WO2021207673A1 - Compositions, méthodes et utilisations pour production de cellules souches hématopoïétiques (csh) - Google Patents

Compositions, méthodes et utilisations pour production de cellules souches hématopoïétiques (csh) Download PDF

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WO2021207673A1
WO2021207673A1 PCT/US2021/026680 US2021026680W WO2021207673A1 WO 2021207673 A1 WO2021207673 A1 WO 2021207673A1 US 2021026680 W US2021026680 W US 2021026680W WO 2021207673 A1 WO2021207673 A1 WO 2021207673A1
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cells
hscs
pan
hdac
class
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Michael Verneris
Seon-hui Shim
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The Regents of the University of Colorodo, a body corporate
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N2501/065—Modulators of histone acetylation
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic 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
    • 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

  • compositions disclosed herein include one or more histone deacetylase inhibitor (HDAC inhibitors, HDACi, HDIs) in combination with embryonic stem (ES) cells, hemogenic endothelium (HE) or induced pluripotent stem (iPS) cells.
  • HDAC inhibitors HDACi, HDIs
  • ES embryonic stem
  • HE hemogenic endothelium
  • iPS induced pluripotent stem
  • embryonic cells cultured under stage I conditions (embryonic body-derived HSC, EB-HSC) generate HSCs derived directly from EBs or induced pluripotent stem (iPS) cells when exposed to one or more HDAC inhibitor.
  • embryonic cells, HEs or iPS cells cultured under stage II conditions in the presence of one or more HDAC inhibitor increase production of EBs or iPS cells giving rise to hemogenic endothelium (HE), progenitor HSCs and/or HSCs.
  • HDAC inhibitors can be at least one of a class II or a pan HDAC inhibitor.
  • HSCs produced from exposure to one or more pan HDAC inhibitor have increased populations of CD34+CD90+ HE cells and/or CD34+CD45+ HSCs.
  • HDAC inhibitors induce production of CD34+CD31+ cells in certain culture systems disclosed herein.
  • Embodiments of the instant disclosure relate to novel compositions and methods for improved production of hematopoietic stem cells (HSCs) using embryonic stem (ES) or induced pluripotent stem (iPS) cell populations.
  • HSCs hematopoietic stem cells
  • ES and iPS cell populations can be used interchangeably for any composition and/or methods and/or uses for the purposes of therapy or as otherwise indicated.
  • compositions disclosed herein include one or more histone deacetylase inhibitor (HDAC inhibitors, HDACi, HDIs) in combination with embryonic stem (ES) cells or induced pluripotent stem (iPS) cells in order to increase production of HSCs.
  • HDAC inhibitors histone deacetylase inhibitors, HDACi, HDIs
  • embryonic cells are part of a stage I (embryonic body-derived HSC, EB-HSC) system which leads to improved production of HSCs derived directly from EBs when exposed to one or more HDAC inhibitor.
  • embryonic cells are part of a stage I (embryonic body-derived HSC, EB-HSC) culture system where HSCs can be produced, derived directly from EBs, when exposed to one or more HDAC inhibitor.
  • ES cells are in stage II where EBs give rise to increased numbers of hemogenic endothelium (HE), HSC progenitors and HSCs in the presence of one or more HDAC inhibitor (HDACi) disclosed herein.
  • HE hemogenic endothelium
  • HDACi HDAC inhibitor
  • HDAC inhibitors are class II or pan.
  • HDAC inhibitors include, but are not limited to, a pan HDAC inhibitor, SAHA (Suberoylanilide hydroxamic acid, also known as Vorinostat) or other HDAC inhibitor.
  • SAHA Suberoylanilide hydroxamic acid, also known as Vorinostat
  • compositions including at least one HDAC class II or pan inhibitor increases production and/or differentiation of hemogenic endothelium (HEs), hematopoietic stem cell (HSC) progenitors and HSCs from ES or iPS cells but not established HSCs.
  • HSC hemogenic endothelium
  • HSC hematopoietic stem cell
  • HSCs produced from exposure to one or more pan HDAC inhibitors disclosed produce increased numbers of CD34+CD31+, CD34+CD90+, CD34+CD43+ and CD34+CD45+ HSC cells compared to cultures not having been exposed to one or more pan HDAC inhibitors.
  • HSCs produced from exposure to one or more pan HDAC inhibitors disclosed herein induce production of HSCs with increased gene expression of certain markers.
  • gene expression of one or more of iPSC-derived CD34+ (iCD34+), c-KIT (CD117), fms-related tyrosine kinase 3 (FLT3) ligand (FLT3LG), and thrombopoietin (TPO) can be upregulated where these genes are markers of HSC lineage maintenance.
  • overexpression of these and other HSC marker genes in combination with HDAC class II or pan inhibitor exposure to cells contemplated herein can induce production of HSCs and/or induce production of HSCs having improved engraftment properties when transplanted into a subject.
  • compositions containing class II HDAC or pan inhibitors can be used to increase production of HSCs for increased and/or improved production of blood cells using a Stage I and/or Stage II culture system as described herein.
  • compositions containing class II HDACi or pan inhibitors and ES, HE or iPS cells can be used to increase production of progenitor cells leading to the increased production of one or more of platelet cells, Natural Killer cells (NK), T cells, B cells, antigen presenting monocyte or macrophages, red blood cells, white blood cells, and other CD34+ HSCs.
  • NK Natural Killer cells
  • T cells T cells
  • B cells antigen presenting monocyte or macrophages
  • compositions disclosed herein can include, but are not limited to, a composition comprising one or more class II HDACi and/or pan inhibitor and optionally, one or more additional agent for inducing production of progenitor cells.
  • compositions disclosed herein can include, but are not limited to, a composition comprising one or more class II HDACi and/or pan inhibitor and/or one or more additional agent or induction of one or more genes for inducing production of progenitor cells including, but not limited to, Homeobox A5 (HOXA5), Homeobox protein A9 (HOXA9), Homeobox protein A10 (HOXA10), runt-related transcription factor 1 (RUNX1), SPI1 (encodes Transcription factor PU.1), ETS-related gene (ERG) and/or Ligand-dependent corepressor (LCoR).
  • compositions disclosed herein can further include RUNX1 or agents for inducing expression of RUNX1.
  • compositions disclosed herein can include one or more HDACi class II or pan inhibitor, in combination with RUNX1.
  • these compositions can be used to increase production and/or differentiation of hemogenic endothelium (HEs), hematopoietic stem cell (HSC) progenitors and HSCs from ES or iPS cells but not established HSCs of use in compositions and methods to treat a health condition.
  • HSCs produced by methods disclosed herein can be used in therapeutic applications to treat a condition or prevent a health condition in a subject.
  • HSCs produced by compositions and methods disclosed herein can be used to treat hematological malignancies, bone marrow failure syndromes and certain genetic disorders for example, such as inherited genetic disorders.
  • genetic disorders contemplated herein can include, but is not limited to, hematopoietic, nervous or immune system-related disorders.
  • the subject is a human.
  • the subject is a mammal such as a pet, livestock, zoo animal, horse or other animal.
  • HSCs generated by exposure to one or more class II HDACi or pan HDAC inhibitor can be derived from an animal to treat that animal or another animal.
  • animal derived HSCs generated by compositions and methods disclosed herein can be used to treat a subject of any species depending on the health condition to be treated and the condition of the subject.
  • HSCs generated by exposure to one or more class II or pan HDAC inhibitor have improved engraftment compared to HSCs not produced by these compositions and methods.
  • HSCs produced by compositions and methods disclosed herein are maintained or survive for longer periods in vivo and/or in vitro than HSCs obtained from other methods or harvested for use.
  • HSCs can be generated by exposure to a composition comprising one or more HDACi inhibitor alone or in combination with overexpression of one or more of Homeobox A5 (HOXA5), Homeobox protein A9 (HOXA9), Homeobox protein A10 (HOXA10), runt-related transcription factor 1 (RUNX1), SPI1 (encodes Transcription factor PU.1), ETS-related gene (ERG) and/or Ligand-dependent corepressor (LCoR) or one or more of Homeobox A5 (HOXA5), Homeobox protein A9 (HOXA9), Homeobox protein A10 (HOXA10), runt-related transcription factor 1 (RUNX1), ETS-related gene (ERG) and/or Ligand-dependent corepressor (LCoR); or one or more of HOXA5, HOXA9, HOXA10, RUNX1, ETS-related gene (ERG) and/or Ligand-dependent corepressor (LCoR); or one or more of
  • Figs.1A-1B illustrates exemplary culture methods, Stage I (1A) and Stage II (1B) for generating human stem cells (HSCs) from embroid bodies and HE precursor cells.
  • HSCs human stem cells
  • Fig.1C represents exemplary FACS plots of cells from a Stage II culturing system in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.1D represents exemplary graphs illustrating different cell populations obtained in Stage II cultures in the presence or absence of a pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.1E represents histogram plots representing expression levels of various genes in differentiated cell fractions (e.g. adherent and non-adherent) obtained from a Stage II culturing system in the presence or absence of a pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.1F represents exemplary FACS plots of HSCs differentiated from iPSCs using a Stage II culturing system in the presence of absence a pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.1G represents exemplary results analyzing gene expression levels in differentiated cell fractions obtained from an iPSC culture system in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.1H represents exemplary results from a colony forming unit assay of stage II differentiated cells in the presence or absence of a pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.2A represents exemplary FACS plots of hemogenic endothelium (HE) cells in a stage II system illustrating expression of various cell surface markers according to certain embodiments disclosed herein.
  • Fig.2B represents exemplary FACS plots and a histogram plot of hemogenic endothelium (HE) cells expressing various markers in a stage II system cultured in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.2C represents exemplary FACS plots and histogram plots of HSCs or HSC precursors differentiated in a Stage II system in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.2D represents exemplary FACS plots and a histogram plot of HSCs differentiated in a Stage I system in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Figs.2E- 2F represent exemplary FACS plots (E) and histogram plots (F) representing certain cell populations of HSC progenitors, hemogenic endothelium (HE) or HSCs differentiated in Stage I or Stage II systems in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.2G represents exemplary FACS plots of HSCs derived from donor umbilical cord blood cells (UCB) in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.2H represent exemplary histogram plots illustrating changes in expression of various genes in UCB-derived HSCs in the presence and absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.3A represents a Venn diagram illustrating overlap of enriched KEGG pathways in Stage I or Stage II culture systems in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.3B represents exemplary results analyzing gene expression levels of various genes associated of varied cellular differentiation including HSC, platelets, monocytes and T/B adaptive immune cells in Stage I or Stage II cultures in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Figs.3C-3E represents exemplary histogram plots of changes in expression of various genes in stage I versus stage II HSC cultures (3C) produced from exposure to an exemplary pan HDAC inhibitor compared to a control stage II HSC culture not exposed to the pan HDAC inhibitor and representative expression of particular target genes (3D) and vascular development genes (3E) according to certain embodiments disclosed herein.
  • Figs.3F-3G represent exemplary analysis of gene expression levels of various genes in Stage I or Stage II cultures in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Figs.4A-4B represent exemplary FACS plots (4A) and cell distribution data for CD34/CD45+ cells (4B) of HSCs prepared in Stage II cultures in the presence or absence of an exemplary HDAC class I inhibitor, a HDAC class II inhibitor, a HDAC class II inhibitor, or a pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Figs.4C-4D represent exemplary histogram plots of changes in expression of various genes relative to untreated controls in HSCs prepared from Stage II cultures, exposed to different HDAC inhibitors compared to controls and collected at an early (4C) and late (4D) time point according to certain embodiments disclosed herein.
  • Figs.5A-5B represent an exemplary Western blot analysis (5A) and histogram plots illustrating post ChiP q-RT-PCR analysis (5B) performed on Stage II differentiated cells in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.6A represents an exemplary Western blot illustrating protein overexpression of an experimental gene compared to a control gene according to certain embodiments disclosed herein.
  • Fig.6B-6C represent exemplary FACS plots (6B) of cells differentiated with the stage II system using a control guide RNA or a CRISPR/dCAS9 overexpression system and histogram plots (6C) of combinations of an exemplary overexpressed in the presence or absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.7A represents an exemplary experimental design for engraftment of hESCs from HSCs derived from compositions and methods disclosed herein in immunodeficient mice to assess engraftment and exemplary FACS plots of cells obtained from bone marrow of the engrafted experimental animals in the presence and absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.7B represents an exemplary plot comparison quantifying percentage of human cells having a particular marker obtained from the bone marrow of engrafted immunosuppressed mice in the presence and absence of an exemplary pan HDAC inhibitor according to certain embodiments disclosed herein.
  • Fig.7C represents an exemplary histogram plot illustrating expression of human HSC cells having a particular gene marker in pooled bone marrow cells in immunosuppressed mice engrafted with hESCs derived HSCs in the absence of a pan HDAC inhibitor, hESCs derived HSCs in the presence of a pan HDAC inhibitor, or a control cell type according to certain embodiments disclosed herein.
  • Terms, unless specifically defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.
  • treatment can refer to obtaining a desired pharmacologic and/or physiologic effect.
  • effects can be prophylactic in terms of completely, or partially preventing a condition or symptom thereof.
  • “individual”, “subject”, “host”, and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • “effective amount” as used herein can refer to a particular amount of a pharmaceutical composition including a therapeutic agent that achieves a clinically beneficial result (e.g., for example, a reduction of symptoms or side effects of the condition).
  • concurrent administration can refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).
  • a composition and/or formulation disclosed herein can be administered concurrently with a standard or known composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION [0046] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments of the invention.
  • compositions and methods for more efficient production of hematopoietic stem cells are disclosed. Due in part to the growing interests and successes in using stem cell derived products for medical applications, there is a need for more efficient processes to produce these cells.
  • HSCs can be used to in cellular immunotherapy, anti-cancer therapies and other therapies.
  • production of stem cells for these uses can include HSCs derived from adult sources (e.g. bone marrow, peripheral blood or cord blood) or from pluripotent sources (e.g. embryonic stem [ES] cells or induced pluripotent stem [iPS] cells).
  • HSCs derived from adult sources (e.g. bone marrow, peripheral blood or cord blood) or from pluripotent sources (e.g. embryonic stem [ES] cells or induced pluripotent stem [iPS] cells).
  • ES embryonic stem
  • iPS induced pluripotent stem
  • target cells of interest to be generated by compositions and methods disclosed herein can be T cells, B cells, natural killer (NK) cells, antigen presenting cells, monocyte/macrophages, red blood cells, platelets and white blood cells, or other CD34+ HSCs or CD35+.
  • pluripotent cells need to initially differentiae into HSCs and then into the lineage of interested as referenced herein.
  • compositions and methods disclosed herein overcome inefficiencies in these processes of differentiation from a pluripotent stem cell to a hematopoietic stem cells to overcome a barrier to the clinical use of pluripotent cell-derived therapies and production of these differentiated populations.
  • the instant disclosure relates to novel compositions including one or more histone deacetylase inhibitor (HDAC inhibitors, HDACi, HDIs) in combination with ES, HE or iPS cells or a combination thereof to enhance and/or increase differentiation of these cells (e.g. increase numbers of HSCs produced by these compositions and methods).
  • HDAC inhibitors histone deacetylase inhibitor
  • HDACi histone deacetylase inhibitor
  • ES cells can be cultured under stage II conditions where EBs give rise to increased populations of hemogenic endothelium (HE) and/or HSCs in the presence of one or more HDACi in order to improve production of HEs, HSC progenitor cells and HSCs.
  • HDACi are class II or pan.
  • compositions containing HDAC class II inhibitors increase production of hemogenic endothelium (HEs), hematopoietic stem cell (HSC) progenitors and HSCs.
  • HSCs produced from exposure to HDAC class II or pan inhibitors have increased populations of CD34+CD144+, CD34+CD90+, CD34+CD31+, CD34+CD43+ and CD34+CD45+ cells compared to ES, HE or iPS cells not exposed to HDAC class II or pan inhibitors.
  • differentiation conditions disclosed herein can include at least one of stage I and stage II systems.
  • compositions and methods disclosed herein where progenitor cells are exposed to one or more class II or pan HDACi lead to improved production of HSCs where a significant number of HSCs are obtained compared to systems not exposed to one or more class II or pan HDACi in stage I or stage II culture systems.
  • compositions and methods lead to improved production of HSCs with improved engraftment.
  • ES, HE or iPS cells cultured under stage II conditions in the presence of one or more HDACi induces production of HSCs as well as inducing production of HSCs having improved engraftment properties compared to ES, HE or iPS cells cultured under stage II conditions in the absence of one or more HDACi.
  • ES, HE or iPS cells conditioned with compositions disclosed herein including at least one HDACi can induce overexpression of genes related to differentiation into HSCs.
  • the overexpressed genes can include, but is not limited to, MkP differentiation related genes.
  • ES, HE or iPS cells conditioned with compositions disclosed herein including at least one HDACi can induce overexpression of genes related to HSC engraftment.
  • overexpressed genes can include, but are not limited to, one or more of HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1 and LCOR or other engraftment or HSC marker-related gene.
  • over- expression of one or more of these genes in combination with HDACi exposure in Stage II culture conditions can lead to improved HSC production.
  • HSCs can be generated by exposure to a composition comprising one or more HDACi inhibitor alone or in combination with overexpression of one or more of HOXA5, HOXA9, HOXA10, RUNX1, SPI1, ERG and/or LCoR or one or more of HOXA5, HOXA9, HOXA10, RUNX1, ERG and/or LCoR; or one or more of HOXA5, HOXA9, HOXA10, RUNX1, ERG and/or LCoR; or other combination or by overexpressing RUNX1.
  • overexpression of one or more of HOXA5, HOXA9, HOXA10, RUNX1, SPI1, ERG and/or LCoR can be induced before, during or after exposure to one or more class II or pan HDACi.
  • HSCs for engraftment or other therapeutic use can be generated by inducing RUNX1 overexpression in HSC progenitor cells (e.g. using Stage II culturing systems) followed by exposure to at least one composition comprising one or more class II or pan HDAC inhibitor where the resulting HSCs can be harvested and used as indicated herein.
  • HSCs produced by methods disclosed herein can be used in therapeutic applications to treat a condition or prevent a health condition in a subject.
  • HSCs produced by methods disclosed herein can be used to treat hematological malignancies, bone marrow related-syndromes, certain genetic disorders for example, such as inherited genetic disorders.
  • genetic disorders contemplated herein can include, but are not limited to, hematopoietic, nervous system or immune system-related disorders.
  • HSCs can be used in therapeutic applications and improved production of these cells can lead to increased availability of HSCs for these therapeutic uses and for further study.
  • HSCs from either UCB, BM, peripheral blood or those differentiated from iPSC/ES/HE cell lines, can be used as a source of cellular immunotherapy or other therapeutic use.
  • methods have been described to differentiate HSCs into a variety of different cell types with therapeutic potential including natural killer cells (NK cells), T cells, B cells, dendritic cells, differentiated endothelial cells and many other non-immune cells such as red blood cells and platelets.
  • NK cells natural killer cells
  • T cells T cells
  • B cells dendritic cells
  • differentiated endothelial cells and many other non-immune cells such as red blood cells and platelets.
  • one advantage of using iPS/ES or HE cells to increase availability of these cell populations is that that these cell lines can grow indefinitely.
  • these cells can be genetically modified to enhance function for a particular targeted therapy; for example, genetically modified to carry a specific gene or other modification.
  • HSCs generated herein can be further genetically modified using any method known in the art to modify the cells for a particular purpose.
  • the instant invention demonstrates improved differentiation of iPS/ES/HE cells to give rise to HSCs which are typically very inefficient processes; thereby, providing a readily derivable viable pool of HSCs of use for therapeutic applications.
  • HSCs or differentiated HSCs generated by compositions and methods disclosed herein can be used to replace cells damaged by chemotherapy or disease.
  • HSCs or differentiated HSCs generated by compositions and methods disclosed herein can be used to serve as a way for a donor's immune system (e.g. original progenitor cells derived from the donor) to fight certain types of cancer and blood-related diseases, such as leukemia, lymphoma, neuroblastoma and multiple myeloma.
  • a donor's immune system e.g. original progenitor cells derived from the donor
  • compositions and methods disclosed herein can be used to generate new cells such as cells to treat Parkinson's disease. rebuild bones and cartilage. repair damaged immune systems. make replacement heart valves in a subject.
  • HSCs generated by compositions and methods disclosed herein can be used to treat cerebral palsy, multiple sclerosis (e.g.
  • compositions comprising one or more HDACis can be used to increase production of HSCs.
  • HDACis which are generally divided into four classes, based on sequence homology to yeast counterparts.
  • ES or iPS cell-containing compositions disclosed herein can include an HDACi.
  • SAHA Suberoylanilide hydroxamic acid
  • hydroxamic acid-based vorinostat also known as Zolinza
  • SAHA is known to inhibit classes I, II and IV histone deacetylases, but not the NAD-dependent class III enzymes.
  • ES, HE, HSE or iPS cell- containing compositions disclosed herein can be exposed or contain an HDACi including, but are not limited to, Entinostat (MS-275), Panobinostat (LBH589), Trichostatin A (TSA), Trichostatin A (TSA), Mocetinostat (MGCD0103), Belinostat (PXD101), Romidepsin (FK228, Depsipeptide), n-buryrate, MS-275, MC-1568, Tubastatin A HCl, Givinostat (ITF2357), Dacinostat (LAQ824), CUDC-101, Quisinostat (JNJ-26481585) 2HCl, Pracinostat (SB939), PCI-34051, Droxinostat, Abexinostat (PCI-24781), RGFP96
  • a pan or class II HDACi comprises one or more of SAHA, LMK235, TSA (Trichostatin A) or a combination thereof.
  • SAHA SAHA
  • LMK235 a pan or class II HDACi
  • TSA Trichostatin A
  • ES cells are derived from the preimplantation-stage embryo. ES cell lines can be cultured indefinitely and can differentiate in vitro to give rise to cells that make up either endoderm, mesoderm or ectoderm through the creation of an embryoid body (EB), which is a three-dimensional mass of cells that partially recapitulates embryogenesis. When EBs are cultured in medium with hematopoietic cytokines, they give rise to two different types of hematopoietic stem cells.
  • EB embryoid body
  • Extended culture of EBs in cytokines can also produce multi-lineage hematopoiesis, called definitive hematopoiesis which is characteristic of hematopoiesis that occurs over the life of the organism.
  • HSCs multi-lineage hematopoietic stem cells
  • progenitor cells in the developing organism first emerge from endothelial cells in the dorsal aorta.
  • VE-cadherin lineage cells labeled within the aorta-gonad-mesonephros region before the definitive hematopoiesis, differentiate to blood cells of all lineages in vivo.
  • Hemogenic endothelium cells produce HSC through a process known as endothelial-to-hematopoietic transition (EHT). Similar to ES cells, induced pluripotent stem cell (iPSC)-derived HSCs can transit through a hemogenic endothelium stage which is transient and infrequent. This cell transition includes switching on a hematopoietic transcriptional program in selected endothelial cells, which then become hemogenic, followed by morphologic changes that lead to the breaking of tight junctions with neighboring endothelial cells and rounding up, to then being released into the blood stream. [0058] HE cells are rare.
  • compositions disclosed herein are capable of differentiating progenitor cells into CD34+CD90+CD45- cells of use in therapeutic treatments. In other embodiments, compositions and methods disclosed herein can be used to produce endothelium.
  • compositions and methods disclosed herein can be used to generate HE cells wherein these cells can be further differentiated into blood stem cells and/or endothelial stem cells.
  • HDACi-containing cultures contain increased numbers of HSCs at later time points, suggesting that this agent prolongs the “stemness” or delays differentiation of these cells.
  • RNA-sequencing demonstrated upregulation of specific genes involved in HSC lineage maintenance, including, but not limited to CD34, c-KIT, FLT3LG and TPO.
  • ES or iPS cell exposure to HDACi increased expression of these genes in both stage I and II systems.
  • Stage II derived HSCs exposed to an HDACi e.g.
  • HDACi e.g. SAHA
  • HDACi e.g. SAHA
  • HE Stage II-derived HSCs cultured with HDACi
  • genetic markers affiliated with platelet cells identified in methods disclosed herein include, but are not limited to, CD9, GP5, GP9 and IL11.
  • HE cells exposed to HDACi induce progenitor cells capable of differentiating into platelets.
  • concentrations of class II or pan HDACi provided to ES, HE or iPS cell populations can be about 1.0 nM (nanomolar) to about 1.0 mM; or about 10 nM to about 500 nM or about 10 nM to about 200 nM.
  • HDACis can be combined with other agents in the cell cultures.
  • agents of use in culturing ES, HE or iPS cell include, but are not limited to media or various growth factors except to the extent that the media and/or growth factors aid in the processes disclosed herein.
  • selected medias of use herein can be devoid of polyvinyl alcohol.
  • media and supplementary factors can include, but are not limited to, one or more of BPEL media (without polyvinyl alcohol), supplemented with about 10 to about 100 ng/mL (e.g.40 ng/mL) SCF, about 1.0 ng/mL to about 100 ng/mL (e.g.40 ng/mL) VEGF, about 1.0 ng/mL to about 100 ng/mL (e.g.30 ng/mL) thrombopoietin, about 1.0 ng/mL to about 100 ng/mL (e.g.30 ng/mL) interleukin-3 (IL-3), and about 1.0 ng/mL to about 100 ng/mL (e.g.30 ng/mL) IL-6.
  • BPEL media without polyvinyl alcohol
  • compositions containing one or more HDACi can include one or more of vascular endothelial growth factor (VEGF), stem cell factor (SCF), interleukin-3 (IL-3) and interleukin-6 (IL-6).
  • VEGF vascular endothelial growth factor
  • SCF stem cell factor
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • Nicotinamide an inhibitor of the NAD-dependent class III HDAC, was tested on UCB-derived CD34+ HSCs and led to hematopoietic progenitor cell expansion, increased HSC numbers, enhanced engraftment in murine models, as well as encouraging results in early human clinical trials.
  • HDACi when introduced to ES or iPS cells influence differentiation of pluripotent stem cells into the HSC lineage of use in mammalian therapies.
  • two hESC human embryonic stem cell differentiation systems were exposed to HDACis in order to assess timing and applicability of exposure and both demonstrated improved production outcomes of HSCs.
  • a stage I culture system embryonic body-derived HSC, EB-HSC
  • HDACi e.g. SAHA
  • a stage II culture system which starts with an EBs and give rise to both hemogenic endothelium (HE) and HSCs was exposed to an HDACi (e.g. SAHA) with an even greater effect on production of HSCs.
  • the HDACi can be a class II or a pan inhibitor.
  • Induced pluripotent stem cell (iPSC)-derived HSCs can transit through a HE stage which is transient and infrequent.
  • iPSC Induced pluripotent stem cell
  • HSCs By differentiating iPSC cells into HSCs and using single cell RNA sequencing, molecularly defined HE was assessed. Further, it was observed in animal studies that forced expression of seven transcription factors (HOXA5, HOXA9, HOXA10, RUNX1, SPI1, ERG and LCOR) was demonstrated to be sufficient to convert HE to HSCs for use in therapeutic methods and further, these cells can durably engraft as demonstrated herein. [0063] In some embodiments, it was demonstrated that HDACis increase generation of HE and their development into HSCs.
  • HDAC inhibitors By adding HDAC inhibitors to cultures, the expression of various transcription factors previously associated with an “engraftable” hESC-derived HSC were observed and their expression was confirmed as HDAC-driven via the presence of acetylated- H3 bound to the promoter of these genes.
  • HDAC inhibitor e.g. class II/pan
  • SAHA the HDAC inhibitor
  • increased gene expression of one or more markers e.g. HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1 and LCOR
  • one or more markers e.g. HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1 and LCOR
  • RUNX1 overexpression alone or in combination with overexpression of one or more other differentiation and/or engraftment related gene in ES, HE and/or iPS cells in combination with one or more HDACi e.g.
  • cells in a stage II culture system can be used to improve HSC production in culture and/or engraftment of HSCs in a subject.
  • cells can be obtained from a subject having a condition such as cancer.
  • the cells can be reprogrammed to iPSCs and then differentiated to HSCs using compositions and methods disclosed herein and then reintroduced to the subject using a BMT.
  • Other conditions are contemplated using this same procedure to administer the reprogrammed and differentiated HSCs to the subject for any disease or condition contemplated herein.
  • iPSCs can be generated and genetically modified to make a universal donor or generally applicable cell population and then differentiated into CD34+ cells.
  • these CD34+ cells can then be differentiated into NK, T-cells, B cells, monocytes, RBC or platelets (essentially any component of the blood) of use to treat a condition using this generally applicable cell populated.
  • Pharmaceutical Compositions [0066] Exemplary methods of administering a composition including HSCs produced by methods disclosed herein can include: engraftment, transplantation through a catheter or other applicable catheter delivery device (e.g.
  • Treatment can refer to any treatment of a condition in a mammal and can include: preventing the condition from occurring in a subject which may be at risk of developing the condition (e.g.
  • treatment can be performed prior to partial and/or complete loss of function of one or more affected tissues and/or organs in a subject.
  • treatment can be administered during symptomatic stages of the condition, before symptoms occur and in some examples after the symptomatic stages of the condition have occurred in a subject.
  • effective amount of HSCs delivered to a subject can refer to a particular number of cells provided to a subject such as a human or other mammal including a pet such as a dog or cat or other pet, horse or livestock.
  • Number of HSC cells needed to treat a subject can depend on the condition to be treated, age of the subject and other pertinent factors. In accordance with these embodiments, the range of number of cells can be a few thousand to tens of millions of cells.
  • HSCs derived herein can be administered systemically to a subject. In other embodiments, HSCs derived herein can be administered directly to a wound or infection site of the subject.
  • compositions disclosed herein can include a pharmaceutically acceptable excipient or carrier for stabilizing the composition and/or cells in the composition.
  • compositions disclosed herein can include a formulation of use to store HSCs produced by compositions and methods disclosed herein in order to permit freezing and thawing of the HSCs for storage and transport, for example. It is contemplated that the HSCs generated herein can be stored for long periods for later expansion and use.
  • a pharmaceutically acceptable excipient or carrier can be used in formulations for delivering cells produced by compositions and methods disclosed herein to a subject in need of such a treatment.
  • cells can be stored in liquid nitrogen or by other quick freezing and storage for example with agents to preserved the integrity of the cells (e.g. DMSO) using very standard conditions.
  • media and expansion systems are described herein.
  • composition disclosed herein can be part of a kit.
  • compositions can include one or more HDACi and can further include progenitor cells.
  • compositions disclosed herein can be present in one or more containers or vials, e.g., single use or multi-use containers or vials and maintained at a predetermined temperature (e.g. freezing temperatures for storage).
  • containers storing cells for administration to a subject can include a single application or more than one application.
  • kits disclosed herein can include one or more HDACi composition for providing to a population of cells for on-site preparation of HSCs for delivery to a subject.
  • compositions and formulations disclosed herein can be stored for administration to a subject in a bag for intravenous delivery or in one or more bolus for administration directly to a site of interest in a subject (e.g. wound or infection).
  • the composition can be diluted in a suitable diluent for administration.
  • the kit or composition can include a single-dose or multiple doses such as multiple vials of HSCs derived herein.
  • kits can further include media for expanding an ES or iPS cell population contemplated herein for further study or therapeutic use.
  • the subject is a mammal (e.g. horse, dog, cat, cow, pig, sheep, goat, rabbit).
  • the subject is a human.
  • the subject is an unborn child (a fetus), a baby, a toddler, a young child, a child or adolescent or teenager.
  • the subject is an adult of 18 years or older.
  • Example 1 HDACi increased both progenitor of HSCs and HSCs from hESCs
  • HDACi HDAC inhibitors
  • stage I system which results in CD34+CD45+cells derived directly from embryonic bodies (EBs) (See for example, Fig.1A).
  • stage II system which initially begins with EBs that are then transferred to flat bottom plates and give rise to both hemogenic endothelium (HE) and CD34+CD45+ cells (See for example, Fig.1B).
  • HDACi in Stage II cultures [0075] In certain exemplary methods, stage II culture systems were used which promote endothelial and hematopoietic cell differentiation, as illustrated in Fig. 1B.
  • FACS analysis was used to assess the percentage of endothelial cells (CD34+CD144+, CD34+CD31+), HSC progenitors (CD34+CD43+) and of HSCs (CD34+CD45+) at the end of a culture period (7+12 days) in the presence of two different HDAC inhibitors, TSA (50 nM) and SAHA (200 nM) and DMSO (control).
  • TSA 50 nM
  • SAHA 200 nM
  • DMSO control
  • HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1 and LCOR were all overexpressed. These seven genes have been associated with HE conversion into engraftable HSCs.
  • SAHA induced expression significantly more than TSA.
  • cycle threshold (Ct) values were normalized to a control gene (e.g. ⁇ -actin) at each time point and the data is presented as relative fold change to a control, DMSO-treated controls.
  • HDACi was analyzed to assess whether it was capable of increasing CD34+CD45+ cell populations from iPSCs. In these methods, two iPSC lines were differentiated with the stage II system.
  • iPSC-derived CD34+CD45+ cells were collected for antibody staining and counting. Similar to ES-derived cells, the percentage and absolute numbers of iPSC-derived CD34+CD45+ cells were significantly increased in the presence of SAHA compared to vehicle control (Fig.1F). These stage II differentiated iPSCs were harvested at day 7+3 and probed for gene expression of certain genes known to be overexpressed using qRT-PCR. In this experiment, using the iPSC-1 line, all tested genes were increased. All of the above seven genes were upregulated in the presence of SAHA. However, in iPSC-2 line, six genes (HOXA5, HOXA9, HOXA10, RUNX1, ERG and LCOR) were upregulated, but SP1 was not (Fig. 1G).
  • CFU-E burst forming unit-erythroid
  • CFU-G CFU–granulocyte
  • CFU-M CFU–macrophage
  • CFU-GM CFU–granulocyte
  • CFU-GEMM CFU– granulocyte, erythroid, macrophage, megakaryocyte
  • stage II culture systems give rise to multiple cell populations, including adherent, fibroblast-appearing cells some of which could be HE.
  • HE As illustrated in Fig.2B, in the DMSO treated controls, ⁇ 20-50% of the adherent cells were HE defined as being CD34+CD90+CD45-. As demonstrated herein, in the presence of SAHA, increased numbers of CD34+CD90+CD45- HE cells were present (Fig.2B), supporting the observation that SAHA increased the differentiation of EB-derived HE. [0079] In other exemplary methods, HEs were analyzed to see if these HDAC inhibitors could produce progenitor of HSCs/HSCs.
  • HE CD34+CD90+
  • progenitors of HSCs CD34+CD43+
  • HSCs CD34+CD45+
  • Fig.2D and Fig.2E illustrates representative FACS plots of hESC differentiation from the stage I and II systems at day 7+6 following treatment with SAHA starting at day 7+0.
  • HDACi on UCB-derived HSCs [0082] In another exemplary method, it was examined whether HDACi had effects on differentiation of different sources of progenitor cells, umbilical cord blood (UCB)-derived HSCs (CD34+ cells).
  • Example 2 Gene analysis of stage I-derived and stage II-derived HSCs
  • an exemplary HDAC inhibitor e.g., SAHA
  • RNA sequencing was performed on FACS sorted CD34+CD45+ HSCs obtained from each culture system generated having DMSO (control) or an HDAC inhibitor (e.g. SAHA).
  • Fig.3 illustrates a gene analysis between stage I-derived and stage II-derived HSCs.
  • HSCs derived with or without SAHA are limited to genes within relatively select pathways, as opposed to broad changes in gene expression (data not shown regarding pathway comparison and overview). It was noted that the hematopoietic cell lineage gene set appeared in all comparisons. Accordingly, based on the enriched KEGG pathway analysis, in an exemplary method, individual gene changes particularly in the hematopoietic cell lineage gene set were investigated. Genes were divided into 4 categories: HSC “stemness”, platelet-related, monocyte-related and adaptive immune (T or B cell)- related.
  • Fig.3B a heatmap demonstrating relative gene expression of these 4 KEGG categories, SAHA treated hESCs demonstrated an increase in c-KIT, FLT3, CD34 and TPO, all of which can facilitate HSC maintenance, within stage I- and stage II-derived HSCs.
  • overexpression of “stemness” genes was highest in stage II cultures treated with the HDACi (e.g. SAHA).
  • HDACi e.g. SAHA
  • platelet related genes were also overexpressed in the stage II HSCs differentiated with SAHA.
  • Stage II HSCs differentiated without SAHA demonstrated an upregulation of monocyte lineage related genes. Increases in the expression of adaptive immunity-related genes were observed, such as CD3 and CD19 in stage I cultures in the controls which was further augmented by SAHA.
  • Fig.3C-Fig.3E are exemplary bar plots that are representative of relative expression of HOXA, HOXB and HOXC cluster genes (Fig.3C), as well as subsets of HOXB4 downstream targets (Fig.3D) and genes related to vascular development (Fig. 3E). It is illustrated that Log2 (fold change) values are relative to the stage I-C condition, with genes ordered by stage II-SAHA values.
  • HOXB4 target genes such as ID4, FLI1, PBX1, ID2, LYL1, MEIS1, GATA2 and RUNX1 are upregulated in the stage II-derived HSCs with SAHA (Fig.3D).
  • FL-HSPCs fetal liver hematopoietic progenitor cells
  • ES-HSPCs hESC-derived HSPCs
  • FL-HSPCs which engrafted efficiently in vivo, showed lower expression of vascular development related genes (NRP1, FLT1, SOX17, VEGFA, HEY1 and HEY2) when compared to ES-HSPCs (Fig.3E).
  • stage II- derived HSCs cultured in SAHA in a similar manner were examined and demonstrated downregulation of these same genes, except for HEY2.
  • Stage II-derived HSCs treated with SAHA also demonstrated enhanced cell adhesion and chemokine signaling by KEGG analysis.
  • Fig.3F and Fig.3G are heat maps illustrating a subset of genes from cell adhesion molecules (Fig.3F) and chemokine signaling (Fig.3G) KEGG pathways. Color bars represent standard gene expression. The genes are ordered by the sum of standardized expression in the stage II-SAHA samples. These latter two pathways are potentially involved in homing and engraftment of HSCs.
  • stage II HSCs tend to be enriched for genes that prolong the stemness of HSCs, over-express HOX cluster genes and downregulate genes associated with vascular development.
  • Example 3 Inhibition of HDAC class II enhances and increases the Stage II-derived HSCs and seven gene overexpression [0086] It is known that SAHA is a pan-HDAC inhibitor. It is unclear whether the exemplary results in the previous Examples are mediated by the inhibition of HDAC I, II, III or a combination.
  • class specific HDAC inhibitors including CI994 (HDAC class I), LMK235 (HDAC class II), 3-TYP (HDAC class III) and SAHA (HDAC pan inhibitor) were tested using the stage II system described above. Briefly, hESCs were first made into spin EBs and differentiated with stage II method, then each HDAC inhibitor was added with the cytokine mixture at day 7+0: CI994200nM (HDAC class I), LMK235100nM (HDAC class II), 3-TYP 20nM (HDAC class III) and SAHA 200nM (HDAC pan inhibitor).
  • Example 4 Acetylated-H3 is bound to the promoter region of the seven genes in the presence of SAHA [0087]
  • acetylation of histones was explored in these processes where an HDACi can inhibit activity of HDAC enzyme, leading to a more relaxed chromatin structure associated with gene transcription. While the previous examples demonstrated up- regulation and expression of the seven gene signature (HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1 and LCOR) by qPCR, these changes were confirmed using Western blot analysis and protein expression.
  • H3 A non-acetylated form of H3 was used as a negative control
  • RPL30 was used as a positive control for acetylated H3
  • ⁇ -satellite was also used as a negative control.
  • Control versus HDACi samples were analyzed for acetylation of histones. It was observed that there was enhanced binding of acetylated histone-H3 to the promoter region of all seven genes, except for HOXA10, as illustrated by histogram plots (See for example, Fig. 5B).
  • a CRISPR/dCAS9 system was used to overexpress the RUNX1 gene in hESCs. Increased expression of RUNX1 protein was confirmed by Western Blot (Fig.6A).
  • Fig.6A Western Blot
  • hESCs more efficiently differentiated into HSCs in the presence of the HDAC inhibitor than using HDAC inhibitor alone yet both efficiently upregulated gene expression.
  • SAHA After treating with an HDACi, SAHA, RUNX1 overexpressed hESCs further enhanced the percentage of CD34+CD31+, CD34+CD43+ and CD34+CD45+ cells when compared to the control guide-RNA treated only with SAHA, as demonstrated by FACS (Fig.6B).
  • HDACi e.g. SAHA
  • Fig.7A depicts the irradiation and injection protocol and also shows five representative FACS plots of human CD45+Mcd45- cells in bone marrow of NSG mice. After 10 ⁇ 12 weeks, a significantly higher percentage of hCD45+ cells were observed in the bone marrow(BM) of mice that received SAHA-treated hESC-derived CD34+ cells (Fig.7B). hCD45 cells were not found in peripheral blood and the spleen (data not shown).
  • hESCs and iPSCs were allowed to differentiate as spin embryoid bodies (EBs) as described in the following method. Briefly, hESCs and iPSCs were plated at 3000 cells and 8000 cells, respectively, per 100 ⁇ L in a round-bottom 96-well plate using serum-free bovine serum albumin polyvinyl alcohol essential lipid (BPEL) media supplemented with 20 ng/mL bone morphogenetic protein 4 (BMP4), 40 ng/mL stem cell factor (SCF), and 20 ng/mL vascular endothelial growth factor (VEGF).
  • BPEL serum-free bovine serum albumin polyvinyl alcohol essential lipid
  • BMP4 bone morphogenetic protein 4
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • EBs were centrifuged to form EBs with 1250 rcf (defined as day 0) and were incubated for 7 days (defined as day 7) to promote mesoderm induction.
  • the stage I differentiation method was used as illustrated in Fig. 1A.
  • EBs were cultured in U- bottom plates during the entire culture period and at day 7, the media was changed to BPEL media (without polyvinyl alcohol), supplemented with 40 ng/mL SCF, 40 ng/mL VEGF, 30 mg/mL thrombopoietin (all from StemCell Technologies, Vancouver, BC, Canada), 30 ng/mL IL-3, and 30 ng/mL IL-6 (both R&D Systems, Minneapolis, MN). After changing media, EBs were cultured for an additional 7 ⁇ 10 days (defined as day 7+7/10). [0092] In another exemplary method to differentiate early endothelial and hematopoietic progenitor cells the Stage II differentiation method was used as illustrate in Fig.
  • EBs were transferred to pre-gelatinized 24-well plates with BPEL media (without polyvinyl alcohol) supplemented with the same cytokines as in Stage I (40 ng/mL SCF(R&D), 40 ng/mL VEGF(R&D), 30 mg/mL thrombopoietin(Stemcell), 30 ng/mL interleukin-3 (IL-3) (e.g. Stemcell), and 30 ng/mL IL-6 (e.g. Stemcell).
  • IL-3 interleukin-3
  • IL-6 e.g. Stemcell
  • HDAC inhibitor EBs were treated at day 7+0 with either DMSO control, 10nM TSA (Trichostatin A from Sigma-Aldrich, St.
  • RNA sequencing [0095] CD34+CD45+ cells from stage I and stage II differentiation at day 7+3 were first sorted from each stage using FACS, then RNA was purified with an RNeasy Micro kit (Qiagen). RNA libraries were sequenced as 151 bp paired-end reads on the Illumina NovaSeq 6000 platform. Read quality was assessed using FastQC v0.11.7 and multiQC v1.7.dev0 both before and after trimming, which was performed using BBDuk v38.70.
  • Cells were rinsed with PBS, suspended in lysis buffer (0.5% NP40, 50 mM Tris-Cl, 150 mM NaCl, 1 mM DTT, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 0.1 M aprotinin, and 1 M pepstatin A) and incubated at 4 o C for 30 min. The cell lysates were then centrifuged at 16300 g for 20 min at 4 o C. An appropriate amount of each supernatant (determined by protein assay) was mixed with 4X sample loading buffer and denatured for 10 min at 70C.
  • lysis buffer 0.5% NP40, 50 mM Tris-Cl, 150 mM NaCl, 1 mM DTT, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 0.1 M aprotinin, and 1 M pepstatin A
  • RNA extracted from cells were fractionated on 4–12% Nu-PAGE Bis-Tris gels (Invitrogen), transferred onto nitrocellulose membranes (Schleicher & Schuell) and incubated with Tris-buffered saline containing 0.1% Tween-20, and 5% non-fat dry milk. The membranes were then incubated with HOX9, RUNX1, LCOR, SPI1, ERG (e.g. Santa Cruz) and Ac-H3 (e.g. Abcam). Quantitative PCR [0098] In certain exemplary methods, a quantitative PCR method was performed on RNA extracted from cells. RNA extraction was performed using an RNAeasy Mini kit (Qiagen).
  • Chromatin immunoprecipitation [0099] In certain exemplary methods, at day 7+6, after differentiating HE-derived HSCs, all fractions of cells were collected and chromatin immunoprecipitation (CHIP) was performed with a SimpleChip enzymatic chromatin IP Kit (CellSignaling Technology). All procedures followed manufacturer’s protocol. Briefly, immunoprecipitation was performed using 10 ⁇ g of pan-acetylated H3 antibody (Abcam) and H3 antibody for non-acetylated control (Cellsignaling). ChIP-DNA templates were amplified with PCR primers for each genes’ promoter region.
  • CHIP chromatin immunoprecipitation
  • HOXA5 represented at NCBI GENE ID 3202, exemplary nucleotide sequence: SEQ ID NO: 27, exemplary polypeptide sequence: SEQ ID NO: 34; HOXA9 represented at NCBI GENE ID 3205, exemplary nucleotide sequence: SEQ ID NO: 28, exemplary polypeptide sequence: SEQ ID NO 35; HOXA10 represented at NCBI GENE ID 3206, exemplary nucleotide sequence: SEQ ID NO: 29, exemplary polypeptide sequence: SEQ ID NO: 36; RUNX1 represented at NCBI GENE ID 861, exemplary nucleotide sequence: SEQ ID NO: 30, exemplary polypeptide sequence: SEQ ID NO: 37; ERG represented at NCBI GENE ID 2078, exemplary nucleotide sequence: SEQ ID NO: 31, exemplary polypeptide sequence: SEQ ID NO: 38; SPI represented at NCBI GENE ID 6688, exemplary nucleotide sequence: SEQ ID NO: 32, exemplary nucleotide sequence: S
  • mice were bred in a barrier facility. Six to 10 week old mice were sublethally irradiated (single dose of 220 rad) before (e.g.12 hours before) receiving a transplantation via intrafemoral injection. After irradiation, uniprim food was supplied to mice to prevent infections. Before transplantation, mice were temporarily sedated with isoflurane.
  • a 27- gauge needle was used to drill the femur.1 x 10 6 human embryonic stem cells (hESCs) derived HSCs in 25 ⁇ L volume were transplanted by intra- femoral injection into the mice. Mice were sacrificed 10-12 weeks post injection to collect bone marrow (BM) mononuclear cells for analysis. Secondary transplantation used the same methods described above.
  • hESCs human embryonic stem cells
  • BM bone marrow
  • COMPOSITIONS and METHODS have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variation may be applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the METHODS described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

Des modes de réalisation de la présente invention concernent de nouvelles compositions et méthodes pour une production améliorée de cellules souches hématopaïétiques (CSH). Dans certains modes de réalisation, les compositions de l'invention comprennent un ou plusieurs inhibiteurs d'histone désacétylase (inhibiteurs de HDAC, HDACi, HDI) en combinaison avec des cellules souches embryonnaires (ES) ou des cellules souches pluripotentes induites (iPS). Dans certains modes de réalisation, l'invention concerne des cellules embryonnaires cultivées dans des conditions d'étape I (CSH dérivées du corps embryonnaire, EB-CSH) ce qui a pour conséquence que les CSH dérivées directement d'EB sont exposées à un ou plusieurs inhibiteurs de HDAC. Dans d'autres modes de réalisation, l'invention concerne des cellules embryonnaires cultivées dans des conditions d'étape II où les EB donnent naissance à des nombres accrus d'endothélium hémogénique (HE) et de CSH en présence d'un ou de plusieurs inhibiteurs de HDAC. Dans certains modes de réalisation, les inhibiteurs de HDAC sont de classe II ou pan. Dans d'autres modes de réalisation, les CSH produites à partir d'une exposition à des inhibiteurs de classe II de HDAC ont des nombres accrus de cellules CSH CD34+/CD90+.
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