WO2018185584A1

WO2018185584A1 - Methods and compositions for treating acute lung injury and respiratory distress syndrome

Info

Publication number: WO2018185584A1
Application number: PCT/IB2018/051847
Authority: WO
Inventors: Yaacob YANAY; Arie EISENKRAFT
Original assignee: Pluristem Ltd.
Priority date: 2017-04-05
Filing date: 2018-03-20
Publication date: 2018-10-11

Abstract

Disclosed herein are methods and compositions comprising adherent stromal cells, derived from placental tissue, for treating subjects suffering from for treating acute lung injury and respiratory distress syndrome (e.g. acute respiratory distress syndrome, pulmonary ARDS, or extrapulmonary ARDS). The compositions include pharmaceutical compositions, which may further include pharmacologically acceptable excipients.

Description

METHODS AND COMPOSITIONS FOR TREATING ACUTE LUNG INJURY AND

RESPIRATORY DISTRESS SYNDROME

FIELD

Disclosed herein are methods and compositions comprising placental-derived adherent stromal cells for treating acute lung injury and respiratory distress syndrome.

BACKGROUND

ARDS is a disease involving increased pulmonary capillary permeability. The consequent accumulation of protein-rich fluid inside the alveoli is the result of the damage to the capillary endothelium and alveolar epithelium; this causes the release of cytokines, producing diffuse alveolar damage. Common risk factors for ARDS are: pneumonia, sepsis, gastric content aspiration, trauma, pancreatitis, inhalation injury, burns, non-cardiogenic shock, drug overdose, transfusion related acute lung injury (TRALI), and drowning. The characteristic pathological features of ARDS have classically been described by three overlapping phases: an exudative or inflammatory phase, a proliferative phase and a fibrotic phase. According to this scheme, initial fluid accumulation is followed, within 72 h, by a variable amount of proliferation of type II alveolar cells and fibroblasts, and new matrix deposition (Umbrello M et al).

SUMMARY

In one embodiment, there is provided a method of treating a respiratory distress syndrome, comprising the step of administering to the subject a pharmaceutical composition comprising placental adherent stromal cells (ASC), thereby treating a respiratory distress syndrome.

In another embodiment, there is provided a pharmaceutical composition for treating respiratory distress syndrome, comprising a therapeutically effective amount of placental ASC.

In another embodiment, there is provided use of placental ASC in the preparation of a medicament for treating respiratory distress syndrome.

In one embodiment, there is provided a method of treating acute lung injury (ALI), comprising the step of administering to the subject a pharmaceutical composition comprising placental adherent stromal cells (ASC), thereby treating ALI. In another embodiment, there is provided a pharmaceutical composition for treating ALI, comprising a therapeutically effective amount of placental ASC.

In another embodiment, there is provided use of placental ASC in the preparation of a medicament for treating ALI.

In certain embodiments, the ASC described herein have been cultured in 2-dimensional (2D) culture, 3-dimensional (3D) culture, or a combination thereof. Non-limiting examples of 2D and 3D culture conditions are provided in the Detailed Description and in the Examples.

Reference herein to "growth" of a population of cells is intended to be synonymous with expansion of a cell population.

Except where otherwise indicated, all ranges mentioned herein are inclusive.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

In the drawings:

FIG. 1 is a diagram of a bioreactor that can be used to prepare the cells. FIG. 2 contains pictures of bone marrow (BM)-derived MSC (top row) or placental cells after adipogenesis assays. Cells were incubated with (left column) or without (right column) differentiation medium. Placental ASC were expanded in SRM (middle 3 rows depict 3 different batches) or in full DMEM (bottom row).

FIG. 3 contains pictures of BM-derived MSC (top row) or placental cells after osteogenesis assays. Cells were incubated with (left column) or without (right column) differentiation medium. Placental ASC were expanded in SRM (middle 3 rows depict 3 different batches) or in full DMEM (bottom row).

FIG. 4 is a perspective view of a carrier (or "3D body"), according to an exemplary embodiment. B is a perspective view of a carrier, according to another exemplary embodiment. C is a cross-sectional view of a carrier, according to an exemplary embodiment.

DETAILED DESCRIPTION

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

Aspects of the invention relate to methods and compositions that comprise placenta- derived adherent stromal cells (ASC). In some embodiments, the ASC may be human ASC, or in other embodiments animal ASC.

In some embodiments, there is provided a method of treating respiratory distress syndrome in a subject in need thereof, comprising the step of administering to the subject a pharmaceutical composition comprising placental adherent stromal cells (ASC), thereby treating respiratory distress syndrome. In some embodiments, the respiratory distress syndrome is acute respiratory distress syndrome (ARDS), which may be, in more specific embodiments, pulmonary ARDS, or extrapulmonary ARDS. In still further embodiments, the ARDS comprises pulmonary edema, which may be, in more specific embodiments, noncardiogenic pulmonary edema; or in other embodiments arterial hypoxemia; or in other embodiments, a combination thereof. In other embodiments, the respiratory distress syndrome is infant respiratory distress syndrome (IRDS).

In still other embodiments, there is provided a method of treating acute lung injury (ALI) in a subject in need thereof, comprising the step of administering to the subject a pharmaceutical composition comprising placental ASC, thereby treating ALI.

In yet other embodiments, there is provided a method of treating pulmonary edema, which may be, in more specific embodiments, noncardiogenic pulmonary edema, comprising the step of administering to the subject a pharmaceutical composition comprising placental ASC. In other embodiments, there is provided a method of treating arterial there is provided a method of treating, comprising the step of administering to the subject a pharmaceutical composition comprising placental ASC.

In certain embodiments, the ARDS follows pneumonia, e.g. within 7 days of the onset of pneumonia. In other embodiments, the ARDS is secondary to pneumonia. In still other embodiments, the ALI follows pneumonia. In other embodiments, the ALI is secondary to pneumonia.

In other embodiments, the ARDS follows sepsis, e.g. within 7 days of the onset of sepsis. In other embodiments, the ARDS is secondary to sepsis. In still other embodiments, the ALI follows sepsis. In other embodiments, the ALI is secondary to sepsis.

In still other embodiments, the ARDS follows a condition selected from gastric content aspiration, trauma, pancreatitis, inhalation injury, burns, non -cardiogenic shock, drug overdose, transfusion related acute lung injury (TRALI), an amniotic fluid embolism, and ischemic reperfusion injury, each of which represents a separate embodiment, e.g. within 7 days of the insult or injury. In other embodiments, the ARDS is secondary to the insult or injury. In still other embodiments, the ALI follows the insult or injury. In other embodiments, the ALI is secondary to the insult or injury.

In yet other embodiments, the ARDS follows drowning e.g. within 7 days of the drowning incident. In other embodiments, the ARDS is secondary to the drowning. In still other embodiments, the ALI follows the drowning. In other embodiments, the ALI is secondary to the drowning. In other embodiments, the respiratory distress syndrome is acute respiratory distress syndrome (ARDS). Except where indicated otherwise, "ARDS" refers to all phases of the disease. In more specific embodiments, the ARDS is selected from the exudative or inflammatory phase, the proliferative phase, and the fibrotic phase of ARDS. In certain embodiments, ARDS is defined according to the Berlin criteria (Ranieri VM et al).

In more specific embodiments, the ARDS may be pulmonary ARDS, e.g. ARDS due to a direct (ARDSp) lung injury, or extrapulmonary ARDS, e.g.. ARDS due to an indirect (ARDSexp) lung injury. Those skilled in the art will appreciate that the prevalent damage in the early stages of ARDSp is intra-alveolar, whereas in ARDSexp, it is the interstitial edema. Moreover, the radiological pattern, by chest x-ray or computed tomography (CT), is typically characterized by prominent consolidation in ARDSp, while in ARDSexp, it is typically characterized by prominent ground-glass opacification. Finally, in ARDSp lung elastance is typically more markedly increased than in ARDSexp, where the main abnormality is typically an increase in chest wall elastance, due to abnormally high intra-abdominal pressure (Pelosi P et al). In still other embodiments, ARDSp coexists with ARDSexp e.g., when one lung with direct injury {e.g. from pneumonia) and the other has indirect injury, e.g. from mediator release from the contralateral pneumonia.

In certain embodiments, the respiratory distress syndrome or ALI co-presents with pulmonary arterial hypertension. In other embodiments, the respiratory distress syndrome or ALI comprises pulmonary fibrosis. In still other embodiments, the respiratory distress syndrome or ALI comprises pulmonary fibrosis and also co-presents with pulmonary arterial hypertension.

Methods of diagnosing respiratory distress syndrome and ALI and determining the effect of therapeutic modalities on respiratory distress syndrome are well known in the art, and are described, for example, in Sweeney and McAuley, Ranieri VM et al, and references cited therein.

In other embodiments, the respiratory distress syndrome or ALI is secondary to pneumonia. In other embodiments, the respiratory distress syndrome or ALI is secondary to hemodynamic shock. In other embodiments, the respiratory distress syndrome or ALI is secondary to thermal burns {e.g. from an explosion). In still other embodiments, the respiratory distress syndrome or ALI is secondary to renal IRI (ischemic reperfusion injury). In yet other embodiments, the respiratory distress syndrome or ALI is secondary to limb IRI, e.g. post-tourniquet or after hemodynamic shock. In more specific embodiments, the respiratory distress syndrome or ALI may result from inflammatory mediators released upon return of blood to ischemic tissue (e.g. after an acute ischemic event or after application of a tourniquet). In still other embodiments, the respiratory distress syndrome or ALI is secondary to trauma, a non-limiting example of which is shrapnel injury. In other embodiments, the respiratory distress syndrome or ALI is secondary to sepsis. In other embodiments, the respiratory distress syndrome or ALI is secondary to gastric content aspiration. In still other embodiments, the respiratory distress syndrome or ALI is secondary to pancreatitis. In other embodiments, the respiratory distress syndrome or ALI is secondary to inhalation injury (e.g. from smoke inhalation or inhalation of chemical irritants). In other embodiments, the respiratory distress syndrome or ALI is secondary to an amniotic fluid embolism. In various other embodiments, the respiratory distress syndrome or ALI is secondary to non-cardiogenic shock, drug overdose, transfusion related acute lung injury (TRALI), or drowning.

In other embodiments, the respiratory distress syndrome is ARDS that is secondary to pneumonia. In various embodiments, the pneumonia may be bacterial, viral, fungal, or parasitic pneumonia. In more specific embodiments, bacterial pneumonia may be caused by infection with Streptococcus pneumoniae, Jirovecii, Haemophilus influenzae, Enterobacteriaceae, Staphylococcus aureus, Legionella pneumophila, Clamydia pneumoniae, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, or Stenotrophompnas maltophilia; viral pneumonia may be caused by infection with Influenza A, Influenza B, a Rhinovirus, RSV, a Parainfluenza virus, a Coronavirus, an Enterovirus, HSV, or CMV; fungal pneumonia may be caused by infection with Pneumocystis, Aspergillus fumigatus; and parasitic pneumonia may be caused by infection with Toxoplasma gondii. In other embodiments, the pneumonia occurs in an immunocompromised subject, and may result e.g. from infection with Pneumocystis jirovecii, Toxoplasma gondii, or Aspergillus fumigatus. Alternatively or in addition, the subject is receiving extracorporeal membrane oxygenation.

In certain embodiments, the respiratory distress syndrome or ALI occurs following surgery (Cutts S et al). In some embodiments, the respiratory distress syndrome or ALI co-presents with Sequential [Sepsis-related] Organ Failure or multiple organ dysfunction syndrome (MODS). In more specific embodiments, the MODS follows a trauma. In certain embodiments, the prior trauma is an unintentional trauma, e.g. resulting from an accident. In other embodiments, the prior trauma is a prior surgery. As will be appreciated by those skilled in the art, Sequential [Sepsis-related] Organ Failure can be diagnosed using the Sequential [Sepsis-related] Organ Failure Assessment (SOFA) score or the quick SOFA (qSOFA) score (Raith EP et al).

In still other embodiments, the respiratory distress syndrome or ALI is associated with presence of pro-inflammatory mediators and/or increased cyclo-oxygenase-2 (COX-2) expression.

In other embodiments, the respiratory distress syndrome or ALI co-presents with systemic inflammatory response syndrome (SIRS). Alternatively or in addition, the respiratory distress syndrome or ALI is co-present with non-specific immune activation. As will be appreciated by those skilled in the art, SIRS can be diagnosed using the SIRS criteria (Seymour CW et al). The effect of therapeutic modalities on intrapulmonary inflammatory responses can be determined in humans or in animal models, e.g. the porcine model described in Hartmann EK et al, Jugg BJ et al, and the references cited therein.

The skilled practitioner will also appreciate that stress-related immune alteration can be readily measured by determining the concentrations of inflammatory cytokines and immune cell subtype compositions. In non-limiting embodiments, serum cytokine concentrations are measured (e.g. as described in Rodrigues CE et al, van Griensven et al, and the references cited therein).

In other embodiments, the respiratory distress syndrome is infant respiratory distress syndrome (IRDS). IRDS is usually found in premature infants at least 6 weeks before their due date, and results from an inability to produce sufficient surfactant.

In various embodiments, the ASC are administered to the subject within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 15 hours, within 18 hours, within 24 hours, within 30 hours, within 36 hours, within 48 hours, within 3 days, within 4 days, within 5 days, within 6 days, within 8 days, within 10 days, within 12 days, or within 20 days of the diagnosis of respiratory distress syndrome, or in other embodiments, ALI. In some embodiments, the described compositions are administered after the subject is stabilized from acute pathologies. In some embodiments, the subject is stabilized by supportive medical care. In more specific embodiments, the described compositions are administered 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 8-24, 10-24, 12-48, 1-48, 2-48, 3-48, 4-48, 5-48, 6-48, 8-48, 10-48, 12-48, 18-48, 24-48, 1-72, 2-72, 3-72, 4-72, 5-72, 6-72, 8-72, 10-72, 12-72, 18- 72, 24-72, or 36-72 hours after the diagnosis of respiratory distress syndrome. In still other embodiments, the described compositions are administered 3-48, 4-48, 5-48, or 6-48 hours after the diagnosis of respiratory distress syndrome, or in other embodiments, ALL

In various embodiments, the aforementioned supportive medical care includes antibiotic treatment for pneumonia and/or treatment, such as ventilation, to sustain vital functions, to improve and ensure an adequate gas exchange, while reducing the probability of causing damage such as by VILI (ventilator induced lung injury). Methods of supportive medical care to sustain life in subjects suffering from respiratory distress syndrome or ALI are well known in the art, and are described, for example, in Umbrello M et al.

In various embodiments, the described cells are able to exert the described therapeutic effects, each of which is considered a separate embodiment, with or without the cells themselves engrafting in the host. For example, the cells may, in various embodiments, be able to exert a therapeutic effect, without themselves surviving for more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, or more than 14 days.

ASC and sources thereof

Except where indicated otherwise herein, the terms "placenta" and "placental tissue", as well as reference to "placenta-derived" cells, refer to any portion of the placenta. Placenta-derived adherent cells may be obtained, in various embodiments, from either fetal or, in other embodiments, maternal regions of the placenta, or in other embodiments, from both regions. More specific embodiments of maternal sources are the decidua basalis and the decidua parietalis. More specific embodiments of fetal sources are the amnion, the chorion, and the villi. In certain embodiments, tissue specimens are washed in a physiological buffer [e.g., phosphate -buffered saline (PBS) or Hank's buffer]. In certain embodiments, the placental tissue from which cells are harvested includes at least one of the chorionic and decidua regions of the placenta, or, in still other embodiments, both the chorionic and decidua regions of the placenta. More specific embodiments of chorionic regions are chorionic mesenchymal and chorionic trophoblastic tissue. More specific embodiments of decidua are decidua basalis, decidua capsularis, and decidua parietalis.

Single-cell suspensions can be made, in other embodiments, by treating the tissue with a digestive enzyme (see below) or/and physical disruption, a non-limiting example of which is mincing and flushing the tissue parts through a nylon filter or by gentle pipetting (e.g. Falcon, Becton, Dickinson, San Jose, CA) with washing medium. In some embodiments, the tissue treatment includes use of a DNAse, a non-limiting example of which is Benzonase from Merck.

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

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

The various media described herein, i.e. the 2D growth medium and the 3D growth medium, may be independently selected from each of the described embodiments relating to medium composition. In various embodiments, any medium suitable for growth of cells in a standard tissue apparatus and/or a bioreactor may be used.

Those skilled in the art will appreciate in light of the present disclosure that cells may be, in some embodiments, extracted from a placenta, for example using physical and/or enzymatic tissue disruption, followed by marker-based cell sorting, and then may be subjected to the culturing methods described herein. In still other embodiments, the cells are a placental cell population that is a mixture of fetal- derived placental ASC (also referred to herein as "fetal ASC" or "fetal cells") and maternal-derived placental ASC (also referred to herein as "maternal ASC" or "maternal cells"), where a majority of the cells are maternal cells. In more specific embodiments, the mixture contains at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, or at least 99.99% maternal cells, or contains between 90-99%, 91-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, 97-99%, 98-99%, 90-99.5%, 91-99.5%, 92-99.5%, 93-99.5%, 94-99.5%, 95-99.5%, 96- 99.5%, 97-99.5%, 98-99.5%, 90-99.9%, 91-99.9%, 92-99.9%, 93-99.9%, 94-99.9%, 95-99.9%, 96-99.9%, 97-99.9%, 98-99.9%, 99-99.9%, 99.2-99.9%, 99.5-99.9%, 99.6-99.9%, 99.7-99.9%, or 99.8-99.9% maternal cells.

Predominantly or completely maternal cell preparations may be obtained by methods known to those skilled in the art, including the protocol detailed in Example 1 and the protocols detailed in PCT Publ. Nos. WO 2007/108003, WO 2009/037690, WO 2009/144720, WO 2010/026575, WO 2011/064669, and WO 2011/132087. The contents of each of these publications are incorporated herein by reference. Predominantly or completely fetal cell preparations may be obtained by methods known to those skilled in the art, including selecting fetal cells via their markers (e.g. a Y chromosome in the case of a male fetus), and expanding the cells.

In other embodiments, the cells are a placental cell population that does not contain a detectable amount of maternal cells and is thus entirely fetal cells. A detectable amount refers to an amount of cells detectable by FACS, using markers or combinations of markers present on maternal cells but not fetal cells, as described herein. In certain embodiments, "a detectable amount" may refer to at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1%.

In still other embodiments, the preparation is a placental cell population that is a mixture of fetal and maternal cells, where a majority of the cells are fetal cells. In more specific embodiments, the mixture contains at least 70% fetal cells. In more specific embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the cells are fetal cells. Expression of CD200, as measured by flow cytometry, using an isotype control to define negative expression, can be used as a marker of fetal cells under some conditions. In yet other embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% of the described cells are fetal cells.

In more specific embodiments, the mixture contains 20-80% fetal cells; 30-80% fetal cells; 40-80% fetal cells; 50-80% fetal cells; 60-80% fetal cells; 20-90% fetal cells; 30-90% fetal cells; 40-90% fetal cells; 50-90% fetal cells; 60-90% fetal cells; 20-80% maternal cells; 30-80% maternal cells; 40-80% maternal cells; 50-80% maternal cells; 60-80% maternal cells; 20-90% maternal cells; 30-90% maternal cells; 40-90% maternal cells; 50-90% maternal cells; or 60-90% maternal cells.

Also described herein are placental cell populations that are produced by expanding a population of adherent stromal cells (ASC) in a medium that contains less than 5% animal serum.

In certain embodiments, the aforementioned medium contains less than 4% animal serum; less than 3% animal serum; less than 2% animal serum; less than 1% animal serum; less than 0.5% animal serum; less than 0.3% animal serum; less than 0.2% animal serum; or less than 0.1% animal serum. In other embodiments, the medium does not contain animal serum. In other embodiments, the medium is a defined medium to which no serum has been added. Low-serum and serum-free media are collectively referred to as "serum-deficient medium/media".

Those skilled in the art will appreciate that reference herein to animal serum includes serum from a variety of species, provided that the serum stimulates expansion of the ASC population. In certain embodiments, the serum is mammalian serum, non-limiting examples of which are human serum, bovine serum (e.g. fetal bovine serum and calf bovine serum), equine serum, goat serum, and porcine serum.

In certain embodiments, the serum-deficient medium is supplemented with factors intended to stimulate cell expansion in the absence of serum. Such medium is referred to herein as serum-replacement medium or SRM, and its use, for example in cell culture and expansion, is well known in the art, and is described, for example, in Kinzebach et al. In other embodiments, the serum-deficient medium contains one or more growth factors. In certain embodiments, the growth factors, individually or, in other embodiments collectively, induce cell expansion in culture. In other embodiments, the growth factors, individually or, in other embodiments collectively, induce cell expansion in culture without differentiation.

In more specific embodiments, the factor(s) contained in the serum-deficient medium is selected from a Fibroblast Growth Factor (FGF), TGF-beta (Uniprot accession no. P01137), transferrin (e.g. serotransferrin or lacto transferrin; Uniprot accession nos. P02787 and P02788), insulin (Uniprot accession no. P01308), EGF (epidermal growth factor; Uniprot accession no. P01133), and/or PDGF (platelet-derived growth factor, including any combination of subunits A and B; Uniprot accession nos. P04085 and P01127), each of which represents a separate embodiment. A non-limiting example of PDGF is PDGF-BB.

Except where indicated otherwise, reference herein to a protein includes all its isoforms functional fragments thereof, and mimetics thereof. Such reference also includes homologues from a variety of species, provided that the protein acts on the target cells in a similar fashion to the homologue from the same species as the target cells. For example, if human cells are being expanded, reference to bFGF would also include any non-human bFGF that stimulates proliferation of human cells. Those skilled in the art will appreciate that, even in the case of human cells, the aforementioned proteins need not be human proteins, since many non-human (e.g. animal) proteins are active on human cells. Similarly, the use of modified proteins that have similar activity to the native forms falls within the scope of the described methods and compositions.

The FGF (fibroblast growth factor) family includes a number of proteins that are described in Imamura. A non-limiting example is bFGF (Uniprot accession no. P09038).

In other embodiments, the serum-deficient medium comprises an FGF and TGF-beta. In still other embodiments, the medium comprises an FGF, TGF-beta, and PDGF. In more specific embodiments, the medium further comprises transferrin, insulin, or both transferrin and insulin. Alternatively or in addition, the medium further comprises oleic acid.

In still other embodiments, the serum-deficient medium comprises an FGF and EGF. In still other embodiments, the medium further comprises transferrin, insulin, or both transferrin and insulin. SRM formulations include MSC Nutristem® XF (Biological Industries); Stempro® SFM and Stempro® SFM-XF (Thermo Fisher Scientific); PPRF-msc6; D-hESFlO; TheraPEAK™ MSCGM-CDTM (Lonza, cat. no. 190632); and MesenCult-XF (Stem Cell Technologies, cat. no. 5429). The StemPro® media contain PDGF-BB, bFGF, and TGF-β, and insulin (Chase et al). The composition of PPRF-msc6 is described in US 2010/0015710, which is incorporated herein by reference. D-hESFlO contains insulin (10 micrograms per milliliter [mcg/ml]); transferrin (5 mcg/ml); oleic acid conjugated with bovine albumin (9.4 mcg/ml); FGF-2 (10 ng/ml); and TGF- pi (5 ng/ml), as well as heparin (1 mg/ml) and standard medium components (Mimura et al).

As provided herein, ASC were expanded in Stempro® SFM-XF. MSC Nutristem® XF was also tested and yielded similar results. Additionally, an in-house medium was produced and tested, containing DMEM/F-12 supplemented with 50 ng/ml PDGF-BB, 15 ng/ml bFGF, and 2 ng/ml TGF-β. This medium yielded similar results to Stempro® SFM-XF. DMEM/F-12 is a known basal medium, available commercially from Thermo Fisher Scientific (cat. no. 10565018).

Another SRM formulation is described in Rajaraman G et al and contains FGF-2 (10 ng/ml); epidermal growth factor (EGF) (10 ng/ml); 0.5% BSA; Insulin (10 mcg/ml); transferrin (5.5 mcg/ml); 6.7 ng/mL sodium selenite, sodium pyruvate (11 mcg/ml); heparin (0.1 mg/ml); 10 nM linolenic acid.

In certain embodiments, the described SRM comprises bFGF (basic fibroblast growth factor, also referred to as FGF-2), TGF-β (TGF-β, including all isotypes, for example ΤϋΡβΙ, ΤϋΡβ2, and ΤΟΡβ3), or a combination thereof. In other embodiments, the SRM comprises bFGF, TGF-β, and PDGF. In still other embodiments, the SRM comprises bFGF and TGF-β, and lacks PDGF-BB. Alternatively or in addition, insulin is also present. In still other embodiments, an additional component selected from ascorbic acid, hydrocortisone and fetuin is present; 2 components selected from ascorbic acid, hydrocortisone and fetuin are present; or ascorbic acid, hydrocortisone and fetuin are all present.

In other embodiments, the described SRM comprises bFGF, TGF-β, and insulin. In additional embodiments, a component selected from transferrin (5 mcg/ml) and oleic acid are present; or both transferrin and oleic acid are present. Oleic acid can be, in some embodiments, conjugated with a protein, a non-limiting example of which is albumin. In some embodiments, the SRM comprises 5-20 ng/ml bFGF, 2-10 ng/ml TGF-β, and 5-20 ng/ml insulin, or, in other embodiments, 7-15 ng/ml bFGF, 3-8 ng/ml TGF-β, and 7-15 ng/ml insulin. In more specific embodiments, a component selected from 2-10 mcg/ml transferrin and 5-20 mcg/ml oleic acid, or in other embodiments, a component selected from 3-8 mcg/ml transferrin and 6-15 mcg/ml oleic acid, or in other embodiments the aforementioned amounts of both components (transferrin and oleic acid) is/are also present.

In yet other embodiments, the described SRM comprises bFGF and EGF. In more specific embodiments, the bFGF and EGF are present at concentrations independently selected from 5-40, 5-30, 5-25, 6-40, 6-30, 6-25, 7-40, 7-30, 7-25, 7-20, 8- , 8-17, 8-15, 8-13, 9-20, 9-17, 9-15, 10-15, 5-20, 5-10, 7-13, 8-12, 9-11, or 10 ng/ml. In certain embodiments, insulin; and/or transferrin is also present. In more specific embodiments, the insulin and transferrin are present at respective concentrations of 5-20 and 2-10; 6-18 and 3-8; or 8-15 and 4-7 mcg/ml. Alternatively or in addition, the SRM further comprises an additional component selected from BSA, selenite (e.g. sodium selenite), pyruvate (e.g. sodium pyruvate); heparin, and Hnolenic acid. In other embodiments 2 or more, or in other embodiments 3 or more, in other embodiments 4 or more, or in other embodiments all 5 of BSA, selenite, pyruvate, heparin, and Hnolenic acid are present. In more specific embodiments, the BSA, selenite, pyruvate, heparin, and hnolenic acid are present at respective concentrations of 0.1-5%, 2-30 ng/mL, 5-25 mcg/ml, 0.05-0.2 mg/ml, and 5-20 nM; or in other embodiments at respective concentrations of 0.2-2%, 4-10 ng/mL, 7-17 mcg/ml, 0.07-0.15 mg/ml, and 7-15 nM; or in other embodiments the aforementioned amounts or 2 or more, or in other embodiments 3 or more, in other embodiments 4 or more, or in other embodiments all 5 of BSA, selenite, pyruvate, heparin, and hnolenic acid are present.

In other embodiments, bFGF, where present, is present at a concentration of 1-40, 1-30, 1- 20, 2-40, 2-30, 2-20, 3-40, 3-30, 3-20, 3-15, 4-30, 4-20, 4-15, 5-30, 5-20, 5-15, 6-14, 7-14, 8-13, 8-12, 9-11, 9-12, about 10, or 10 nanograms per milliliter (ng/ml).

In other embodiments, EGF, where present, is present at a concentration of 1-40, 1-30, 1- 20, 2-40, 2-30, 2-20, 3-40, 3-30, 3-20, 3-15, 4-30, 4-20, 4-15, 5-30, 5-20, 5-15, 6-14, 7-14, 7-25, 7-22, 8-25, 8-22, 9-21, 10-20, 8-13, 8-12, 9-11, 9-12, about 10, or 10 ng/ml.

In other embodiments, TGF-β, where present, is present at a concentration of 1-25, 2-25,

3- 25, 4-25, 5-25, 1-20, 1-15, 1-10, 1-8, 1-7, 1-6, 1-5, 2-20, 2-15, 2-10, 3-20, 3-15, 3-10, 3-8, 3-7,

4- 8, 4-7, 4-6, 4.5-5.5, about 5, or 5 ng/ml. In other embodiments, PDGF, where present, is present at a concentration of 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-8, 2- 7, 2-6, 2-5, 2-4, 3-50, 3-40, 3-30, 3-20, 3-15, 3-10, 3-8, 3-7, 3-6, 3-5, 3-4, 4-40, 4-30, 4-20, 5-40, 5-30, 5-20, 5-15, 5-12, 5-10, 10-20, 10-18, 10-16, or 10-15, 2-20, about 2, about 3, about 5, about 10, about 15, about 20, 2, 3, 5, 10, 15, or 20 ng/mL.

In still other embodiments, the ASC are expanded in a multi-step process, including the steps of (a) incubating a population of ASC in a serum-deficient medium, thereby obtaining a first expanded cell population; and (b) incubating the first expanded cell population in a second medium, wherein the second medium contains at least 10% animal serum.

The aforementioned second medium, in some embodiments, contains an animal serum content of 5-25%, 6-25%, 7-25%, 8-25%, 9-25%, 10-25%, 11-25%, 12-25%, 13-25%, 14-25%, 15-25%, 10-24%, 10-23%, 10-22%, 10-21%, 10-20%, 11-19%, 12-18%, 13-17%, 16-24%, 17- 23%, or 18-22%. In other embodiments, the second medium contains at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% animal serum. In certain embodiments, the second medium does not contain added growth factors, other than those present in the animal serum added thereto.

In still other embodiments, the described methods are preceded by an earlier step wherein cells are cultured in serum-containing medium, prior to culturing in a serum-deficient medium. The serum-containing medium can be, in certain embodiments, any standard growth medium. Non-limiting examples, for exemplary purposed only, are DMEM + 10% FBS and DMEM + 5% human serum. In certain embodiments, the optional step is performed for 1-3 passages, 1-2 passages, or a single passage. In other embodiments, the optional step is performed for 2-5 population doublings, or in other embodiments 2-20, 2-15, 2-10, 2-8, 2-6, or 2-5 doublings. Those skilled in the art will appreciate that it may be difficult to determine an exact population doubling level (PDL) between extraction of cells from tissue and the first passage. In such case, if necessary the population doublings at this first stage may be estimated. Typical population doubling values prior to the first passage are below 5, often ranging from 2-5 (e.g. 1-3 passages, or in other embodiments 2-10 doublings) following their extraction from the source tissue. In certain embodiments, the incubation is followed by expansion in serum-deficient medium, which is, in some embodiments, in turn followed by further expansion in serum-containing medium. As provided herein, the initial use of serum-containing medium, for example after extraction, facilitates, in some scenarios, initial attachment and expansion of cells after their extraction. In certain embodiments, the earlier step is performed on a 2D substrate.

In certain embodiments, the aforementioned step of incubating the ASC population in serum-free medium, or in other embodiments in SRM, is performed for at least 12, at least 15, at least 17, at least 18, 12-30, 12-25, 15-30, 15-25, 16-25, 17-25, or 18-25 doublings.

In other embodiments, the ASC population is incubated in serum-free medium, or in other embodiments in SRM, for a defined number of passages, for example 1-4, 1-3, 1-2, 2-4, or 2-3; or a defined number of population doublings, for example at least 4, at least 5, at least 6, at least 7, at least 8, 4-10, 4-9, 4-8, 5-10, 5-9, or 5-8. The cells are then cryopreserved, then subjected to additional culturing in SRM. In some embodiments, the additional culturing in SRM is performed for at least 6, at least 7, at least 8, at least 9, at least 10, 6-20, 7-20, 8-20, 9-20, 10-20, 6-15, 7-15, 8-15, 9-15, or 10-15 population doublings.

In some embodiments, the step of incubating an ASC population in serum-deficient medium is performed on a 2D substrate; and at least a portion of the subsequent step (incubating the expanded cell population in a serum-containing medium) is performed on a 3D substrate. In certain embodiments, the 3D substrate is in a bioreactor. Alternatively or in addition, the 3D substrate is a synthetic adherent material. These embodiments of methods may be freely combined with any of the described embodiments of bioreactors, adherent materials, and/or 3D carriers and substrates. In still other embodiments, the aforementioned subsequent step is initiated on a 2D substrate for a duration of at least 2, at least 3, at least 4, at least 5, at least 6, 2-10, 3-10, 4-10, 5- 10, 2-8, 3-8, 4-8, or 5-8 cell doublings, before performing additional expansion in a serum- containing medium on a 3D substrate. The 2D substrate on which the subsequent step is initiated may be the same or different from the 2D substrate on which the described earlier step was performed, where applicable.

Other culture embodiments

In other embodiments, the placental ASC are cultured in the presence of extracts, or in other embodiments CM, from ischemic cells. Non-limiting example of such protocols are described in Cha et al. In yet other embodiments, the placental ASC are cultured, or in other embodiments incubated, under hypoxic conditions. Methods for hypoxia preconditioning are well known in the art; non-limiting examples of such methods include treatment with 0.1 -0.3% O2, treatment with 0.5% O2, e.g. for 24 hours; treatment with a 1 % O2 and 5% CO2 atmosphere, in some embodiments in glucose-free medium, e.g. for 24 hours; culturing at 2%, 3% or 5% O2 for 1-7 days; culturing at 5% O2 for 2 days; and culturing in 95% O². Also encompassed, in other embodiments, are regimens of hypoxia preconditioning (which may be 1-5%, e.g. about 2.5% O2), reoxygenation (e.g. at ambient conditions, which may be 15-25%, e.g. about 21 % O2), and further hypoxia preconditioning (which may be 1-5%, e.g. about 2.5% O2); for example as described in Hu C and Li L, Liu J et al, Sun J et al, Boyette LB et al, Kheirandish M et al., Kim YS et al., Barros S et al, Waszak P et al, and the references cited therein.

In yet other embodiments, the placental ASC are subjected to pharmacological preconditioning, non-limiting examples of which are treatment with Deferoxamine (DFO), polyribocytidylic acid, and other toll-like receptor-3 (TLR3) agonists. Protocols for pharmacological preconditioning are well known in the art; non-limiting examples of such methods are described in Najafi R et al, Qiu Y et al, Liu X et al, and Hu C and Li L, and the references cited therein.

In yet other embodiments, the placental ASC are subjected to preconditioning with one or more hormones, non-limiting examples of which are oxytocin, melatonin, all-trans retinoic acid, SDF-1/CXCR4 (Uniprot Accession No. P61073), Oncostatin M (Uniprot Accession No. P13725), and TGF-beta-1 (Uniprot Accession No. P01137), interferon-gamma (Uniprot Accession No. P01579), and migration inhibitory factor (Uniprot Accession No. P14174). Protocols for hormone preconditioning are well known in the art; non-limiting examples of such methods are described in Noiseux N et al, Tang Y et al, Pourjafar M et al, Lan YW et al, Li D et al, Duijvestein M et al, Xia W, Hu C and Li L, ,and the references cited therein.

In still other embodiments, the placental ASC are subjected to preconditioning with laser light, pulsed electromagnetic fields (PEMF), or nanoparticles and/or microparticles (e.g. silica particles). Protocols for such treatments are well known in the art, and non-limiting examples are described in Yin K et al, Urnukhsaikhan E et al, Kim KJ et al, and the references cited therein.

Surface markers and additional characteristics of ASC In some embodiments, the ASC express some or all of the following markers: CD 105 (UniProtKB Accession No. P17813), CD29 (UniProtKB Accession No. P05556), CD44 (UniProtKB Accession No. P I 6070), CD73 (UniProtKB Accession No. P21589), and CD90 (UniProtKB Accession No. P04216). In some embodiments, the ASC do not express some or all of the following markers: CD3 (e.g. UniProtKB Accession Nos. P09693 [gamma chain] P04234 [delta chain] , P07766 [epsilon chain] , and P20963 [zeta chain]), CD4 (UniProtKB Accession No. P01730), CD l lb (UniProtKB Accession No. PI 1215), CD 14 (UniProtKB Accession No. P08571), CD 19 (UniProtKB Accession No. P15391), and/or CD34 (UniProtKB Accession No. P28906). In more specific embodiments, the ASC also lack expression of CD5 (UniProtKB Accession No. P06127), CD20 (UniProtKB Accession No. PI 1836), CD45 (UniProtKB Accession No. P08575), CD79-alpha (UniProtKB Accession No. B5QTD1 ), CD80 (UniProtKB Accession No. P33681), and/or HLA-DR (e.g. UniProtKB Accession Nos. P04233 [gamma chain], P01903 [alpha chain], and P0191 1 [beta chain]). The aforementioned, non-limiting marker expression patterns were found in certain maternal placental cell populations that were expanded on 3D substrates. All UniProtKB entries mentioned in this paragraph were accessed on July 7, 2014. Those skilled in the art will appreciate that the presence of complex antigens such as CD3 and HLA-DR may be detected by antibodies recognizing any of their component parts, such as, but not limited to, those described herein.

In some embodiments, the ASC possess a marker phenotype that is distinct from bone marrow-mesenchymal stem cells (BM-MSC). In certain embodiments, the ASC are positive for expression of CD 10 (which occurs, in some embodiments, in both maternal and fetal ASC); are positive for expression of CD49d (which occurs, in some embodiments, at least in maternal ASC); are positive for expression of CD54 (which occurs, in some embodiments, in both maternal and fetal ASC); are bimodal, or in other embodiments positive, for expression of CD56 (which occurs, in some embodiments, in maternal ASC); and/or are negative for expression of CD 106. Except where indicated otherwise, bimodal refers to a situation where a significant percentage (e.g. at least 20%) of a population of cells express a marker of interest, and a significant percentage do not express the marker.

In certain embodiments, over 90% of the ASC are positive for CD29, CD90, and CD54. In other embodiments, over 85% of the described cells are positive for CD29, CD73, CD90, and CD 105. In yet other embodiments, less than 3% of the described cells are positive for CD 14, CD 19, CD31, CD34, CD39, CD45RA (an isotype of CD45), HLA-DR, Glycophorin A, and CD200; less than 6% of the cells are positive for GlyA; and less than 20% of the cells are positive for SSEA4. In more specific embodiments, over 90% of the described cells are positive for CD29, CD90, and CD54; and over 85% of the cells are positive for CD73 and CD 105. In still other embodiments, over 90% of the described cells are positive for CD29, CD90, and CD54; over 85% of the cells are positive for CD73 and CD 105; less than 6% of the cells are positive for CD 14, CD 19, CD31, CD34, CD39, CD45RA, HLA-DR, GlyA, CD200, and GlyA; and less than 20% of the cells are positive for SSEA4. The aforementioned, non-limiting marker expression patterns were found in certain maternal placental cell populations that were expanded on 3D substrates.

In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the ASC; and the cells do not differentiate into adipocytes, under conditions where mesenchymal stem cells would differentiate into adipocytes. In some embodiments, as provided herein, the conditions are incubation of adipogenesis induction medium, for example a solution containing 1 mcM dexamethasone, 0.5 mM 3-Isobutyl-l-methylxanthine (IBMX), 10 mcg/ml insulin, and 100 mcM indomethacin, on days 1, 3, 5, 9, 11, 13, 17, 19, and 21; and replacement of the medium with adipogenesis maintenance medium, namely a solution containing 10 mcg/ml insulin, on days 7 and 15, for a total of 25 days. In yet other embodiments, each of CD34, CD45, CD19, CD14 and HLA-DR is expressed by less than 3% of the cells; and the cells do not differentiate into adipocytes, after incubation under the aforementioned conditions. In other embodiments, each of CD73, CD29, and CD105 is expressed by more than 90% of the cells, each of CD34, CD45, CD 19, CD 14 and HLA-DR is expressed by less than 3% of the cells; and the cells do not differentiate into adipocytes, after incubation under the aforementioned conditions. In still other embodiments, a modified adipogenesis induction medium, containing 1 mcM dexamethasone, 0.5 mM IBMX, 10 mcg/ml insulin, and 200 mcM indomethacin is used, and the incubation is for a total of 26 days. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art. The aforementioned, non-limiting phenotypes and marker expression patterns were found in certain maternal placental cell populations that were expanded on 3D substrates.

In yet other embodiments, the placental MSC do not express Neutrophil gelatinase- associated lipocalin (LCN2; Uniprot Accession No. P80188). "Positive" expression of a marker indicates a value higher than the range of the main peak of a fluorescence-activated cell sorting (FACS) isotype control histogram; this term is synonymous herein with characterizing a cell as "express"/" expressing" a marker. "Negative" expression of a marker indicates a value falling within the range of the main peak of an isotype control histogram; this term is synonymous herein with characterizing a cell as "not express'V'not expressing" a marker. "High" expression of a marker, and term "highly express[es]" indicates an expression level that is more than 2 standard deviations higher than the expression peak of an isotype control histogram, or a bell-shaped curve matched to said isotype control histogram.

In still other embodiments, the majority, in other embodiments over 60%, over 70%, over 80%, or over 90% of the expanded cells express CD29, CD73, CD90, and CD105. In yet other embodiments, less than 20%, 15%, or 10% of the described cells express CD3, CD4, CD34, CD39, and CD106. In yet other embodiments, less than 20%, 15%, or 10% of the described cells highly express CD56. In various embodiments, the cell population may be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, or less than 5% positive for CD200. In other embodiments, the cell population is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98%, more than 99%, or more than 99.5% positive for CD200. In certain embodiments, more than 50% of the cells express, or in other embodiments highly express, CD141 (thrombomodulin; UniProt Accession No. P07204), or in other embodiments SSEA4 (stage-specific embryonic antigen 4, an epitope of ganglioside GL- 7 (IV³ NeuAc 2 → 3 GalGB4); Kannagi R et al), or in other embodiments both markers. Alternatively or in addition, more than 50% of the cells express HLA-A2 (UniProt Accession No. P01892). The aforementioned, non-limiting marker expression patterns were found in certain fetally-derived placental cell populations that were expanded on 3D substrates. The Uniprot Accession Nos. mentioned in the paragraph were accessed on accessed on February 8, 2017.

In other embodiments, each of CD29, CD73, CD90, and CD105 is expressed by more than 80% of the cells that have been expanded; and the cells do not differentiate into osteocytes, after incubation for 17 days with a solution containing 0.1 mcM dexamethasone, 0.2 mM ascorbic acid, and 10 mM glycerol-2-phosphate, in plates coated with vitronectin and collagen. In yet other embodiments, each of CD34, CD39, and CD106 is expressed by less than 10% of the cells; less than 20% of the cells highly express CD56; and the cells do not differentiate into osteocytes, after incubation under the aforementioned conditions. In other embodiments, each of CD29, CD73, CD90, and CD 105 is expressed by more than 90% of the cells, each of CD34, CD39, and CD 106 is expressed by less than 5% of the cells; less than 20%, 15%, or 10% of the cells highly express CD56, and/or the cells do not differentiate into osteocytes, after incubation under the aforementioned conditions. In still other embodiments, the conditions are incubation for 26 days with a solution containing 10 mcM dexamethasone, 0.2 mM ascorbic acid, 10 mM glycerol-2- phosphate, and 10 nM Vitamin D, in plates coated with vitronectin and collagen. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art. In yet other embodiments, less than 20%, 15%, or 10% of the described cells highly express CD56. In various embodiments, the cell population may be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, or less than 5% positive for CD200. In other embodiments, the cell population is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98%, more than 99%, or more than 99.5% positive for CD200. In certain embodiments, greater than 50% of the cells highly express CD 141, or in other embodiments SSEA4, or in other embodiments both markers. In other embodiments, the cells highly express CD141. Alternatively or in addition, greater than 50% of the cells express HLA-A2. The aforementioned, non-limiting phenotypes and marker expression patterns were found in certain fetally-derived placental cell populations that were expanded on 3D substrates.

In other embodiments, each of CD29, CD73, CD90, and CD105 is expressed by more than 80% of the cells that have been expanded; and the cells do not differentiate into adipocytes, after incubation in adipogenesis induction medium, namely a solution containing 1 mcM dexamethasone, 0.5 mM IB MX, 10 mcg/ml insulin, and 100 mcM indomethacin, on days 1, 3, 5, 9, 11, 13, 17, 19, and 21 ; and replacement of the medium with adipogenesis maintenance medium, namely a solution containing 10 mcg/ml insulin, on days 7 and 15, for a total of 25 days. In yet other embodiments, each of CD34, CD39, and CD106 is expressed by less than 10% of the cells; less than 20% of the cells highly express CD56; and the cells do not differentiate into adipocytes, after incubation under the aforementioned conditions. In other embodiments, each of CD29, CD73, CD90, and CD 105 is expressed by more than 90% of the cells, each of CD34, CD39, and CD 106 is expressed by less than 5% of the cells; less than 20%, 15%, or 10% of the cells highly express CD56; and the cells do not differentiate into adipocytes, after incubation under the aforementioned conditions. In still other embodiments, a modified adipogenesis induction medium, containing 1 mcM dexamethasone, 0.5 mM IB MX, 10 mcg/ml insulin, and 200 mcM indomethacin is used, and the incubation is for a total of 26 days. The aforementioned solutions will typically contain cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art. In various embodiments, the cell population may be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, or less than 5% positive for CD200. In other embodiments, the cell population is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98%, more than 99%, or more than 99.5% positive for CD200. In certain embodiments, greater than 50% of the cells highly express CD141, or in other embodiments SSEA4, or in other embodiments both markers. In other embodiments, the cells highly express CD 141. Alternatively or in addition, greater than 50% of the cells express HLA-A2. The aforementioned, non-limiting phenotypes and marker expression patterns were found in certain fetally-derived placental cell populations that were expanded on 3D substrates.

In still other embodiments, the placental ASC, after culturing on a 3D substrate, do not express TGFB3 (Transforming growth factor, beta 3; Uniprot Accession No. A5YM40) at a significant level. Expression at a significant level may refer, in some embodiments, to expression at least 50% above background levels, or in other embodiments, to expression at least 2-fold that of background levels. In other embodiments, the placental ASC, after culturing on a 3D substrate, express TGFB3 at a level at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15- fold, or 20-fold lower than BM-MSC cultured under the same conditions. In certain embodiments, lack of TGFB3 expression correlates with lack of potential to differentiate into chondrocytes. In other embodiments, the placental ASC, after culturing on a 3D substrate, do not express BMP2 (Bone morphogenetic protein 2; Uniprot Accession No. P12643) at a high level (e.g. at least 10- fold lower than BM-MSC cultured under the same conditions. In still other embodiments, the placental ASC, after culturing on a 3D substrate, express BMP2 at a level at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, or 20-fold lower than BM-MSC cultured under the same conditions. In certain embodiments, lack of high BMP2 expression correlates with lack of potential to differentiate into osteoblasts. In other embodiments, the placental ASC, after culturing on a 3D substrate, do not express PPARG (Peroxisome proliferator-activated receptor gamma; Uniprot Accession No. P37231) at a high level (e.g. at least 10-fold lower than BM-MSC cultured under the same conditions. In still other embodiments, the placental ASC, after culturing on a 3D substrate, express PPARG at a level at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, or 20-fold lower than BM-MSC cultured under the same conditions. In certain embodiments, lack of high PPARG expression correlates with lack of potential to differentiate into adipocytes. In this regard, culturing on a 3D substrate, refers, in some embodiments, to culturing for 5 days on a polyester non-woven fibrous matrix. In other embodiments, the term refers to spheroid culture, e.g. as described by Cha et al. Uniprot Accession Numbers in this paragraph were accessed on February 27, 2018.

Additionally or alternatively, the ASC secrete or express (as appropriate in each case) IL-6 (UniProt identifier P05231), IL-8 (UniProt identifier P10145), eukaryotic translation elongation factor 2 (EEEF2), reticulocalbin 3, EF-hand calcium binding domain (RCN₂), and/or calponin 1 basic smooth muscle (CNN1). In more specific embodiments, greater than 50%, in other embodiments greater than 55%, in other embodiments greater than 60%, in other embodiments greater than 65%, in other embodiments greater than 70%, in other embodiments greater than 75%, in other embodiments greater than 80%, in other embodiments greater than 85%, in other embodiments greater than 90%, in other embodiments greater than 95%, in other embodiments greater than 96%, in other embodiments greater than 97%, in other embodiments greater than 98%, in other embodiments greater than 99%, of the cells express or secrete at least one, in other embodiments at least 2, in other embodiments at least 3, in other embodiments at least 4, in other embodiments all five of the aforementioned proteins.

Reference herein to "secrete"/ "secreting"/ "secretion" relates to a detectable secretion of the indicated factor, above background levels in standard assays. For example, 0.5 x 10⁶ fetal or maternal ASC can be suspended in 4 ml medium (DMEM + 10% fetal bovine serum (FBS) + 2 mM L-Glutamine), added to each well of a 6 well-plate, and cultured for 24 hrs in a humidified incubator (5% CO2, at 37°C). After 24h, DMEM is removed, and cells are cultured for an additional 24 hrs in 1 ml RPMI 1640 medium + 2 mM L-Glutamine + 0.5% HSA. The CM is collected from the plate, and cell debris is removed by centrifugation.

In still other embodiments, the ASC secrete Flt-3 ligand (Fms-related tyrosine kinase 3 ligand; Uniprot Accession No. P49772), stem cell factor (SCF; Uniprot Accession No. P21583), IL-6 (Interleukin-6; UniProt identifier P05231), or combinations thereof, each of which represents a separate embodiment. In certain embodiments, the ASC secrete levels of Flt-3 ligand, SCF, IL- 6, or in other embodiments combinations thereof, that are at least 2-, 3-, 4-, 5-, 6-, 8-, 10-, 12-, 15- , or 20-fold higher than that expressed or secreted by ASC of placenta tissue grown on a 2D substrate. ASC grown on a 3D substrate secrete higher levels of Flt-3 ligand, SCF, and IL-6 than ASC grown on a 2D substrate, as provided in PCT Application Publ. No. WO/2007/108003, which is fully incorporated herein by reference in its entirety. Uniprot entries in this and the following 2 paragraphs were accessed on February 26, 2017.

According to some embodiments, the described ASC are capable of suppressing an immune reaction in the subject. Methods of determining the immunosuppressive capability of a cell population are well known to those skilled in the art, and exemplary methods are described in Example 3 of PCT Publication No. WO 2009/144720, which is incorporated herein by reference in its entirety. For example, a mixed lymphocyte reaction (MLR) may be performed. In an exemplary, non-limiting MLR assay, cord blood (CB) mononuclear cells, for example human cells or cells from another species, are incubated with irradiated cord blood cells (iCB), peripheral blood-derived monocytes (PBMC; for example human PBMC or PBMC from another species), in the presence or absence of a cell population to be tested. CB cell replication, which correlates with the intensity of the immune response, can be measured by a variety of methods known in the art, for example by ³H-thymidine uptake. Reduction of the CB cell replication when co-incubated with test cells indicates an immunosuppressive capability. Alternatively, a similar assay can be performed with peripheral blood (PB)-derived MNC, in place of CB cells. Alternatively or in addition, secretion of pro-inflammatory and anti-inflammatory cytokines by blood cell populations (such as CB cells or PBMC) can be measured when stimulated (for example by incubation with non-matched cells, or with a non-specific stimulant such as PHA), in the presence or absence of the ASC. In certain embodiments, for example in the case of human ASC, as provided in WO 2009/144720, when 150,000 ASC are co-incubated for 48 hours with 50,000 allogeneic PBMC, followed by a 5-hour stimulation with 1.5 meg of LPS, the amount of IL-10 secretion by the PBMC is at least 120%, at least 130%, at least 150%, at least 170%, at least 200%, or at least 300% of the amount observed with LPS stimulation in the absence of ASC.

In other embodiments, each of CD73, CD29, and CD 105 is expressed by more than 90% of the described ASC; and the cells inhibit T cell proliferation. In yet other embodiments, each of CD34, CD19, and CD14 is expressed by less than 3% of the cells; and the cells inhibit T cell proliferation. In other embodiments, each of CD73, CD29, and CD 105 is expressed by more than 90% of the cells, each of CD34, CD19, and CD14 is expressed by less than 3% of the cells; and the cells inhibit T cell proliferation.

In still other embodiments, the ASC secrete immunoregulatory factor(s). In certain embodiments, the ASC secrete a factor selected from TNF-beta (UniProt identifier P01374) and Leukemia inhibitory factor (LIF; UniProt identifier PI 5018). In other embodiments, the ASC secrete a factor selected from MCP-1 (CCL2), Osteoprotegerin, MIF (Macrophage migration inhibitory factor; Uniprot Accession No. P14174), GDF-15, SDF-1 alpha, GROa (Growth- regulated alpha protein; Uniprot Accession No. P09341), beta2-Microglobulin, IL-6, IL-8 (UniProt identifier P10145), TNF-beta, ENA78/CXCL5, eotaxin/CCLl l (Uniprot Accession No. P51671), and MCP-3 (CCL7). In certain embodiments, the ASC secrete MCP-1, Osteoprotegerin, MIF, GDF-15, SDF-1 alpha, GROa, beta2-Microglobulin, IL-6, IL-8, TNF-beta, and MCP-3, which were found to be secreted by maternal cells. In other embodiments, the ASC secrete MCP- 1, Osteoprotegerin, MIF, GDF-15, SDF-1 alpha, beta2-Microglobulin, IL-6, IL-8, ENA78, eotaxin, and MCP-3, which were found to be secreted by fetal cells. All Swissprot and UniProt entries in this paragraph were accessed on March 23, 2017.

In yet other embodiments, the ASC secrete anti-fibrotic factor(s). In certain embodiments, the ASC secrete a factor selected from Serpin El (Plasminogen activator inhibitor 1 ; Uniprot Accession No. P05121) and uPAR (Urokinase plasminogen activator surface receptor; Uniprot Accession No. Q03405). In other embodiments, the ASC secrete factors that facilitate. In still other embodiments, the ASC secrete Serpin El and uPAR, which were found to be secreted by maternal and fetal cells. All Swissprot and UniProt entries in this paragraph were accessed on April 3, 2017.

In other embodiments, the ASC secrete a factor(s) that promotes extracellular matrix (ECM) remodeling. In certain embodiments, the ASC secrete a factor selected from TIMP1, TIMP2, MMP-1, MMP-2, and MMP-10. In other embodiments, the ASC secrete TIMP1, TIMP2, MMP-1, MMP-2, and MMP-10, which were found to be secreted by maternal cells. In still other embodiments, the ASC secrete TIMP1, TIMP2, MMP-1, and MMP-10, which were found to be secreted by fetal cells.

In other embodiments, the described ASC exhibit a spindle shape when cultured under 2D conditions. According to some embodiments, the ASC express CD200, while in other embodiments, the ASC lack expression of CD200. In still other embodiments, less than 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, or 2%, 1%, or 0.5% of the adherent cells express CD200. In yet other embodiments, greater than 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the adherent cells express CD200.

In still other embodiments, the cells may be allogeneic, or in other embodiments, the cells may be autologous. In other embodiments, the cells may be fresh or, in other embodiments, frozen (for example, cryo-preserved).

Additional method characteristics for preparation of ASC

In certain embodiments, the described ASC have been subject to a 3D incubation, as described further herein. In more specific embodiments, the ASC have been incubated in a 2D adherent-cell culture apparatus, prior to the step of 3D culturing. In some embodiments, cells (which have been extracted, in some embodiments, from placenta) are then subjected to prior step of incubation in a 2D adherent-cell culture apparatus, followed by the described 3D culturing steps.

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

The terms "three-dimensional culture" and "3D culture" refer to a culture in which the cells are exposed to conditions that are compatible with cell growth and allow the cells to grow in a 3D orientation relative to one another. The term "three-dimensional [or 3D] culture apparatus" refers to an apparatus for culturing cells under conditions that are compatible with cell growth and allow the cells to grow in a 3D orientation relative to one another. Such apparatuses will typically have a 3D growth surface (also referred to as a "three-dimensional substrate" or "3D substrate"), in some embodiments comprising an adherent material, which is present in the 3D culture apparatus, e.g. the bioreactor. Certain, non-limiting embodiments of 3D culturing conditions suitable for expansion of adherent stromal cells are described in PCT Application Publ. No. WO/2007/108003, which is fully incorporated herein by reference in its entirety.

In various embodiments, "an adherent material" refers to a material that is synthetic, or in other embodiments naturally occurring, or in other embodiments a combination thereof. In certain embodiments, the material is non-cytotoxic (or, in other embodiments, is biologically compatible). Alternatively or in addition, the material is fibrous, which may be, in more specific embodiments, a woven fibrous matrix, a non-woven fibrous matrix, or any type of fibrous matrix. In still other embodiments, the material exhibits a chemical structure such as charged surface exposed groups, which allows cell adhesion. Non-limiting examples of adherent materials which may be used in accordance with this aspect include a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, a poly-L-lactic acid, and an inert metal fiber. Other embodiments include Matrigel™, an extra-cellular matrix component (e.g., Fibronectin, Chondronectin, Laminin), and a collagen. In more particular embodiments, the material may be selected from a polyester and a polypropylene. Non-limiting examples of synthetic adherent materials include polyesters, polypropylenes, polyalkylenes, polyfluorochloroethylenes, polyvinyl chlorides, polystyrenes, polysulfones, cellulose acetates, and poly-L-lactic acids, glass fibers, ceramic particles, and an inert metal fiber, or, in more specific embodiments, polyesters, polypropylenes, polyalkylenes, polyfluorochloroethylenes, polyvinyl chlorides, polystyrenes, polysulfones, cellulose acetates, and poly-L-lactic acids.

The length of the described 3D culturing, in other embodiments, is at least 4 days; between

4- 12 days; in other embodiments between 4-11 days; in other embodiments between 4-10 days; in other embodiments between 4-9 days; in other embodiments between 5-9 days; in other embodiments between 5-8 days; in other embodiments between 6-8 days; or in other embodiments between 5-7 days. In other embodiments, the 3D culturing is performed for 5-15 cell doublings, in other embodiments 5-14 doublings, in other embodiments 5-13 doublings, in other embodiments

5- 12 doublings, in other embodiments 5-11 doublings, in other embodiments 5-10 doublings, in other embodiments 6-15 cell doublings, in other embodiments 6-14 doublings, in other embodiments 6-13 doublings, or in other embodiments 6-12 doublings, in other embodiments 6- 11 doublings, or in other embodiments 6-10 doublings. In some embodiments, the described lengths describes the total time in a 3D substrate culture apparatus, including the expansion and induction stages.

In certain embodiments, 3D culturing can be performed in a 3D bioreactor. In some embodiments, the 3D bioreactor comprises a container for holding medium and a 3D attachment substrate disposed therein, and a control apparatus, for controlling pH, temperature, and oxygen levels and optionally other parameters. The terms attachment substrate and growth substrate are interchangeable. In certain embodiments, the attachment substrate is in the form of carriers, which comprise, in more specific embodiments, a surface comprising a synthetic adherent material. Alternatively or in addition, the bioreactor contains ports for the inflow and outflow of fresh medium and gases. Except where indicated otherwise, the term "bioreactor" excludes decellularized organs and tissues derived from a living being.

Examples of bioreactors include, but are not limited to, a continuous stirred tank bioreactor, a CelliGen Plus® bioreactor system (New Brunswick Scientific (NBS) and a BIO FLO 310 bioreactor system (New Brunswick Scientific (NBS).

As provided herein, a 3D bioreactor is capable, in certain embodiments, of 3D expansion of ASC under controlled conditions (e.g. pH, temperature and oxygen levels) and with growth medium perfusion, which in some embodiments is constant perfusion and in other embodiments is adjusted in order to maintain target levels of glucose or other components. Furthermore, the cell cultures can be directly monitored for concentrations of glucose, lactate, glutamine, glutamate and ammonium. The glucose consumption rate and the lactate formation rate of the adherent cells enable, in some embodiments, measurement of cell growth rate and determination of the harvest time.

In some embodiments, a continuous stirred tank bioreactor is used, where a culture medium is continuously fed into the bioreactor and a product is continuously drawn out, to maintain a time- constant steady state within the reactor. A stirred tank bioreactor with a fibrous bed basket is available for example from New Brunswick Scientific Co., Edison, NJ). Additional bioreactors that may be used, in some embodiments, are stationary-bed bioreactors; and air-lift bioreactors, where air is typically fed into the bottom of a central draught tube flowing up while forming bubbles, and disengaging exhaust gas at the top of the column. Additional possibilities are cell- seeding perfusion bioreactors with polyactive foams [as described in Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)] and radial-flow perfusion bioreactors containing tubular poly-L- lactic acid (PLLA) porous scaffolds [as described in Kitagawa et al., Biotechnology and Bioengineering 93(5): 947-954 (2006). Other bioreactors which can be used are described in U.S. Pat. Nos. 6,277, 151; 6,197,575; 6, 139,578; 6,132,463; 5,902,741 ; and 5,629,186, which are incorporated herein by reference. A "stationary-bed bioreactor" refers to a bioreactor in which the cellular growth substrate is not ordinarily lifted from the bottom of the incubation vessel in the presence of growth medium. For example, the substrate may have sufficient density to prevent being lifted and/or it may be packed by mechanical pressure to present it from being lifted. The substrate may be either a single body or multiple bodies. Typically, the substrate remains substantially in place during the standard perfusion rate of the bioreactor. In certain embodiments, the substrate may be lifted at unusually fast perfusion rates, for example greater than 200 rpm.

Another exemplary, non-limiting bioreactor, the Celligen 310 Bioreactor, is depicted in Fig. 1. A Fibrous-Bed Basket (16) is loaded with polyester disks (10). In some embodiments, the vessel is filled with deionized water or isotonic buffer via an external port ( 1 [this port may also be used, in other embodiments, for cell harvesting]) and then optionally autoclaved. In other embodiments, following sterilization, the liquid is replaced with growth medium, which saturates the disk bed as depicted in (9). In still further embodiments, temperature, pH, dissolved oxygen concentration, etc., are set prior to inoculation. In yet further embodiments, a slow initial stirring rate is used to promote cell attachment, then the stirring rate is increased. Alternatively or addition, perfusion is initiated by adding fresh medium via an external port (2). If desired, metabolic products may be harvested from the cell-free medium above the basket (8). In some embodiments, rotation of the impeller creates negative pressure in the draft-tube (18), which pulls cell-free effluent from a reservoir (15) through the draft tube, then through an impeller port (19), thus causing medium to circulate (12) uniformly in a continuous loop. In still further embodiments, adjustment of a tube (6) controls the liquid level; an external opening (4) of this tube is used in some embodiments for harvesting. In other embodiments, a ring sparger (not visible), is located inside the impeller aeration chamber (11), for oxygenating the medium flowing through the impeller, via gases added from an external port (3) , which may be kept inside a housing (5), and a sparger line (7). Alternatively or in addition, sparged gas confined to the remote chamber is absorbed by the nutrient medium, which washes over the immobilized cells. In still other embodiments, a water jacket (17) is present, with ports for moving the jacket water in (13) and out (14).

In certain embodiments, a perfused bioreactor is used, wherein the perfusion chamber contains carriers. The carriers may be, in more specific embodiments, selected from macrocarriers, microcarriers, or both together. Non-limiting examples of microcarriers that are available commercially include alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex, GE Healthcare), collagen-based (Cultispher, Percell), and polystyrene-based (SoloHill Engineering) microcarriers. In certain embodiments, the microcarriers are packed inside the perfused bioreactor.

In some embodiments, the carriers in the perfused bioreactor are packed, for example forming a packed bed, which is submerged in a nutrient medium. Alternatively or in addition, the carriers may comprise an adherent material. In other embodiments, the surface of the carriers comprises an adherent material, or the surface of the carriers is adherent. In still other embodiments, the material exhibits a chemical structure such as charged surface exposed groups, which allows cell adhesion. Non-limiting examples of adherent materials which may be used in accordance with this aspect include a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, a poly-L-lactic acid, and an inert metal fiber. In more particular embodiments, the material may be selected from a polyester and a polypropylene. In various embodiments, an "adherent material" refers to a material that is synthetic, or in other embodiments naturally occurring, or in other embodiments a combination thereof. In certain embodiments, the material is non-cytotoxic (or, in other embodiments, is biologically compatible). Non-limiting examples of synthetic adherent materials include polyesters, polypropylenes, polyalkylenes, polyfluorochloroethylenes, polyvinyl chlorides, polystyrenes, polysulfones, cellulose acetates, and poly-L-lactic acids, glass fibers, ceramic particles, and an inert metal fiber, or, in more specific embodiments, polyesters, polypropylenes, polyalkylenes, polyfluorochloroethylenes, polyvinyl chlorides, polystyrenes, polysulfones, cellulose acetates, and poly-L-lactic acids. Other embodiments include Matrigel™, an extra-cellular matrix component (e.g., Fibronectin, Chondronectin, Laminin), and a collagen.

In other embodiments, cells are produced using a packed-bed spinner flask. In more specific embodiments, the packed bed may comprise a spinner flask and a magnetic stirrer. The spinner flask may be fitted, in some embodiments, with a packed bed apparatus, which may be, in more specific embodiments, a fibrous matrix; or in more specific embodiments, a non-woven fibrous matrix. In other embodiments, the fibrous matrix comprises polyester, or comprises at least about 50% polyester. In still other embodiments, the non-woven fibrous matrix comprises polyester, or comprises at least about 50% polyester.

In still other embodiments, the matrix is similar to the Celligen™ Plug Flow bioreactor which is, in certain embodiments, packed with Fibra-cel® carriers (or, in other embodiments, other carriers). The spinner is, in certain embodiments, batch fed (or in other alternative embodiments fed by perfusion), fitted with one or more sterilizing filters, and placed in a tissue culture incubator. In further embodiments, cells are seeded onto the scaffold by suspending them in medium and introducing the medium to the apparatus. In still further embodiments, the stirring speed is gradually increased, for example by starting at 40 RPM for 4 hours, then gradually increasing the speed to 120 RPM. In certain embodiments, the glucose level of the medium may be tested periodically (i.e. daily), and the perfusion speed adjusted maintain an acceptable glucose concentration, which is, in certain embodiments, between 400-700 mgUiter, between 450-650 mgUiter, between 475-625 mgUiter, between 500-600 mgUiter, or between 525-575 mgUiter. In yet other embodiments, at the end of the culture process, carriers are removed from the packed bed, washed with isotonic buffer, and processed or removed from the carriers by agitation and/or enzymatic digestion.

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

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

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

In other embodiments, over 5 x 10⁵, over 7 x 10⁵, over 8 x 10⁵, over 9 x 10⁵, over 10⁶, over 1.5 x 10⁶, over 2 x 10⁶, over 3 x 10⁶, over 4 x 10⁶, or over 5 x 10⁶ viable cells are removed per milliliter of the growth medium in the bioreactor. In still other embodiments over between 5 x 10⁵ - 1.5 x 10⁷, between 7 x 10⁵ - 1.5 x 10⁷, between 8 x 10⁵ - 1.5 x 10⁷, between 1 x 10⁶ - 1.5 x 10⁷, between 5 x 10⁵ - 1 x 10⁷, between 7 x 10⁵ - 1 x 10⁷, between 8 x 10⁵ - 1 x 10⁷, between 1 x 10⁶ - 1 x 10⁷, between 1.2 x 10⁶ - 1 x 10⁷, or between 2 x 10⁶ - 1 x 10⁷viable cells are removed per milliliter of the growth medium in the bioreactor. In other embodiments, incubation of ASC may comprise microcarriers, which may, in certain embodiments, be inside a bioreactor. Microcarriers are well known to those skilled in the art, and are described, for example in US Patent Nos. 8,828,720, 7,531,334, 5,006,467, which are incorporated herein by reference. Microcarriers are also commercially available, for example as Cytodex™ (available from Pharmacia Fine Chemicals, Inc.), Superbeads (commercially available from Flow Labs, Inc.), and DE-52 and DE-53 (commercially available from Whatman, Inc.). In certain embodiments, the ASC may be incubated in a 2D apparatus, for example tissue culture plates or dishes, prior to incubation in microcarriers. In other embodiments, the ASC are not incubated in a 2D apparatus prior to incubation in microcarriers. In certain embodiments, the microcarriers are packed inside a bioreactor.

In some embodiments, with reference to Figs. 4A-B, and as described in WO/2014/037862, published on March 13, 2014, which is incorporated herein by reference in its entirety, grooved carriers 30 are used for proliferation and/or incubation of ASC. In various embodiments, the carriers may be used following a 2D incubation (e.g. on culture plates or dishes), or without a prior 2D incubation. In other embodiments, incubation on the carriers may be followed by incubation on a 3D substrate in a bioreactor, which may be, for example, a packed-bed substrate or microcarriers; or incubation on the carriers may not be followed by incubation on a 3D substrate. Carriers 30 can include multiple two-dimensional (2D) surfaces 12 extending from an exterior of carrier 30 towards an interior of carrier 30. As shown, the surfaces are formed by a group of ribs 14 that are spaced apart to form openings 16, which may be sized to allow flow of cells and culture medium (not shown) during use. With reference to Fig. 4C, carrier 30 can also include multiple 2D surfaces 12 extending from a central carrier axis 18 of carrier 30 and extending generally perpendicular to ribs 14 that are spaced apart to form openings 16, creating multiple 2D surfaces 12. In some embodiments, carriers 30 are "3D bodies" as described in WO/2014/037862; the contents of which relating to 3D bodies are incorporated herein by reference.

In certain embodiments, the described carriers (e.g. grooved carriers) are used in a bioreactor. In some, the carriers are in a packed conformation.

In still other embodiments, the material forming the multiple 2D surfaces comprises at least one polymer. Suitable coatings may, in some embodiments, be selected to control cell attachment or parameters of cell biology. In certain embodiments, further steps of purification or enrichment for ASC may be performed. Such methods include, but are not limited to, cell sorting using markers for ASC and/or, in various embodiments, mesenchymal-like ASC.

Cell sorting, in this context, refers to any procedure, whether manual, automated, etc., that selects cells on the basis of their expression of one or more markers, their lack of expression of one or more markers, or a combination thereof. Those skilled in the art will appreciate that data from one or more markers can be used individually or in combination in the sorting process.

In more particular embodiments, cells may be removed from a 3D matrix while the matrix remains within the bioreactor. In certain embodiments, at least about 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, or at least 30% of the cells are in the S and G2/M phases (collectively), at the time of harvest from the bioreactor. Cell cycle phases can be assayed by various methods known in the art, for example FACS detection. Typically, in the case of FACS, the percentage of cells in S and G2/M phase is expressed as the percentage of the live cells, after gating for live cells, for example using a forward scatter/side scatter gate. Those skilled in the art will appreciate that the percentage of cells in these phases correlates with the percentage of proliferating cells. In some cases, allowing the cells to remain in the bioreactor significantly past their logarithmic growth phase causes a reduction in the number of cells that are proliferating.

In certain embodiments, the harvesting process comprises agitation. In certain embodiments, the agitation utilizes vibration, for example as described in PCT International Application Publ. No. WO 2012/140519, which is incorporated herein by reference. In certain embodiments, during harvesting, the cells are agitated at 0.7-6 Hertz, or in other embodiments 1- 3 Hertz, during, or in other embodiments during and after, treatment with a protease, optionally also comprising a calcium chelator. In certain embodiments, the carriers containing the cells are agitated at 0.7-6 Hertz, or in other embodiments 1-3 Hertz, while submerged in a solution or medium comprising a protease, optionally also comprising a calcium chelator. Non-limiting examples of a protease plus a calcium chelator are trypsin, or another enzyme with similar activity, optionally in combination with another enzyme, non-limiting examples of which are Collagenase Types I, II, III, and IV, with EDTA. Enzymes with similar activity to trypsin are well known in the art; non-limiting examples are TrypLE™, a fungal trypsin-like protease, and Collagenase, Types I, II, III, and IV, which are available commercially from Life Technologies. Enzymes with similar activity to collagenase are well known in the art; non-limiting examples are Dispase I and Dispase II, which are available commercially from Sigma- Aldrich. In still other embodiments, the cells are harvested by a process comprising an optional wash step, followed by incubation with collagenase, followed by incubation with trypsin. In various embodiments, at least one, at least two, or all three of the aforementioned steps comprise agitation. In more specific embodiments, the total duration of agitation during and/or after treatment with protease plus a calcium chelator is between 2-10 minutes, in other embodiments between 3-9 minutes, in other embodiments between 3-8 minutes, and in still other embodiments between 3-7 minutes. In still other embodiments, the cells are subjected to agitation at 0.7-6 Hertz, or in other embodiments 1-3 Hertz, during the wash step before the protease and calcium chelator are added. Alternatively or in addition, the ASC are expanded using an adherent material in a container, which is in turn disposed within a bioreactor chamber; and an apparatus is used to impart a reciprocating motion to the container relative to the bioreactor chamber, wherein the apparatus is configured to move the container in a manner causing cells attached to the adherent material to detach from the adherent material. In more specific embodiments, the vibrator comprises one or more controls for adjusting amplitude and frequency of the reciprocating motion. Alternatively or in addition, the adherent material is a 3D substrate, which comprises, in some embodiments, carriers comprising a synthetic adherent material.

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

Non-limiting examples of base media useful in 2D and 3D culturing include Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), FIO(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5 A Medium, Medium Ml 99 (M199E-with Earle's sale base), Medium M199 (M199H-with Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with non-essential amino acids), among numerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. In certain embodiments, DMEM is used. These and other useful media are available from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel, among others.

In some embodiments, the medium may be supplemented with additional substances. Non- limiting examples of such substances are serum, which is, in some embodiments, fetal serum of cows or other species, which is, in some embodiments, 5-15% of the medium volume. In certain embodiments, the medium contains 1-5%, 2-5%, 3-5%, 1-10%, 2-10%, 3-10%, 4-15%, 5-14%, 6- 14%, 6-13%, 7-13%, 8-12%, 8-13%, 9-12%, 9-11%, or 9.5%-10.5% serum, which may be fetal bovine serum, or in other embodiments another animal serum. In still other embodiments, the medium is serum-free.

Alternatively or in addition, the medium may be supplemented by growth factors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g. B-glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g., growth hormone, erythropoietin, thrombopoietin, interleukin 3, interleukin 7, macrophage colony stimulating factor, c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, insulin-like growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, ciliary neurotrophic factor, platelet-derived growth factor, and bone morphogenetic protein.

In yet other embodiments, the placental ASC are grown as spheroids as part of the preparation process. Examples of conditions that favor spheroid formation ("spheroid conditions") are well known to those skilled in the art. Non-limiting examples of such conditions include incubation in hydrogels (e.g. polyethylene glycol hydrogels), for example as described in Cha et al and the references cited therein. In more specific embodiments, the cells are cultured in spheroid conditions following 2D culture and prior to bioreactor culture, from which the subcellular fractions are isolated; or the cells are cultured in spheroid conditions following 2D culture, from which the subcellular fractions are isolated; or the cells are cultured in spheroid conditions following 2D culture and prior to additional 2D culture, which is then followed by bioreactor culture, from which the subcellular fractions are isolated; or the cells are cultured in spheroid conditions following 2D culture and prior to additional 2D culture, from which the subcellular fractions are isolated, each of which represents a separate embodiment. In still other embodiments, the cells are cultured in spheroid conditions following 2D culture and prior to bioreactor culture, after which the cells are isolated from the bioreactor and are used for therapeutic applications; or the cells are cultured in spheroid conditions following 2D culture, after which the cells are isolated from the spheroid conditions and are used for therapeutic applications; or the cells are cultured in spheroid conditions following 2D culture and prior to additional 2D culture, which is then followed by bioreactor culture, from which the cells are isolated are used for therapeutic applications; or the cells are cultured in spheroid conditions following 2D culture and prior to additional 2D culture, from which the cells are isolated are used for therapeutic applications, each of which represents a separate embodiment.

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

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

In yet other embodiments, the placental ASC are immortalized, thereby generating a cell line. The cell line is used, in other embodiments, to form an optionally frozen cell bank, which is, in turn, used therapeutically as described herein. Methods for cell immortalization are well known in the art. Non-limiting examples of such methods are described, for example, in the handbook titled General Guidelines for Cell Immortalization, published by Applied Biologic Materials Inc. (Richmond, BC, Canada); in Andaloussi, EL et al; and the references cited therein.

In other embodiments, conditioned medium (CM) derived from the described ASC is utilized in the described methods, for example post-incubation medium from the described tissue culture incubation or bioreactor incubation. In yet other embodiments, there is provided a pharmaceutical composition comprising the CM, which may be, in some embodiments, indicated for the described therapeutic indications. Those skilled in the art will appreciate that, in certain embodiments, various bioreactors may be used to prepare CM, including but not limited to plug- flow bioreactors, and stationary-bed bioreactors (Kompier R et al. Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells. Enzyme Microb Technol. 1991. 13(10):822-7.) For example, CM is produced as a by-product of the described methods for cell expansion. The CM in the bioreactor can be removed from the bioreactor or otherwise isolated. In other embodiments, the described expanded cells are removed from the bioreactor and incubated in another apparatus (a non-limiting example of which is a tissue culture apparatus), and CM from the cells is collected.

In yet other embodiments, extracellular vesicles, e.g. exosomes, secreted by the described ASC are used in the described methods and compositions. Methods of isolating exosomes are well known in the art, and include, for example, immuno-magnetic isolation, for example as described in Clayton A et al, 2001; Mathias RA et al, 2009; and Crescitelli R et al, 2013.

In certain embodiments, the described methods comprise isolation of exosomes, for example as described in Conde-Vancells et al and Koga et al, or the references cited therein. One such protocol, provided solely for purposes of exemplification, involved centrifuging samples for 30 min at 1500 x g to remove large cellular debris. The resultant supernatants are subjected to filtration on 0.22 μπι pore filters, followed by ultra-centrifugation at 10 000 x g and 100 000 x g for 30 and 60 min, respectively. The resulting pellets are suspended in PBS, pooled, and again ultracentrifuged at 100 000 x g for 60 min. The final pellet (containing vesicles) is suspended in 150 μΤ of PBS, aliquoted and stored at -80°C. For higher-purity preparations, exosomes can be further purified on sucrose-containing gradients (e.g. a 30% sucrose cushion), e.g. as described in Thery C et al. Vesicle preparations are diluted in PBS and under-layered on top of a density cushion composed of pH-buffered 30% sucrose (optionally containing deuterium oxide (D2O)), around pH 7.4, forming a visible interphase. The samples are ultracentrifuged at 100 000 x g at 4°C for 75 min in a swinging bucket rotor, and the gradient is withdrawn in aliquots from the bottom. Vesicles contained in the 30% sucrose/D₂0 cushion are collected, diluted in buffered solution, and optionally centrifuged at 100 000 x g to concentrate the contents. Kits for exosome isolation are available commercially, non-limiting examples of which are ExoQuick® reagents, ExoMAX Opti enhancer, and ExoFLOW products, all of which can be obtained from System Biosciences (Palo Alto, CA).

In some embodiments, the exosomes or other extracellular vesicles are harvested from a 3D bioreactor in which the ASC have been incubated. Alternatively or in addition, the cells are cryopreserved, and then are thawed, after which the exosomes are isolated. In some embodiments, after thawing, the cells are cultured in 2D culture, from which the exosomes are harvested.

Pharmaceutical compositions

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

In other embodiments, compositions are provided herein that comprise ASC or CM in combination with an excipient, e.g., a pharmacologically acceptable excipient. In certain embodiments, any of the described compositions further comprises a pharmacologically acceptable excipient. In further embodiments, the excipient is an osmoprotectant or cryoprotectant, an agent that protects cells from the damaging effect of freezing and ice formation, which may in some embodiments be a permeating compound, non-limiting examples of which are dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, formamide, propanediol, poly-ethylene glycol, acetamide, propylene glycol, and adonitol; or may in other embodiments be a non-permeating compound, non-limiting examples of which are lactose, raffinose, sucrose, trehalose, and d- mannitol. In other embodiments, both a permeating cryoprotectant and a non-permeating cryoprotectant are present. In other embodiments, the excipient is a carrier protein, a non-limiting example of which is albumin. In still other embodiments, both an osmoprotectant and carrier protein are present; in certain embodiments, the osmoprotectant and carrier protein may be the same compound. Alternatively or in addition, the composition is frozen. The cells may be any embodiment of ASC mentioned herein, each of which is considered a separate embodiment.

Provided in addition are pharmaceutical compositions, comprising the described placental ASC, in the absence of non-placental cell types.

Also provided are pharmaceutical compositions, comprising the described placental ASC- derived CM, in the absence of CM derived from other cell types. In other embodiments, there are provided pharmaceutical compositions, comprising the described exosomes.

Since non-autologous cells may in some cases induce an immune reaction when administered to a subject, several approaches may be utilized according to the methods provided herein to reduce the likelihood of rejection of non-autologous cells. In some embodiments, these approaches include either suppressing the recipient immune system or encapsulating the non- autologous cells in immune-isolating, semipermeable membranes before transplantation. In some embodiments, this may be done regardless of whether the ASC themselves engraft in the host. For example, the majority of the cells may, in various embodiments, not survive after engraftment for more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, or more than 14 days.

Examples of immunosuppressive agents that may be used in the methods and compositions provided herein include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF- alpha blockers, biological agents that antagonize one or more inflammatory cytokines, and Nonsteroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, and tramadol.

One may, in various embodiments, administer the pharmaceutical composition in a systemic manner (as detailed hereinabove). Alternatively, one may administer the pharmaceutical composition locally, for example, via injection of the pharmaceutical composition directly into an exposed or affected tissue region of a patient. In other embodiments, the cells are administered intravenously (IV), subcutaneously (SC), by the intraosseous route (e.g. by intraosseous infusion), or intraperitoneally (IP), each of which is considered a separate embodiment. In other embodiments, the ASC or composition is administered intramuscularly; while in other embodiments, the ASC or composition is administered systemically. In this regard, "intramuscular" administration refers to administration into the muscle tissue of a subject; "subcutaneous" administration refers to administration just below the skin; "intravenous" administration refers to administration into a vein of a subject; "intraosseous" administration refers to administration directly into bone marrow; and "intraperitoneal" administration refers to administration into the peritoneum of a subject. In still other embodiments, the cells are administered intratracheally, intrathecally, by inhalation, or intranasally. In certain embodiments, lung-targeting routes of administration may utilize cells encapsulated in liposomes or other barriers to reduce entrapment within the lungs.

In still other embodiments, the pharmaceutical composition is administered intralymphatically, for example as described in United States Patent No. 8,679,834 in the name of Eleuterio Lombardo and Dirk Buscher, which is hereby incorporated by reference.

In other embodiments, for injection, the described cells may be formulated in aqueous solutions, e.g. in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer, optionally in combination with medium containing cryopreservation agents.

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

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.

The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be, in some embodiments, chosen by the individual physician in view of the patient's condition.

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

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

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

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

The described compositions may, if desired, be packaged in a container that is accompanied by instructions for administration. The container may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. In other embodiments, the described ASC are suitably formulated as a pharmaceutical composition which can be suitably packaged as an article of manufacture. Such an article of manufacture comprises a packaging material which comprises a label describing a use in treating a disease or disorder or therapeutic indication that is mentioned herein. In other embodiments, a pharmaceutical agent is contained within the packaging material, wherein the pharmaceutical agent is effective for the treatment of a disorder or therapeutic indication that is mentioned herein. In some embodiments, the pharmaceutical composition is frozen.

It is clarified that each embodiment of the described ASC may be freely combined with each embodiment relating to a therapeutic method or pharmaceutical composition.

Furthermore, each embodiment of the described exosomes may be freely combined with each embodiment relating to a therapeutic method or pharmaceutical composition.

In still other embodiments, the described CM is used in any of the described therapeutic methods. Each embodiment of CM may be freely combined with each embodiment relating to a therapeutic method or pharmaceutical composition.

Subjects

In certain embodiments, the subject treated by the described methods and compositions is a human. In some embodiments, the subject is an adult subject, e.g. a subject over the age of 25. In other embodiments, the subject is an elderly subject, for example a subject over 60, over 65, over 70, over 75, over 80, 60-85, 65-85, or 70-85 years in age. In other embodiments, the subject is a preterm infant. In still other embodiments, the subject suffers from a condition selected from pneumonia, sepsis, gastric content aspiration, trauma, pancreatitis, inhalation injury, burns, non- cardiogenic shock, drug overdose, transfusion-related acute lung injury (TRALI), VILI (ventilator induced lung injury), an amniotic fluid embolism, and ischemic reperfusion injury. In other embodiments, the subject may be an animal. In some embodiments, treated animals include domesticated animals and laboratory animals, e.g., non-mammals and mammals, for example non- human primates, rodents, pigs, dogs, and cats. In certain embodiments, the subject may be administered with additional therapeutic agents or cells.

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

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

EXAMPLES

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

EXAMPLE 1: CULTURING AND PRODUCTION OF ADHERENT PLACENTAL CELLS

Placenta-derived cell populations containing over 90% maternally-derived cells were prepared as described in Example 1 of International Patent Application WO 2016/098061, in the name of Esther Lukasiewicz Hagai et al, published on June 23, 2016, which is incorporated herein by reference in its entirety.

Osteogenesis and adipogenesis assays were performed on placental cells that were prepared as described in the previous paragraph, and on BM adherent cells. In osteogenesis assays, over 50% of the BM cells underwent differentiation into osteocytes, while none of the placental-derived cells exhibited signs of osteogenic differentiation. In adipogenesis assays, over 50% of the BM- derived cells underwent differentiation into adipocytes. In contrast, none of the placental-derived cells exhibited morphological changes typical of adipocytes. These experiments were performed as described in Example 2 of WO 2016/098061, which is incorporated herein by reference.

EXAMPLE 2: CULTURE OF PLACENTAL CELLS IN SERUM-FREE MEDIUM

METHODS

Overview: The manufacturing process consisted of 3 stages, followed by downstream processing steps: Stage 1, the intermediate cell stock (ICS) production, contains the following steps:

1. Extraction of ASCs from the placenta. Initial incubation was in serum-containing

medium.

2. 2-dimensional cell growth ("2D" growth in flasks) for 3 passages in serum-free medium (typically about 4-10 population doublings after the first passage).

3. Cell concentration, formulation, filling and cryopreservation.

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

1. 2D cell growth of the thawed ICS in serum-free medium for 2 additional passages (3/1 and 3/2) (typically about 10-14 population doublings after thawing).

Stage 3, the additional culture steps in the presence of serum, contains the following steps:

1. 2D cell growth of the thawed ICS in serum-containing medium for 1 additional passage.

In some cases, cells were switched to serum-containing medium for the final 3 days of passage 3/2. In either case, the total number of population doublings after adding serum- containing medium was typically about 3-8.

2. 3D cell growth in a bioreactor for up to 10 additional doublings.

The downstream processing steps included harvest from flasks or bioreactor/s, cell concentration, washing, formulation, filling and cryopreservation.

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

Step 1-1— Extraction of Adherent Stromal Cells (ASCs)

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

Within 36 hours of the delivery, the placenta (apart from the amnion and chorion) was placed with the maternal side facing upwards and was cut into pieces, which were washed thoroughly with isotonic buffer) containing gentamicin. • The washed pieces were incubated for 1-3 hours with collagenase and DNAse in isotonic buffer.

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

• The cells were suspended in culture medium, seeded in flasks, and incubated at 37°C in a tissue culture incubator under humidified conditions supplemented with 5% CO2.

• After 2 days, cells were washed twice with Phosphate-Buffered Saline (PBS), and the culture medium was replaced with StemPro® MSC SFM XenoFree medium (serum-free and xeno- free culture medium [SFM-XF]) (ThermoFisher Scientific, catalog no. A10675-01; hereinafter "StemPro® medium"), and CellStart™ cell attachment solution was added.

• Cells were incubated in StemPro® medium until the end of the first passage. Step 1-2— Initial 2- Dim ensio rial Culturing

• Passage 1: Cells were detached using trypsin, centrifuged, and seeded at a culture density of 3.16 ± 0.5 x 10³ cells/cm² in tissue culture flasks, in gentamicin-free StemPro® medium in the presence of CellStart™.

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

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

Following the final passage, the resulting cell suspension was centrifuged and re-suspended in culture medium at a final concentration of 20-40 x 10⁶ cells/milliliter (mL). The cell suspension was diluted 1: 1 with 2D Freezing Solution (20% DMSO, 80% FBS), and the cells were cryopreserved in 10% DMSO, 40% FBS, and 50% DMEM. The temperature was reduced in a controlled rate freezer (l°C/min down to -80°C, followed by 5°C/min down to -120°C), and the cells were stored in a liquid nitrogen freezer to produce the ICS. Production of Cell Product

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

The ICS was thawed, diluted with and cultured in StemPro® medium until 60 90% confluence (typically 4-7 days after seeding), and cultured for 2 additional passages (referred to as passages 3/1 and 3/2 respectively; again passaging when reaching 60 90% confluence), then were harvested for seeding in the bioreactor.

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

Each bioreactor contained Fibra-cel^® carriers (New Brunswick Scientific) made of polyester and polypropylene, and StemPro® medium.

The culture medium in the bioreactor/s was kept at the following conditions: temp: 37±1°C, Dissolved Oxygen (DO): 70±20% and pH 7.4±0.4. Filtered gases (Air, C0₂, N₂ and 0₂) were supplied as determined by the control system in order to maintain the target DO and pH values.

After seeding, the medium was agitated with stepwise increases in the speed, up to 150- 200 RPM by 24 hours. Perfusion was initiated several hours after seeding and was adjusted on a daily basis in order to keep the glucose concentration constant at approximately 550mg\liter.

Cell harvest was performed at the end of the growth phase (approximately day 6). Bioreactors were washed for 1 minute with pre-warmed sterile PBS, and cells were detached.

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

In some experiments, the cell suspension underwent concentration and washing, using suspension solution (5% w/v human serum albumin [HSA] in isotonic solution) as the wash buffer, and diluted 1: 1 with 2X 3D-Freezing solution (20% DMSO v/v and 5% HSA w/v in isotonic solution) to a final concentration of 10-20x10⁶ cells/ml, in isotonic solution containing 10% DMSO v/v and 5% HSA w/v. The temperature of the vials was gradually reduced, and the vials were stored in a gas-phase liquid nitrogen freezer.

Bone marrow migration assay. ASC were suspended in full DMEM at a concentration of 1 x 10⁶ cells per 4 ml. medium. An aliquot of cell suspension containing 1 x 10⁶ cells was added to each well of a 6-well plate and incubated for 24 hr. Cells were then washed with PBS and incubated in chemotaxis buffer (Roswell Park Memorial Institute [RPMI] with 5% albumin) for another 24 hrs., after which the CM was collected and centrifuged at 1500 rpm for 5 min, and the supernatant was retained.

Mouse BM cells were suspended at 10 x 10⁶ cells/ml in chemotaxis buffer, and 100 mcL per well of the cell suspension was added to the upper compartment of 24-well Transwell® plates. 0.5 ml. CM from ASC, collected as described in the previous paragraph, was added to the lower compartment, and wells were incubated for 24 hr. at 37 °C, in a 5% CC -containing incubator. The upper compartments were gently removed, medium from the lower compartments were removed, the wells were washed, and the wash buffer was combined with the removed medium. Cells were counted, and the percentage of migration was defined as the number of migrated cells divided by the total number of cells added to the well.

RESULTS

Placental cells were extracted and expanded in serum-free (SF) medium for 3 passages. Cell characteristics of several batches were assessed (Table 1). The cells exhibited a significant ability to enhance hematopoiesis in a bone marrow migration (BMM) assay.

Table 1. Characteristics of placental cells expanded in SF medium. PDL refers to population doubling level— in this case, the number of doublings since passage 1.

Total cell

BATCH GROUP Passage growth size PDL

(days) (μιη)

1 7 22 NA

B 2 14 18.2 2.1

3 20 19.2 6.1

1 7 NA NA

PD230414SFM NA 2 14 NA 2.3

3 19 16.2 5.7

1 7 NA NA

PD040514SFM NA 2 14 NA 2.7

3 18 15.6 6.5

1 7 NA NA

PD260514SFM NA 2 13 NA 2.9

3 17 15.8 6.6

1 6 NA NA

PD180814SFM NA 2 10 NA 2.1

3 16 16.7 5.3

1 8 NA NA unfiltered 2 14 NA 2.1

3 20 17 5.6

PD220914SFM

1 8 NA NA filtered 2 14 NA 2

3 20 17.8 5.1

1 9 NA NA

PD271014SFM filtered 2 15 NA 2.1

3 21 17 5.1

Average P 3 19.1 17.55 6.12

%CV P 3 8 9 11 EXAMPLE 3: OSTEOCYTE AND ADIPOCYTE DIFFERENTIATION ASSAYS

ASC were prepared as described in Example 1. BM adherent cells were obtained as described in WO 2016/098061 to Esther Lukasiewicz Hagai and Rachel Ofir, which is incorporated herein by reference in its entirety. Osteogenesis and adipogenesis assays were performed as described in WO 2016/098061.

Osteocyte induction. Incubation of BM-derived adherent cells in osteogenic induction medium resulted in differentiation of over 50% of the BM cells, as demonstrated by positive alizarin red staining. On the contrary, none of the placental-derived cells exhibited signs of osteogenic differentiation.

Next, a modified osteogenic medium comprising Vitamin D and higher concentrations of dexamethasone was used. Over 50% of the BM cells underwent differentiation into osteocytes, while none of the placental-derived cells exhibited signs of osteogenic differentiation.

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

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

EXAMPLE 4: FURTHER OSTEOCYTE AND ADIPOCYTE DIFFERENTIATION ASSAYS

ASC were prepared as described in Example 2. Adipogenesis and Osteogenesis were assessed using the STEMPRO® Adipogenesis Differentiation Kit (GIBCO, Cat# A1007001) and the STEMPRO® Osteogenesis Differentiation Kit (GIBCO, Cat# A1007201), respectively.

RESULTS

Adipogenesis and Osteogenesis of placental cells grown in SRM or in full DMEM were tested. Groups are shown in Table 2.

Table 2: experimental groups Group Product Batch

Al BM derived MSC (positive control) BM-122

Bl ASC grown in SRM PD220914SFMS3 R001 B1.2

CI ASC grown in SRM R050115 R01

Dl ASC grown in SRM R280115 R01

El ASC grown in full DMEM PT041011R36

In adipogenesis assays, BM-MSCs treated with differentiation medium stained positively with Oil Red O (Fig. 2). By contrast, 2/3 of the SRM batches exhibited negligible staining, and the other SRM batch, as well as the full DMEM-grown cells, did not exhibit any staining at all. Thus, in certain embodiments, placental cells grown in SRM or full DMEM do not have significant adipogenic potential.

In osteogenesis assays, BM-MSCs treated with differentiation medium stained positively with Alizarin Red S (Fig. 3). By contrast, none of the placental cells batches grown in SRM or full DMEM exhibited staining. Thus, placental cells grown in SRM or full DMEM do not have significant osteogenic potential.

EXAMPLE 5: THE EFFECT OF ASC ON ACUTE LUNG INJURY

Acute lung injury is induced in ICR mice (Harlan Sprague Dawley Inc.) by treatment with lipopolysaccharide (LPS) from E. coli, by oropharyngeal aspiration (OA) instillation, as described in Lakatos HF et al. Four hours later, 1 x 10^A6 ASC are administered intravenously. 48 hours after LPS administration, pulmonary inflammation and injury are determined by bronchoalveolar lavage (BAL), RNA extraction, histology and lung wet/dry weight measurements.

EXAMPLE 6: THE EFFECT OF ASC ON ACUTE LUNG INJURY IN HUMAN SUBJECTS

Human subjects suffering from acute lung injury are administered 150 x 10^A6 ASC. Progression or resolution of acute lung injury is followed by chest radiography, and monitoring of PiCCO (Pulse Contour Cardiac Output), Pa0₂/Fi02 ratio, and positive end expiratory pressure (PEEP), as described in VM et al, and the references cited therein. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

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

REFERENCES

(Additional references may be cited in text)

Andaloussi, EL et al, Extracellular vesicles: biology and emerging therapeutic opportunities. Nat.

Rev. Drug. Discov. 12, 347-357 (2013).

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Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of treating respiratory distress syndrome in a subject in need thereof, comprising administering to said subject a pharmaceutical composition comprising placental adherent stromal cells (ASC), thereby treating respiratory distress syndrome.

2. The method of claim 1, wherein said respiratory distress syndrome is acute respiratory distress syndrome (ARDS).

3. The method of claim 2, wherein said ARDS is pulmonary ARDS.

4. The method of claim 2, wherein said ARDS is extrapulmonary ARDS.

5. The method of claim 1, wherein said respiratory distress syndrome is infant respiratory distress syndrome (IRDS).

6. A method of treating acute lung injury (ALI) in a subject in need thereof, comprising administering to said subject a pharmaceutical composition comprising placental adherent stromal cells (ASC), thereby treating ALI.

7. The method of any of claims 1-6, wherein said respiratory distress syndrome or ALI follows pneumonia.

8. The method of any of claims 1-6, wherein said respiratory distress syndrome or ALI follows sepsis.

9. The method of any of claims 1-6, wherein said respiratory distress syndrome or ALI follows a condition selected from gastric content aspiration, trauma, pancreatitis, inhalation injury, burns, non-cardiogenic shock, drug overdose, transfusion related acute lung injury (TRALI), VILI (ventilator induced lung injury), an amniotic fluid embolism, and ischemic reperfusion injury.

10. The method of any of claims 1-6, wherein said respiratory distress syndrome or ALI follows drowning.

11. The method of any of claims 1-10, wherein said placental ASC have been incubated in a 3D culture apparatus.

12. The method of claim 11, further comprising the subsequent step of harvesting said placental ASC by removing said placental ASC from said 3D culture apparatus.

13. The method of claim 11 or 12, wherein said placental ASC have been incubated in a 2D adherent-cell culture apparatus, prior to said incubation in a 3D culture apparatus.

14. The method of any of claims 11-13, wherein said 3D culture apparatus comprises a bioreactor.

15. The method of any of claims 11-14, wherein said 3D culture apparatus comprises a synthetic adherent material.

16. The method of claim 15, wherein said synthetic adherent material is a fibrous matrix.

17. The method of claim 16, wherein said synthetic adherent material is selected from the group consisting of a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, a poly-L-lactic acid, and an inert metal fiber.

18. The method of any of claims 11-17, wherein said 3D culture apparatus comprises microcarriers.

19. The method of any of claims 1-10, wherein said placental ASC have been incubated in carriers, wherein said each of said carriers include multiple 2D surfaces extending from an exterior of said carrier towards an interior of said carrier.

20. The method of claim 19, wherein said placental ASC have been incubated in a 2D adherent- cell culture apparatus, prior to said incubation in said carriers.

21. The method of claim 19 or 20, wherein said incubation in said carriers takes place inside a bioreactor.

22. The method of claim 19, wherein said placental ASC are incubated in a 3D adherent-cell culture apparatus, following said incubation in said carriers.

23. The method of any of claims 1-22, wherein said placental ASC express a marker selected from the group consisting of CD73, CD90, CD29 and CD 105.

24. The method of any of claims 1-23, wherein said placental ASC do not express a marker selected from the group consisting of CD3, CD4, CDl lb, CD14, CD19, and CD34.

25. The method of any of claims 1-23, wherein said placental ASC do not express a marker selected from the group consisting of CD3, CD4, CD34, CD39, and CD106.

26. The method of claim 25, wherein less than 50% of said placental ASC express CD200.

27. The method of claim 25, wherein more than 50% of said placental ASC express CD200.

28. The method of any of claims 25-28, wherein more than 50% of said placental ASC express CD141.

29. The method of any of claims 25-29, wherein more than 50% of said placental ASC express SSEA4.

30. The method of any of claims 25-30, wherein more than 50% of said placental ASC express HLA-A2.

31. The method of any of claims 1 -30, wherein the placental ASC secrete Flt-3 ligand or stem cell factor (SCF).

32. The method of any of claims 1-31, wherein the ASC secrete a factor selected from TNF-beta and LIF.

33. The method of any of claims 1-31, wherein the ASC secrete a factor selected from Serpin El, uPAR, MMP-1, MMP-2, MMP-10, Follistatin, MIF, and LIF.

34. The method of any of claims 1-33, wherein the cells are administered intramuscularly, intravenously, subcutaneously, or intraperitoneally.

35. The method of any of claims 1-33, wherein the cells are administered intratracheally, by inhalation, or intranasally.