Methods of producing erythroid cells and / or red blood cells

Abstract

This disclosure provides a method for producing erythrocytic stem cells and / or erythrocytes, comprising culturing hematopoietic stem cells (HSCs) or erythrocytic cells together with a population of immortalized mesenchymal stem cells (MSCs) or a conditioned medium obtained from such immortalized MSCs, wherein the immortalized MSCs are genetically engineered to have viable genes. A method for manufacturing blood products for transfusion and a method for increasing hemoglobin synthesis are also provided.

Description

Technical Field

[0001] This disclosure pertains to the field of red blood cell production. Specifically, engineered stem cells containing at least one viable gene are used to produce erythropoietin-like cells and / or red blood cells.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 63 / 024,176, filed May 13, 2020, the entire contents of which are incorporated herein by reference for all purposes. Background Technology

[0004] Although blood transfusions are widely used in various clinical therapies, clinical blood supplies are limited, and the supply of blood for transfusions depends on volunteer donations. The gradual decline in birth rates is leading to a decrease in the eligible donor population, and a global blood shortage is projected (Transfusion 2010; 50:584-588). Furthermore, transfusion-related infectious diseases remain a significant problem. Fortunately, because the culture medium used for cell expansion can be automatically replaced, it is possible to obtain large quantities of target cells beyond laboratory levels.

[0005] The discovery of technologies for large-scale in vitro production of red blood cells (RBCs) is important for alternative sources of RBC production. Feeder-free culture in bioreactor systems enables manufacturers to develop xenogeneic, cost-effective culture protocols for large-scale in vitro cell generation, offering significant advantages for clinical applications (Tissue Engineering. Part CMethods 2011; 17:1131-1137, Biomaterials 2005; 26:7481-7503). However, the total number of mature red blood cells or the final RBC enucleation rate after leukopenia is not yet clear. The reproducibility and feasibility of these results should be demonstrated before practical application.

[0006] Therefore, methods for large-scale production of red blood cells are urgently needed for therapeutic applications. Summary of the Invention

[0007] This disclosure relates to providing a suitable microenvironment and matrix, such as mesenchymal stem cells (MSCs), to induce erythrocyte production and RBC enucleation.

[0008] In one embodiment, this disclosure provides a method for producing erythroblasts and / or erythrocytes, comprising culturing hematopoietic stem cells or erythroblasts together with a population of immortalized mesenchymal stem cells (MSCs) or a conditioned medium obtained from immortalized MSCs, wherein the immortalized MSCs are genetically engineered with viable genes.

[0009] In some embodiments, the cell count of HSCs or erythroblasts is in the range of approximately 100:1 to approximately 1:100, approximately 80:1 to approximately 1:80, approximately 70:1 to approximately 1:70, approximately 60:1 to approximately 1:60, approximately 50:1 to approximately 1:50, approximately 40:1 to approximately 1:40, approximately 30:1 to approximately 1:30, approximately 20:1 to approximately 1:20, approximately 18:1 to approximately 1:18, approximately 16:1 to approximately 1:16, approximately 14:1 to approximately 1:14, approximately 12:1 to approximately 1:12, approximately 10:1 to approximately 1:10, approximately 10:1 to approximately 1:8, approximately 10:1 to approximately 1:6, approximately 10:1 to approximately 1:4, approximately 10:1 to approximately 1:2, and approximately 10:1 to approximately 1:1.

[0010] In some embodiments, HSC is CD34 + HSC. In another variant, HSC is preferably derived from human umbilical cord blood.

[0011] In some embodiments, the surviving gene is the Akt gene or the hepatocyte growth factor (HGF) gene. Preferably, the surviving gene is the Akt gene.

[0012] In some embodiments, immortalized MSCs are immortalized using human telomerase reverse transcriptase (hTERT).

[0013] In one embodiment, the mesenchymal stem cells described herein are umbilical cord mesenchymal stem cells (UMSC), adipose-derived mesenchymal stem cells (ADSC), or bone marrow mesenchymal stem cells (BMSC).

[0014] In some embodiments, the immortalized MSC is CD146. + IGF1R + .

[0015] In some embodiments, immortalized MSCs are subjected to hypoxia treatment.

[0016] In one embodiment, the method described herein includes enhancing HSC proliferation by co-culturing HSCs with immortalized MSCs or conditioned medium obtained from immortalized MSCs. In some embodiments, the HSC proliferation line is enhanced by co-culturing HSCs with immortalized MSCs or conditioned medium obtained from immortalized MSCs for 0.5 to 8 days, such as 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days; preferably 2 to 6 days, such as 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days; more preferably 3 to 5 days, such as 3 days, 3.5 days, 4 days, 4.5 days, or 5 days.

[0017] In some embodiments, the method further comprises culturing the HSC with at least one of the following: stem cell factor (SCF), fms-like tyrosine kinase 3 (Flt-3), interleukin-3 (IL-3), vitamin C, and dexamethasone.

[0018] In one embodiment, the method described herein includes inducing HSC differentiation into erythroblasts by culturing HSCs together with immortalized MSCs or conditioned medium obtained from immortalized MSCs. In some embodiments, HSCs are cultured together with immortalized MSCs or conditioned medium obtained from immortalized MSCs to induce HSC differentiation into erythroblast lines for 5 to 20 days, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days; preferably 8 to 16 days, such as 8, 9, 10, 11, 12, 13, 14, 15, or 16 days; more preferably 10 to 15 days, such as 10, 11, 12, 13, 14, or 15 days.

[0019] In some embodiments, the method further comprises culturing HSCs with at least one of the following: SCF, erythropoietin (EPO), granulocyte-macrophage community-stimulating factor (GM-CSF), Flt-3, dexamethasone, IL-3, vitamin C, and platelet-rich plasma (PRP).

[0020] In one embodiment, the method described herein includes promoting erythroblast differentiation and maturation by co-culturing erythroblasts with immortalized MSCs or conditioned medium obtained from immortalized MSCs. In some embodiments, the culturing of erythroblasts with immortalized MSCs or conditioned medium obtained from immortalized MSCs to promote erythroblast differentiation and maturation is performed for 0.5 to 8 days, such as 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days; preferably 2 to 6 days, such as 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days; more preferably 2 to 5 days, such as 2 days, 3 days, 4 days, or 5 days.

[0021] In some embodiments, the method further comprises culturing erythroblasts with at least one of the following: heparin, transferrin, SCF, EPO, and vitamin C.

[0022] In some embodiments, the concentration of SCF in the culture medium used to culture HSCs or erythroblasts is in the following ranges: about 10 ng / mL to about 1,000 ng / mL; about 20 ng / mL to about 800 ng / mL; about 30 ng / mL to about 600 ng / mL; about 40 ng / mL to about 400 ng / mL; about 50 ng / mL to about 300 ng / mL; about 60 ng / mL to about 250 ng / mL; about 80 ng / mL to about 200 ng / mL; about 80 ng / mL to about 150 ng / mL. In some embodiments, the concentration of Flt3 in the culture medium used to culture HSCs or erythroblasts is in the following ranges: about 10 ng / mL to about 1,000 ng / mL; about 20 ng / mL to about 800 ng / mL; about 30 ng / mL to about 600 ng / mL; about 40 ng / mL to about 400 ng / mL; about 50 ng / mL to about 300 ng / mL; about 60 ng / mL to about 250 ng / mL; about 80 ng / mL to about 200 ng / mL; about 80 ng / mL to about 150 ng / mL. In some embodiments, the concentration of IL-3 in the culture medium used to culture HSCs or erythroblasts is in the following ranges: about 1 ng / mL to about 100 ng / mL; about 2 ng / mL to about 80 ng / mL; about 4 ng / mL to about 60 ng / mL; about 6 ng / mL to about 40 ng / mL; about 8 ng / mL to about 35 ng / mL; about 10 ng / mL to about 30 ng / mL; about 12 ng / mL to about 25 ng / mL; about 15 ng / mL to about 25 ng / mL. In some embodiments, the concentration of vitamin C in the culture medium used for culturing HSCs or erythroblasts is in the following ranges: about 5 μM to about 200 μM; about 8 μM to about 150 μM; about 10 μM to about 120 μM; about 15 μM to about 100 μM; about 20 μM to about 80 μM; about 25 μM to about 60 μM; about 25 μM to about 40 μM; about 25 μM to about 35 μM. In some embodiments, the concentration of dexamethasone in the culture medium used for culturing HSCs or erythroblasts is in the following ranges: about 0.1 μM to about 10 μM; about 0.2 μM to about 8 μM; about 0.3 μM to about 6 μM; about 0.4 μM to about 4 μM; about 0.5 μM to about 3 μM; about 0.6 μM to about 2 μM; about 0.8 μM to about 1.5 μM; about 0.8 μM to about 1.2 μM.In some embodiments, the concentration of EPO in the culture medium used to culture HSCs or erythroblasts is in the following ranges: about 0.1 IU / mL to about 20 IU / mL; about 0.2 IU / mL to about 18 IU / mL; about 0.5 IU / mL to about 16 IU / mL; about 0.8 IU / mL to about 14 IU / mL; about 1 IU / mL to about 12 IU / mL; about 2 IU / mL to about 10 IU / mL; about 3 IU / mL to about 9 IU / mL; about 4 IU / mL to about 8 IU / mL. In some embodiments, the concentration of GM-CSF in the culture medium used for culturing HSCs or erythroblasts is in the following ranges: about 1 ng / mL to about 50 ng / mL; about 2 ng / mL to about 45 ng / mL; about 4 ng / mL to about 40 ng / mL; about 6 ng / mL to about 35 ng / mL; about 8 ng / mL to about 30 ng / mL; about 10 ng / mL to about 25 ng / mL; about 12 ng / mL to about 25 ng / mL; about 13 ng / mL to about 20 ng / mL. In some embodiments, the concentration of PRP in the culture medium used for culturing HSCs or erythroblasts is in the following ranges: about 1% to about 100%; about 2% to about 80%; about 3% to about 60%; about 4% to about 40%; about 5% to about 35%; about 6% to about 30%; about 7% to about 20%; about 8% to about 15%. In some embodiments, the concentration of heparin in the culture medium used to culture HSCs or erythroblasts is in the following ranges: about 0.1 U / mL to about 20 U / mL; about 0.2 U / mL to about 18 U / mL; about 0.5 U / mL to about 16 U / mL; about 0.8 U / mL to about 14 U / mL; about 1 U / mL to about 12 U / mL; about 2 U / mL to about 10 U / mL; about 3 U / mL to about 9 U / mL; about 4 U / mL to about 8 U / mL. In some embodiments, the concentration of transferrin in the culture medium used to culture HSCs or erythroblasts is in the following ranges: about 10 μg / mL to about 2,000 μg / mL; about 50 μg / mL to about 1,800 μg / mL; about 100 μg / mL to about 1,600 μg / mL; about 200 μg / mL to about 1,400 μg / mL; about 300 μg / mL to about 1,300 μg / mL; about 40 μg / mL to about 1,200 μg / mL; about 500 μg / mL to about 1,000 μg / mL; about 600 μg / mL to about 900 μg / mL.

[0023] In one embodiment, the method described herein includes enhancing HSC proliferation by culturing HSCs with immortalized MSCs or conditioned medium obtained from immortalized MSCs; inducing HSC differentiation into erythroblasts by culturing HSCs with immortalized MSCs or conditioned medium obtained from immortalized MSCs; and promoting erythroblast differentiation and maturation by culturing erythroblasts with immortalized MSCs or conditioned medium obtained from immortalized MSCs.

[0024] In one embodiment, this disclosure provides a method for manufacturing blood products for blood transfusion, comprising producing erythroblasts and / or erythrocytes by means of the methods described herein.

[0025] In one embodiment, this disclosure provides a method for increasing hemoglobin synthesis, comprising producing erythroblasts and / or erythrocytes by means of the methods described herein.

[0026] In some embodiments, the hemoglobin is adult hemoglobin.

[0027] Simple Explanation of the Diagram

[0028] Figure 1A The results of adipocyte, chondrocyte, and osteocyte differentiation of hTERT-ADSC-Akt and hTERT-ADSC are presented.

[0029] Figure 1B This study presents the results of plastid construction for AKT transduction and the analysis of hTERT-ADSC-Akt and hTERT-ADSC using Western ink dot assay, ELISA, and flow cytometry.

[0030] Figure 1C The results of VEGF secretion by hTERT-ADSC-Akt, hTERT-ADSC, hTERT-ADSC-Akt pretreated with hypoxia (H2O), and hTERT-ADSC pretreated with hypoxia (H2O) are presented according to ELISA at 24, 48, and 72 hours.

[0031] Figure 1D CD34 cells cultured from day 5 to day 21, with or without conditioned medium. + The results of cell proliferation.

[0032] Figure 2A Shown from CB CD34 + The result of cells producing red blood cells in vitro on an industrial scale.

[0033] Figure 2B The results of cell proliferation and differentiation into erythroblast lineage based on flow cytometry analysis are presented.

[0034] Figure 2C The results show the proliferation and differentiation of cells from stem cells into erythroblast lineages based on Wright-Giemsa cell staining.

[0035] Figure 2D The results of cell staining using Wright-Giemsa staining are shown from day 1 to day 21.

[0036] Figure 3A The results show the hemoglobin content of differentiated cells from day 18 to day 21.

[0037] Figure 3B Photographs showing differentiated cells from day 18 to day 21.

[0038] Figure 3C Results demonstrating cell vitality.

[0039] Figure 3D Demonstrating the enucleated RBC rate (CD235a) based on flow cytometry. + / NucRed - The result of ).

[0040] Figure 4A The results of examining hemoglobin subtypes and the hemoglobin expression of cultured erythroblasts and PBs using flow cytometry are presented.

[0041] Figure 4B The results show the markers of red blood cells such as RBCs and the content of hemoglobin after culture.

[0042] Figure 5 Demonstrates CFSE under a confocal microscope when adult peripheral blood RBCs (pRBCs) or cRBCs labeled with CFSE are injected into NOD / SCID or nude mice treated with CL2MDP-liposomes. + Results of cRBC percentage. Detailed Implementation

[0043] Unless otherwise defined, all scientific or technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains. One of ordinary skill in the art can understand and practice this disclosure using any methods and materials similar to or equivalent to those described herein.

[0044] Unless otherwise indicated, all figures used in this specification and the claims to represent quantities of ingredients, reaction conditions, etc., are to be understood as being modified by the term "about" in all cases. Therefore, unless indicated to the contrary, the numerical parameters set forth in this disclosure and the claims are approximate values ​​and may vary depending on the desired characteristics sought by this disclosure.

[0045] The term "a / an" shall mean one or more of the objects described in this disclosure. The term "and / or" means one or both of the alternatives. The term "a cell" or "the cell" may include a plurality of cells.

[0046] As used in this article, "erythroblasts" contain a nucleus until the cell expels its nucleus and enters circulation as anucleate red blood cells / erythrocytes.

[0047] The term "in vitro" generally refers to the external environment of a living organism, such as experiments conducted in an artificial environment formed outside the organism. The term "in vitro" typically describes procedures, tests, and experiments performed outside a living organism.

[0048] As used in this article, “immortification” refers to the induction, promotion, or realization of cell viability, cell survival, and / or cell proliferation.

[0049] As used herein, the term "stem cell" refers to cells in an undifferentiated or partially differentiated state that possess self-renewal properties and the developmental potential to naturally differentiate into more differentiated cell types, without any specific implied meaning regarding developmental potential (i.e., totipotency, pluripotency, multipotency, etc.). Self-renewal means that stem cells can proliferate and produce more of these stem cells while maintaining their developmental potential. Therefore, the term "stem cell" refers to any subpopulation of cells that, under certain conditions, possess the developmental potential to differentiate into a more specific or differentiated phenotype, and in some cases retain the ability to proliferate without substantially differentiating.

[0050] As used in this article, the term “originating from” should be understood to indicate that a particular sample or sample group is derived from a specified species, but not necessarily directly from a specified source.

[0051] In the context of cell ontogenesis, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is a cell that further develops in the developmental pathway compared to the cell being compared. Thus, stem cells can differentiate into lineage-restricted precursor cells (such as HSCs), which in turn can further differentiate into other types of precursor cells in the pathway (such as erythroblasts), and then differentiate into terminally differentiated cells, which play a characteristic role in certain tissue types and may or may not retain the ability to proliferate further.

[0052] The term "genetically engineered" or "genetic engineering" in cells refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels within cells. Genetic material can be in the form of DNA or RNA. It can be transferred into cells via various methods, including viral transduction and non-viral transfection. Following genetic engineering, the expression levels of certain genes within the cell can be permanently or temporarily altered.

[0053] The term "transduction" or "transduce" refers to the delivery of genetic material into cells using a virus, which can be an integrating or non-integrating virus. The integrating virus used in this disclosure can be a lentivirus or a retrovirus. An integrating virus allows its coding gene to be integrated into transduced cells infected with viral particles. Non-integrating viruses can be adenoviruses or Sendai viruses. Non-viral methods, such as transfecting cells with DNA or RNA material, can also be used in this disclosure. DNA material can be in the form of PiggyBac, microcircular vectors, or epiplastic plasmids. RNA material can be in the form of mRNA or miRNA.

[0054] The term "expression vector" refers to a reagent that carries a foreign gene into a cell for expression without degradation. In this disclosure, expression vectors may include plastids, viral vectors, and artificial chromosomes.

[0055] To induce erythrocyte production and RBC enucleation, it is crucial to prepare a suitable microenvironment. Recently, CB-derived CD34 cells co-cultured on xenogeneic (mouse) stromal cells have been shown to be effective. + Large-scale expansion of RBCs was achieved using human cells (Nat Biotechnol. 2005;23:69-74). However, for human applications, animal-derived cells replaced with human stromal cells should be established. CD34 was observed in the hTERT stromal co-culture system compared to liquid cultures without feeder cells. +Cell proliferation rate and erythroblast enucleation rate were significantly increased (Nat Biotechnol. 2006;24:1255-6).

[0056] This disclosure reveals the use of immortalized MSCs with viable genetic modifications to optimize culture strategies for producing CB CD34. + A continuous triphasic co-culture system for large-scale in vitro production of human erythrocytes. Therefore, this disclosure provides a method for producing erythroblasts and / or erythrocytes, comprising co-culturing hematopoietic stem cells or erythroblasts with a population of immortalized mesenchymal stem cells (MSCs) or conditioned medium obtained from immortalized MSCs, wherein the immortalized MSCs are genetically engineered to have viable genes.

[0057] The mesenchymal stem cells used in this disclosure can be obtained from various sources, preferably from the umbilical cord, adipose tissue, or bone marrow. Depending on the source, the mesenchymal stem cells are umbilical cord mesenchymal stem cells (UMSC), adipose-derived mesenchymal stem cells (ADSC), or bone marrow mesenchymal stem cells (BMSC). In some embodiments of this disclosure, MSCs are isolated and purified from the umbilical cord and are referred to as "umbilical cord MSCs" or "UMSC". In some embodiments, the UMSCs of this disclosure have been determined to exhibit the same surface marker selection as MSCs isolated from other organisms and to show comparable activity.

[0058] The immortalized MSCs disclosed herein are modified to express Akt or HGF. As used herein, the term "modified to express" means the transfer of a foreign gene or gene fragment into mesenchymal stem cells to enable them to express the foreign gene or gene fragment. Preferably, this modification does not alter the differentiation potential of the immortalized MSCs. In another instance, this modification is preferably a stable modification, and the expression can be persistent or inducible. The immortalized MSCs disclosed herein, modified to express Akt or HGF, still possess similar pluripotent differentiation potential to commonly used immortalized MSCs or normal MSCs without Akt or HGF transduction, such as, but not limited to, adipogenesis, chondrogenesis, osteogenic formation, and angiogenesis.

[0059] Protein kinase B (PKB), also known as Akt, is a serine / threonine-specific protein kinase that plays a crucial role in various cellular processes, such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Akt regulates cell survival and metabolism by binding to and modulating many downstream effectors, such as nuclear factor-κB, Bcl-2 family proteins, the master lysosomal regulator TFEB, and murine two-microsome 2 (MDM2). Akt can directly and indirectly promote growth factor-mediated cell survival. It has been found that hypoxia pretreatment of transplanted cells (briefly cultured cells before transplantation) protects human brain endothelial cells from ischemic apoptosis by activating the Akt-dependent pathway (Am J Transl Res. 2017; 9: 664-673).

[0060] Hepatocyte growth factor (HGF), or dispersing factor (SF), is a paracrine cell growth, activity, and morphogenesis factor. Secreted by mesenchymal cells, it primarily targets and acts on epithelial and endothelial cells, and also on hematopoietic precursor cells and T cells. HGF regulates cell growth, cell activity, and morphogenesis by activating the tyrosine kinase signaling cascade after binding to the proto-oncogenic c-Met receptor. HGF is secreted by mesenchymal cells and acts as a multifunctional cytokine on cells primarily derived from epithelial cells.

[0061] The method of modifying immortalized MSCs with Akt or HGF is not limited. Preferably, Akt or HGF is transduced via transposon or lentivirus; more preferably, the transposon is the piggyBac transposon. Results show that the piggyBac transposon can effectively and stably transfect MSCs, and the gene modification of piggyBac does not change the DNA copy number or configuration of MSCs.

[0062] In some embodiments, the immortalized stem cells used in any of the methods described herein contain agents that induce cell immortality.

[0063] In some embodiments, immortalized cell lines are generated by treating cells with an immortalization agent. In some embodiments, the immortalization agent comprises a transgenic gene that expresses or overexpresses a polypeptide that induces cell immortalization. In some embodiments, the immortalization agent comprises a polypeptide that induces cell immortalization. In some embodiments, the polypeptide that induces cell immortalization is an oncogenic peptide. An oncogenic peptide is any suitable class of peptides that induce cell immortalization. For example, in some embodiments, suitable oncogenic peptides that induce cell immortalization are: growth factors and / or mitogens (e.g., PDGF-derived growth factors, such as c-Sis); receptor tyrosine kinases, specifically constitutively active receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR), coagulation cell-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and HER2 / neu); cytoplasmic tyrosine kinases (e.g., the Src family of tyrosine kinases, Syk...). -ZAP-70 family and BTK family); cytoplasmic serine / threonine kinases and their regulatory subunits (e.g., Raf kinase, cyclin-dependent kinase, Akt family members); regulatory GTPases (e.g., Ras protein); transcription factors (e.g., Myc and HIF-1a); telomerase reverse transcriptase (e.g., TERT or hTERT); and / or factors that activate other oncoplasmic peptides (e.g., cyclins, including cyclins A, B, D and / or E, such as cyclins D1 and D3). In some embodiments, the tumor peptide is Myc, HIF-1a, Notch-1, Akt, hTERT or cyclin. In some embodiments, the tumor peptide is a functional fragment, homolog or analog of any oncoplasmic peptide that induces cell viability, cell survival and / or cell proliferation, such as Myc, HIF-1a, Notch-1, Akt, hTERT or cyclin, preferably a functional fragment, homolog or analog of hTERT.

[0064] The immortalized MSCs disclosed herein contain expression vectors comprising the Akt or HGF gene. In addition to the Akt or HGF sequence, the vectors disclosed herein also contain one or more control sequences for regulating the expression of the polynucleotides disclosed herein. The manipulation of the isolated polynucleotides before their insertion into the vector may be desired or necessary depending on the expression vector used. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, the control sequences particularly include promoters, leader sequences, polyadenylated sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, a suitable promoter is selected based on host cell selection.

[0065] The recombinant expression vectors disclosed herein are disclosed together with one or more expression regulation regions, such as promoters and terminators, origins of replication, etc., depending on the type of host to which they are intended to be introduced. Non-limiting examples of constitutive promoters include SFFV, CMV, PKG, MDNU3, SV40, Ef1a, UBC, and CAGG.

[0066] The various nucleic acids and control sequences described herein are conjugated together to produce recombinant expression vectors, which include one or more convenient restriction sites allowing insertion or substitution of the polynucleotides disclosed herein at such sites. Alternatively, in some embodiments, the polynucleotides disclosed herein are expressed by inserting the polynucleotide or a nucleic acid construct containing the sequence into a suitable vector for expression. In some embodiments involving the generation of expression vectors, a coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression. The recombinant expression vector can be any suitable vector (e.g., plastid or virus) that can suitably undergo a recombinant DNA procedure and elicit expression of the polynucleotides disclosed herein. The choice of vector will generally depend on the compatibility of the vector with the host cell in which the vector is introduced. The vector can be a linear or closed circular plastid. In one embodiment, the vector is a viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, alpha virus vectors, and the like. In one embodiment, the viral vector is a lentiviral vector. Lentiviral vector systems are based on or derived from oncogenic retroviruses (retroviral subgroups containing MLV) and lentiviruses (retroviral subgroups containing HIV). Examples of such viruses include, but are not limited to, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Alternatively, other retroviruses, such as murine leukemia virus (MLV), may be considered as the basis for the vector backbone.

[0067] In some embodiments, the immortalized MSCs disclosed herein have been tested in various differentiation analyses to determine their compatibility with conventional MSCs isolated from other locations in the mammalian body. Differentiation analyses include adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation. In some embodiments, differentiation analyses further include neuronal cell differentiation.

[0068] In some embodiments disclosed herein, Akt-modified hTERT-MSCs are used to optimize the culture strategy to produce a continuous triphase co-culture system with hTERT-MSC-Akt for use with CB CD34. + Human red blood cells are produced on a large scale in vitro. To induce erythrocyte production and RBC enucleation, it is important to prepare a suitable microenvironment with sufficient interleukin supplements and matrix (such as mesenchymal stem cells (MSCs)).

[0069] Preferably, the immortalized MSCs as described in this disclosure are subjected to hypoxia treatment. In one embodiment of this disclosure, hypoxia pretreatment of Akt-modified immortalized MSCs induces greater VEGF secretion in conditioned medium compared to Akt-free immortalized MSCs.

[0070] In one embodiment of this disclosure, via a co-culture system with MSCs or with umbilical cord blood-derived CD34 + HSC as the starting material and a combination of derivatized conditioned medium for liquid culture were used to amplify erythroblasts in vitro. Under optimal conditions of erythroblast proliferation and differentiation, cultured for more than 25 days, more than 10 [units of erythroblasts] were produced within 25 days. 6 -10 7 Double expansion. Homogeneous erythrocytic lines were characterized by cell morphology and flow cytometry. Additionally, conditioned medium or CD146 cells carrying Akt (hTERT-ADSC-Akt) were added. + IGF1R + Immortalized MSCs were co-cultured to improve terminal erythroblast maturation. Cultured erythroblasts underwent several maturation events, including size reduction, increased expression of blood group glycoprotein A (CD235a), and nuclear condensation, resulting in the expulsion of pyknotted nuclei in up to 80% or more of the cells. Importantly, they possess the ability to express the adult definitive β-hemoglobin chain (HbA) upon further maturation. The oxygen balance profile of cord blood differentiated erythroblasts (RBCs) was comparable to that of normal RBCs. The high number and purity of erythroblasts and RBCs derived from cord blood make this method suitable for providing a basis for future production of RBCs usable for transfusion.

[0071] In one embodiment, erythroblasts are derived from HSCs that have been expanded and differentiated in vitro or in vitro. In some embodiments, erythroblasts include hematopoietic precursor cells, such as CD34 cells. + cell.

[0072] In one embodiment, erythroblasts are obtained from blood. Erythroblasts obtained from blood or from HSCs expanded and differentiated in vitro or in vitro can be used for further erythrocyte production.

[0073] In some embodiments, the immortalized HSC was successfully maintained continuously as an immortalized ESC strain.

[0074] In one embodiment, the method described herein includes a first phase of enhancing HSC proliferation by culturing HSCs together with immortalized MSCs or conditioned medium obtained from immortalized MSCs. In some embodiments, the first phase of the method further includes culturing HSCs together with at least one of the following: stem cell factor (SCF), fms-like tyrosine kinase 3 (Flt-3), interleukin-3 (IL-3), vitamin C, and dexamethasone.

[0075] In one embodiment, the method described herein includes a second stage of inducing HSC differentiation into erythroblasts by culturing HSCs together with immortalized MSCs or conditioned medium derived from immortalized MSCs. In some embodiments, the second stage of the method further includes culturing HSCs together with at least one of the following: SCF, erythropoietin (EPO), granulocyte-macrophage community-stimulating factor (GM-CSF), Flt-3, dexamethasone, IL-3, vitamin C, and platelet-rich plasma (PRP).

[0076] In one embodiment, the method described herein includes a third stage of promoting erythroblast differentiation and maturation by culturing erythroblasts with immortalized MSCs or conditioned medium obtained from immortalized MSCs. In some embodiments, the third stage of the method further includes culturing erythroblasts with at least one of the following: heparin, transferrin, SCF, EPO, and vitamin C.

[0077] In one embodiment of this disclosure, via a co-culture system with MSCs or with umbilical cord blood-derived CD34 + HSC as the starting material and a combination of derivatized conditioned medium for liquid culture were used to amplify erythroblasts in vitro. Under optimal conditions of erythroblast proliferation and differentiation, cultured for more than 25 days, more than 10 [units of erythroblasts] were produced within 25 days. 6 -10 7 Double expansion. Homogeneous erythrocytic lines were characterized by cell morphology and flow cytometry. Additionally, conditioned medium or CD146 cells carrying Akt (hTERT-ADSC-Akt) were added. + IGF1R + Immortalized MSCs were co-cultured to improve terminal erythroblast maturation. Cultured erythroblasts underwent several maturation events, including size reduction, increased expression of blood group glycoprotein A (CD235a), and nuclear condensation, resulting in the expulsion of pyknotted nuclei in up to 80% of the cells. Importantly, they possess the ability to express the adult definitive β-hemoglobin chain (HbA) upon further maturation. The oxygen balance profile of cord blood differentiated erythroblasts (RBCs) was comparable to that of normal RBCs. The high number and purity of erythroblasts and RBCs derived from cord blood make this method suitable for providing a basis for future production of RBCs usable for transfusion.

[0078] In one embodiment, erythroblasts are derived from HSCs that have been expanded and differentiated in vitro or in vitro. In some embodiments, erythroblasts include hematopoietic precursor cells, such as CD34 cells. + cell.

[0079] In one embodiment, erythroblasts are obtained from blood. Erythroblasts obtained from blood or from HSCs expanded and differentiated in vitro or in vitro can be used for further erythrocyte production.

[0080] In some embodiments, the immortalized HSC was successfully maintained continuously to become the immortalized ESC strain.

[0081] As used herein, conditioned media refers to media conditioned for the culture of immortalized MSCs. Such conditioned media contain molecules secreted by immortalized MSCs, including unique gene products. Such conditioned media and any combination of molecules contained therein (especially proteins or peptides) can be used to treat diseases. They can be used to supplement or replace the activity of immortalized MSCs, for example, for the purpose of producing erythrocytic and / or erythrocytes.

[0082] In one embodiment, this disclosure provides a method for manufacturing blood products for blood transfusion, comprising the method of producing erythrocytes and / or red blood cells as described herein.

[0083] In one embodiment, this disclosure provides a method for increasing hemoglobin synthesis, comprising the method for producing erythroblasts and / or erythrocytes as described herein.

[0084] It should be understood that if any prior art disclosure is cited in this document, such citation does not constitute an acknowledgment that such disclosure constitutes part of the common general knowledge in the art.

[0085] Although the disclosure has been provided in considerable detail with the aid of illustrations and examples for the purpose of clear understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of this disclosure. Therefore, the foregoing descriptions and examples should not be considered limiting.

[0086] Example

[0087] Methods and materials:

[0088] CD34 + Cell isolation and collection

[0089] Healthy adult volunteers provided cord blood (CB) samples (type O) from normal full-term deliveries. To obtain CB CD34... + We isolated low-density monocytes from CB cells using sucrose-sodium diatrizoate (SIGMA®) centrifugation, and then purified CB CD34+ cells from monocytes using a Mini-MACS column (MILTENYI®) via supermagnetic beads. The isolated CD34+ cells... +Cell purity is in the range of 90% to 99%, as determined by flow cytometry using anti-human CD34 mAb (BD®) bound to phycoerythrin (PE).

[0090] Preparation, separation and characterization of primary UMSCs

[0091] Human umbilical cord tissue without calcium 2+ and Mg 2+ Wash three times with PBS (DPBS, LIFE TECHNOLOGY®). Mechanically cut along the midline with scissors, separating the umbilical artery, veins, and outlining membrane from Wharton's jelly (WJ). Then, extensively cut the gelatinous contents into pieces smaller than 0.5 cm. 3 Small pieces of explants were treated with type 1 collagenase (SIGMA®, St Louis, USA) and cultured at 37°C in a humidified atmosphere of 95% air / 5% CO2 for 3 hours. The explants were then cultured in DMEM containing 10% fetal bovine serum (FCS) and antibiotics at 37°C in a humidified atmosphere of 95% air / 5% CO2. They were kept undisturbed for 5–7 days to allow cell migration from the explants. The cell morphology of umbilical cord-derived mesenchymal stem cells (UMSCs) became uniformly spindle-shaped in the culture after 4–8 passages, and specific surface molecules from WJ cells were characterized by flow cytometry. Cells were isolated using PBS containing 2 mM EDTA, washed with PBS (SIGMA®) containing 2% BSA and 0.1% sodium azide, and cultured with individual antibodies bound to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE), including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC, and HLA-DR (BD®, PHARMINGEN®). Cells were then analyzed using a Becton Dickinson flow cytometer (BD®).

[0092] Plasma Structure

[0093] Akt cDNA (0.1 µg) (pCMV6-myc-DDK-Akt, ORIGENE®) from Akt plasmids was transferred to pIRES (CLONTECH®) or pSF-CMV-CMV-SbfI (OXFORD GENETICS®) using specific restriction enzyme ligands (EcoR1, Nhe1, BamH1, and Not1) to construct the pSF-Akt-GFP construct.

[0094] Construction of the piggyBac transposon system for stabilizing cell lines

[0095] The piggyBac vector pPB-CMV-MCS-EF1α-RedPuro, containing multiple selection sites (MCS), piggyBac terminal repeats (PB-TR), a core insulator (CI), and a puromycin select marker (BSD) (fused with human EF1α-driven RFP), was used as the base vector (SYSTEM BIOSCIENCES®). A DNA fragment containing Akt (from pSF-Akt) was amplified by PCR and then selectively colonized into the pPB-CMV-MCS-EF1α-RedPuro vector, preceding the EF1α coding region. Detailed information about the vector construct (pPB-Akt) is shown in [link to documentation]. Figure 1B To generate stable hTERT-ADSC-Akt cells, the pPB-Akt plasmids were co-transfected into hTERT-ADSC (SCRC-4000™, ATCC) using the piggyBac transposase expression vector (SYSTEM BIOSCIENCES®) via electroporation (AMAXA NUCLEOFECTOR II®, Lonza). Stable transfected cells were selected in the presence of puromycin.

[0096] Total protein extraction, Western ink spot method and ELISA

[0097] Cells were lysed in a buffer containing 320 mM sucrose, 5 mM HEPES, 1 μg / mL antifibrinolytic peptide, and 1 μg / mL aprotinin. The lysate was centrifuged at 13,000 g for 15 min. The resulting aggregates were resuspended in sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue, and 50 mM DTT) and subjected to SDS-polyacrylamide gel electrophoresis (4–12%). The gel was then transferred to a Hybond-P Nylon membrane. The membrane was then incubated with an appropriately diluted antibody: Akt (1:200, NOVUS BIOLOGICALS®). Membrane blocking, primary and secondary antibody incubation, and chemiluminescence reactions were performed on each antibody according to the manufacturer's protocol. The intensity of each band was measured using a Kodak® Digital Science 1D image analysis system (EASTMAN KODAK®). In addition, the total amount of VEGF and HGF (Quantikine ELISA kit, R&D®) in the culture medium was measured according to the manufacturer's instructions. Optical density was measured using a spectrophotometer (MOLECULAR DEVICES®), and a standard curve was generated using the SOFTmax program (MOLECULAR DEVICES®).

[0098] In vitro differentiation analysis

[0099] For adipocyte differentiation, cells were cultured in a medium containing low-glucose DMEM, 1× ITS (SIGMA®), 1 mg / ml LABSA (SIGMA®), 1 mM hydrocortisone (SIGMA), 60 mM indomethacin (SIGMA®), 0.5 mM isobutylmethylxanthine (SIGMA®), and 10% horse serum (INVITROGEN®). To assess adipogenic differentiation, cells were stained at room temperature with 0.3% Oil Red O (SIGMA®) as an indicator of intracellular lipid accumulation for 10 minutes and counterstained with hematoxylin. For chondrocyte differentiation, cells were cultured in a medium containing 90% high-glucose DMEM, 10% FBS, 1× ITS, 1 mg / ml LABSA, 50 nM dexamethasone, and 60 pM transforming growth factor-β1 (TGF-β1) (R&D SYSTEMS®). Osteogenic differentiation was performed using Alcian Blue 8GX for the proteoglycan-rich cartilage matrix and 1% Sirius Red F3B for the collagen matrix (SIGMA®). APSC confluent monolayer cultures grown in high-glucose DMEM containing 10% FCS, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mg / ml L-ascorbic acid 2-phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone were subjected to osteogenic differentiation. Osteogenesis was assessed using Alizarin Red S staining (1%) to detect calcium mineralization.

[0100] Preparation of MSC-derived conditioned medium

[0101] CD146 was placed in a culture flask. + IGF1R + hTERT-ADSC-Akt (1×10 6 Cells were grown to 80-90% confluence. They were then conditioned with 10 mL of serum-free CellGenix SCGM (CELLGENIX®). After 24 hours, the conditioned medium was collected and sterilized using a 0.2 mm syringe filter (THERMO FISHER®). The prepared conditioned medium was kept at -80°C until use.

[0102] hypoxia program

[0103] Cells cultured at 37°C in a 5% CO2 humidified incubator were treated under normoxic (21% O2) or various hypoxic conditions (1%, 3%, and 5% O2) at different time points (24 hours, 48 ​​hours, or 72 hours). Hypoxic cultures were cultured in a dual-gas incubator (JOUAN INC, Winchester, Virginia) equipped with an O2 probe to regulate N2 gas content. Trypan blue exclusion assay was used to assess cell number and viability.

[0104] Cytokine array

[0105] Whole protein was extracted using a lysis buffer supplemented with a mixture of protease and phosphatase inhibitors (INVITAGEN). The cytokine content of 100 mg of exosomal protein was tested according to the manufacturer's instructions using a Human Intercytokine Array (R&D SYSTEMS®). In short, the exosomal lysate was mixed with a detection antibody mixture and incubated overnight at 4°C with a membrane containing 40 different anti-cytokine capture antibodies. After incubation with the antibiotic streptavidin-HRP, the membrane was incubated with a chemiluminescent substrate and exposed to X-rays. Protein pixel density was quantified using ImageJ 1.47 software.

[0106] CD34 collected from umbilical cord blood (CB) and isolated + cell

[0107] Umbilical cord CB (CB) samples (type O) were collected. This study was approved by the hospital's ethics review board (IRB). CD34+ cells were isolated from CBs using a Mini-MACS column (MILTENYI®) via selection with supermagnetic microbeads and anti-CD34 mAb. The isolated CD34+ cells were measured by flow cytometry (BD®). + Cell purity.

[0108] CB CD34 + Cell culture in cell-free systems or on hTERT-ADSC-Akt (Phase 1)

[0109] In order to cultivate CB CD34 in the first stage of culture (days 1-4) + Cell expansion of HSCs, CB CD34 at 37°C in 5% CO2 + Cells (1×10) 5 (Number of cells / mL) were inoculated into a cell-free system containing conditioned medium, which had been spread onto a 75 cm³ plate containing 10 mL of serum-free SCGM (CELLGENIX®). 2In the CORNING® flask, the SCGM contained albumin and insulin, supplemented with 100 ng / mL recombinant human stem cell factor (SCF, GIBCO®), 1 µM dexamethasone (Dex, SIGMA®), 30 µM vitamin C (Vit-C, SIGMA®), and 1 ng / mL recombinant human interleukin-3 (IL-3, GIBCO®). The culture medium was partially replenished every two days.

[0110] HSCs were cultured to amplify and differentiate erythroblasts on hTERT-ADSC-Akt (stages two and three).

[0111] On day 8, to facilitate erythroblast expansion, cells (1 to 2 × 10⁻⁶) were... 6 (cells / mL) at 75 cm 2 The cells were maintained in CellGenix SCGM (CELLGENIX®) conditioned medium (with or without hTERT-ADSC-Akt) for 12–14 days in flasks (CORNING®) or Hyperflask (CORNING®). The CellGenix SCGM medium was supplemented with 100 ng / mL recombinant human stem cell factor (SCF, GIBCO®), 6 U / mL recombinant human erythropoietin (EPO, SIGMA®), 1 ng / mL IL-3 (GIBCO®), 30 µM vitamin C (Vit-C, SIGMA®), 5% platelet-rich plasma (PRP, AVENTACELL®), 15 ng / mL GM-CSF (GIBCO®), 100 ng / mL Flt3 (GIBCO®), and 1 µM dexamethasone (SIGMA®) (stage two). Subsequently, differentiated and enucleated (stage three) erythroblasts were seeded onto a monolayer of CD146. + IGF1R + hTERT-ADSC-Akt (1×10 6Cells were induced to differentiate in a newly regenerated (half) differentiation medium supplemented with CellGenix SCGM (CELLGENIX®) containing EPO (10 U / mL), SCF (100 ng / mL), transferrin (700 μg / mL, SIGMA®), 30 µM vitamin C (Vit-C, SIGMA®), and heparin (5 U / mL, SIGMA®) for 3 days. For leukocyte filtration, the cultured cells were then purified using a 60 mL leukocyte-depleting filter (Immuguard III-RC, TERUMO®). After filtration, the filter was washed twice and resuspended in 25 mL of CellGenix SCGM (CELLGENIX®). Cells were centrifuged at 1600 rpm for 5 min to obtain compressed RBCs. As previously described, the cultured cells were collected and stored at 4°C in a solution based on citrate-dextrose adenine (CPDA-1) preservative for 4 weeks.

[0112] Flow cytometry

[0113] To analyze cell surface marker expression, cells were separated in PBS containing 2 mM EDTA, washed with PBS containing BSA (2%) and sodium azide (0.1%), and then incubated with antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) until analysis. As a control, cells were stained with mouse IgG1 isotype control antibody. Antibody systems against CD34, CD36, CD45, CD71, CD146, IGF1R, and CD235a used for flow cytometry were purchased from BD Biosciences. Cells were analyzed using FACScan (BD®), CellQuest Analysis (BD BIOSCIENCES®), and FlowJo software v.8.8 (TREESTAR Inc.). Results are expressed as the percentage of positively stained cells relative to the total number of cells. For quantitative comparison of surface protein expression, fluorescence intensity for each sample is presented as median fluorescence intensity (MFI). Nuclei were stained with NucRed Live647 (NucRed, INVITROGEN®). From CD235a on days 18-21 + / NucRed - The kernel removal rate was partially calculated. Data were analyzed using FACScan (BD®), CellQuest Analysis (BD BIOSCIENCES®), and FlowJo v.8.8 (TREESTAR®).

[0114] Cell counting and morphological analysis of cultured cells

[0115] Cell number and morphology were assessed using an automated cell counter Z1 (BECKMAN COULTER®) and Wright-Giemsa staining (SIGMA®).

[0116] Hemoglobin content detection and oxygen dissociation curve

[0117] Hemoglobin (Hb) levels in cultured cells and red blood cells (RBCs) from healthy volunteers were quantified using Drabkin's reagent (SIGMA®) at 540 nm brightness. To measure hemoglobin status using flow cytometry, cells were fixed, permeabilized, and labeled with fetal hemoglobin-FITC (Hb-F, BD®) and hemoglobin β-PE (Hb-β, Santa Cruz). The oxygen dissociation curve of Hb in RBCs was measured using a Hemox-Analyzer (TCS SCIENTIFIC CORP).

[0118] Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0119] Cultured red blood cells (RBCs) were collected and evaluated to determine the RNA expression levels of ε-hemoglobin, γ-hemoglobin, β-hemoglobin, ζ-hemoglobin, and α-hemoglobin. Total RNA was isolated using the RNeasy mini kit (QIAGEN®), and complementary DNA (cDNA) was obtained using Superscript 3 First-strand for RT-PCR Synthesis (LIFE TECHNOLOGIES®). Quantitative PCR analysis was performed using gene-specific primers and probes in an Mx3000P (AGILENT TECHNOLOGIES®) instrument.

[0120] Hemoglobin (Hb) analysis by HPLC

[0121] To determine the ratio of Hb A to F, high-performance liquid chromatography (HPLC) at 610 nm was used to measure erythroblast lysates, CD34-derived RBCs, and CBs spectrophotometrically on a cation exchange TSK gel G7 HSi column (SIGMA®). Washed cell aggregates were analyzed using the Bio-Rad Variant II dual-program (BIO-RAD LABORATORIES®) according to the manufacturer's instructions.

[0122] In vivo mouse studies

[0123] Eight-week-old NOD / SCID or NSG mice were used. Before culturing RBCs (cRBCs) for injection, mice were intravenously injected twice (days -3 and 1) with CL2MDP-liposome (FORMUMAX®) to deplete macrophages. CFSE (LIFETECHNOLOGIES®)-labeled cRBCs (1.5 × 10⁻⁶) were then injected. 8 (1.5 × 10⁻⁶) or adult peripheral RBCs (pRBCs) (1.5 × 10⁻⁶) 8 (Number of cells) were injected into the femoral vein of mice. At 10, 20, 40, 60, 120, 240, 480, and 720 minutes post-inoculation, heparinized peripheral blood was aspirated from the retroorbital vein of NOD / SCID mice, once daily for 3–5 days thereafter. Cells were counted and double-stained with anti-human CD71, anti-human CD235a, and NucRed Live 647 nucleic acid dye (NUCRED®), and analyzed by flow cytometry. Blood transfusions were also administered to mice not treated with CL2MDP-liposomes (controls) and analyzed to assess the effect of murine macrophages on the inoculated cells.

[0124] Example 1: Optimization of culture protocols for expanding human erythrocytes from autologous hematopoietic stem cells

[0125] A three-stage protocol for autologous cord blood (CB) CD34 was developed using a standard culture medium formulation. + Cell proliferation and differentiation into human erythrocytes in vitro.

[0126] To isolate hematopoietic stem cells and collect them for CD34 + The volume of the selected CB sample was 95 ± 7.8 mL (n = 8). Separated CD34 + The purity and cell count of the cells were 95.5 ± 2.1% and 3.1 ± 0.3 × 10⁻⁶, respectively. 6 CD34 as assessed by 7-aminoactinomycin D (7-AAD) + The cell viability was 97.6 ± 0.4%.

[0127] CD34 + The ratio of cell count to cell count of immortalized MSCs (hTERT-ADSC-Akt or hTERT-ADSC) was approximately 10:1.

[0128] To demonstrate the advantages of hTERT-ADSC-Akt and its potential for stem cell autologous renewal, the mesenchymal differentiation among adipocytes, chondrocytes, and osteocytes was similar between hTERT-ADSC and hTERT-ADSC-Akt. Figure 1ACompared to hTERT-ADSC, a significant increase in the performance of Akt and p-Akt was observed in hTERT-ADSC-Akt. Figure 1B Importantly, CD146 is present in the hTERT-ADSC-Akt group. + IGF1R + The horizontal enhancement of the dry surface markers above ( Figure 1B Consistently, according to ELISA, hypoxic pretreatment with hTERT-ADSC-Akt induced more VEGF secretion in conditioned medium compared to hTERT-ADSC. Figure 1C ).

[0129] To demonstrate that the conditioned medium enhanced cell proliferation in step 1 (days 1 to 4), isolated CD34 cells were... + Cell expansion for 4 days to increase CD34 + The amount of hematopoietic stem cells (HSCs). CellGenix SCGM (CELLGENIX®) conditioned medium with hTERT-ADSC-Akt supplemented with 100 ng / ml SCF, 100 ng / mL Flt3, 20 ng / ml IL-3, 30 µM Vit-C, and 1 µM Dex induced approximately 30 ± 1.6 times higher amplification compared to unconditioned medium. Figure 1D ).

[0130] To facilitate the differentiation of HSCs induced and expanded in step 2 (days 5 to 18) into erythroblast lineages, we optimized the combination and concentration of growth factors, with or without hTERT-ADSC-Akt conditioned medium, for the in vitro generation of human erythroblast precursor cells. This medium included CellGenix SCGM (CELLGENIX®) supplemented with 100 ng / ml SCF, 6 IU / ml EPO, 10 ng / mL GM-CSF, 100 ng / mL Flt3, 1 µM dexamethasone, and 20 ng / ml IL-3 for erythroblast differentiation. Figure 1D Importantly, the addition of 5% human platelet-rich plasma (PRP) significantly increased cell yield.

[0131] To promote further differentiation and maturation of cultured erythroblasts in step 3 (days 19 to 21), cultured erythroblasts co-cultured with hTERT-ADSC-Akt were cultured in CellGenix SCGM (CELLGENIX®) supplemented with heparin (5 IU / ml), transferrin (700 µg / ml), SCF (100 ng / ml), and EPO (10 IU / ml) to obtain a higher level of total red blood cell count. Figure 1D SCF, EPO, GM-CSF, Flt3, and IL-3 showed significant amplification of cultured erythrocytic cells with 5% PRP.

[0132] Example 2: From CD34 + Cell amplification and expansion of human red blood cells

[0133] The optimization strategies mentioned above were used to perform self-CBCD34 culture on the Hyperflask culture system (CORNING®). + Cells produce erythrocytes in vitro on an industrial scale. This is achieved using approximately 100-120 liters of culture medium at a concentration of 1×10⁻⁶ cells / mL. 5 Cells / mL CBCD34 + It can produce 2.9 × 10⁻⁶ kernels with a kernel removal rate of 55.0%. 11 Total red blood cells (RBC). CD34 + The ratio of cell count to immortalized MSC cell count was approximately 10:1. The in vitro magnification of the total cells that slowly expanded during the initial culture period (Step 1, Day 1 to Day 4) is shown in the growth curve. Figure 2A Next, in step 2 (days 5 to 18), the cells maintain a high proliferation rate until the exponential growth phase. Figure 2A By day 12 and day 15, the cells had expanded to approximately 2.9 × 10⁻⁶. 6 Doubled to 8.9×10 7 The number of cells increased several times. Finally, in step 3, total cell production achieved a slow expansion rate, reaching approximately 2 × 10⁻⁶ by day 21-22. 8 times (1.4-2.53 10 8 The steady-state phase (times) was observed. Compared to the unconditioned medium, the culture protocol using hTERT-ADSC-Akt conditioned medium showed a greater expansion in cell yield (times). Figure 2A If culture is maintained, cell growth will decrease relative to cell differentiation and death observed from day 22-23 (data not shown).

[0134] Morphological examination of cell proliferation and differentiation into erythrocytic lineages from stem cells was performed using Wright-Giemsa staining and flow cytometry. Initially, as expected, erythrocytic markers CD71 and CD235a showed low levels, while high levels of HSC markers (CD34 and CD45) were observed from isolated CD34. + Cellular expression (day 0) Figures 2B-2C Gradually, CD34 + The percentage decreased significantly to approximately 1%-2% after 21 days of differentiation. Figures 2B-2C Conversely, CD235a expression gradually increases and remains at a high level after cell differentiation. Figures 2B-2C In differentiating cells, CD71 levels rapidly increased to a peak on day 8, and then continuously decreased after the differentiation process. Figures 2B-2C Finally, fully differentiated cells showed strong expression of CD235a (90.1% ± 6.2%) and weak expression of CD71 (54.0% ± 7.2%) on day 21. Figures 2B-2C Cell staining using Wright-Giemsa staining sequentially revealed cell morphology from the initial proerythroblasts to enucleated RBCs; erythrocytic phenotype was observed in this population. Figure 2D ).

[0135] Example 3: Enhanced erythroblast proliferation and maturation

[0136] The hemoglobin content of differentiated cells gradually increased from day 18 to day 21 (from 17.6 ± 2.2 pg / cell to 30.3 ± 1.8 pg / cell) to reach approximately the normal human RBC content (27-33 pg / cell). Figure 3A Furthermore, the increased hemoglobin synthesis following cell differentiation causes the color of cell aggregates to change from white-light pink to red after centrifugation. Figure 3B ).

[0137] Good cell morphology was observed during the immature stage up to day 11, but dead cells were observed starting from day 18. Cell viability on the final culture day showed intact cell membranes. Figure 3C Compared to no co-culture, co-culture of red blood cells with hTERT-ADSC-Akt significantly increased the enucleated RBC rate (CD235a) as measured by flow cytometry. + / NucRed - The average value up to day 21 was 54-65%. Figure 3D ).

[0138] Example 4: Adult hemoglobin with higher oxygen-carrying capacity

[0139] To examine hemoglobin subtypes using flow cytometry, although CB CD34+ cells mainly expressed both fetal hemoglobin (Hb-F) and adult hemoglobin (Hb-β), cultured RBCs in the hTERT-ADSC-Akt group showed a higher predominance of Hb-β compared to hTERT-ADSC, reaching 84.3±5.2% on day 21, which was significantly higher than that of normal adult peripheral blood (PB). Figure 4A Very few Hb-F positive cells were found, and Hb-β was also present. + Hb-F - The average proportion begins to increase from day 21. Figure 4A ).

[0140] To facilitate long-term storage of cultured RBCs, they were collected on day 28 and stored at 4°C in a preservative solution (CPDA-1) for 4 weeks. During storage, erythroblast markers and hemoglobin levels remained unchanged. Figure 4B ).

[0141] Example 5: Maturation of cultured red blood cells (cRBCs) in the NOD / SCID model

[0142] To investigate whether cultured red blood cells (cRBCs) mature in vivo, we injected CFSE-labeled adult peripheral blood RBCs (pRBCs) or cRBCs collected on days 21-23 into NOD / SCID or nude mice treated with CL2MDP-liposomes. Within 3 days post-injection, CFSE was detected in the peripheral blood of mice in both RBC groups. + cell( Figure 5 Three days after injection, according to confocal microscopy, CFSE + The percentage of cRBCs gradually decreased and remained similar to CFSE in mouse circulation. + The same degree of pRBC.

[0143] Although this disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications, and variations are considered to fall within the scope of this disclosure.

Claims

1. A method for producing red blood cells, the method comprising: (1) Hematopoietic stem cell (HSC) proliferation, wherein HSC proliferation is enhanced by culturing the HSCs together with a population of immortalized mesenchymal stem cells (MSCs) or a conditioned medium containing the immortalized MSCs; (2) Inducing the differentiation of the HSCs into erythroblasts by culturing the HSCs together with immortalized MSCs or a conditioned medium containing immortalized MSCs; and (3) Red blood cells are obtained by inducing enucleation of the erythroblasts by culturing them together with immortalized MSCs or a conditioned medium containing immortalized MSCs, wherein the cell count of the erythroblasts is in the range of 100:1 to 1:1 compared with the cell count of the immortalized MSCs. The immortalized MSCs mentioned above are adipose-derived mesenchymal stem cells (ADSCs) that express human telomerase reverse transcriptase (hTERT) and are genetically engineered to express the Akt gene.

2. The method of claim 1, wherein the immortalized MSC is CD146. + IGF1R + .

3. The method of claim 1, wherein the immortalized MSC is subjected to hypoxia treatment.

4. The method of claim 1, wherein the conditioned medium in step (3) further comprises at least one of the following: heparin, transferrin, SCF, EPO and vitamin C.

5. The method of claim 1, wherein the HSC is CD34. + HSC.

6. The method of claim 1, wherein the HSC is derived from human umbilical cord blood.

7. The method of claim 1, wherein the conditioned medium in step (1) further comprises at least one of the following: stem cell factor (SCF), fms-like tyrosine kinase 3 (Flt-3), interleukin-3 (IL-3), vitamin C, and dexamethasone.

8. The method of claim 1, wherein the conditioned medium in step (2) further comprises at least one of the following: SCF, erythropoietin (EPO), granulocyte-macrophage community-stimulating factor (GM-CSF), Flt-3, dexamethasone, IL-3, vitamin C and platelet-rich plasma (PRP).

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