[go: up one dir, main page]

WO2018183653A1 - Procédé de génération et d'utilisation de cellules cd34 + dérivées de fibroblastes - Google Patents

Procédé de génération et d'utilisation de cellules cd34 + dérivées de fibroblastes Download PDF

Info

Publication number
WO2018183653A1
WO2018183653A1 PCT/US2018/025106 US2018025106W WO2018183653A1 WO 2018183653 A1 WO2018183653 A1 WO 2018183653A1 US 2018025106 W US2018025106 W US 2018025106W WO 2018183653 A1 WO2018183653 A1 WO 2018183653A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hemangioblast
population
fibroblasts
mammalian
Prior art date
Application number
PCT/US2018/025106
Other languages
English (en)
Inventor
Jalees Rehman
Asrar Malik
Original Assignee
The Board Of Trustees Of The University Of Illinois
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The University Of Illinois filed Critical The Board Of Trustees Of The University Of Illinois
Publication of WO2018183653A1 publication Critical patent/WO2018183653A1/fr

Links

Classifications

    • 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
    • A61K35/33—Fibroblasts
    • 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
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Terminally differentiated somatic cells have been used to generate induced pluripotent stem cells (iPSCs) via overexpression of a defined set of transcription factors.
  • the most widely used set of reprogramming factors, Oct4 (Octamer-binding transcription factor 4), Sox2 (SRY-Box 2), Klf4 (Kruppel Like Factor 4) and c-Myc (MYC Proto- Oncogene) collectively referred to as "OSKM, " was identified initially by screening 24 pre-selected factors in mouse embryonic fibroblasts (MEFs; Takahashi & Yamanaka (2006) Cell 126 ( 4 ) : 663-76 ) .
  • fibroblasts can be directly reprogrammed into hepatocyte-like cells, cardiomyocytes , neurons, endothelial cells (ECs) and erythroid progenitors. See, e.g., Capellera-Garcia, et al. (2016) Cell Rep. 15 ( 11 ): 2550-62.
  • One approach relies on de-differentiation in which Oct-4, KLF4, Sox2, and c-Myc are expressed to partially de-differentiate fibroblasts, followed by applying endothelial growth factor VEGF to induce terminal endothelial differentiation, thereby avoiding formation of iPSCs (Kurian, et al. (2013) Nat. Methods 10:77-83; Li, et al. (2013) Arterioscler. Thromb . Vase. Biol. 33:1366-75; Margariti, et al . (2012) Proc. Natl. Acad. Sci . USA 109:139793-8; US 2014/0162366).
  • the second approach relies on targeted over-expression of embryonic transcription factors known to regulate formation of endothelial cells.
  • neither approach has been shown to be coupled to the generation of erythroblasts , which are critical for oxygen transport into ischemic tissue.
  • This invention provides a method for producing endothelial cells by contacting a population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells with Soxl7 or Soxl8 and inducing endothelial cell differentiation, e.g., by culturing the population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells for about 10 days in endothelial cell growth medium supplemented with Bone Morphogenetic Protein 4 and Vascular Endothelial Growth Factor to produce endothelial cells.
  • the mammalian fibroblast-derived, hemangioblast-like CD34 + cells are produced by contacting mammalian fibroblasts with 0ct4, Klf4, Sox2 and c-Myc; culturing the mammalian fibroblasts under conditions to generate a population of hemangioblast-like CD34 + cells; and isolating the population of hemangioblast-like CD34 + cells from the mammalian fibroblasts.
  • This invention also provides a method of treating ischemia or regenerating tissue, which involves the steps of contacting a population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells with Soxl7 or Soxl8; and administering to a subject in need of treatment an effective amount of the population of mammalian fibroblast- derived, hemangioblast-like CD34 + cells.
  • the population of mammalian fibroblast- derived, hemangioblast-like CD34 + cells is produced by contacting mammalian fibroblasts, e.g., autologous mammalian fibroblasts, with 0ct4, Klf4, Sox2 and c-Myc; culturing the mammalian fibroblasts under conditions to generate a population of hemangioblast-like CD34 + cells; and isolating the population of hemangioblast-like CD34 + cells from the mammalian fibroblasts.
  • mammalian fibroblasts e.g., autologous mammalian fibroblasts, with 0ct4, Klf4, Sox2 and c-Myc.
  • This invention further provides a method for producing erythroblasts , which involves the steps of contacting a population of mammalian fibroblasts with an inhibitor of Soxl7 or Soxl8 expression; reprogramming the mammalian fibroblasts of (a) to produce a population of hemangioblast-like CD34 + cells; isolating the population of hemangioblast-like CD34 + cells from the mammalian fibroblasts; and inducing erythroblast differentiation, e.g., by culturing the population of fibroblast-derived, hemangioblast-like CD34 + cells for about 4 days in basal medium supplemented with Stem Cell Factor, Interleukin-3 , Erythropoietin, Insulin-like Growth Factor 1 and FMS-like tyrosine kinase 3 ligand.
  • the step of reprogramming the mammalian fibroblasts is achieved by contacting the mammalian fibroblasts with Oct4, Klf4, Sox2 and c-Myc; and culturing the mammalian fibroblasts under conditions to generate a population of hemangioblast-like CD34 + cells.
  • this invention provides a method of treating an anemia by contacting a population of mammalian fibroblasts, e.g., autologous mammalian fibroblasts, with an inhibitor of Soxl7 or Soxl8 expression; reprogramming the mammalian fibroblasts to produce a population of hemangioblast-like CD34 + cells; isolating the population of hemangioblast-like CD34 + cells from the mammalian fibroblasts; and administering to a subject with an anemia an effective amount of the population of fibroblast-derived, hemangioblast-like CD34 + cells thereby treating the subject's anemia.
  • mammalian fibroblasts e.g., autologous mammalian fibroblasts
  • an inhibitor of Soxl7 or Soxl8 expression e.g., Soxl7 or Soxl8 expression
  • the step of reprogramming the mammalian fibroblasts involves contacting the mammalian fibroblasts with 0ct4, Klf4, Sox2 and c-Myc; and culturing the mammalian fibroblasts under conditions to generate a population of hemangioblast-like CD34 + cells.
  • FIG. 1 provides a schematic of method for converting human dermal fibroblasts (Fib) into induced endothelial cells and induced erythroblasts.
  • FIG. 2 shows the permeability of iECs in response to thrombin.
  • Trans-endothelial electrical resistance TER
  • Thrombin 3.5 pg/ml;0.5 unit
  • FIG. 6 shows that overexpression of Soxl7 and Soxl8 increased endothelial induction, as represented by VE- cadherin, CD31 and Flkl expression, when compared to control GFP overexpression.
  • Data are presented as mean ⁇ SE. *P ⁇ 0.05 compared to control (scramble shRNA or GFP); # P ⁇ 0.05 compared to cells overexpressing Soxl8; S P ⁇ 0.05 compared to cells overexpressing Soxl7.
  • Methods for generating multipotent mesodermal progenitors from de-differentiated adult fibroblasts have now been developed to provide a means of forming new blood vessels along with hematopoietic cells including erythroblasts .
  • the methods involve reprogramming of human fibroblasts into endothelial cells and erythroblasts via the generation of intermediate hemangioblast-like cells.
  • the cells generated in this manner have the potential to oxygenate ischemic tissue.
  • fibroblasts were first partially de-differentiated to generate the multipotent CD34 + hemangioblast-like cells (as opposed to pluripotent stem cells) via expression of OKSM factors and then subsequently converted into functional endothelial cells, iECs, and erythroblasts/erythroblasts by modulating the expression of Soxl7 and/or Soxl8.
  • the generated iECs expressed the prototypic endothelial cell surface proteins, proliferated and formed tube-like structures, and responded normally to inflammatory stimuli by increasing expression of cell surface adhesion molecules.
  • Fibroblast-derived human iECs also crucially restored endothelial barrier function underscoring their reparative potential.
  • this invention provides methods for producing endothelial cells and/or erythroblasts by modulating the expression/level of Soxl7 and/or Soxl8 in mammalian fibroblast-derived, hemangioblast-like CD34 + cells.
  • SRY-related HMG-box (Sox) proteins are a family of DNA-binding transcription factors.
  • Sox family members Sox7, Soxl7, and Soxl8 are highly related and constitute the Sox subgroup F (SoxF) .
  • Soxl7 and Soxl8 participate in various developmental processes and biologic activities, such as vascular development (Matsui, et al . (2006) J. Cell Sci. 119(Pt. 17 ): 3513-26) .
  • Soxl7 also plays an important role in fetal hematopoiesis in the yolk sac and fetal liver, especially in the maintenance of fetal and neonatal hematopoietic stem cells (HSCs), but not adult HSCs (Kim, et al. (2007) Cell 130(3) : 70-83) .
  • HSCs fetal and neonatal hematopoietic stem cells
  • Overexpression of Soxl7 has also been shown to confer fetal HSC characteristics onto adult hematopoietic progenitors (He, et al . (2011) Genes Dev. 25 ( 15 ): 1613-27 ) .
  • Sox7 and Soxl8 are transiently expressed in hemangioblasts and hematopoietic precursors, respectively, at the onset of blood specification. Sustained expression of Sox7 and Soxl8, but not Soxl7, in early hematopoietic precursors from mouse embryonic stem cells and embryos enhances their proliferation while blocking their maturation (Gandillet, et al . (2009) Blood 114 (23) : 4813-22; Serrano, et al . (2010) Blood 115 ( 19 ): 3895- 98) .
  • Modulation of Soxl7 and/or Soxl8 expression includes increasing the expression/level of Soxl7 and/or Soxl8 and decreasing the expression/level of Soxl7 and/or Soxl8 protein.
  • Increases in Soxl7/Soxl8 expression/levels can be achieved by transfecting a cell with a gene encoding Soxl7/Soxl8 or transducing a cell with Soxl7/18 protein using nucleic acid and protein sequences well known in the art.
  • nucleic acid molecules encoding human Soxl7 and Soxl8 are readily available under GENBANK Accession Nos . NM_022454 and NM_018419, respectively.
  • protein sequences for human Soxl7 and Soxl8 are known in the art and available under GENBANK Accession Nos. NP_071899 and NP_060889, respectively.
  • Plasmids, inducible vectors, lentiviral vectors, adenoviral vectors, and AAV vectors designed to express the Soxl7 and Soxl8 proteins in mammalian cells are readily available from commercial sources such as GenScript, Vector BioLabs, and Addgene .
  • Expression Soxl7 and/or Soxl8 can occur transiently (e.g., using a non-integrative or episomal nucleic acid) or stably in a cell.
  • Episomal or non-integrative nucleic acid molecules are not part of the chromosomal DNA and replicate independently thereof. Thus, during "transient expression" the transfected gene is not transferred to the daughter cell during cell division.
  • a transfected gene Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell.
  • a selection advantage may be a resistance towards a certain toxin that is presented to the cell.
  • Transient increases in Soxl7 and/or Soxl8 levels can also be achieved by protein transduction.
  • Protein transduction refers to the internalization from the external environment of proteins into a cell. As such, when a cell is transduced with a protein, the protein transits the cell membrane so as to pass from the external environment of the cell into the cell, e.g., into the cytoplasm of the cell. Protein transduction can be achieved by fusing a protein of interest, e.g., Soxl7 or Soxl8, to a protein transduction domain (PTD), which facilitates transport of the protein of interest into the cell.
  • PTD protein transduction domain
  • PTDs may include short cationic peptides (e.g., 5 to 100 amino acids, or from 5 to 25 amino acids in length) that can bind to the cell surface through electrostatic attachment to the cell membrane and be taken up by the cell by membrane translocation (Kabouridis (2003) Trends Biotech.
  • a given transduction protein molecule may include a single PTD or multiple PTDs (e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer or larger multimer) or different PTDs can be conjugated to the protein of interest.
  • PTDs include, but are not limited to, PTDs derived from human immunodeficiency virus 1 (HIV-I) TAT (Ruben, et al . (1989) J. Virol. 63:1-8); the herpes virus tegument protein VP22 (Elliott & O'Hare (1997) Cell 88:223- 233) ; the homeotic protein of Drosophila melanogaster Antennapedia (Antp) protein (Penetratin PTD; Derossi, et al. (1996) J. Biol. Chem.
  • PTDs can also be used including, but not limited to, transportan (Pooga, et al . (1988) FASEB J. 12:67-77), MAP (Oehlke, et al. (1998) Biochim. Biophys. Acta. 1414:127-139), KALA ( yman, et al.
  • PTD peptides and variant PTDs also are provided in, for example, US 2005/0260756, US 2006/0178297, US 2006/0100134, US 2006/0222657, US 2007/0161595, US 2007/0129305, EP 1867661, WO 2000/062067, WO 2003/035892, WO 2007/097561 and WO 2007/053512.
  • Transduction proteins can be fabricated using any convenient protocol.
  • the PTD can be conjugated to a protein of interest using any convenient protocol, such as, for example, conjugation by recombinant means or by chemical coupling.
  • the linkage of the components in the conjugate can be by any convenient method, so long as the attachment of the linker moiety to the protein of interest does not substantially impede the desired activity of the protein of interest.
  • Linkers and linkages that are suitable for chemically linked conjugates include, but are not limited to, disulfide bonds, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups .
  • Decreases in Soxl7/Soxl8 expression/levels can be achieved using an inhibitor of Soxl7 or Soxl8 expression.
  • An "inhibitor of Soxl7 or Soxl8 expression” refers to a molecule the measurably decreases the mRNA or protein levels of Soxl7 or Soxl8 by at least 30%, 40%, 50% 60%, 70%, 80%, 90% or 100% as compared to a cell not contacted with said inhibitor.
  • Suitable inhibitors of Soxl7 or Soxl8 expression include inhibitory RNAs such as antisense, ribozymes, short interfering nucleic acid (siNA) , short interfering RNA (siRNA) , double-stranded RNA (dsRNA) , micro-RNA (miRNA) , and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against Soxl7 or Soxl8 nucleic acid sequences.
  • inhibitory RNAs such as antisense, ribozymes, short interfering nucleic acid (siNA) , short interfering RNA (siRNA) , double-stranded RNA (dsRNA) , micro-RNA (miRNA) , and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against Soxl7 or Soxl8 nucleic acid sequences.
  • miR-141 has been shown to downregulate the expression of So
  • siRNA molecules include the sequences 5 ' -GGACCGCACGGAAUUUGAA (SEQ ID NO: 5), 5' -GCAUGACUCCGGUGUGAAU (SEQ ID NO: 6) and 5'-
  • an shRNA or siRNA is used to inhibit the expression of Soxl7 or Soxl8.
  • a method for producing endothelial cells by contacting a population of mammalian fibroblast-derived, hemangioblast- like CD34 + cells with Soxl7 or Soxl8 and inducing endothelial cell differentiation.
  • this invention provides a method for producing erythroblasts by contacting a population of mammalian fibroblasts with an inhibitor of Soxl7 or Soxl8 expression; reprogramming said mammalian fibroblasts to produce a population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells; isolating the population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells from the mammalian fibroblasts; and inducing erythroblast differentiation .
  • hemangioblast-like refers to a cell that is phenotypically similar to a hemangioblast and is capable of differentiating into both a blood lineage cell, in particular a erythroblast, and a vascular endothelial cell.
  • a "population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells” refers to a population of hemangioblast-like CD34 + cells derived from mammalian fibroblasts. The term "derived from,” when referring to cells, indicates that the cells were obtained from the stated source at some point in time.
  • hemangioblast-like CD34 + cells are derived from mammalian fibroblasts by reprogramming the mammalian fibroblasts to produce a population of hemangioblast-like CD34 + cells.
  • reprogramming refers to the process of partially de-differentiating a terminally differentiated cell (i.e., a fibroblast) into a cell exhibiting multipotent characteristics, i.e., a cell that can develop into more than one cell type, but is more limited than pluripotent cells.
  • Fibroblasts refers to a type of cell in connective tissue that produces the extracellular matrix and collagen but do not produce bone, bone minerals, or cartilage. Fibroblasts are large, flat, elongated (spindle-shaped) cells and may be defined as having high expression of FSP- 1, collagen III, and/or collagen type 15. Fibroblasts of use in this invention can be readily isolated from the skin of a mammal, such as a human, non-human, primate, mouse, rat, rabbit, horse, pig, cow, dog, cat, or other companion animal. In certain embodiments the fibroblasts are adult human dermal fibroblasts.
  • Mammalian fibroblasts which are typically devoid of CD34 expression, can be reprogrammed or partially dedifferentiated to CD34 + cells using any suitable method described in the art.
  • mammalian fibroblast-derived, hemangioblast-like CD34 + cells are obtained by contacting mammalian fibroblasts with Oct4, Sox2, Klf4 and c-Myc (i.e., the OSKM quartet); culturing the mammalian fibroblasts under conditions to generate a population of hemangioblast-like CD34 + cells; and isolating the population of hemangioblast-like CD34 + cells from the mammalian fibroblasts.
  • the step of contacting mammalian fibroblasts with the OSKM quartet can carried out by conventional transfection or protein transduction approaches.
  • polycistronic lentiviral vectors have been used to deliver the OSKM quartet to somatic cells in a single lentiviral construct reducing the number of genomic insertions (Kim, et al . (2009) Stem Cells 27(3):543-9; Chang, et al. (2009) Stem Cells 27 (5) : 1042-9) .
  • OSKM reprogramming factors have been delivered with minimal or total absence of genetic modifications by using LoxP sites and Cre-induced excision and piggyBac transposon excision of integrated reprogramming vector sequences (Soldner, et al. (2009) Stem Cells 136 (5) : 964-77; Kaji, et al . (2009) Nature 458 (7239) : 771-5) , or an oriP/EBNAl-based episomal vector (Yu, et al. (20090 Science 324 (5928 ): 797-801) .
  • poly-arginine tagged Oct4, Sox2, Klf4 and c-Myc proteins have been found to readily enter cells and translocate into the nucleus (Zhou, et al . (2009) Cell Stem Cell 4(5): 381-4).
  • protein-induced pluripotent stem cells have been obtained from human newborn fibroblasts after several rounds of treatment with cell extracts of HEK293 cell lines expressing poly-arginine tagged OSKM genes (Kim, et al. (2009) Cell Stem Cell 4(6):472-6).
  • the mammalian fibroblasts are further contacted with miR302-367 , Lin28 and/or an shRNA against p53 to facilitate CD34 + generation. See, e.g. , Kurian, et al . (2013) Nature Methods 10:77-83.
  • the mammalian fibroblasts are cultured under conditions to generate a population of hemangioblast-like CD34 + cells.
  • the terms "culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,” etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. The term does not imply that all cells in the culture survive or grow or divide, as some may naturally senesce, etc.
  • Cells are typically cultured in media, which can be changed during the course of the culture.
  • the mammalian fibroblasts are cultured at a temperature typically at about 37 °C with a C0 2 level of typically around 5% in an appropriate media (including salts, buffer, and nutrients) for a time sufficient for OSKM quartet to de-differentiate the fibroblasts to hemangioblast-like CD34 + cells.
  • an appropriate media including salts, buffer, and nutrients
  • OSKM quartet including salts, buffer, and nutrients
  • Suitable basal media for culturing the fibroblasts include, but are not limited to, DMEM, F12, or a combination thereof, supplemented with, e.g., serum or a serum replacement, L-glutamine, ⁇ -mercaptoethanol , and nonessential amino acids.
  • CD34 is a cell surface glycoprotein
  • the presence of hemangioblast-like CD34 + cells i.e., cells with increased expression of CD34 compared to fibroblasts, can be readily isolated by conventional cell sorting techniques.
  • the population of hemangioblast-like CD34 + cells are isolated from the parental mammalian fibroblasts ⁇ i.e., CD34 ⁇ cells).
  • Cell sorting techniques of use include, for example, fluorescence-activated cell sorting (FACS) , magnet- activated cell sorting (MACS), and panning.
  • FACS fluorescence-activated cell sorting
  • MCS magnet- activated cell sorting
  • hemangioblast-like CD34 + cells are cultured in cell growth medium appropriate for endothelial cell growth ⁇ i.e., endothelial cell growth media) .
  • the endothelial cell growth medium includes endothelial cell growth factors such as BMP4 (Bone Morphogenetic Protein 4) and VEGF (Vascular Endothelial Growth Factor) .
  • BMP4 Bisphogenetic Protein 4
  • VEGF Vascular Endothelial Growth Factor
  • the course or duration of endothelial cell differentiation is typically in the range of 6 and 20 days, e.g., at least 6, 8, 10, 12 or 14 days and can optionally be carried out in the presence of collagen (e.g., the cells are grown on collagen plates) .
  • the hemangioblast-like CD34 + cells are cultured for about 10 days in endothelial cell growth medium.
  • the erythroblast growth medium includes a basal medium ⁇ e.g., IMDM and Ham's F12) supplemented with EPO, a key regulator in ex vivo erythropoiesis methodologies, along with other growth factors such as SCF (Stem Cell Factor) , IL-3
  • the course or duration of erythroblast differentiation is typically in the range of 3 and 7 days, e.g., at least 3, or 4 days and can optionally be carried out in the presence of collagen (e.g., the cells are grown on collagen plates) .
  • the hemangioblast-like CD34 + cells are cultured for about 4 days and erythroblasts are collected as a non-adherent cell fraction.
  • the methods of this invention are typically carried out with a plurality of hemangioblast-like CD34 + cells, to form a population of cells that, over the course of differentiation, includes increasing numbers of endothelial cells or erythroblasts.
  • the percentage of endothelial cells or erythroblasts in the population of cells increases to about 50-100% over the course of differentiation, e.g., more than 65, 75, 80, 85, 90, 95 or higher percentage endothelial cells or erythroblasts.
  • the endothelial cells or erythroblasts can be further separated from the population using the methods described herein and prepared for storage (e.g., in freezing media) or therapeutic application.
  • Endothelial cell surface markers that can be used for separation include VE-cadherin, endoglin (CD105), vWF, CD31, and Z01 (tight junction protein) , though negative selection can also be used (e.g., alpha-S A or other non-endothelial cell marker) .
  • endothelial cells can be assessed for expression of endothelial markers VE- cadherin, CD31, FLK1, and vWF.
  • erythroblasts can be assessed for expression of erythroid marker glycophorin CD235a, as well as RUNXl, PU.l, GATAl, human hemoglobin beta and hemoglobin gamma.
  • Standard methods known in the art may be used to determine the detectable expression, low expression or lack thereof of the various markers discussed herein.
  • Suitable methods include, but are not limited to, immunocytochemistry, immunoassays, flow cytometry, such as FACS, and polymerase chain reaction (PCR) , such as reverse transcription PCR (RT-PCR) .
  • Suitable immunoassays include, but are not limited to, western blot analysis, enzyme- linked immunoassays (ELISA) , enzyme-linked immunosorbent spot assays (ELISPOT assays) , enzyme multiplied immunoassay techniques, radioallergosorbent (RAST) tests, radioimmunoassays, radiobinding assays and immunofluorescence.
  • Western blot analysis, ELISAs and RT- PCR are all quantitative and can be used to measure the level of expression of the various markers if present.
  • Antibodies and fluorescently-labelled antibodies for all of the various markers discussed herein are commercially- available .
  • This invention also provides methods for treating a disease or condition or regenerating tissue in a subject wherein generation of endothelial cells and/or erythroblasts would provide a benefit.
  • diseases or conditions include, e.g., ischemia and anemia.
  • one aspect of the invention provides a method for treating ischemia or regenerating tissue by contacting a population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells with Soxl7 or Soxl8; and administering to a subject in need thereof an effective amount of the population of mammalian fibroblast-derived, hemangioblast-like CD34 + cells.
  • Representative ischemic diseases or conditions include, but are not limited to, limb ischemia, congestive heart failure, cardiac ischemia, kidney ischemia and ESRD, stroke, and ischemia of the eye.
  • Representative uses for tissue regeneration include bone regeneration, cardiac regeneration, vascular regeneration, and neural regeneration, as well as tissue engineering and tissue repair .
  • the invention provides a method for treating an anemia by contacting a population of mammalian fibroblasts with an inhibitor of Soxl7 or Soxl8 expression; reprogramming said mammalian fibroblasts to produce a population of hemangioblast-like CD34 + cells; isolating the population of hemangioblast-like CD34 + cells from the mammalian fibroblasts; and administering to a subject with an anemia an effective amount of the population of fibroblast-derived, hemangioblast-like CD34 + cells.
  • anemia associated with kidney disease e.g., end-stage renal disease
  • anemia of chronic disease e.g., anemia of cancer
  • chemotherapy-induced anemia e.g., iron deficiency anemia
  • anemia associated with sickle cell disease e.g., sickle cell disease, and the like.
  • the mammalian fibroblasts used in the methods of the invention are obtained from a donor source (allogenic) or are autologous mammalian fibroblasts, e.g., obtained via skin biopsy.
  • compositions described herein can be administered as a pharmaceutically or physiologically acceptable preparation or composition containing a physiologically acceptable carrier, excipient, or diluent, and administered to the tissues of the recipient subject, including humans and non-human animals.
  • Representative compositions can be prepared by resuspending the cells in a suitable liquid or solution such as sterile physiological saline or other physiologically acceptable injectable aqueous liquids.
  • suitable liquid or solution such as sterile physiological saline or other physiologically acceptable injectable aqueous liquids.
  • the amounts of the components to be used in such compositions can be routinely determined by those having skill in the art.
  • the cells or compositions thereof can be administered by placement of cell suspensions onto absorbent or adherent material, e.g., a collagen sponge matrix, and insertion of cell-containing material into or onto the site of interest.
  • the cells can be administered by parenteral routes of injection, including subcutaneous, intravenous, intramuscular, and intrasternal .
  • Other modes of administration include, but are not limited to, intranasal, intrathecal, intracutaneous, percutaneous, enteral, and sublingual.
  • administration of the cells can be mediated by endoscopic surgery.
  • the cells or compositions thereof is in sterile solution or suspension or can be resuspended in pharmaceutically- and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e., blood) of the recipient subject.
  • aqueous or oleaginous vehicles which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e., blood) of the recipient subject.
  • excipients suitable for use include water, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures .
  • the amounts or quantities, as well as the routes of administration used, are determined on an individual basis, and correspond to the amounts used in similar types of applications or indications known to those of skill in the art.
  • the cells or compositions thereof can be administered to body tissues, including liver, pancreas, lung, salivary gland, blood vessel, bone, skin, cartilage, tendon, ligament, brain, hair, kidney, muscle, cardiac muscle, nerve, skeletal muscle, joints, and limbs.
  • the number of cells in a cell suspension or composition and the mode of administration may vary depending on the site and condition being treated. As non- limiting examples, in accordance with the present invention, about 3.5xl0 7 to 3.0xl0 8 cells are injected to effect tissue repair. Consistent with the Examples disclosed herein, a skilled practitioner can modulate the amounts and methods of cells-based treatments according to requirements, limitations, and/or optimizations determined for each case.
  • Dishes (100 mm) were plated with Plat-A cells, one dish per plasmid, each encoding 0CT4, KLF4, SOX2, and c-MYC (i.e., OKSM, all obtained from Addgene) , as well as green fluorescent protein (GFP) as a control.
  • GFP green fluorescent protein
  • Fugene® HD transfection reagent (Roche) and 10 ⁇ g of the appropriate plasmid DNA were added to one 100 mm dish cultured with Plat-A cells. The virus-containing medium was collected and filtered from dishes of transfected Plat-A cells on Day 1 and Day 2 for titration.
  • the medium of human fibroblasts was replaced with virus-containing medium on Day 1 and Day 2. On Day 3 the medium was switched to DMEM/F12 (Life Technologies) , 20% KnockOutTM serum replacement (Life Technologies), 10 ng/ml basic fibroblast growth factor (bFGF; R&D Systems) , 1 rtiM GlutaMAXTM (L-alanyl-L-glutamine) , 0.1 mM non-essential amino acids, and 55 ⁇ ⁇ -mercaptoethanol with medium changes every day for 5 days to continue the de- differentiation phase.
  • the fibroblasts transduced by OKSM and maintained in this medium were referred to as "dedifferentiated fibroblasts" or "De-Diff-Fib . " The protocol is shown in FIG. 1. All cell lines were maintained in an incubator (37°C, 5% C0 2 ) .
  • Hemangioblast-Like Cells from De ⁇ differentiated Fibroblasts .
  • Hemangioblast-like cells were identified by the presence of CD34, a marker of human progenitors, including endothelial progenitor cells and hematopoietic progenitors (Sidney, et al . (2014) Stem Cells 32:1380-1389).
  • CD34 a marker of human progenitors, including endothelial progenitor cells and hematopoietic progenitors (Sidney, et al . (2014) Stem Cells 32:1380-1389).
  • Hemangioblast-like CD34 + cells generated during de-differentiation of fibroblasts were isolated with anti-CD34-conjugated magnetic beads (Miltenyi) according to manufacturer's instructions with slight modifications.
  • EGM-2 endothelial medium
  • EGM-2 endothelial medium
  • BMP4 bone morphogenetic protein 4
  • VEGF Vascular endothelial growth factor
  • FIG. 1 Primary human aortic endothelial cells or lung microvascular endothelial cells were purchased from Lonza and cultured in EGM-2 medium as human endothelial control cells. All cell lines were maintained in an incubator (37°C, 5% C0 2 ) .
  • SCF Stem Cell Factor
  • IL-3 Interleukin-3
  • EPO Erythropoietin
  • IGF-1 Insulinlike Growth Factor 1
  • FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 ligand (Flt3- L; PeproTech) (FIG. 1) .
  • FMS-like tyrosine kinase 3 ligand Flt3- L; PeproTech
  • FIG. 1 FMS-like tyrosine kinase 3 ligand
  • EPO is a key regulator in ex vivo erythropoiesis methodologies along with other growth factors such as SCF, IL-3, IGF-1, and Fit-3. See Singh, et al . (2014) Adv. Regen . Med. Article ID 426520. Unlike adherent endothelial cells, erythroblasts detached upon generation and were collected as non-adherent cell fraction at Day 4 for characterization.
  • iECs adult human endothelial cells
  • ECs adult human endothelial cells
  • Affymetrix® GeneChip® Human Transcriptome Array 2.0 The analysis was performed in the R environment for statistical computing using the Affy and Limma packages.
  • the 11 Affymetrix CEL files were pre- processed by Robust MultiChip Analysis.
  • the normalized intensity in each cell type was paired with the normalized intensity of the remaining samples and analyzed with the Limma package.
  • a linear model was fitted to the design matrix for the samples using Limma, and differentially expressed genes identified for each pairwise comparison using a moderated t statistic, which applies an empirical Bayes method to borrow information across genes.
  • MatrigelTM Plug in vivo Angiogenesis Assay To test the ability of iECs to form blood vessels, an in vivo MatrigelTM plug assay was performed. Immune-deficient NOD- SCID mice (NOD . Cq-Prkdcscid I12rgtmlWj 1/SzJ; Jackson Laboratory) were used to avoid the rejection of human iECs. Anesthesia was induced using a mixture of xylazine (Rompun® 2%, Bayer) at 10 mg/kg body weight and ketamine (Imalgene 1000®, Merial) at 100 mg/kg body weight in NaCl at 0.9% i.p.
  • hemangioblast-like cells Prior to injection, hemangioblast-like cells were harvested using 0.05% trypsin/EDTA (Invitrogen) . A total of lxlO 6 cells (either fibroblast-derived hemangioblast-like cells, control human fibroblasts, or control human primary ECs) were suspended in 250 ⁇ of cold MatrigelTM (MatrigelTM basement-membrane matrix from BD adjusted to 9.8 mg/ml with phosphate buffered saline (PBS) ) .
  • PBS phosphate buffered saline
  • mice were then injected subcutaneously in the abdomen of mice, carefully positioning the needle between the epidermis and the muscle layer. Seven days later, mice were sacrificed and the MatrigelTM plugs were removed by a wide excision of the skin, including the connective tissues.
  • Surgical anesthesia was maintained using 1% isoflurane delivered through a vaporizer with air connected in series to rodent ventilator.
  • a dose of buprenorphine sustained release (0.1 mg/kg s.c.) was administered pre-operatively .
  • left thoracotomy was performed by 1 cm careful incision along sternum and 1 mm to the left from a midline between the 2 nd and 4 th rib in layers (skin, pectoral muscles and ribs avoiding mammary arteries). Having the heart in view, the pericardium was removed to ligate the left main coronary artery with 8-0 Prolene® suture 1-2 mm below the ostium.
  • fibroblast-derived, hemangioblast-like CD34 + cells or control fibroblasts suspended in MatrigelTM were injected into the border zone of the infarct at 2-3 different areas using a gastight 1710 syringe (Hamilton).
  • the cells were pre-incubated with 2.5 pg/ml Dil stain (1,1* -Dioctadecyl-3 , 3 , 3 ' , 3 ' -
  • mice Tetramethylindocarbocyanine Perchlorate; Thermo Fisher Scientific) for 5 minutes.
  • the chest cavity was then closed in three layers: intercostal muscles-interruptive, pectoral muscles and skin-continuous with the 7-0 nylon sutures.
  • the thorax was drained with a PE-10 cannula inserted into incision upon closure to evacuate the chest cavity and reestablish proper intrapleural pressure.
  • mice were weaned from anesthesia and placed in the warming pad until ambulatory and then returned to the appropriate housing facility. The mouse surgeon was blinded to the nature of the injected cells.
  • Transthoracic Echocardiography Evaluation of regional cardiac function based on left ventricle (LV) thickness and morphology was performed by transthoracic echocardiography (ECG) . Mice were anesthetized by 3.5% isoflurane and placed a heated platform (VisualSonics , Inc.) where ECG (lead II), heart rate, respiratory waveform and respiratory rate were monitored and displayed on the ultrasound's CPU display. Then, three views were recorded with the sample volume in the proper modes (B-mode, M-mode, pulsed Doppler or tissue Doppler) : 1. The parasternal long axis [B-mode and/or M-mode]; 2.
  • FS Fractional shortening
  • EF ejection fraction
  • TaqMan® Gene Expression Assays from Applied Biosystems were used to detect human hemoglobin beta and human hemoglobin gamma with the primer/probe sets of hemoglobin beta (Hs00747223_gl) and hemoglobin gamma
  • the slides were permeabilized and blocked with 5% normal goat serum, 0.1% TritonTM X-100 in PBS for 1 hour at room temperature. The slides were then incubated with primary antibody at 4°C overnight. For immunofluorescence, fluorescent Alexa FluorTM 488 dye and Alexa FluorTM 568 dye (Invitrogen, Carlsbad, CA) labeled secondary antibodies were applied to identify the primary antibodies.
  • the slides were mounted in ProLong® Gold antifade reagent with DAPI (Invitrogen) . Florescence images were acquired using Zeiss ApoTome microscope with 20X, 40X and 60X objectives.
  • Confocal image acquisition was performed using a Zeiss LSM 780 laser scanning microscope (Carl Zeiss Jena) with 20X, 40X and 60X water objectives.
  • Primary antibodies used were mouse anti-human CD31 (DAKO, Denmark) , goat anti-VE-cadherin (Santa Cruz Biotech) , rat anti-mouse TER 119/Erythroid cells (BD Pharmingen) , mouse anti-human CD235a (Glycophorin A; R&D Systems) , mouse anti- human CD233 (Thermo Fisher Scientific) .
  • Isotype controls were Alexa FluorTM 647 mouse IgGlk (BD Biosciences), Alexa FluorTM 647 mouse IgGlk (BD Biosciences), PE mouse IgGlk (BD Pharmigen) , PE mouse IgG2bk (BD Biosciences), FITC mouse IgGlk (BD Pharmigen) and APC mouse IgG2ak (BD Biosciences).
  • Cell proliferation was assayed by flow cytometry using the APC BrdU flow cytometry kit (BD Biosciences) according to manufacturer's instructions.
  • Transendothellal Electrical Resistance Measurement Assessment of responses of iEC to inflammatory stimuli was performed by measuring thrombin-induced increase in endothelial permeability according to known methods (Tiruppathi, et al . (2000) Proc. Natl. Acad. Sci. USA 97:7440-7445).
  • Human iECs and control human ECs (5. OxIO cm 2 ) were cultured on gelatin-coated gold electrodes for 1 day. The smaller electrode and larger counter electrode were connected to a phase-sensitive lock- in amplifier to monitor the voltage.
  • a constant current of 1 ⁇ was supplied by a 1 V, 4000 Hz AC signal connected serially to a 1 ⁇ resistor between the smaller electrode and the larger counter electrode.
  • telomerase activity was determined using the telomeric repeat amplification protocol (TRAP) .
  • TRAP assay was performed using the TRAPeze® Telomerase Detection kit (Millipore) and a Cy5 fluorescently labeled TS primer according to established protocols (Herbert, et al . (2006) Nat. Protoc. 1:1583- 1590). Densitometry of telomerase-specific ladder and internal standard were quantified using ImageJ and used to calculate relative telomerase activity compared to an internal standard control.
  • Tube Formation Assay A cell suspension containing 0.3xl0 5 /well of either ECs (control) or iECs was placed on top of wells coated with MatrigelTM (10 mg/ml; BD MatrigelTM Basement Membrane Matrix) in a 24-well plate (Nunc) . Rearrangement of cells and formation of capillary structures were observed at 24 hours.
  • Soxl7 Plasmid Constructs r Retrovirus or Lentivirus Packaging, and Infection.
  • Small double-stranded hairpin siRNAs targeting Soxl7 were designed by BLOCK-iTTM RNAi Designer ( Invitrogen) , synthesized by Integrated DNA Technologies (IDT), and inserted into a pLL3.7 lentiviral vector.
  • the target sequences of Soxl7 siRNAs were Soxl7-l: 5' -GGACCGCACGGAATTTGAA (SEQ ID NO: 36); Soxl7-2: 5'- GCATGACTCCGGTGTGAAT (SEQ ID NO:37); Soxl7-3: 5'- CCGCGGTATATTACTGCAA (SEQ ID NO:38).
  • LipofectamineTM 2000 was used to transfect pLL3.7 lentiviral vector to HEK293T cells (seed 2.0xl0 6 /6-well dish 1 day prior) .
  • the medium containing lentiviruses was collected and filtered two days after transfection.
  • the iECs was incubated with the virus cocktail supplemented with 8 ⁇ g/ml polybrene for 6-8 hours.
  • Soxl8 siRNA lentivectors i023437a, i023437b, i023437c
  • scrambled siRNA GFP lentivectors LV015-G
  • Soxl8 siRNA lentivectors or scrambled siRNA GFP lentivectors was performed according to Applied Biological Materials' instructions.
  • full- length Soxl7 (pMXs-Soxl7) and Soxl8 (pMXs-Soxl8) plasmids were purchased from Addgene .
  • Fugene® HD transfection reagent (Roche) was used to transfect Plat-A cells ( 1.0xl0 6 /6-well dish) . The medium containing the retrovirus was collected and filtered two days after transfection.
  • iECs were incubated with a virus cocktail supplemented with 4 ⁇ g/ml polybrene for 6-8 hours .
  • MethCultTM H4034 Optimum with Recombinant Cytokines and EPO were purchased from Stemcell Technologies.
  • a colony forming assay was performed according to the manufacturer instructions to evaluate the colony-forming function of generated erythroblasts . Briefly, 3xl0 3 induced erythroblasts or fibroblasts were collected, washed and then mixed in 3x1300 ⁇ MethCultTM. The suspended cells were dispensed into petri dishes using a syringe and blunt-end needle. The dishes were then incubated for 14 days in a humidified incubator at 37 °C and 5% C0 2 . The erythroid colonies were evaluated and counted using an inverted microscope and gridded scoring dishes. The colonies were plucked for further assays.
  • OCT4, KLF4, SOX2, c-MYC were expressed in human fibroblasts for 2 days as described herein (FIG. 1) .
  • the medium was replaced with bFGF and other constituents for 5 days to generate CD34 + progenitor cells.
  • the fibroblast-derived cells generated by this protocol were referred to as "de-differentiated fibroblasts" or "De- Diff-Fib” or "hemangioblast-like cells”. Endothelial or erythroid growth factors were then applied to differentiate the De-Diff-Fib cells into endothelial cells or erythroblasts (FIG. 1) .
  • CD34 + cells were isolated from the de-differentiated fibroblast population using magnetic bead sorting, and the cells were exposed first to endothelial lineage induction medium composed of standard endothelial growth medium supplemented with endothelial differentiation factors VEGF and BMP-4 for 10 days.
  • endothelial lineage induction medium composed of standard endothelial growth medium supplemented with endothelial differentiation factors VEGF and BMP-4 for 10 days.
  • endothelial lineage induction medium composed of standard endothelial growth medium supplemented with endothelial differentiation factors VEGF and BMP-4 for 10 days.
  • endothelial lineage induction medium composed of standard endothelial growth medium supplemented with endothelial differentiation factors VEGF and BMP-4 for 10 days.
  • endothelial cell markers VE- cadherin, vWF, CD31 and Flkl progressively increased by about 15- to 40-fold.
  • iECs induced endothelial cells
  • VE-cadherin trans-interactions between cells stabilizes VE-cadherin at cell membrane and promotes formation of adherens junctions (Daneshjou, et al . (2015) J. Cell Biol. 208:23-32)
  • iECs were co-cultured with primary adult human ECs . Confocal imaging demonstrated that this co-culturing increased membrane localization of VE-cadherin in iECs such that iECs formed characteristic adherens junctions via homotypic interactions .
  • TER trans- endothelial electrical resistance
  • VCAMl adhesion molecules CD106
  • CD62E E-selectin
  • telomeres As the ability of ECs to proliferate in required for vascular regeneration, proliferation of iECs was assessed. This analysis demonstrated that 20-30% of iECs were labeled with BrdU, a value similar to proliferation of primary human ECs. As cell expansion induces cell senescence and results in cell cycle arrest due to continuous shortening of telomeres (Armanios (2013) J. Clin. Invest. 123:996- 1002; Blasco (2007) Nat. Chem. Biol. 3:640-649; Rando & Chang (2012) Cell 148:46-57), it was determined whether the generated iECs represented an aging cell population.
  • telomere activity was determined since it maintains telomere length and protects against premature senescence in embryonic and neonatal cells (Armanios (2013) J. Clin. Invest. 123:996-1002; Blasco (2007) Nat. Chem. Biol. 3:640- 649; Rando & Chang (2012) Cell 148:46-57). It was observed that telomerase activity of iECs, as assessed by telomeric repeat amplification protocol (TRAP) , was significantly greater as compared in adult fibroblasts or primary adult human ECs (FIG. 3) . Importantly, despite higher telomerase activity, iECs did not show significantly elevated expression of the pluripotency transcription factor 0CT4 or the proto-oncogene c- YC.
  • TRAP telomeric repeat amplification protocol
  • Fibroblast-derived, hemangioblast-like CD34 + progenitors were cultured in erythroblast induction medium for 4 days to assess generation of erythroblasts. Unlike iECs, which remained adherent to the substrate, emerging erythroblasts detached from the CD34 + cell layer. Hema 3TM staining Cytospin® preparations of induced erythroblasts showed varied morphology reflecting different stages of maturation from basophilic erythroblasts, polychromatic erythroblasts to mature erythroblasts.
  • Cytospin® preparations were stained for CD235a (GPA, Glycophorin A) and CD233 (band 3 of SLC4A1, solute carrier family 4 member 1) .
  • a few enucleated cells were also CD233 + , indicating transition to mature erythroblasts.
  • Flow cytometry quantification demonstrated that 4.2+0.8% of the cells undergoing erythroblast induction were CD235a + .
  • Erythroid colony-forming-cell (CFC) assay demonstrated formation of distinct erythroid colonies (about 35 colonies/10000 cells vs. 0 colonies for fibroblasts) .
  • gene expression analysis showed expression of human hemoglobin beta and hemoglobin gamma confirming the erythroid nature of the generated human cells (FIG. 4) .
  • Gene expression of hematopoietic transcriptional factors, RUNXl and Pu.l, as well as erythroblast transcription factor GATA1 Hewitt, et al. (2014) Curr. Opin. Hematol. 21:155-164; Takeuchi, et al. (2015) Proc. Natl. Acad. Sci.
  • vascular developmental genes were assessed in fibroblasts, hemangioblast-like cells, iECs, and primary adult ECs. Only Soxl7 and Soxl8 showed a pattern suggestive of their mechanistic role in endothelial fate transition. It was observed that the transcriptional regulator Soxl8 (a member of Sry-transcription factor family) was significantly up-regulated in hemangioblast- like cells, and even more so in iECs but was minimally expressed in the parent fibroblasts. Soxl7, another member of the Sry transcription factor family, showed similar upregulation in iECs.
  • the transcription factor ETV2 (also known as ER71), which when over-expressed induces fibroblast-to-endothelial transition (Han, et al . (2014) Circulation 130:1168-1178; Morita, et al . (2015) Proc . Natl. Acad. Sci. USA 112:160-165), was not significantly up-regulated during lineage conversion of hemangioblast- like cells to iECs.
  • Soxl7 and Soxl8 were required for conversion of hemangioblast-like cells to iECs.
  • Soxl7 and Soxl8 expression gradually increased during the 10 Day induction period. Immunoblot analysis confirmed upregulation of Soxl7 and Soxl8 in iECs.
  • lentiviral shRNAs were used to deplete Soxl7 or Soxl8 during the endothelial induction phase. Using multiple shRNA constructs, a Soxl7 shRNA and Soxl8 shRNA were identified that achieved significant Soxl7 depletion and Soxl8 depletion, respectively.
  • Soxl7 prevented differentiation of hemangioblast-like cells to iECs as demonstrated by suppression of endothelial genes (VE- cadherin, CD31 and Flkl), both at mRNA and protein levels when compared to control shRNA.
  • Depletion of Soxl8 also inhibited upregulation of VE-cadherin and CD31 but did not significantly affect Flkl levels.
  • Soxl7 and Soxl8 also function as key switches suppressing the transition of hemangioblast-like cells to erythroblasts. This analysis indicated that the expression of Soxl7/Soxl8 increased in hemangioblast-like cells when compared to the parent fibroblasts but progressively decreased during the induction of erythroblasts, consistent with the role of Soxl7/Soxl8 in favoring endothelial fate conversion at the hemangioblast-like cell bifurcation. To explore the regulatory role of Soxl7 and Soxl8 in the induction of erythroblasts, Soxl7 and Soxl8 were depleted by shRNA, respectively, in fibroblasts before de-differentiation.
  • Soxl7 or Soxl8 increased the expression of hematopoietic transcription factors RUNXl and Pu .1 as well as erythropoiesis-specific regulator GATA1 during erythroblast induction.
  • Flow cytometry also confirmed increased erythropoiesis upon Soxl7 depletion trending towards increased erythropoiesis.
  • Soxl7 or Soxl8 significantly increased the formation of colonies using the in vitro erythroid CFC assay (about 40-50 colonies/10000 cells for Soxl7 or Soxl8 shRNA vs.
  • the newly formed human blood vessels also contained both human erythroblasts and murine host erythroblasts, indicating communication between the mouse vasculature and newly formed vessels derived from iECs.
  • MatrigelTM plugs containing primary human ECs showed presence of erythroblasts derived from the host mouse and cells were negative for human CD235a, indicating that transplanted adult human ECs were incapable of giving rise to erythroblasts .
  • a mouse myocardial infarction (MI) model was used to assess the regenerative potential of adult human hemangioblast-like cells in which MatrigelTM suspension containing either hemangioblast-like cells or control fibroblasts were injected in the peri-infarct area immediately distal to coronary artery ligation. Masson trichrome staining of cross section of hearts on Day 14 post-MI showed reduced fibrosis in hearts injected with hemangioblast-like cells. Heart weight/body weight ratio was also decreased in mice receiving hemangioblast-like cells, indicating suppression of adverse myocardial remodeling after infarction.
  • Echocardiography demonstrated significantly improved cardiac contractility, as measured by ejection fraction and fractional shortening at 1 and 2 weeks post-MI in hearts receiving hemangioblast-like cells when compared to hearts which received control fibroblasts (Table 2).
  • Hemangioblast-like cell implantation also improved morphologic and hemodynamic indexes as assessed by echocardiography (Table 2) .
  • LVIDs LV internal diameter at end of systole
  • LVIDd LV internal diameter at end of distole
  • LVESV LV end systolic volume
  • LVEDV LV end diastolic volume
  • Fib fibroblast
  • HLC hemangioblast-like cells.
  • Immunofluorescence staining demonstrated that implantation of hemangioblast-like cells resulted in formation of human CD31 + blood vessels carrying human CD235a + erythroblasts , indicating that implanted cells contributed to vascular regeneration and human erythropoiesis .
  • Gene expression analysis of infarcted tissue implanted with CD34 + cells demonstrated expression of human CD31 and human VE-cadherin in contrast to the area implanted with control human fibroblasts.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Vascular Medicine (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés de reprogrammation de fibroblastes de mammifère en cellules progénitrices mésodermiques CD34+ de type hémangioblaste et de génération de cellules endothéliales et d'érythroblastes par modulation de l'expression de Sox17 ou Sox18, ainsi que des procédés d'utilisation des cellules pour former des vaisseaux sanguins et des érythroblastes.
PCT/US2018/025106 2017-03-29 2018-03-29 Procédé de génération et d'utilisation de cellules cd34 + dérivées de fibroblastes WO2018183653A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762478387P 2017-03-29 2017-03-29
US62/478,387 2017-03-29

Publications (1)

Publication Number Publication Date
WO2018183653A1 true WO2018183653A1 (fr) 2018-10-04

Family

ID=63676850

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/025106 WO2018183653A1 (fr) 2017-03-29 2018-03-29 Procédé de génération et d'utilisation de cellules cd34 + dérivées de fibroblastes

Country Status (1)

Country Link
WO (1) WO2018183653A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020146874A1 (fr) 2019-01-11 2020-07-16 Figene, Llc Cellules fibroblastes régénératrices
WO2021133790A1 (fr) * 2019-12-26 2021-07-01 Figene, Llc Prévention et traitement d'une insuffisance rénale par administration de fibroblastes et produits associés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140162366A1 (en) * 2012-08-31 2014-06-12 Salk Institute For Biological Studies Generation of vascular progenitor cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140162366A1 (en) * 2012-08-31 2014-06-12 Salk Institute For Biological Studies Generation of vascular progenitor cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AKSOY, I ET AL.: "Sox Transcription Factors Require Selective Interactions with Oct4 and Specific Transactivation Functions to Mediate Reprogramming", STEM CELLS, vol. 31, no. 12, December 2013 (2013-12-01), pages 2632 - 2646, XP055556430 *
KELLY, PF ET AL.: "Stem Cell Collection and Gene Transfer in Fanconi Anemia", MOLECULAR THERAPY, vol. 15, no. 1, January 2007 (2007-01-01), pages 211 - 219, XP055402484 *
NOBUHISA, I ET AL.: "Sox17-Mediated Maintenance of Fetal Intra-Aortic Hematopoietic Cell Clusters", MOLECULAR AND CELLULAR BIOLOGY, vol. 34, no. 11, 24 March 2014 (2014-03-24), pages 1976 - 1990, XP055516924 *
ZHANG, L ET AL.: "SOX17 R egulates conversion of numan Fibroblasts into Endothellal and Erythroblasts by Dedifferentiation Into CD 34+ Progenitor Cells", CIRCULATION, vol. 135, no. 25, 20 June 2017 (2017-06-20), pages 2505 - 2523, XP055556440 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020146874A1 (fr) 2019-01-11 2020-07-16 Figene, Llc Cellules fibroblastes régénératrices
JP2022517784A (ja) * 2019-01-11 2022-03-10 フィジーン、エルエルシー 線維芽細胞再生細胞
WO2021133790A1 (fr) * 2019-12-26 2021-07-01 Figene, Llc Prévention et traitement d'une insuffisance rénale par administration de fibroblastes et produits associés

Similar Documents

Publication Publication Date Title
EP3327118B1 (fr) Procédé d'induction de cellules endothéliales vasculaires
EP3117839B1 (fr) Composition pour induire la transdifférenciation directe de cellules somatiques en cellules progénitrices vasculaires, et son utilisation
Yu et al. Stimulation of somatic cell reprogramming by ERas-Akt-FoxO1 signaling axis
JP5846558B2 (ja) 多能性幹細胞から骨格筋前駆細胞への分化誘導方法
US20240002811A1 (en) Methods for Generation for Pluripotent and Multipotent Cells
CN104520422B (zh) 由人羊水来源的细胞生成功能性的和持久的内皮细胞
KR101861171B1 (ko) 투석된 혈청이 있는 심근세포 배지
US8815593B2 (en) Induction of human embryonic stem cell derived cardiac pacemaker or chamber-type cardiomyocytes by manipulation of neuregulin signaling
JP5751548B2 (ja) イヌｉＰＳ細胞及びその製造方法
CN105492597A (zh) 利用hmga2制备由非神经元细胞重编程的诱导神经干细胞的方法
JP2022101564A (ja) より未分化状態への細胞の誘導方法
CA3216719A1 (fr) Procedes de generation de cellules endotheliales corneennes matures
Piñeiro-Ramil et al. Immortalizing mesenchymal stromal cells from aged donors while keeping their essential features
CN110291190A (zh) 骨骼肌细胞及其诱导方法
WO2018183653A1 (fr) Procédé de génération et d'utilisation de cellules cd34 + dérivées de fibroblastes
JP2023502062A (ja) 細胞をリプログラミングするための方法
JP2013009742A (ja) 多能性幹細胞の増殖を抑制するための組成物及び方法
EP4310176A1 (fr) Péricyte ayant un gène de facteur de croissance fibroblastique basique (bfgf) introduit dans celui-ci
US20240316115A1 (en) Method for producing vascular endothelial growth factor (vegf)- highly expressing pericyte-like cell
US20200248141A1 (en) Composition and kit for differentiating cancer associated fibroblasts into macrophages, and method of using the same
Lou Tissue Engineered Cardiac Muscle Patches with Human Pluripotent Stem Cells Enhance the Repairing Efficacy of Infarcted Cardiac Muscle in Mouse Model
Lou Human Pluripotent Stem Cells and Patches Enhance the Repairing Efficacy of Infarcted Cardiac Muscle in Mouse Model
Cavaco Understanding the mechanisms at the onset of LAMA2-congenital muscular dystrophy
Cannata et al. Efficiently Promote Myogenesis in Induced Pluripotent Stem Cells
Nefele et al. MICAL2 is essential for myogenic lineage commitment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18776602

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18776602

Country of ref document: EP

Kind code of ref document: A1