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WO2018185584A1 - Procédés et compositions pour traiter une lésion pulmonaire aiguë et un syndrome de détresse respiratoire - Google Patents

Procédés et compositions pour traiter une lésion pulmonaire aiguë et un syndrome de détresse respiratoire Download PDF

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Publication number
WO2018185584A1
WO2018185584A1 PCT/IB2018/051847 IB2018051847W WO2018185584A1 WO 2018185584 A1 WO2018185584 A1 WO 2018185584A1 IB 2018051847 W IB2018051847 W IB 2018051847W WO 2018185584 A1 WO2018185584 A1 WO 2018185584A1
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Prior art keywords
cells
asc
placental
respiratory distress
distress syndrome
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PCT/IB2018/051847
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English (en)
Inventor
Yaacob YANAY
Arie EISENKRAFT
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Pluristem Ltd.
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Publication of WO2018185584A1 publication Critical patent/WO2018185584A1/fr

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48Reproductive organs
    • A61K35/50Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

Definitions

  • compositions comprising placental-derived adherent stromal cells for treating acute lung injury and respiratory distress syndrome.
  • 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.
  • TRALI transfusion related acute lung injury
  • 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).
  • 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.
  • ASC placental adherent stromal cells
  • a pharmaceutical composition for treating respiratory distress syndrome comprising a therapeutically effective amount of placental ASC.
  • placental ASC in the preparation of a medicament for treating respiratory distress syndrome.
  • a method of treating acute lung injury comprising the step of administering to the subject a pharmaceutical composition comprising placental adherent stromal cells (ASC), thereby treating ALI.
  • a pharmaceutical composition for treating ALI comprising a therapeutically effective amount of placental ASC.
  • placental ASC in the preparation of a medicament for treating ALI.
  • the ASC described herein have been cultured in 2-dimensional (2D) culture, 3-dimensional (3D) culture, or a combination thereof.
  • 2D and 3D culture conditions are provided in the Detailed Description and in the Examples.
  • 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).
  • BM bone marrow
  • placental cells after adipogenesis assays.
  • 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.
  • aspects of the invention relate to methods and compositions that comprise placenta- derived adherent stromal cells (ASC).
  • ASC placenta- derived adherent stromal cells
  • the ASC may be human ASC, or in other embodiments animal ASC.
  • 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.
  • the respiratory distress syndrome is acute respiratory distress syndrome (ARDS), which may be, in more specific embodiments, pulmonary ARDS, or extrapulmonary ARDS.
  • 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.
  • the respiratory distress syndrome is infant respiratory distress syndrome (IRDS).
  • 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.
  • ALI acute lung injury
  • a method of treating pulmonary edema comprising the step of administering to the subject a pharmaceutical composition comprising placental ASC.
  • a method of treating arterial comprising the step of administering to the subject a pharmaceutical composition comprising placental ASC.
  • 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.
  • 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.
  • 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.
  • the ARDS is secondary to the insult or injury.
  • the ALI follows the insult or injury. In other embodiments, the ALI is secondary to the insult or injury.
  • 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).
  • 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.
  • ARDSp direct lung injury
  • ARDSexp extrapulmonary ARDS
  • ARDSexp indirect lung injury
  • the prevalent damage in the early stages of ARDSp is intra-alveolar, whereas in ARDSexp, it is the interstitial edema.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • TRALI transfusion related acute lung injury
  • the respiratory distress syndrome is ARDS that is secondary to pneumonia.
  • the pneumonia may be bacterial, viral, fungal, or parasitic pneumonia.
  • 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
  • the pneumonia occurs in an immunocompromised subject, and may result e.g. from infection with Pneumocystis jirovecii, Toxoplasma gondii, or Aspergillus fumigatus.
  • the subject is receiving extracorporeal membrane oxygenation.
  • 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).
  • SOFA Sequential [Sepsis-related] Organ Failure Assessment
  • qSOFA quick SOFA
  • the respiratory distress syndrome or ALI is associated with presence of pro-inflammatory mediators and/or increased cyclo-oxygenase-2 (COX-2) expression.
  • COX-2 cyclo-oxygenase-2
  • the respiratory distress syndrome or ALI co-presents with systemic inflammatory response syndrome (SIRS).
  • SIRS systemic inflammatory response syndrome
  • the respiratory distress syndrome or ALI is co-present with non-specific immune activation.
  • 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.
  • stress-related immune alteration can be readily measured by determining the concentrations of inflammatory cytokines and immune cell subtype compositions.
  • serum cytokine concentrations are measured (e.g. as described in Rodrigues CE et al, van Griensven et al, and the references cited therein).
  • the respiratory distress syndrome is infant respiratory distress syndrome (IRDS).
  • IRDS infant respiratory distress syndrome
  • IRDS is usually found in premature infants at least 6 weeks before their due date, and results from an inability to produce sufficient surfactant.
  • 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.
  • the described compositions are administered after the subject is stabilized from acute pathologies. In some embodiments, the subject is stabilized by supportive medical care.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Placenta and placental tissue 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.
  • tissue specimens are washed in a physiological buffer [e.g., phosphate -buffered saline (PBS) or Hank's buffer].
  • a physiological buffer e.g., phosphate -buffered saline (PBS) or Hank's buffer.
  • 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.
  • 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.
  • the placental tissue is optionally minced, followed by enzymatic digestion.
  • 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.
  • perfusion refers to the act of pouring or passaging a fluid over or through an organ or tissue.
  • 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.
  • 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.
  • the placenta is kept chilled prior to harvest of the cells.
  • 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.
  • placental cells are, in certain embodiments, allowed to adhere to the surface of an adherent material to thereby isolate adherent cells.
  • 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.
  • any medium suitable for growth of cells in a standard tissue apparatus and/or a bioreactor may be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • "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%.
  • 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.
  • the mixture contains at least 70% fetal cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • ASC adherent stromal cells
  • 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.
  • the medium does not contain animal serum.
  • 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".
  • animal serum includes serum from a variety of species, provided that the serum stimulates expansion of the ASC population.
  • 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.
  • the serum-deficient medium is supplemented with factors intended to stimulate cell expansion in the absence of serum.
  • 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.
  • the serum-deficient medium contains one or more growth factors.
  • the growth factors individually or, in other embodiments collectively, induce cell expansion in culture.
  • the growth factors individually or, in other embodiments collectively, induce cell expansion in culture without differentiation.
  • 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.
  • FGF Fibroblast Growth Factor
  • TGF-beta Uniprot accession no. P01137
  • transferrin e.g. serotransferrin or lacto transferrin
  • insulin Uniprot accession no. P01308
  • EGF epidermal growth factor
  • 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).
  • 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.
  • the serum-deficient medium comprises an FGF and EGF.
  • 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; TheraPEAKTM MSCGM-CDTM (Lonza, cat. no. 190632); and MesenCult-XF (Stem Cell Technologies, cat. no. 5429).
  • the StemPro® media contain PDGF-BB, bFGF, and TGF- ⁇ , and insulin (Chase et al).
  • 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).
  • ASC were expanded in Stempro® SFM-XF.
  • MSC Nutristem® XF was also tested and yielded similar results.
  • 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- ⁇ . 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.
  • FGF-2 10 ng/ml
  • EGF epidermal growth factor
  • BSA 0.5%
  • 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 10 nM linolenic acid.
  • the described SRM comprises bFGF (basic fibroblast growth factor, also referred to as FGF-2), TGF- ⁇ (TGF- ⁇ , including all isotypes, for example ⁇ , ⁇ 2, and ⁇ 3), or a combination thereof.
  • FGF-2 basic fibroblast growth factor
  • TGF- ⁇ TGF- ⁇ , including all isotypes, for example ⁇ , ⁇ 2, and ⁇ 3
  • the SRM comprises bFGF, TGF- ⁇ , and PDGF.
  • the SRM comprises bFGF and TGF- ⁇ , and lacks PDGF-BB.
  • insulin is also present.
  • 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.
  • the described SRM comprises bFGF, TGF- ⁇ , and insulin.
  • 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.
  • the SRM comprises 5-20 ng/ml bFGF, 2-10 ng/ml TGF- ⁇ , and 5-20 ng/ml insulin, or, in other embodiments, 7-15 ng/ml bFGF, 3-8 ng/ml TGF- ⁇ , and 7-15 ng/ml insulin.
  • the described SRM comprises bFGF and EGF.
  • 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.
  • insulin; and/or transferrin is also present.
  • 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.
  • the SRM further comprises an additional component selected from BSA, selenite (e.g. sodium selenite), pyruvate (e.g. sodium pyruvate); heparin, and Hnolenic acid.
  • BSA selenite
  • pyruvate e.g. sodium pyruvate
  • heparin e.g. sodium pyruvate
  • Hnolenic acid e.g. sodium selenite
  • all 5 of BSA, selenite, pyruvate, heparin, and Hnolenic acid are present.
  • 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.
  • bFGF 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).
  • 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.
  • TGF- ⁇ where present, is present at a concentration of 1-25, 2-25,
  • PDGF 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.
  • 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%.
  • the second medium contains at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% animal serum.
  • the second medium does not contain added growth factors, other than those present in the animal serum added thereto.
  • 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.
  • the optional step is performed for 1-3 passages, 1-2 passages, or a single passage.
  • 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.
  • 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.
  • serum-containing medium for example after extraction, facilitates, in some scenarios, initial attachment and expansion of cells after their extraction.
  • the earlier step is performed on a 2D substrate.
  • the aforementioned step of incubating the ASC population in serum-free medium 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.
  • 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.
  • 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.
  • 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.
  • the 3D substrate is in a bioreactor.
  • the 3D substrate is a synthetic adherent material.
  • 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.
  • the placental ASC are cultured in the presence of extracts, or in other embodiments CM, from ischemic cells.
  • CM ischemic cells
  • 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.
  • 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
  • further hypoxia preconditioning which may be 1-5%, e.g.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • PEMF pulsed electromagnetic fields
  • 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).
  • the ASC do not express some or all of the following markers: CD3 (e.g. UniProtKB Accession Nos.
  • 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
  • CD34 UniProtKB Accession No. P28906
  • 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]).
  • 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.
  • CD3 and HLA-DR may be detected by antibodies recognizing any of their component parts, such as, but not limited to, those described herein.
  • the ASC possess a marker phenotype that is distinct from bone marrow-mesenchymal stem cells (BM-MSC).
  • 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.
  • 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.
  • over 90% of the ASC are positive for CD29, CD90, and CD54.
  • over 85% of the described cells are positive for CD29, CD73, CD90, and CD 105.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • adipogenesis induction medium for example a solution containing 1 mcM dexamethasone, 0.5 mM 3-Isobutyl-l-methylxanthine (IBMX), 10 mc
  • 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.
  • 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.
  • 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.
  • 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.
  • FACS fluorescence-activated cell sorting
  • “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 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.
  • 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.
  • 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.
  • 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 (IV 3 NeuAc 2 ⁇ 3 GalGB4); Kannagi R et al), or in other embodiments both markers.
  • more than 50% of the cells express HLA-A2 (UniProt Accession No. P01892).
  • HLA-A2 UniProt Accession No. P01892
  • 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.
  • 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.
  • 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.
  • 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.
  • cell culture medium such as DMEM + 10% serum or the like, as will be appreciated by those skilled in the art.
  • less than 20%, 15%, or 10% of the described cells highly express CD56.
  • 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.
  • 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.
  • the cells highly express CD141.
  • 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.
  • 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.
  • adipogenesis induction medium namely a solution containing 1 mcM dexamethasone, 0.5 mM IB MX, 10 mcg/ml insulin, and 100 mcM indomethacin
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • greater than 50% of the cells highly express CD141, or in other embodiments SSEA4, or in other embodiments both markers.
  • the cells highly express CD 141.
  • 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.
  • 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.
  • 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.
  • lack of TGFB3 expression correlates with lack of potential to differentiate into chondrocytes.
  • 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.
  • 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.
  • lack of high BMP2 expression correlates with lack of potential to differentiate into osteoblasts.
  • 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.
  • PPARG Peroxisome proliferator-activated receptor gamma
  • 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.
  • lack of high PPARG expression correlates with lack of potential to differentiate into adipocytes.
  • culturing on a 3D substrate refers, in some embodiments, to culturing for 5 days on a polyester non-woven fibrous matrix.
  • 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.
  • 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 (RCN 2 ), and/or calponin 1 basic smooth muscle (CNN1).
  • IL-6 UniProt identifier P05231
  • IL-8 UniProt identifier P10145
  • EEEF2 eukaryotic translation elongation factor 2
  • RCN 2 reticulocalbin 3
  • CNN calponin 1 basic smooth muscle
  • Reference herein to "secrete”/ "secreting”/ “secretion” relates to a detectable secretion of the indicated factor, above background levels in standard assays.
  • 0.5 x 10 6 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.
  • 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.
  • 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.
  • 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.
  • a mixed lymphocyte reaction may be performed.
  • cord blood (CB) mononuclear cells for example human cells or cells from another species
  • CB cord blood
  • iCB irradiated cord blood cells
  • PBMC peripheral blood-derived monocytes
  • 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 3 H-thymidine uptake. Reduction of the CB cell replication when co-incubated with test cells indicates an immunosuppressive capability.
  • PB peripheral blood
  • PB peripheral blood
  • secretion of pro-inflammatory and anti-inflammatory cytokines by blood cell populations 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.
  • 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.
  • each of CD73, CD29, and CD 105 is expressed by more than 90% of the described ASC; and the cells inhibit T cell proliferation.
  • each of CD34, CD19, and CD14 is expressed by less than 3% of the cells; and the cells inhibit T cell proliferation.
  • 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.
  • the ASC secrete immunoregulatory factor(s).
  • the ASC secrete a factor selected from TNF-beta (UniProt identifier P01374) and Leukemia inhibitory factor (LIF; UniProt identifier PI 5018).
  • 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.
  • 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.
  • 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.
  • the ASC secrete anti-fibrotic factor(s).
  • 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).
  • Serpin El Plasminogen activator inhibitor 1 ; Uniprot Accession No. P05121
  • uPAR Ultrakinase plasminogen activator surface receptor
  • the ASC secrete factors that facilitate.
  • 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.
  • the ASC secrete a factor(s) that promotes extracellular matrix (ECM) remodeling.
  • the ASC secrete a factor selected from TIMP1, TIMP2, MMP-1, MMP-2, and MMP-10.
  • the ASC secrete TIMP1, TIMP2, MMP-1, MMP-2, and MMP-10, which were found to be secreted by maternal cells.
  • the ASC secrete TIMP1, TIMP2, MMP-1, and MMP-10, which were found to be secreted by fetal cells.
  • the described ASC exhibit a spindle shape when cultured under 2D conditions.
  • the ASC express CD200, while in other embodiments, the ASC lack expression of CD200.
  • less than 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, or 2%, 1%, or 0.5% of the adherent cells express CD200.
  • greater than 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the adherent cells express CD200.
  • 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).
  • the described ASC have been subject to a 3D incubation, as described further herein.
  • the ASC have been incubated in a 2D adherent-cell culture apparatus, prior to the step of 3D culturing.
  • 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.
  • 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.
  • apparatuses for 2D culture are cell culture dishes and plates. Included in this definition are multi-layer trays, such as Cell FactoryTM, manufactured by NuncTM, 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”.
  • 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.
  • 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.
  • an adherent material refers to a material that is synthetic, or in other embodiments naturally occurring, or in other embodiments a combination thereof.
  • the material is non-cytotoxic (or, in other embodiments, is biologically compatible).
  • 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.
  • 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 MatrigelTM, an extra-cellular matrix component (e.g., Fibronectin, Chondronectin, Laminin), and a collagen.
  • 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
  • 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
  • the described lengths describes the total time in a 3D substrate culture apparatus, including the expansion and induction stages.
  • 3D culturing can be performed in a 3D bioreactor.
  • 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.
  • the attachment substrate is in the form of carriers, which comprise, in more specific embodiments, a surface comprising a synthetic adherent material.
  • 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.
  • 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).
  • 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.
  • controlled conditions e.g. pH, temperature and oxygen levels
  • 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.
  • 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.
  • a continuous stirred tank bioreactor 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.
  • 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.
  • 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.
  • the substrate remains substantially in place during the standard perfusion rate of the bioreactor.
  • the substrate may be lifted at unusually fast perfusion rates, for example greater than 200 rpm.
  • FIG. 1 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).
  • 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.
  • the liquid is replaced with growth medium, which saturates the disk bed as depicted in (9).
  • temperature, pH, dissolved oxygen concentration, etc. are set prior to inoculation.
  • a slow initial stirring rate is used to promote cell attachment, then the stirring rate is increased.
  • perfusion is initiated by adding fresh medium via an external port (2).
  • metabolic products may be harvested from the cell-free medium above the basket (8).
  • 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.
  • adjustment of a tube (6) controls the liquid level; an external opening (4) of this tube is used in some embodiments for harvesting.
  • 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).
  • sparged gas confined to the remote chamber is absorbed by the nutrient medium, which washes over the immobilized cells.
  • a water jacket (17) is present, with ports for moving the jacket water in (13) and out (14).
  • a perfused bioreactor wherein the perfusion chamber contains carriers.
  • the carriers may be, in more specific embodiments, selected from macrocarriers, microcarriers, or both together.
  • 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.
  • the microcarriers are packed inside the perfused bioreactor.
  • the carriers in the perfused bioreactor are packed, for example forming a packed bed, which is submerged in a nutrient medium.
  • the carriers may comprise an adherent material.
  • the surface of the carriers comprises an adherent material, or the surface of the carriers is adherent.
  • 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.
  • the material may be selected from a polyester and a polypropylene.
  • an "adherent material” refers to a material that is synthetic, or in other embodiments naturally occurring, or in other embodiments a combination thereof.
  • the material is non-cytotoxic (or, in other embodiments, is biologically compatible).
  • 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 MatrigelTM, an extra-cellular matrix component (e.g., Fibronectin, Chondronectin, Laminin), and a collagen.
  • cells are produced using a packed-bed spinner flask.
  • 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.
  • the fibrous matrix comprises polyester, or comprises at least about 50% polyester.
  • the non-woven fibrous matrix comprises polyester, or comprises at least about 50% polyester.
  • the matrix is similar to the CelligenTM 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.
  • cells are seeded onto the scaffold by suspending them in medium and introducing the medium to the apparatus.
  • the stirring speed is gradually increased, for example by starting at 40 RPM for 4 hours, then gradually increasing the speed to 120 RPM.
  • the glucose level of the medium may be tested periodically (i.e.
  • 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.
  • carriers are removed from the packed bed, washed with isotonic buffer, and processed or removed from the carriers by agitation and/or enzymatic digestion.
  • 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,
  • between 1-20 x 10 6 cells per gram (gr) of carrier (substrate) are seeded, or in other embodiments 1.5-20 x 10 6 cells / gr carrier, or in other embodiments 1.5-18 x 10 6 , or in other embodiments 1.8-18 x 10 6 , or in other embodiments 2-18 x 10 6 , or in other embodiments 3-18 x 10 6 , or in other embodiments 2.5-15 x 10 6 , or in other embodiments 3-15 x 10 6 , or in other embodiments 3-14 x 10 6 , or in other embodiments 3-12 x 10 6 , or in other embodiments 3.5-12 x 10 6 , or in other embodiments 3-10 x 10 6 , or in other embodiments 3-9 x 10 6 , or in other embodiments 4-9 x 10 6 , or in other embodiments 4-8 x 10 6 , or in other embodiments 4-7 x 10 6 , or in other embodiments 4.5-6.5 x 10 6 cells / gr carrier.
  • 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.
  • 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.
  • 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.
  • over 5 x 10 5 , over 7 x 10 5 , over 8 x 10 5 , over 9 x 10 5 , over 10 6 , over 1.5 x 10 6 , over 2 x 10 6 , over 3 x 10 6 , over 4 x 10 6 , or over 5 x 10 6 viable cells are removed per milliliter of the growth medium in the bioreactor.
  • 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 CytodexTM (available from Pharmacia Fine Chemicals, Inc.), Superbeads (commercially available from Flow Labs, Inc.), and DE-52 and DE-53 (commercially available from Whatman, Inc.).
  • 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.
  • the microcarriers are packed inside a bioreactor.
  • grooved carriers 30 are used for proliferation and/or incubation of ASC.
  • the carriers may be used following a 2D incubation (e.g. on culture plates or dishes), or without a prior 2D incubation.
  • 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.
  • carriers 30 are "3D bodies" as described in WO/2014/037862; the contents of which relating to 3D bodies are incorporated herein by reference.
  • the described carriers are used in a bioreactor.
  • the carriers are in a packed conformation.
  • 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.
  • 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.
  • cells may be removed from a 3D matrix while the matrix remains within the bioreactor.
  • 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.
  • 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.
  • 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.
  • the harvesting process comprises agitation.
  • the agitation utilizes vibration, for example as described in PCT International Application Publ. No. WO 2012/140519, which is incorporated herein by reference.
  • 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.
  • 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 TrypLETM, 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.
  • the cells are harvested by a process comprising an optional wash step, followed by incubation with collagenase, followed by incubation with trypsin.
  • at least one, at least two, or all three of the aforementioned steps comprise agitation.
  • 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.
  • 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.
  • 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.
  • the vibrator comprises one or more controls for adjusting amplitude and frequency of the reciprocating motion.
  • the adherent material is a 3D substrate, which comprises, in some embodiments, carriers comprising a synthetic adherent material.
  • HBSS Hank's Balanced Salt Solution
  • 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, CM
  • the medium may be supplemented with additional substances.
  • additional 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.
  • 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.
  • the medium is serum-free.
  • 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.
  • growth factors e.g. ascorbic acid
  • cytokines e.g. B-glycerophosphate
  • steroids e.g. dexamethasone
  • hormones e.g., growth hormone, erythropoietin, thrombopoietin,
  • the placental ASC are grown as spheroids as part of the preparation process.
  • spheroid conditions include incubation in hydrogels (e.g. polyethylene glycol hydrogels), for example as described in Cha et al and the references cited therein.
  • 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.
  • 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.
  • Such components may be antibiotics, antimycotics, albumin, amino acids, and other components known to the art for the culture of cells.
  • the cells and the culture medium are substantially xeno-free, i.e., devoid of any animal contaminants.
  • the culture medium can be supplemented with a serum-replacement, human serum and/or synthetic or recombinantly produced factors.
  • 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.
  • CM conditioned medium
  • CM conditioned medium
  • a pharmaceutical composition comprising the CM, which may be, in some embodiments, indicated for the described therapeutic indications.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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 ⁇ of PBS, aliquoted and stored at -80°C.
  • exosomes can be further purified on sucrose-containing gradients (e.g. a 30% sucrose cushion), e.g. as described in Thery C et al.
  • sucrose-containing gradients e.g. a 30% sucrose cushion
  • 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.
  • D2O deuterium oxide
  • 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).
  • the exosomes or other extracellular vesicles are harvested from a 3D bioreactor in which the ASC have been incubated.
  • the cells are cryopreserved, and then are thawed, after which the exosomes are isolated.
  • the cells after thawing, the cells are cultured in 2D culture, from which the exosomes are harvested.
  • 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.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent.
  • a pharmaceutically acceptable carrier does not cause significant irritation to a subject.
  • 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.
  • the pharmaceutical carrier is an aqueous solution of saline.
  • compositions are provided herein that comprise ASC or CM in combination with an excipient, e.g., a pharmacologically acceptable excipient.
  • an excipient e.g., a pharmacologically acceptable excipient.
  • any of the described compositions further comprises a pharmacologically acceptable excipient.
  • 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.
  • DMSO dimethyl sulfoxide
  • glycerol glycerol
  • ethylene glycol formamide
  • propanediol poly-ethylene glycol
  • acetamide propanediol
  • propylene glycol poly-ethylene glycol
  • adonitol or may in other embodiments be a non-perme
  • both a permeating cryoprotectant and a non-permeating cryoprotectant are present.
  • the excipient is a carrier protein, a non-limiting example of which is albumin.
  • both an osmoprotectant and carrier protein are present; in certain embodiments, the osmoprotectant and carrier protein may be the same compound.
  • the composition is frozen.
  • the cells may be any embodiment of ASC mentioned herein, each of which is considered a separate embodiment.
  • compositions comprising the described placental ASC, in the absence of non-placental cell types.
  • compositions comprising the described placental ASC- derived CM, in the absence of CM derived from other cell types.
  • pharmaceutical compositions comprising the described exosomes.
  • non-autologous cells may in some cases induce an immune reaction when administered to a subject
  • 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.
  • immunosuppressive agents examples 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).
  • methotrexate cyclophosphamide
  • cyclosporine cyclosporine A
  • chloroquine hydroxychloroquine
  • sulfasalazine sulphasalazopyrine
  • gold salts gold salts
  • D-penicillamine leflunomide
  • azathioprine ana
  • 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.
  • 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.
  • the ASC or composition is administered intramuscularly; while in other embodiments, the ASC or composition is administered systemically.
  • 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
  • intraperitoneal administration refers to administration into the peritoneum of a subject.
  • the cells are administered intratracheally, intrathecally, by inhalation, or intranasally.
  • lung-targeting routes of administration may utilize cells encapsulated in liposomes or other barriers to reduce entrapment within the lungs.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer
  • 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.
  • 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.
  • 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.
  • therapeutic methods 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.
  • 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.
  • 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.
  • 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.
  • 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 may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • 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.
  • 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.
  • the pharmaceutical composition is frozen.
  • each embodiment of the described ASC may be freely combined with each embodiment relating to a therapeutic method or pharmaceutical composition.
  • each embodiment of the described exosomes may be freely combined with each embodiment relating to a therapeutic method or pharmaceutical composition.
  • 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.
  • the subject treated by the described methods and compositions is a human.
  • the subject is an adult subject, e.g. a subject over the age of 25.
  • 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.
  • the subject is a preterm infant.
  • 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.
  • the subject may be an animal.
  • treated animals include domesticated animals and laboratory animals, e.g., non-mammals and mammals, for example non- human primates, rodents, pigs, dogs, and cats.
  • the subject may be administered with additional therapeutic agents or cells.
  • 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.
  • 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.
  • 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.
  • Stage 1 The manufacturing process consisted of 3 stages, followed by downstream processing steps: Stage 1, the intermediate cell stock (ICS) production, contains the following steps:
  • 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).
  • Stage 2 the thawing of the ICS and initial further culture steps, contains the following step:
  • Stage 3 the additional culture steps in the presence of serum, contains the following steps:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 10 3 cells/cm 2 in tissue culture flasks, in gentamicin-free StemPro® medium in the presence of CellStartTM.
  • Step 1-3 Cell Concentration, Washing, Formulation, Filling and Cryopreservation
  • the resulting cell suspension was centrifuged and re-suspended in culture medium at a final concentration of 20-40 x 10 6 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.
  • 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, C0 2 , N 2 and 0 2 ) were supplied as determined by the control system in order to maintain the target DO and pH values.
  • 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
  • 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 10-20x10 6 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 10 6 cells per 4 ml. medium. An aliquot of cell suspension containing 1 x 10 6 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.
  • RPMI Roswell Park Memorial Institute
  • Mouse BM cells were suspended at 10 x 10 6 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.
  • 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.
  • 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.
  • BMM bone marrow migration
  • PDL Population doubling level— in this case, the number of doublings since passage 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • BM-MSCs treated with differentiation medium stained positively with Oil Red O (Fig. 2).
  • Fig. 2 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.
  • placental cells grown in SRM or full DMEM do not have significant adipogenic potential.
  • 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 A 6 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.
  • LPS lipopolysaccharide
  • OA oropharyngeal aspiration
  • Kannagi R et al Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J.
  • Oncostatin M-preconditioned mesenchymal stem cells alleviate bleomycin-induced pulmonary fibrosis through paracrine effects of the hepatocyte growth factor. Stem Cells Transl Med. 2017; 6: 1006-17.
  • TLR3 preconditioning enhances the therapeutic efficacy of umbilical cord mesenchymal stem cells in TNBS-induced colitis via the TLR3-Jagged-1 -Notch- 1 pathway. Mucosal Immunol. 2017; 10: 727-42.

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Abstract

L'invention concerne des procédés et des compositions comprenant des cellules stromales adhérentes, dérivées de tissu placentaire, pour traiter des sujets souffrant d'une lésion pulmonaire aiguë et d'un syndrome de détresse respiratoire (par exemple, le syndrome de détresse respiratoire aiguë, le SDRA pulmonaire ou le SDRA extrapulmonaire). Les compositions comprennent des compositions pharmaceutiques, qui peuvent en outre comprendre des excipients pharmacologiquement acceptables.
PCT/IB2018/051847 2017-04-05 2018-03-20 Procédés et compositions pour traiter une lésion pulmonaire aiguë et un syndrome de détresse respiratoire WO2018185584A1 (fr)

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WO2021202570A1 (fr) * 2020-03-30 2021-10-07 Children's Medical Center Corporation Utilisation de cellules souches pour le traitement d'une inflammation excessive
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11542328B2 (en) 2008-11-14 2023-01-03 The Brigham And Women's Hospital, Inc. Therapeutic and diagnostic methods relating to cancer stem cells
US11542473B2 (en) 2016-10-21 2023-01-03 Amniotics Ab Methods and compositions for generating hematopoietic cells
CN112261943B (zh) * 2018-06-05 2024-11-15 Medipost株式会社 用于预防或治疗炎症性疾病的包含间充质干细胞作为有效成分的药物组合物
CN112261943A (zh) * 2018-06-05 2021-01-22 Medipost株式会社 用于预防或治疗炎症性疾病的包含间充质干细胞作为有效成分的药物组合物
US11446334B2 (en) 2019-10-18 2022-09-20 Amniotics Ab Use of term amniotic fluid cells for the treatment of acute and chronic respiratory diseases
WO2021076043A1 (fr) * 2019-10-18 2021-04-22 Amniotics Ab Utilisation de cellules de liquide amniotique à terme pour le traitement de maladies respiratoires aiguës et chroniques
EP4114419A4 (fr) * 2020-03-06 2024-04-17 Figene, LLC Traitement du syndrome de détresse respiratoire aiguë induite par un virus par des fibroblastes ainsi que des fibroblastes activés par des tlr
WO2021181394A1 (fr) * 2020-03-12 2021-09-16 Pluristem Ltd. Procédés et compositions pour le traitement d'infections virales et de séquelles de celles-ci
US20210283189A1 (en) * 2020-03-12 2021-09-16 Pluristem Ltd. Methods and compositions for treating viral infections
WO2021202570A1 (fr) * 2020-03-30 2021-10-07 Children's Medical Center Corporation Utilisation de cellules souches pour le traitement d'une inflammation excessive
WO2021224766A1 (fr) * 2020-05-05 2021-11-11 Sibcell Biotech Ab Méthodes de traitement d'une maladie à coronavirus
WO2021226602A1 (fr) * 2020-05-08 2021-11-11 Celularity Inc. Cellule souche adhérente dérivée du placenta (pda) pour le traitement d'adultes atteints d'une insuffisance respiratoire aiguë associée au sras-cov-2 et du sdra (covid-19)
WO2022249167A1 (fr) * 2021-05-23 2022-12-01 Bonus Therapeutics Ltd. Compositions de cellules ayant une capacité thérapeutique améliorée
US12435308B2 (en) 2021-11-05 2025-10-07 Amniotics Ab Immunomodulation by amniotic fluid mesenchymal stem cells

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