicts safranin-O-stained sections of rat patellar tendons injured by collagenase and treated with stromal cells. Proteoglycan content (red staining [or darker staining in black and white]) at labeled insertion sites was greater in treated tendons (panel A) than in untreated tendons (panel B).

FIG. 7 depicts picrosirius-stained tendon sections imaged by polarized microscopy. Four weeks after treatment with cells, treated samples (panel A) demonstrated greater collagen content at the tendon insertion site than untreated controls (panel B).

FIG. 8 depicts imaging analysis of proteoglycan staining of tendons treated with stromal cells or saline. Treated tendons demonstrated a greater mean area of proteoglycan content at each time point.

FIG. 9 depicts imaging analysis of collagen content of tendons treated with stromal cells or saline. Treated tendons demonstrated a greater mean area of collagen content at each time point.

FIG. 10 depicts results of gene expression analysis of inflammatory cytokines IL-6 (panel A), ILlb (panel B), MMP3 (panel C), and MMP13 (panel D) performed on rat tendons harvested 4 days or 1, 2, or 4 weeks following injection of stromal cells after tendon injury with collagenase.

DETAILED DESCRIPTION

Provided herein are methods of treating injured tendons, using adherent cells derived from placenta. In certain embodiments, the adherent cells are adherent stromal cells.

The principles and operation of the invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

As is described herein, placenta-derived adherent cells were expanded (in other words, cultured in order to increase their numbers) and were found to be viable, following cryo- preservation, as evidenced for example by adherence and re-population assays. Flow cytometry analysis of placenta-derived adherent cells revealed a distinct marker expression pattern as is described further herein.

In some embodiments, there is provided a method of increasing the proteoglycan content in an injured tendon in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the adherent stromal cells.

In other embodiments is provided a method of increasing the collagen content in an injured tendon in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the adherent stromal cells.

In other embodiments is provided a method of decreasing inflammation in an injured tendon in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the adherent stromal cells.

In other embodiments is provided a composition comprising adherent stromal cells for increasing proteoglycan content in an injured tendon.

In other embodiments is provided a composition comprising adherent stromal cells for increasing collagen content in an injured tendon.

In other embodiments is provided a composition comprising adherent stromal cells for decreasing inflammation in an injured tendon. In further embodiments, the adherent stromal cells increase the expression levels of IL-6, ILlb, MMP3, or MMP13 in the injured tendon. In other, the cells increase the levels of IL-6 and ILlb; IL-6 and MMP3; IL-6 and MMP13; ILlb and MMP3; ILlb and MMP13; MMP3 and MMP13; IL-6, ILlb, and MMP3; IL-6, ILlb, and MMP13; IL-6, ILlb, and MMP13; ILlb, MMP3, and MMP13; or IL-6, ILlb, MMP3, and MMP13.

In other embodiments, there is provided a method of increasing the expression levels of IL-6, ILlb, MMP3, and/or MMP13 in an injured tendon in a subject in need thereof. In other embodiments is provided a method of increasing the levels of IL-6 and ILlb; IL-6 and MMP3; IL-6 and MMP13; ILlb and MMP3; ILlb and MMP13; MMP3 and MMP13; IL-6, ILlb, and MMP3; IL-6, ILlb, and MMP13; IL-6, ILlb, and MMP13; ILlb, MMP3, and MMP13; or IL-6, ILlb, MMP3, and MMP13, in an injured tendon in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the adherent stromal cells.

In some embodiments, the described injured tendon is a patellar tendon.

Alternatively or in addition, the stromal cells are administered within 6 days, in other embodiments within 5 days, in other embodiments within 4 days, in other embodiments within 3 days, in other embodiments within 2 days, or in other embodiments within 1 day of the injury.

Unless indicated otherwise herein, the terms "placenta", "placental tissue", and the like refer to any portion of the placenta. Placenta-derived adherent cells may be obtained, in various embodiments, from either fetal or, in other embodiments, maternal parts of the placenta, or in other embodiments, from both parts. More specific embodiments of maternal sources are the decidua basalis and the decidua parietalis. More specific embodiments of fetal sources are the amnion, the chorion, and the villi. Tissue specimens are washed in a physiological buffer [e.g., phosphate -buffered saline (PBS) or Hank's buffer]. Single-cell suspensions are made, in certain embodiments, by treating the tissue with a digestive enzyme (see below) or/and mincing and flushing the tissue parts through a nylon filter or by gentle pipetting (Falcon, Becton, Dickinson, San Jose, CA) with washing medium. In some embodiments, the tissue treatment includes use of a DNAse, a non-limiting example of which is Benzonase from Merck.

In other embodiments, at least 70% of the described cells are from the maternal portion of the placenta. In yet other embodiments, at least 70% of the described cells are from the fetal portion of the placenta. Placental cells may be obtained from a full-term or pre-term placenta. In some embodiments, residual blood is removed from the placenta before cell harvest. This may be done by a variety of methods known to those skilled in the art, for example by perfusion. The term "perfuse" or "perfusion" as used herein refers to the act of pouring or passaging a fluid over or through an organ or tissue. In certain embodiments, the placental tissue may be from any mammal, while in other embodiments, the placental tissue is human. A convenient source of placental tissue is a post-partum placenta (e.g., less than 10 hours after birth), however, a variety of sources of placental tissue or cells may be contemplated by the skilled person. In other embodiments, the placenta is used within 8 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, or within 1 hour of birth. In certain embodiments, the placenta is kept chilled prior to harvest of the cells. In other embodiments, prepartum placental tissue is used. Such tissue may be obtained, for example, from a chorionic villus sampling or by other methods known in the art. Once placental cells are obtained, they are, in certain embodiments, allowed to adhere to an adherent material (e.g., configured as a surface) to thereby isolate adherent cells. In some embodiments, the donor is 35 years old or younger, while in other embodiments, the donor may be any woman of childbearing age.

Placenta-derived adherent stromal cells can be propagated, in some embodiments, using 2D or 3D culturing conditions, or in other embodiments, using a combination of 2D and 3D culturing conditions. Conditions for propagating adherent stromal cells in 2D and 3D culture are further described hereinbelow and in the Examples section which follows.

The phrase "two-dimensional culture" refers to a culture in which the cells are exposed to conditions that are compatible with cell growth and allow the cells to grow in a monolayer. Such apparatuses will typically have flat growth surfaces, in some embodiments comprising an adherent material, which may be flat or curved. Non-limiting examples of apparatuses for 2D culture are cell culture dishes and plates. Included in this definition are multi-layer trays, such as Cell Factory™, manufactured by Nunc™, provided that each layer supports monolayer culture. It will be appreciated that even in 2D apparatuses, cells can grow over one another when allowed to become overconfluent. This does not affect the classification of the apparatus as "two-dimensional".

The phrase "three-dimensional culture" refers to a culture in which the cells are exposed to conditions that are compatible with cell growth and allow the cells to grow in a 3D orientation relative to one another. Such apparatuses will typically have a 3D matrix of growth surfaces, in some embodiments comprising an adherent material. In various embodiments, "an adherent material" refers to a material that is synthetic, naturally occurring, or a combination thereof. In certain embodiments, the material is non- cytotoxic (or, in other embodiments, is biologically compatible). In still other embodiments, the material exhibits a chemical structure such as charged surface exposed groups, which allows cell adhesion. Non-limiting examples of adherent materials which may be used in accordance with this aspect include a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, Matrigel™, an extra-cellular matrix component (e.g., Fibronectin, Chondronectin, Laminin), a collagen, a poly-L-lactic acid, and an inert metal fiber. In more particular embodiments, the material may be selected from a polyester and a polypropylene.

According to an embodiment of the present invention, 3D culturing is performed for at least 4 doublings, at least 5 doublings, at least 6 doublings, at least 7 doublings, at least 8 doublings, at least 9 doublings, or at least 10 doublings. In certain embodiments, cells are typically passaged when the culture reaches about 70-90% confluence, typically after 3-5 days (e.g., 1-3 doublings).

In some embodiments 3D culturing can be performed in a 3D bioreactor. In some embodiments, the 3D bioreactor comprises a container for holding a 3-dimensional attachment surface and a control apparatus, for controlling pH, temperature, and oxygen levels and optionally other parameters. Alternatively or in addition, the bioreactor contains ports for the inflow and outflow of fresh medium and gases.

Examples of such bioreactors include, but are not limited to, a plug-flow bioreactor, a continuous stirred tank bioreactor, a stationary-bed bioreactor, a CelliGen Plus® bioreactor system (New Brunswick Scientific (NBS) or a BIOFLO 310 bioreactor system (New Brunswick Scientific (NBS).

As provided herein, a 3D bioreactor is capable of 3D expansion of adherent stromal cells under controlled conditions (e.g. pH, temperature and oxygen levels) and with growth medium perfusion, which in some embodiments is constant perfusion and in other embodiments is adjusted in order to maintain target levels of glucose or other parameters,. Furthermore, the cell cultures can be directly monitored for concentrations of glucose, lactate, glutamine, glutamate and ammonium. The glucose consumption rate and the lactate formation rate of the adherent cells enable, in some embodiments, measurement of cell growth rate and determination of the harvest time. In some embodiments, a continuous stirred tank bioreactor is used, where a culture medium is continuously fed into the bioreactor and a product is continuously drawn out, to maintain a time-constant steady state within the reactor. A stirred tank bioreactor with a fibrous bed basket is available for example from New Brunswick Scientific Co., Edison, NJ). Additional bioreactors that may be used, in some embodiments, are stationary-bed bioreactors; and air-lift bioreactors, where air is typically fed into the bottom of a central draught tube flowing up while forming bubbles, and disengaging exhaust gas at the top of the column. Additional possibilities are cell-seeding perfusion bioreactors with polyactive foams [as described in Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)] and radial-flow perfusion bioreactors containing tubular poly-L-lactic acid (PLLA) porous scaffolds [as described in Kitagawa et al., Biotechnology and Bioengineering 93(5): 947-954 (2006). Other bioreactors which can be used are described in U.S. Pat. Nos. 6,277,151; 6,197,575; 6,139,578; 6,132,463; 5,902,741; and 5,629,186, which are incorporated herein by reference.

Another exemplary bioreactor, the Celligen 310 Bioreactor, is depicted in Figure 1. A Fibrous-Bed Basket (16) is loaded with polyester disks (10). In some embodiments, the vessel is filled with deionized water or isotonic buffer via an external port (1 [this port may also be used, in other embodiments, for cell harvesting]) and then optionally autoclaved. In other embodiments, following sterilization, the liquid is replaced with growth medium, which saturates the disk bed as depicted in (9). In still further embodiments, temperature, pH, dissolved oxygen concentration, etc., are set prior to inoculation. In yet further embodiments, a slow stirring initial rate is used to promote cell attachment, then agitation is increased. Alternatively or addition, perfusion is initiated by adding fresh medium via an external port (2). If desired, metabolic products may be harvested from the cell-free medium above the basket (8). In some embodiments, rotation of the impeller creates negative pressure in the draft-tube (18), which pulls cell-free effluent from a reservoir (15) through the draft tube, then through an impeller port (19), thus causing medium to circulate (12) uniformly in a continuous loop. In still further embodiments, adjustment of a tube (6) controls the liquid level; an external opening (4) of this tube is used in some embodiments for harvesting. In other embodiments, a ring sparger (not visible), is located inside the impeller aeration chamber (11), for oxygenating the medium flowing through the impeller, via gases added from an external port (3) , which may be kept inside a housing (5), and a sparger line (7). Alternatively or in addition, sparged gas confined to the remote chamber is absorbed by the nutrient medium, which washes over the immobilized cells. In still other embodiments, a water jacket (17) is present, with ports for moving the jacket water in (13) and out (14). In some embodiments, cells are produced using a packed-bed spinner flask. In more specific embodiments, the packed bed may comprise a spinner flask and a magnetic stirrer. The spinner flask may be fitted, in some embodiments, with a packed bed apparatus similar to the Celligen™ Plug Flow bioreactor which is, in certain embodiments, packed with Fibra-cel® (or, in other embodiments, other carriers). The spinner is, in certain embodiments, batch fed (or in other alternative embodiments fed by perfusion), fitted with one or more sterilizing filters, and placed in a tissue culture incubator. In further embodiments, cells are seeded onto the scaffold by suspending them in medium and introducing the medium to the apparatus. In still further embodiments, the agitation speed is gradually increased, for example by starting at 40 RPM for 4 hours, then gradually increasing the speed to 120 RPM. In certain embodiments, the glucose level of the medium may be tested periodically (i.e. daily), and the perfusion speed adjusted maintain an acceptable glucose concentration. In yet other embodiments, at the end of the culture process, carriers are removed from the packed bed, washed with isotonic buffer, and processed or removed from the carriers by agitation and/or enzymatic digestion.

In certain embodiments, the bioreactor is seeded at a concentration of between 10,000 - 2,000,000 cells / ml of medium, in other embodiments 20,000-2,000,000 cells / ml, in other embodiments 30,000-1,500,000 cells / ml, in other embodiments 40,000-1,400,000 cells / ml, in other embodiments 50,000-1,300,000 cells / ml, in other embodiments 60,000-1,200,000 cells / ml, in other embodiments 70,000-1,100,000 cells / ml, in other embodiments 80,000- 1,000,000 cells / ml, in other embodiments 80,000-900,000 cells / ml, in other embodiments 80,000-800,000 cells / ml, in other embodiments 80,000-700,000 cells / ml, in other embodiments 80,000-600,000 cells / ml, in other embodiments 80,000-500,000 cells / ml, in other embodiments 80,000-400,000 cells / ml, in other embodiments 90,000-300,000 cells / ml, in other embodiments 90,000-250,000 cells / ml, in other embodiments 90,000-200,000 cells / ml, in other embodiments 100,000-200,000 cells / ml, in other embodiments 110,000- 1,900,000 cells / ml, in other embodiments 120,000-1,800,000 cells / ml, in other embodiments 130,000-1,700,000 cells / ml, in other embodiments 140,000-1,600,000 cells / ml.

In still other embodiments, between 1-20 x 10⁶ cell / gr carrier are seeded, or in other embodiments 1.5-20 x 10⁶ cell / gr carrier, or in other embodiments 1.5-18 x 10⁶ cell / gr carrier, or in other embodiments 1.8-18 x 10⁶ cell / gr carrier, or in other embodiments 2-18 x 10⁶ cell / gr carrier, or in other embodiments 3-18 x 10⁶ cell / gr carrier, or in other embodiments 2.5-15 x 10⁶ cell / gr carrier, or in other embodiments 3-15 x 10⁶ cell / gr carrier, or in other embodiments 3-14 x 10⁶ cell / gr carrier, or in other embodiments 3-12 x 10⁶ cell / gr carrier, or in other embodiments 3.5-12 x 10⁶ cell / gr carrier, or in other embodiments 3-10 x 10⁶ cell / gr carrier, or in other embodiments 3-9 x 10⁶ cell / gr carrier, or in other embodiments 4-9 x 10⁶ cell / gr carrier, or in other embodiments 4-8 x 10⁶ cell / gr carrier, or in other embodiments 4-7 x 10⁶ cell / gr carrier, or in other embodiments 4.5-6.5 x 10⁶ cell / gr carrier.

In certain embodiments, the harvest from the bioreactor is performed when at least about 10%, in other embodiments at least 12%, in other embodiments at least 14%, in other embodiments at least 16%, in other embodiments at least 18%, in other embodiments at least 20%, in other embodiments at least 22%, in other embodiments at least 24%, in other embodiments at least 26%, in other embodiments at least 28%, or in other embodiments at least 30%, of the cells are in the S and G2/M phases (collectively), as can be assayed by various methods known in the art, for example FACS detection. Typically, in the case of FACS, the percentage of cells in S and G2/M phase is expressed as the percentage of the live cells, after gating for live cells, for example using a forward scatter/side scatter gate. Those skilled in the art will appreciate that the percentage of cells in these phases correlates with the percentage of proliferating cells. In some cases, allowing the cells to remain in the bioreactor significantly past their logarithmic growth phase causes a reduction in the number of cells that are proliferating.

In other embodiments, the length of culturing in the bioreactor is at least 4 days; between 4-12 days; in other embodiments between 4-11 days; in other embodiments between 4-10 days; in other embodiments between 4-9 days; in other embodiments between 5-9 days; in other embodiments between 5-8 days; in other embodiments between 6-8 days; or in other embodiments between 5-7 days.

In certain embodiments, further steps of purification or enrichment for adherent stromal cells may be performed. Such methods include, but are not limited to, FACS using adherent stromal cell marker expression. In other embodiments, the described cells have not been subject to any type of cell sorting in the process used to isolate them.

Those skilled in the art will appreciate that a variety of isotonic buffers may be used for washing cells and similar uses. Hank's Balanced Salt Solution (HBSS; Life Technologies) is only one of many buffers that may be used.

Non-limiting examples of base media useful in 2D and 3D culturing include Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), FIO(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton- Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle' s salt base), Dulbecco's Modified Eagle Medium (DMEM- without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium Ml 99 (M199E-with Earle' s sale base), Medium Ml 99 (M199H-with Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with Earle' s salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with non essential amino acids), among numerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. In certain embodiments, DMEM is used. These and other useful media are available from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel, among others.

In some embodiments, the medium may be supplemented with additional substances. Non-limiting examples of such substances are serum, which is, in some embodiments, fetal serum of cows or other species. Alternatively or in addition, the medium may be supplemented by growth factors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g. B- glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g., growth hormone, erythropoietin, thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophage colony stimulating factor, c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, insulin-like growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, cilary neurotrophic factor, platelet-derived growth factor, and bone morphogenetic protein.

It will be appreciated that additional components may be added to the culture medium. Such components may be antibiotics, antimycotics, albumin, amino acids, and other components known to the art for the culture of cells.

It will also be appreciated that in certain embodiments, when the described adherent stromal cells are intended for administration to a human subject, the cells and the culture medium (e.g., with the above described medium additives) are substantially xeno-free, i.e., devoid of any animal contaminants e.g., mycoplasma. For example, the culture medium can be supplemented with a serum-replacement, human serum and/or synthetic or recombinantly produced factors.

In some embodiments, the described cells do not differentiate into osteocytes, under conditions where "classical" mesenchymal stem cells would differentiate into osteocytes. In some embodiments, the conditions are incubation with a solution containing 0.1 mcM dexamethasone, 0.2 mM ascorbic acid, and 10 mM glycerol-2-phosphate, in plates coated with vitronectin and collagen, for 17 days. In still other embodiments, the conditions are incubation with a solution containing 10 mcM dexamethasone, 0.2 mM ascorbic acid, 10 mM glycerol-2-phosphate, and ΙΟηΜ Vitamin D, in plates coated with vitronectin and collagen, for 26 days. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art.

In other embodiments, the described cells do not differentiate into adipocytes, under conditions where mesenchymal stem cells would differentiate into adipocytes. In some embodiments, the conditions are incubation of adipogenesis induction medium, namely a solution containing 1 mcM dexamethasone, 0.5 mM 3-Isobutyl-l-methylxanthine (IBMX), 10 mcg/ml insulin, and 100 mcM indomethacin, added fresh on days 1, 3, 5, 9, 11, 13, 17, 19, and 21, while the medium is replaced with adipogenesis maintenance medium, namely a solution containing 10 mcg/ml insulin, on days 7 and 15, for a total of 25 days. In still other embodiments, a modified adipogenesis induction medium, containing 1 mcM dexamethasone, 0.5 mM IBMX, 10 mcg/ml insulin, and 200 mcM indomethacin is used, and the incubation is for a total of 26 days. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art.

In certain embodiments, in vitro, the described cells stimulate endothelial cell proliferation, or in another embodiment inhibit T cell proliferation, or in another embodiment both. In other embodiments, in vivo, the cells stimulate angiogenesis, or in another embodiment exhibit immunosuppressive activity (in some embodiments, particularly for T cell responses), and or in another embodiment support hematopoietic stem cell engraftment, or in other embodiments any 2 of the above in vivo characteristics, or in other embodiments all 3 of the above in vivo characteristics. Each combination is considered to be a separate embodiment.

As provided herein, the described cells suppress the immune reaction of human cord blood mononuclear cells in a mixed lymphocyte reaction (MLR) assay, thus exhibiting T cell suppression activity and supporting hematopoietic stem cell engraftment. In certain embodiments, the cells are capable of suppressing immune reaction in a subject. In certain embodiments, the immunosuppressive activity comprises an inhibition of T cell proliferation.

According to some embodiments, the adherent stromal cells are capable of suppressing an immune reaction in the subject. Methods of determining the immunosuppressive capability of a cell population are well known to those skilled in the art. For example, a mixed lymphocyte reaction (MLR) may be performed. In an exemplary, non- limiting MLR assay, cord blood (CB) mononuclear cells, for example human cells or cells from another species, are incubated with irradiated cord blood cells (iCB), peripheral blood- derived monocytes (PBMC; for example human PBMC or PBMC from another species), in the presence or absence of a cell population to be tested. CB cell replication, which correlates with the intensity of the immune response, can be measured by a variety of methods known in the art, for example by H-thymidine uptake. Reduction of the immune response when co- incubated with test cells indicates an immunosuppressive capability. Alternatively, a similar assay can be performed with peripheral blood (PB)-derived MNC, in place of CB cells. As an additional alternative, secretion of pro-inflammatory and anti-inflammatory cytokines upon co-culture with CB cells or PBMC can be measured, in the presence or absence of the sample cell population.

As also provided herein in the Example section, the described adherent cells are capable, in certain embodiments, of increasing proteoglycan content in injured tendons. In other embodiments, the cells are capable of increasing collagen content in injured tendons. In still other embodiments, the cells are capable of decreasing inflammation in injured tendons. In yet other embodiments, the cells are capable of increasing both proteoglycan content and collagen content in injured tendons. In yet other embodiments, the cells are capable of both increasing proteoglycan content and decreasing inflammation in injured tendons. In other embodiments, the cells are capable of both increasing collagen content and decreasing inflammation in injured tendons. In still other embodiments, the cells are capable of increasing proteoglycan content, increasing collagen content, and decreasing inflammation in injured tendons

In other embodiments, the described cells exhibit a spindle shape when cultured under 2D conditions. Alternatively or additionally, the cells may express a marker or a collection of markers (e.g. surface marker) characteristic of mesenchymal stem cells. Examples of stromal cell surface markers include but are not limited to CD 105 (UniProtKB Accession No. P17813), CD29 (UniProtKB Accession No. P05556), CD44 (UniProtKB Accession No. P16070), CD73 (UniProtKB Accession No. P21589), and CD90 (UniProtKB Accession No. P04216). Examples of markers expected to be absent from stromal cells are CD3 (UniProtKB Accession Nos. P09693 [gamma chain] P04234 [delta chain], P07766 [epsilon chain], and P20963 [zeta chain]), CD4 (UniProtKB Accession No. P01730), CD34 (UniProtKB Accession No. P28906), CD45 (UniProtKB Accession No. P08575), CD80 (UniProtKB Accession No. P33681), CD19 (UniProtKB Accession No. P15391), CD5 (UniProtKB Accession No. P06127), CD20 (UniProtKB Accession No. PI 1836), CD11B (UniProtKB Accession No. PI 1215), CD14 (UniProtKB Accession No. P08571), CD79-alpha (UniProtKB Accession No. B5QTD1), and HLA-DR (UniProtKB Accession Nos. P04233 [gamma chain], P01903 [alpha chain], and P01911 [beta chain]). All UniProtKB entries were accessed on July 7, 2014. Those skilled in the art will appreciate that the presence of complex antigens such as CD3 and HLA-DR may be detected by antibodies recognizing any of their component parts, such as, but not limited to, those described herein.

Other stromal cell markers include but are not limited to D7-fib.

In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells. In still other embodiments, each of CD44, CD73, CD29, and CD105 is expressed by more than 90% of the cells. In yet other embodiments, each of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells. In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells, and each of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells. In other embodiments, each of CD44, CD73, CD29, and CD105 is expressed by more than 90% of the cells, and each of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells.

In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells; and the cells do not differentiate into osteocytes, after incubation with a solution containing 0.1 mcM dexamethasone, 0.2 mM ascorbic acid, and 10 mM glycerol-2- phosphate, in plates coated with vitronectin and collagen, for 17 days. In yet other embodiments, each of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells; and the cells do not differentiate into osteocytes, after incubation under the aforementioned conditions. In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells, and of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells; and the cells do not differentiate into osteocytes, after incubation under the aforementioned conditions. In still other embodiments, the conditions are incubation with a solution containing 10 mcM dexamethasone, 0.2 mM ascorbic acid, 10 mM glycerol-2-phosphate, and ΙΟηΜ Vitamin D, in plates coated with vitronectin and collagen, for 26 days. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art.

In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells; and the cells do not differentiate into adipocytes, after incubation in adipogenesis induction medium, namely a solution containing 1 mcM dexamethasone, 0.5 mM 3-Isobutyl-l-methylxanthine (IB MX), 10 mcg/ml insulin, and 100 mcM indomethacin, on days 1, 3, 5, 9, 11, 13, 17, 19, and 21; and replacement of the medium with adipogenesis maintenance medium, namely a solution containing 10 mcg/ml insulin, on days 7 and 15, for a total of 25 days. In yet other embodiments, each of CD34, CD45, CD19, CD14 and HLA- DR is expressed by less than 3% of the cells; and the cells do not differentiate into adipocytes, after incubation under the aforementioned conditions. In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells, each of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells; and the cells do not differentiate into adipocytes, after incubation under the aforementioned conditions. In still other embodiments, a modified adipogenesis induction medium, containing 1 mcM dexamethasone, 0.5 mM IBMX, 10 mcg/ml insulin, and 200 mcM indomethacin is used, and the incubation is for a total of 26 days. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art.

In more specific embodiments, greater than 50%, in other embodiments greater than 55%, in other embodiments greater than 60%, in other embodiments greater than 65%, in other embodiments greater than 70%, in other embodiments greater than 75%, in other embodiments greater than 80%, in other embodiments greater than 85%, in other embodiments greater than 90%, in other embodiments greater than 95%, in other embodiments greater than 96%, in other embodiments greater than 97%, in other embodiments greater than 98%, in other embodiments greater than 99% of the adherent stromal cells express a marker selected from CD73, CD90, CD29, and CD105, or in other embodiments 2 or more of these markers, or in other embodiments 3 or more of these markers, or in other embodiments all four of these markers.

According to some embodiments, the adherent cells express CD200. In still other embodiments, less than 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, or 2%, 1%, or 0.5% of the adherent cells express CD200. In yet other embodiments, greater than 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the adherent cells express CD200.

According to some embodiments, greater than 50%, in other embodiments greater than 55%, in other embodiments greater than 60%, in other embodiments greater than 65%, in other embodiments greater than 70%, in other embodiments greater than 75%, in other embodiments greater than 80%, in other embodiments greater than 85%, in other embodiments greater than 90%, in other embodiments greater than 95%, in other embodiments greater than 96%, in other embodiments greater than 97%, in other embodiments greater than 98%, in other embodiments greater than 99% of the adherent stromal cells do not express a marker selected from CD3, CD4, CD45, CD80, HLA-DR, CDl lb, CD14, CD19, CD34, and CD79-alpha, or in other embodiments 2 or more of these markers, or in other embodiments 3 or more of these markers, or in other embodiments 4 or more of these markers, or in other embodiments 5 or more of these markers, or in other embodiments 6 or more of these markers, or in other embodiments 7 or more of these markers, or in other embodiments 8 or more of these markers, or in other embodiments 9 or more of these markers, or in other embodiments all ten of these markers.

In certain embodiments, the described cells express or secrete (as appropriate for each protein) SCF, Flt-3, H2A histone family (H2AF), and/or Aldehyde dehydrogenase X (ALDH X). In more specific embodiments, greater than 50%, in other embodiments greater than 55%, in other embodiments greater than 60%, in other embodiments greater than 65%, in other embodiments greater than 70%, in other embodiments greater than 75%, in other embodiments greater than 80%, in other embodiments greater than 85%, in other embodiments greater than 90%, in other embodiments greater than 95%, in other embodiments greater than 96%, in other embodiments greater than 97%, in other embodiments greater than 98%, in other embodiments greater than 99% of the cells express or secrete at least one, in other embodiments at least 2, in other embodiments at least 3, in other embodiments all four of the aforementioned proteins.

Additionally or alternatively, the cells secrete or express IL-6, eukaryotic translation elongation factor 2 (EEEF2), reticulocalbin 3, EF-hand calcium binding domain (RCN₂), and/or calponin 1 basic smooth muscle (CNN1). In more specific embodiments, greater than 50%, in other embodiments greater than 55%, in other embodiments greater than 60%, in other embodiments greater than 65%, in other embodiments greater than 70%, in other embodiments greater than 75%, in other embodiments greater than 80%, in other embodiments greater than 85%, in other embodiments greater than 90%, in other embodiments greater than 95%, in other embodiments greater than 96%, in other embodiments greater than 97%, in other embodiments greater than 98%, in other embodiments greater than 99%, of the cells express or secrete at least one, in other embodiments at least 2, in other embodiments at least 3, in other embodiments at least 4, in other embodiments all five of the aforementioned proteins. Additionally or alternatively, the cells express low amounts of heterogeneous nuclear ribonucleoprotein HI (Hnrphl), CD44 antigen isoform 2 precursor, 3 phosphoadenosine 5 phosphosulfate synthase 2 isoform a (Papss2), and/or ribosomal protein L7a (rpL7a). In more specific embodiments, greater than 50%, in other embodiments greater than 55%, in other embodiments greater than 60%, in other embodiments greater than 65%, in other embodiments greater than 70%, in other embodiments greater than 75%, in other embodiments greater than 80%, in other embodiments greater than 85%, in other embodiments greater than 90%, in other embodiments greater than 95%, in other embodiments greater than 96%, in other embodiments greater than 97%, in other embodiments greater than 98%, of the cells do not express or secrete at least one, in other embodiments at least 2, in other embodiments at least 3, in other embodiments all four of the aforementioned proteins.

In certain embodiments, the described cells have not been transfected is one or more therapeutic factors. In other embodiments, the cells have not been transfected.

The subject may be, in various embodiments, any mammal with one or more injured tendons, including e.g. human or domesticated animals including, but not limited to, horses (i.e. equine), cattle, goat, sheep, pig, dog, cat, camel, alpaca, llama and yak.

According to an embodiment of the present teachings, the described adherent cells may be used to treat conditions such as tendon injuries produced by degeneration resulting from over-strain of tendons, tendinitis, tendinosis, tenosynovitis, and the like, in horses and other subjects in need thereof.

In various embodiments, the cells may be allogeneic, or in other embodiments, autologous. In other embodiments, the cells may be fresh or, in other embodiments, frozen (e.g., cryo-preserved).

In certain embodiments, the subject may be administered with additional therapeutic agents or cells.

Since non- autologous cells may in some cases induce an immune reaction when administered to the body, several approaches have been developed to reduce the likelihood of rejection of non- autologous cells. These include either suppressing the recipient immune system or encapsulating the non- autologous cells in immune-isolating, semipermeable membranes before transplantation.

Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine ( sulphas alazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF. alpha, blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox- 2 inhibitors and tramadol.

In any of the methods described herein, the cells can be administered as a part of a pharmaceutical composition that further comprises one or more pharmaceutically acceptable carriers. Hereinafter, the term "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered cells. Examples, without limitations, of carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water. In some embodiments, the pharmaceutical carrier is an aqueous solution of saline.

One may, in various embodiments, administer the pharmaceutical composition in a systemic manner (as detailed hereinabove). Alternatively, one may administer the pharmaceutical composition locally, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient, such as, in non-limiting embodiments, intramuscular administration or administration directly into the injured joint. In other embodiments, the cells are administered intravenously (IV), subcutaneously (SC), or intraperitoneally (IP), each of which is considered a separate embodiment.

In other embodiments, for injection, the described cells may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer, optionally in combination with medium containing cryopreservents that is used for freezing the cells.

For any preparation used in the described methods, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. Preferably, a dose is formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be, in some embodiments, chosen by the individual physician in view of the patient's condition.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or, in other embodiments, a plurality of administrations, with a course of treatment lasting from several days to several weeks or, in other embodiments, until alleviation of the disease state is achieved.

In certain embodiments, following transplantation, the majority of the cells, in other embodiments more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the cells are no longer detectable within the subject 1 month after administration.

Compositions including the described preparations formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

The described compositions may, if desired, be packaged in a container that is accompanied by instructions for administration. The container may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

The described adherent cells are, in some embodiments, suitably formulated as pharmaceutical compositions which can be suitably packaged as an article of manufacture. Such an article of manufacture comprises a packaging material which comprises a label for use in treating an injured tendon, as described herein.

A typical dosage of the adherent stromal cells used alone might range, in some embodiments, from about 10 million to about 500 million cells per administration. For example, the dosage can be, in some embodiments, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 million cells or any amount in between these numbers. It is further understood that a range of adherent stromal cells can be used including from about 10 to about 500 million cells, from about 100 to about 400 million cells, from about 150 to about 300 million cells. Accordingly, disclosed herein are methods of treating an injured tendon in a subject in need thereof, the method comprising administering to said subject a therapeutically or prophylactically effective amount of adherent stromal cells, wherein the dosage administered to the subject is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 million cells or, in other embodiments, between 150 million to 300 million cells. Adherent stromal cells, compositions comprising adherent stromal cells, and/or medicaments manufactured using adherent stromal cells can be administered, in various embodiments, in a series of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1-10, 1-15, 1-20, 2- 10, 2-15, 2-20, 3-20, 4-20, 5-20, 5-25, 5-30, 5-40, or 5-50 injections, or more.

Also disclosed herein are kits and articles of manufacture that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits and articles of manufacture can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods, including adherent stromal cells. In another aspect, the kits and articles of manufacture may comprise a label, instructions, and packaging material, for example for treating an injured tendon or for other therapeutic indications mentioned herein.

Additional objects, advantages, and novel features of the invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate certain embodiments in a non-limiting fashion.

EXAMPLE 1

PRODUCTION AND CULTURING OF ADHERENT CELLS FROM PLACENTA

Overview: The manufacturing process for the final cell product consists of two stages: Stage 1, the intermediate cell stock (ICS) production, contains the following steps:

1. Extraction of ASCs from the placenta. 2. 2-dimensional cell growth for up to 12 population doublings.

3. Cell concentration, formulation, filling and cryopreservation.

Stage 2, the thawing of the ICS and further culture, contains the following steps:

1. 2-dimensional cell growth of the thawed ICS for up to 8 additional doublings.

2. 3-dimensional cell growth in bioreactor/s and harvest from bioreactor/s up to 10

additional doublings.

3. Downstream processing: cell concentration, washing, formulation, filling and

cryopreservation.

The procedure included periodic testing of the growth medium for sterility and contamination.

Production of ICS

Step 1-1 - Extraction of Adherent Stromal Cells (ASC's)

Placentas were obtained from donors up to 35 years old, who were pre- screened for hepatitis B, hepatitis C, HIV-1 and HIV-2, HTLV-1 and HTLV-2, and syphilis. The donor placenta was maintained sterile and cooled until the initiation of the extraction process.

Within 4 hours of the delivery, the placenta was placed with the maternal side facing upwards and was cut into pieces (sized ~lcm ), which were washed thoroughly with isotonic buffer) containing gentamicin.

• The washed pieces were incubated for 3 hours with collagenase and DNAse in isotonic buffer.

• Culture medium (DMEM], 10% filtered FBS and L-Glutamine) supplemented with gentamicin, was added, and the digested tissue was coarsely filtered through a sterile stainless steel sieve and centrifuged.

• The cells were suspended in culture medium, seeded in flasks, and incubated at 37°C in a tissue culture incubator under humidified conditions supplemented with 5% C0₂.

• After 2-3 days, cells were washed twice with Phosphate-Buffered Saline (PBS), and the culture medium was replaced.

• Cells were incubated for an additional 4-5 days prior to the first passage. Step 1-2 - Initial 2-Dimensional Culturing

• Passage 1: Cells were detached using trypsin, centrifuged, and seeded at a culture density of 3+0.2x10 3 cells/cm 2 in tissue culture flasks, in culture medium lacking gentamicin.

• Subsequent Passages: When the culture reached 60-90% confluence, cells were passaged as described above.

Step 1-3 - Cell Concentration, Washing, Formulation, Filling and Cryopreservation

Following the final passage, the resulting cell suspension was centrifuged and re- suspended in culture medium at a final concentration of 20-40 x 10⁶ cells/milliliter (mL). The cell suspension was diluted 1: 1 with Freezing Solution (20% DMSO, 80% FBS), and the cells were cryopreserved in 10% DMSO, 40% FBS, 50% Full DMEM. The temperature was reduced in a controlled rate freezer (l°C/min down to -80°C followed by 5°C/min down to - 120°C), and the cells were stored in a liquid nitrogen freezer to produce the ICS.

Production of Cells

Step 2-1: Additional Two-Dimensional (2D) Cell Culturing.

The ICS was thawed, diluted with culture medium, and cultured for up to 10 additional doublings, passaging when reaching 60 90% confluence, then were harvested for seeding in the bioreactor.

Step 2-2: Three Dimensional (3D) Cell Growth in Bioreactor/s

From the cell suspension, 1 or 2 bioreactors were seeded. Each bioreactor contained FibraCel^® carriers (New Brunswick Scientific) made of polyester and polypropylene, and culture medium. 450-600 x 10⁶ cells were seeded into each 5-liter bioreactor.

The growth medium in the bioreactor/s was kept at the following conditions: temp: 37+l°C, Dissolved Oxygen (DO): 70+10% and pH 7.3+0.2. Filtered gases (Air, C0₂, N₂ and 0₂) were supplied as determined by the control system in order to maintain the target DO and pH values.

After seeding, the medium was agitated with stepwise increases in the speed, up to 200 RPM by 24 hours. Perfusion was initiated several hours after seeding and was adjusted on a daily basis in order to keep the glucose concentration constant at approximately 550mg\liter. Cell harvest was performed at the end of the growth phase (approximately day 6). Bioreactors were washed for 1 minute with pre-warmed sterile PBS, and cells were detached.

Step 2-3: Downstream Processing: Cell Concentration, Washing, Formulation, Filling and Cryopreservation

The cell suspension underwent concentration and washing, using suspension solution (5% w/v human serum albumin [HSA] in isotonic solution) as the wash buffer, and diluted 1: 1 with 3D-Freezing solution (20% DMSO v/v and 5% HSA w/v in isotonic solution) to a concentration of 10-20xl0⁶ cells/ml. The temperature of the vials was gradually reduced, and the vials were stored in a gas-phase liquid nitrogen freezer.

EXAMPLE 2

OSTEOCYTE AND ADIPOSE DIFFERENTIATION ASSAYS METHODS

Bone marrow adherent cells - Bone marrow (BM) adherent cells were obtained from aspirated sterna marrow of hematologic ally healthy donors undergoing open-heart surgery or BM biopsy. Marrow aspirates were diluted 3-fold in HBSS) and subjected to Ficoll-Hypaque (Robbins Scientific Corp. Sunnyvale, CA) density gradient centrifugation. Thereafter, marrow mononuclear cells (<1.077 gm/cm ) were collected, washed 3 times in HBSS, and resuspended in growth media [DMEM (Biological Industries, Beit Ha'emek, Israel) supplemented with 10% FCS (GIBCO BRL), 10^"4 M mercaptoethanol (Merck, White House Station, NJ), Pen-Strep-Nystatin mixture (100 U/ml: 100 μg/ml: 1.25 un/ml; Beit Ha'Emek), 2 mM L-glutamine (Beit Ha'Emek)]. Cells from individual donors were incubated separately in tissue culture flasks (Corning, Acton, MA) at 37 °C (5% C0₂) with weekly change of culture media. Cells were passaged every 3-4 days using 0.25% trypsin-EDTA (Beit Ha'Emek). Following 2-40 passages, when reaching 60-80% confluence, cells were collected for analysis.

Table 1: Osteogenesis medium components

dexamethasone 1 mM 1 μΐ 0.1 μΜ

Ascorbic Acid-2 -Phosphate solution 0.1 M 20 μΐ 0.2 mM

Glycerol-2-Phosphate Solution 1 M 100 μΙ_ ΙΟ ηιΜ

L-glutamine X 100 100 μΐ X 1

Pen & Strep X 100 100 μΐ X 1

Induction of osteogenesis

Placenta-derived cells or bone marrow (BM)-derived cells were plated (200,000 cells per well) in 1 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) in wells coated with a coating mixture containing 12 μg/ml vitronectin and 12 μg/ml collagen, which was provided with the Millipore Mesenchymal Stem Cell Osteogeneis Kit. Cells were grown until 100% confluent (typically overnight) before adding osteogenic differentiation medium.

On differentiation day 1, growth medium was aspirated and replaced with 1 ml osteogenesis induction medium, which was replaced with fresh medium every 2-3 days for 14-17 days. Osteocytes were fixed and stained with Alizarin Red Solution.

In other experiments, a modified osteogenesis induction medium was used, having the components listed in Table 2, including Vitamin D, for 26 days.

Table 2: Osteogenesis medium components

Induction of adipogenesis

Adipogenesis was carried out according to the instructions provided with the Chemicon Adipogenesis Kit (cat no. scr020, Millipore, MA, USA)

Adipogenesis induction medium

Adipogenesis induction and maintenance medium were freshly prepared prior to every medium exchange, using the components depicted in Tables 3 and 4, below.

Table 3: Adipogenesis induction medium components

Table 4: Adipogenesis maintenance medium components

Cell Growth

Placenta-derived or BM-derived cells were plated (200,000 cells per well) in 1 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma- Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) and were grown until 100% confluent (typically overnight) before initiating adipogenesis differentiation.

On differentiation day 1, growth medium was aspirated and replaced with 1 ml adipogenesis induction medium, which was replaced with fresh induction or maintenance medium every 2-3 days for a total of 25 days, according to the schedule in Table 5. Table 5: Adipogenesis differentiation schedule

On day 25, adipocytes were fixed and stained with oil red solution.

Modified adipogenesis induction medium

The modified adipogenesis induction medium contained the components depicted in Table 6, and was used for a total of 26 days.

Table 6: Adipogenesis induction medium components

RESULTS

Osteocyte induction. Incubation of BM-derived adherent cells in osteogenic induction medium resulted in differentiation of over 50% of the BM cells, as demonstrated by positive alizarin red staining. On the contrary, none of the placental-derived cells exhibited signs of osteogenic differentiation. Next, a modified osteogenic medium comprising Vitamin D and higher concentrations of dexamethasone was used. Over 50% of the BM cells underwent differentiation into osteocytes, while none of the placental-derived cells exhibited signs of osteogenic differentiation.

Adipocyte induction. Adipocyte differentiation of placenta- or BM-derived adherent cells in adipocyte induction medium resulted in differentiation of over 50% of the BM-derived cells, as demonstrated by positive oil red staining and by typical morphological changes (e.g. accumulation of oil droplets in the cytoplasm). In contrast, none of the placental-derived cells differentiated into adipocytes.

Next, a modified medium containing a higher indomethacine concentration was used. Over 50% of the BM-derived cells underwent differentiation into adipocytes. In contrast, none of the placental-derived cells exhibited morphological changes typical of adipocytes.

EXAMPLE 3

Marker expression on placenta-derived stromal cells.

FACS analysis of membrane markers - cells were stained with monoclonal antibodies as previously described. In short, 400,000-600,000 cells were suspended in 1 ml flow cytometer buffer in a 5 ml test tube and incubated for 15minutes at room temperature (RT), in the dark, with each of the following monoclonal antibodies (MAbs): FITC- conjugated anti-human CD29 MAb (eBioscience), PE-conjugated anti human CD73 MAb (Becton Dickinson), PE-conjugated anti human CD 105 MAb (eBioscience), PE-conjugated anti human CD90 MAb (Becton Dickinson), FITC-conjugated anti-human CD45 MAb (IQProducts), PE-conjugated anti-human CD19 MAb (IQProducts), PE-conjugated anti human CD 14 MAb (IQProducts), FITC-conjugated anti human HLA-DR MAb (IQProduct), PE-conjugated anti human CD34 MAb (IQProducts), FITC-conjugated anti human CD31 MAb (eBioscience), FITC-conjugated anti human KDR MAb (R&D systems), anti human fibroblasts marker (D7- FIB) MAb(ACRIS), FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugated anti-human CD86 MAb (BD), PE conjugated anti-human CD200 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD), FITC-conjugated anti-human HLA- ABC MAb (BD), Isotype IgGl FITC-conjugated (IQ Products), Isotype IgGl PE-conjugated (IQ Products). Cells were washed twice with flow cytometer buffer, resuspended in 500 microliters (mcl) flow cytometer buffer, and analyzed by flow cytometry. Negative controls were prepared with relevant isotype fluorescence molecules.

Mixed L ymphocyte fteac/ion (ML/Ϊ)

2 x 10⁵ peripheral blood (PB) derived MNC (from donor A) were stimulated with equal amount of irradiated (3000 Rad) PB derived MNCs (from donor B). Increasing amounts of stromal cells were added to the cultures. Three replicates of each group were seeded in 96- well plates. Cells were cultured in RPMI 1640 medium containing 20% FBS. Plates were pulsed with 1 microCurie (mcC) H-thymidine during the last 18 hrs. of the 5-day culturing. Cells were harvested over a fiberglass filter and thymidine uptake was quantified with scintillation counter.

For CFSE staining, PB-MNC cells were stained for CFSE (Molecular Probes) for proliferation measurement before culturing. Cells were collected after 5 days and the intensity of CFSE staining was detected by Flow Cytometry.

EL/SA

MNCs (isolated from peripheral blood) were stimulated with 5 microgram (mcg)/ml ConA (Sigma), 0.5 mcg/ml LPS (SIGMA), or 10 mcg/ml PHA (SIGMA) in the presence of stromal cells under a humidified 5% C0₂ atmosphere at 37 °C. Supernatants were collected and subjected to cytokine analysis using ELISA kits for IFN-gamma (DIACLONE), TNFa (DIACLONE) and IL-10 (DIACLONE).

Expression of cellular markers on isolated cells - the surface antigens expressed by the isolated cells were examined using monoclonal antibodies. Results indicated that the cells were characterized by the positive markers: CD73, CD29 and CD105 and the negative markers: CD34, CD45, CD19, CD14 and HLA-DR. More specifically, all the positive markers were expressed by more than 90% of the cells, and all the negative markers were expressed by less than 3% of the cells.

Furthermore, the cells did not express endothelial markers as shown by negative staining for the two endothelial markers CD31 and KDR. However, expression of a fibroblast-typical marker, D7-fib, was evident.

EXAMPLE 4

I mm u n ogen icity and immunomodulatory properties of placenta-derived stromal cells

In order to examine the immunogenicity of the obtained cells, the expression of co- stimulatory molecules on the cells was measured. FACS analysis demonstrated the absence of CD80, CD86 and CD40 on the cell membranes. Moreover, the cells expressed low levels HLA class I molecules, as detected by staining for HLA A/B/C. The expression of stimulatory and co-stimulatory molecules was similar to bone marrow (BM) derived MSCs.

To further investigate the immunogenicity and the immunomodulatory properties of the cells, Mixed Lymphocyte Reaction (MLR) tests were performed. The cells were shown to both escape allorecognition and reduce T cell proliferation, as measured by thymidine incorporation. Furthermore, the reduction in lymphocyte proliferation was dose dependent with the number of stromal cells. The stromal cells also reduced lymphocyte proliferation following mitogenic stimuli, such as Concavalin A (Con A) and Phytohemagglutinin (PHA), and non-specific stimulation by anti-CD3 and anti-CD28.

In order to investigate the mechanism of action of modulation of lymphocyte proliferation, and to see if this action is mediated via cell to cell interaction or cytokines secretion, PB-derived Mononuclear cells (MNCs) were stimulated by PHA using the transwell method (which prevents cell-to-cell contact but enables the diffusion of cytokines between the two compartments). The inhibition of proliferation was maintained even in this assay, showing that cell-to-cell contact was not necessary for the inhibition.

EXAMPLE 5

Cytokine secretion by placenta-derived stromal cells

As described, the isolated stromal cells reduce the proliferation rate of lymphocytes. Further investigation of the cytokines secreted by lymphocytes in response to the stromal cells was performed to elucidate the mechanism of action of these cells. Culturing of PB-derived mononuclear (MN) cells with stromal cells slightly reduced IFN-gamma secretion and dramatically reduced TNF-alpha secretion by the MN cells, even in the presence of low amounts of stromal cells. In addition, following lipopoly saccharide (LPS) stimulation, the stromal cells increased secretion of IL-10 by PB-derived MNCs, while decreasing their secretion of TNF-alpha, in a dose dependent manner.

EXAMPLE 6

TREATMENT OF PATHOLOGIES REQUIRING CONNECTIVE TISSUE REGENERATION AND/OR REPAIR

1. Treating pathologies requiring tendon regeneration and/or repair using adherent cells

The effect of adherent placental-derived stromal cells on the healing of tendons was examined in a rat collagenase-induced patellar tendon injury model.

METHODS Surgical Procedure (Mini-open injection)

Sixty-one male Sprague Dawley rats (weight 300-350 grams) were used for this study. Following isoflurane inhalation anesthesia, a 5-mm incision was made medial to the knee bilaterally, and the patellar tendons were identified. A 30-gauge needle was inserted into the distal third of the tendon, and 30 microliter (mcL) (250 units) of collagenase was injected into both patellar tendons. The skin was then closed using a single interrupted 4-0 nylon suture. Six days after collagenase injection, the patellar tendons were exposed again bilaterally, and 100 mcL of either saline or a cell suspension containing stromal cells (2.0xl0⁶ cells in 100 mcL suspension) was delivered into the collagenase injection site.

Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) Labeling of Cells

Six days after collagenase injection, six rats underwent patellar tendon injection with stromal cells (2xl0⁶ cells in 100 mcL suspension) labeled with CFSE stain. The rats were then sacrificed 4 days, 7 days, 2 weeks, or 4 weeks after injection of the cells, and the patellar tendon tissues were isolated, harvested, and then cryopreserved in Tissue-Tek® O.C.T™ compound via liquid nitrogen freezing. Cryostat sectioning of the samples was then performed, and tissue sections were visualized by fluorescent microscopy. In order to discern tissue architecture, some frozen tissue sections were briefly fixed with 0.4% paraformaldehyde, and then stained with hematoxylin and eosin.

Analysis of effects of stromal cells on tendon injury

Thirty-nine rats received bilateral injections of collagenase (250 IU) into the patellar as described above. Six days after injury by injection of collagenase, the patellar tendons were randomly injected with either stromal cells (2xl0⁶ cells in 100 mcL suspension) or 100 mcL of saline. Thirteen rats were then euthanized at each designated study time point (1, 2, or 4 weeks). After sacrifice, the specimens were analyzed biomechanically and histologically.

i) Biomechanical Analysis

Bilateral tibia-patellar tendon— patella constructs were harvested from ten rats per time point and frozen at -80° C until testing. Prior to testing, all the soft tissues were dissected, leaving the repaired tendon with its attachments. The tibia and patella were then placed in a custom-designed clamp to ensure secure fixation. Specimens were then mounted on a MTS machine in a special-designed jig so that tension was aligned along the long axis of the tendon. A pre-load of 1 N was applied and then the specimens were pre-conditioned for five cycles. The specimens were then loaded in uniaxial tension to failure at a rate of 16.7 microns/sec. Ultimate load-to-failure (N) was defined as the maximal force achieved on the load-elongation curve. Linear stiffness (N/mm) was determined from the linear portion of the load-deformation curve.

ii) Histological Analysis

Bilateral tibia-patellar tendon-patella constructs were harvested from 3 rats per time point for histological analysis. The patellar tendon tissues were fixed in 10% neutral-buffered formalin, trimmed, and then decalcified in formic acid bone decalcifier (Immunocal). The tissues were then embedded in paraffin and sectioned. Two sequential 5 pm sections were mounted and stained. The slides were then examined with an Olympus BH-2 light microscope (Olympus Optical Co. Ltd., Tokyo, Japan), and images were digitized and captured using a camera system. The tendons were the examined for cellularity, collagen organization, and abnormal deposition of proteoglycans, using hematoxylin and eosin (H&E) and safranin-0 stains, respectively. Polarized light microscopy was used to examine picrosirius red-stained sections in order to determine tissue birefringence, which is a measure of collagen content and organization.

Hi) Assessment of Gene Expression Profiles

At 4, 7, 14, and 28 days following either stromal cell treatment or saline treatment, bilateral rat patellar tendons were harvested from 4 animals per time point and placed in Tri- Reagent (Sigma). The samples were then stored at -80°C for subsequent RNA extraction. Total RNA was isolated from the samples using the TRIzol® Reagent (Invitrogen, Carlsbad, CA) extraction method. Equal amounts of mRNA were then reverse-transcribed using the Bio-Rad iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA). The cDNA products were amplified and quantified by RT-PCR using iQ™ SYBR® Green Supermix on a MyiQ®™ Single-Color Real Time PCR Detection System machine (both Bio-Rad, Hercules, CA). All reactions were cycled 40 times in triplicate. Relative expression levels were calculated based on delta-Ct values (the difference in the threshold cycle between the gene of interest and the housekeeping gene GAPDH). Primers were designed for mouse GAPDH, as well as for the following musculoskeletal tissue markers: tendon (CollA2, Col3A2, Scleraxis), MMP-3, MMP-13, VEGF, TGF-beta, IL-lb, and IL-6.

RESULTS

i) Gross patellar tendon appearance following injury and treatment

Both treated and untreated tendons appeared inflamed at 1 and 2 weeks following collagenase injury (Figure 2, panels A-B). Tissues appeared bulbous and fibrotic, consistent with scarring following acute injury. No consistent difference in prevalence or severity of inflammation was seen between the two groups at 1, 2, and 4 weeks (Figure 2). The gross appearance of tendons in both groups began to resemble normal tendon appearance at 4 weeks (Figure 2, panel C). No adverse soft tissue reaction was seen in treated samples.

ii) Viability of stromal cells after injection in injured rate tendons

Fluorescent microscopy demonstrated cluster-like signals indicative of viable CFSE- labeled stromal cells until the last measured timepoint, at 4 weeks following injection (Figure 3; fluorescent microscopy images of CFSE-labeled cells at 4 days [panel A], 2 weeks [panel B], and 4 weeks [panel C] following cell administration; clusters of fluorescent cell-like signals are encircled with dotted lines). The prevalence of fluorescent signals appeared to decrease over time. Normal tendon collagen architecture is highly ordered and demonstrates autofluorescence, but following the acute collagenase injury, the autofluorescence was diminished, due to disruption of normal collagen architecture. By 4 weeks, however, areas of linear fluorescent signals were visible, indicative of progressive tendon healing and re- establishment of normal tendon tissue architecture.

in) Biomechanical properties

Both treated and untreated patellar tendons demonstrated improving tissue load-to- failure and stiffness over the 4-week study period, indicative of tissue healing over time.

Stromal cell treatment produced an early beneficial effect on tendon healing. Treated patellar tendons demonstrated a significantly higher load-to-failure at the 2-week time point following cell injection when compared to untreated patellar tendons (77.01+10.51 N for treated tendons [left bar for each timepoint] versus 58.87+11.97 N for untreated [right bar for each time point], p=0.012; Figure 4, outlined box). Treated tendons also had a higher mean tendon load-to-failure (83.74+15.34 N for treated tendons versus 78.19+21.74 N for untreated, Figure 4) and stiffness (31.44+6.06 N/mm for treated [left bar for each time point] versus 27.91+6.57 N/mm for untreated [right bar for each timepoint]; Figure 5) at the 4-week timepoint.

Cell-treated tendons exhibited higher proteoglycan and collagen content during healing.

Cell-treated tendons exhibited higher proteoglycan deposition at the tendon-proximal tibia attachment site following treatment. The higher proteoglycan content is consistent with a more mature tendon attachment interface, as indicated in Safranin-O-stained sections of rat patellar tendons, where greater proteoglycan content was observed in treated tendons (Figure 6, panel A; red [dark in black and white] proteoglycan staining in insertion area (labeled "insertion site") is more prominent than untreated tendons (Figure 6, panel B). Polarized microscopy of picrosirius-stained tendon sections also demonstrated greater areas of birefringence along tendon insertion site in treated patellar tendons (Figure 7, treated samples shown in panel A, with birefringent area encircled with dotted line; untreated samples shown in panel B; with non-birefringent area encircled in dotted line). This demonstrates greater collagen content and a more mature healing interface in the treated tendons.

Image analysis of safranin-0 and picrosirius slides demonstrated a trend toward greater mean areas of proteoglycan deposition (Figure 8) and collagen content (Figure 9) along the tendon-insertion sites of treated tendons at 1, 2, and 4 weeks after treatment relative to untreated tendons.

v) Gene Expression Analysis Following Injection of stromal cells after tendon injury Gene expression analysis of harvested patellar tendons indicated that stromal cells influence the levels of inflammatory cytokines levels during the early phase of healing following tendon injury (Figure 10). Stromal cells produced increases in expression levels of inflammatory cytokines IL-6, ILlb, MMP3, and MMP13 (Figures 10A-D, respectively). These findings are consistent with cluster-like inflammatory cells seen in early treated samples. These inflammatory cytokines may affect the rate of tendon remodeling and healing following injury in this model.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the invention.

REFERENCES

(Additional References are cited in text)

Bruder SP, et al. 1998. The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am. 80(7):985-96 Carstanjen B, Desbois C, Hekmati M, and Behr L. Successful engraftment of cultured autologous mesenchymal stem cells in a surgically repaired soft palate defect in an adult horse. Can J Vet Res. 2006 April; 70(2): 143-147.

Chao Wan, Qiling He, Gang Li, 2006. Allogenic peripheral blood derived mesenchymal stem cells (MSCs) enhance bone regeneration in rabbit ulna critical-sized bone defect model. Journal of Orthopaedic Research 24 (4) 610-618.

Dominici et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The

International Society for Cellular Therapy position statement. Cytotherapy. 2006;

8(4):315-7.

Gordon et al, Tendon Regeneration Using Mesenchymal Stem Cells.p313-320 in Tendon

Injuries. Springer London. 2005.

Horwitz et al., 1999. Transplantability and therapeutic effects of bone marrow derived mesenchymal cells in children with osteogenesis imperfecta. Nat. Med. 5:309-313. Horwitz et al., 2002. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. PNAS 99(13)8932-8937.

Livingston, T. L. 2003 Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration. Journal of Materials Science: Volume 14

(3):211-218.

Young et al.1998. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res. 16(4):406-13.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of increasing a proteoglycan content or collagen content in an injured tendon in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of adherent stromal cells derived from placenta, thereby increasing the proteoglycan content or collagen content in an injured tendon in the subject.

2. Use of adherent stromal cells derived from placenta for the manufacture of a medicament for increasing a proteoglycan content or collagen content in an injured tendon.

3. A composition comprising adherent stromal cells derived from placenta, for increasing a proteoglycan content or collagen content in an injured tendon.

4. A method of deceasing inflammation following an acute tendon injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of adherent stromal cells derived from placenta, thereby decreasing inflammation following an acute tendon injury in the subject.

5. Use of adherent stromal cells derived from placenta for the manufacture of a medicament for deceasing inflammation following an acute tendon injury.

6. A composition comprising adherent stromal cells derived from placenta, for deceasing inflammation following an acute tendon injury.

7. The method, use, or article of manufacture of any one of claims 1-6, wherein said adherent stromal cells increases expression levels of IL-6, ILlb, MMP3, or MMP13 in said tendon.

8. The method, use, or article of manufacture of any one of claims 1-6, wherein said adherent stromal cells are obtained from a three-dimensional (3D) culture.

9. The method, use, or article of manufacture of claim 8, wherein said three- dimensional (3D) culture comprises a 3D bioreactor.

10. The method, use, or article of manufacture of claim 8, wherein culturing of said cells in said 3D culture is effected under perfusion.

11. The method, use, or article of manufacture of claim 8, wherein culturing conditions of said three-dimensional culture comprise an adherent material selected from the group consisting of a polyester and a polypropylene.

12. The method, use, or article of manufacture of any one of claims 1-6, wherein culturing of said cells is performed for at least 3 days.

13. The method, use, or article of manufacture of any one of claims 1-6, wherein said adherent stromal cells express a marker selected from the group consisting of CD73, CD90, CD29 and CD105.

14. The method, use, or article of manufacture of any one of claims 1-6, wherein said adherent stromal cells do not express a marker selected from the group consisting of CD3, CD4, CD45, CD80, HLA-DR, CDl lb, CD14, CD19, CD34 and CD79-alpha.

15. The method, use, or article of manufacture of any one of claims 1-3, wherein the tendon injury is selected from tendon degeneration and tendon strain.

16. The method, use, or article of manufacture of any one of claims 4-6, wherein the tendon injury is accompanied by a condition selected from tendinitis, tendinosis, and tenosynovitis.

17. The method, use, or article of manufacture of any one of claims 1-6, wherein said tendon is a patellar tendon.