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WO2014113593A1 - Procédés et compositions pour préparer des substituts sanguins - Google Patents

Procédés et compositions pour préparer des substituts sanguins Download PDF

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Publication number
WO2014113593A1
WO2014113593A1 PCT/US2014/011898 US2014011898W WO2014113593A1 WO 2014113593 A1 WO2014113593 A1 WO 2014113593A1 US 2014011898 W US2014011898 W US 2014011898W WO 2014113593 A1 WO2014113593 A1 WO 2014113593A1
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cell
blood
blood substitute
cells
erythroblasts
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Jonathon S. BARTON
Cristopher D. GEILER
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ADVANCED DIAGNOSTIC TECHNOLOGIES LLC
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ADVANCED DIAGNOSTIC TECHNOLOGIES LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • 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
    • C12N2501/125—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
    • C12N2501/14—Erythropoietin [EPO]
    • 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
    • C12N2501/50—Cell markers; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Embodiments of the present invention include methods and compositions relating to blood substitutes. Some embodiments include contacting a CD34+ cell with a recombinant c-Myc protein, a Toll-like receptor 3 (TLR3) activator, and a calpain inhibitor, thereby obtaining a population of red blood cell precursor cells.
  • a CD34+ cell with a recombinant c-Myc protein, a Toll-like receptor 3 (TLR3) activator, and a calpain inhibitor, thereby obtaining a population of red blood cell precursor cells.
  • TLR3 Toll-like receptor 3
  • Recombinant erythropoietin can be used to stimulate the endogenous production of red blood cells(RBCs) but it is not helpful for the acute or sub-acutely anemic patient. Additional alternatives include intravenous hemoglobin solutions which have been used with limited success in South Africa.
  • Some embodiments of the methods and compositions provided herein include a method of preparing a blood substitute comprising: contacting a cell medium comprising a red blood cell precursor cell with a recombinant c-Myc protein, and a Toll-like receptor 3 (TLR3) activator, thereby obtaining a population of red blood cell precursor cells.
  • a method of preparing a blood substitute comprising: contacting a cell medium comprising a red blood cell precursor cell with a recombinant c-Myc protein, and a Toll-like receptor 3 (TLR3) activator, thereby obtaining a population of red blood cell precursor cells.
  • TLR3 Toll-like receptor 3
  • Some methods also include contacting the population of red blood cell precursor cells with an agent selected from the group consisting of stem cell factor (SCF), erythropoietin (EPO), insulin- like growth factor 1 (IGF-1), IL-3, and dexamethasone (DXM), thereby inducing differentiation of the population of red blood cell precursor cells to a population of erythrob lasts.
  • SCF stem cell factor
  • EPO erythropoietin
  • IGF-1 insulin- like growth factor 1
  • IL-3 IL-3
  • DXM dexamethasone
  • Some methods also include contacting the cell medium comprising a red blood cell precursor cell with a calpain inhibitor.
  • the red blood cell precursor cell is selected from the group consisting of a hematopoietic stem cell, an embryonic stem cell, and induced pluripotent stem cell.
  • the red blood cell precursor cell comprises a CD 34+ hematopoietic stem cell.
  • contacting the cell medium comprises adding about 50 nM to about 1000 nM recombinant c-Myc protein to the cell culture medium.
  • the recombinant c-Myc protein is added to the cell medium at least once every two days. In some embodiments, the recombinant c-Myc protein is added to the cell medium at least daily. In some embodiments, the recombinant c-Myc protein is added to the cell medium at least twice daily. In some embodiments, the recombinant c-Myc protein is added to the cell medium at least three times daily.
  • the c-Myc protein comprises a membrane penetrating polypeptide. In some embodiments, the c-Myc protein comprises c-Myc-Rl 1.
  • the Toll-like receptor 3 (TLR3) activator comprises polyinosinic-polycytidylic acid (poly (I:C)). In some embodiments, contacting the cell culture with the TLR3 activator comprises adding between about 100 ng/ml to about 1 mg/ml poly (I:C) to the cell medium. In some embodiments, the poly (I:C) is added to the cell medium at least daily.
  • the calpain inhibitor is selected from the group consisting of ALLN, and Z-VAD-FMK. In some embodiments, contacting the cell with the calpain inhibitor comprises adding about 10 ⁇ ALLN to the cell medium.
  • Some embodiments also include contacting the cell medium with a protein selected from the group consisting of SOX2, HOXB4, and GATA1.
  • the cell is human.
  • Some embodiments of the methods and compositions provided herein include a population of erythroblasts prepared by any one of the methods provided herein.
  • Some embodiments of the methods and compositions provided herein include a blood substitute comprising a population of erythroblasts prepared by any one of the methods provided herein.
  • the population of erythroblasts comprises a volume at least about 500 ml. In some embodiments, the population of erythroblasts comprises a volume at least about 1 L. In some embodiments, the population of erythroblasts comprises a volume at least about 5 L. In some embodiments, the population of erythroblasts comprises a volume at least about 10 L. In some embodiments, the population of erythroblasts comprises a volume equivalent to about 2 units of blood. In some embodiments, the population of erythroblasts comprises a volume equivalent to about 10 units of blood. In some embodiments, the population of erythroblasts comprises a volume equivalent to about 20 units of blood.
  • the density of erythroblasts is at least about 1 X 10 6 cells/ml. In some embodiments, the density of erythroblasts is at least about 4 X 10 6 cells/ml.
  • the erythroblasts are derived from cultured red blood cell precursor cells.
  • the erythroblasts are derived from a population of precursor cells from a single individual.
  • Some methods and compositions provided herein include a method of treating a patient with a blood substitute comprising transfusing a patient with any one of the blood substitutes provided herein.
  • Some methods and compositions provided herein include a method of treating a patient with a blood substitute comprising: obtaining a red blood cell precursor cell from a subject; preparing a blood substitute according to any one of the methods provided herein; and transfusing the patient with the population of erythroblasts.
  • the patient and the subject are the same individual.
  • the patient is human.
  • Some methods and compositions provided herein include a blood substitute comprising a population of erythroblasts derived from cultured red blood cell precursor cells from a single individual, wherein the volume of the blood substitute is at least about 500 ml and the density of the erythroblasts is at least about 1 X 10 6 cells/ml.
  • the red blood cell precursor cells comprise hematopoietic stem cells. In some embodiments, the red blood cell precursor cells comprise CD 34+ hematopoietic stem cells.
  • the volume of the blood substitute is at least about 1 L. In some embodiments, the volume of the blood substitute is at least about 5 L. In some embodiments, the volume of the blood substitute is at least about 10 L. In some embodiments, the volume of the blood substitute is equivalent to about 2 units of blood. In some embodiments, the volume of the blood substitute is equivalent to about 10 units of blood. In some embodiments, the volume of the blood substitute is equivalent to about 20 units of blood.
  • the density of the erythroblasts is at least about 1 X 10 6 cells/ml. In some embodiments, the density of the erythroblasts is at least about 4 X 10 cells/ml.
  • Some embodiments also include a second population of erythroblasts derived from a second individual.
  • Some embodiments also include one or more agents selected from the group consisting of a recombinant c-Myc protein, a Toll-like receptor 3 (TLR3) activator, and a calpain inhibitor.
  • TLR3 Toll-like receptor 3
  • Some methods and compositions provided herein include a population of erythrocytes obtained by any one of the methods provided herein.
  • Some methods and compositions provided herein include a kit for preparing a blood substitute comprising: an affinity matrix for enriching for CD34+ cells from a subject, wherein the affinity matrix includes an anti-CD34+ antibody; a recombinant c-Myc protein; and a Toll-like receptor 3 (TLR3) activator.
  • an affinity matrix for enriching for CD34+ cells from a subject wherein the affinity matrix includes an anti-CD34+ antibody; a recombinant c-Myc protein; and a Toll-like receptor 3 (TLR3) activator.
  • TLR3 Toll-like receptor 3
  • Some embodiments also include a calpain inhibitor.
  • the recombinant c-Myc protein comprises c-Myc-Rl 1.
  • the TLR3 activator comprises polyinosinic-polycytidylic acid (poly (I:C)).
  • the calpain inhibitor comprises ALLN.
  • kits for transfusing a patient with a blood substitute comprising: a container containing a blood substitute, wherein the blood substitute is comprises any one of the populations of erythroblasts provided herein.
  • the container is selected from the group consisting of ajar, cylinder, and bag.
  • FIG. 1 is a graph of total number of viable cells over time. The larger circles indicate lymphocyte counts based on fluorescent activated cell sorting, where after 14 days, 5.7% remained.
  • FIG. 2 is a graph of number of viable cells over time. Cells were treated with c-Myc protein, c-Myc and poly(LC), or control.
  • FIG. 3 depicts photomicrographs of CD34+ cells (10X).
  • Panel A depicts CD34+ treated with c-Myc protein
  • panel B depicts CD34+ cells treated with c-Myc protein and poly(LC)
  • panel C depicts CD34+ cells treated with SOX2 protein and poly(LC)
  • panel D depicts untreated control CD34+ cells.
  • FIG. 4 is a graph of number of viable cells over time.
  • Mononuclear cells were treated as follows: ( ⁇ ) 6 U/mL erythropoietin; ( ⁇ ) 8 U/mL erythropoietin, and 300 ng/niL poly(LC); (A) 8 U/mL erythropoietin, 300 ng/mL poly(LC), and 400 nM c-Myc-Rl 1; or (X) 8 U/mL erythropoietin, 300 ng/mL poly(LC), and 800 nM cMyc-Rl 1.
  • Embodiments of the present invention include methods and compositions relating to blood substitutes. Some embodiments include contacting a CD34+ cell with a recombinant c-Myc protein, a Toll-like receptor 3 (TLR3) activator, and a calpain inhibitor, thereby obtaining a population of red blood cell precursor cells.
  • a recombinant c-Myc protein such as CD34+ hematopoietic stem cells.
  • TLR3 Toll-like receptor 3
  • calpain inhibitor a calpain inhibitor
  • Applicants have discovered methods and compositions to induce high proliferation rates in red blood cell precursors, such as CD34+ hematopoietic stem cells. The high proliferation rates are useful to achieve sufficient numbers of red blood cell precursors for subsequent differentiation, and use of the differentiated cells in a blood substitute.
  • red blood cell precursors have been an obstacle to producing clinically relevant quantities of cells to prepare a blood substitute.
  • Methods that use embryonic stem cells, cord blood, viral induced pluripotent stem cells, mouse feeder cells, and media containing animal products may not be useful to treat clinical patients.
  • pretreatment of a donor with G-CSF to mobilize red blood cell precursors such as CD34+ limits the commercial market potential and wide spread acceptance.
  • some embodiments of the methods and compositions provided herein induce high proliferation rates without genetic manipulation of the red blood cell precursor cell.
  • high proliferation rates of red blood cell precursors are achieved by contacting the cell with a recombinant c-Myc protein, a TLR3 inhibitor and a calpain inhibitor.
  • red blood cell precursors such as CD34+ hematopoietic stem cells are obtained by mobilization of the donor by granulocyte- colony stimulating factor (G-CSF) or leukapheresis.
  • G-CSF granulocyte- colony stimulating factor
  • the high proliferation rates achieved by the methods and compositions provided herein do not need the larger number of red blood cell precursor cells of prior methods in order to achieve populations with sufficient numbers of cells.
  • HEMA Human Erythroid Massive Amplification
  • Human Erythroid Massive Amplification (HEMA) culture includes in vitro culturing of isolated primitive red blood cell precursor cells, such as CD34+ hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. Cells in the culture medium are stimulated to proliferate and differentiate into red blood cells.
  • Adult mammalian hematopoietic stem cells are typically capable of restricted in vitro expansion.
  • Optimized HEMA cultures include Iscove's Modified Dulbecco's Medium (MDM) with 2% bovine serum albumin, recombinant human insulin, human transferrin (iron-saturated), 2- mercaptoethanol, 100 ng/ml recombinant human SCF/C-Kit ligand, 2 U/ml recombinant human EPO, 40 ng/ml recombinant human IGF-1, 10 ng/ml recombinant human IL-3, 1 ⁇ dexamethasone, 40 ⁇ g/ml human LDL, and 100 U/lOOug/ml penicillin/streptomycin, 0.25 ⁇ / ⁇ 1 amphotericin B, and 1 ⁇ Estradiol.
  • MDM Modified Dulbecco's Medium
  • bovine serum albumin 2% bovine serum albumin
  • recombinant human insulin recombinant human insulin
  • human transferrin iron-saturated
  • Optimized HEMA culture conditions include up to daily passage and feedings and maintaining cellular concentrations less the 10 5 cell per ml and maintenance in a sterile incubator at 37°C with C0 2 5% and 0 2 at or less than 21%. Given that the best demonstrated expansion rates range from 10 2 - to 10 5 - fold increase over 16 to 20 days, and that 10 12 red blood cells are needed for a unit of blood, clinically significant amounts of blood cannot be produced by typical culture methods.
  • Hematopoietic stem cells and erythroid cell precursor cells have a limited ability for self-renewal. Additionally, erythroid cells die excessively in culture.
  • Some embodiments of the methods and composition provided herein include a process for producing large amounts of cells that would otherwise exhibit apoptosis at high densities.
  • cellular mechanisms are modulated to amplify cellular expansion.
  • differentiation of hematopoietic stem cells and erythroid precursor cells is delayed at a growth stage with a high expansion capability.
  • cells are stimulated to stay in the growth phase of the cell cycle within optimized HEMA culture conditions.
  • the growth stage is the proerythroblast stage.
  • Some embodiments also include altering cellular mechanisms to limit cellular death by apoptosis in vitro.
  • Some embodiments of the methods and compositions provided herein include the exogenous addition of a transcription factor protein to the optimized HEMA culture at supra-physiological concentrations ranging from 50 nM to 400 nM.
  • the transcription factor protein is c-Myc.
  • c-Myc protein is part of a fusion protein that includes a cell permeable protein to improve transport into the cellular nucleus.
  • An example of a c-Myc fusion protein is c-Myc-Rl l which includes a full length c-Myc protein fused at its C-terminus to eleven arginine (Rl l) cell penetrating peptide that enables penetration across a cell membrane.
  • c-Myc-Rl 1 stimulates proliferation of the precursor cells.
  • incorporation of c-Myc-Rl 1 into the cell nucleus is further catalyzed by stimulating the toll-like receptor 3 (TLR3).
  • TLR3 toll-like receptor 3
  • TLR3 may be stimulated with poly(LC).
  • 300 ng/ml poly(LC) may be added to an optimized HEMA culture to produce the open chromatin configuration.
  • addition of c-Myc-Rl l and poly(LC) induce a highly proliferative state of the HSC and proerythroblast cells.
  • the highly proliferative state is temporary due to the short half-life of the c-Myc in a cell. Once cellular levels of c-Myc are depleted, the high proliferation is pondered and the culture returns to the previous slow rate of growth.
  • high levels of cellular c-Myc-Rl 1 are maintained by the addition of a calpain inhibitor.
  • the calpain inhibitor slows the degradation of cellular c-Myc- Rl l .
  • Examples of calpain inhibitors include calpain inhibitor I (ALLN).
  • 10 ⁇ ALLN is added to an optimized HEMA culture.
  • agents to inhibit or reduce levels of apoptosis can be added to an optimized HEMA culture.
  • agents to inhibit or reduce levels of apoptosis include Z-VAD-FMK, human recombinant Be 12, and Bcl-xl.
  • the c-Myc-Rl 1 protein is a regulator of cellular proliferation and inhibits exit from the growth phase of the cell cycle and prevents differentiation and favors cell proliferation.
  • the transcription factor c-Myc-Rl 1 is added to the culture media on a start date (e.g., day 0) of the culture and at a concentration of 50 nM to 400 nM.
  • the c-Myc-Rl 1 is then re-added at a routine frequency in a range of 3 times a day to every other day, (e.g., 3 times, 2 times or 1 time per day, or every other day) until a target amplification is achieved.
  • the activity of c-Myc is used to limit or delay cellular differentiation of the Hematopoietic stem cells and maintain the proerythroblast cell stage which is much more proliferative than more mature stages [0050] It is believed that the c-Myc transcription factor can be more efficiently incorporated into a nucleus with an open chromatin confirmation.
  • the open chromatin configuration is simultaneously induced by the addition to the culture media of a toll-like receptor 3 stimulator.
  • the process could use poly(LC) that is added to the culture media daily at the concentration of 300 ng/ml.
  • the open chromatin confirmation is achieved by toll-like receptor3 stimulation which is achieved by adding poly(LC) 300 ng/ml to the culture media, preferably on a daily basis, although it is contemplated that the TLR3 stimulator can be added 4 times, 3 times, 2 times or 1 time per day, or every other day.
  • toll-like receptor3 stimulation which is achieved by adding poly(LC) 300 ng/ml to the culture media, preferably on a daily basis, although it is contemplated that the TLR3 stimulator can be added 4 times, 3 times, 2 times or 1 time per day, or every other day.
  • the c-Myc transcription factor protein has a short half-life (e.g., approximately 20-30 minutes) and is deactivated by proteases including calpains.
  • a calpain inhibitor added to the culture will inhibit the breakdown of c-Myc increasing the half-life of c-Myc added to the optimized HEMA culture.
  • the calpain inhibitor added to the culture will also inhibit cellular death mechanisms.
  • the specific protein transcription factor c-Myc-Rl l is used to alter cellular function in human erythroid amplification culture for the purpose of increasing cellular proliferation for the clinical application of amplifying production of erythroid cells.
  • transcription factors could be used to increase cellular proliferation such as SOX2, HOXB4, or GATAl .
  • cellular function will be altered without the use of genetic manipulation (i.e., adding genes to culture cells).
  • the half-life of the c-Myc-Rl l transcription factor protein is increased by the addition of the calpain inhibitor (e.g., 10 ⁇ ALLN, Z-VAD-FMK, etc.) to the culture medium.
  • the calpain inhibitor should be added to the culture at a routine frequency, for example, 4 times, 3 times, 2 times or 1 time per day, or every other day.
  • Different embodiments may increase the proliferation of not only hematopoietic stem cells in an in vitro culture, but of any multipotent adult stem cell.
  • the process could be adapted for use with endothelial stem cells, ectodermal (neural) stem cells, endodermal and mesodermal adult stem cells.
  • the process can produce blood that is type 0-, which any patient can accept for a blood transfusion.
  • a blood substitute includes a physiological solution comprising a population of cultured red blood cells.
  • a blood substitute can be prepared by obtaining a red blood cell precursor from a subject.
  • the subject is a mammal, such as a human.
  • the red blood cell precursor can include a hematopoietic stem cell, an embryonic stem cell, and induced pluripotent stem cell.
  • the hematopoietic stem cell is a CD34+ hematopoietic stem cell.
  • CD34+ may be enriched or isolated from whole blood using methods known in the art.
  • a population of red blood cell precursor is obtained from the red blood cell precursor cell by adding a recombinant c-Myc protein to a cell medium comprising the red blood cell precursor to increase the proliferation rate of the red blood cell precursor.
  • the cell medium can also include a Toll-like receptor 3 (TLR3) activator.
  • the cell medium can also include a calpain inhibitor.
  • the recombinant c-Myc protein includes a membrane-penetrating peptide.
  • Membrane -penetrating peptides include short peptides having an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • a membrane -penetrating peptide includes the Rl l peptide.
  • the recombinant c-Myc protein comprises a recombinant c-Myc-Rl 1 protein which is a full-length c-Myc protein fused at its C-terminus to 11 arginine (Rl l) cell penetrating peptide (Zhou et al. Cell Stem Cell, May, 2009, incorporated by reference in its entirety).
  • the recombinant c-Myc protein is added to the cell medium at a concentration of, at a concentration greater than, or at a concentration greater than about 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, and 1000 nM.
  • the recombinant c- Myc protein is added to the cell medium at a concentration of, at a concentration less than, or at a concentration less than about, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , and 10 ⁇ , or a range defined by any of the preceding values, including but not limited to 1 nM to 10 ⁇ , 50 nM to 1000 nM, 50 nM to 900 nM, 200 nM to 900 nM, 200 nM to 900 nM, and 100 nM to 900 nM.
  • the recombinant c-Myc protein is added to the cell medium at least once every four days, at least once every three days, at least once every two days, at least daily, at least twice daily, at least three times daily or at least four times daily. In some embodiments, the recombinant c-Myc protein is added to the cell medium once every four days, once every three days, once every two days, daily, twice daily, three times daily or four times daily.
  • a Toll-like receptor 3 (TLR3) activator is added to the cell medium to increase the proliferation rate of the red blood cell precursor.
  • the TLR3 activator comprises polyinosinic-polycytidylic acid (poly (I:C)) (Fortier ME., et al, Am. J. Physiol. Regul. Integr. Comp. Physiol. 287 (4): R759-66, incorporated by reference in its entirety).
  • the TLR3 activator is added to the cell medium at a concentration of, at a concentration greater than, or at a concentration greater than about 1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500 ng/ml, and 1000 ng/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, 20 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, 200 ⁇ g/ml, 500 ⁇ g/ml, 1000 ⁇ g/ml, 2000 ⁇ g/ml, and 5000 ⁇ g/ml, or a range defined by any two of the preceding values, including but not limited to 1 ng/ml to 5000 ⁇ g/ml, 10 ng/ml to 1000 ⁇ g/ml, 10 ng/ml to 500 ⁇ g/ml, and 20 ng/ml, or
  • the TLR3 activator is added to the cell medium at least once every four days, at least once every three days, at least once every two days, at least daily, at least twice daily, at least three times daily or at least four times daily. In some embodiments, the TLR3 activator is added to the cell medium once every four days, once every three days, once every two days, daily, twice daily, three times daily or four times daily.
  • a calpain inhibitor is added to the cell medium to increase the proliferation rate of the red blood cell precursor.
  • Calpain inhibitor include ALLN (calpain inhibitor 1), calpain inhibitor 2, calpain inhibitor peptide, calpain inhibitor 3, Z-VAD-FMK.
  • recombinant Bcl2, or recombinant Bcl-xl is added to the cell medium to increase the proliferation rate of the red blood cell precursor.
  • the calpain inhibitor is added to the cell medium at a concentration of, at a concentration greater than, or at a concentration greater than about 1 ⁇ , 5 ⁇ , 10 ⁇ , 20 ⁇ , 50 ⁇ , and 100 ⁇ , or a range defined by any two of the preceding values, including but not limited to 1 ⁇ to 100 ⁇ , 5 ⁇ to 100 ⁇ , 10 ⁇ to 100 ⁇ , 20 ⁇ to 100 ⁇ , and 50 ⁇ to 100 ⁇ .
  • the calpain inhibitor is added to the cell medium at least once every four days, at least once every three days, at least once every two days, at least daily, at least twice daily, at least three times daily or at least four times daily.
  • the calpain inhibitor is added to the cell medium once every four days, once every three days, once every two days, daily, twice daily, three times daily or four times daily.
  • Some embodiments of the methods and compositions provided herein include inducing differentiation of a population of red blood cell precursor cells. Some such embodiments include inducing differentiation of a population of red blood cell precursor cells to obtain a population of erythrocytes. Methods to induce differentiation of red blood cell precursor cells include (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines (Lapillonne H. et al., Haematol (2010) vol. 95 no.
  • red blood cell precursor cells are exposed to short pulses of cytokines favorable for erythroid differentiation (Olivier E., et al., Stem Cells Trans Med (2012) vol. 1 no. 8 604-614, incorporated by reference in its entirety).
  • inducing differentiation of a population of red blood cell precursor cells includes contacting the population of red blood cell precursor cells with an agent including stem cell factor (SCF), erythropoietin (EPO), insulin- like growth factor 1 (IGF-1), IL-3, and dexamethasone (DXM).
  • SCF stem cell factor
  • EPO erythropoietin
  • IGF-1 insulin-like growth factor 1
  • IL-3 IL-3
  • DXM dexamethasone
  • Some of the methods and compositions provided herein also include adding an additional protein to the cell medium comprising a red blood cell precursor.
  • additional protein examples include recombinant transcription factors such as SOX2, HOXB4, and GATA1.
  • the recombinant protein includes a membrane penetrating peptide, such as the Rl 1 peptide.
  • a blood substitute can be prepared by the methods provided herein.
  • the blood substitute provided herein can be prepared at volumes that greatly exceed the volume of blood that may be obtained through conventional donations by an individual.
  • Blood substitutes can include a population of erythroblasts at a useful physiological density.
  • the density of erythroblasts is, the density of erythroblasts is can be greater than, or the density of erythroblasts is can be greater than about 1 X 10 4 cell/ml, 1 X 10 5 cell/ml, 1 X 10 6 cell/ml, and 1 X 10 7 cell/ml, or a range defined by any two of the preceding values, including but not limited to 1 X 10 4 cell/ml to 1 X 10 7 cell/ml, 1 X 10 5 cell/ml to 1 X 10 7 cell/ml, and 1 X 10 5 cell/ml to 1 X 10 8 cell/ml.
  • the blood substitute can include a population of erythroblasts with a volume that is, a volume of at least, or a volume that is at least about 500 ml, 1 L, 2 L, 3L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, and 10 L, preferably at a physiological density and/ or a density recited herein.
  • the blood substitute can include a population of erythroblasts with a volume equivalent to, a volume equivalent to at least, or a volume equivalent to at least about 2 units of blood, 3 units of blood, 4 units of blood, 5 units of blood, 6 units of blood, 7 units of blood, 8 units of blood, 9 units of blood, 10 units of blood, and 20 units of blood.
  • the erythroblasts or their precursor cells were not genetically altered, for example by transfection with genetic material, including but not limited to DNA or R A.
  • a blood substitute comprising a population of erythroblasts derived from cultured red blood cell precursor cells from a single individual.
  • a blood substitute can include a second population of erythroblasts derived from a second individual.
  • a blood substitute can include one or more agents selected from the group consisting of a recombinant c-Myc protein, a Toll-like receptor 3 (TLR3) activator, and a calpain inhibitor.
  • the one or more agents are at substantially reduced levels.
  • exogenous c-myc is substantially cleared, or absent, from a substitute blood composition before use as substitute blood.
  • Some of the methods and compositions provided herein include methods of treating a patient with a blood substitute.
  • a patient can include an individual in need of a blood transfusion. Some such embodiments include obtaining a red blood cell precursor cell from a subject, and preparing a blood substitute by the methods provided herein. Some embodiments also include transfusing the patient with the blood substitute.
  • the subject and the patient are the same individual. In some embodiments, the subject and the patient are different individuals.
  • kits for preparing a blood substitute can include a kit for enriching for CD34+ cells from a subject, and a recombinant c-Myc protein.
  • a kit for enriching for CD34+ cells from a subject includes an affinity matrix for enriching for CD34+ cells from a subject, wherein the affinity matrix includes an anti-CD34+ antibody.
  • the antibody is linked to a magnetic bead.
  • the c- Myc protein includes a membrane -penetrating peptide, such as Rl l .
  • the recombinant c-Myc is c-Myc-Rl 1.
  • a kit can also include a TLR3 activator, such as poly(LC); and a calpain inhibitor, such as ALLN.
  • kits for transfusing a patient with the blood substitute provided herein include a kit for transfusing a patient with the blood substitute provided herein.
  • the kit can include a container containing a blood substitute.
  • the blood substitute is prepared by the methods provided herein.
  • the container is ajar, cylinder, or bag.
  • Example 1 isolation and culture of CD34+ cells from peripheral blood.
  • a 10 ml sample of non-mobilized peripheral venous blood was obtained from a normal adult human in a sodium heparin blood drawing tube.
  • whole blood samples were diluted in a 1 : 1 mixture of PBS.
  • Peripheral blood mononuclear cells were isolated from whole blood sample with Ficoll paque plus density gradient. Samples were centrifuged at 800g for 35 minutes.
  • the buffy coat / MNC fraction was isolated and diluted in a 1 : 1 mixture with PBS 2% FBS.
  • the MNC fraction was cultured and lymphocytes depleted using a lymphotoxic drug, 1 ⁇ g/ml to 1 mg/ml Cyclosporin A.
  • CD34+ cells were isolated using the EasySep®Human CD34 Selection Kit cat#18056(StemCellTech Vancouver, Canada) for positive selection of CD34+ cells.
  • the selection kit includes Tetrameric Antibody Complexes recognizing CD34 and dextran-coated magnetic particles. Typically, at least 1 X 10 4 CD34+ cells were isolated from 10 ml whole blood.
  • CD34+ cells 5 X 10 6 cells/mL were cultured in serum-free medium (StemSpan; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with EPO (6 U/mL Prospectbio, East Brunswick,NJ), Dexamethasone (1 X 10 6 M; Sigma, St Louis, MO), insulin- like growth factor 1 (IGF-1; 40 ng/niL; Prospectbio, East Brunswick,NJ), SCF (100 ng/mL; Prospectbio, East Brunswick,NJ), and lipids (40 mg/mL cholesterol-rich lipid mix; Sigma). Homogeneous cultures of erythroid progenitors established after several days were kept in the same medium at about 2 X 10 6 cells/mL by daily partial medium changes.
  • Example 2 effects of polyinosinic-polycytidylic acid (poly(I:Q) on erythroblast proliferation
  • CD34+ erythroblasts were isolated from a peripheral blood sample as described in Example 1. Populations of CD34+ erythroblasts were treated with (1) 300 ng/ml poly(LC) and 20 mM Dlisocitrate, (2) 1 ⁇ g/ml poly(LC), or (3) carrier only. The number of cells in each treated population was measured over time. FIG. 1.
  • cells treated with either 300 ng/ml poly(LC) and 20 mM Dlisocitrate, or 1 ⁇ g/ml poly(LC) showed a significantly greater viable cell count over untreated control cells.
  • cells treated with 300 ng/ml poly(LC) and 20 mM Dlisocitrate showed a 15 X 10 4 -fold increase in viable cells between days 0 and 20.
  • untreated control cells showed a 6.5 X 10 4 -fold increase in viable cells between days 0 and 20.
  • NF- ⁇ factors decline as early erythroid progenitor cells undergo erythropoiesis progresses and differentiate.
  • Poly(LC) increases NF- ⁇ levels which maintain early erythroid progenitor cells in a highly proliferative state. Maintaining NF-KB levels also increases transcription of survival genes which protect against apoptosis during the expansion of the erythroblast culture.
  • Example 3 treatment of CD34+ cells c-Myc protein and poly(I:C)
  • Recombinant c-Myc-Rl 1 protein includes the c-Myc polypeptide with eleven arginine residues that enhance transport of the recombinant protein across the cell membrane.
  • CD34+ cells were cultured in StemSpan medium supplemented with SCF, EPO, IGF-1, IL-3, DXM, and LDL.
  • CD34+ cells were plated in 12-well plates at a density of 10 4 cells/ml, and passaged daily for 5 days.
  • CD34+ cells were treated with 100 nM c-Myc-Rl l; 100 nM c- Myc-Rl 1 and 300 ng/ml poly(I:C); SOX2-R11 protein and poly(I:C); or control. Viable cells were counted at days 0, 3, and 5, and cultures were photographed at Day 5.
  • FIG. 2 and FIG. 3. There was no significant increase in proliferation rates for cells treated with SOX2-R11 protein and poly(LC) over control cells, however, treatment with c-Myc-Rl l increased proliferation rates of cells.
  • Treatment of CD34+ cells with with c-Myc-Rl l with poly(I:C) increased proliferation of cells about 100-fold greater than untreated control cells.
  • the c-Myc-Rl l protein has a short half-life of about 38 minutes and should be cleared from a cell culture without additional intervention.
  • To test for clearance of the c-Myc-Rl 1 protein addition of exogenous c-Myc-Rl 1 protein to a CD34+ cell culture is stopped.
  • the levels of exogenous c-Myc-Rl 1 in the culture is measured using a monoclonal anti-c-Myc antibody (Sigma Aldrich St Louis MO) and indirect immunofluorescent staining of cultured cells. The levels of exogenous c-Myc-Rl 1 is much reduced over time.
  • a mononuclear cell fraction was obtained from whole blood. The number of CD34+ cells in the fraction was measured. The fraction was cultured in the following media: Media: 100 mL Stemspan media with growth factors: 100 ng/niL Stem Cell Factor, 10 ng/ml IL3, 40 ng/mL IGF-1, 15 ng/niL BMP4, 500 ⁇ g/ml Holotransferin, 1 X 10 "6 Dexamethasone, 1 X 10 "6 Estradiol, 1.0 mg/100 mL chemically defined lipid, and 1 ⁇ g/mL Cyclosporin A. Cultures were maintained in a high humidity incubator with 5% C0 2 and 0 2 at %5 at 37°C.
  • Samples from the cultured fraction were treated as follows: (1) 6 U/mL erythropoietin; (2) 8 U/mL erythropoietin, and 300 ng/mL poly(LC); (3) 8 U/mL erythropoietin, 300 ng/mL poly(LC), and 400 nM c-Myc-Rl l; or (4) 8 U/mL erythropoietin, 300 ng/mL poly(LC), and 800 nM cMyc-Rl l . The total number of viable cells in each sample was measured over time.
  • Example 6 delayed maturation of c-Myc-Rl 1 treated cells
  • a mononuclear cell fraction was obtained from whole blood. Samples from the cultured fraction were treated as follows: (1) 400 nM c-Myc-Rl 1 and 300 ⁇ g/ml poly(LC); or (2) untreated control. Flow Cytometry was used to determine the immunophenotype of the cultured cells. Cells were analyzed for the presence of the CD71, CDl 17, and CD235a.
  • CD71 is the transferrin receptor molecule which is present on all cells that have committed to the erythroid cell line.
  • CD235a is the molecule Glycophorins A and becomes present on the cell surface as it matures from the early Pronormoblast to the more mature Basophilic Normoblast.
  • CDl 17 is the c-kit molecule also known as Stem Cell Factor receptor molecule, and is present on hematopoietic stem cells and early Burst Forming Unit- erythroid (BFU-E) progenitors but is gradually lost as the cell reaches the PolychromatophiUic Normoblast and completely missing from the Orthochromic Normoblast stage. Results are summarized in TABLE 2.
  • Example 7 differentiation of CD34+ cells to erythroblasts
  • CD34+ cells are treated with one or more factors including stem cell factor (SCF), erythropoietin (EPO), insulin- like growth factor 1 (IGF-1), IL-3, dexamethasone (DXM).
  • SCF stem cell factor
  • EPO erythropoietin
  • IGF-1 insulin- like growth factor 1
  • IL-3 IL-3
  • DXM dexamethasone
  • CD36 which is associated with cells that have converted into early erythroid progenitors
  • CD235a which is associated with basophilic, polychromatic and orthochromatic erythroblasts
  • E-cadherin which is associated with pronormoblasts
  • glycophorin which is associated with orthochrmic/poly chromatic normoblasts.

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Abstract

La présente invention concerne, dans certains modes de réalisation, des procédés et des compositions se rapportant à des substituts sanguins. Certains modes de réalisation consistent à mettre en contact une cellule CD34+ avec une protéine c-Myc recombinante, un activateur du récepteur 3 de type Toll (TLR3), et un inhibiteur de la calpaïne, ce qui permet d'obtenir une population de cellules précurseurs d'érythrocyte.
PCT/US2014/011898 2013-01-18 2014-01-16 Procédés et compositions pour préparer des substituts sanguins Ceased WO2014113593A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020120098A1 (en) * 1996-08-27 2002-08-29 David Bell Enhanced stimulation of erythropoiesis
US6586192B1 (en) * 1998-05-29 2003-07-01 Thomas Jefferson University Compositions and methods for use in affecting hematopoietic stem cell populations in mammals
US7790458B2 (en) * 2004-05-14 2010-09-07 Becton, Dickinson And Company Material and methods for the growth of hematopoietic stem cells
US20110003387A1 (en) * 2009-07-02 2011-01-06 Abbot Stewart Method of producing erythrocytes without feeder cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020120098A1 (en) * 1996-08-27 2002-08-29 David Bell Enhanced stimulation of erythropoiesis
US6586192B1 (en) * 1998-05-29 2003-07-01 Thomas Jefferson University Compositions and methods for use in affecting hematopoietic stem cell populations in mammals
US7790458B2 (en) * 2004-05-14 2010-09-07 Becton, Dickinson And Company Material and methods for the growth of hematopoietic stem cells
US20110003387A1 (en) * 2009-07-02 2011-01-06 Abbot Stewart Method of producing erythrocytes without feeder cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HATTANGADI, SHILPA M. ET AL.: "From stem cell to red cell : regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications", BLOOD, vol. 118, no. 24, 8 December 2011 (2011-12-08), pages 6258 - 6268 *
PRIES, RALPH ET AL.: "Induction of c-Myc-dependent cell proliferation through toll-like receptor 3 in head and neck cancer", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 21, no. 2, February 2008 (2008-02-01), pages 209 - 215 *

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