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WO2019113139A1 - Système de culture d'éosinophiles faisant appel à des cellules souches pluripotentes induites humaines - Google Patents

Système de culture d'éosinophiles faisant appel à des cellules souches pluripotentes induites humaines Download PDF

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Publication number
WO2019113139A1
WO2019113139A1 PCT/US2018/063953 US2018063953W WO2019113139A1 WO 2019113139 A1 WO2019113139 A1 WO 2019113139A1 US 2018063953 W US2018063953 W US 2018063953W WO 2019113139 A1 WO2019113139 A1 WO 2019113139A1
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days
monolayer
ipsc
media
cells
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Patricia C. FULKERSON
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Cincinnati Childrens Hospital Medical Center
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Cincinnati Childrens Hospital Medical Center
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    • 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
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2305—Interleukin-5 (IL-5)
    • 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
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • Eosinophils are produced in the bone marrow from CD34-expressing eosinophil lineage-committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. Differentiation of EoPs to mature eosinophils is a multistage process requiring the coordinated activity of multiple regulators. However, the developmental program that orchestrates eosinophil maturation is not well delineated. Studies focused on EoP maturation are inherently difficult to perform due to the rarity of EoPs in the bone marrow under homeostatic conditions; they comprise -0.05-0.1% of total murine bone marrow cells.
  • eosinophil culture systems that contain myeloid cell forming complexes (MCFCs).
  • the MCFCs may be used to shed MCFC derived cells, including, for example, myeloid progenitor cells, myeloid precursor cells, and combinations thereof.
  • the disclosed MCFC derived cells may be differentiated into eosinophils.
  • methods of deriving eosinophils from an iPSC cell BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG 1 depicts an eosinophil differentiation schematic in human iPSC cultures.
  • Induced stem cells (iPSCs) cultured in feeder-free conditions are initially induced to generate spherical embryoid bodies (EBs).
  • the EBs are culture under conditions to form endothelial- like stromal cells and then myeloid cell-forming complexes that contain CD45+CD34+ hematopoietic progenitor cells.
  • the CD34+ progenitors are subjected to prolonged IL-3 and IL-5 exposure to continuously generate terminally differentiated CDllb+Siglec-8+ eosinophils for 5-6 weeks.
  • FIGS 2A-2B depict hematopoietic progenitors generated from human iPSCs.
  • FIG 2B depicts cytospins of sorted CD45+CD34+ cells consistent with hematopoietic progenitors.
  • FIGS 3A and 3B Eosinophils generated from human iPSCs.
  • FIG 3A depicts quantities by week of harvest of CD45+Siglec-8+ eosinophils harvested from the
  • FIG 4 depicts eosinophils generated from three unique human iPSC lines. Representative dot blots showing Siglec-8 surface staining of CD45+ cells generated from iPSCs cultured for hematopoietic and eosinophil differentiation in IL-3 and IL-5 are shown. Each dot blot represents a unique iPSC line. Percentage of gated, live CD45+ cells is shown in the Siglec-8 quadrant. DETAILED DESCRIPTION
  • the term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.
  • “about” may mean within 1 or more than 1 standard deviation, per the practice in the art.
  • “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.
  • the term may mean within an order of magnitude, for example, within 5-fold, or within 2-fold, of a value.
  • Applicant has developed an embryoid body-based culture system that yields mature eosinophils starting from induced pluripotent stem cells, which is believed to be useful as a model system to identify critical regulators of eosinophil development, among other uses which will be understood in view of the instant disclosure.
  • Eosinophils derived from iPS cells via an embryoid body (EB)-based culture system has not been described.
  • iPSC induced pluripotent stem cell
  • the disclosed culture contains multiple cell types (stromal cells as well as hematopoietic cells) which results in improved differentiation compared to existing differentiation protocols.
  • This culture system is adapted from a hematopoietic differentiation protocol designed to generate neutrophils and monocytes on a large scale (Lachmann N, Stem Cell Reports. 2015).
  • the method of embryoid body (EB) formation and the sequential addition of particular cytokines, IL-3 and then IL-5, to specifically expand the eosinophil lineage are modified for the disclosed culture system.
  • hematopoietic differentiation of iPSCs takes approximately 4 weeks in culture (FIG 1).
  • spherical embryoid bodies may be generated from iPSC colonies, manually transferred onto tissue culture plates and cultivated in media to promote hematopoietic differentiation.
  • Myeloid cell— forming complexes may be formed and are supported in the culture by endothelial-like stromal cells.
  • MCFCs continuously shed myeloid progenitor and precursor cells for about 4-6 weeks, and these cells further differentiate into eosinophils.
  • MCFCs cultured in IL-3 or IL-3 and IL-5 shed CD45 + CD34 + hematopoietic progenitors that may be recoverable during the first three weeks of the culture (FIG 2A, highest production weeks 2-3).
  • IL-5 resulted in an increased number of hematopoietic progenitors that are capable of producing eosinophil colonies in semisolid medium (data not shown).
  • the cell morphology of sorted CD45 + CD34 + cells is consistent with hematopoietic progenitors (FIG 2B).
  • Hematopoietic differentiation in the presence of IL-3 or IL-3 and IL-5 resulted in the continuous production of CDl lb + Siglec-8 + cells over 5 weeks (FIG 3A, highest production weeks 3-5).
  • the cell morphology of the Sigelc-8 + cells was consistent with eosinophil peroxidase— containing mature eosinophils (FIG 3B).
  • Applicant has found that the generation of terminally differentiated eosinophils has been successful from multiple iPSC lines (FIG 4).
  • eosinophil culture systems that comprising myeloid cell forming complexes (MCFCs) capable of shedding MCFC derived cells.
  • the MCFC derived cells may include, for example, myeloid progenitor cells, myeloid precursor cells, and combinations thereof.
  • the MCFC derived cells may be, in certain aspects, capable of differentiating into eosinophils.
  • the MCFCs may be derived from embryoid bodies, wherein the embryoid bodies are derived from induced pluripotent stem cells (iPSCs).
  • the iPSCs may be derived from an individual having an eosinophil-related disorder, or an individual known to have a genotype associated with an eosinophil-related disorder.
  • the method may comprise the steps of a. culturing an iPSC on a semi-solid medium for a period of time sufficient to form an iPSC monolayer;
  • EB spherical embryoid body
  • the semi-solid medium may comprise a basement membrane matrix.
  • the iPSC cells of step a may be grown to confluence, for example, a confluence of about 70% to about 80%, prior to addition of the EB-media.
  • step a may be carried out for a period of time of from about 24 hours to about 150 hours, or from about 48 hours to about 120 hours, or about 72 to about 96 hours.
  • step a may be carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days.
  • the semi-solid medium containing iPSC cells of step a may be mechanically disrupted to disturb the monolayer into fragments, for example, via mechanical disruption. This may be accomplished be via a needle used to score the iPSC monolayer in a grid.
  • the iPSC monolayer of step (b) may be cultured in the presence of EB -media that promotes differentiation of the iPSCs.
  • the EB media may comprise, for example, less than about 10 ng/mL, or less than about 9 ng/mL or less than about 8 ng/mL, or less than about 7 ng/mL, or less than about 6 ng/mL, or less than about 5 ng/mL, or less than about 4 ng/mL, or less than about 3 ng/mL, or less than about 2 ng/mL, or less than about 1 ng/mL, or is substantially free of low basic fibroblast growth factor (bLGL).
  • the EB media may be replaced daily.
  • the iPSC monolayer may be contacted with an enzyme during the fragmentation step in an amount and for a period of time sufficient to disrupt the attachment of the monolayer to the culture plates.
  • exemplary enzymes include, for example, dispase, collagenase, or a combination thereof.
  • the fragmentation step may be carried out for a period of time of from about 2 days to about 10 days, or from about 4 days to about 8 days, or from about 5 days to about 6 days.
  • the monolayer fragments of step (b) may be cultured in the presence of EB-media.
  • the EB media may comprise, for example, less than about 10 ng/mL, or less than about 9 ng/mL or less than about 8 ng/mL, or less than about 7 ng/mL, or less than about 6 ng/mL, or less than about 5 ng/mL, or less than about 4 ng/mL, or less than about 3 ng/mL, or less than about 2 ng/mL, or less than about 1 ng/mL, or is substantially free of low basic fibroblast growth factor (bLGL).
  • the EB media may be replaced daily.
  • the monolayer fragments may be cultured with movement.
  • the movement may be provided by an orbital shaker.
  • the EB may be cultured on a surface comprising a gelatin coating.
  • the EB media of step c may be replaced at least once a day.
  • step c may be carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days.
  • the EB may be cultured in a myeloid media comprising IL-3 in an amount sufficient to promotes hematopoietic differentiation without serum, optionally further comprising IL-5.
  • the culture period of step d may be for a period of time sufficient to allow formation of a myeloid-cell-forming-complex (MCFC), for example, wherein the MCFC produces immature myeloid cells expressing CD45, CD34, Siglec8, or combinations thereof.
  • the step (d) may be carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days.
  • the MCFCs of step (d) are in suspension.
  • any of steps a through e may be carried out without the use of a feeder cell.
  • IL-3 may be present at an amount of from about 15 to about 40 ng/mL, or from about 20 to about 30 ng/mL or about 25 ng/mL; in one aspect, IL-5 may be present at an amount of from about 10 to about 100 ng/mL, or from about 25 to 75 ng/mL, or about 50 ng/mL. IL-3 may be present at an amount necessary for hematopoietic
  • MCFC formation MCFC differentiation
  • amount may be empirically determined by one of ordinary skill in the art in view of the inventive concept identified by Applicant herein.
  • a method of identifying genes or gene polymorphisms that regulate eosinophil development and/or function is disclosed.
  • the eosinophils of the disclosed culture may be used to identify genes and or polymorphisms that relate to or impact eosinophil development and/or function.
  • a method of testing a drug intended to affect an eosinophil may comprise the step of contacting a drug of interest with the eosinophil culture of claim 1 , further comprising the step of determining an outcome following contact with the drug, wherein the outcome may be selected from eosinophil number, gene expression, eosinophil function, eosinophil phenotype, or a combination thereof.
  • Human iPSCs are cultured in mTEASR media (StemCElls) using matrigel- coated 6 wells plates. When cells reached 70-80 % confluency, media is changed to embryoid body (EB) -promoting media (EB Media) that can include DMEM, 20% of FBS (Hyclone Defined) and a non-essential amino acid solution or another serum free media that supports EB formation, such as X-VIVO medium (Lonza). EB media is replaced daily for 96 hours. Cells are treated with enzymes, such as dispase or collagenase, to loosen their attachment to the plate, then scored in a grid format using a 25-gauge needle to disturb the monolayer into fragments.
  • enzymes such as dispase or collagenase
  • the fragments can be generated by plating iPSCs on feeder cells followed by disruption via enzymatic digestion.
  • EB formation is induced by cultivating the fragments in ultra- low binding plates in suspension on an orbital shaker (80 rpm) for 5 days - 7 days.
  • EBs are manually transferred onto gelatin-coated 6-wells plates.
  • Each well contains 15-20 EBs maintained in APELTM2 media ( STEMCELL ) supplemented with IL-3 (Myeloid media) or IL-3 and IL-5 (Eos media) for 7 days to form myeloid-cell-forming-complexes (MCFC). The media is fully replaced after 7 days.
  • Cells in suspension are harvested weekly thereafter.
  • the MCFCs contain CD34-expressing progenitor cells that generate immature eosinophils and other myeloid lineage cells. Cells are analyzed by flow cytometry to detect surface expression of CD45 (hematopoietic marker), CD34 (progenitor cell marker) and Siglec-8 (mature eosinophil marker). The immature myeloid cells can be collected and with continued exposure for an additional 10 days to IL-3 and IL-5 (or IL-5 alone) in IMDM culture media (Gibco) with added FBS and 2-mercaptoethanol to generate terminally
  • the EB Media is DMEM (Gibco), 20% FBS (Hyclone Defined), non-essential amino acids or X-VIVO (Lonza) and the Myeloid Media is APELTM2 Medium (STEMCELL), IL-3.
  • DMEM Gibco
  • FBS Hyclone Defined
  • non-essential amino acids or X-VIVO (Lonza)
  • Myeloid Media is APELTM2 Medium (STEMCELL), IL-3.
  • APELTM2 Medium STEMCELL
  • IL-3and IL-5 IL-5.

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Abstract

L'invention concerne des systèmes de culture d'éosinophiles qui contiennent des complexes formant des cellules myéloïdes (MCFC). Les MCFC peuvent être utilisés pour excréter des cellules dérivées de MCFC, comprenant, par exemple, des cellules progénitrices myéloïdes, des cellules précurseurs myéloïdes et des combinaisons de celles-ci. Les cellules dérivées de MCFC selon l'invention peuvent être différenciées en éosinophiles. L'invention concerne également des procédés de dérivation d'éosinophiles à partir d'une cellule souche pluripotente induite.
PCT/US2018/063953 2017-12-05 2018-12-05 Système de culture d'éosinophiles faisant appel à des cellules souches pluripotentes induites humaines Ceased WO2019113139A1 (fr)

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US201762594818P 2017-12-05 2017-12-05
US62/594,818 2017-12-05

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WO2019113139A1 true WO2019113139A1 (fr) 2019-06-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113493767A (zh) * 2020-04-01 2021-10-12 北京大学 利用人多潜能干细胞体外制备嗜酸性粒细胞

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009086596A1 (fr) * 2008-01-08 2009-07-16 The University Of Queensland Procédé de production d'une population de cellules
US20100016410A1 (en) * 2006-05-17 2010-01-21 Wagner Lori A Methods and Compositions Related to Eosinophil Regulation
WO2017070337A1 (fr) * 2015-10-20 2017-04-27 Cellular Dynamics International, Inc. Procédés de différenciation dirigée de cellules souches pluripotentes en cellules immunes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100016410A1 (en) * 2006-05-17 2010-01-21 Wagner Lori A Methods and Compositions Related to Eosinophil Regulation
WO2009086596A1 (fr) * 2008-01-08 2009-07-16 The University Of Queensland Procédé de production d'une population de cellules
WO2017070337A1 (fr) * 2015-10-20 2017-04-27 Cellular Dynamics International, Inc. Procédés de différenciation dirigée de cellules souches pluripotentes en cellules immunes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LACHMANN, N. ET AL.: "Large-Scale Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells Provides Granulocytes or Macrophages for Cell Replacement Therapies", STEM CELL REPORTS, vol. 4, 10 February 2015 (2015-02-10), pages 282 - 296, XP055384440 *
YAMAGUCHI, Y. ET AL.: "Purified Interleukin 5 Supports The Terminal Differentiation And Proliferation Of Murine Eosinophilic Precursors", J. EXP. MED., vol. 167, January 1988 (1988-01-01), pages 43 - 56, XP55614443 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113493767A (zh) * 2020-04-01 2021-10-12 北京大学 利用人多潜能干细胞体外制备嗜酸性粒细胞
CN113493767B (zh) * 2020-04-01 2024-04-26 北京大学 利用人多潜能干细胞体外制备嗜酸性粒细胞

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