WO2014089397A1 - Compositions and methods of treating and preventing pulmonary fibrosis - Google Patents

Abstract

The present invention provides methods of treating and preventing pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). Methods of the invention include administering to a subject with or at risk for pulmonary fibrosis an isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage, wherein the viability of said cells is at least 80% and the composition contains: a) about 5-75% viable CD90⁺ cells with the remaining cells in said composition being CD45⁺; b) less than 2 μg/ml of bovine serum albumin; c) less than 1 mg/ml of a enzymatically active harvest reagent; and d) substantially free of mycoplasma, endotoxin, and microbial contamination.

Description

COMPOSITIONS AND METHODS OF TREATING AND PREVENTING

PULMONARY FIBROSIS

RELATED APPLICATIONS

[01] This application claims the benefit of U.S. Provisional Application No. 61/734,262, filed on December 6, 2012, the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[02] The present invention relates to compositions of mixed cell populations and their use in vivo for the treatment and prevention of pulmonary fibrosis.

BACKGROUND OF THE INVENTION

[03] Regenerative medicine harnesses, in a clinically targeted manner, the ability of regenerative cells, e.g., stem cells and/or progenitor cells (i.e., the unspecialized master cells of the body), to renew themselves indefinitely and develop into mature specialized cells. Stem cells are found in embryos during early stages of development, in fetal tissue and in some adult organs and tissue. Embryonic stem cells (hereinafter referred to as "ESCs") are known to become many if not all of the cell and tissue types of the body. ESCs not only contain all the genetic information of the individual but also contain the nascent capacity to become any of the 200+ cells and tissues of the body. Thus, these cells have tremendous potential for regenerative medicine. For example, ESCs can be grown into specific tissues such as heart, lung or kidney which could then be used to repair damaged and diseased organs. However, ESC derived tissues have clinical limitations. Since ESCs are necessarily derived from another individual, i.e., an embryo, there is a risk that the recipient's immune system will reject the new biological material. Although immunosuppressive drugs to prevent such rejection are available, such drugs are also known to block desirable immune responses such as those against bacterial infections and viruses.

[04] Moreover, the ethical debate over the source of ESCs, i.e., embryos, is well- chronicled and presents an additional and, perhaps, insurmountable obstacle for the foreseeable future.

[05] Adult stem cells (hereinafter interchangeably referred to as "ASCs") represent an alternative to the use of ESCs. ASCs reside quietly in many non-embryonic tissues, presumably waiting to respond to trauma or other destructive disease processes so that they can heal the injured tissue. Notably, emerging scientific evidence indicates that each individual carries a pool of ASCs that may share with ESCs the ability to become many if not all types of cells and tissues. Thus, ASCs, like ESCs, have tremendous potential for clinical applications of regenerative medicine.

[06] ASC populations have been shown to be present in one or more of bone marrow, skin, muscle, liver and brain. However, the frequency of ASCs in these tissues is low. For example, mesenchymal stem cell frequency in bone marrow is estimated at between 1 in 100,000 and 1 in 1,000,000 nucleated cells Thus, any proposed clinical application of ASCs from such tissues requires increasing cell number, purity, and maturity by processes of cell purification and cell culture.

[07] Although cell culture steps may provide increased cell number, purity, and maturity, they do so at a cost. This cost can include one or more of the following technical difficulties: loss of cell function due to cell aging, loss of potentially useful cell populations, delays in potential application of cells to patients, increased monetary cost, increased risk of contamination of cells with environmental microorganisms during culture, and the need for further post-culture processing to deplete culture materials contained with the harvested cells.

[08] More specifically, all final cell products must conform with rigid requirements imposed by the Federal Drug Administration (FDA). The FDA requires that all final cell products must minimize "extraneous" proteins known to be capable of producing allergenic effects in human subjects as well as minimize contamination risks. Moreover, the FDA expects a minimum cell viability of 70%, and any process should consistently exceed this minimum requirement.

[09] While there are existing methods and apparatus for separating cells from unwanted dissolved culture components and a variety of apparatus currently in clinical use, such methods and apparatus suffers from a significant problem - cellular damage caused by mechanical forces applied during the separation process, exhibited, for instance, by a reduction in viability and biological function of the cells and an increase in free cellular DNA and debris. Furthermore, significant loss of cells can occur due to the inability to both transfer all the cells into the separation apparatus as well as extract all the cells from the apparatus. In addition, for mixed cell populations, these methods and apparatus can cause a shift in cell profile due to the preferential loss of larger, more fragile subpopulations.

[010] Thus, there is a need in the field of cell therapy, such as tissue repair, tissue regeneration, and tissue engineering, for cell compositions that are ready for direct patient administration with substantially high viability and functionality, and with substantial depletion of materials that were required for culture and harvest of the cells. Furthermore, there are needs for reliable processes and devices to enable production of these compositions that are suitable for clinical implementation and large-scale commercialization of these compositions as cell therapy products.

[Oil] One area in which there is a need for cell therapy is pulmonary fibrosis. Lung injury may progress to chronic fibrotic lung disease, or pulmonary fibrosis. Injurious stimuli may be of exogenous origin or environmental origin, but can also be endogenous such as in interstitial lung diseases of widely differing etiologies, including autoimmune diseases as well as idiopathic pulmonary fibrosis (IPF). IPF is a progressive and generally fatal disease of the lung and currently has no known cause or treatment. IPF is characterized by the progressive and irreversible destruction of lung architecture caused by scar formation that leads to organ failure, disruption of gas exchange, and death from respiratory failure. IPF affects approximately 128,000 people and causes 40,000 fatalities per year. Of the 128,000 people afflicted with IPF, 40,000 die due to the disease and 48,000 new cases are diagnosed annually. There is no current evidence of long-term survival with current IPF therapies (such as anti-inflammatory and cytotoxic agents) and lung transplant is currently the only therapy shown to prolong survival. Even then, lung transplant has poor outcomes compared to other organ transplants with a 5-year survival rate of only 55%. As such, there is no known effective therapy for pulmonary fibrosis, and the disease almost always results in a fatal outcome. A potential therapeutic for treating or preventing pulmonary fibrosis would therefore serve an orphan drug market with high unmet medical need. This invention addresses this need.

SUMMARY OF THE INVENTION

[012] The invention provides a method of treating or alleviating a symptom of pulmonary fibrosis in a subject in need thereof. In some embodiments, the subject suffers from scleroderma, liver cirrhosis, kidney fibrosis, and/or cystic fibrosis (e.g., in addition to pulmonary fibrosis). The invention also provides a method of treating or alleviating a symptom of scleroderma, liver cirrhosis, kidney fibrosis, and/or cystic fibrosis in a subject in need thereof. The method comprises administering to the subject an isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage, wherein the viability of said cells is at least 80% and the composition contains: about 5-75% viable CD90⁺ cells with the remaining cells in said composition being CD45⁺; less than 2 μg/ml of bovine serum albumin; less than 1 mg/ml of a enzymatically active harvest reagent; and is substantially free of mycoplasma, endotoxin, and microbial contamination. The isolated cell composition for tissue repair is also referred to herein as the tissue repair cell (TRC) composition.

[013] The cells of the composition are derived from mononuclear cells. The mononuclear cells are derived from bone marrow, peripheral blood, umbilical cord blood or fetal liver.

[014] In another embodiment, the cells of the composition are in a pharmaceutical-grade electrolyte solution suitable for human administration. In certain aspects of the invention, the composition is substantially free of horse serum and/or fetal bovine serum.

[015] In certain aspects of the invention, at least 10% of the CD90⁺ cells of the composition co-express CD15. Alternatively or in addition, the CD45⁺ cells of the composition are CD14⁺, CD34⁺ or VEGFR1 ⁺.

[016] In certain embodiments of the method, the total number of viable cells in the composition is 1 x 10⁶ to 500 x 10⁶ (e.g., between 35 million and 300 million). For example, the composition contains an average of 1 x 10⁶ to 500 x 10⁶ viable cells, e.g., 1 x 10⁶ to 500 x 10⁶ viable cells, 1 x 10⁶ to 250 x 10⁶ viable cells, 2 x 10⁶ to 250 x 10⁶ viable cells, 3 x 10⁶ to 250 x 10⁶ viable cells, 4 x 10⁶ to 250 x 10⁶ viable cells, 5 x 10⁶ to 250 x 10⁶ viable cells, 5 x 10⁶ to 100 x 10⁶ viable cells, 5 x 10⁶ to 50 x 10⁶ viable cells, 5 x 10⁶ to 10 x 10⁶ viable cells, 8 x 10⁶ to 250 x 10⁶ viable cells, 8 x 10⁶ to 100 x 10⁶ viable cells, 8 x 10⁶ to 50 x 10⁶ viable cells, 8 x 10⁶ to 10 x 10⁶ viable cells, 1 x 10⁶ to 100 x 10⁶ viable cells, 1 x 10⁶ to 50 x 10⁶ viable cells, 1 x 10⁶ to 10 x 10⁶ viable cells, or 1 x 10⁶ to 5 x 10⁶ viable cells. In some embodiments, the composition contains an average of between 1 x 10⁶ to 180 x 10⁶ viable cells, e.g. , 90- 180 x 10⁶ viable cells.

[017] In certain aspects, the cells are in a volume equal to or less than 15 milliliters, 10 milliliters, 7.5 milliliters, or 5 milliliters.

[018] In another embodiment, the composition is administered by injection at one or more sites, including intramuscular injection or endotracheal injection. In other embodiments, the composition is administered by intravenous injection or infusion.

[019] The invention also features a method in which the clinical goal is alleviation of a symptom of pulmonary fibrosis, reduced rate of disease progression, or increased survival. In other embodiments, the invention also features a method in which a clinical goal is alleviation of a symptom of scleroderma, reduced rate of disease progression, or increased survival. [020] Symptoms of pulmonary fibrosis include but are not limited to shortness of breath, disruption of gas exchange, abnormal breath sounds, fatigue, chest discomfort, chronic dry cough, loss of appetite, aching muscles and joints, rapid weight loss, and blue-colored skin around the mouth or fingernails.

[021] In some embodiments, a reduced rate of disease progression is determined by comparing one or more symptoms in the treated subject to one or more symptoms in an untreated subject, wherein the untreated subject is also diagnosed with pulmonary fibrosis, and wherein fewer or less severe symptoms or disease indicators in the treated subject indicates a reduced rate of disease progression. In one aspect of the invention, the reduced rate of disease progression leads to an increased recovery rate.

[022] In other embodiments, a reduced rate of disease progression is determined by comparing one or more symptoms in a subject diagnosed with pulmonary fibrosis prior to treatment with the symptoms at a timepoint after starting treatment (e.g., by administering the subject with a TRC composition of the invention). In cases where the subject presents with fewer or less severe symptoms post-treatment than pre-treatment, the subject has a reduced rate of disease progression. Alternatively, a reduction in rate of disease progression is determined by comparing one or more disease indicators in a subject diagnosed with pulmonary fibrosis pre-treatment versus after starting treatment (e.g., with a TRC

composition of the invention). In cases where the subject presents with fewer or less severe disease indicators after treatment than before treatment, the subject has a reduced rate of disease progression.

[023] Disease indicators of pulmonary fibrosis comprise a profibrotic inflammatory response, dysregulated fibrogenesis, abnormalities in bronchial or alveolar structure (e.g., thickened alveolar septae), and scarring pattern in a lung. A profibrotic inflammatory response is characterized by increases in inflammatory cytokine expression, leukocyte accumulation, alveolitits, release of pro-inflammatory mediators, or recruitment of inflammatory cells to lesions. Dysregulated fibrogenesis is characterized by fibroblast proliferation or differentiation of fibroblasts to myofibroblasts, fibroblastic cell infiltration into the lung, presence of or an increase in fibrotic lesions, increased expression of collagen in the lung, unchecked synthesis of extracellular matrix proteins, or abnormal deposition of extracellular matrix proteins.

[024] For example, disease indicators comprise an increase in expression level (mRNA or protein) of inflammatory cytokines and/or chemokines in a lung of a subject suffering from pulmonary fibrosis compared to a lung from a healthy subject. Exemplary inflammatory cytokines and/or chemokines include TNF-a and Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP- 1). Other disease indicators include an increase in expression (mRNA or protein) of myofibroblast differentiation markers, such as a-smooth muscle actin (a-SMA), in a lung of a subject suffering from pulmonary fibrosis compared to a lung from a healthy subject. Disease indicators also include increased fibrotic content, e.g., as measured by total hydroxyproline content or expression level (mRNA or protein) of a collagen protein such as type I collagen a2 chain (COL1A2), in a lung of a subject suffering from pulmonary fibrosis compared to a lung from a healthy subject.

[025] In some embodiments, the TRCs (e.g., at a therapeutically effective dose) are effective in reducing the rate of disease progression and/or alleviating a symptom of pulmonary fibrosis without causing adverse side effects, such as acute inflammatory responses. In other embodiments, intravenous administration of the TRCs (e.g., at a therapeutically effective dose) reduces the rate of disease progression and/or alleviates a symptom of pulmonary fibrosis while causing minimal adverse side effects, such as acute inflammatory responses.

[026] Administration of a therapeutically effective dose of TRCs reduces the expression level (at the mRNA or protein level) of an inflammatory cytokine or chemokine (or fragment thereof) in the lung of a subject suffering from pulmonary fibrosis, e.g., by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater compared to the expression level of the inflammatory cytokine or chemokine prior to administration.

Preferably, administration of a therapeutically effective dose of TRCs reduces the expression level of an inflammatory cytokine or chemokine in the lung of a subject in suffering from pulmonary fibrosis by at least 50% compared to the expression level of the inflammatory cytokine or chemokine prior to administration. Alternatively, administration of a

therapeutically effective dose of TRCs reduces the expression level of an inflammatory cytokine or chemokine (or fragment thereof) in the lung of a subject suffering from

pulmonary fibrosis to a level that is 5-fold or less, 4-fold or less, 3-fold or less, 2-fold or less, 100% or less, 90% or less, 80%, or less, 70% or less of the expression level of the

inflammatory cytokine or chemokine (or fragment thereof) in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis). Exemplary chemokines or cytokines include but are not limited to TNF-a and CCL2 (MCP- 1).

[027] In some embodiments, administration of a therapeutically effective dose of TRCs reduces the expression level (mRNA or protein) of a myofibroblast differentiation marker (or fragment thereof) in the lung of a subject in suffering from pulmonary fibrosis, e.g., by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater, compared to the expression level of the myofibroblast differentiation marker prior to administration. Preferably, administration of a therapeutically effective dose of TRCs reduces the expression level of a myofibroblast differentiation marker in the lung of a subject in suffering from pulmonary fibrosis by at least 50% compared to the expression level of the myofibroblast differentiation marker prior to administration. Alternatively, administration of a therapeutically effective dose of TRCs reduces the expression level of myofibroblast differentiation marker (or fragment thereof) in the lung of a subject suffering from pulmonary fibrosis to a level that is 5-fold or less, 4-fold or less, 3-fold or less, 2-fold or less, 100% of less, 90% or less, 80%, or less, 70% or less of the expression level of the myofibroblast differentiation marker (or fragment thereof) in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis). Exemplary myofibroblast differentiation markers include but are not limited to a-SMA.

[028] In some embodiments, administration of a therapeutically effective dose of TRCs reduces the total hydroxyproline content in the lung of a subject in suffering from pulmonary fibrosis, e.g., by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater compared to the total hydroxyproline content in the lung prior to administration. Preferably, administration of a therapeutically effective dose of TRCs reduces the total hydroxyproline content in the lung of a subject in suffering from pulmonary fibrosis by at least 50% compared to the total hydroxyproline content in the lung prior to administration. Alternatively, administration of a therapeutically effective dose of TRCs reduces the total hydroxyproline content in the lung of a subject suffering from pulmonary fibrosis to a level that is 5-fold or less, 4-fold or less, 3-fold or less, 2-fold or less, 100% of less, 90% or less, 80%, or less, 70% or less of the total hydroxyproline content in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis). Total hydroxyproline content is determined by standard methods commonly known in the art.

[029] In some embodiments, administration of a therapeutically effective dose of TRCs reduces the expression level (mRNA or protein) of an extracellular matrix (ECM) protein (or fragment thereof) in the lung of a subject suffering from pulmonary fibrosis, e.g., by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater compared to the expression level of the ECM protein prior to administration. Preferably, administration of a therapeutically effective dose of TRCs reduces the expression level of an ECM protein in the lung of a subject in suffering from pulmonary fibrosis by at least 50% compared to the expression level of the ECM protein prior to administration. Alternatively, administration of a therapeutically effective dose of TRCs reduces the expression level of an ECM protein in the lung of a subject suffering from pulmonary fibrosis to a level that is 5- fold or less, 4-fold or less, 3-fold or less, 2-fold or less, 100% of less, 90% or less, 80%, or less, 70% or less of the the expression level of the ECM protein in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis). Exemplary ECM proteins include but are not limited to Fibronectin, proteoglycans, and collagen. Exemplary collagen proteins include but are not limited to type I or type II collagen (e.g., type I collagen a2 chain, or COL1A2). The expression level of ECM proteins can be detected by standard methods in the art, e.g., staining for an ECM protein in a sample of the lung(s).

[030] In some embodiments, administration of a therapeutically effective dose of TRCs reduces one or more disease indicators, e.g., thickening of alveolar septae, fibroblastic cell infiltration, loss of normal alveolar architecture, the number of or extent of fibrotic lesions, and inflammatory cell infiltration, in a lung of a subject suffering from pulmonary fibrosis.

[031] For example, the number of fibroblastic cells and/or inflammatory cells that have infiltrated the lung of a subject suffering from pulmonary fibrosis is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 40-fold, or more after initiating treatment (e.g., with TRCs) compared to pre- treatment. Alternatively, the number of fibroblastic cells and/or inflammatory cells that have infiltrated the lung of a subject suffering from pulmonary fibrosis is reduced to a number that is 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, or 1.5-fold that of, or 100%, 90%, 80%, 70%, 60%, or 50% or less of, the number of fibroblastic cells and/or inflammatory cells in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis).

[032] For example, the number of fibrotic lesions and/or the diameter of a fibrotic lesion in a lung of a subject suffering from pulmonary fibrosis is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 40-fold, or more after initiating treatment (e.g., with TRCs) compared to pre-treatment.

Alternatively, the number of fibrotic lesions and/or the diameter of a fibrotic lesion in a lung of a subject suffering from pulmonary fibrosis is reduced to a number that is 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, or 1.5-fold that of, or 100%, 90%, 80%, 70%, 60%, or 50% or less of, the number of fibrotic lesions and/or the diameter of a fibrotic lesion in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis).

[033] For example, the thickness of an alveolar septae in the lung of a subject suffering from pulmonary fibrosis is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 40-fold, or more after initiating treatment (e.g., with TRCs) compared to pre-treatment. Alternatively, the thickness of the alveolar septae in the lung of a subject suffering from pulmonary fibrosis is reduced to a number that is 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, or 1.5-fold that of, or 100%, 90%, 80%, 70%, 60%, or 50% or less of, the thickness of the alveolar septae in a healthy lung (e.g., a lung of a subject not suffering from pulmonary fibrosis).

[034] The number of fibroblastic cells and/or inflammatory cells that have infiltrated a lung, the thickness of alveolar septae, the number and/or diameter of fibrotic lesions in a lung are determined by standard methods in the art, e.g., by observing the morphology of a lung tissue sample and/or by staining a lung tissue sample for a marker. A lung tissue sample is obtained by methods commonly known in the art, e.g., by surgery or by bronchoscopy.

[035] Increased survival is determined by comparing the prognosis for survival in the subject from a time period prior to administration of the composition to the prognosis for survival in the subject following administration of the composition, wherein an increase in predicted survival time indicates that the treatment increased survival of the subject following administration of the composition.

[036] The invention further features a method of increasing survival in a subject diagnosed with pulmonary fibrosis, comprising administering TRCs to the subject. The survival is increased in the treated subject when compared to an untreated subject, wherein the untreated subject is also diagnosed with pulmonary fibrosis. In certain aspects of the invention the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[037] In some embodiments, the subject suffers from scleroderma (e.g. , in addition to pulmonary fibrosis). The invention also features a method of increasing survival in a subject diagnosed with scleroderma, comprising administering TRCs to the subject. The survival is increased in the treated subject when compared to an untreated subject, wherein the untreated subject is also diagnosed with scleroderma. In certain aspects of the invention the

scleroderma is limited systemic scleroderma or diffuse systemic scleroderma.

[038] In some embodiments, the subject suffers from advanced pulmonary fibrosis. For example, the subject suffers from one or more of the following: cyanosis (blue-colored skin, e.g., around the mouth, or in fingernails), clubbing of the fingers (e.g., enlarged fingertips), shortness of breath without exercise (e.g., while eating, talking, or resting), low blood oxygen levels (hypoxemia) compared to a healthy subject not suffering from pulmonary fibrosis and/or scleroderma, pulmonary hypertension, respiratory failure, a collapsed lung, an enlarged heart compared to a healthy subject not suffering from pulmonary fibrosis and/or scleroderma, heart failure, fluid accumulation in a body part such as the abdomen or leg, and/or prominent pulsations in a neck vein. In some embodiments, a subject suffering from advanced pulmonary fibrosis has no option for treatment or alleviation of symptoms other than lung transplantation. In some embodiments, the subject has been treated with an antiinflammatory agent, where the agent was ineffective in treating or alleviating a symptom of pulmonary fibrosis and/or scleroderma. In some embodiments, the subject suffers from advanced pulmonary fibrosis and scleroderma.

[039] The invention also features a method of preventing or delaying onset of pulmonary fibrosis in a subject at risk for developing pulmonary fibrosis (e.g. , a subject suffering from scleroderma and/or rheumatoid arthritis), comprising administering TRCs to the subject. The onset of pulmonary fibrosis is delayed in the treated subject when compared to an untreated subject, wherein the untreated subject is also at risk for developing pulmonary fibrosis. In certain aspects of the invention, the subject at risk has suffered an injury to a lung, is 40 years old or older, smokes or has smoked cigarettes, has been exposed to a toxin or pollutant that can damage the lung, has undergone radiation treatment, has taken a chemotherapy drug, has taken a heart medication, has taken an antibiotic, or has a family history of pulmonary fibrosis. The toxin or pollutant includes but is not limited to metal dust, wood dust, stone dust, sand dust, grain dust, asbestos fiber, and bird or animal dropping. Exemplary chemotherapy drugs include but are not limited to bleomycin, methotrexate, carmustine, busulfan, and cyclophosphamide. Exemplary heart medications include but are not limited to amiodarone and propranolol. Exemplary antibiotics include but are not limited to

nitrofurantoin, amphotericin B, sulfonamides, and sulfasalazine.

[040] In certain aspects of the invention, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[041] In certain embodiments of the method, the viability of the TRCs is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater. The total number of viable cells in the composition is 1 x 10⁶ to 500 x 10⁶ (e.g. , 35 million to 300 million) and in volume equal to or less than 25 ml, 20 ml, 15 ml, 10 ml, 7.5 ml, 5 ml or less. At least 5% of the viable cells in the composition are CD90⁺. For example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more are CD90⁺. In some aspects at least 5%, 10%, 15%, 20%, 50% or more of the CD90⁺ co-express CD15. Preferably, the cells are about 5-75% viable CD90⁺ with the remaining cells in the composition being CD45⁺. The CD45⁺ cells are CD14⁺, CD34⁺ or VEGFR1 ⁺.

[042] The composition is substantially free of components used during the production of the cell composition, e.g., cell culture components such as bovine serum albumin, horse serum, fetal bovine serum, enzymatically active harvest reagent (e.g., trypsin) and substantially free of mycoplasma, endotoxin, and microbial contamination . Preferably, the composition contain 10, 5, 4, 3, 2, 1, 0.1, 0.05 or less μg/ml bovine serum albumin and 5, 4, 3, 2, 1, 0.1, 0.05 mg/ml enzymatically active harvest reagent.

[043] This composition and methods of making this composition are provided in

International Application No. PCT/US2007/023302, Publication No. WO 2008/054825, the contents of which are incorporated herein in their entirety.

[044] 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 present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[045] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[046] Figures 1 A-E are a series of images of H&E (hematoxylin and eosin) stained lung sections from mice treated as indicated and harvested at the indicated time points. Fig. 1A shows lung morphology at 20X magnification. Fig. IB shows lung morphology at 40X magnification. Fig. 1C shows lung morphology at 100X magnification. Fig. ID shows lung morphology at 200X magnification. Fig. IE shows lung morphology at 400X magnification.

[047] Figure 2 is a series of images depicting lung morphology in Masson-trichrome (staining for collagen) stained lung sections from day 28 mice treated as indicated.

[048] Figure 3 is a graph depicting effects of indicated treatments on body weight (means are shown for each group).

[049] Figures 4A-B are graphs depicting effects of indicated treatments on lung

hydroxyproline content. Fig. 4A depicts effects of indicated treatments on total amount of lung hydroxyproline. Fig. 4B depicts effects of indicated treatments on lung hydroxyproline content as a % of respective saline treated controls.

[050] Figure 5 is a graph depicting effects of indicated treatments on lung COL1A2 mRNA analyzed by real time PCR. [051] Figures 6A-B are graphs depicting effects of indicated treatments on lung CCL2 levels. Fig. 6A depicts effects of indicated treatments on lung CCL2 mRNA levels. Fig. 6B depicts effects of indicated treatments on lung CCL2 mRNA levels relative to respective saline controls.

[052] Figures 7A-B are graphs depicting effects of indicated treatments on lung TNFa levels. Fig. 7A depicts effects of indicated treatments on lung TNFa mRNA levels. Fig. 7B depicts effects of indicated treatments on lung TNFa mRNA levels relative to respective saline controls.

[053] Figure 8 is a graph depicting effects of indicated treatments on lung a-SMA mRNA levels on day 14.

DETAILED DESCRIPTION OF THE INVENTION

[054] The present invention is based on the discovery of compositions and methods of producing cells for cell therapy. The compositions are a mixed population of cells that are enhanced in stem and progenitor cells that are uniquely suited to human administration. These cells are referred to herein as "Tissue Repair Cells" or "TRCs." The methods and data presented herein demonstrate that TRCs are useful for the prevention and suppression of pulmonary fibrosis, e.g., IPF, in patients/subjects who suffer from a lung injury that may lead to a fibrotic response.

Tissue Repair Cells (TRCs)

[055] The use of marrow-derived stem cells in the treatment of damaged and diseased tissue has increased exponentially in the last decade. See, e.g., Wold et al. Congest. Heart Fail. 10.6(2004):292-301; Patel et al. Cell Transplant. 16.9(2007):875-878; Liew et al. Stem Cell Research and Therapy 3(2012):28-42; and van Koppen et al. Plos One 7.6(2012):e38746. The TRCs described herein are differentiated in several ways from previously available cellular therapies. The TRCs are a patient- specific, expanded multicellular therapy, manufactured using a highly automated, fully closed cell-processing system. As described herein, the manufacturing technology selectively expands mesenchymal cells and other mononuclear cells by up to several hundred fold over that found in the patient' s bone marrow, while retaining many of the hematopoietic cells, collected from only a small sample (60 ml) of the patient's bone marrow.

[056] The TRCs have several features that are critical for the success in treating patients suffering from complex, multi-factorial, severe and chronic diseases. For example, in some embodiments, the TRCs are patient- specific (autologous). In these cases, the patient's own cells are utilized— these cells are accepted by the patient's immune system, thereby allowing the cells to differentiate and integrate into existing tissues. This characteristic of the TRCs eliminates both the risk of rejection and the risk of having to use immunosuppressive therapy pre- or post-therapy. In addition, the TRCs are expanded cell populations. A small amount of bone marrow from a patient (approximately 60 ml) is significantly expanded, resulting in the expansion of a number of certain cell types, primarily CD90+ mesenchymal cells and mononuclear cells, to far more than are present in the patient's own bone marrow (e.g., up to 300 times the number of these cells compared with the starting bone marrow). The multiple cell types in the TRCs, which are normally found in bone marrow but in different quantities, possess several functions required for tissue repair and regeneration. Additionally, the TRC therapies are minimally invasive. The aspiration procedure for taking bone marrow can be performed in an outpatient setting and takes approximately 15 minutes. For diseases such as critical limb ischemia (CLI), the administration of TRCs is also performed in an outpatient setting in a single procedure lasting approximately 20 minutes. See, e.g., US 2010/0100108, incorporated herein by reference.

[057] The TRCs are also safe. Bone marrow and bone marrow-like therapies have been used safely and efficaciously in medicine for over three decades. The TRCs leverage this body of scientific study and medical experience. Further, of the nearly 200 patients who have been treated in recent clinical trials (over 400 patients safely treated since the start of clinical trials), there have been no apparent safety issues associated with TRC treatment. See, e.g., Powell et al. J. Vascular Surg. 54.4(2011): 1032-1041; Marston et al. Circulation

124(2011):A8547; Power et al. Mol. Therapy 20.(2012): 1280-1286; Patel et al. J. Cardiac Fail. 17.S8(2011):S58; Clinical Trial NCT01670981; Clinical Trial NCT01020968; Clinical Trial NCT01483898; Clinical Trial NCT00468000; and Clinical Trial NCT00765518.

[058] The highly reproducible and robust Good Manufacturing Practices (GMP) manufacturing system utilized to produce TRCs represents an innovation in the field of cell therapy. See, e.g., Gastens et al. Cell Transplant. 16.7(2007):685-696; Dennis et al. Stem Cells 25.10(2007):2575-2582; and Jaroscak et al. Blood 101.12(2003):5061-5067. For example, the manufacturing process is conducted in a highly- automated, fully-closed, and rigorously controlled system. This controlled system is scalable and reproducible. In some embodiments, production is done under current GMP guidelines required by the US Food and Drug Administration with a current annual capacity to treat up to 3,000 patients.

[059] Isolation, purification, characterization, and culture of TRCs is further described in WO 2008/054825, the contents of which are incorporated by reference its entirety. [060] TRCs contain a mixture of cells of hematopoietic, mesenchymal and endothelial cell lineage produced from mononuclear cells. The mononuclear cells are isolated from adult, juvenile, fetal or embryonic tissues. For example, the mononuclear cells are derived from bone marrow, peripheral blood, umbilical cord blood or fetal liver tissue. TRCs are produced from mononuclear cells, for example by an in vitro culture process which results in a unique cell composition having both phenotypic and functional differences compared to the mononuclear cell population that was used as the starting material. Additionally, the TRCs have both high viability and low residual levels of components used during their production.

[061] The viability of the TRCs is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. Viability is measured by methods known in the art such as trypan blue exclusion. This enhanced viability makes the TRC population more effective in tissue repair, as well as enhances the shelf-life and cryopreservation potential of the final cell product.

[062] By components used during production is meant, but not limited, to culture media components such as horse serum, fetal bovine serum and enzyme solutions for cell harvest. Enzyme solutions include trypsins (animal-derived, microbial-derived, or recombinant), various collagenases, alternative microbial-derived enzymes, dissociation agents, general proteases, or mixtures of these. Removal of these components provide for safe

administration of TRC to a subject in need thereof.

[063] Preferably, the TRC compositions of the invention contain less than 10, 5, 4, 3, 2, 1 mg/ml bovine serum albumin; less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5 mg/ml harvest enzymes (as determined by enzymatic activity) and are substantially free of mycoplasma, endotoxin and microbial (e.g., aerobic, anaerobic and fungi) contamination.

[064] By substantially free of endotoxin is meant that there is less endotoxin per dose of TRCs than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of TRCs.

[065] By substantially free for mycoplasma and microbial contamination is meant as negative readings for the generally accepted tests know to those skilled in the art. For example, mycoplasma contamination is determined by subculturing a TRC product sample in broth medium and distributed over agar plates on day 1, 3, 7, and 14 at 37 °C with appropriate positive and negative controls. The product sample appearance is compared microscopically, at lOOx, to that of the positive and negative control. Additionally, inoculation of an indicator cell culture is incubated for 3 and 5 days and examined at 600x for the presence of mycoplasma as by epifluorescence microscopy using a DNA-binding fluorochrome. The product is considered satisfactory if the agar and/or the broth media procedure and the indicator cell culture procedure show no evidence of mycoplasma contamination.

[066] The sterility test to establish that the product is free of microbial contamination is based on the U.S. Pharmacopedia Direct Transfer Method. This procedure requires that a pre-harvest medium effluent and a pre-concentrated sample be inoculated into a tube containing tryptic soy broth media and fluid thioglycoUate media. These tubes are observed periodically for a cloudy appearance (turbidity) for a 14 day incubation. A cloudy appearance on any day in either medium indicate contamination, with a clear appearance (no growth) testing substantially free of contamination.

[067] The ability of cells within TRCs to form clonogenic colonies compared to BM-MNCs was determined. Both hematopoietic (CFU-GM) and mesenchymal (CFU-F) colonies were monitored (Table 1). As shown in Table 1, while CFU-F were increased 280-fold, CFU-GM were slightly decreased by culturing.

Table 1

[068] The cells of the TRC composition have been characterized by cell surface marker expression. Table 2 shows the typical phenotype measured by flow cytometry for starting BM MNCs and TRCs. These phenotypic and functional differences highly differentiate TRCs from the mononuclear cell starting compositions.

Table 2

[069] Markers for hematopoietic, mesenchymal, and endothelial lineages were examined. Most hematopoietic lineage cells, including CD l ib myeloid, CD14auto- monocytes, CD34 progenitor, and CD3 lymphoid, are decreased slightly, while CD14auto+ macrophages, are expanded 81-fold. The mesenchymal cells, defined by CD90⁺ and CD105+/166+/45-/14- have expansions up to 373-fold. Cells that may be involved in vascularization, including mature vascular endothelial cells (CD 144/146) and CXCR4/VEGFR1+ supportive cells have expansions between 6- to 21 -fold.

[070] Although most hematopoietic lineage cells do not expand in these cultures, the final product still contains close to 80% CD45+ hematopoietic cells and approximately 20% CD90⁺ mesenchymal cells.

[071] TRCs are highly enriched for CD90⁺ cells compared to the mononuclear cell population from which they are derived. The cells in the TRC composition are at least 5%, 10%, 25%, 50%, 75%, or more CD90⁺. The remaining cells in the TRC composition are CD45⁺. Preferably, the cells in the TRC composition are about 5-75% viable CD90⁺. In various aspects, at least 5%, 10%, 15% , 20%, 25%, 30%, 40%, 50%, 60% or more of the CD90⁺ are also CD15⁺ (Table 3). In addition, the CD90⁺ are also CD 105 ⁺.

Table 3

[072] In contrast, the CD90⁺ population in bone marrow mononuclear cells (BMMNC) is typically less than 1% with the resultant CD45⁺ cells making up greater than 99% of the nucleated cells in BMMNCs Thus, there is a significant reduction of many of the mature hematopoietic cells in the TRC composition compared to the starting mononuclear cell population (Table 2).

[073] This unique combination of hematopoietic, mesenchymal and endothelial stems cells are not only distinct from mononuclear cells but also other cell compositions currently being used in cell therapy. Table 4 demonstrates the cell surface marker profile of TRC compared to mesenchymal stem cells and adipose derived stem cells. (Deans RJ, Moseley AB. 2000. Exp. Hematol. 28: 875-884; Devine SM. 2002. J Cell Biochem Supp 38: 73-79; Katz AJ, et al. 2005. Stem Cells. 23:412-423; Gronthos S, et al. 2001. J Cell Physiol 189:54-63; Zuk PA, et al. 2002. Mol Biol Cell. 13: 4279-95.) [074] For example, mesenchymal stem cells (MSCs) are highly purified for CD90⁺ (greater than 95% CD90⁺), with very low percentage CD45⁺ (if any). Adipose-derived stem cells are more variable but also typically have greater than 95% CD90⁺, with almost no CD45⁺ blood cells as part of the composition. There are also Multi-Potent Adult Progenitor Cells

(MAPCs), which are cultured from BMMNCs and result in a pure CD90 population different from MSCs that co-expresses CD49c. Other stem cells being used are highly purified cell types including CD34⁺ cells, AC133⁺ cells, and CD34⁺lin^" cells, which by nature have little to no CD90⁺ cells as part of the composition and thus are substantially different from TRCs.

[075] Cell marker analysis have also demonstrated that the TRCs isolated according to the methods of the invention have higher percentages of CD14⁺ auto⁺, CD34⁺ and VEGFR⁺ cells.

Table 4

[076] Each of the cell types present in a TRC population have varying immunomodulatory properties. Monocytes/macrophages (CD45⁺, CD14⁺) inhibit T cell activation, as well as showing indoleamine 2,3-dioxygenase (IDO) expression by the macrophages. (Munn D.H. and Mellor A.L., Curr Pharm Des., 9:257-264 (2003); Munn D.H., et al. J Exp Med., 189: 1363-1372 (1999); Mellor A.L. and Munn D.H., J. Immunol., 170:5809-5813 (2003); Munn D H., et al., J. Immunol., 156:523-532 (1996)). Monocytes and macrophages regulate inflammation and tissue repair. (Duffield J.S., Clin Sci (Lond), 104:27-38 (2003); Gordon, S.; Nat. Rev. Immunol., 3:23-35 (2003); Mosser, D.M., J. Leukoc. Biol., 73:209-212 (2003); Philippidis P., et al., Circ. Res., 94: 119-126 (2004). These cells also induce tolerance and transplant immunosuppression. (Fandrich F et al. Hum. Immunol., 63:805-812 (2002)). Regulatory T-cells (CD45⁺ CD4⁺ CD25⁺) regulate innate inflammatory response after injury. (Murphy T.J., et al., J. Immunol., 174:2957-2963 (2005)). The T-cells are also responsible for maintenance of self tolerance and prevention and suppression of autoimmune disease. (Sakaguchi S. et al, Immunol. Rev., 182: 18-32 (2001); Tang Q., et al, J. Exp. Med., 199: 1455-1465 (2004)) The T-cells also induce and maintain transplant tolerance (Kingsley C.I., et al. J. Immunol., 168: 1080-1086 (2002); Graca L., et al, J. Immunol., 168:5558-5565 (2002)) and inhibit graft versus host disease (Ermann J., et al., Blood, 105:2220-2226 (2005); Hoffmann P., et al., Curr. Top. Microbiol. Immunol., 293:265-285 (2005); Taylor P. A., et al., Blood, 104:3804-3812 (2004). Mesenchymal stem cells (CD45⁺ CD90⁺ CD105⁺) express IDO and inhibit T-cell activation (Meisel R., et al, Blood, 103:4619-4621 (2004); Krampera M., et al., Stem Cells, (2005)) as well as induce anti-inflammatory activity (Aggarwal S. and Pittenger M.F., Blood, 105: 1815-1822 (2005)).

[077] TRCs also show increased expression of programmed death ligand 1 (PDLl).

Increased expression of PDLl is associated with production of the anti-inflammatory cytokine IL-10. PDLl expression is associated with a non-inflammatory state. TRCs have increased PDLl expression in response to inflammatory induction, showing another aspect of the anti-inflammatory qualities of TRCs.

[078] TRCs, in contrast to BM MNCs also produce at least five distinct cytokines and one regulatory enzyme with potent activity both for wound repair and controlled down-regulation of inflammation Specifically, TRCs produce 1) Interleukin-6 (IL-6), 2) Interleukin-10 (IL- 10), 3) vascular endothelial growth factor (VEGF), 4) monocyte chemoattractant protein- 1 (MCP-1) and, 5) interleukin-1 receptor antagonist (IL-lra). The characteristics of these five cytokines is summarized in Table 5, below.

Table 5

level of the antigen presenting cell.

[079] Additional characteristics of TRCs include a failure to spontaneously produce, or very low-level production of certain pivotal mediators known to activate the Thl inflammatory pathway including interleukin-alpha (IL-l ), interleukin-beta (IL-Ιβ) interferon-gamma (IFN-γ) and most notably interleukin-12 (IL-12). Importantly, the TRCs neither produce these latter Thl -type cytokines spontaneously during medium replacement or perfusion cultures nor after intentional induction with known inflammatory stimuli such as bacterial lipopolysaccharide (LPS). TRCs produced low levels of IFN-γ only after T-cell triggering by anti-CD3 mAb. Finally, the TRCs produced by the current methods produce more of the anti-inflammatory cytokines IL-6 and IL-10 as well as less of the inflammatory cytokine IL- 12.

[080] Moreover, TRCs are inducible for expression of a key immune regulatory enzyme designated indoleamine-2,-3 dioxygenase (IDO). The TRCs according to the present invention express higher levels of IDO upon induction with interferon-γ. IDO has been demonstrated to down-regulate both nascent and ongoing inflammatory responses in animal models and humans (Meisel R., et al, Blood, 103:4619-4621 (2004); Munn D.H., et al, J. Immunol., 156:523-532 (1996); Munn D.H., et al. J. Exp. Med. 189: 1363-1372 (1999); Munn D.H. and Mellor A.L., Curr. Pharm. Des., 9:257-264 (2003); Mellor A.L. and Munn D.H., J. Immunol., 170:5809-5813 (2003)).

[081] As discussed above, TRCs are highly enriched for a population of cells that co- express CD90 and CD 15.

[082] CD90 is present on stem and progenitor cells that can differentiate into multiple lineages. These cells are a heterogeneous population of cells that are at different states of differentiation. Cell markers have been identified on stem cells of embryonic or fetal origin that define the differentiation state of the cell. One of these markers, SSEA-1, also referred to as CD15, is found on mouse embryonic stem cells, but is not expressed on human embryonic stem cells. It has however been detected in neural stem cells in both mice and human. CD 15 is also not expressed on purified mesenchymal stem cells derived from human bone marrow or adipose tissue (Table 6). Thus, the cell population in TRCs that co-expresses both CD90 and CD 15 is a unique cell population and may define a the stem-like state of the CD90 adult- derived cells.

[083] Accordingly, in another aspect of the invention the cell population expressing both CD90 and CD 15 may be further enriched. By further enriched is meant that the cell composition contains 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or 100% CD90⁺ CD15⁺ cells. TRCs can be further enriched for CD90⁺ CD15⁺ cells by methods known in the art such as positive or negative selection using antibodies direct to cell surface markers. The TRCs that have been further enriched for CD90⁺ CD15⁺ cells are particularly useful in cardiac repair and regeneration.

Table 6

Methods of Production of TRCs

[084] TRCs are isolated from any mammalian tissue that contains bone marrow

mononuclear cells (BM MNC). Suitable sources for BM MNC is peripheral blood, bone marrow, umbilical cord blood or fetal liver. Blood is often used because this tissue is easily obtained. Mammals include for example, a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse or a pig.

[085] The culture method for generating TRCs begins with the enrichment of BM MNC from the starting material {e.g., tissue) by removing red blood cells and some of the polynucleated cells using a conventional cell fractionation method. For example, cells are fractionated by using a FICOLL® density gradient separation. The volume of starting material needed for culture is typically small, for example, 40 to 50 mL, to provide a sufficient quantity of cells to initiate culture. However, any volume of starting material may be used.

[086] Nucleated cell concentration is then assessed using an automated cell counter, and the enriched fraction of the starting material is inoculated into a biochamber (cell culture container). The number of cells inoculated into the biochamber depends on its volume. TRC cultures which may be used in accordance with the invention are performed at cell densities of from 10⁴ to 10⁹ cells per ml of culture. When a Aastrom Replicell Biochamber is used 2-3 x 10 total cells are inoculated into a volume of approximately 280 mL.

[087] Prior to inoculation, a biochamber is primed with culture medium. Illustratively, the medium used in accordance with the invention comprises three basic components. The first component is a media component comprised of IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy's Medium, or an equivalent known culture medium component. The second is a serum component which comprises at least horse serum or human serum and may optionally further comprise fetal calf serum, newborn calf serum, and/or calf serum.

Optionally, serum free culture mediums known in the art may be used. The third component is a corticosteroid, such as hydrocortisone, cortisone, dexamethasone, solumedrol, or a combination of these, preferably hydrocortisone. The culture medium further comprises B7H3 polypeptides, VSIG4 polypeptides or a combination of both. When the Aastrom Replicell Biochamber is used, the culture medium consists of IMDM, about 10% fetal bovine serum, about 10% horse serum, about 5 μΜ hydrocortisone, and 4mM L-Glutamine. The cells and media are then passed through the biochamber at a controlled ramped perfusion schedule during culture process. The cells are cultured for 2, 4, 6, 8, 10, 12, 14, 16 or more days. Preferably, the cells are cultured for less than 12 days. Not to be bound by theory, but it is thought that the addition of B7H3 polypeptides, VSIG4 polypeptides or both will allow for the rapid expansion of TRCs, in particular the CD45⁺, CD31⁺, CD14⁺, and auto ⁺ cell population. This rapid expansion will greatly reduce culturing time which is a particular advantage when manufacturing cell suitable for transplantation into humans.

[088] For example, when used with the Aastrom Replicell System Cell Cassette, the cultures are maintained at 37 °C with 5% C0₂ and 20% 0₂.

[089] These cultures are typically carried out at a pH which is roughly physiologic, i.e. 6.9 to 7.6. The medium is kept at an oxygen concentration that corresponds to an oxygen- containing atmosphere which contains from 1 to 20 vol. percent oxygen, preferably 3 to 12 vol. percent oxygen. The preferred range of 0₂ concentration refers to the concentration of 0₂ near the cells, not necessarily at the point of 0₂ introduction which may be at the medium surface or through a membrane.

[090] Standard culture schedules call for medium and serum to be exchanged weekly, either as a single exchange performed weekly or a one-half medium and serum exchange performed twice weekly. Preferably, the nutrient medium of the culture is replaced, preferably perfused, either continuously or periodically, at a rate of about 1 ml per ml of culture per about 24 to about 48 hour period, for cells cultured at a density of from 2xl0⁶ to lxlO⁷ cells per ml. For cell densities of from lxlO⁴ to 2xl0⁶ cells per ml the same medium exchange rate may be used. Thus, for cell densities of about 10 cells per ml, the present medium replacement rate may be expressed as 1 ml of medium per 10 cells per about 24 to about 48 hour period. For cell densities higher than 10 cells per ml, the medium exchange rate may be increased proportionality to achieve a constant medium and serum flux per cell per unit time

[091] A method for culturing bone marrow cells is described in Lundell, et al., "Clinical Scale Expansion of Cryopreserved Small Volume Whole Bone Marrow Aspirates Produces Sufficient Cells for Clinical Use," J. Hematotherapy (1999) 8: 115-127 (which is incorporated herein by reference). Bone marrow (BM) aspirates are diluted in isotonic buffered saline (Diluent 2, Stephens Scientific, Riverdale, NJ), and nucleated cells are counted using a Coulter ZM cell counter (Coulter Electronics, Hialeah, FL). Erythrocytes (non-nucleated) are lysed using a Manual Lyse (Stephens Scientific), and mononuclear cells (MNC) are separated by density gradient centrifugation (Ficoll-Paque^® Plus, Pharmacia Biotech, Uppsala, Sweden) (specific gravity 1.077) at 300g for 20 min at 25°C. BM MNC are washed twice with long- term BM culture medium (LTBMC) which is Iscove's modified Dulbecco's medium

(IMDM) supplemented with 4 mM L-glutamine 9GIBCO BRL, Grand Island, NY), 10% fetal bovine serum (FBS), (Bio-Whittaker, Walkersville, MD), 10% horse serum (GIBCO BRL), 20 μg/ml vancomycin (Vancocin^® HC1, Lilly, Indianapolis, IN), 5 μg/ml gentamicin

(Fujisawa USA, Inc., Deerfield, IL), and 5 μΜ hydrocortisone (Solu-Cortef^®, Upjohn, Kalamazoo, MI) before culture.

Cell Storage

[092] After culturing, the cells are harvested, for example using trypsin, and washed to remove the growth medium. The cells are resuspended in a pharmaceutical grade electrolyte solution, for example Isolyte (B. Braun Medical Inc., Bethlehem, PA) supplemented with serum albumin.

[093] Alternatively, the cells are washed in the biochamber prior to harvest using the wash harvest procedure described below. Optionally after harvest the cells are concentrated and cryopreserved in a biocompatible container, such as 250 ml cryocyte freezing containers (Baxter Healthcare Corporation, Irvine, CA) using a cryoprotectant stock solution containing 10% DMSO (Cryoserv, Research Industries, Salt Lake City, UT), 10% HSA (Michigan Department of Public Health, Lansing, MI), and 200 μg/ml recombinant human DNAse (Pulmozyme^®, Genentech, Inc., South San Francisco, CA) to inhibit cell clumping during thawing. The cryocyte freezing container is transferred to a precooled cassette and cryopreserved with rate-controlled freezing (Model 1010, Forma Scientific, Marietta, OH). Frozen cells are immediately transferred to a liquid nitrogen freezer (CMS-86, Forma Scientific) and stored in the liquid phase. Preferred volumes for the concentrated cultures range from about 5 mL to about 15 ml. More preferably, the cells are concentrated to a volume of 7.5 mL.

Post-culture

[094] When harvested from the biochamber the cells reside in a solution that consists of various dissolved components that were required to support the culture of the cells as well as dissolved components that were produced by the cells during the culture. Many of these components are unsafe or otherwise unsuitable for patient administration. To create cells ready for therapeutic use in humans it is therefore required to separate the dissolved components from the cells by replacing the culture solution with a new solution that has a desired composition, such as a pharmaceutical-grade, injectable, electrolyte solution suitable for storage and human administration of the cells in a cell therapy application.

[095] A significant problem associated with many separation processes is cellular damage caused by mechanical forces applied during these processes, exhibited, for instance, by a reduction in viability and biological function of the cells and an increase in free cellular DNA and debris. Additionally, significant loss of cells can occur due to the inability to both transfer all the cells into the separation apparatus as well as extract all the cells from the apparatus.

[096] Separation strategies are commonly based on the use of either centrifugation or filtration. An example of centrifugal separation is the COBE 2991 Cell Processor (COBE BCT) and an example of a filtration separation is the CYTOMATE® Cell Washer (Baxter Corp) (Table 7). Both are commercially available state-of-the-art automated separation devices that can be used to separate (wash) dissolved culture components from harvested cells. As can be seen in Table 7, these devices result in a significant drop in cell viability, a reduction in the total quantity of cells, and a shift in cell profile due to the preferential loss of the large and fragile CD14⁺auto⁺ subpopulation of TRCs.

Table 7

cell viability

Average post-

83% 71 % 81 % separation cell viability

Average reduction in

18% 69% Not available CD14⁺Auto⁺ frequency

Average cell recovery 73% 74% Not available

[097] These limitations in the art create difficulties in implementing manufacturing and production processes for creating cell populations suitable for human use. It is desirable for the separation process to minimize damage to the cells and thereby result in a cell solution that is depleted of unwanted dissolved components while retaining high viability and biological function with minimal loss of cells. Additionally, it is important to minimize the risk of introducing microbial contaminants that will result in an unsafe final product. Less manipulation and transfer of the cells will inherently reduce this risk.

[098] The invention described in this disclosure overcomes all of these limitations in the current art by implementing a separation process to wash the cells that minimizes exposure of the cells to mechanical forces and minimizes entrapment of cells that cannot be recovered. As a result, damage to cells (e.g. reduced viability or function), loss of cells, and shift in cell profile are all minimized while still effectively separating unwanted dissolved culture components. In a preferred implementation, the separation is performed within the same device that the cells are cultured in which eliminates the added risk of contamination by transfer and separation using another apparatus. The wash process according to the invention is described below.

Wash Harvest

[099] As opposed to conventional culture processes where cells are removed (harvested) from the biochamber followed by transfer to another apparatus to separate (wash) the cells from culture materials, the wash-harvest technique reverses the order and provides a unique means to complete all separation (wash) steps prior to harvest of the cells from the biochamber.

[0100] To separate the culture materials from the cells, a new liquid of desired composition (or gas) may be introduced, preferably at the center of the biochamber and preferably at a predetermined, controlled flow rate. This results in the liquid being displaced and expelled along the perimeter of the biochamber, for example, through apertures, which may be collected in the waste bag. [0101] In some embodiments of the invention, the diameter of the liquid space in the biochamber is about 33 cm, the height of the liquid space is about 0.33 cm and the flow rates of adding rinsing and/or harvesting fluids to the biochamber is about 0.03 to 1.0 volume exchanges (VE) per minute and preferably 0.50 to about 0.75 VE per minute. This substantially corresponds to about 8.4 to about 280 mL/min and preferably 140 to about 210 ml/min. The flow rates and velocities, according to some embodiments, aid in insuring that a majority of the cultured cells are retained in the biochamber and not lost into the waste bag and that an excessively long time period is not required to complete the process. Generally,

4 8

the quantity of cells in the chamber may range from 10 to 10 cell/mL. For TRCs, the quantity may range fromlO⁵ to 10⁶ cells/mL, corresponding to 30 to 300 million total cells for the biochamber dimensions above. Of course, one of skill in the art will understand that cell quantity changes upon a change in the biochamber dimensions

[0102] According to some embodiments, in harvesting the cultured cells from the

biochamber, the following process may be followed, and is broadly outlined in Table 8, below. The solutions introduced into the biochamber are added into the center of the biochamber. The waste media bag 76 may collect corresponding fluid displaced after each step where a fluid or gas is introduced into the biochamber. Accordingly, after cells are cultured, the biochamber is filled with conditioned culture medium (e.g., IMDM, 10% FBS, 10% Horse Serum, metabolytes secreted by the cells during culture) and includes between about 30 to about 300 million cells. A 0.9% NaCl solution ("rinse solution") may then be introduced into the biochamber at about 140 to 210 mL per minute until about 1.5 to about 2.0 liters of total volume has been expelled from the biochamber (Step 1).

[0103] While a single volume exchange for introduction of a new or different liquid within the biochamber significantly reduces the previous liquid within the biochamber, some amount of the previous liquid will remain. Accordingly, additional volume exchanges of the new/different liquid will significantly deplete the previous liquid.

[0104] Optionally, when the cells of interest are adherent cells, such as TRCs, the rinse solution is replaced by harvest solution. A harvest solution is typically an enzyme solution that allows for the detachment of cells adhered to the culture surface. Harvest solutions include for example 0.4% Trypsin/EDTA in 0.9% NaCl that may be introduced into the biochamber at about 140 to 210 mL per minute until about 400 to about 550 ml of total volume has been delivered (Step 2). Thereafter, a predetermined period of time elapses (e.g., 13-17 minutes) to allow enzymatic detachment of cells adhered to the culture surface of the biochamber (Step 3). [0105] Isolyte (B Braun) supplemented with 0.5% HSA may be introduced at about 140 to 210 mL per minute until about 2 to about 3 liters of total volume has been delivered, to displace the enzyme solution (Step 4).

[0106] At this point, separation of unwanted solutions (culture medium, enzyme solution) from the cells is substantially complete.

[0107] To reduce the volume collected, some of the Isolyte solution is preferably displaced using a gas (e.g., air) which is introduced into the biochamber at a disclosed flow rate (Step 5). This may be used to displace approximately 200 to 250 cc of the present volume of the biochamber.

[0108] The biochamber may then be agitated to bring the settled cells into solution (Step 6). This cell suspension may then be drained into the cell harvest bag 70 (or other container) (Step 7). An additional amount of the second solution may be added to the biochamber and a second agitation may occur in order to rinse out any other residual cells (Steps 8 & 9). This final rinse may then be added to the harvest bag 70 (Step 10).

Table 8

Therapeutic Methods

[0109] Tissue Repair Cells (TRCs) are useful for the treatment and prevention of pulmonary fibrosis. TRCs prevent or decrease the severity of the disease by expediting the recovery and healing process and leading to faster termination of the fibrotic response. In certain embodiments of the methods described herein, administration of a TRC composition delays or prevents the progression of pulmonary fibrosis over a period of time, thereby decreasing the severity of and improving the survival rate of patients with the disease. In other embodiments, administration of a TRC composition improves a symptom of pulmonary fibrosis, thereby improving the quality of life for the individual.

Pulmonary fibrosis

[0110] Pulmonary fibrosis is the scarring or thickening of the lungs that leads to organ failure, disruption of gas exchange, and death from respiratory failure. Patients with pulmonary fibrosis suffer from symptoms, such as shortness of breath, abnormal breath sounds called crackles, fatigue, chest discomfort, chronic dry and hacking coughs, loss of appetite, aching muscles and joints, and rapid weight loss. Pulmonary fibrosis patients with advanced disease (i.e. , late stage disease) sometimes have cyanosis (blue-colored skin, e.g., around the mouth, or in fingernails, as an effect of low oxygen) or clubbing of the fingers (e.g., enlarged fingertips). In addition, or alternatively, patients with advanced disease have shortness or breath without exercise (e.g., while eating, talking, or resting), low blood oxygen levels (hypoxemia) compared to a healthy subject not suffering from pulmonary fibrosis, pulmonary hypertension, one or more fibrotic lesions in a lung (e.g. , detected by CT scan or x-ray imaging), an enlarged heart compared to that of a healthy subject not suffering from pulmonary fibrosis, heart failure, fluid accumuluation in body parts such as the abdomen or leg, and/or prominent pulsations in neck veins. In some cases, pulmonary fibrosis patients with advanced disease currently have no option for treatment or alleviation of symptoms other than lung transplantation.

[0111] Tests that are used for the diagnosis of pulmonary fibrosis include bronchoscopy with transbronchial lung biopsy, chest x-ray, chest CT scan, surgical lung biopsy, measurements of blood oxygen levels, pulmonary function tests, and exercise tests. In some embodiments, diagnostic tests reveal disease indicators that indicate disease progression. Exemplary disease indicators include but are not limited to abnormalities in bronchial or alveolar architecture, scarring pattern in a lung, a profibrotic inflammatory response, and dysregulated fibrogenesis. In some aspects, the profibrotic inflammatory response includes increases in cytokine expression (e.g., CCL2 and TNFa), leukocyte accumulation, alveolitis, release of proinflammatory mediators, and recruitment of inflammatory cells to lesions. In some aspects, dysregulated fibrogenesis includes an increase in a-smooth muscle actin (a-SMA) expression in lung, fibroblast proliferation, differentiation of fibroblasts to myofibroblasts, unchecked synthesis of extracellular matrix proteins, and abnormal deposition of extracellular matrix proteins (e.g., collagen). [0112] Idiopathic pulmonary fibrosis (IPF) is pulmonary fibrosis in which the cause is unknown. IPF is a chronic progressive lung disease with unknown natural history that progresses to end stage disease and respiratory failure. IPF is a devastating fibrotic disease of the lung that has no effective therapy to reverse or delay the natural course of the disease and usually results in a fatal outcome. The pathophysiology of IPF is thought to be a disorder of fibroblast proliferation. See, e.g., Wynn et al. Nature Med. 18.7(2012): 1028-1040. IPF is characterized by the progressive and irreversible destruction of lung architecture caused by scar formation due to repeated epithelial injury. This scarring progresses to chronic fibrotic lung disease that inexorably leads to end stage lung disease, organ failure, disruption of gas exchange, and death from respiratory failure. Repair of damaged tissue is a fundamental biological mechanism that allows for the ordered replacement of dead or damaged cells after injury, a process critically important for survival. However, if this process becomes dysregulated, it can lead to the development of a permanent fibrotic scar, which is

characterized by the excess accumulation of extracellular matrix components (fibronectin, proteoglycans, and interstitial collagens) at the site of tissue injury.

[0113] Injurious stimuli may be of exogenous origin or environmental origin, but can also be endogenous such as interstitial lung diseases of widely differing etiologies, including autoimmune diseases as well as idiopathic pulmonary fibrosis. Lung fibrosis can also develop from multiple causes including viral infection and exposure to radiotherapy, and chemotherapeutic drugs. Fibrosis can also occur in bone marrow transplant recipients that suffer from chronic graft versus host disease and in a subset of individuals with chronic inflammatory diseases like scleroderma and rheumatoid arthritis. See, e.g., Kelly et al. Am. J. Respir. Crit. Care Med. 166(2002):510-513; Denham et al. Radiotherapy and Oncology 63.2(2002): 129- 145; Chen et al. Nat. Rev. Cancer 5.2(2005): 102-12; Wolff et al. Bone marrow transplant. 29(2002):357-360; and Young et al. Rheumatology 46(2007):350-357.

[0114] In some embodiments, the TRCs of the invention are administered to a subject suffering from pulmonary fibrosis that also suffers from scleroderma and/or rheumatoid arthritis.

[0115] IPF, a particularly severe form of pulmonary fibrosis has an unknown etiology and primarily occurs in older adults. Current therapies are inadequate, resulting in a poor prognosis with an estimated survival of 2-5 years from the time of diagnosis of IPF.

Estimated mortality rates are 64.3 deaths per million in men and 58.4 deaths per million in women. Currently, no effective treatments are available or indicated to treat IPF, and the disease almost always has a fatal outcome. See, e.g., Walter et al. Proc. Am. Thorac. Soc. 3(2006):330-338. Lung transplant is the only therapy that has been shown to prolong survival in advanced IPF. See, e.g., Walter et al. Proc. Am. Thorac. Soc. 3(2006):330-338. The wait for lung transplantation is long (-46 months), and lung transplantation is limited by supply to 1,400 patients per year, of which approximately half have IPF. As such, there is a high mortality rate in patients on the transplant waiting list. Unfortunately, lung

transplantation has poor outcomes compared to other organs with 5-year survival at only 55%.

[0116] Recent studies have demonstrated success with administration of mesenchymal stem cells (MSCs) in various animal models of lung injury and fibrosis. See, e.g., Rojas et al. Am. J. Respir. Cell Mol. Biol. 33(2005): 145- 152; Abreu et al. Intensive Care Medicine

37(2011): 1421-1431 ; and Ortiz et al. Proc. Natl. Acad. Sci. 100.14(2003):8407-8411.

Protection appears to be independent of stable significant engraftment of the administered MSCs and perhaps mediated by paracrine mechanisms that are not fully understood.

However, the drawback to treatment with MSC is the apparent limited window of treatment. A study has shown that delaying MSC treatment in a mouse model of bleomycin-induced pulmonary fibrosis eliminated the ability of the MSCs to alter the progression of disease. See Ortiz et al. Proc. Natl. Acad. Sci. 100.14(2003):8407-8411. Patients with IPF are usually diagnosed and referred late in the course of the disease; therefore, the use of MSC therapy may be limited.

[0117] A subject at risk for developing pulmonary fibrosis, e.g., IPF, includes but is not limited to a subject who has suffered a lung injury, is older in age (e.g., 40, 45, 50, 55, 60, 65, 70, or 75 years old or older), smokes or has smoked cigarettes, has been exposed to a toxin or pollutant that can damage lungs (e.g., metal dust, wood dust, stone dust, sand dust, grain dust, asbestos fiber, bird or animal dropping), has undergone a radiation treatment (e.g., a radiation treatment to the chest), has taken a chemotherapy drug (e.g., bleomycin, methotrexate, carmustine, busulfan, or cyclophosphamide), has taken a heart medication (e.g., amiodarone or propranolol), has taken an antibiotic (e.g., nitrofurantoin, amphotericin B, sulfonamides, or sulfasalazine), or has a family history of pulmonary fibrosis.

[0118] In addition to respiratory failure and collapsed lungs, hypoxia caused by pulmonary fibrosis can lead to pulmonary hypertension, which can lead to heart failure. Pulmonary fibrosis also increases the risk for pulmonary emboli. There are currently no treatments for pulmonary fibrosis. In severe cases, lung transplantation is the only option. About two-thirds of pulmonary fibrosis patients die within five years. Anti-inflammatory agents have only had limited success in reducing the fibrotic process. [0119] Although the IPF patient population is forecasted to be 128,000 patients, those accurately diagnosed and managed is likely to be about 50%. The reasons for this include misdiagnosis early in the disease, lack of access to pulmonology specialty care, small numbers of patients, and lack of approved therapies. For those who are being actively managed, the therapeutic agents to treat the underlying inflammation and fibrosis are poor and uncertain. The National Institutes of Health (NIH) is conducting the Prednisone, Azathioprine, and N- acetylcysteine (PANTHER)-IPF study and stopped what was considered to be the current standard of care (although not indicated) triple therapy arm of prednisone, azathioprine and N- acetylcysteine due to worse outcomes than placebo. The active versus placebo arm has 11% vs. 1% death and 29% vs. 8% hospitalizations and 31% vs. 9% serious adverse events (SAEs). These findings further highlight the need for investment in well- researched and developed therapies.

[0120] Consequently, there has been a growing and, prior to the present invention, an unmet need for a composition and method to treat and prevent pulmonary fibrosis, e.g., IPF.

[0121] The invention provides a patient- specific therapy for the treatment of pulmonary fibrosis by targeting both the inflammatory and fibrotic processes of the disease to reduce the progression of pulmonary fibrosis, e.g., IPF. The TRCs are an expanded autologous multicellular therapy that contains a mixture of cell types cultured from bone marrow mononuclear cells. The cell types in the TRCs possess functions required for tissue remodeling, immune-modulation, and promotion of angiogenesis. As described herein, in some embodiments, a 12-day process is used to generate TRCs. This process significantly expands the number of certain cell types, primarily CD90+ mesenchymal cells, CD 14+ monocytes and alternately activated macrophages to far more than are present in the patient' s own bone marrow— up to 300 times the number compared to the starting bone marrow.

[0122] Clinical trials as well as in vitro and in vivo studies have demonstrated that the TRCs reduce the inflammatory response, produce anti-inflammatory cytokines, and reduce collagen deposition in tissue. For example, the TRCs have the ability to elicit an anti-inflammatory response. Due to these anti-inflammatory effects, the TRCs are a promising effective treatment for pulmonary fibrosis, e.g., IPF, as well as other disease states with similar inflammatory and fibroproliferative pathology. The studies described herein examined the effects of TRCs on bleomycin-induced pulmonary fibrosis and provided evidence that TRC treatment caused a reduction in the rate of fibrotic response, a reduction in fibroblast differentiation, and a reduction in fibrosis compared to control groups. [0123] The invention provides a method of treating or preventing pulmonary fibrosis, e.g., IPF, in a subject, wherein the subject presents one or more risk factors for or one or more symptoms of pulmonary fibrosis, e.g., IPF. The method includes administering TRCs to the subject.

[0124] According to the methods of the invention, TRCs are delivered to pulmonary fibrosis patients using the procedures provided herein.

[0125] Successful treatment of pulmonary fibrosis achieves a clinical goal. Exemplary clinical goals include but are not limited to alleviation of a symptom of pulmonary fibrosis, reduction of the rate of disease progression, increased recovery rate, termination of disease progression, and increased survival.

[0126] The TRC composition is administered by endotracheal, intramuscular, intradermal, or intravenous injection at one or more sites. Preferably, the composition is administered by endotracheal or intravenous injection. Alternatively, the composition is administered by intramuscular injection at one or more sites (e.g. , approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) sites. The TRC composition may be delivered through a wide range of needle sizes, from large 16 gauge needles to very small 30 gauge needles, as well as very long 28 gauge catheters for minimally invasive procedures. In some embodiments, the TRC composition is administered (e.g., by endotracheal or intravenous injection/infusion) every 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, weeks.

[0127] The cells of the composition are derived from mononuclear cells. These mononuclear cells are derived from bone marrow, peripheral blood, umbilical cord blood or fetal liver.

[0128] Optionally, the cells of the composition are in formulated or provided in a

pharmaceutical-grade electrolyte solution suitable for human administration. The

composition is substantially free of horse serum and/or fetal bovine serum.

[0129] In certain aspects of the invention, at least 10% of the CD90⁺ cells of the composition co-express CD15. Alternatively, or in addition, the CD45⁺ cells of the composition are CD14⁺, CD34⁺ or VEGFR1 ⁺.

[0130] The total number of viable cells in the composition is 1 x 10⁶ to 500 x 10⁶ (e.g., between 35 million and 300 million). For example, the composition contains an average of 1 x 10⁶ to 500 x 10⁶ viable cells, e.g., 1 x 10⁶ to 500 x 10⁶ viable cells, 1 x 10⁶ to 250 x 10⁶ viable cells, 2 x 10⁶ to 250 x 10⁶ viable cells, 3 x 10⁶ to 250 x 10⁶ viable cells, 4 x 10⁶ to 250 x 10⁶ viable cells, 5 x 10⁶ to 250 x 10⁶ viable cells, 5 x 10⁶ to 100 x 10⁶ viable cells, 5 x 10⁶ to 50 x 10⁶ viable cells, 5 x 10⁶ to 10 x 10⁶ viable cells, 8 x 10⁶ to 250 x 10⁶ viable cells, 8 x 10⁶ to 100 x 10⁶ viable cells, 8 x 10⁶ to 50 x 10⁶ viable cells, 8 x 10⁶ to 10 x 10⁶ viable cells, 1 x 10⁶ to 100 x 10⁶ viable cells, 1 x 10⁶ to 50 x 10⁶ viable cells, 1 x 10⁶ to 10 x 10⁶ viable cells, or 1 x 10⁶ to 5 x 10⁶ viable cells. In some embodiments, the composition contains an average of between 90-180 x 10⁶ viable cells. The cells may be suspended in a volume of equal to or less than 15 milliliters, equal to or less than 10 milliliters, equal to or less than 7.5 milliliters, or equal to or less than 5 milliliters.

[0131] For example, a therapeutically effective dose of TRCs contains 1 x 10⁶ to 500 x 10⁶ {e.g. , 35-350 x 10⁶, 90-80 x 10⁶, or 10-180 x 10⁶) viable cells in a volume of 15 milliliters or less {e.g. , 10 milliliters or less, 7.5 milliliters or less, or 5 milliliters or less).

[0132] The invention further provides a method of alleviating one or more symptom in a subject diagnosed with pulmonary fibrosis, including administering TRCs to the subject.

[0133] The invention provides a method of reducing the rate of disease progression in a subject diagnosed with pulmonary fibrosis, including administering TRCs to the subject. A reduction in rate of disease progression is determined by comparing one or more symptoms in the treated subject to one or more symptoms in an untreated subject that also is diagnosed with pulmonary fibrosis. In cases where the treated subject presents with fewer or less severe symptoms than the untreated subject, the treated subject has a reduced rate of disease progression. Alternatively, a reduction in rate of disease progression is determined by comparing one or more disease indicators in the treated subject to one or more disease indicators in an untreated subject that also is diagnosed with pulmonary fibrosis. In cases where the treated subject presents with fewer or less severe disease indicators than the untreated subject, the treated subject has a reduced rate of disease progression.

[0134] In other embodiments, a reduction in rate of disease progression is determined by comparing one or more symptoms in a subject diagnosed with pulmonary fibrosis prior to treatment with the symptoms at a timepoint after starting treatment {e.g., by administering to the subject a TRC composition of the invention). For example, a symptom is assessed in the subject at least 12 hours {e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks, 1.5 years, 2 years, 3 years, 4 years, or more) after starting treatment. In cases where the subject presents with fewer or less severe symptoms post-treatment than pre-treatment, the subject has a reduced rate of disease progression. Alternatively, a reduction in rate of disease progression is determined by comparing one or more disease indicators in a subject diagnosed with pulmonary fibrosis prior to treatment versus after starting treatment {e.g., with a TRC composition of the invention). For example, a disease indicator is assessed in the subject at least 12 hours (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks, 1.5 years, 2 years, 3 years, 4 years, or more) after starting treatment. In cases where the subject presents with fewer or less severe disease indicators after treatment than before treatment, the subject has a reduced rate of disease progression. In one aspect, a reduction in rate of disease progression leads to an increase in recovery rate. The invention provides a method of increasing survival in a subject diagnosed with pulmonary fibrosis, including administering TRCs to the subject. Optionally, the survival is increased in the treated subject when compared to an untreated subject, wherein the untreated subject is also diagnosed with pulmonary fibrosis.

[0135] The invention provides a method of preventing or delaying onset of pulmonary fibrosis in a subject at risk for developing pulmonary fibrosis, including administering TRCs to the subject. In certain aspects of this method, pulmonary fibrosis is prevented from the time of administration of the composition until the passage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 years. In certain aspects, the onset of pulmonary fibrosis is delayed in the treated subject when compared to an untreated subject that also is at risk for developing pulmonary fibrosis.

[0136] In certain embodiments of the methods provided herein, the composition is administered to a subject who presents one or more symptom of pulmonary fibrosis, in combination with another therapy. For instance, the composition is administered in combination with one or more anti-inflammatory agents, one or more immunosuppressive agents, or oxygen therapy.

[0137] The studies described herein used a well-established rodent model of pulmonary fibrosis. In summary, pulmonary fibrosis was induced in NOD/SCID and C57BL/6 mice by endotracheal injection of the antitumor antibiotic, bleomycin, on day 0. Two days following bleomycin treatment, mice were treated with TRCs or vehicle by endotracheal injection. Treatment with TRCs did not ameliorate the early effects of bleomycin treatment on lung injury and inflammation as assessed morphologically. TRC administration led to an enhanced acute response; however the TRC-treated groups appeared to have a significant diminution of cytokine induction and fibrosis. The enhanced acute response observed in these studies was likely due to the mixed population of cells in the TRC preparation. The early acute exacerbation by TRC co-treatment can likely be diminished by a lower dose of cells without diminishing the reduction in overall fibrosis. In some cases, the timing of the administration can also reduce the undesirable acute exacerbation side effects. Taken together, the TRCs have the superior and unexpected properties of having an anti-fibrotic and anti-inflammatory effect late in the pulmonary fibrosis disease process, e.g., in advanced disease.

[0138] IPF is a specific lung manifestation of a broader disease etiology called scleroderma. The impact of scleroderma causes many other direct and secondary diseases, which are, most times, also orphan and areas of high unmet need. The effects of scleroderma can be found throughout the body organ systems including the vascular, skin, heart, GI, kidney, brain and others. Complications associated with scleroderma include liver, lung, kidney, heart, and digestive tract problems. For example, scleroderma-associated complications include esophageal motility disorder, heartburn, gastroesophageal reflux (GERD), constipation, diarrhea, fecal incontinence, pulmonary fibrosis, pulmonary hypertension, hypertension, kidney fibrosis, renal failure, fibrosis of the heart, heart failure, pericarditis, liver cirrhosis, osteoporosis, hypothyroidism, open sores, or nerve damage. Liver cirrhosis is a disease in which healthy liver tissue is replaced by scar tissue, which prevents the liver from

functioning properly. A number of diseases lead to cirrhosis— these diseases cause injury and death of liver cells, and the resulting inflammation and repair process leads to scar tissue formation. In patients with advanced cirrhosis, liver transplantation is often necessary.

Kidney fibrosis is a common final manifestation of a large number of chronic kidney diseases. As chronic kidney diseases progress, widespread tissue scarring leads to the destruction of kidney parenchyma and end-stage renal failure. The tissue scarring in the kidney is caused by build-up of extracellular matrix (ECM) components. Scleroderma is an autoimmune disease characterized by hardening (i.e. , fibrosis) and/or tightening of the skin and connective tissues. The cause of scleroderma is unknown, but increased expression of collagen in the skin and connective tissues leads to symptoms of the disease.

[0139] Skin symptoms of scleroderma can include fingers or toes that turn blue or white in response to hot and cold temperatures (Raynaud's phenomenon), hair loss, skin hardness and thickening, skin that is darker or lighter than normal, stiffness and tightness skin of fingers, hands, and forearm, small white lumps beneath the skin that sometimes ooze a white substance, sores (ulcers) on the fingertips or toes, or tight and mask-like skin on the face. Bone and muscle symptoms include joint pain; numbness and pain in the feet; pain, stiffness, and swelling of fingers and joints; or wrist pain. Breathing problems can result from scarring in the lungs and can include dry cough, shortness of breath, or wheezing. Digestive tract problems can include bloating after meals, constipation, diarrhea, difficulty swallowing, esophageal reflux or heartburn, or problems controlling stools. [0140] Limited systemic scleroderma is a type of scleroderma in which fibrosis and/or tightening of skin and connective tissues occurs in mainly the hands, arms, and face.

Progession of limited systemic scleroderma can lead to calcinosis, Raynaud' s phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia, and/or pulmonary arterial hypertension. Diffuse systemic scleroderma is another type of scleroderma that progresses rapidly and affects a large area of the skin as well as internal organs (e.g., the kidney, esophagus, heart, and/or lung). For this type of scleroderma, the five-year survival rate is about 70%, and the 10-year survival rate is about 55%, and death often occurs from lung, heart, and kidney complications.

[0141] Scleroderma can be diagnosed by detecting hard, tight, and/or thick skin.

Alternatively or in addition, scleroderma is diagnosed by using blood tests (e.g. , to detect for anti-nuclear antibodies, rheumatoid factor antibody levels, or erythrocyte sedimentation rate (ESR)). For example, a rheumatoid factor antibody level of 40 ug/mL or higher (e.g. , 40 ug/mL, 50 ug/mL, 60 ug/mL, 70 ug/mL, 80 ug/mL, 100 ug/mL, 150 ug/mL, 200 ug/mL, 300 ug/mL, 400 ug/mL or higher) can indicate that the subject suffers from scleroderma. For example, an ESR of 10 mrn/hr, 12 mrn/hr, 15 mm/hr, 20 mrn/hr, 25 mrn/hr, 30 mrn/hr, 35 mrn/hr, 40 mm/hr, 45 mm/hr, 50 mm/hr or greater can indicate that the subject suffers from scleroderma. For example, a detectable level of anti-nuclear antibodies can indicate that the subject suffers from scleroderma.

[0142] Other methods of diagnosing scleroderma include chest x-rays (e.g. , to detect fibrosis in the skin and/or an organ in the chest), a CT scan of the lungs (e.g. , to detect fibrosis in the lungs), an echocardiogram (e.g. , to detect reduced heart function), and/or skin biopsy (e.g. , to detect for increased collagen levels and/or fibrotic morphology).

[0143] As the TRCs of the invention have anti-inflammatory and anti-fibrotic effects in a pulmonary fibrosis model, the TRCs are also promising as an effective therapy for other diseases that have inflammatory and fibrotic components, such as cystic fibrosis, scleroderma (e.g., limited systemic scleroderma and/or diffuse systemic scleroderma), liver cirrhosis, kidney fibrosis, or other disorders associated with scleroderma.

[0144] Cystic fibrosis (CF) is a hereditary disease that causes sticky, thick mucus to build up in the lungs, digestive tract, and other areas of the body. In CF, a mutation in the cystic fibrosis transmembrane conductance regulator (CTFR) gene causes a defect in the ability to regulate movement of chloride and sodium ions across epithelial membranes (e.g. , the alveolar epithelia). This leads to the production of abnormally thick and sticky mucus, which builds up in the lungs and the pancreas. The build-up of mucus results in lung infections, lung inflammation, difficulty breathing, sinus infections, hemoptysis (coughing up blood), pulmonary hypertension, heart failure, hypoxia, respiratory failure, poor growth, and digestive problems. As lung function declines in subjects suffering from CF, lung transplantation becomes necessary. Thus, there is also a need for a therapy to treat or alleviate a symptom of CF.

[0145] In some embodiments, the TRCs are administered to a subject suffering from scleroderma, liver cirrhosis, kidney fibrosis, and/or cystic fibrosis. In some embodiments, the TRCs (e.g., at a therapeutically effective dose) prevents, treats, and/or alleviates a symptom of scleroderma, liver cirrhosis, kidney fibrosis, and/or cystic fibrosis. In other embodiments, the TRCs (e.g., at a therapeutically effective dose) increases the survival of a subject suffering from scleroderma, liver cirrhosis, kidney fibrosis, and/or cystic fibrosis, and/or reduces the rate of disease progression.

[0146] In some embodiments, administration of a therapeutically effective dose of TRCs reduces the expression level (at the mRNA or protein level) of a collagen protein (or fragment thereof) in a portion of the skin or in an organ of a subject in suffering from scleroderma, e.g., by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater compared to the expression level of the collagen prior to administration. Preferably, administration of a therapeutically effective dose of TRCs reduces the expression level of a collagen protein in a portion of the skin or in an organ of a subject suffering from scleroderma by at least 50% compared to the expression level of the collagen protein prior to administration. Alternatively, administration of a therapeutically effective dose of TRCs reduces the expression level of a collagen protein in a portion of the skin or in an organ of a subject in suffering from scleroderma to a level that is 5-fold or less, 4-fold or less, 3-fold or less, or 2-fold or less that of, or 100% of less, 90% or less, 80%, or less, 70% or less of the expression level of the collagen protein in the skin or organ of a healthy subject (e.g., not suffering from scleroderma). Exemplary collagen proteins include but are not limited to type I or type II collagen (e.g., type I collagen a2 chain, or COL1A2). The expression level of collagen proteins can be detected by standard methods in the art, e.g., staining for collagen protein in a sample of skin or organ.

[0147] Although MSCs have been shown to be effective in models of pulmonary fibrosis, delaying administration of MSCs has been shown to eliminate the effectiveness of treatment. See, e.g., Rojas et al. Am. J. Respir. Cell Mol. Bio. 33(2005): 145-152. Patients are usually diagnosed late in their disease course. Therefore, the use of MSC therapy might be limited, while the mixed cell population of the TRCs are likely more effective late in the disease course, e.g., during advanced disease. The superior ability of the TRCs to effectively treat this disease at a late stage would create a change in clinical practices and give hope to patients suffering with this fatal disease. In some embodiments, the invention provides a method of treating pulmonary fibrosis, e.g., IPF, in a patient that has been diagnosed late in his or her disease course or that suffers from late stage or advanced pulmonary fibrosis, by administering a TRC composition (e.g., at a therapeutically effective dose) described herein.

[0148] For example, the methods include administering a TRC composition (e.g., at a therapeutically effective dose) to a subject suffering from advanced pulmonary fibrosis. In some embodiments, a subject suffering from advanced pulmonary fibrosis has one or more of the following symptoms: cyanosis (blue-colored skin, e.g., around the mouth, or in fingernails, as an effect of low oxygen), clubbing of the fingers (e.g., enlarged fingertips), shortness of breath without exercise (e.g., while eating, talking, or resting), low blood oxygen levels (hypoxemia) compared to a healthy subject not suffering from pulmonary fibrosis, pulmonary hypertension, one or more fibrotic lesions in a lung (e.g., detected by CT scan or x-ray imaging), respiratory failure, a collapsed lung, an enlarged heart, heart failure, fluid accumuluation in body parts such as the abdomen or leg, and/or prominent pulsations in neck veins. In some cases, a subject suffering from advanced pulmonary fibrosis has no option for treatment or alleviation of symptoms other than lung transplantation.

[0149] In some embodiments, the TRC composition (e.g., at a therapeutically effective dose) treats or alleviates a symptom of pulmonary fibrosis in a subject suffering from advanced pulmonary fibrosis. In other embodiments, the TRC composition (e.g., at a therapeutically effective dose) reduces the rate of disease progression or increases the survival time of a subject suffering from advanced pulmonary fibrosis.

Pharmaceutical Administration and Dosage Forms

[0150] The described TRCs can be administered as a pharmaceutically or physiologically acceptable preparation or composition containing a physiologically acceptable carrier, excipient, or diluent, and administered to the tissues of the recipient organism of interest, including humans and non-human animals. TRC-containing composition can be prepared by resuspending the cells in a suitable liquid or solution such as sterile physiological saline or other physiologically acceptable injectable aqueous liquids. The amounts of the components to be used in such compositions can be routinely determined by those having skill in the art. An exemplary formulation of the TRC-containing composition is Ixmyelocel-T, for which clinical trial results have been published, for e.g., in Marston, W. et al. Circulation 2011; 124: Abstract 8547, the contents of which are incorporated herein by reference. [0151] The TRCs can be administered by parenteral routes of injection, including

endotracheal, subcutaneous, intravenous, intramuscular, and intrasternal. Other modes of administration include, but are not limited to, intranasal, intrathecal, intracutaneous, and percutaneous. In one embodiment of the present invention, administration of the TRCs can be mediated by endoscopic surgery.

[0152] For injectable administration, the composition is in sterile solution or suspension or can be resuspended in pharmaceutically- and physiologically- acceptable aqueous or oleaginous vehicles, which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e. blood) of the recipient. Non-limiting examples of excipients suitable for use include water, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures. The amounts or quantities, as well as the routes of administration used, are determined on an individual basis, and correspond to the amounts used in similar types of applications or indications known to those of skill in the art.

[0153] Consistent with the present invention, the TRC can be administered to body tissues, including lung, blood vessel, muscle, skeletal muscle, joints, and limb.

[0154] The number of cells in a TRC suspension and the mode of administration may vary depending on the site and condition being treated. As non-limiting examples, in accordance with the present invention, a dose (e.g. , a therapeutically effective dose) of about 1 x 10⁶ to about 500 x 10⁶ TRCs (e.g. , about 35 to about 300xl0⁶ TRCs) are injected to effect tissue repair. Consistent with the Examples disclosed herein, a skilled practitioner can modulate the amounts and methods of TRC -based treatments according to requirements, limitations, and/or optimizations determined for each case.

[0155] In preferred embodiments, the TRC pharmaceutical composition comprises between about 8 and 54% CD90⁺ cells and between about 46 and 92% CD45⁺ cells. The TRC pharmaceutical composition preferably contains between about 35x10⁶ and 300x10⁶ viable nucleated cells and between about 7xl0⁶ and 75x10⁶ viable CD90⁺ cells. The TRC pharmaceutical compositional preferably has less than 0.5 EU/ml of endotoxin and no bacterial or fungal growth. In preferred embodiments, a dosage form of TRCs is comprised within 4.7-7.3 mL of pharmaceutically acceptable aqueous carrier. The preferred suspension solution is Multiple Electrolyte Injection Type 1 (USP/EP). Each 100 mL of Multiple Electrolyte Injection Type 1 contains 234 mg of Sodium Chloride, USP (NaCl); 128 mg of Potassium Acetate, USP (C₂H₃KO₂); and 32 mg of Magnesium Acetate Tetrahydrate (Mg(C₂H₃0₂)₂*4H₂0). It contains no antimicrobial agents. The pH is adjusted with hydrochloric acid. The pH is 5.5 (4.0 to 8.0). The Multiple Electrolyte Injection Type 1 is preferably supplemented with 0.5% human serum albumin (USP/EP). Preferably, the TRC pharmaceutical composition is stored at 0-12 °C, unfrozen.

Indications and Modes of Delivery for TRCs

[0156] TRCs may be manufactured and processed for delivery to patients using the described processes where the final formulation is the TRCs with all culture components substantially removed to the levels deemed safe by the FDA. It is critical for the cells to have a final viability greater than 70%, however the higher the viability of the final cell suspension the more potent and efficacious the final cell dose will be, and the less cellular debris (cell membrane, organelles and free nucleic acid from dead cells), so processes that enhance cell viability while maintaining the substantially low culture and harvest components, while maintaining closed aseptic processing systems are highly desirable.

[0157] The dosage, timing, and frequency of administration of TRCs will be determined based on the nature of the fibrotic response and may be varied to maximize the recovery and healing process while minimizing any side effects of TRC treatment. In some cases, an early acute exacerbation by TRC treatment (e.g., TRC+bleomycin co-treatment in the pulmonary fibrosis rodent model) is diminished by a lower dose of instilled cells without diminishing the positive effects on overall fibrosis. Moreover, timing of the instillation after the acute bleomycin-induced injury can also reduce this undesirable acute exacerbation. Further experiments described herein evaluate these possibilities by optimizing the dosage, timing and frequency of administration of cells, and/or to distinguish effects of purified MSCs from that of whole bone marrow cells. Advantages of the cell expansion technique reported herein include the need to collect a relatively small amount of bone marrow under local anesthesia. Alternative techniques require harvesting up to 500-600 mL of bone marrow under general anesthesia. Other potential advantages of TRCs are that the expansion process enriches for the cell lineages thought to be important for angiogenesis and neovascularization and may reverse the suppressive effects of chronic medical conditions on bone marrow progenitors that may impair their regenerative function (Lawall H. et al. Thromb Haemost. 2010 Mar 31; 103(4): 696-709).

[0158] The invention will be further illustrated in the following non-limiting examples. EXAMPLES

Example 1: Effect of expanded human bone marrow cells on a murine bleomycin- induced lung fibrosis model

[0159] The chemotherapeutic agent, bleomycin, is known to cause lung injury and fibrosis in numerous species, including humans, and this has been exploited in studies in animal models of human fibrotic lung disease. Studies of bleomycin-induced pulmonary fibrosis in animals, and rodents especially, have shed light on the importance of several key cells, extracellular matrix components and mediators, such as cytokines and chemokines. In rodents, bleomycin administration results in an acute pulmonary injury accompanied by an acute inflammatory response characterized by increases in inflammatory cytokine expression and leukocyte accumulation. This is followed subsequently by activation and proliferation of fibroblasts and deposition of extracellular matrix. Typically, rodents that are endotracheally challenged with bleomycin exhibit cell death of pneumocytes and endothelial cells 0-1 days post challenge, possibly due to the direct effects of bleomycin on those cells (stage 1); a profibrotic inflammatory response with acute alveolitis 2-3 days post challenge and intense interstitial inflammation 4-12 days post challenge, due to the release of pro-inflammatory mediators and the recruitment of inflammatory cells to the lesion (stage 2); dysregulated fibrogenesis, due to fibroblast proliferation and differentiation to myofibroblasts, and the unchecked synthesis and deposition of extracellular matrix proteins, 10-days to three weeks post challenge (stage 3). Protocol

[0160] The studies were undertaken using a well-established rodent model of pulmonary fibrosis. The model was induced in NOD/SCID and C57BL/6 or CBA/J mice by endotracheal injection of the antitumor antibiotic, bleomycin on day 0. At indicated times (depending on experimental design) beginning before and/or after injection, certain groups of mice were treated by endotracheal injection with bone marrow derived cultured cells, at escalating doses and varying frequency. Control mice received vehicle (saline) only or, in select experiments, normal murine lung fibroblasts. Animals were monitored for body weight and on the indicated days after induction of fibrosis, they were euthanized for analysis of pulmonary inflammation, cytokine expression and fibrosis.

[0161] Mice were randomly divided into 6 groups of 10 animals each for evaluation of the effects of cell administration on control (saline-injected) and bleomycin-induced tissue, and cellular alterations were related to the fibrotic response. The different groups were treated as follows: groups 1-2 received endotracheal injection of saline plus cell media (SAL+ Vehicle) or TRC (SAL+TRC), respectively. Groups 3-4 received bleomycin endotracheally plus injections of media (BLM+ Vehicle) or TRC (BLM+TRC). Saline or bleomycin treatment was done on day 0, and the media or TRC given on one day later. At the indicated time points, the lungs from a designated subgroup were rapidly harvested and quickly frozen in liquid nitrogen for mRNA (as a measure of cytokine and extracellular matrix gene expression) studies. The lungs from another subgroup were used for hydroxyproline

(colorimetric) and protein/cytokine (ELISA) analyses, while the remaining animals were used for morphological analysis, including routine histopathology and immunohistochemical analysis where indicated. Total body weight and survival were also recorded.

[0162] Quantitative data on lung hydroxyproline content, cytokine/chemokine expression (ELISA) and mRNA analysis by real time PCR were evaluated for statistically- significant differences using ANOVA and Scheffe' s test. This initial evaluation of these parameters allows an adequate evaluation of the efficacy of TRC for treatment of pulmonary fibrosis, with respect to both fibrosis (hydroxyproline and histopathological analyses) and

inflammation (cytokine/chemokine and histopathological analyses) in this animal model. Results

Morphology

[0163] Fibrosis was evaluated by morphological analysis of routine H&E (hematoxylin and eosin) and Masson-trichrome (staining for collagen) stained lung tissue sections from 1-2 animals per group. The lungs from these animals were inflated with formalin and after fixation were embedded in paraffin and sectioned for the indicated stains. The sections were evaluated at 20, 40, 100, 200 and 400X magnification as indicated in Figures 1A-E and 2. The results showed the expected normal lung architecture in the SAL+Vehicle control group samples (data not shown), which were macro scopically indistinguishable from lungs of the SAL+TRC group. However microscopically, especially at higher magnification (e.g., >40X), scattered mononuclear inflammatory cells were more frequently encountered in the latter (SAL-TRC group) when compared to the SAL- Vehicle samples at days 7 and 14 (Figures 1C-E). By day 28, there was no discernible difference between the SAL- Vehicle and SAL- TRC lung sections microscopically.

[0164] Bleomycin treatment caused an increase in inflammatory cell infiltration, both polymorphonuclear and mononuclear, especially on days 7 and 14 (Figures 1A-E); which was followed by fibrotic changes on days 14 and 28 as manifested by thickened alveolar septae, fibroblastic cell infiltration and loss of normal alveolar architecture. In the BLM+TRC lung sections, similar changes were present, although the lesions were smaller and appeared less cellular than the BLM+ Vehicle lungs. The fibrotic lesions appeared looser or less dense on day 28 relative to the BLM+Vehicle sections. However on day 7, the inflammatory infiltrate appeared comparable, although the BLM+TRC lungs exhibited microscopic hemorrhage that was not apparent in the BLM+Vehicle lungs. Moreover the inflammatory infiltrate appeared denser in some areas of the BLM+TRC lung sections relative to those in the BLM+Vehicle sections. Lungs from two mice were available for the BLM+Vehicle and BLM+TRC groups. The BLM+Vehicle samples from the two mice showed similar changes, while one of the BLM+TRC lungs showed much less fibrosis than the other. Only sections from one animal were shown in Figures 1A-E.

[0165] Further morphological evaluation was undertaken using Masson-trichrome stained lung sections to evaluate presence of collagen, which stains blue. Only sections from day 28 were shown since fibrosis would be optimal at this time point. The results (Figure 2) confirmed the observations from the H&E stained slides. Abnormal and extensive collagen (stained blue) deposition indicative of fibrosis was evident in the BLM- Vehicle lung sections, but appeared less intense and extensive in the BLM-TRC lungs from 2 different animals in this group, especially in the #2 sample (Figure 2).

Body Weight

[0166] Bleomycin treatment acutely causes lung injury resulting in loss of appetite and body weight over the first week with gradual recovery over the next 2-3 weeks. This was observed in the BLM+Vehicle group (Figure 3) and consistent with the injury and fibrosis pattern seen morphologically (Figures 1A-E and 2). TRC treatment did not significantly alter this weight loss pattern, and had no significant effect on the SAL treated control group as well. The body weight loss appeared to be more steep for the BLM+TRC group, but the recovery was somewhat faster (note slope of red line after day 7).

Lung Collagen

[0167] Lung collagen was evaluated by assaying for total hydroxyproline content (a measure of total collagen content) and type I collagen a2 chain (COL1A2) mRNA levels (since fibrotic lesions are composed predominantly of interstitial collagens, namely collagens I and III). Bleomycin treatment caused a significant increase (p<0.05, ANOVA with post hoc Scheffe's test) in lung hydroxyproline content that appeared to be unaffected by TRC co- treatment (Figure 4A). However, the SAL+TRC group exhibited a higher level of

hydroxyproline, such that the increase induced by bleomycin treatment was insignificant. This difference was more readily apparent when the results were expressed as a percentage of the respective saline control mean values (Figure 4B). Thus, bleomycin caused a significant increase in lung hydroxyproline, which became statistically insignificant when the animals were also treated with TRC (Figures 4A-B). The relative increase in hydroxyproline was not significantly different between vehicle and TRC-treated groups.

[0168] When lung COL1A2 mRNA levels were analyzed, TRC co-treatment caused a significant decrease in the bleomycin-induced elevation of COL1A2 mRNA on day 28, but not at the day 14 time point (Figure 5). This reduction at the later time point indicates a disruption of progression of fibrosis by the TRC treatment, which possibly leads to the decreased elevation of hydroxyproline content in this group at the time of sacrifice on day 28. Cytokine Expression

[0169] Bleomycin-induced fibrosis typically caused induction of lung CCL2 (MCP-1) and TNFa expression, both of which had been shown to play important pathogenic roles in this animal model. Evaluation of CCL2 mRNA by real time PCR revealed induction of expression by bleomycin, which appeared to be unaffected by TRC treatment at the day 7 time point (Figures 6A-B). However, this induction was significantly suppressed by TRC co- treatment at the day 14 time point. This was especially apparent when the results were expressed as the relative level induced by bleomycin treatment (Figure 6B).

[0170] Similar effects were observed on lung TNFa mRNA levels, although the absolute level of bleomycin-induced expression was lower than that observed with CCL2 mRNA (Figures 7A-B). Similar significant induction of TNFa mRNA by bleomycin was observed in both vehicle and TRC-treated groups on day 7, but on day 14 only the induction in the vehicle group was significant (Figure 7A). The pattern was especially evident when bleomycin induced induction was expressed as the relative ratio to the respective saline- treated control groups (Figure 7,B). Thus in both these cases (i.e., CCL2 and TNFa), induction of cytokine by bleomycin treatment declined more rapidly with TRC treatment.

[0171] As a measure of myofibroblast differentiation, the level of lung a-smooth muscle actin (a-SMA) mRNA was determined. Expression of this actin isoform is commonly used as an indicator of myofibroblast differentiation, which is a key factor in fibrosis and its progression. The results showed the expected elevation in a-SMA expression by bleomycin treatment in the vehicle treated control group (Figure 8). This increase was abolished by TRC co-treatment.

Conclusion

[0172] Treatment with TRC did not ameliorate the early effects of bleomycin treatment on lung injury and inflammation as assessed morphologically. This correlated with the lack of discernible effect on induced matrix (COL1A2) gene expression on day 14, and the lack of significant effects on cytokine expression on day 7. However, the TRC treated groups appeared to have a significant diminution of cytokine induction on day 14. a-SMA, an indicator of myofibroblast differentiation, was also suppressed by TRC treatment at the day 14 time point. Moreover, matrix gene expression was reduced on day 28 by TRC co- treatment. This correlated morphologically with evidence of less extensive fibrosis in the TRC-treated groups.

[0173] The totality of the results indicate that, while TRC in combination with bleomycin treatment caused a greater acute response, these human bone marrow cells dampen the fibrotic response at later time points. This indicates either a more rapid recovery or healing process or more rapid termination of the fibrotic response as a result of TRC treatment.

Example 2: Ability of the TRCs to reduce inflammation and fibrosis in the murine bleomycin-induced pulmonary fibrosis model is dependent on dose and route of administration

[0174] In preliminary studies, an acute inflammatory response was seen with TRC treatment. To determine whether a different dose will reduce or ameliorate this side effect, a dose response will be performed to determine the dose that eliminates the acute inflammation but that still retains the reduction in late-inflammation and reduction in fibrosis. Previous studies with TRC used endotracheal administration. Here, the use of intravenous (IV) administration is also tested to determine whether IV administration eliminates the initial inflammation side effect. In some cases, IV is a more clinically useful method of drug administration compared to endotracheal administration.

Experimental Design and Methods

[0175] Mice are randomly divided into groups of 30 animals each for evaluation of the effects of cell administration on control and bleomycin-induced tissue and cellular alterations related to the fibrotic response. Seven, fourteen and twenty-one days following bleomycin treatment, the lungs from a designated subgroup are rapidly harvested and quickly frozen in liquid nitrogen for mRNA analysis (e.g., of markers such as COL1A2, TNF-a and a-SMA). The lungs from another subgroup are used for hydroxyproline (e.g., by standard colorimetric methods) and protein/cytokine (e.g., ELISA) analysis, while the remaining animals are used for morphological analysis, including routine histopathology and immunohistochemical (IHC) analysis as well as IHC staining to determine TRC tissue engraftment. Total body weight and survival are also recorded. The treatment groups are shown in Table 9.

Table 9: Experimental Groups

ET=endo tracheal .

Data Analysis and Interpretation

[0176] Quantitative data on lung hydroxyproline content, cytokine/chemokine expression (ELISA), and mRNA analysis by real time PCR are evaluated for statistically- significant differences using ANOVA and Scheffe's tests. This initial evaluation of these parameters allows for an adequate evaluation of the efficacy of TRCs for the treatment of pulmonary fibrosis, with respect to both fibrosis (hydroxyproline and histopathological analysis) and inflammation (cytokine/chemokine and histopathological analysis) in this animal model. Criteria for acceptance is a 50% reduction in inflammatory cytokine expression and fibrotic content in the lung following treatment with TRCs.

[0177] Alternatively or in addition to reducing the dose, the route of administration is varied. For example, IV dosing can prevent irritation of the lung tissue and eliminate the acute inflammatory response. Experiments are performed to optimize the dose of TRC for IV administration as well as for endotracheal administration in order to retain the reduction in fibrosis while avoiding the acute inflammation side effect.

[0178] In some embodiments, a reduction in TRC dose and/or IV administration eliminates the acute inflammation side effect observed in the preliminary studies. Also, in some cases, IV administration at a higher dose than administered endotracheally reduces the level of inflammatory cytokines and fibrosis observed at a late stage of disease progression in the pulmonary fibrosis model. Example 3: TRCs are effective in reducing fibrosis in the IPF model when administered late in the disease process.

[0179] Patients are typically diagnosed late in the disease process. Therefore, a therapy that is effective during this stage of the disease is highly desirable. The dose and route of administration of TRCs is optimized as described above. The effectiveness of TRC therapy is compared with that of MSCs in the bleomycin-induced lung fibrosis model. Optimal treatment timing and routes of administration of MSCs based on the scientific literature are used. The treatment groups are shown in Table 10.

Table 10: Experimental Groups

Experimental Design and Methods

[0180] Mice are randomly divided into groups of 30 animals each for evaluation of the effects of cell administration on control and bleomycin-induced tissue and cellular alterations related to the fibrotic response. Seven, fourteen and twenty-one days following bleomycin treatment, the lungs from a designated subgroup are rapidly harvested and quickly frozen in liquid nitrogen for mRNA analysis (COL1A2, TNF-a and a-SMA). The lungs from another subgroup are used for hydroxyproline {e.g., via standard colorimetric assays) and protein/cytokine (e.g., ELISA) analysis, while the remaining animals are used for

morphological analysis including routine histopathology and immunohistochemical (IHC) analysis as well as IHC staining to determine TRC tissue engraftment. Total body weight and survival are also recorded.

Data Analysis

[0181] Quantitative data on lung hydroxyproline content, cytokine/chemokine expression (ELISA), and mRNA analysis by real time PCR are evaluated for statistically- significant differences using ANOVA and Scheffe's tests. This initial evaluation of these parameters allows for an adequate evaluation of the efficacy of TRCs for treatment of pulmonary fibrosis, with respect to both fibrosis (hydroxyproline and histopathological analysis) and inflammation (cytokine/chemokine and histopathological analysis) in this animal model. Criteria for acceptance is a 50% reduction in inflammatory cytokine expression and fibrotic content in the lung following treatment with TRC.

[0182] In some cases, a time course of TRC treatment is undertaken to determine the latest time of treatment and the appropriate route of administration that is still therapeutically effective. In some embodiments, the TRCs are effective in reducing fibrosis in the bleomycin-induced pulmonary fibrosis model when administered late in the disease process. A positive effect in this study is a 50% reduction in fibrosis. In some embodiments, a greater reduction in fibrosis is observed after treatment with TRCs than with mesenchymal stem cells.

Example 4: Experimental methods utilizing vertebrate animals

[0183] Female NOD/SCID mice (20-25 g. body weight) are used in the experiments described herein. Bleomycin (BLM)-induced pulmonary fibrosis is induced by endotracheal injection following tracheostomy under ketamine anesthesia. Select groups also receive commercial preparations of cultured human bone marrow cells (TRCs, or ixmyelocel-T, Aastrom Biosciences, Inc, Ann Arbor, MI) by endotracheal or intravenous injections. At indicated time points, control and treated animals are sacrificed by exsanguination by transection of the abdominal aorta while under ketamine anesthesia. Samples of lung tissue and blood are then collected for isolation of cells and immunochemical, biochemical and molecular biological analyses. Mice are used as models of pulmonary fibrosis because the murine bleomycin model is a well-established model of human lung injury and fibrosis, and has been extensively used in past studies of fibrosis. [0184] Animals are anesthetized by intraperitoneal injection of ketamine and Xylazine. For studies in which pulmonary fibrosis is induced, animals are anesthetized during the endotracheal administration of BLM. Every effort is made to minimize discomfort and pain in these animals by the careful use of anesthetics and humane handling by qualified laboratory personnel, and under the supervision of professional veterinarians of the ULAM. For euthanasia, animals are anesthetized with ketamine/Xylazine and then exsanguinated by transection of the abdominal aorta, which is in compliance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

OTHER EMBODIMENTS

[0185] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS We claim:

1. A method of treating or alleviating a symptom of pulmonary fibrosis in a subject in need thereof, comprising administering to the subject an isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage, wherein the viability of said cells is at least 80% and the composition contains:

a) about 5-75% viable CD90⁺ cells with the remaining cells in said composition being CD45⁺;

b) less than 2 μg/ml of bovine serum albumin;

c) less than 1 mg/ml of a enzymatically active harvest reagent; and

d) substantially free of mycoplasma, endotoxin, and microbial contamination.

2. The method of claim 1, wherein the cells of the composition are derived from mononuclear cells.

3. The method of claim 2, wherein the mononuclear cells are derived from bone marrow, peripheral blood, umbilical cord blood or fetal liver.

4. The method of claim 1, wherein said cells of the composition are in a pharmaceutical- grade electrolyte solution suitable for human administration.

5. The method of claim 1, wherein at least 10% of the CD90⁺ cells of the composition co-express CD 15.

6. The method of claim 1, wherein the CD45⁺ cells of the composition are CD14⁺, CD34⁺ or VEGFR1⁺.

7. The method of claim 1, wherein said composition is substantially free of horse serum and/or fetal bovine serum.

8. The method of claim 1, wherein the total number of viable cells in the composition is 35 million to 300 million.

9. The method of claim 8, wherein the cells are in a volume less than 15 milliliters.

10. The method of claim 8, wherein the cells are in a volume less than 10 milliliters.

11. The method of claim 8, wherein the cells are in a volume less than 7.5 milliliters.

12. The method of claim 1, wherein the composition is administered by intramuscular injection at one or more sites.

13. The method of claim 1, wherein the composition is administered by endotracheal injection at one or more sites.

14. The method of claim 1, wherein the composition is administered by intravenous injection.

15. The method of claim 1, wherein the composition comprise an average of between 1- 180 x 10⁶ viable cells.

16. The method of claim 1, wherein the composition comprises an average of between 90- 180 x 10⁶ viable cells.

17. The method of claim 1, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

18. A method of increasing survival in a subject diagnosed with pulmonary fibrosis, comprising administering to the subject an isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage, wherein the viability of said cells is at least 80% and the composition contains:

a) about 5-75% viable CD90⁺ cells with the remaining cells in said composition being CD45⁺;

b) less than 2 μg/ml of bovine serum albumin;

c) less than 1 mg/ml of a enzymatically active harvest reagent; and

d) substantially free of mycoplasma, endotoxin, and microbial contamination.

19. The method of claim 18, wherein the survival is increased in the treated subject when compared to an untreated subject, wherein the untreated subject is also diagnosed with pulmonary fibrosis.

20. The method of claim 18, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

21. The method of claim 1 or 18, wherein the subject suffers from advanced pulmonary fibrosis.

22. The method of claim 21, wherein the subject suffers from one or more of the following: cyanosis, clubbing of a finger, an enlarged fingertip, shortness of breath without exercise, hypoxemia compared to a healthy subject not suffering from pulmonary fibrosis, pulmonary hypertension, one or more fibrotic lesions in a lung, respiratory failure, a collapsed lung, an enlarged heart compared to a healthy subject not suffering from pulmonary fibrosis, heart failure, fluid accumulation in a body part, and prominent pulsations in a neck vein.

23. A method of preventing or delaying onset of pulmonary fibrosis in a subject at risk for developing pulmonary fibrosis, comprising administering to the subject an isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage, wherein the viability of said cells is at least 80% and the composition contains:

a) about 5-75% viable CD90⁺ cells with the remaining cells in said composition being CD45⁺;

b) less than 2 μg/ml of bovine serum albumin;

c) less than 1 mg/ml of a enzymatically active harvest reagent; and

d) substantially free of mycoplasma, endotoxin, and microbial contamination.

24. The method of claim 23, wherein the onset of pulmonary fibrosis is delayed in the treated subject when compared to an untreated subject, wherein the untreated subject is also at risk for developing pulmonary fibrosis.

25. The method of claim 23, wherein the subject at risk has suffered an injury to a lung, is 40 years old or older, smokes or has smoked cigarettes, has been exposed to a toxin or pollutant that can damage the lung, has undergone radiation treatment, has taken a chemotherapy drug, has taken a heart medication, has taken an antibiotic, or has a family history of pulmonary fibrosis.

26. The method of claim 25, wherein the toxin or pollutant is selected from the group comprising metal dust, wood dust, stone dust, sand dust, grain dust, asbestos fiber, and bird or animal dropping.

27. The method of claim 25, wherein the chemotherapy drug is selected from the group comprising bleomycin, methotrexate, carmustine, busulfan, and cyclophosphamide.

28. The method of claim 25, wherein the heart medication is selected from the group comprising amiodarone and propranolol.

29. The method of claim 25, wherein the antibiotic is selected from the group comprising nitrofurantoin, amphotericin B, sulfonamides, and sulfasalazine.

30. The method of claim 23, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

31. The method of claim 1, 18, or 23, wherein the subject additionally suffers from scleroderma.