WO2014113593A1 - Methods and compositions for preparing blood substitutes - Google Patents

Abstract

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.

Description

METHODS AND COMPOSITIONS FOR PREPARING BLOOD SUBSTITUTES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/754,350 filed January 18, 2013 entitled "IMPROVED IN- VITRO PRODUCTION OF ERYTHROID CELLS FROM HEMATOPOIETIC STEM CELLS", the contents of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] 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.

BACKGROUND OF THE INVENTION

[0003] There exists a significant unmet need for a viable alternative to allogenic blood transfusion treatment for the anemic patient. The safety and adequacy of the national blood supply continues to be a national concern and a public health priority. Every two seconds someone in the U.S. needs blood and more than 38,000 blood donations are needed every day. A total of 30 million blood components are transfused each year in the U.S. and 70 million units globally. This figure unfortunately falls short of the global need for transfused blood by 110 million units every year. Even when blood supplies are adequate, finding blood for certain patients may be difficult. Other challenges include chronically transfused patients who may become immunized to polymorphic cell surface proteins and transfusion requiring patients with Myelofibrosis. Few alternative treatment options exist for the anemic patient. 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.

[0004] Patients often require blood supplies to treat various conditions. Typically, anemic patients are treated by allogenic transfusions of blood received from others. However, allogenic blood transfusions are often dependent on factors that are unrelated to the underlying treatment. For instance, an ample supply of donated blood must be available to perform allogenic blood transfusions. This is problematic because it requires that there be sufficient blood donors and that the donated blood is suitable for transfusion. Allogenic blood transfusions have the potential to transmit infection or create transfusion reactions. Thus, there exists a significant unmet need for an alternative treatment to allogenic blood transfusions for anemic patients.

SUMMARY OF THE INVENTION

[0005] 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.

[0006] 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.

[0007] Some methods also include contacting the cell medium comprising a red blood cell precursor cell with a calpain inhibitor.

[0008] In some embodiments, 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. In some embodiments, the red blood cell precursor cell comprises a CD 34+ hematopoietic stem cell.

[0009] In some embodiments, contacting the cell medium comprises adding about 50 nM to about 1000 nM recombinant c-Myc protein to the cell culture medium.

[0010] In some embodiments, 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.

[0011] In some embodiments, the c-Myc protein comprises a membrane penetrating polypeptide. In some embodiments, the c-Myc protein comprises c-Myc-Rl 1. [0012] In some embodiments, 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.

[0013] In some embodiments, 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 μΜ ALLN to the cell medium.

[0014] Some embodiments also include contacting the cell medium with a protein selected from the group consisting of SOX2, HOXB4, and GATA1.

[0015] In some embodiments, the cell is human.

[0016] Some embodiments of the methods and compositions provided herein include a population of erythroblasts prepared by any one of the methods provided herein.

[0017] 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.

[0018] In some embodiments, 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.

[0019] In some embodiments, the density of erythroblasts is at least about 1 X 10⁶ cells/ml. In some embodiments, the density of erythroblasts is at least about 4 X 10⁶ cells/ml.

[0020] In some embodiments, the erythroblasts are derived from cultured red blood cell precursor cells.

[0021] In some embodiments, the erythroblasts are derived from a population of precursor cells from a single individual. [0022] 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.

[0023] 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.

[0024] In some embodiments, the patient and the subject are the same individual.

[0025] In some embodiments, the patient is human.

[0026] 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 10⁶ cells/ml.

[0027] In some embodiments, 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.

[0028] In some embodiments, 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.

[0029] In some embodiments, the density of the erythroblasts is at least about 1 X 10⁶ cells/ml. In some embodiments, the density of the erythroblasts is at least about 4 X 10 cells/ml.

[0030] Some embodiments also include a second population of erythroblasts derived from a second individual.

[0031] 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.

[0032] Some methods and compositions provided herein include a population of erythrocytes obtained by any one of the methods provided herein. [0033] 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.

[0034] Some embodiments also include a calpain inhibitor. In some embodiments, the recombinant c-Myc protein comprises c-Myc-Rl 1. In some embodiments, the TLR3 activator comprises polyinosinic-polycytidylic acid (poly (I:C)). In some embodiments, the calpain inhibitor comprises ALLN.

[0035] Some methods and compositions provided herein include a kit 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. In some embodiments, the container is selected from the group consisting of ajar, cylinder, and bag.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

DETAILED DESCRIPTION

[0040] 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. 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.

[0041] Achieving high proliferation rates for red blood cell precursors has 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. In addition, 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.

[0042] Advantageously, some embodiments of the methods and compositions provided herein induce high proliferation rates without genetic manipulation of the red blood cell precursor cell. In some embodiments, 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. Typically, 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. However, 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.

[0043] Blood can be produced in vitro for eventual use in transfusions and other treatments. 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 μΜ dexamethasone, 40 μg/ml human LDL, and 100 U/lOOug/ml penicillin/streptomycin, 0.25 μ /ηι1 amphotericin B, and 1 μΜ Estradiol. Optimized HEMA culture conditions include up to daily passage and feedings and maintaining cellular concentrations less the 10⁵ cell per ml and maintenance in a sterile incubator at 37°C with C0₂ 5% and 0₂ at or less than 21%. Given that the best demonstrated expansion rates range from 10²- to 10⁵- fold increase over 16 to 20 days, and that 10¹² red blood cells are needed for a unit of blood, clinically significant amounts of blood cannot be produced by typical culture methods.

[0044] 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. In some embodiments, cellular mechanisms are modulated to amplify cellular expansion. In some embodiments, differentiation of hematopoietic stem cells and erythroid precursor cells is delayed at a growth stage with a high expansion capability. In some embodiments, cells are stimulated to stay in the growth phase of the cell cycle within optimized HEMA culture conditions. In some embodiments, the growth stage is the proerythroblast stage. Some embodiments also include altering cellular mechanisms to limit cellular death by apoptosis in vitro.

[0045] 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. In some embodiments, the transcription factor protein is c-Myc. In some embodiments, 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.

[0046] In some embodiments, incorporation of c-Myc-Rl 1 into the cell nucleus is further catalyzed by stimulating the toll-like receptor 3 (TLR3). Without being bound to any particular mode of operation, it is believed that stimulation of the TLR3 induces an open chromatin configuration of the cellular nucleus. In some embodiments, TLR3 may be stimulated with poly(LC). In some embodiments, 300 ng/ml poly(LC) may be added to an optimized HEMA culture to produce the open chromatin configuration. [0047] In some embodiments 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 quelled and the culture returns to the previous slow rate of growth. In some embodiments, 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). In some embodiments, 10 μΜ ALLN is added to an optimized HEMA culture. In some embodiments, agents to inhibit or reduce levels of apoptosis can be added to an optimized HEMA culture. In some embodiments, agents to inhibit or reduce levels of apoptosis include Z-VAD-FMK, human recombinant Be 12, and Bcl-xl.

[0048] The use of a protein transcription factor to alter cellular mechanisms and amplify hematopoietic stem cell production of erythroid cells in culture is an improvement over currently existing technology that uses transfection of genetic material, DNA, or RNA, which typically results in permanent cancer-like function. Thus, although the changes to the cellular mechanisms induce a temporary highly proliferative state of the HSC and proerythroblast cells, the duration of this state terminates after ceasing the addition of exogenous c-Myc. Moreover, the process does not cause permanent effects to the cells and does not require genetic manipulation. This is an improvement because genetic manipulation to alter cellular functions has been demonstrated to cause cancer and may have unforeseen future effects on the cellular function, and thus, cannot be translated into clinical medicine. In some embodiments, the method does not include the use of transfection of genetic material, including but not limited to DNA, or RNA.

[0049] 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. In some embodiments, 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. Thus, 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. In some embodiments, the open chromatin configuration is simultaneously induced by the addition to the culture media of a toll-like receptor 3 stimulator. For example, 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. Thus, improved penetration of the cells with culture results when c- Myc transcription factor is attached to a cell permeable protein. As the c-Myc protein transcription factor has more efficient access to a cellular nucleus with an open chromatin configuration, this is achieved by toll-like receptor3 stimulation.

[0051] 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. In some embodiments, 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. Alternatively other transcription factors could be used to increase cellular proliferation such as SOX2, HOXB4, or GATAl . In this way, 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 μΜ 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.

[0052] Different embodiments may increase the proliferation of not only hematopoietic stem cells in an in vitro culture, but of any multipotent adult stem cell. For example, the process could be adapted for use with endothelial stem cells, ectodermal (neural) stem cells, endodermal and mesodermal adult stem cells. Also, the process can produce blood that is type 0-, which any patient can accept for a blood transfusion. By identifying a gene associated with cancer, and utilizing its protein transcription factor (as a product of the gene), the process can achieve the increased production of blood without genetic manipulation.

Methods of preparing a blood substitute

[0053] Some embodiments of the methods and compositions provided herein relate to preparing blood substitutes. In some embodiments, a blood substitute includes a physiological solution comprising a population of cultured red blood cells. In some embodiments, a blood substitute can be prepared by obtaining a red blood cell precursor from a subject. In some embodiments, 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. In some embodiments, the hematopoietic stem cell is a CD34+ hematopoietic stem cell. In some embodiments, CD34+ may be enriched or isolated from whole blood using methods known in the art. Examples methods include use of anti-CD34+ antibodies, cell sorting methods, and affinity methods. 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. In some embodiments, the cell medium can also include a Toll-like receptor 3 (TLR3) activator. In some embodiments, the cell medium can also include a calpain inhibitor.

[0054] In some embodiments, 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. In some embodiments, a membrane -penetrating peptide includes the Rl l peptide. In some such embodiments, 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).

[0055] In some embodiments, 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. In some embodiments, 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 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, and 10 μΜ, or a range defined by any of the preceding values, including but not limited to 1 nM to 10 μΜ, 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. In some embodiments, 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.

[0056] In some embodiments, a Toll-like receptor 3 (TLR3) activator is added to the cell medium to increase the proliferation rate of the red blood cell precursor. In some embodiments, 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). In some embodiments, 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 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 500 μg/ml, 1000 μg/ml, 2000 μg/ml, and 5000 μg/ml, or a range defined by any two of the preceding values, including but not limited to 1 ng/ml to 5000 μg/ml, 10 ng/ml to 1000 μg/ml, 10 ng/ml to 500 μg/ml, and 20 ng/ml to 200 μg/ml. In some embodiments, 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.

[0057] In some embodiments, 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. In some embodiments, recombinant Bcl2, or recombinant Bcl-xl is added to the cell medium to increase the proliferation rate of the red blood cell precursor. In some embodiments, 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 μΜ, 5 μΜ, 10 μΜ, 20 μΜ, 50 μΜ, and 100 μΜ, or a range defined by any two of the preceding values, including but not limited to 1 μΜ to 100 μΜ, 5 μΜ to 100 μΜ, 10 μΜ to 100 μΜ, 20 μΜ to 100 μΜ, and 50 μΜ to 100 μΜ. In some embodiments, 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. In some embodiments, 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.

[0058] 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. 10 1651-1659; and Kobari L., et al., Haematol (2012) vol. 97 no. 12 1795-1803, incorporated by reference in their entireties). In some embodiments, 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). In some embodiments, 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).

[0059] Some of the methods and compositions provided herein also include adding an additional protein to the cell medium comprising a red blood cell precursor. Examples of such proteins include recombinant transcription factors such as SOX2, HOXB4, and GATA1. In some embodiments, the recombinant protein includes a membrane penetrating peptide, such as the Rl 1 peptide.

[0060] Some of the methods and compositions provided herein include a blood substitute. In some embodiments, a blood substitute can be prepared by the methods provided herein. Advantageously, 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. In some embodiments, 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 10⁴ cell/ml, 1 X 10⁵ cell/ml, 1 X 10⁶ cell/ml, and 1 X 10⁷ cell/ml, or a range defined by any two of the preceding values, including but not limited to 1 X 10⁴ cell/ml to 1 X 10⁷ cell/ml, 1 X 10⁵ cell/ml to 1 X 10⁷ cell/ml, and 1 X 10⁵ cell/ml to 1 X 10⁸ cell/ml. In some embodiments, 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. In some embodiments, 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. In a preferred embodiment, 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.

[0061] Some embodiments of the 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. In some embodiments, a blood substitute can include a second population of erythroblasts derived from a second individual. In some embodiments, 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. In some embodiments, the one or more agents are at substantially reduced levels. In some embodiments, exogenous c-myc is substantially cleared, or absent, from a substitute blood composition before use as substitute blood.

Methods of treatment

[0062] 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. In some embodiments, the subject and the patient are the same individual. In some embodiments, the subject and the patient are different individuals.

Kits

[0063] Some embodiments of the methods and compositions provided herein include a kit for preparing a blood substitute. Some such kits can include a kit for enriching for CD34+ cells from a subject, and a recombinant c-Myc protein. In some embodiments, 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. In some embodiments, the antibody is linked to a magnetic bead. In some embodiments, the c- Myc protein includes a membrane -penetrating peptide, such as Rl l . In some embodiments, the recombinant c-Myc is c-Myc-Rl 1. In some embodiments, a kit can also include a TLR3 activator, such as poly(LC); and a calpain inhibitor, such as ALLN.

[0064] Some embodiments of the methods and compositions provided herein include a kit for transfusing a patient with the blood substitute provided herein. In some embodiments, the kit can include a container containing a blood substitute. In some embodiments, the blood substitute is prepared by the methods provided herein. In some embodiments, the container is ajar, cylinder, or bag.

EXAMPLES

Example 1— isolation and culture of CD34+ cells from peripheral blood.

[0065] A 10 ml sample of non-mobilized peripheral venous blood was obtained from a normal adult human in a sodium heparin blood drawing tube. For the isolation of MNC fraction from whole blood, 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 μg/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 10⁴ CD34+ cells were isolated from 10 ml whole blood. [0066] For the initial expansion of CD34+ cells, 5 X 10⁶ 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 10⁶ 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 10⁶ cells/mL by daily partial medium changes.

Example 2— effects of polyinosinic-polycytidylic acid (poly(I:Q) on erythroblast proliferation

[0067] This example demonstrates that addition of poly(LC) to a culture of erythroblasts significantly increased rates of 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 μg/ml poly(LC), or (3) carrier only. The number of cells in each treated population was measured over time. FIG. 1.

[0068] After 20 days, cells treated with either 300 ng/ml poly(LC) and 20 mM Dlisocitrate, or 1 μg/ml poly(LC) showed a significantly greater viable cell count over untreated control cells. In particular, cells treated with 300 ng/ml poly(LC) and 20 mM Dlisocitrate, showed a 15 X 10⁴-fold increase in viable cells between days 0 and 20. In contrast, untreated control cells showed a 6.5 X 10⁴-fold increase in viable cells between days 0 and 20.

[0069] The levels of NF-κΒ factors decline as early erythroid progenitor cells undergo erythropoiesis progresses and differentiate. Poly(LC) increases NF-κΒ 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)

[0070] This example demonstrates the unexpected enhanced proliferation rates achieved by treating CD34+ cells with a recombinant c-Myc-Rl 1 protein and poly(LC). Recombinant c-Myc-Rl l 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.

[0071] CD34+ cells were plated in 12-well plates at a density of 10⁴ 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. FIG. 2.

Example 4— clearance of exogenous c-Myc protein from cell cultures

[0072] 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.

Example 5— expansion and differentiation of CD34+ cells

[0073] 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 μg/ml Holotransferin, 1 X 10^"6 Dexamethasone, 1 X 10^"6 Estradiol, 1.0 mg/100 mL chemically defined lipid, and 1 μg/mL Cyclosporin A. Cultures were maintained in a high humidity incubator with 5% C0₂ and 0₂ 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. The fold increase between CD34+ cells at day 0 and number of erythroblasts at day 21, and fold increase between number of mononuclear cells at day 0 and number of erythroblasts at day 21 were calculated. TABLE 1 and FIG. 4 summarize the results.

TABLE 1

[0074] As time progresses, certain cells in the mononuclear cell fraction die, and certain cell populations in the fraction, such as CD34+ cells, proliferate and undergo differentiation to erythroblasts. Cells treated with 8 U/mL EPO; 300 ng/mL poly(LC); 400 nM c-Myc-Rl 1 had the greatest fold- increases in total cell numbers.

Example 6— delayed maturation of c-Myc-Rl 1 treated cells

[0075] 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 μg/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.

TABLE 2

[0076] For treated cell at day 23, 81% of the cells remain in an immature state (CD71+,CD117+), despite the age of culture. The majority of these immature cells; 66.4% were Basophillic Normoblasts. The untreated control cells at day 10, 50.2% were CD71+,CD117+. An extrapolation of the data predicts that treated cells comprise a population that remains in an immature, less differentiated state that retains a high proliferation state. In other words, these results support delaying differentiation of the erythroid line such that the culture spends more time in a more immature and highly proliferative state in order to amplify the expansion potential of the culture.

Example 7— differentiation of CD34+ cells to erythroblasts

[0077] 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). Cellular commitment and differentiation of cell cultures is measured using markers including CD36 which is associated with cells that have converted into early erythroid progenitors (proerythroblast); CD235a which is associated with basophilic, polychromatic and orthochromatic erythroblasts; E-cadherin which is associated with pronormoblasts; and glycophorin which is associated with orthochrmic/poly chromatic normoblasts.

[0078] The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0079] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0080] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. 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.

2. The method of claim 1, further comprising 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 erythroblasts.

3. The method of any one of claims 1-3, further comprising contacting the cell medium comprising a red blood cell precursor cell with a calpain inhibitor.

4. The method of any one of claims 1-4, wherein 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.

5. The method of any one of claims 1-4, wherein the red blood cell precursor cell comprises a CD34+ hematopoietic stem cell.

6. The method of any one of claims 1-5, wherein contacting the cell medium comprises adding about 50 nM to about 1000 nM recombinant c-Myc protein to the cell culture medium.

7. The method of claim 6, wherein the recombinant c-Myc protein is added to the cell medium at least once every two days.

8. The method of claim 6, wherein the recombinant c-Myc protein is added to the cell medium at least daily.

9. The method of claim 6, wherein the recombinant c-Myc protein is added to the cell medium at least twice daily.

10. The method of claim 6, wherein the recombinant c-Myc protein is added to the cell medium at least three times daily.

11. The method of any one of claims 1-10, wherein the c-Myc protein comprises a membrane penetrating polypeptide.

12. The method of any one of claims 1-11, wherein the c-Myc protein comprises c-Myc-Rl l .

13. The method of any one of claims 1-12, wherein the Toll-like receptor 3 (TLR3) activator comprises polyinosinic-polycytidylic acid (poly (I:C)).

14. The method of claim 13, wherein 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.

15. The method of claim 13, wherein the poly (I:C) is added to the cell medium at least daily.

16. The method of any one of claims 1-15, wherein the calpain inhibitor is selected from the group consisting of ALLN, and Z-VAD-FMK.

17. The method of claim 16, wherein contacting the cell with the calpain inhibitor comprises adding about 10 μΜ ALLN to the cell medium.

18. The method of any one of claims 1-17, further comprising contacting the cell medium with a protein selected from the group consisting of SOX2, HOXB4, and GATAl .

19. The method of any one of claims 1-18, wherein the cell is human.

20. A population of erythroblasts prepared by the method of any one of claims 1-

19.

21. A blood substitute prepared by the method of any one of claims 1-19.

22. The blood substitute of claim 21, wherein the population of erythroblasts comprises a volume at least about 500 ml.

23. The blood substitute of any one of claims 21-22, wherein the population of erythroblasts comprises a volume at least about 1 L.

24. The blood substitute of any one of claims 21-23, wherein the population of erythroblasts comprises a volume at least about 5 L.

25. The blood substitute of any one of claims 21-24, wherein the population of erythroblasts comprises a volume at least about 10 L.

26. The blood substitute of claim 21, wherein the population of erythroblasts comprises a volume equivalent to at least about 2 units of blood.

27. The blood substitute of claim 26, wherein the population of erythroblasts comprises a volume equivalent to at least about 10 units of blood.

28. The blood substitute of any one of claims 26-27, wherein the population of erythroblasts comprises a volume equivalent to at least about 20 units of blood.

29. The blood substitute of any one of claims 21-28, wherein the density of erythroblasts is at least about 1 X 10⁶ cells/ml.

30. The blood substitute of any one of claims 21-29, wherein the density of erythroblasts is at least about 4 X 10⁶ cells/ml.

31. The blood substitute of any one of claims 21-30, wherein the erythroblasts are derived from cultured red blood cell precursor cells.

32. The blood substitute of any one of claims 21-31, wherein the erythroblasts are derived from a population of precursor cells from a single individual.

33. A method of treating a patient with a blood substitute comprising transfusing a patient with the blood substitute of any one of claims 21-32.

34. 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 claims 1-19; and transfusing the patient with the population of erythroblasts.

35. The method of claim 34, wherein the patient and the subject are the same individual.

36. The method of any one of claims 34-35, wherein the patient is human.

37. 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 10⁶ cells/ml.

38. The blood substitute of claim 37, wherein the red blood cell precursor cells comprises hematopoietic stem cells.

39. The blood substitute of any one of claims 37-38, wherein the red blood cell precursor cells comprises CD34+ hematopoietic stem cells.

40. The blood substitute of any one of claims 37-39, wherein the volume of the blood substitute is at least about 1 L.

41. The blood substitute of any one of claims 37-40, wherein the volume of the blood substitute is at least about 5 L.

42. The blood substitute of any one of claims 37-41, wherein the volume of the blood substitute is at least about 10 L.

43. The blood substitute of any one of claims 37-39, wherein the volume of the blood substitute is equivalent to at least about 2 units of blood.

44. The blood substitute of any one of claims 37-39, wherein the volume of the blood substitute is equivalent to at least about 10 units of blood.

45. The blood substitute of any one of claims 37-39, wherein the volume of the blood substitute is equivalent to at least about 20 units of blood.

46. The blood substitute of any one of claims 37-45, wherein the density of the erythroblasts is at least about 1 X 10⁶ cells/ml.

47. The blood substitute of any one of claims 37-45, wherein the density of the erythroblasts is at least about 4 X 10⁶ cells/ml.

48. The blood substitute of any one of claims 37-47, further comprising a second population of erythroblasts derived from a second individual.

49. The blood substitute of any one of claims 37-48, comprising 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.

50. 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.

51. The kit of claim 50, further comprising a calpain inhibitor.

52. The kit of any one of claims 50-51, wherein the recombinant c-Myc protein comprises c-Myc-Rl 1.

53. The kit of any one of claims 50-52, wherein the TLR3 activator comprises polyinosinic-polycytidylic acid (poly (I:C)).

54. The kit of any one of claims 50-53, wherein the calpain inhibitor comprises

ALLN.

55. A kit for transfusing a patient with a blood substitute comprising:

a container containing a blood substitute, wherein the blood substitute is comprises the population of erythroblasts of claim 20

56. The kit of claim 55, wherein the container is selected from the group consisting of ajar, cylinder, and bag.