WO2019075101A1 - Tissus dérivés ex vivo comprenant des multicouches de cellules souches dérivées de tissu adipeux et utilisations de ceux-ci - Google Patents

Tissus dérivés ex vivo comprenant des multicouches de cellules souches dérivées de tissu adipeux et utilisations de ceux-ci Download PDF

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WO2019075101A1
WO2019075101A1 PCT/US2018/055265 US2018055265W WO2019075101A1 WO 2019075101 A1 WO2019075101 A1 WO 2019075101A1 US 2018055265 W US2018055265 W US 2018055265W WO 2019075101 A1 WO2019075101 A1 WO 2019075101A1
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tissue
derived
adipose
vivo
stem cells
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PCT/US2018/055265
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Joan OLIVA VILANA
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Los Angeles Biomedical Research Institute At Harbor-Ucla Medical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • Embodiments of the invention relate to methods of making and using ex vivo derived tissues (e.g. , multilayer cell sheets).
  • the ex vivo derived tissues disclosed herein comprise human adipose stem cells or differentiated cells derived therefrom.
  • the ex vivo derived tissues disclosed herein can be used, in certain embodiments, to repair damaged tissues or organs and to treat certain conditions.
  • an ex vivo derived tissue comprising two or more monolayers of viable, adipose-derived stem cells.
  • the ex vivo derived tissue comprises two, three or four layers of cells.
  • the adipose-derived stem cells are human cells.
  • the ex vivo derived tissues comprise undifferentiated or differentiated adipose-derived stem cells.
  • an ex vivo derived tissue comprising two or more monolayers of viable, adipose-derived human stem cells, where the ex vivo derived tissue does not include a scaffold.
  • the ex vivo derived tissues disclosed herein consist essentially of undifferentiated ADSCs and/or differentiated ADSCs and therefore do not include scaffolds or supports.
  • an ex vivo derived tissue comprising two or more monolayers of viable, undifferentiated human adipose-derived stem cells, wherein at least 70%, at least 80%, at least 90%, or substantially all of the stem cells are undifferentiated, and at least 70%, at least 80%, at least 90%, or substantially all of the stem cells express CD29, CD73 and CD 105 on their cell surface.
  • an ex vivo derived tissue comprising two or more monolayers of viable, undifferentiated human adipose-derived stem cells, wherein at least 70%, at least 80%, at least 90%, or substantially all of the stem cells are undifferentiated, and at least 70%, at least 80%, at least 90%, or substantially all of the stem cells do not express CD45R and CD 19 on their cell surface. In certain embodiments, at least 70%, at least 80%, at least 90%, or substantially all of the stem cells express E- Cadherin.
  • an ex vivo derived tissue comprising two or more monolayers of viable, differentiated adipose-derived stem cells, wherein at least 70%, at least 80% or substantially all of the differentiated cells are adipocytes.
  • an ex vivo derived tissue comprising two or more monolayers of viable, differentiated adipose-derived stem cells, wherein at least 70%, at least 80% or substantially all of the differentiated cells are chondrocytes.
  • an ex vivo derived tissue comprising two or more monolayers of viable, differentiated adipose-derived stem cells, wherein at least 70%, at least 80% or substantially all of the differentiated cells are osteoblasts.
  • ex vivo derived tissues wherein undifferentiated adipose-derived stem cells are grown on a tissue culture substrate for a prolonged period of time (e.g., for at least 5 days, or at least 8 days) prior to harvesting the resulting ex vivo derived tissue.
  • a prolonged period of time e.g., for at least 5 days, or at least 8 days
  • a method of making ex vivo derived tissue consisting essentially of undifferentiated or differentiated human adipose-derived stem cells, where the ex vivo derived tissue is grown in the absence of a scaffold or feeder layer for 5 to 10 days prior to harvesting the resulting ex vivo derived tissue.
  • the damaged tissue may be cardiac tissue, bone, neuro tissue, liver tissue, skin or pancreatic tissue.
  • Fig. 1 shows a Scheme (Scheme 1) for growth and differentiation of human adipose-derived stem cells.
  • Fig. 1A-C shows a timeline of undifferentiated hADSC ex vivo derived tissues culture.
  • Fig. 1 A shows a time line to engineer adipocyte ex vivo derived multilayer tissue (multilayer cell sheet).
  • Fig. IB shows a time line to engineer osteoblast ex vivo derived multilayer tissue (multilayer cell sheet).
  • Fig. 1C shows a time line for engineering chondrocyte ex vivo derived multilayer tissue (multilayer cell sheet).
  • Fig. 2 shows a summary of undifferentiated human adipose-derived stem cell (hADSC) sheets.
  • hADSC undifferentiated human adipose-derived stem cell
  • Fig. 2A shows a hADSC monolayer, when the adipocyte, osteoblast and chondrocyte differentiation started (scale bar: 889 ⁇ ).
  • Fig. 2B shows a hADSC multilayer ex vivo derived tissue on the day of the harvesting. The density of cells in Fig. 2B is much higher than found in the monolayer of Fig. 2A.
  • Fig. 2C shows a harvesting steps of the undifferentiated multilayer hADSC ex vivo derived tissue.
  • Fig. 2D shows H&E staining showing the morphology of the undifferentiated hADSC cell tissue.
  • Fig. 2E shows immunostaining of the cell tissue illustrating expression of positive markers of hADSC cells (CD29, CD73, and CD105), and negative for CD 19 and CD45R.
  • Fig. 3 shows a summary of the adipocyte multilayer hADSC ex vivo derived tissues.
  • Fig. 3A shows a monolayer of adipocyte hADSC ex vivo derived tissue, the last day of treatment (scale bar: 889 ⁇ ).
  • Fig. 3B shows a multilayer of adipocyte hADSC, the last day of treatment. Density of adipocyte in the multilayer ex vivo derived tissue is much higher than monolayer.
  • Fig. 3C shows steps for harvesting the adipocyte multilayer hADSC ex vivo derived tissue.
  • Fig. 3D shows H&E staining shows the morphology of the adipocyte hADSC ex vivo derived tissue.
  • Fig. 3E shows immunostaining of the adipocyte ex vivo derived tissue shows that the ex vivo derived tissue increases the expression of adipocyte markers such as SREBP1, PPARg and AcetylCoA
  • Fig. 4 shows oil red staining of adipocyte hADSC monolayers cell sheets, the last day of treatment.
  • Fig. 4B shows oil red staining of osteogenic hADSC multilayer ex vivo derived tissues, the day of harvesting.
  • Fig. 4C shows the rate of differentiation of hADSC into adipocyte, comparing the monolayer to multilayer hADSC cell sheets.
  • Multilayer hADSC ex vivo derived tissues differentiate faster than monolayer, into adipocyte.
  • Fig. 5 shows a summary of the osteogenic hADSC multilayer ex vivo derived tissue.
  • Fig. 5C shows a step for harvesting the osteogenic multilayer hADSC ex vivo derived tissue.
  • H&E staining shows the morphology of the osteogenic multilayer hADSC ex vivo derived tissue.
  • Fig. 5E shows immunostaining of the adipocyte ex vivo derived tissue shows that the ex vivo derived tissue increases the expression of adipocyte markers such as osteocalcin.
  • Fig. 6 shows a summary of a differentiated chondrocyte hADSC multilayer tissue.
  • Fig. 6C shows steps for harvesting the chondrocyte multilayer hADSC ex vivo derived tissue.
  • Fig. 6D show H&E staining showing the morphology of the chondrogenic multilayer hADSC ex vivo derived tissue.
  • Fig. 6E shows immunostaining of the chondrogenic ex vivo derived tissue shows that the ex vivo derived tissue increases the expression of chondrocyte markers such as Aggrecan and SPARC.
  • Fig. 7A-D Histocompatibility antigen HLA-A was detected in undifferentiated, adipocyte, osteogenic and chondrocyte hADSC multilayer ex vivo derived tissues.
  • Fig. 7E-H Histocompatibility antigen HLA-DR was not detected in the undifferentiated, adipocyte, osteogenic and chondrocyte hADSC multilayer ex vivo derived tissues.
  • stem cells Because of the ethical issues regarding the use of human embryonic stem cells, laboratories and researchers focused on identifying different sources of adult stem cells.
  • researchers started to use single cell suspensions of adult stem cell derived from various tissues. The resulting undifferentiated or differentiated adult stem cells were directly injected into an area or location where they need to exert a curative effect.
  • the majority of injected stem cells tend to migrate away from the target location in the body resulting in low efficacy for this type of treatment. Further, injected stem cells tend to undergo apoptosis and display low viability after injection (e.g., from 0.01% to 6%).
  • stem cells tend to migrate to certain preferred organs, mainly targeting the lungs.
  • stem cells are typically injected in the hepatic artery in an attempt to decrease the diffusion and migration of the stem cells into ectopic organs.
  • Grafting a layer of cells can overcome the random migration of the cells and can also increase the treatment efficacy of the cells by targeting the cells to a specific desired area.
  • the inventors herein have discovered methods of growing multilayer cell tissues comprising adipose-derived stem cells.
  • the multilayer tissues disclosed herein are not grown as separate monolayers and later compiled. Further, in certain embodiments, the multilayer tissues disclosed herein do not require growth of monolayers on support frameworks or expensive pre-treatment of tissue culture substrates.
  • An adipose-derived stem cell is a pluripotent mesenchymal stem cell with the potential of self-renewal and the potential to differentiate into any type of cell, such as adipocytes, myocytes, and neurons, etc.
  • adipose -derived stem cell adipose -derived stem cell (ADSC) tissues.
  • the tissues disclosed herein may be undifferentiated or differentiated.
  • the inventors herein have discovered a novel method of growing adipose-derived stem cells in culture, where the cells form a multilayer tissue that can be harvested for grafting.
  • the invention provides an ex vivo derived tissue comprising two or more monolayers of viable, adipose-derived stem cells.
  • the adipose-derived stem cells disclosed herein can be obtained or derived from any suitable mammal.
  • the adipose-derived stem cells are mammalian-derived stem cells.
  • the adipose-derived stem cells are human-derived stem cells.
  • the adipose-derived stem cells are human cells.
  • the adipose-derived stem cells are obtained from the adipose tissue of a human.
  • an ex vivo derived tissue comprises, consists of, or consists essentially of adipose-derived human stem cells. In some embodiments, an ex vivo derived tissue comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of undifferentiated adipose derived human stem cells. In some embodiments, an ex vivo derived tissue consists of, or consists essentially of undifferentiated adipose derived human stem cells.
  • an ex vivo derived tissue comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of differentiated adipose derived human stem cells.
  • an ex vivo derived tissue consists of, or consists essentially of differentiated adipose derived human stem cells.
  • a monolayer of adipose-derived stem cells comprises or consist of adipose-derived stem cells organized into a flexible horizontal plane comprising a vertical dimension substantially equivalent to the diameter of a single adipose -derived stem cell.
  • the ex vivo derived tissues disclosed herein comprise at least two, at least three, at least four, at least five or at least ten monolayers of cells and can be grown on any suitable tissue culture substrate, or in the absence of a substrate.
  • tissue culture substrates include various polymer plastics (e.g. , polypropylene, polystyrene, polycarbonate, and the like) and glass.
  • Each layer e.g.
  • each monolayer) of an ex vivo derived tissue disclosed herein often comprises at least 2 x 10 5 , at least 5 x 10 5 , at least 1 x 10 6 , at least 2.5 x 10 6 , at least 5 x 10 6 , or at least 10 x 10 6 of adipose-derived stem cells per cm 2 .
  • an ex vivo derived tissue disclosed herein comprises at least 1 x 10 6 , at least 2.5 x 10 6 , at least 5 x 10 6 , at least 10 x 10 6 , at least 50 x 10 6 , at least 100 x 10 6 , at least 250 x 10 6 , or at least 500 x 10 6 adipose- derived stem cells per cm2.
  • an ex vivo derived tissue disclosed herein comprises at least 1 x 10 6 , at least 2.5 x 10 6 , at least 5 x 10 6 , at least 10 x 10 6 , at least 50 x 10 6 , at least 100 x 10 6 , at least 250 x 10 6 , or at least 500 x 10 6 adipose-derived stem cells.
  • the ex vivo derived tissues disclosed herein comprise or consist of multiple monolayers of adipose-derived stem cells organized into a flexible horizontal plane comprising a vertical dimension substantially equivalent to the diameter of at least 2, at least 3, at least 4, at least 5 or at least 10 single adipose -derived stem cells.
  • the ex vivo derived tissues disclosed herein comprise or consist of multiple monolayers of adipose-derived stem cells organized into a flexible horizontal plane comprising a vertical dimension substantially equivalent to the diameter of 1 to 10, 2 to 20, 2 to 15, 2 to 10, 2 to 8 or 2 to 5 single adipose-derived stem cells.
  • the ex vivo derived tissues disclosed herein comprise or consist of 1 to 10, 2 to 20, 2 to 15, 2 to 10, 2 to 8 or 2 to 5 monolayers of adipose-derived stem cells organized into a flexible horizontal plane wherein each layer comprises a vertical dimension or thickness substantially equivalent to the diameter of a single adipose- derived stem cells.
  • the ex vivo derived tissues disclosed herein comprise or consist of 1 to 10, 2 to 20, 2 to 15, 2 to 10, 2 to 8 or 2 to 5 monolayers of adipose-derived stem cells organized into a flexible horizontal plane wherein each layer comprises a vertical dimension or thickness of about 0.8 to about 2 mm, about 0.9 mm to about 2 mm, about 0.9 mm to about 1.8 mm, about 0.9 to about 1.5 mm, or about 1.0 mm to about 1.5 mm.
  • the ex vivo derived tissues disclosed herein comprise or consist of 2 to 20, 2 to 15, 2 to 10, 1 to 10, 2 to 8 or 2 to 5 monolayers of adipose-derived stem cells organized into a flexible horizontal plane wherein each layer comprises a vertical dimension or thickness of about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.8 mm, about 1.9 mm or about 2.0 mm. Accordingly, in certain embodiments, each layer comprises a vertical dimension or thickness of about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.8 mm, about 1.9 mm or about 2.0 mm. Accordingly, in certain
  • the average, nominal or absolute thickness or vertical diameter of an ex vivo derived tissue disclosed herein is about 2 mm to about 40 mm, about 2 mm to about 20 mm, about 2 mm to about 15 mm, about 2 mm to about 10 mm, about 3 mm to about 10 mm, or about 3 mm to about 8 mm
  • the horizontal dimensions of an ex vivo derived tissue can be any suitable shape or size.
  • the horizontal dimensions of an ex vivo derived tissue disclosed herein is square, circular or rectangular.
  • the horizontal dimensions of an ex vivo derived tissue disclosed herein is at least 0.1 cm 2 , at least 0.5 cm 2 , at least 1 cm 2 , at least 2 cm 2 , at least 5 cm 2 , at least 10 cm 2 , at least 100 cm 2 , at least 200 cm 2 , at least 500 cm 2 , or at least 1,000 cm 2 .
  • the horizontal dimensions of an ex vivo derived tissue disclosed herein is about 1 cm 2 to about 1,000 cm 2 , about 1 cm 2 to about 100 cm 2 , about 1 cm 2 to about 10 cm 2 , or about 5 cm 2 to about 10 cm 2 .
  • the average, largest, nominal or absolute diameter of an ex vivo derived tissue is in the range of about 1 cm to about 5000 cm, about 5 cm to about 5000 cm, about 1 cm to about 1000 cm, about 5 cm to about 1000 cm, about 1 cm to about 500 cm, about 5 cm to about 500 cm, about 0.5 cm to about 200 cm, about 1 cm to about 200 cm, about 0.5 cm to about 50 cm, about 1 cm to about 50 cm, about 5 cm to about 50 cm, about 0.5 cm to about 10 cm, about 1 cm to about 10 cm, about 0.1 cm to about 5 cm, or about 0.1 cm to about 0.9 cm.
  • the average, largest, nominal or absolute diameter of an ex vivo derived tissue is about 1 cm, about 2 cm, about 4 cm, about 6 cm about 10 cm, about 20 cm, about 50 cm, about 100 cm, about 500 cm, about 1000 cm or larger.
  • the minimum diameter of an ex vivo derived tissue is about 0.1 cm, about 0.5 cm, about 1 cm, about 2 cm, about 4 cm, about 6 cm or about 10 cm.
  • the maximum diameter of an ex vivo derived tissue is about 10 cm, about 50 cm, about 100 cm, about 200 cm, about 500 cm, about 1000 cm or about 10,000 cm.
  • an ex vivo derived tissue disclosed herein comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% viable cells.
  • Cell viability can be determined by any suitable method.
  • an ex vivo derived tissue comprises undifferentiated adipose-derived stem cells.
  • undifferentiated adipose-derived stem cells express CD29, CD73 and/or CD105.
  • at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% of the undifferentiated adipose-derived stem cells of an ex vivo tissue disclosed herein express CD29, CD73 and/or CD 105.
  • undifferentiated adipose- derived stem cells do not express detectable amounts of CD45R and/or CD19.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, or 100% of the undifferentiated adipose-derived stem cells of an ex vivo tissue disclosed herein express CD29, CD73 and CD105, and do not express detectable levels of CD19 or CD45R.
  • an ex vivo derived tissue comprises, or consists essentially of,
  • an ex vivo derived tissue comprises adipose-derived stem cells that are differentiated into adipocytes.
  • Adipocytes often express Acetyl-CoAl, SREBP1 and PPARg.
  • an ex vivo derived tissue comprises, or consists essentially of, differentiated adipose-derived stem cells, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, or 100% of the cells of the tissue express Acetyl-CoAl, SREBP1 and PPARg.
  • an ex vivo derived tissue comprises adipose-derived stem cells that are differentiated into a cell-type selected from osteoblasts, cardiomyocytes, chondrogenic cells, neurons, hepatocytes, and pancreatic beta-cells.
  • an ex vivo derived tissue comprises, or consists essentially of,
  • an ex vivo derived tissue comprises, or consists essentially of, differentiated adipose -derived stem cells, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, or 100% of the cells of the tissue express osteocalcin.
  • an ex vivo derived tissue comprises, or consists essentially of,
  • an ex vivo derived tissue comprises, or consists essentially of, differentiated adipose-derived stem cells, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, or 100% of the cells of the tissue express aggrecan, SPARC and Collagen II.
  • adipose-derived stem cells of an ex vivo derived tissue disclosed herein express E-Cadherin. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% of adipose-derived stem cells of an ex vivo tissue disclosed herein express E-Cadherin.
  • the ex vivo derived tissues disclosed herein do not include a substrate. In certain embodiments, the ex vivo derived tissues disclosed herein do not include a scaffold. Scaffolds that are associated with ex vivo derived tissues can trigger unwanted immunoreactions when grafted into a mammalian subject.
  • a scaffold refers to an organic bio-material provided to cells or cell monolayers to promote the formation of a tissue, where the scaffold provides a support that often enhances the stability and strength of cell-to-cell contacts within the resulting tissue and where the tissue and scaffold are intended to be grafted into a living subject.
  • Non-limiting examples of scaffolds include collagen, silk, alginate, gel foam, hydrogel, fibrin, fibrin poly (ester-urethane), various polymers (e.g. , polyethylene glycol and poly(lactic-co- glycolic acid) (PLGA)), ceramic, hyaluronan or other natural or synthetic biomaterials.
  • Hi-density plastics such are polycarbonates, polyethylene, polypropylene, glass products and other supports that are traditionally used to culture cells in vitro are not scaffolds as defined herein.
  • the term "scaffold” as used herein excludes any endogenous biomaterial that is expressed on the surface of, or secreted from an ex vivo derived tissue disclosed herein.
  • an ex vivo derived tissue disclosed herein does not comprise a cell feeder layer or cell seeder layer.
  • the ex vivo derived tissues disclosed herein consist essentially of undifferentiated ADSCs and/or differentiated ADSCs and therefore do not include scaffolds, feeder cells, or seeder cells.
  • an ex vivo derived tissue consisting essentially of undifferentiated ADSCs and/or differentiated ADSCs may include organic materials that are expressed by or secreted by the ADSCs of the ex vivo derived tissue.
  • disclosed herein is a method of making an ex vivo derived tissue comprising adipose-derived stem cells.
  • a method of making an ex vivo derived tissue comprising undifferentiated adipose-derived stem cells comprises seeding a single-cell suspension of undifferentiated adipose-derived stem cells onto a tissue culture substrate and allowing the cells to grow from about 7 to 15 days, 8 to 12 days, or about 10 to 11 days.
  • a method of making an ex vivo derived tissue comprising undifferentiated adipose-derived stem cells comprises seeding a single-cell suspension of undifferentiated adipose-derived stem cells onto a tissue culture substrate and allowing the cells to grow from at least 5 days, at least 8 days, at least 10 days or at least 12 days.
  • a single-cell suspension of undifferentiated adipose-derived stem cells can be obtained from the adipose tissue of a suitable mammal, for example a human. Any growth media suitable for growing stem cells can be used.
  • undifferentiated adipose-derived stem cells and/or an ex vivo derived tissue comprising adipose-derived stem cells is grown in MESEN-PRO RSTM Medium (Life Technology, Waltham, MA).
  • An ex vivo derived tissue comprising undifferentiated adipose-derived stem cells can be harvested after at least 5 days, after at least 6 days, after at least 7 days or after at least 8 days of seeding a single-cell suspension of undifferentiated adipose-derived cells onto a suitable tissue culture substrate.
  • undifferentiated adipose-derived stem cells of an ex vivo derived tissue are induced to differentiate into a suitable cell type thereby providing an ex vivo derived tissue comprising differentiated adipose-derived stem cells.
  • a monolayer of undifferentiated adipose- derived stem cells, or a multilayer ex vivo derived tissue comprising undifferentiated adipose-derived stem cells is induced to differentiate into a suitable cell type, which monolayer or tissue is then grown into an ex vivo derived tissue comprising, or consisting essentially of, differentiated cells. Any suitable media for inducing differentiation can be used.
  • undifferentiated cells or ex vivo derived tissue comprising undifferentiated cells can be treated with STEMPRO® Adipogenesis Differentiation Kit (Gibco, Life Technology, Waltham, MA).
  • STEMPRO® Adipogenesis Differentiation Kit Gibco, Life Technology, Waltham, MA.
  • an ex vivo derived tissue comprising, consisting of, or consisting essentially of
  • undifferentiated stem cells is treated with a media configured to induce differentiation of undifferentiated cells into adipocytes, where the treatment is for 8 to 30 days, 8 to 20 days, 10 to 20 days or for about 19 or 20 days.
  • an ex vivo derived tissue comprising, consisting of, or consisting essentially of undifferentiated stem cells is contacted with a media configured to induce differentiation of undifferentiated stem cells into adipocytes.
  • the resulting differentiated ex vivo derived tissue is harvested about 8 to 30 days, 8 to 20 days, 10 to 20 days or about 19 or 20 days later, where the harvested, differentiated, ex vivo derived tissue comprises, consists of, or consists essentially of adipocytes.
  • an ex vivo derived tissue comprising, consisting of, or consisting essentially of undifferentiated stem cells is treated with a media configured to induce differentiation of undifferentiated cells into chondrocytes, where the treatment is for 9 to 30 days, 10 to 25 days, 15 to 25 days or for about 15 to 23 days.
  • an ex vivo derived tissue comprising, consisting of, or consisting essentially of undifferentiated stem cells is contacted with a media configured to induce differentiation of undifferentiated stem cells into chondrocytes.
  • the resulting differentiated ex vivo derived tissue is harvested about 9 to 30 days, 10 to 25 days, 15 to 25 days or about 15 to 23 days later, where the harvested, differentiated, ex vivo derived tissue comprises, consists of, or consists essentially of chondrocytes.
  • a STEMPRO® for differentiating adipose-derived stem cells into chondrocytes.
  • Chondrogenesis Differentiation Kit (Gibco, Life Technology) can be used.
  • an ex vivo derived tissue comprising, consisting of, or consisting essentially of undifferentiated stem cells is treated with a media configured to induce differentiation of undifferentiated cells into osteoblasts, where the treatment is for 20 to 45 days, 20 to 40 days, 25 to 35 days or for about 28 to 30 days.
  • an ex vivo derived tissue comprising, consisting of, or consisting essentially of undifferentiated stem cells is contacted with a media configured to induce differentiation of undifferentiated stem cells into osteoblasts.
  • the resulting differentiated ex vivo derived tissue is harvested about 20 to 45 days, 20 to 40 days, 25 to 35 days or about 28 to 30 days later, where the harvested, differentiated, ex vivo derived tissue comprises, consists of, or consists essentially of osteoblasts.
  • a STEMPRO® Osteogenesis Differentiation Kit for differentiating adipose-derived stem cells into osteoblasts, a STEMPRO® Osteogenesis Differentiation Kit (Gibco, Life Technology) can be used.
  • a method of repairing tissue damage in a subject refers to a mammalian animal. Any suitable mammal can be treated by a method described herein.
  • mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g. , mouse, rat, rabbit, guinea pig).
  • a mammal is a human.
  • a mammal can be any age or at any stage of development (e.g. , an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a mammal can be an animal disease model.
  • a subject is human in need of a treatment disclosed herein.
  • a subject is a human having organ damage or tissue damage.
  • a method of repairing tissue damage in a subject comprises administering an ex vivo derived tissue to a damaged tissue of a subject in need thereof. Any suitable tissue that is damaged can be treated by a method disclosed herein.
  • a damaged tissue can be cardiac tissue, bone tissue, skin tissue, neuro tissue, liver tissue, kidney tissue, brain tissue, stomach tissue or pancreatic tissue, for example.
  • a method of repairing tissue damage comprising directly contacting the damaged tissue with an ex vivo derived tissue disclosed herein.
  • An ex vivo derived tissue may be used in a variety of applications including, but not limited to, promoting repair of and/or regeneration of damaged cardiac muscle, promoting repair and healing during cardiac surgery (e.g. by-pass surgery or heart valve replacement), and promoting healing and repair of damaged smooth muscle, cardiac muscle, skeletal muscle, connective tissue, bone, skin or brain tissue.
  • an ex vivo derived tissue disclosed herein is directly contacted with or adhered to a damaged tissue.
  • an ex vivo derived tissue is placed and/or adhered directly onto a damaged tissue.
  • an ex vivo derived tissue adheres to a damaged tissue by natural cellular attachment.
  • an ex vivo derived tissue is adhered to a damaged tissue.
  • Non-limiting means for direct adherence between an ex vivo derived tissue and a damaged tissue includes a biological glue, a surgical glue (e.g., a fibrin glue or 2-octyl cyanoacrylate ("DERMABOND", Ethicon, Inc., Somerville, N.J.)), a synthetic glue, a laser and/or a laser dye, a hydrogel, a polysaccharide, a hemostatic agent and/or a sealant.
  • a biological glue e.g., a fibrin glue or 2-octyl cyanoacrylate (“DERMABOND”, Ethicon, Inc., Somerville, N.J.)
  • DERMABOND 2-octyl cyanoacrylate
  • synthetic glue e.g., a laser and/or a laser dye, a hydrogel, a polysaccharide, a hemostatic agent and/or a sealant.
  • hemostatic agents and sealants include “SURGICAL” (oxidized cellulose), “ACTIFOAM” (collagen), “FIBRX” (light-activated fibrin sealant), “BOHEAL” (fibrin sealant), “FIBROCAPS” (dry powder fibrin sealant), polysaccharide polymers p-GlcNAc (“SYVEC” patch; Marine Polymer Technologies), Polymer 27CK (Protein Polymer Tech.). Medical devices and apparatus for preparing autologous fibrin sealants from 120 ml of a patient's blood in the operating room in one and one-half hour are also known (e.g., Vivostat System).
  • a method of attaching an ex vivo derived tissue to a damaged tissue includes use of a suitable sutures, non-limiting examples of which include 5-0, 6-0 and 7-0 proline sutures (Ethicon Cat. Nos. 8713H, 8714H and 8701H), poliglecaprone, polydioxanone, polyglactin or other suitable non-biodegradable or biodegradable suture material.
  • a suitable sutures non-limiting examples of which include 5-0, 6-0 and 7-0 proline sutures (Ethicon Cat. Nos. 8713H, 8714H and 8701H), poliglecaprone, polydioxanone, polyglactin or other suitable non-biodegradable or biodegradable suture material.
  • damage of bone and cartilage of articulation limbs can be repaired using ex vivo derived tissue that are differentiated into chondrocytes and osteoblasts.
  • ex vivo derived tissue that are differentiated into chondrocytes and osteoblasts.
  • hADSC ex vivo derived tissues that are differentiated into chondrocytes can be placed on the top of hADSC ex vivo derived tissues differentiated into osteoblasts before grafting on a damaged articulation area.
  • Skin wounds e.g. , in diabetic patients
  • Human ADSC ex vivo derived tissues will decrease local inflammation caused by scaffolds, frameworks and artificial supports. Differentiated human ADSC ex vivo derived tissues can also induce healing by releasing factors involved in tissue repair.
  • Human ADSC cells were purchased from Life Technology (Life Technology, Waltham, MA). The cells were cultured and passaged following the manufacturer (Life Technology, Waltham, MA), using the culture media MesenPRO RSTM Medium (Life Technology, Waltham, MA). Human ADSC were seeded at 2.2xl0 4 cells/cm 2 , for amplification. Human ADSCs at passage 3 were used for the differentiation and harvesting protocol (Scheme 1).
  • Human ADSCs were seeded in 35 mm dishes (Corning, Inc. Corning, NY), at 2.2xl0 4 cells/cm 2 . Human ADSC differentiation was done following the instruction of manufacturer (Life Technology, Waltham, MA). Adipogenesis treatment on hADSC started when the hADSC reached confluence, 2 days after initial seeding, and when hADSC are forming a multilayer ex vivo derived tissue, 10 days after initial seeding (Scheme 1). We used the StemPro® Adipogenesis Differentiation Kit, StemPro® Osteogenesis Differentiation Kit and StemProTM Chondrogenesis Differentiation Kit (Life Technology, Waltham, MA).
  • hADSC already a multilayer ex vivo derived tissue, harvested from the 35 mm dish (Fig. 1, Scheme 1).
  • Adipocyte hADSC multilayer ex vivo derived tissue was harvested 19 days after the initial seeding.
  • Osteogenic hADSC multilayer ex vivo derived tissue was harvested 30 days after the initial seeding.
  • Chondrocyte hADSC multilayer ex vivo derived tissue was harvested 15-23 days after the initial seeding.
  • Multilayer ex vivo derived tissues were harvested using PVDF membrane and forceps.
  • PVDF membranes were cut in a donut shape (PVDF, Hydrophilic, 5.0 ⁇ , 47 mm, white, plain, 100) (Millipore, Burlington, MA) with the following size: 16 mm outer diameter and 9 mm inner diameter.
  • the culture media was removed, and the cell sheets were washed with phosphate-buffered saline (PBS). The PBS was removed and replaced by fresh PBS. Forceps were used to detach the edges of the cells tissue sheets and wrapped on the PVDF membrane placed in the center of the cell sheet.
  • PBS phosphate-buffered saline
  • the whole PVDF/cell sheet was lifted with the forceps and placed in another 60mm cell culture dish (Corning, Inc. Corning, NY), containing PBS.
  • the cell tissue sheets were unwrapped, and they were fixed in 10% Neutral Buffered Formalin for Immunohistochemistry staining.
  • Engineered ex vivo derived tissue were fixed in 10% neutral buffered formalin, embedded in paraffin and the tissue sections were then stained with H&E or used for immunofluore scent staining using CD19, CD73, AcetylCoAl (NovusBio, Littleton, CO), CD29, HLA-A, HLA-DR (Abeam, Cambridge, MA), CD45R, CD105, Osteocalcin, SREBP1 (ThermoFisher Scientific, Carlsbad, CA), E-Cadherin (BD
  • Alexa Fluor® 488 donkey anti-mouse conjugated second antibodies Alexa Fluor® 488 donkey anti-rabbit conjugated second antibodies
  • Alexa Fluor® 488 donkey anti-rat conjugated second antibodies were used.
  • Propidium Iodine Invitrogen, Eugene, OR was used to stain nuclear DNA.
  • a Nikon 400 fluorescent microscope was used to analyze the slides (Nikon Inc., Melville, NY).
  • H&E staining showed that the undifferentiated hADSC multilayer ex vivo derived tissue formed multiple layers of cells (Fig 2D).
  • Fig 2D the formation of the multilayer ex vivo derived tissue didn't affect the expression of hADSC markers: CD19-, CD45R-, CD29+, CD73+, CD105+ (Fig 2E).
  • Undifferentiated monolayer and multilayer hADSC ex vivo derived tissue were treated with adipocyte, osteogenic and chondrocyte culture media, provided by the manufacturer, following the timing described in scheme 1.
  • hADSC monolayer Two days after reaching confluence, hADSC monolayer were treated with adipocyte culture media, and 10 days later, monolayer hADSC adipocyte ex vivo derived tissues were stained with oil red (Fig 3A). Red oil staining showed the presence of adipocyte all over the hADSC monolayer cell sheet.
  • Undifferentiated multilayer hADSC ex vivo derived tissues started to differentiate into adipocyte, 10 days after starting the cell culture (Fig. 1, Scheme 1).
  • adipocyte multilayer hADSC ex vivo derived tissues were harvested (Fig 3C) or stained with red oil to identify the adipocyte (total of 20 days in culture) (Fig 4B), compared to the adipocyte monolayer cell sheets (Fig 4A). Oil red staining showed higher number of adipocyte in the multilayer compared to the adipocyte monolayer, confirming the observation in the cell culture (Fig 4A-C). This might be due to the higher number of hADSC in the cell sheet; however, in Figure 4C, we noticed that hADSC multilayer ex vivo derived tissues differentiate much faster and in larger numbers than monolayer of hADSC, in only 24h of adipocyte treatment.
  • adipocyte hADSC ex vivo derived tissue multilayers confirming the differentiation of hADSC into adipocyte.
  • Undifferentiated multilayer hADSC could not have been differentiated into osteogenic multilayer hADSC ex vivo derived tissue, because hADSC cells detached and died during the osteogenic treatment (Data not shown).
  • osteogenic multilayer of hADSC ex vivo derived tissue could be engineered from a hADSC monolayer because during the osteogenic treatment, hADSC were still growing and increased the cells density.
  • Twenty -eight days after the beginning the osteogenic differentiation, osteogenic multilayer hADSC ex vivo derived tissue were harvested using a PVDF membrane and forceps (Fig 5C). H&E staining showed the morphology of the ex vivo derived tissue, formed by few layers of cells (Fig 5D). The identity of cells was confirmed by immunohistostaining with osteocalcin, a specific marker of the osteoblast (Fig 5E).
  • Multilayer chondrocyte ex vivo derived tissue was engineered when the hADSC monolayer cell sheet was treated with the chondrocyte differentiation culture media (StemPro Chondrogenesis
  • Aggrecan is a protein expressed mainly in immature chondrocyte, which was detected in low levels in the chondrocyte hADSC multilayer ex vivo derived tissue.
  • SPARC protein was strongly stained in the chondrocyte hADSC multilayer ex vivo derived tissue, indicating the secretion of SPARC by the osteoblast for the mineralization of the extracellular matrix.
  • HLA-A and HLA-DR were analyzed on the hADSC tissues.
  • ADSC expresses histocompatibility antigen HLA-A but not HLA-DR.
  • the formation of the multilayer hADSC and the culture conditions didn't affect the expression of HLA-A and HLA-DR.
  • HLA-A is still expressed in undifferentiated, adipocyte, osteogenic chondrocyte hADSC multilayer (Fig 7A-D), and that HLA-DR was not (Fig 6D-I). This results indicates that hADSC multilayer ex vivo derived tissue can be used for autologous or allograft on patients.
  • Multilayer hADSC ex vivo derived tissue was successfully engineered, which can overcome problems of ectopic migration of ADSCs, and can improve the efficacy of grafting treatments.
  • Another advantage of the multilayer ex vivo derived tissue disclosed herein is that these ex vivo derived tissues can differentiate much faster than an undifferentiated monolayer of adipose derived stem cells. This positive result provides for shorter treatment times compared to monolayer sheets.
  • the multilayer tissues described herein provide for more efficient delivery of millions of ADSCs directly to a damaged organ or tissue.
  • Previously described methods of generating multilayer tissues requires the use of expensive biologically derived supports, or frameworks, synthetic scaffolds, seeder layers of cells and/or the use of expensive coating reagents.
  • the ex vivo generated tissues disclosed herein can be generated within days, without the use of expensive scaffolds, frameworks or reagents and are therefore less expensive, and easier to make.
  • a human subject is presented with third degree burns of his chest and arms. Ex vivo derived tissues described herein are applied to the burned exposed area and are allowed time to repair and/or heal the damaged areas.
  • a human subject is presented with damage to cardiac tissue as a result of a massive heart attack.
  • ex vivo derived tissues described herein are applied to the burned exposed area and are allowed time to repair and/or heal the damaged areas.
  • a system having at least one of A, B, and C would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.).
  • a convention analogous to "at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g. , "a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.).

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Abstract

La présente invention concerne des tissus dérivés ex vivo générés à partir de cellules souches dérivées de tissu adipeux ainsi que des procédés de fabrication et d'utilisation de ceux-ci. La présente invention concerne en outre des procédés de réparation ou de cicatrisation de tissus ou d'organes endommagés et de traitement de certaines affections.
PCT/US2018/055265 2017-10-10 2018-10-10 Tissus dérivés ex vivo comprenant des multicouches de cellules souches dérivées de tissu adipeux et utilisations de ceux-ci WO2019075101A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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US20080226692A1 (en) * 2005-02-28 2008-09-18 Masato Sato Cultured Cell Sheet, Production Method Thereof, and Application Method Thereof
WO2015129902A1 (fr) * 2014-02-28 2015-09-03 国立大学法人金沢大学 Feuille de cellules souches dérivées de tissu adipeux, ayant un potentiel de différenciation osseuse et son procédé de production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080226692A1 (en) * 2005-02-28 2008-09-18 Masato Sato Cultured Cell Sheet, Production Method Thereof, and Application Method Thereof
WO2015129902A1 (fr) * 2014-02-28 2015-09-03 国立大学法人金沢大学 Feuille de cellules souches dérivées de tissu adipeux, ayant un potentiel de différenciation osseuse et son procédé de production

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