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WO2019049939A1 - Procédés et compositions pour la propagation de cellules - Google Patents

Procédés et compositions pour la propagation de cellules Download PDF

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WO2019049939A1
WO2019049939A1 PCT/JP2018/033067 JP2018033067W WO2019049939A1 WO 2019049939 A1 WO2019049939 A1 WO 2019049939A1 JP 2018033067 W JP2018033067 W JP 2018033067W WO 2019049939 A1 WO2019049939 A1 WO 2019049939A1
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egfl7
cells
stem cells
itgb3
cell
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Yousef Mahmmoud SALAMA
GEB HEISSIG Beate HATTORI
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University of Tokyo NUC
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University of Tokyo NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone

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  • the field of the invention generally relates to methods and compositions for expanding cells, such as stem cells, hematopoietic stem cells, and progenitor cells.
  • HSPCs hematopoietic stem and progenitor cells
  • the present invention is directed to a mutant RGDdel protein such as that described below.
  • the mutant RGDdel protein comprises or consists of a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity to an epidermal growth factor-like protein 7 (Egfl7) polypeptide and lacks the RGD domain of the Egfl7 polypeptide.
  • the Egfl7 polypeptide is human Egfl7.
  • the mutant RGDdel protein comprises or consists of a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity to SEQ ID NO: 1.
  • the present invention is directed to a composition comprising one or more Egfl7 proteins and one or more Itgb3 inhibitors, wherein said one or more Egfl7 proteins may include a mutant RGDdel protein as disclosed herein.
  • the composition further comprises one or more Notch activators.
  • the composition further comprises Flt-3 ligand, Kit ligand, one or more growth factors such as those listed below.
  • the composition further comprises a culture medium.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the present invention is directed to a method for expanding stem cells, which comprises increasing expression of epidermal growth factor-like protein 7 (Egfl7) in the stem cells and/or contacting the stem cells with one or more Egfl7 proteins or compositions as described herein.
  • the one or more Egfl7 proteins may include a mutant RGDdel protein as described herein.
  • the method further comprises contacting the stem cells with one or more Notch activators.
  • the method further comprises inhibiting or preventing the stem cells from undergoing cell differentiation by contacting the stem cells with one or more Itgb3 inhibitors.
  • the method further comprises allowing the stem cells to undergo cell differentiation by removing any Itgb3 inhibitors in contact with the stem cells and/or contacting the stem cells with one or more Itgb3 activators.
  • the present invention is directed to a method for treating a disease or condition in a subject, which comprises administering to the subject a composition as described herein or transplanting in to the subject stem cells that have been expanded as described herein.
  • the disease or condition is selected from one of those listed below.
  • the subject is mammalian.
  • the subject is human.
  • the subject is in need thereof, e.g., a subject who is diagnosed as suffering from a disease or condition selected from one of those listed below.
  • a kit comprising one or more Egfl7 proteins, wherein said one or more Egfl7 proteins may include a mutant RGDdel protein as described herein, packaged together with an Itgb3 inhibitor, a Notch activator, a culture medium, and/or a drug delivery device.
  • the one or more Notch activators employed in the compositions and methods are selected from those disclosed below.
  • the one or more Itgb3 inhibitors employed in the compositions and methods are selected from those disclosed below.
  • the Itgb3 activator employed in the compositions and methods are selected from those disclosed below.
  • the stem cells employed in the compositions and methods are selected from those disclosed below.
  • the present invention is directed to a plurality of stem cells having been expanded using the methods or compositions described herein.
  • stem cells expanded and/or maintained in an undifferentiated state using the methods described herein exhibit long-term engraftment when transplanted into a subject.
  • Embodiment 1 A method for expanding cells with Notch and ⁇ 3 integrin (Itgb3) signaling pathways, comprising culturing the cells under conditions sufficient to downregulate the Itgb3 signaling pathway and to upregulate the Notch signaling pathway.
  • Itgb3 ⁇ 3 integrin
  • Embodiment 2 The method according to Embodiment 1, wherein the Itgb3 signaling pathway is regulated by one or more Itgb3 inhibitors.
  • Embodiment 3 The method according to Embodiment 2, wherein the Itgb3 inhibitors are selected from the group consisting of cilengitide, disintegrins, JSM6427; GRGDSP; Integrin Antagonists 27, GLPG0187, Cyclo(-RGDfK), Arg-Gly-Asp-Ser, CWHM-12; P11; Echistatin, ⁇ 1 isoform; LM609; Eptifibatide acetate, Tirofiban, Hydrochloride; GR 144053 trihydrochloride, and neutralizing antibodies against human integrin beta 3: e.g., clone 25E11, clone 27.1 (VNR-1), clone PM6/13, and clone 23C6.
  • the Itgb3 inhibitors are selected from the group consisting of cilengitide, disintegrins, JSM6427; GRGDSP; Integrin Antagonists 27, GLPG0187, Cyclo(-
  • Embodiment 4 The method according to Embodiment 1, wherein Itgb3 expressed on the cells is knocked out or knocked down in the cells.
  • Embodiment 5 The method according to Embodiment 1, wherein the Notch signaling pathway is upregulated by one or more Notch activators.
  • Embodiment 6 The method according to Embodiment 5, wherein the Notch activators are selected from the group consisting of Epidermal growth factor-like domain 7 (Egfl7) with or without an Arg-Gly-Asp (RGD) domain; Delta-like-1(Dll1); Jagged-1; and Delta1ext IgG, Jagged-1-IgG; Phenethyl isothiocyanate; resveratrol; active fragment of human Jag-1 protein (aa 188-204); Reelin; N-methylhemeanthidine chloride (NMHC); valpronic acid; Notch 1 agonist; and Notch 2 agonist.
  • Egfl7 Epidermal growth factor-like domain 7
  • RGD Arg-Gly-Asp
  • Embodiment 7 The method according to Embodiment 6, wherein Egfl7 is a mutant lacking the RGD domain.
  • Embodiment 8 The method according to Embodiment 1, wherein the cells are cultured in the presence of one or more agents which promote Egfl7 binding to Itgb3; one or more humanized neutralizing antibodies against the RGD region in Itgb3; one or more small molecules that prevent binding of Egfl7 or Notch activating agents to the RGD region in Itgb3; siRNA to block Itgb3; RGD peptide or RGD containing peptides; one or more signal transducer and activator of transcription 3 (STAT3) inhibitors; Flt-3 ligand; thrombopoietin (TPO); kit ligand (stem cell factor) interleukin-3 (IL-3), IL-1, IL-6, IL-5, IL-7, IL-8, IL-9, and any growth promoting hematopoietic growth factor (including G-CSF, GM-CSF), leukemic inhibitory factor, macrophage colony-stimulating factor, and/or Garcinol or other
  • Embodiment 9 The method according to Embodiment 1, wherein the cells are stem cells, progenitor cells, CD34+ cells from primary tissues, or somatic or pluripotent cells that have been generated by transduction of specific transcription factors (human fibroblasts, embryonic and adult endothelial cells (after transcription factor addition).
  • specific transcription factors human fibroblasts, embryonic and adult endothelial cells (after transcription factor addition).
  • Embodiment 10 The method according to Embodiment 9, wherein the stem cells are selected from hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells.
  • Embodiment 11 The method according to Embodiment 10, wherein the self-renewal capacity and immaturity without differentiation are maintained in the stem cells.
  • Embodiment 12 The method according to Embodiment 9, wherein the stem cells are maintained in an undifferentiated state (e.g., in the G0 cell cycle phase).
  • Embodiment 13 The method according to Embodiment 9, wherein the stem and progenitor cells are selected from the group consisting of human cord blood CD34+ cells, bone marrow derived CD34+ cells, peripheral blood derived human CD34+ cells, early thymic progenitor cells, thymocytes, hematopoietic progenitor cells, bone marrow cells, bone marrow, umbilical cord blood, placental blood, peripheral blood, placental tissues, Wharton's jelly; fetal or neonatal blood, and erythroid progenitors.
  • the stem and progenitor cells are selected from the group consisting of human cord blood CD34+ cells, bone marrow derived CD34+ cells, peripheral blood derived human CD34+ cells, early thymic progenitor cells, thymocytes, hematopoietic progenitor cells, bone marrow cells, bone marrow, umbilical cord blood, placental blood, peripheral blood, placental
  • Embodiment 14 The method according to Embodiment 1, wherein the cells are cultured in the presence of Flt-3 ligand.
  • Embodiment 15 A composition comprising one or more Itgb3 inhibitors and/or one or more Notch activators, including an Egfl7 mutant protein lacking an RGD domain.
  • Embodiment 16 A kit for expanding cells with Notch and Itgb3 signaling pathways, comprising the composition according to Embodiment 15.
  • Embodiment 18 An Egfl7 mutant protein lacking an RGD domain.
  • Embodiment 19 A cell expansion factor comprising one or more Itgb3 inhibitors and/or one or more Notch activators, including an Egfl7 mutant protein lacking an RGD domain.
  • Embodiment 20 A method for the treatment or prevention of a disease associated with the Notch signaling pathway in a subject, comprising downregulating Itgb3 signaling pathway and/or upregulating Notch signaling pathway.
  • Embodiment 21 The method according to Embodiment 20, wherein the disease is selected from the group consisting of: i) hematopoietic pancytopenia/neutropenia, leukopenia, neutropenia in cancer patients treated with chemotherapy; ii) diseases resulting from a failure or dysfunction of normal blood cell production and maturation (e.g., hyperproliferative stem cell disorders, aplastic anemia, pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia, and Blackfan-Diamond syndrome due to drugs, radiation, or idiopathic infection); iii) hematopoietic malignancies (e.g., acute lymphoblastic (lymphocytic) leukemia, chronic lymphocytic leukemia, Burkitt’s lymphoma, acute myeloid leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, myelo
  • Figure 1A- Figure 1C Show high expression of Egfl7 in the most immature hematopoietic stem cells and endothelial cells, and that Egfl7 is upregulated after myelosuppression such as the chemotherapeutic drug 5-fluorouracil.
  • Figure 1B is a graph showing Egfl7 expression on indicated FACS-isolated subpopulations.
  • Murine BM-derived KSL cells were FACS-isolated into distinct cell population based on differential antigen expression profile: GMP (Lin - c-Kit + Sca-1 - CD16/32 + CD34 + ), CMP (Lin - c-Kit + Sca-1 - CD16/32 + CD34 + ) and MEP (Lin- c-Kit + Sca-1 - CD16/32 + CD34 - ).
  • GMP Long - c-Kit + Sca-1 - CD16/32 + CD34 +
  • CMP Lin - c-Kit + Sca-1 - CD16/32 + CD34 +
  • MEP Lin- c-Kit + Sca-1 - CD16/32 + CD34 -
  • Figure 1C is a graph showing the fold increase in Egfl7 gene expression on BM cell samples isolated from 5-FU treated mice (n-2/time point).
  • Figure 2A- Figure 2G Show Egfl7 expands phenotypic HSCs.
  • Figure 2A is a graph showing the fold increase of Egfl7 expression in liver tissues after virus administration by qPCR with normalization using ⁇ -actin (left panel) and a Western blot analysis of human Egfl7 and ⁇ -actin at indicated time points (right panel).
  • Figure 2D are graphs showing the KSL cell frequency, increase, and total numbers in cultures treated with or without Egfl7.
  • KSL cell frequency left panel
  • KSL fold increase compared to non-EC containing cultures
  • the total number of KSL per well right panel
  • HUVEC cells were grown to confluency.
  • FACS-sorted CD34-KSL cells derived from GFP mice (1 x 10 5 /well) were added.
  • CD34-KSL cells retrieved from BM of recombinant Egfl7 treated mice or controls on Day 5 were co-stained with Hoechst blue and the cell cycle status of KSL cells was quantified by cells that efflux the Hoechst 33342 dye.
  • Figure 2G is a graph providing the number of human CD34 + cells as determined by FACS per well.
  • Figure 2H is a graph showing the percentage of murine CD34-KSL cells after 5 days in culture.
  • CD34-KSL cells were cultured with different concentrations of recombinant murine Egfl7 for 5 days in alpha-MEM +TKF in 10% fetal bovine serum (FBS). The percentage of KSL cells was determined in retrieved cells after 5 days in culture. p* ⁇ 0.05; T-test.
  • Figure 3A- Figure 3D Show impaired proliferation of murine Egfl7 KD KSL cells. Egfl7 was knocked down in murine GFP+ CD34-KSL cells transduced with lentiviral shvector.
  • CD34-KSL cells were not infected with virus (mock) or infected with scramble shRNA (Scr) or lentivirus shvector to knockdown Egfl7 (KDEgfl7).
  • Figure 3B shows the percentage of CD34-KSL in recovered cells in vitro.
  • Figure 3D are graphs providing the percent of donor chimerism in the indicated cells after engraftment.
  • Recipient peripheral (PB) was analyzed after 8 weeks for the presence of donor CD45.1 cells.
  • Horizontal lines represent the mean engraftment levels for each group.
  • Figure 4A- Figure 4F Show Itgb3 inhibition amplifies Egfl7-mediated HSC expansion.
  • Figure 4C is a graph showing the frequency of KSL cells after 5 days culture of highly purified CD34 - KSL cells (>97%) with cytokines (TKF) in the presence of recombinant Egfl7 with Itgb3 inhibitor (CGD) or without (unt).
  • TKF cytokines
  • Figure 4D shows the DNA sequence of RGDdel (bottom sequence).
  • the top sequence is SEQ ID NO: 2
  • the bottom sequence is SEQ ID NO: 3
  • the codons encoding the deleted “RDG” sequence is SEQ ID NO: 4.
  • Figure 5A- Figure 5C Show Egfl7 through ⁇ 3 integrin (Itgb3) enhances c-Kit signaling and upregulates Egfl7 in HEL cells.
  • Figure 5B shows the phosphorylation levels of ITGB3 (P-Tyr747), STAT-3, JAK-2, AKT, and ERK1/2 as determined by Western blotting of Scr transfected and Itgb3 KD HEL cells cultured for 4 hrs on plates coated with recombinant Egfl7, Fibronectin (FN), or BSA as determined by Western blotting.
  • Figure 5C shows the protein expression of c-Kit, Egfl7, and FAK in Scr HEL or Itgb3 KD HEL cells cultured for 24 hrs in plates coated with recombinant Egfl7, FN, or BSA.
  • ⁇ -actin served as a loading control (2 independent experiments). Data shown represent means ⁇ s.e.m., P ⁇ 0.05. All comparisons were one-tailed t tests.
  • Figure 6A- Figure 6F Show c-Kit deficiency enhances Egfl7-driven HSC expansion.
  • Figure 6A and Figure 6B are graphs showing the increase in c-Kit expression by qPCR on HEL cells cultured with recombinant Egfl7 (Figure 6A), and on total BMMNCs ( Figure 6B) isolated from AdEgfl7 or AdNull treated mice.
  • c-Kit gene expression as fold increase compared to non-virus injected controls on BM cells by qPCR; Gene normalization using ⁇ -actin (B; n 2).
  • Figure 6E are graphs showing the in vitro KSL cell frequency (left graph) and absolute number (right graph) per total culture after FACS of BMMNCs derived from Kit w /Kit W-v and wt mice.
  • FIG. 6F is a graph showing the frequency of KSL cells.
  • Figure 7A- Figure 7G Show Egfl7 in the absence of Itgb3 activates Notch leading to HSC expansion
  • Figure 7D shows the percentage of cultured KSL cells.
  • Figure 7E is a Western blot of the indicated proteins. Mice were injected with AdEgfl7 or AdNull. BMMNCs of wildtype mice treated with or without vectors were isolated by Day 3. Two independent experiments.
  • FIG 7G is a Western blot of the intracellular domain Notch-IC (NICD) in BMMNCs of c-Kit, Itgb3 deficient, and control mice were injected with virus (AdEgfl7) or without (AdNull) at Day 5. Loading control: ⁇ -actin. Data shown represent means ⁇ s.e.m., P ⁇ 0.05. All comparisons were one-tailed t tests.
  • Figure 8A and Figure 8B Show Egfl7 is upregulated after irradiation and expands immature thymic progenitors.
  • C57/BL6 mice were injected intravenously (i.v.) with 2 x 10 9 PFU/mouse of AdEgfl7 or AdNull (no transgene) on Day 0.
  • Egfl7 expression was determined in liver cell lysates by Western blotting 3 days after injection.
  • Representative FACS blots of CD4 and CD8 stained thymocytes isolated from vector-treated and non-treated mice were obtained and analyzed. Values are the mean ⁇ SEM of duplicate data points.
  • Figure 9A- Figure 9D Show Egfl7 expands early thymic progenitors and thymic ECs after irradiation. Thymocytes were isolated from nonirradiated or irradiated mice injected with AdEgfl7 or AdNull.
  • Figure 9A is a graph showing the percentage of thymocyte subpopulations from nonirradiated mice.
  • the first bars are WT
  • the middle bars are AdNull
  • the last bars are AdEgfl7.
  • Figure 10A- Figure 10C Show enhanced Flt3-Flt3 ligand signaling after Egfl7 overexpression. Values are mean ⁇ SEM. *p ⁇ 0.05, ** p ⁇ 0.01 for all experiments.
  • Figure 10A is a graph showing the fold change in Egfl7, Flt3 ligand, Flt3, and Hes-1 mRNA induction measured by qPCR in thymocytes isolated from AdNull and AdEgfl7 or untreated animals at Day 3. Adenovirus-treated HUVEC cells were collected after 24 hrs. Proteins were immunoblotted with the indicated antibodies.
  • FIG 10B is a graph showing the amount of Flt3 ligand in plasma of mice.
  • Flt3 expression in indicated thymic cell populations isolated from AdEgfl7 treated mice with or without irradiation in vivo was determined by FACS.
  • FACS histograms showing Flt3 expression on CD44 + c-Kit + CD25 - ETPs, and Lin - c-Kit + CD31 + EC were also examined (data not shown).
  • Figure 11A- Figure 11C Show pharmacological inhibition of Flt3 prevents Egfl7-mediated ETP and EC expansion.
  • C57Bl/6 mice were injected with adenovirus expressing Egfl7 or no transgene and cotreated daily with Flt3 inhibitor. Values are mean ⁇ SEM. *p ⁇ 0.05, ** p ⁇ 0.01 for all experiments.
  • Figure 11C is a graph showing the percentage of CD44 + c-Kit + CD25 - ETP.
  • Epidermal growth factor-like domain-containing protein 7 binds stem cell-associated receptors such as the platelet-derived growth factor-beta receptor, integrin ⁇ 3 (Itgb3) receptors, and Notch receptors. Itgb3 and Notch receptors are expressed on hematopoietic stem and progenitor cells (HSPCs), including human HSPCs.
  • HSPCs refer to hematopoietic stem cells (HSCs) and/or hematopoietic progenitor cells (HPCs).
  • HPCs include early thymic progenitor cells (ETPs).
  • Egfl7 in the presence of ⁇ 3 integrin (Itgb3) increases stem cell proliferation and differentiation by blocking Notch signaling.
  • Egfl7 in the absence of Itgb3 enhances stem cell self-renewal by activating Notch signaling.
  • knockdown of Egfl7 with miR126 (which is located in intron 7 of Egfl7) in stem cells led to impaired HSC survival by downregulating c-Kit expression on stem cells.
  • overexpression of Egfl7 led to stem cell expansion in Itgb3 + cells.
  • the experiments herein indicate that (1) when Egfl7 is added to stem cells, the stem cells proliferate and differentiate in vivo and in vitro; (2) when a Notch activator, such as Egfl7, plus an Itgb3 inhibitor are added to stem cells, the stem cells proliferate but do not differentiate, and the stem cells exhibit long-term hematopoietic reconstitution after engraftment. That is, the experiments herein indicate that the combination of Egfl7 and activation of the Itgb3 signaling pathway blocks the Notch signaling pathway and leads to stem cell proliferation and differentiation, whereas the combination of Egfl7 plus (a) an Itgb3 inhibitor activates the Notch pathway and leads to stem cell proliferation without differentiation.
  • the present invention provides methods of inducing stem cells, such as HSPCs, to proliferate and differentiate, which comprise increasing Egfl7 expression in the stem cells and/or contacting the stem cells with an amount of exogenous Egfl7.
  • the present invention provides methods of maintaining stem cells in an undifferentiated state (e.g., in the G0 cell cycle phase) or inducing the stem cells to proliferate without differentiation, which comprise (a) increasing Egfl7 expression in the stem cells, contacting the stem cells with one or more Egfl7 proteins, and/or contacting the stem cells with a Notch activator other than an Egfl7 protein, and (b) inhibiting the Itgb3 signaling pathway by contacting the stem cells with an Itgb3 inhibitor.
  • the stem cells are contacted with an Itgb3 activator.
  • the stem cells are induced to proliferate and differentiate in vitro.
  • the expanded stem cells are transplanted in a subject, and then the stem cells are induced to differentiate in vivo by increasing Egfl7 expression in the transplanted stem cells, administering to the subject a therapeutically effective amount of an Egfl7 protein (such as human Egfl7) and/or an Itgb3 activator.
  • an Egfl7 protein such as human Egfl7
  • stem cells which may be endogenous, allogenic, or autologous to a subject, are induced to proliferate without differentiation in vivo in the subject by (a) increasing Egfl7 expression in the stem cells, administering a therapeutically effective amount of Egfl7 to the subject, and/or administering a therapeutically effective amount of a Notch activator (other than an Egfl7 protein) to the subject, and (b) administering a therapeutically effective amount of an Itgb3 inhibitor to the subject.
  • the stem cells after the stem cells are expanded in the subject, the stem cells are induced to differentiate by ceasing administration of the Itgb3 inhibitor, and optionally administering a therapeutically effective amount of an Itgb3 activator to the subject.
  • Notch activators in addition to Egfl7 proteins, include: Delta like canonical Notch ligand 1 (Dll1) (NP_005609 or AAG09716); Jagged-1 (AAC52020.1); Delta1ext IgG (see Varnum-Finney et al.
  • Itgb3 inhibitors include: small molecule inhibitors (e.g., JSM6427, Tirofiban, GR 144053 trihydrochloride); Cilengitide (EDM121974, Merck Darmstad); agents that block or antagonize the RGD binding site of Itgb3 (e.g., GLPG0187, GRGDSP peptide, Eptifibatide, P11 (CAS 848644-86-0), Integrin Antagonists 27 (CAS 593274-97-6)), antibodies that block or antagonize the RGD binding site of Itgb3 (e.g., LM609 (#ab190147, Abcam), 25E11 (#MAB1957, Millipore Sigma), 27.1 (VNR-1) (#MAB1876, Millipore Sigma), PM6/13 (#CBL479, Millipore Sigma), and 23C6 (#ab20143, Abcam); siRNAs (e.g., sc-29375, shRNA plasmi
  • Itgb3 activators include: fibronectin, vitronectin, thrombospondin, pleiotrophin, and the like.
  • Stem cells are generally categorized as embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), cord blood and amniotic fluid stem cells.
  • stem cells include HSPCs, CD34+ cells obtained from tissues, iPSCs made by transducing somatic cells with one or more reprogramming factors such as transcription factors Oct4 (Pou5f1), Sox2, cMyc, Klf4, and the like.
  • Such stem cells may be obtained from bone marrow (BM), umbilical cord blood, placental blood, peripheral blood, placental tissues, Wharton's jelly, and fetal or neonatal blood.
  • Specific examples of stem cells include early thymic progenitor cells, thymocytes, and erythroid progenitor cells.
  • the stem cells are human stem cells.
  • Egfl7 on cells such as stem cells
  • Egfl7 expression vectors may be used to overexpress Egfl7 in target cells.
  • the subject may be administered an Egfl7 expression vector that expresses Egfl7 or a mutated Egfl7 protein that lacks the RGD domain of the corresponding wildtype Egfl7 protein.
  • a mutant RGDdel protein has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1.
  • a mutant RGDdel protein comprises or consists of SEQ ID NO: 1.
  • the present invention provides methods of maintaining stem cells in an undifferentiated state (e.g., in the G0 cell cycle phase) or inducing the stem cells to proliferate without differentiation, which comprise contacting the stem cells with a mutant RGDdel protein.
  • the methods described herein may be used to expand cells, such as stem cells and HSPCs, in vitro for allogenic or autologous transplantation in subjects.
  • Subjects who may be in need of such treatment include those suffering from hematological diseases, cancers, immunodeficiencies, and the like.
  • stem cells obtained from umbilical cord blood (UCB) and expanded in vitro in an undifferentiated state and then the expanded cells may be transplanted in a subject in need thereof.
  • the methods described herein are used to expand engineered T cells, which may express chimeric antigen receptors against given antigens such as tumor-associated antigens and HIV antigens, and then the expanded cells are transplanted in a subject.
  • the cells to be transplanted in a subject are contacted with exogenous Egfl7 by, e.g., culturing the cells in culture medium that is supplemented with Egfl7 or by overexpressing Egfl7 in feeder layer cells (e.g., endothelial cells like (human umbilical vein endothelial cells (HUVECs) or fibroblasts) using an Egfl7 expression vector.
  • feeder layer cells e.g., endothelial cells like (human umbilical vein endothelial cells (HUVECs) or fibroblasts
  • expression of , the cells to be transplanted in a subject are contacted with a mutant RGDdel protein.
  • HSPCs such as ETPs and ECs in subjects suffering from irradiation-induced myelosuppression
  • the methods disclosed herein may be used to treat subjects suffering from irradiation-induced myelosuppression, HSC associated diseases such as sickle cell anemia, T cell deficiencies resulting from HIV infection, chemotherapy, organ-transplantation, bone marrow transplantation, and aging.
  • the treatment methods comprise increasing Egfl7 expression in the HSPCs in the subject and/or administering to the subject a therapeutically effective amount of one or more Egfl7 proteins.
  • the treatment methods comprise administering to the subject a therapeutically effective amount of one or more mutant RGDdel proteins.
  • the treatment methods further comprise administering to the subject a therapeutically effective amount of an Itgb3 inhibitor.
  • cells, e.g., stem cells, in a subject are induced to proliferate in vivo by administering to the subject an exogenous amount of Egfl7 or a mutated Egfl7 protein that lacks the RGD domain of the corresponding wildtype Egfl7 protein.
  • cells, e.g., stem cells, in a subject are induced to proliferate in vivo by administering to the subject an expression vector that expresses Egfl7 or a mutant RGDdel protein.
  • the cells in a subject that are induced to proliferate are endogenous cells.
  • the cells in a subject that are induced to proliferate are exogenous cells that have been transplanted in the subject.
  • the exogenous cells are autologous.
  • the exogenous cells are allogenic.
  • the exogenous cells are syngeneic.
  • kits comprising one or more Notch activators (e.g., Egfl7 proteins such as human Egfl7 or a mutant RGDdel protein) and
  • compositions of the present invention include one or more Egfl7 therapeutic agents.
  • an “Egfl7 therapeutic agent” refers to a compound or composition that increases the expression of Egfl7 in stem cells (such as HSPCs) and Egfl7 proteins (such as human Egfl7 (NP_958854) and analogs and homologs thereof, and mutant RGDdel proteins).
  • the compositions comprise a mutant RGDdel protein.
  • the compositions comprise a purified or concentrated amount of one or more Egfl7 proteins such as human Egfl7.
  • the compositions may further comprise an Itgb3 inhibitor.
  • compositions may further comprise a Notch activator.
  • compositions according to the present invention further comprise Flt-3 ligand, Kit ligand (also known as Stem cell factor (SCF)), one or more growth factors, Garcinol, and/or one or more histone acetyltransferase inhibitors.
  • growth factors include: Bone morphogenetic proteins (BMPs), Ciliary neurotrophic factor (CNTF), Leukemia inhibitory factor (LIF), Colony-stimulating factors (e.g. Macrophage colony-stimulating factor (m-CSF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF)), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factors, GDNF family of ligands (e.g., Glial cell line-derived neurotrophic factor (GDNF), Neurturin, Persephin, Artemin), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin and Insulin-like growth factors, Interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7
  • composition refers to a composition suitable for pharmaceutical use in a subject.
  • a pharmaceutical composition generally comprises an effective amount or a therapeutically effective amount of an active agent, e.g., one or more Egfl7 therapeutic agents (such as human Egfl7 and/or a mutant RGDdel protein), and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions according to the present invention may further include one or more supplementary agents. Examples of suitable supplementary agents include Itgb3 inhibitors, myelosuppressive agents, growth factors, cytokines, and the like.
  • myelosuppressive agents include Peginterferon alfa-2a, Interferon alfa-n3, Peginterferon alfa-2b, Aldesleukin, Gemtuzumab ozogamicin, Interferon alfacon-1, Rituximab, Ibritumomab tiuxetan, Tositumomab, Alemtuzumab, Bevacizumab, L-Phenylalanine, Bortezomib, Cladribine, Carmustine, Amsacrine, Chlorambucil, Raltitrexed, Mitomycin, Bexarotene, Vindesine, Floxuridine, Tioguanine, Vinorelbine, Dexrazoxane, Sorafenib, Streptozocin, Gemcitabine, Teniposide, Epirubicin, Chloramphenicol, Lenalidomide, Altretamine, Zidovudine, Cisplatin
  • Egfl7 therapeutic agents such as human Egfl7 and/or a mutant RGDdel protein
  • a subject is mammalian, more preferably, the subject is human.
  • Preferred pharmaceutical compositions are those comprising at least one Egfl7 therapeutic agent (such as human Egfl7 and/or a mutant RGDdel protein) in a therapeutically effective amount and a pharmaceutically acceptable vehicle.
  • an “effective amount” refers to a dosage or amount sufficient to produce a desired result.
  • the desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount, e.g., long-term survival, effective prevention of a disease state, and the like.
  • a “therapeutically effective amount” refers to an amount that may be used to treat, prevent, or inhibit a given disease or condition, in a subject as compared to a control, such as a placebo. Again, the skilled artisan will appreciate that certain factors may influence the amount required to effectively treat a subject, including the degree of the disease or condition, previous treatments, the general health and age of the subject, and the like. Nevertheless, therapeutically effective amounts may be readily determined by methods in the art.
  • a therapeutically effective amount of an Egfl7 protein ranges from about 0.01 to about 10 mg/kg body weight, about 0.01 to about 3 mg/kg body weight, about 0.01 to about 2 mg/kg, about 0.01 to about 1 mg/kg, or about 0.01 to about 0.5 mg/kg body weight for parenteral formulations.
  • Therapeutically effective amounts for oral administration may be up to about 10-fold higher. It should be noted that treatment of a subject with a therapeutically effective amount may be administered as a single dose or as a series of several doses. The dosages used for treatment may increase or decrease over the course of a given treatment.
  • Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using dosage-determination tests and/or diagnostic assays in the art. Dosage-determination tests and/or diagnostic assays may be used to monitor and adjust dosages during the course of treatment.
  • compositions of the present invention may be formulated for the intended route of delivery, including intravenous, intramuscular, intra peritoneal, subcutaneous, intraocular, intrathecal, intraarticular, intrasynovial, cisternal, intrahepatic, intralesional injection, intracranial injection, infusion, and/or inhaled routes of administration using methods known in the art.
  • compositions according to the present invention may include one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • adjuvants e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents
  • liposomal formulations e.g., nanoparticles, dispersions, suspensions, or emulsions
  • sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular Egfl7 therapeutic agent used.
  • a “pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration.
  • UDP-NF National Formulary
  • unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration.
  • Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington: The Science and Practice of Pharmacy. 20th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference.
  • compositions of the present invention may be provided in dosage unit forms.
  • a “dosage unit form” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the one or more Egfl7 therapeutic agents calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the given Egfl7 therapeutic agent and desired therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of Egfl7 therapeutic agents according to the instant invention and compositions thereof can be determined using cell cultures and/or experimental animals and pharmaceutical procedures in the art. For example, one may determine the lethal dose, LC 50 (the dose expressed as concentration x exposure time that is lethal to 50% of the population) or the LD 50 (the dose lethal to 50% of the population), and the ED 50 (the dose therapeutically effective in 50% of the population) by methods in the art.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Egfl7 therapeutic agents which exhibit large therapeutic indices are preferred. While Egfl7 therapeutic agents that result in toxic side-effects may be used, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and, thereby, reduce side-effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
  • Preferred dosages provide a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • Therapeutically effective amounts and dosages of one or more Egfl7 therapeutic agents can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a dosage suitable for a given subject can be determined by an attending physician or qualified medical practitioner, based on various clinical factors.
  • cell “expansion” refers to an increase in an amount of cells by cell proliferation.
  • sequence identity refers to the percentage of nucleotides or amino acid residues that are the same between sequences, when compared and optimally aligned for maximum correspondence over a given comparison window, as measured by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).
  • Software for performing BLAST (e.g., BLASTP and BLASTN) analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov).
  • the comparison window can exist over a given portion, e.g., a functional domain, or an arbitrarily selection a given number of contiguous nucleotides or amino acid residues of one or both sequences. Alternatively, the comparison window can exist over the full length of the sequences being compared. For purposes herein, where a given comparison window (e.g., over 80% of the given sequence) is not provided, the recited sequence identity is over 100% of the given sequence. Additionally, for the percentages of sequence identity of the proteins provided herein, the percentages are determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and the default parameters available at blast.ncbi.nlm.nih.gov/Blast.cgi. See also Altschul, et al. (1997), Nucleic Acids Res. 25:3389-3402; and Altschul, et al. (2005) FEBS J. 272:5101-5109.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.
  • protein protein
  • polypeptide peptide
  • peptide are used interchangeably to refer to two or more amino acids linked together. Groups or strings of amino acid abbreviations are used to represent peptides. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus.
  • Mutant RGDdel proteins may be made using methods known in the art including chemical synthesis, biosynthesis or in vitro synthesis using recombinant DNA methods, and solid phase synthesis. See, e.g., Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol. 12, J. K. Setlow ed., Plenum Press, NY, pp. 1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2ed. Pierce, Rockford, IL, which are herein incorporated by reference.
  • Mutant RGDdel proteins may be purified using protein purification techniques known in the art such as reverse phase high-performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See, e.g., Olsnes and Pihl (1973) Biochem. 12(16):3121-3126; and Scopes (1982) Protein Purification, Springer-Verlag, NY, which are herein incorporated by reference.
  • mutant RGDdel proteins may be made by recombinant DNA techniques known in the art.
  • polynucleotides that encode mutant RGDdel proteins are contemplated herein.
  • the polypeptides and polynucleotides are isolated.
  • an “isolated” compound refers to a compound that is isolated from its native environment.
  • an isolated polynucleotide is a one which does not have the bases normally flanking the 5’ end and/or the 3’ end of the polynucleotide as it is found in nature.
  • an isolated polypeptide is one which does not have its native amino acids, which correspond to the full-length polypeptide, flanking the N-terminus, C-terminus, or both.
  • an isolated fragment of Egfl7 refers to an isolated polypeptide that consists of only a portion of Egfl7 or comprises some, but not all, of the amino acid residues of Egfl7, and may include non-native amino acids, i.e., amino acids that are different from the amino acids found at the corresponding positions of Egfl7, at its N-terminus, C-terminus, or both.
  • Egfl7 therapeutic agents are substantially purified.
  • a “substantially purified” compound refers to a compound that is removed from its natural environment and/or is at least about 60% free, preferably about 75% free, and more preferably about 90% free, and most preferably about 95-100% free from other macromolecular components or compounds with which the compound is associated with in nature or from its synthesis.
  • antibody refers to naturally occurring and synthetic immunoglobulin molecules and immunologically active portions thereof (i.e., molecules that contain an antigen binding site that specifically bind the molecule to which antibody is directed against).
  • antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments.
  • molecules which are described by the term “antibody” herein include: single chain Fvs (scFvs), Fab fragments, Fab’ fragments, F(ab’)2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • a compound e.g., receptor or antibody “specifically binds” a given target (e.g., ligand or epitope) if it reacts or associates more frequently, more rapidly, with greater duration, and/or with greater binding affinity with the given target than it does with a given alternative, and/or indiscriminate binding that gives rise to non-specific binding and/or background binding.
  • a given target e.g., ligand or epitope
  • background binding refer to an interaction that is not dependent on the presence of a specific structure (e.g., a given epitope).
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals.
  • the subject is a mammal. In some embodiments of the present invention, the subject is a human.
  • diagnosis refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g., paper or electronic media), another party, e.g., a patient, of the diagnosis.
  • prognosis refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g., paper or electronic media), another party, e.g., a patient, of the prognosis.
  • any subset of A, B, C, and D for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).
  • composition comprises or consists of A
  • the phrase “comprises or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A or consists of A.
  • the sentence “In some embodiments, the composition comprises or consists of A” is to be interpreted as if written as the following two separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists of A.”
  • a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself.
  • the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C.” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.”
  • Example 1 - HSCs Express Egfl7 Epidermal growth factor-like domain 7 (Egfl7) affects endothelial cells (ECs), neuronal and embryonic stem cell behavior.
  • Egfl7 Epidermal growth factor-like domain 7
  • ECs endothelial cells
  • marrow niche To define the hematopoietic and stromal cell types within the bone marrow niche, primary cells and cell line were tested by quantitative polymerase chain reaction (qPCR).
  • High Egfl7 expression was detected on the human bone marrow microvascular endothelial cell line, BMEC-1; human umbilical vein endothelial cells (HUVECs, positive control); the human macrophage cell line, THP-1; and the human erythroleukemica cell line, HEL, while low Egfl7 expression was found on the murine stromal cell line, MS-5 ( Figure 1A).
  • qPCR analysis of murine primary bone marrow (BM) cells revealed that Egfl7 expression was highest in the most primitive hematopoietic stem cell (HSC) fraction, the CD34 - c-Kit + Sca1 + Lin neg (KSL) cells, and was gradually downregulated on FACS-purified hematopoietic progenitors like common lymphoid progenitors (CLP), megakaryocyte erythroid progenitors (MEP), common myeloid progenitors (CMP), and granulocyte macrophage progenitors (GMP), which were identified by the antigen marker profile and total BM (Figure 1B). Egfl7 protein expression on CD34-KSL cells was confirmed on FACS-sorted KSL cells by immunofluorescence (data not shown).
  • Egfl7 can be up-regulated within the BM after myelosuppressive stress.
  • C57/Bl6 mice were treated with 5-FU.
  • Egfl7 mRNA expression was high in BM cells ( Figure 1C).
  • BMMNCs BM mononuclear cells
  • Egfl7 is an EC-derived factor, that can block Notch signaling on neuronal stem cells.
  • ECs express Notch ligands that promote proliferation and prevent exhaustion of long term-HSCs
  • KSL expansion was quantified after Egfl7 treatment in EC-co-cultures ( Figure 2D). KSL expansion was high in EC co-cultures, but KSL expansion was further amplified in Egfl7 supplemented EC co-cultures ( Figure 2D).
  • Egfl7 expanded KSL cells To further characterize Egfl7 expanded KSL cells, various progenitor and stem cell assays were carried out. Administration of recombinant Egfl7 augmented the frequency of BM-derived GMP and MEP progenitor sub-fractions (Figure 2E).
  • HSCs are normally maintained in an undifferentiated state (e.g., in the G0 cell cycle phase), with this quiescence protecting the cells against loss of self-renewal capacity.
  • Cell-cycle analysis revealed that more KSL cells derived from Egfl7-treated mice were in the S-phase (15% of KSL cells derived from recombinant Egfl7-treated mice) of the cell cycle, while only 3% of control KSL cells were positive in S-phase of the cell cycle (Hoechst blue positive) indicating that Egfl7 enhances cell cycle entry (Figure 2F).
  • Egfl7 is a growth factor for human CD34 + cells
  • cord blood derived CD34 + cells were placed alone or with various concentrations of murine recombinant Egfl7 in cultures with the cytokine cocktail Thrombopoietin, Kit ligand, and FMS-like tyrosine kinase 3 (TKF).
  • Recombinant Egfl7 at a concentration of 300 ng/ml increased the number of CD34 - KSL cells in 5-day cultures ( Figure 2G), and expanded murine CD34 - KSL cells (Figure 2H).
  • Example 3 - Egfl7 Increases In Vitro Survival of HSCs To functionally explore the role of endogenous Egfl7 in regulating HSCs fate, shRNA lentivirus strategies were applied to knockdown (KD) Egfl7 in KSL cells.
  • CD34-KSL cells were transduced with shRNA targeting Egfl7 or scramble siRNA (Scr).
  • Scr scramble siRNA
  • CD34-KSL cells were resorted and were subjected to in vitro assays or transplantation in mice. An aliquot of transduced cells was maintained and analyzed for transduction efficiency. Mean transduction efficiency was 60%.
  • Egfl7 KD in GFP + resorted KSL cells after 4 days culture was confirmed by qPCR (Figure 3A). After the 4 days culture, the number of recovered Egfl7 KD KSL cells was lower when compared to Scr KSL cells ( Figure 3B). Less colonies generated after 10 days in methylcellulose-based cultures were found when Egfl7 KD KSL cells had been plated ( Figure 3C). These data suggest that Egfl7 increases survival of HSCs.
  • CRU competitive repopulation units
  • Egfl7 KD or Scr KSL cells were also transplanted into ablated Ly5.1 mice with 1 x 10 5 competitor BMMNCs.
  • Recipient peripheral blood (PB) was analyzed for test cell versus competitor cell contribution 8 weeks after transplantation.
  • Egfl7 KD KSL cells were transplanted into wild-type recipient mice.
  • Egfl7 KD and Scr KSL cells showed a similar CRU ability with three-lineage cell differentiation potential and normal KSL recovery (Figure 3D), indicating that the observed survival disadvantage of Egfl7 KD KSL cells in vitro was compensated in vivo.
  • Example 4 Egfl7-Mediated HSC Expansion Requires Itgb3 Signaling Egfl7 enhances Itgb3 signaling on ECs.
  • Treatment of HEL cells with recombinant human Egfl7 increased Itgb3 mRNA expression after 24 hrs ( Figure 4A), indicating that Egfl7 upregulates one of its own receptors.
  • Murine BM-derived CD34-KSL cells express Itgb3 as determined by FACS (data not shown).
  • the percentage of Itgb3 expressing KSL cells increased in BMMCs by Day 5 after AdEgfl7 injection in mice ( Figure 4B).
  • Egfl7 can bind to platelet derived growth factor-receptor ⁇ , Itgb3, or Notch receptors.
  • HSCs like murine KSL cells express at the two Egfl7 receptors Itgb3 and Notch1, and 2. Often integrins recognize the Arg-Gly-Asp motif (RGD) in their ligands.
  • RGDdel Arg-Gly-Asp motif
  • SEQ ID NO: 1 YRPGRRVCAVRAHGDPVSESFVQRVYQPFLTTCDGHRACSTYRTIYRTAYRRSPGLAPARPRYACCPGWKRTSGLPGACGAAICQPPCRNGGSCVQPGRCRCPAGWTCQSDVDERSARRGGCPQRCINTAGSYWCQCWEGHSLSADGTLCVPKGGPPRVAPNPTGVDSAMKEEVQRLQSRVDLLEEKLQLVLAPLHSLASQALEHGLPDPGSLLVHSFQQLGRIDSLSEQISFLEEQLGSCSCKKDS
  • Figure 4D aligns the wildtype sequence and the AdRGDdel mutant sequence to exemplify the RDG deletion as follows:
  • RGD Deletion (codons encoding the RGD domain) SEQ ID NO: 4: CGGGGTGAC
  • Egfl7 full-form and fibronectin (FN, a known ligand for Itgb3), but not the Egfl7 deletion mutant protein (RGDdel) coating of culture plates enhanced adhesion of Lin- cells ( Figure 4E), indicating that the newly generated RGDdel protein was unable to bind KSL cells, but other Itgb3 ligands could ( Figure 4E).
  • FN fibronectin
  • RGDdel Egfl7 deletion mutant protein
  • Example 5 Egfl7 Enhances c-Kit Signaling in Itgb3 Expressing Cells It was hypothesized that Egfl7 binding to Itgb3 regulates GATA-2 and c-Kit expression.
  • the highly expressing Itgb3 erythroleukemic cell line HEL was chosen for analysis to further delineate the mechanism how Itgb3 (also known as CD61) alters cell proliferation of Egfl7.
  • Itgb3 KD was achieved in HEL cells using siRNA. Itgb3 KD was confirmed in transfected cells by qPCR and Western blot (Figure 5A and Figure 5B). Indeed, Itgb3 KD HEL cells showed a decreased GATA-2 ( Figure 5A) and c-Kit expression ( Figure 5B) when compared to Scr HEL cells by qPCR and/or Western blotting.
  • HEL cells were cultured on FN (positive control for Itgb3 activation), BSA (bovine serum albumin, negative control), or recombinant Egfl7 (Figure 5B and Figure 5C).
  • FN positive control for Itgb3 activation
  • BSA bovine serum albumin, negative control
  • Egfl7 Figure 5B and Figure 5C
  • Cells cultured in the presence of Egfl7 and FN, but not BSA (control) showed enhanced Itgb3 signaling as determined by Tyr747 phosphorylation of human Itgb3 by Western blotting (Figure 5B).
  • Itgb3 KD HEL cells when compared to Scr HEL cells showed lower expression of Egfl7 on the mRNA and protein level ( Figure 5A and Figure 5B). This suggests that Egfl7 amplifies itself in an Itgb3 dependent manner (autocrine amplification loop).
  • Example 6 Egfl7 Upregulates c-Kit Expression and Enhances HSC Survival
  • c-Kit expression was determined in hematopoietic cells after in vitro and in vivo exposure to Egfl7.
  • c-Kit expression was upregulated on Egfl7-treated HEL cells ( Figure 6A).
  • BM cells from AdEgfl7-treated, but not from AdNull-treated mice showed higher c-Kit expression ( Figure 6B). Higher c-Kit expression was found on KSL cells derived from AdEgfl7-treated, but not AdNull-treated mice as determined by FACS ( Figure 6C).
  • Kit-Kit W-v mice have a mutation in the c-Kit receptor leading to a functionally inactive c-Kit receptor, which allows mice to survive.
  • Egfl7 and mutant RGDdel proteins enhance stem cell expansion rather than cell differentiation when c-Kit signaling is blocked. These data indicate that Egfl7 biases KSL cells to self-renew rather than differentiation in the absence of c-Kit.
  • Example 7 - Egfl7 in the Absence of Itgb3 Activates Notch and Flt Signaling in HSCs As disclosed herein, Egfl7 mediates HSC self-renewal and expansion in the absence of Itgb3. Egfl7 antagonizes Notch receptor/ligand interaction by either binding to the receptor or its corresponding ligand in certain cells. Egfl7 can antagonize Notch signaling in neuronal stem cells. Notch1 receptor is expressed on immature CD34 + hematopoietic lymphoid, myeloid, and erythroid precursors.
  • Hes-1 Hairy enhancer of split-1
  • GSI gamma secretase inhibitor
  • FMS-like tyrosine kinase 3 (Flt3) gene expression was found in total BMMNCs of AdEgfl7 treated mice, and on KSL cells extracted 5 days after virus injection as determined by qPCR and by FACS analysis ( Figure 7C).
  • Flt3 inhibitor treatment prevented Egfl7-mediated KSL expansion ( Figure 7D), indicating Flt3 signaling is important for Egfl7-mediated HSC expansion in vitro.
  • Notch-IC is translocated into the nucleus and forms a complex with the DNA-binding protein RBPj and induces expression of downstream effectors such as the transcriptional repressor genes Hes1 and Hes5.
  • Blockade of Notch signaling was found in AdEgfl7 derived wildtype mice, as evidenced by downregulation of Hes-1 and NICD in BMMNCs and KSL cells ( Figure 7F- Figure 7G).
  • Egfl7 blocked Notch signaling in wildtype (wt) cells or c-Kit -/- BMMNCs as demonstrated by impaired Hes-1 mRNA expression and a reduction in NICD using Western blot analysis ( Figure 7E- Figure 7G). In contrast, Egfl7 in Itgb3 -/- cells activated Notch signaling ( Figure 7G).
  • Egfl7 is expressed at rather low levels under steady state conditions in the BM niche. Endogenous Egfl7 expression in HSC maintains high c-Kit expression, while Flt3 expression is rather low. Following stress like myelosuppression, Egfl7 increases in the niche, which not only can amplify its own expression, but also upregulates Itgb3 on HSC. Dependent on the Itgb3 expression status of the cell, Egfl7 changes the expression of stem cell-active receptor like c-Kit and Flt3. Egfl7 in Itgb3 positive HSCs up-regulates c-Kit and Flt3 expression, while blocking Notch signaling, which ultimately drives HSC expansion, and differentiation. In contrast, in low expressing Itgb3 positive HSCs, a characteristic of quiescent HSC, Egfl7 activates the Notch pathway causing HSC expansion and self-renewal.
  • Example 9 Egfl7 Augments Early Thymic Progenitors and ECs
  • C57/BL6 mice were injected with AdEgfl7 or AdNull intravenously.
  • Egfl7 overexpression was confirmed in liver cell lysates from adenovirus-injected mice by Western blotting.
  • Thymic T cell differentiation that can be divided into discrete stages characterized by the expression pattern of CD4 and CD8 was evaluated.
  • CD4 and CD8 double negative (DN) cells are the early T cell progenitors, that can differentiate into CD4 and CD double positive (DP) and then give rise to CD4 or CD8 single-positive (SP) cells.
  • the increase in the DN fraction was due to an accumulation of DN1 thymocytes (Figure 9A) that also harbors the most primate thymic progenitor ETP population (Lin low CD44 + CD25 - c-kit + ), a fraction that was increased in thymocytes of AdEgfl7-treated mice ( Figure 9B).
  • Example 11 Egfl7 Upregulates Flt3 Receptor on ECs Whether Egfl7 mediates cellular changes in the thymus by altering the Flt3/Flt3-ligand (Flt3L) pathway was investigated. Thymic tissues retrieved from mice after AdEgfl7 injection showed high Egfl7, Flt3 and Flt3L, but low Hes1 expression by qPCR ( Figure 10A). Egfl7 and Flt3 up-regulation and Hes1 down-regulation was confirmed by Western blotting in AdEgfl7 infected HUVECs (data not shown).
  • Flt3 expression increased in thymic ETP, and ECs derived from AdEgfl7 treated or irradiated mice as determined by FACS ( Figure 10C).
  • Example 12 - Egfl7 Expands ETP and ECs by Activating Flt3 Signaling
  • Flt3 inhibition prevented Egfl7-mediated ETP and thymic EC expansion in vivo ( Figure 11A and Figure 11B).
  • Egfl7 may enhance the lympho-stromal signaling that may be important for thymus organogenesis and regeneration for maintaining a pool of thymic progenitor cells.
  • Egfl7 induced in cells e.g., after irradiation blocks intracellular Notch signaling in the cells and thereby leads to expansion of ETPs and ECs through upregulation of Flt3 receptor the release of Flt3 ligand.
  • mice C57BL/6 mice (6-8 weeks old) were purchased from SLC, Inc. (Shizuoka, Japan). Animal experiments were conducted in accordance with the guidelines and approval of the Institutional Animal Care and Usage Committee at the Institute of Medical Science, The University of Tokyo. WBB6F1/Kit-Kitw-v/Slc (c-Kit -/- ) and wildtype control mice were purchased from Nihon Clear. Itgb3-/- mice on a c57/Bl6 background were kindly provided by Terumasa Umemoto (Kumamoto University, Japan). All human cell-associated experiments were approved under a protocol from the ethics committee of The Institute of Medical Science, The University of Tokyo.
  • mice Groups of whole-body irradiated mice (6-8 wks old; 2 Gy using 137 C) were given AdEgfl7 or AdNull in the tail vein. Thymic recovery was determined 3 days after irradiation of the mice. At Day 0, mice were irradiated with a single dose of 2 Gy.
  • Adenoviral vectors expressing Egfl7 or containing no transgene were kindly provided by Matthias Friedrich and Dirk Dikic (Institute of Biochemistry II, Johann Wolfgang Goethe University School of Medicine) (Picuric et al., 2009; Schmidt et al., 2009).
  • this replication-deficient adenovirus is based on adenovirus type 5, which lacks the EIA, E1B, and E3 regions of the virus and contains the SRa promoter, human Egfl7 cDNA, and SV40 poly(A) signal sequences inserted into the El-deleted region.
  • AdEgfl7 or AdNull was co-injected with the ⁇ 3 inhibitor Itgb3 (EMD 121974) (Selleckchem) by i.p. at a concentration of 1 mg/kg for three times per week.
  • mice received Flt3 inhibitor (Tandutinib) (30 mg/kg) or PBS orally daily at Days 0, 1, and 2.
  • c-Kit + Sca-1 + Lin - (KSL) cells were sorted and tranduced using shRNAs targeting Egfl7 and cultured in 96-well plates (4 x 10 2 cells per well) with serum-free medium (S-CLONE SF-O3; Iwai North America Inc., Cat. No. SJU: 1303) supplemented with thrombopoietin (TPO, 50 ng/mL), Kit ligand (KitL, 100 ng/mL), and Flt-3 ligand (Flt-3L, 50 ng/mL) (PeproTech, Rocky Hill, NJ).
  • S-CLONE SF-O3 serum-free medium
  • Kit ligand Kit ligand
  • Flt-3 ligand Flt-3L, 50 ng/mL
  • PBMCs were cultured further for 4 days.
  • the lentivirus-transfected KSL cells (2 x 10 2 ) were mixed with normal BM cells (5 x 10 5 ) and transplanted into lethally irradiated mice (9.5 Gy) retro-orbital.
  • Peripheral blood cells of the recipient mice were analyzed 2 months after transplantation.
  • the percentage of donor-derived lineage contributions in PBMCs was assessed using antibodies against CD45.2, Gr-1/CD11b, B220, CD19, CD4, and CD8. Total chimerism of more than 1% for all antibodies tested using PBMCs was considered as long-term reconstitution.
  • the frequencies were determined using FlowJo software (TreeStar, Ashland, OR).
  • HEL cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin (P/S). HEL cells (1 x 10 5 ) were seeded in 24-well plates for 24 hours (hrs). HEL cells were transiently transfected by using Lipofectamine RNAiMAX (Invitrogen).
  • IMDM Modified Dulbecco’s Medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • siRNA targeting ⁇ 3 integrin was designed using BLOCK-iT TM RNAi Designer (Invitrogen) and the sense and antisense sequences are as follows:
  • Itgb3 Sense SEQ ID NO: 5 CCAAGACUCAUAUAGCAUU
  • Control Antisense SEQ ID NO: 8 AAUUCUCGUAUAUGAGUGG
  • shRNAs was designed using BLOCK-iT TM RNAi Designer.
  • shRNAs targeting Egfl7 Fasmac
  • Egfl7 Top Strand shRNA SEQ ID NO: 9: CACCGCTTGTGGAGCAGCAATATGCCGAAGCATATTGCTGCTCCACAAGC
  • Egfl7 Bottom Strand shRNA SEQ ID NO: 10: AAAAGCTTGTGGAGCAGCAATATGCTTCGGCATATTGCTGCTCCACAAGC
  • Bottom Strand Scramble SEQ ID NO: 12: AAAAGGAGACGGAGGATAGTCTTTTCGAAGACTATCCTCCGTCTCC were cloned into CS-Ubc-GFP vector. Gene knockdown efficiency in LSK cells was quantified by qRT-PCR.
  • Vesicular stomatitis virus glycoprotein-pseudotyped lentivirus was prepared as previously described via a three-plasmid system (Target vector, pMDL, and vesicular stomatitis virus glycoprotein envelope plasmid) by co-transfection of 293T cells using polyethylenimine (PEI, Takara, Japan). Viral supernatant was collected 48 hrs later, cleared, and stored at -80 °C. Viral titration was performed using 293T cells.
  • Adhesion Lin- cells were cultured for 4 hrs on the described pre-coated decellularized 96 well microplates (IWAKI, Japan). Then, cells were fixed with 4% formaldehyde in PBS for 10 minutes (mins), followed by a washing step using 0.1% BSA/Dulbecco’s Modified Eagle’s Medium (DMEM) buffer. Then, cells were stained with 0.5% crystal violet for 10 mins, washed with tap water. 2% SDS was added to dried plates. The plate was incubated at room temperature (RT) for 30 mins, and the absorbance was detected at 550 nm using a microplate reader (Molecular Devices, USA).
  • RT room temperature
  • Thymi were minced, and thymus single cell suspensions were blocked with 2% FBS, washed and stained.
  • Murine BM cells were obtained after flushing femurs and tibiae.
  • Murine KSL analysis by FACS PBMNCs and BMMNCs were incubated with the following antibodies: CD45-Pacific Blue (clone 30-F11; BioLegend, San Diego CA), CD11b-FITC (clone M1/70; BD Biosciences, San Jose, CA), F4/80-PE (clone BM8; BioLegend), Gr1-APC (clone RB6-8C5; BD Biosciences), c-kit (CD117)-APC (clone 2B8; BioLegend), biotinylated lineage cocktail antibodies (Miltenyi Biotec), Sca-1-PE.Cy7 (clone D7; eBioscience), CD16/32 (FC ⁇ RII)-PE (clone 93; eBioscience), CD34-FITC (clone RAM34; BD Biosciences), CD61-FITC (BD Biosciences).
  • CD45- Pacific Blue clone 30-F11; BioLegend, San Diego CA
  • BMMNCs were stained using a lineage cell depletion kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). After running through 2 MACS columns (Miltenyi Biotec) more than 90% of the separated cells were lineage negative (Lin - ) by FACS.
  • thymic EC isolation thymocytes were stained with CD45 and CD31 magnetic beads and isolated by MACS (Miltenyi Biotec).
  • FACS sorting MACS-isolated Lin- cells were stained with above-mentioned specific antibodies. FACS sorting was performed to isolate the following cell populations: CD34 - c-kit + Sca-1 + Lin - (CD34 - KSL) cells, CD34 + KSL - cells.
  • ETP Proliferation assay 1 x 10 3 FACS sorted ETP cells per well were cultured in Dulbecco’s modified Eagle’s medium containing 20% FBS supplemented recombinant mouse Kit ligand (PeproTech, 50 ng/ml), mouse IL-7 (PeproTech, 50 ng/ml), mouse Flt3 ligand (PeproTech, 50 ng/ml) with or without recombinant Egfl7 (Abnova, 300 ng/ml) at the start of the culture.
  • the Flt3 inhibitor (Tandutinib, ChemScene) was added at a concentration of 25 uM to indicated cultures. On Day 3 of culture, cells were counted, and the percentage of ETP was determined using FACS.
  • Murine KSL Proliferation assay Liquid suspension cultures of bone marrow murine CD34 - KSL cells (200 per 96-well plate) were supplemented with Iscove’s modified Dulbecco’s medium plus 10% FBS, 1% P/S, 50 ng/ml thrombopoietin, 100 ng/ml Kit ligand and 50 ng/ml Flt-3 ligand (“TKF”) with or without recombinant murine Egfl7 at indicated concentration for the indicated time. If the Egfl7 concentration was not specifically mentioned, recombinant Egfl7 was added at 300 ng/ml (Abnova).
  • Inhibitors for Itgb3 (Cilengitide; 5 ⁇ M), and Flt3 (Tandutinib, ChemScene; 25uM) were added at Day 0. Cells after 5 days in culture were collected and either analyzed by FACS or samples were prepared for qPCR and Western Blot.
  • Colony Formation Assays Colony formation was examined with the use of Methocult medium (Stemcell Technologies, Vancouver, British Columbia, Canada).
  • Op9-Dll1 and HSC co-cultures Op9 mouse fibroblasts and Op9-Delta-like-1 (Dll1) cells (provided by RIKEN, Japan) were maintained using Minimum Essential Medium-Alpha ( ⁇ -MEM) culture medium supplemented with 10% FBS, and 1% P/S.
  • ⁇ -MEM Minimum Essential Medium-Alpha
  • KSL cells were co-cultured for 1 week (initial cell input 5 x 10 2 cells) with OP9 stromal cells in a 12-well plate containing ⁇ -MEM (Sigma) supplemented with 10% FBS (Invitrogen, Carlsbad, CA). The co-cultures were performed in the presence or absence of Flt3 inhibitor (Tandutinib, ChemScene; 25 uM). The KSL cells were collected after 1 week and assayed by FACS.
  • h ⁇ -actin SEQ ID NO: 13 GACGACATGGAGAAAATCTG (forward primer)
  • SEQ ID NO: 14 AGGTCTCAAACATGATCTGG (reverse primer)
  • SEQ ID NO: 17 CCAACCGTGAAAAGATGACC (forward primer)
  • SEQ ID NO: 18 ACCAGAGGCATACAGGGACA (reverse primer)
  • mHes1 SEQ ID NO: 21: GTGGGTCCTAACGCAGTGTC (forward primer)
  • mFLT3 SEQ ID NO: 23: GCCTCATTTCCTTGTGAACAG (forward primer)
  • hItgb3 SEQ ID NO: 29: CGCTAAATTTGAGGAAGAACG (forward primer)
  • hC-kit SEQ ID NO: 31: TCAGCAAATGTCACAACAACC (forward primer)
  • Membranes were stained with secondary antibody conjugated with horseradish peroxidase (Nichirei, rabbit-HRP or goat-HRP), and developed with the ECL Plus detection system (Amersham Life Science, RPN2132) using image analyzer Image-Quant LAS4000 (GE-healthcare).
  • ELISA Flt3 ligand plasma levels were measured using commercially available ELISA kits (R&D Systems Inc., Minneapolis, MN) according to the manufacturer’s protocol.
  • Thymi were embedded in OCT compound (Sakura), and frozen. Tissue sections (5 ⁇ m) were cut with an OM cryostat (HM500; Microm) and collected onto Superfrost/Plus slides (Fisher Scientific). For immunohistochemical staining of mouse thymus, 4% formaldehyde fixed cryo-sections were blocked with 5% BSA in PBS solution, and stained overnight at 4 °C with goat anti-Egfl7 (Santa Cruz Biotech, sc-34416), anti-Flt3 (proteintech, 21049-1-AP), and anti-CD31 (Santa Cruz Biotech, sc-28188).
  • tissue sections were incubated for 1 hr at RT with Alexa Fluor 488 rabbit anti-goat IgG, Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 donkey anti-rabbit IgG, respectively. Sections were counterstained with DAPI.
  • the Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells. Nature Cell Biology 15, 916-925. de Koning et al. (2016) Strategies before, during, and after hematopoietic cell transplantation to improve T-cell immune reconstitution. Blood 128, 2607. de Leeuw et al. (2014) Attenuation of microRNA-126 expression that drives CD34+38- stem/progenitor cells in acute myeloid leukemia leads to tumor eradication. Cancer Research 74, 2094-2105. Delgado et al.
  • Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins and promotes cell motility. Cancer Research 62, 5358-5364. Hartmann et al. (2016) MicroRNA-Based Therapy of GATA2-Deficient Vascular Disease. Circulation 134, 1973-1990. Hassanein et al. (2016) FLT3 Inhibitors for Treating Acute Myeloid Leukemia. Clinical Lymphoma Myeloma and Leukemia 16, 543-549. Hattori et al.
  • Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment. Nature Medicine 8, 841-849. Heissig et al. (2002) Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109, 625-637. Heissig et al. (2007) The plasminogen fibrinolytic pathway is required for hematopoietic regeneration. Cell Stem Cell 1, 658-670. Heissig et al. (2009) Contribution of the fibrinolytic pathway to hematopoietic regeneration. Journal of Cellular Physiology 221, 521-525. Heissig et al.
  • Solanilla et al. (2000) Expression of Flt3-ligand by the endothelial cell. Leukemia 14, 153-162. Soncin et al. (2003) VE-statin, an endothelial repressor of smooth muscle cell migration. The EMBO Journal 22, 5700-5711. Stier et al. (2002) Notch1 activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome. Blood 99, 2369-2378. Sun et al. (2010) [miR-126 modulates the expression of epidermal growth factor-like domain 7 in human umbilical vein endothelial cells in vitro].
  • Nan fang yi ke da xue xue bao Journal of Southern Medical University 30, 767-770. Tadokoro et al. (2014) Control of self-renewal activity of hematopoietic stem cells by spred1. Experimental Hematology 42, S61. Thoren et al. (2008) Kit regulates maintenance of quiescent hematopoietic stem cells. J Immunol 180, 2045-2053. Tsai and Orkin (1997) Transcription Factor GATA-2 Is Required for Proliferation/Survival of Early Hematopoietic Cells and Mast Cell Formation, But Not for Erythroid and Myeloid Terminal Differentiation. Blood 89, 3636. Umemoto et al.

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Abstract

L'invention concerne des procédés et des compositions pour la propagation et/ou la conservation de cellules souches dans un état indifférencié.
PCT/JP2018/033067 2017-09-06 2018-09-06 Procédés et compositions pour la propagation de cellules Ceased WO2019049939A1 (fr)

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WO2022011284A1 (fr) * 2020-07-10 2022-01-13 Ohio State Innovation Foundation Peptide de type facteur de croissance épidermique 7 et utilisations associées
CN114410642A (zh) * 2021-12-31 2022-04-29 苏州大学 一种急性t淋巴细胞白血病药物靶点及其应用

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WO2015179633A1 (fr) * 2014-05-22 2015-11-26 Fred Hutchinson Cancer Research Center Expansion médiée par lilrb2 et notch de cellules précurseurs hématopoïétiques

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DE102007019162A1 (de) * 2007-04-20 2008-10-23 Johann Wolfgang Goethe-Universität Frankfurt am Main Verwendung von EGFL7 zur Modulation von Zellen
WO2015179633A1 (fr) * 2014-05-22 2015-11-26 Fred Hutchinson Cancer Research Center Expansion médiée par lilrb2 et notch de cellules précurseurs hématopoïétiques

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SALAMA, Y. ET AL.: "EGFL7 recruits quiescent HSCs into active cell cycle and expands HSCs", REGENERATIVE MEDICINE, vol. 13, no. 0-47-1, 2014, pages 246 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022011284A1 (fr) * 2020-07-10 2022-01-13 Ohio State Innovation Foundation Peptide de type facteur de croissance épidermique 7 et utilisations associées
CN114410642A (zh) * 2021-12-31 2022-04-29 苏州大学 一种急性t淋巴细胞白血病药物靶点及其应用
CN114410642B (zh) * 2021-12-31 2023-01-17 苏州大学 一种急性t淋巴细胞白血病药物靶点及其应用

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