Method for producing erythrocytes from canine hematopoietic or erythroid progenitor cells through in vitro culture

Abstract

The present invention relates to a method for differentiating canine hematopoietic stem cells into erythrocytes through in vitro culture, and a method for producing canine erythrocytes using same. According to the method, erythrocytes can be effectively produced from canine hematopoietic stem cells by using cytokines at specific concentrations.

Description

Method for producing red blood cells from canine hematopoietic stem cells or erythroid progenitor cells through in vitro culture

[0001] The present invention relates to a method for culturing canine hematopoietic stem cells or erythroid progenitor cells in vitro to differentiate and proliferate them into erythrocytes, and a method for producing canine erythrocytes through the same.

[0002] Blood transfusions in dogs play a crucial role in veterinary medicine, managing anemia and acute blood loss due to trauma or surgery. Blood donations are the only source of blood transfusions. Despite the widespread availability of advanced veterinary care, the demand for blood products continues to grow. However, the supply of blood from companion animals is limited, and ethical concerns surrounding blood donations are increasing, leading to a growing need for in vitro-produced red blood cells.

[0003] In humans, research on in vitro production has been ongoing for many years due to the shortage of blood donations, and clinical trials are currently underway. However, in dogs, even basic red blood cell production methods have not been studied, the related reagents are not abundant, and the appropriate culture medium is not known. A study by Leberbauer et al. (2005) demonstrated that up to 1x10 erythroid stem cells can be produced from umbilical cord blood mononuclear cells (MNCs). 9 They demonstrated that it can be expanded to the stomach. Researchers successfully produced red blood cells (RBCs) with simultaneous maturation and very high enucleation rates, making them indistinguishable from peripheral blood RBCs (Ref. 1). In 2011, the French research team Douay et al. reported the first case of transfusing 2 mL of blood cultured with CD34+ cells from autologous blood samples into a human recipient (Ref. 2). Recently, a clinical trial of blood transfusions using laboratory-grown RBCs (Recovery and survival of stem cell originated red cells: RESTORE) was attempted in the United Kingdom.

[0004] In contrast, studies on erythropoiesis, cell proliferation, differentiation, and hematopoiesis using mononuclear cells in dogs are relatively limited. H.-J. Kolb et al. investigated the immunophenotype of canine hematopoietic progenitor cells and explored potential therapeutic applications and disease models (Ref. 3).

[0005] In most cases, these studies aimed to understand immune responses and cell functions in various contexts by culturing immune cells in vitro. SK Kim et al. demonstrated that NK cell subtypes in dogs exhibit phenotypic switching and elucidated NK cell biology and potential therapeutic applications through in vitro monocyte culture (Reference 4).

[0006] Despite this, no studies have yet been conducted on in vitro culture of erythrocytes derived from monocytes. Similar to human erythrocytes, canine erythrocytes maintain a smooth, concave surface, and their average diameter increases slightly with age (Ref. 5). Cross-species erythrocyte metabolomic studies have shown that canine erythrocytes exhibit stronger antioxidant capacity and energy metabolism properties (Ref. 6). However, data and results related to in vitro studies of canine erythrocytes are currently lacking.

[0007] In the above situation, the inventors of the present invention studied a method for producing canine red blood cells, and confirmed that canine mononuclear cells can be effectively differentiated into red blood cells by controlling the concentration of canine EPO, thereby completing the present invention.

[0008] Accordingly, one object of the present invention is to provide a method for producing red blood cells by differentiating and proliferating canine hematopoietic stem cells or erythroid progenitor cells into red blood cells.

[0009] Another object of the present invention is to provide red blood cells produced by the above method.

[0010] To achieve the above object, one aspect of the present invention provides a method for producing canine red blood cells in vitro, comprising the following steps:

[0011] A step of obtaining hematopoietic stem cells or erythroid progenitor cells from cells of canine origin; and

[0012] A step of culturing the acquired hematopoietic stem cells or erythroid progenitor cells in a medium containing erythropoietin (EPO).

[0013] In the present invention, the canine-derived cells may be blood-derived cells, bone marrow-derived cells, umbilical cord blood-derived cells, or stem cells. Furthermore, the stem cells may be, but are not limited to, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPS cells, iPSCs).

[0014] Methods known in the art to which the present invention pertains can be used to isolate hematopoietic stem cells or erythroid progenitor cells from the canine cells. For example, if the canine cells are blood-derived cells, hematopoietic stem cells can be isolated using Ficoll or the like. If the canine cells are stem cells, the stem cells can be cultured in a medium that induces differentiation into hematopoietic stem cells / erythroid progenitor cells to isolate hematopoietic stem cells or erythroid progenitor cells.

[0015] The term erythroid precursor cells used in the present invention refers to all cells involved in the process of erythrocyte formation except for mature erythrocytes, and refers to erythroid cells before enucleation. For example, the erythroid precursor cells may be CD34+ cells. In addition, the erythroid precursor cells may be selected from the group consisting of burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, and reticulocyte.

[0016] The term "hematopoietic stem cells" used in the present invention refers to cells that account for approximately 0.1% of the blood of a normal person and possess the ability to produce all blood cells. Hematopoietic stem cells are found throughout the body and are mass-produced in the bone marrow. In the present invention, hematopoietic stem cells refer to cells capable of producing blood lineage cells through differentiation or other methods.

[0017] According to one specific example of the present invention, the hematopoietic stem cells may be mononuclear cells (MNCs). Mononuclear cells are peripheral blood cells with round nuclei and are composed of lymphocytes (T cells, B cells, NK cells) and monocytes.

[0018] In the present invention, the erythropoietin may be included in the medium at a concentration of 20 to 80 ng / ml, and preferably at a concentration of 30 to 50 ng / ml.

[0019] The present inventors confirmed the effect of erythropoietin concentration on the differentiation of canine monocytes into erythrocytes, and confirmed that increasing the concentration of erythropoietin caused differences in cell proliferation rate, enucleation, erythrocyte maturation, and lineage differentiation (Fig. 5). In particular, increasing the concentration of EPO did not increase cell proliferation and erythroid differentiation efficiency, and the present inventors confirmed that cell proliferation and erythroid differentiation efficiency were the best when canine recombinant EPO was used at a concentration of 40 ng / ml (Figs. 5 and 7).

[0020] Accordingly, in the present invention, the erythropoietin may be a canine erythropoietin, and may be added to the medium at a concentration of 5 to 80 ng / ml, preferably 20 to 60 ng / ml, and most preferably 40 ng / ml.

[0021] The present inventors also confirmed the influence of human erythropoietin, and confirmed that erythrocytes can be induced from canine mononuclear cells, although with lower efficiency than canine erythropoietin. Therefore, the erythropoietin may be human erythropoietin.

[0022] In the present invention, the medium may additionally contain transferrin, insulin, penicillin / streptomycin, and amino acids in addition to cytokines.

[0023] Additionally, the medium may further include stem cell factor (SCF) and interleukin-3 (IL3) as cytokines in addition to erythropoietin. The stem cell factor may be included in the medium at a concentration of 10 to 100 ng / ml, and interleukin-3 may be included in the medium at a concentration of 1 to 10 ng / ml.

[0024] The present inventors confirmed that when canine mononuclear cells were cultured in a medium containing erythropoietin, the cell number temporarily decreased in the early stages of culture, but from the 10th day of culture, erythrocytes without nuclei were observed (Fig. 2), and the cell number reached its maximum on the 17th day of culture (Fig. 5). Therefore, in the present invention, the culturing step may be performed for 3 to 30 days to allow sufficient cell proliferation beyond the period of cell number decrease. Preferably, the culturing step may be performed for 7 to 25 days.

[0025]

[0026] Another aspect of the present invention provides red blood cells produced according to the above method.

[0027] The inventors measured the oxygen equilibrium curves of red blood cells produced using the method of the present invention and red blood cells isolated from dogs, confirming that the oxygen-Hb binding and dissociation curves were similar for both types of red blood cells (Fig. 13b). These results indicate that red blood cells produced using the method of the present invention can produce hemoglobin and can be used for transfusions in canines.

[0028] According to the method of the present invention, by controlling the concentration of canine EPO, canine hematopoietic stem cells can be effectively differentiated into red blood cells, and canine red blood cells can be produced.

[0029] Figure 1 schematically illustrates a method for differentiating canine mononuclear cells into red blood cells according to an example of the present invention.

[0030] Figure 2 shows the results of confirming the cell pellet by harvesting cells on the 10th and 14th days of culture during the process of differentiating canine mononuclear cells into erythrocytes.

[0031] Figure 3 shows the results of observing cell morphological changes during the process of differentiating canine monocytes into erythrocytes (crEPO 40 ng / ml): red arrows - erythrocytes (RBCs); and yellow arrows - orthochromatic erythroblasts.

[0032] Figure 4 schematically shows the morphological changes of cells during the erythropoiesis process in humans and dogs.

[0033] Figure 5 is a graph showing the number of proliferating cells while differentiating canine monocytes into erythrocytes using media containing different concentrations of crEPO.

[0034] Figure 6 is a graph showing cell viability while differentiating canine monocytes into erythrocytes using media containing different concentrations of crEPO.

[0035] Figure 7 is a graph showing the number of erythroid cells on day 17 of culture while differentiating canine monocytes into erythrocytes using media containing different concentrations of crEPO: RBC = erythrocyte; ortho = orthochromatic erythroblast; pro-baso-poly = proerythroblast, basophilic erythroblast, polychromatic erythroblast; and others = other cells.

[0036] Figure 8 is a graph showing the percentage of erythroid cells according to the culture period while differentiating canine monocytes into erythrocytes using a medium containing 40 ng / ml of crEPO: RBC-erythrocytes; ortho-orthochromatic erythroblasts; pro-baso-poly-; and others: other cells.

[0037] Figure 9 is a graph showing the number of proliferating cells while differentiating canine monocytes into erythrocytes using media containing different concentrations of crEPO or human EPO.

[0038] Figure 10 is a graph showing cell viability while differentiating canine monocytes into erythrocytes using media containing different concentrations of crEPO or human EPO.

[0039] Figure 11 is a graph showing the number of erythroid cells on day 17 of culture while differentiating canine monocytes into erythrocytes using media containing different concentrations of crEPO or human EPO: RBC-erythrocytes; ortho-orthochromatic erythroblasts; pro-baso-poly-; and others: other cells.

[0040] Figure 12 shows the results of analyzing the expression of erythroid markers on days 0, 10 to 13, and 15 to 17 of culture while differentiating canine monocytes into erythrocytes using a medium containing crEPO 40 ng / ml or 80 ng / ml: a-Alpha Hemoglobin Stabilizing Protein (AHSP); b-EPO receptor (EPOR); c-Kruppel-like factor (KLF1); and d-Transferrin Receptor 1 (TFRC).

[0041] Figure 13 shows the results of confirming the function of erythrocytes produced from canine mononuclear cells through in vitro culture: a - Appearance of cell pellet on day 18 of culture; b - Oxygen-Hb binding and dissociation curves of canine PBMCs cultured in vitro for 18 days and canine erythrocytes on day 1; c - Confirmation of the expression of hemoglobin α in canine PBMCs cultured in vitro for 17 days.

[0042] Figure 14 shows the results of comparing the amino acid sequences of human EPO and canine EPO.

[0043] Figure 15 shows the results of comparing the amino acid sequences of human EPOR and dog EPOR.

[0044] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0045]

[0046] Experimental method

[0047] Isolation of mononuclear cells (MNCs) from canine peripheral blood

[0048] Peripheral blood of dogs used in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University. Whole blood and Ficoll were mixed in a 1:1 ratio using SepMate (SepMate) using Ficoll-Paque (1.078 g / ml, Cytiva, #17144003). TM The plasma supernatant and monocyte layer were separated into PBMC Isolation Tubes (STEMCELL technologies) and centrifuged at 500 x g for 35 min at room temperature. After confirming the plasma supernatant and monocyte layer, only the monocyte layer was collected. The collected monocyte layer was diluted in PBS containing 10% FBS and washed by centrifugation at 800 x g for 20 min at room temperature. The separated cells were collected to determine the monocyte number.

[0049]

[0050] cell culture

[0051] Mononuclear cells (PBMNCs) isolated from the peripheral blood of dogs FBS (HyClone TM ) StemSpan containing 30% and basic supplements TM(STEMCELL technologies) medium. Basic supplements included Holo-Transferrin (200 ng / ml, Sigma-Aldrich, T4132), insulin (1 µl / ml), 1% penicillin / streptomycin, and 1% amino acids. Cytokines used were canine recombinant EPO (3816-CE 010 / C, R&D systems), canine recombinant SCF (Canine recombinant SCF: 10–100 ng / ml; 2278-SC 025 / CF, R&D systems), and human recombinant IL3 (Human recombinant IL3 (human rIL3): 1–10 ng / ml; 203-IL-050, R&D systems), and cytokine conditions varied depending on the specific experiment. Cell culture medium was replaced with fresh medium at 90% volume every 2–3 days.

[0052]

[0053] Cell morphology analysis using Wright-Giemsa staining

[0054] 2-4x10 cultured erythroid lineage cells 4 The cells were smeared on slides using a cell centrifuge (Cellspin; Hanil Science Industrial, Incheon, Korea), stained with Wright-Giemsa (Sigma), and cell morphology was observed.

[0055]

[0056]

[0057] Western blot

[0058] The expression level of canine hemoglobin α was analyzed using Western blotting. Cultured cells were collected, washed with PBS, and lysed in radioimmunoprecipitation assay (RIPA, Sigma) buffer. The separated proteins were quantified using the Pierce BCA protein kit (Thermo Scientific). A certain amount of protein was electrophoresed on 10% SDS-PAGE and transferred to a PVDF membrane (Milipore Corporation, MA). The membrane containing the transferred protein was blocked with 5% skim milk and then incubated with primary antibody (Anti-Hemoglobin antibody, ab231732) and secondary antibodies. Protein detection was performed using the ChemiDoc XRS+ system (Bio-RAD, Hercules, CA).

[0059]

[0060] Functional analysis of in vitro cultured canine erythrocytes

[0061] On the 18th day of culture, the oxygen affinity of hemoglobin was determined using canine erythrocytes. The oxygen equilibrium curve was measured using a Hemox-Analyzer (Hemox-Analyzer, TCS, Medical Products Division, Southampton, PA).

[0062]

[0063] Experimental results

[0064] Erythropoietic characteristics of differentiated canine PBMNCs

[0065] After isolating the dog PBMNCs, 1x10 6Cells were seeded at a density of 10 cells / ml, and proliferation and differentiation were observed for 3 weeks. Platelets were present in PBMNCs for the first 6–7 days, but they gradually disappeared. From day 0 to day 6, the percentage of seeded cells decreased to 20% (Fig. 5). However, proliferation began thereafter, and from day 10, adenocytes were observed, and the cells began to turn red (Fig. 2). The maximum proliferation compared to the initially seeded cells occurred on days 16–17 of proliferation, and the proliferation rate varied among donors. Cell viability also remained generally high (Fig. 6).

[0066] The membranes of mature differentiated erythrocytes were maintained intact, and the differentiation stage of the cells could be confirmed by Wright-Giemsa staining (Figs. 3 and 4).

[0067]

[0068] Optimal cytokine concentration

[0069] To determine the optimal conditions for proliferation and differentiation of erythroid lineages, the concentrations of canine recombinant EPO (crEPO) were investigated. The concentrations of crEPO were set at 10, 20, 40, 80, and 160 ng / ml, and cultured for approximately 20 days.

[0070] Regardless of cytokine concentration, cell numbers decreased until day 7 after inoculation, after which cell proliferation began and total cell numbers increased. A concentration of 40 ng / ml of cEPO was used as a reference, but no significant differences in cell morphology or proliferation rates were observed at any cEPO concentration during the first 7 days.

[0071] From day 13 of culture, cell aggregation was observed at low concentrations of 10 ng / ml and 20 ng / ml, and cells began to form island-like structures (data not shown). From day 14 to 15 of culture, differences in enucleation, erythroid maturation, and lineage differentiation began to appear. After day 15 of culture, the cell proliferation rate of the crEPO 20 ng / ml group decreased, and the cell number of the crEPO 40 ng / ml group increased 1.35-fold (Fig. 5). In addition, the cell number of the crEPO 10 ng / ml group was 3.18-fold lower than that of the crEPO 40 ng / ml group and 2.34-fold lower than that of the crEPO 20 ng / ml group.

[0072] On the 17th day of culture, the erythrocyte percentage was 36.8% (n=7) in the 40 ng / ml crEPO group, 15.12% (n=2) in the 20 ng / ml crEPO group, and 17.94% (n=2) in the 10 ng / ml crEPO group. The total erythrocyte count and cell count were highest in the 40 ng / ml crEPO group. Compared with the 40 ng / ml crEPO group, the 20 ng / ml and 10 ng / ml crEPO groups showed similarly low proliferation and enucleation rates (Fig. 7).

[0073] Even when high concentrations of crEPO were used, there were no morphological differences in the cells. The cell proliferation rates of the crEPO 80 ng / ml and crEPO 160 ng / ml groups decreased compared to the 40 ng / ml crEPO group during the first 7 days (Fig. 5). In the crEPO 80 ng / ml group, differentiation progressed similarly to the crEPO 40 ng / ml group, with the highest number of erythrocytes observed on the 17th day of culture, but cell viability decreased thereafter.

[0074] However, higher concentrations of crEPO did not guarantee higher average cell proliferation rates. While higher concentrations of crEPO tended to slightly increase the erythroid percentage, the highest percentage of erythroid lineages, relative to the final cell count, was observed at crEPO 40 ng / ml (Fig. 7).

[0075]

[0076] We compared the concentrations of canine EPO and human EPO cytokines.

[0077] In terms of cell proliferation rate, enucleation, and erythrocyte production, canine EPO 40 ng / ml showed a high proliferation rate, and accordingly, a high erythrocyte production rate. Next in order were human EPO 40 ng / ml for good proliferation rate and erythrocyte production, and it was confirmed that erythrocytes were produced even when human EPO was used. The difference in proliferation rate between the canine EPO and human EPO groups widened from 1.61 to 2.83 times between days 18 and 21 of culture (Fig. 9).

[0078] Comparison of the amino acid sequences of human EPO and canine EPO revealed an identity of 81.21% and a similarity of 93.93%, while the sequences of human EPOR and canine EPOR were confirmed to have an identity of 82.64% and 91.53%, respectively (Figs. 14 and 15).

[0079]

[0080]

[0081] Canine erythroid lineage differentiation and cell morphology

[0082] The differentiation and maturation of canine erythroblasts can be divided into several distinct stages, which are common to the differentiation process of mammalian cells.

[0083] Proerythroblasts, the earliest stage of development, are relatively large cells with a high nucleus-to-nucleoplasm ratio, the presence of ribosomes, and a high nucleus-to-nucleoplasm ratio. As they transform into basophilic erythroblasts, they decrease in size, and increased hemoglobin synthesis imparts more acidophilic properties to the cytoplasm. Cells at this stage exhibit a more condensed chromatin structure than in previous stages, and nuclear size begins to decrease.

[0084] In the next stage, polychromatic erythroblasts, cell size decreases further and hemoglobin accumulates further. Furthermore, hemoglobin and residual RNA mix, resulting in a pinkish cytoplasm in Wright-Giemsa staining. This stage marks the peak of hemoglobin synthesis before the cell prepares for enucleation.

[0085] Finally, orthochromatic erythroblasts are the smallest of the erythrocyte precursor cells, possessing a dense, condensed nucleus and a high hemoglobin content. Typically, the nucleus is oriented toward one side of the cytoplasm, preparing for enucleation and terminal erythrocyte differentiation. Enucleation then leads to the formation of reticulocytes, which have lost their nuclei and ultimately become red blood cells (RBCs). During this stage, cell size decreases, and morphological changes lead to the transition to functional erythrocytes, ultimately enabling the cells to transport oxygen efficiently.

[0086]

[0087] When canine PBMNCs were cultured in vitro and morphological changes following erythropoiesis were observed, transformation of canine PBMNCs from proerythroblasts to eosinophilic erythroblasts and erythrocytes was confirmed using Wright-Giemsa staining. When the concentration of EPO cytokine and the degree of differentiation were confirmed, optimal differentiation into erythrocytes according to cell proliferation rate was achieved at crEPO 40 ng / ml (Figs. 5 to 11).

[0088]

[0089]

[0090] Identification of erythrocyte-specific mRNA expression and erythrocyte markers

[0091] To confirm the mRNA expression of erythrocyte-specific markers, the expression of Alpha Hemoglobin Stabilizing Protein (AHSP) (ref. 9), EPO receptor (EPOR) (ref. 10), Kruppel-like factor 1 (KLF1) (ref. 11), and Transferrin receptor 1 (TFRC) (ref. 12) was examined.

[0092] From the start of the study, mRNA samples were collected starting from day 7, when proliferation began after administration of crEPO at concentrations of 40 ng / ml and 80 ng / ml. The expression of erythrocyte-specific markers was evaluated in the early stage, days 10–13 (Figs. 2 and 5), when the pellet began to turn red, and in the late stage, days 15–17 (Fig. 2), when the number of enucleated erythrocytes increased and cell viability reached 80%. The mRNA expression levels of AHSP, EPOR, KLF1, and TFRC were observed to increase at both concentrations (crEPO 40 ng / ml and 80 ng / ml).

[0093]

[0094] Since there are no existing results analyzing the expression of erythrocyte markers during the erythropoiesis process in dogs, the inventors compared the mRNA expression results with the mRNA expression patterns in humans and mice.

[0095]

[0096] - AHSP

[0097] It is known that the expression of AHSP (α-hemoglobin stabilizing protein) increases when erythrocytes are produced in human PBMNCs (Ref. 13).

[0098] Expression of AHSP (α-hemoglobin stabilizing protein) during the culture period was confirmed, and as differentiation progressed, expression of AHSP mRNA increased, similar to the erythropoiesis process derived from human PBMNC, and this was confirmed at both 40 ng / ml and 80 ng / ml of crEPO (Fig. 12a).

[0099] In humans, the highest proportion of polychromatic and orthochromatic erythroblasts was observed between days 8 and 10, correlating with the peak of AHSP expression. Conversely, during erythropoiesis from canine PBMCs, the proportion of polychromatic and orthochromatic erythroblasts was higher at day 17 than at day 10 (Fig. 8). This comparison suggests that AHSP expression increases with differentiation rate in dogs, similar to humans.

[0100]

[0101] - EPOR

[0102] EPOR expression has been reported to increase during the transition from burst-forming unit-erythroid (BFU-E) to colony-forming unit-erythroid (CFU-E) in humans and mice (ref. 14). In dogs, EPOR expression is known to be upregulated in chronic hypoxic lung disease, inducing polycythemia as a compensatory mechanism to improve oxygen supply (ref. 15). Furthermore, EPOR is known to progressively decrease during erythroblast maturation in a mouse model (ref. 16).

[0103] Although there has been no previous study on EPOR in canine erythropoiesis, we confirmed that EPOR expression was reduced at a concentration of 40 ng / ml crEPO in mature erythroblasts (Fig. 12b).

[0104]

[0105] - KLF1 and TFRC

[0106] KLF1 plays a crucial role in determining the lineage commitment of early megakaryocyte-erythroid progenitors (MEPs), and is expressed in the erythroid lineage to suppress megakaryocyte lineage commitment. In the present study, KLF1 expression increased during erythroid cell proliferation and then relatively decreased with maturation (Fig. 12c).

[0107]

[0108] TFRC is abundant in the cell membrane and disappears after nucleus removal in mature erythrocytes (Ref. 17). It is also involved in the regulation of iron uptake (Ref. 16) and is expressed not only in erythrocytes but also in other hematopoietic lineages (Ref. 18). In the present invention, we confirmed that mRNA expression increased during canine erythropoiesis (Fig. 12d). Although TFRC is not an erythrocyte-specific marker, its enhanced expression suggests that it may play a role in iron uptake.

[0109] However, as mRNA expression decreased with enucleation of mixed hematopoietic lineage cells and erythroid cells, it was difficult to identify significant trends in mRNA expression across culture days and erythroid markers. Although mRNA expression was slightly higher in the crEPO 80 ng / ml group than in the 40 ng / ml group, the difference was not statistically significant.

[0110]

[0111] Hemoglobin detection and oxygen balance analysis

[0112] Hemoglobin (Hb) α expression was confirmed by Western blot and general PCR.

[0113] Two 17-day-old canine donor samples were compared, and the protein expression of Hb α was measured relative to GAPDH. The results showed that Hb α was expressed (Fig. 12c).

[0114] Additionally, the oxygen equilibrium curves of canine erythrocytes (day 1) and canine PBMCs (crEPO 40 ng / ml) cultured in vitro for 18 days were measured using a Hemox-analyzer. The results showed that the oxygen-Hb binding and dissociation curves of in vitro-cultured canine PBMCs and day 1 canine erythrocytes were similar. The P50 values ​​were 21.03 and 20.25, respectively (Fig. 13b). These results indicate that canine PBMC erythrocytes cultured in vitro can produce mature hemoglobin.

[0115]

[0116]

[0117] conclusion

[0118] Despite the increasing number of companion dogs, research on artificial blood for dogs is virtually nonexistent, and exploration of differentiation processes remains minimal. The present inventors successfully produced canine erythrocytes differentiated in vitro for the first time, investigated cell proliferation and differentiation characteristics, and evaluated the functional properties of the cultured erythrocytes.

[0119] In initial experiments, attempts were made to isolate canine CD34+ cells (refs. 19, 20) from canine PBMCs, similar to human cells, and differentiate them into specific erythroid lineages. However, CD34+ cell isolation was unsuccessful (data not shown), and proliferation was minimal during a 7-day culture period. Furthermore, although currently available reagents are labeled for canine use, they are less effective than human reagents. CD34+, a cell marker commonly used to classify erythroid lineages in human PBMNCs, did not work effectively with canine cells, and suitable markers (CD71, CD235a) for determining maturation status were not available.

[0120] Despite the difficulty in differentiating canine erythrocytes in vitro, the inventors of the present invention directly induced erythrocyte differentiation of canine PBMCs by controlling cytokine concentrations, and as a result, were able to produce erythrocytes in vitro in dogs.

[0121] When comparing the proliferation and differentiation rates of erythroid lineages according to the concentration of canine EPO cytokine, proliferation was low at low concentrations of crEPO 40 ng / ml or less, the expression rates of other hematopoietic lineage cells increased, and erythrocytes were produced. On the other hand, at concentrations of crEPO 40 ng / ml or more, erythrocytes were well produced, and mRNA expression markers were also well expressed. However, since the proliferation and enucleation rates did not significantly differ between crEPO 40 ng / ml and high-concentration-dependent concentrations, the optimal concentration was set at crEPO 40 ng / ml. In a comparative experiment with human EPO, the concentration of human EPO 40 ng / ml (3 to 15 times higher than the utilization activity value of human EPO) showed the second highest proliferation and differentiation graphs of crEPO 40 ng / ml, but when comparing the relative erythrocyte production, it did not exceed the proliferation rate of crEPO 40 ng / ml (2.83 times).

[0122] Analysis of the P50 curves for oxygen binding-dissociation for functional analysis of erythrocytes revealed similar curves between in vitro-cultured canine erythrocytes and those derived from PB. This confirmed that in vitro-cultured canine erythrocytes function better than peripheral blood erythrocytes.

[0123]

[0124] [References]

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[0126] 2) doi: 10.1182 / blood-2011-06-36203

[0127] 3) Immune phenotype of canine hematopoietic progenitor cells, https: / doi.org / 10.1111 / j.1399-0039.1997.tb02901.x

[0128] 4) Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes, doi:10.3389 / fimmu.2018.00841

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[0130] 6) ZooMics: comparative metabolomics of red blood cells from dogs, cows, horses and donkeys during refrigerated storage for up to 42 days, doi: 10.2450 / 2022.0118-22

[0131] 7) England, S., McGrath, K., Frame, J., & Palis, J. (2011). Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus. Blood, 117(9), 2708-2717. https: / doi.org / 10.1182 / blood-2010-07-299743

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Claims

1. A step of obtaining hematopoietic stem cells or erythroid progenitor cells from cells derived from a dog; and A method for producing red blood cells in a dog in vitro, comprising the step of culturing acquired hematopoietic stem cells or erythroid progenitor cells in a medium containing erythropoietin (EPO).

2. A method for producing canine red blood cells in vitro, wherein the canine-derived cells in paragraph 1 are blood-derived cells, bone marrow-derived cells, cord blood-derived cells, or stem cells.

3. A method for producing red blood cells in a dog in vitro, wherein the hematopoietic stem cells in paragraph 1 are mononuclear cells.

4. A method for producing red blood cells in vitro in the first paragraph, wherein the erythropoietin is included in the medium at a concentration of 20 to 80 ng / ml.

5. A method for producing red blood cells in vitro in the fourth paragraph, wherein the erythropoietin is included in the medium at a concentration of 30 to 50 ng / ml.

6. A method for producing canine red blood cells in vitro, wherein the erythropoietin in the first paragraph is a canine erythropoietin.

7. A method for producing dog red blood cells in vitro, wherein the erythropoietin in the first paragraph is a human erythropoietin.

8. A method for producing canine red blood cells in vitro, wherein the medium further comprises transferrin, insulin, penicillin / streptomycin and amino acids in the first paragraph.

9. A method for producing canine red blood cells in vitro, wherein the culturing step in paragraph 1 is performed for 3 to 30 days.

10. A method for producing canine red blood cells in vitro, wherein the canine erythropoietin (EPO) is included in the medium at a concentration of 5 to 80 ng / ml in the 6th paragraph.

11. A method for producing canine red blood cells in vitro, wherein the medium further comprises stem cell factor (SCF) and interleukin-3 in the first paragraph.

12. A method for producing red blood cells in vitro in claim 11, wherein the stem cell factor (SCF) is included in the medium at a concentration of 10 to 100 ng / ml.

13. A method for producing red blood cells in vitro in claim 11, wherein interleukin-3 is included in the medium at a concentration of 1 to 10 ng / ml.

14. Red blood cells produced according to any one of the methods in clauses 1 to 13.

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