CN107663515B - Method for directionally preparing human red blood cells and preparation - Google Patents
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Abstract
The invention relates to a method for directionally preparing human red blood cells and a preparation. The method comprises three parts of hematopoietic stem cell amplification, induced erythrocyte directed differentiation and erythrocyte enucleation maturation, wherein a hematopoietic stem cell amplification formula is used at the early stage, a formula for promoting erythrocyte induced differentiation is adopted at the middle stage, and a formula for promoting erythrocyte enucleation maturation is adopted at the late stage, so that the hematopoietic stem cells are amplified and differentiated into erythrocytes in vitro in large quantity.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a method and a preparation for directionally preparing human red blood cells.
Background
Stem cells are a group of cells with self-renewal and multipotentiality. The stem cells can be classified into totipotent stem cells, pluripotent stem cells and unipotent stem cells according to the differentiation potential of the stem cells. Among them, Hematopoietic Stem Cells (HSCs) are pluripotent stem cells, which are a group of primitive hematopoietic cells mainly present in bone marrow, peripheral blood, and umbilical cord blood. Hematopoietic stem cells are the only source of various blood cells in the body, and ultimately produce a variety of blood functional cells including red blood cells, white blood cells, platelets, and the like.
Erythrocytes (Erythrocytes) account for 94-97% of the total number of blood cells and are the most important vehicle for the transport of oxygen through the blood in the human body. The reduction of the number of the red blood cells can cause the oxygen deficiency of the organism and the damage of functional organs, and directly threatens the life safety of people in serious cases. Hematopoietic dysfunction and surgical, tumor, traumatic blood loss and the like all cause erythroblast reduction, and are clinically solved at present mainly by infusing blood donated by volunteers. Although annual blood supply of domestic blood stations is increasing in recent years, clinical needs cannot be met, and hepatitis B and HIV virus infection events caused by emergency blood transfusion often occur, so that the problem of safe, stable and sufficient blood sources is urgently needed to be solved.
The present inventors have invented a novel ex vivo technique for efficiently expanding hematopoietic stem cells, which can obtain efficiently expanded HSCs by using the method developed by the present inventors for combining the expansion medium with other components and various human cytokines (Chinese patent application 201410066574.9; 201410066590.8).
In the above-described techniques previously developed by the present inventors, although hematopoietic stem cells that have been efficiently expanded can already be obtained, further studies have been required to further induce differentiation of hematopoietic stem cells into mature erythrocytes under in vitro conditions.
Disclosure of Invention
The invention aims to provide a method and a preparation for directionally preparing human red blood cells.
In a first aspect of the present invention, there is provided a composition for promoting the induction of differentiation of erythrocytes, comprising:
in a preferred embodiment, the composition for promoting the induction of differentiation of erythrocytes comprises:
in another aspect of the present invention, there is provided a composition for promoting the enucleated maturation of erythrocytes, comprising:
in a preferred embodiment, the composition for promoting the enucleated maturation of erythrocytes comprises:
in another aspect of the present invention, there is provided a use of the composition for promoting differentiation induction of erythrocytes, for promoting differentiation induction of erythrocytes; or used for preparing a culture medium for promoting the induction and differentiation of the red blood cells.
In another aspect of the invention, the use of the composition for promoting the enucleated maturation of erythrocytes is provided, which is used for promoting the enucleated maturation of erythrocytes; or used for preparing a culture medium for promoting the enucleation maturation of the red blood cells.
In another aspect of the present invention, there is provided a medium for promoting differentiation induction of erythrocytes, comprising: IMDM medium or DMEM/F12 medium; and said composition for promoting the induction of differentiation of erythrocytes; preferably, the medium further comprises fetal bovine serum.
In a preferred embodiment, the medium for promoting the induced differentiation of the erythrocytes has a final concentration of 5-25% (v/v) of fetal bovine serum; more preferably 10-20% (v/v).
In another aspect of the present invention, there is provided a culture medium for promoting the enucleated maturation of erythrocytes, comprising: IMDM medium or DMEM/F12 medium; and said composition for promoting the enucleated maturation of erythrocytes.
In another aspect of the present invention, there is provided a kit comprising: the composition for promoting the differentiation induction of the red blood cells and/or the composition for promoting the enucleation maturation of the red blood cells; or
The kit comprises: the culture medium for promoting the differentiation induction of the red blood cells and/or the culture medium for promoting the enucleation maturation of the red blood cells.
In a preferred embodiment, the kit further comprises: a composition comprising or a hematopoietic stem cell expansion medium comprising the following (preferably, the hematopoietic stem cell expansion medium uses Stemspan medium as a basal medium):
stem cell factor: 50-500 ng/ml;
flt 3-ligand: 50-500 ng/ml; and
platelet factor: 3-200 ng/ml.
In another preferred embodiment, the hematopoietic stem cell expansion medium comprises:
stem cell factor: 60-300 ng/ml; preferably 80-150 ng/ml;
flt 3-ligand: 60-300 ng/ml; preferably 80-150 ng/ml;
platelet factor: 5-150 ng/ml; preferably 6-100 ng/ml.
In another preferred embodiment, the kit further comprises: IMDM medium or DMEM/F12 medium.
In another aspect of the present invention, there is provided a method for the directed preparation of human red blood cells, the method comprising:
(1) culturing the human hematopoietic stem/progenitor cells by using the culture medium for promoting the induction and differentiation of the red blood cells; preferably, said human hematopoietic stem/progenitor stem cells are CD34+ human hematopoietic stem/progenitor stem cells;
(2) and (2) culturing the cells obtained in the step (1) by using the culture medium for promoting the enucleation maturation of the erythrocytes to obtain the human erythrocytes.
In a preferred embodiment, in step (1), the human hematopoietic stem/progenitor cells are expanded human hematopoietic stem/progenitor cells, and the expansion is performed using a hematopoietic stem cell expansion medium, which comprises:
stem cell factor: 50-500 ng/ml;
flt 3-ligand: 50-500 ng/ml; and
platelet factor: 3-200 ng/ml.
In another preferred example, in step (1), during the culture period, the method further comprises the step of replacing 5-8 times of fresh culture medium for promoting the differentiation induction of the red blood cells; or step (1), during the culture period, the method also comprises the step of replacing 1-2 times of fresh culture medium for promoting the enucleated maturation of the red blood cells.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 is a photograph of a roller bottle culture system used in the examples of the present invention.
FIG. 2 shows the total cell proliferation fold at different time points in 21 days after the in vitro expansion and directional differentiation of human cord blood-derived hematopoietic stem cells into erythrocytes.
FIG. 3, centrifugation of roller bottle cultured red blood cells.
FIG. 4 flow cytometry analysis of the expression of cell surface markers CD71, CD235a, CD34, CD45, CD133 during in vitro expansion differentiation culture.
FIG. 5A is a graph of cell Giemsa reyi staining at different time points during in vitro culture.
FIG. 5B, in vitro culture procedure, at various time points, a small number of cells were centrifuged in an EP tube to pellet the cells.
Fig. 6A, standard curve of hemoglobin measurement.
FIG. 6B, hemoglobin content of cells measured at different time points.
FIG. 7, change in cellular protooncogene expression level.
Fig. 8A, the LDS + ratio (i.e., the ratio of nucleated cells) of the reinfused cells, which were measured by flow-testing blood from the mice after reinfusion (first to third days).
FIG. 8B, blood flow assay of mice taken after reinfusion (first to third days), reinfusion cell CFSE + ratio and CD71+ cell ratio therein.
Detailed Description
The present inventors have made extensive studies to develop a method and a preparation for directed preparation of human erythrocytes, wherein hematopoietic stem/progenitor cells are efficiently and massively expanded by using a hematopoietic stem cell expansion medium while maintaining the original dry characteristics, and then induced to differentiate by using a erythrocyte induced differentiation medium, and finally cultured by using an erythrocyte enucleation maturation medium to obtain mature erythrocytes.
On the basis of the technology of expanding a large number of hematopoietic stem/progenitor cells developed by the inventor in the earlier stage, the inventor further adjusts a cell culture scheme, realizes the efficient expansion and the induction of the hematopoietic stem/progenitor cells to erythroid differentiation through three stages of in vitro culture differentiation, and verifies the function, the safety and the effectiveness of the obtained erythrocytes through a series of in vitro and in vivo experiments.
As used herein, the term "comprising" or "includes" comprising "," consisting (made) predominantly of … … ", consisting essentially of … …", and "consisting of … …".
Composition and culture medium
The inventors have optimized the medium for the directed preparation of human red blood cells. According to different stages of the hematopoietic stem cell expansion and induction culture, a culture medium suitable for different stages is provided.
Promotion ofComposition and culture medium for inducing differentiation of red blood cells
The composition for promoting the red blood cell to induce differentiation comprises: transferrin, inositol, folic acid, insulin, stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, granulocyte macrophage stimulating factor, and erythropoietin.
The components are added into a basic culture medium in a proper proportion, so that a proper environment is provided for the hematopoietic stem/progenitor cells to induce and differentiate into the red blood cells, and the directed differentiation of the hematopoietic stem/progenitor cells into the red blood cells is promoted. As a preferred embodiment of the present invention, the amounts of the respective components used for preparing the medium for promoting the induction of differentiation of erythrocytes according to the present invention are shown in Table 1.
TABLE 1
The components of the formulation of table 1 were dissolved in a cell culture medium (basal medium) to obtain the erythrocyte differentiation induction medium, thereby providing a suitable environment for the induction of differentiation from hematopoietic stem cells to erythrocytes. The cell culture medium can be selected from IMDM medium or similar cell culture medium, such as DMEM/F12 medium.
Composition and culture medium for promoting red blood cell enucleation maturation
The composition for promoting the enucleation maturation of the red blood cells comprises: human plasma albumin, D-mannitol, adenine, disodium hydrogen phosphate dodecahydrate and mifepristone. The components are added into a cell growth culture solution in a proper proportion, so that a proper environment can be provided for enucleation and maturation of erythrocytes. As a preferred mode of the present invention, the amounts of the respective components used for preparing the culture medium for promoting the enucleated maturation of erythrocytes according to the present invention are shown in Table 2.
TABLE 2
The components of the formula of table 2 were dissolved in cell culture medium (basal medium) to provide the culture medium for enucleated maturation of erythrocytes, thereby providing a preferred simplified medium formulation for enucleated maturation of erythrocytes.
Composition and culture medium for promoting hematopoietic stem cell expansion
In order to obtain a sufficient amount of hematopoietic stem cells for inducing erythrocytes, the present invention also provides a suitable hematopoietic stem cell expansion medium. The amounts of the respective components used for preparing the hematopoietic stem cell expansion medium are shown in Table 3.
TABLE 3
| Components | Content (wt.) | Preferred amount of | More preferred amount |
| Stem cell factor | 50-500ng/ml | 60-300ng/ml | 80-150ng/ml |
| flt 3-ligand | 50-500ng/ml | 60-300ng/ml | 80-150ng/ml |
| Platelet factor | 3-200ng/ml | 5-150ng/ml | 6-100ng/ml |
The various components used to formulate the medium are readily available to those skilled in the art, e.g., commercially available, or may be obtained by artificial synthesis or recombinant expression.
Culture method
The culture method comprises three parts of hematopoietic stem cell amplification, induction of erythrocyte directed differentiation and erythrocyte enucleation maturation: the hematopoietic stem cell expansion formula is used in the early stage, the formula for promoting the induction and differentiation of the red blood cells is adopted in the middle stage, and the formula for promoting the enucleation and maturation of the red blood cells is adopted in the late stage, so that the in-vitro large-scale expansion and differentiation of the hematopoietic stem cells into the red blood cells are realized.
Accordingly, the present invention provides a method for expanding cultured hematopoietic stem cells, the method comprising: (1) culturing the human hematopoietic stem/progenitor cells by using the culture medium for promoting the induction and differentiation of the red blood cells; (2) and (2) culturing the cells obtained in the step (1) by using the culture medium for promoting the enucleation maturation of the erythrocytes to obtain the human erythrocytes. Preferably, the human hematopoietic stem/progenitor stem cells are CD34+ human hematopoietic stem/progenitor stem cells. During the culture, fresh medium may be replaced according to the culture conditions.
In the present invention, the hematopoietic stem/progenitor cells may be those derived from human, from umbilical cord abandoned in hospitals (e.g. umbilical cord of newborn), or from hematopoietic stem/progenitor cells that have been expanded or commercialized by the prior art. For example, the present inventors have expanded human hematopoietic stem/progenitor cells in previous studies, and these in vitro expanded human hematopoietic stem/progenitor cells can be used.
In the present invention, the hematopoietic stem/progenitor cells are not particularly limited with respect to blood type, and may be umbilical cord blood derived from blood type A, blood type B or blood type O. Preferably, the hematopoietic stem/progenitor cells from blood O can be universally applied to the people needing blood transfusion clinically, can cover most people for transfusion, and can reduce the process of blood type detection particularly in emergency treatment and preparation.
In the embodiment of the invention, the technology for preparing the universal blood by using the umbilical cord blood stem cells is established by first obtaining the hematopoietic stem cells and then adjusting the cell growth factors and other components in the culture medium in stages to realize the process of expanding and differentiating the hematopoietic stem cells into red blood cells. The early stage is a hematopoietic stem cell expansion medium mainly for promoting proliferation, the middle stage is an erythropoiesis medium mainly for inducing differentiation, and the late stage is a serum-free enucleation medium mainly for maturation differentiation. The invention utilizes a roller bottle culture system to induce and differentiate hematopoietic stem cells into red blood cells with 2 hundred million times increased total cell number within 21 days of in vitro culture, and verifies the characteristics and functions of the obtained red blood cells through a series of in vitro experiments (cell staining, flow measurement of cell surface markers, hemoglobin content measurement and the like) and in vivo experiments of mice.
Several main features and advantages of the present invention are as follows: firstly, the human cell growth factor is adopted in the whole culture system, exogenous gene expression is not introduced, and exogenous cells such as feeder cells and the like are not involved, so that the interference of the exogenous gene on the genome stability of the original stem cells and the transplantation potential safety hazard related to the exogenous cells are avoided. Secondly, the artificial blood produced by the stem cells is closer to natural blood, and has the characteristics of high efficiency, sufficient source, safety, stability and convenient use, and the red blood cells prepared in a laboratory are convenient to be monitored by a standard operation process, and the propagation of some serious infectious diseases through a conventional blood transfusion way is also avoided. Thirdly, the invention realizes the in vitro pilot scale industrial preparation of the red blood cells by using the roller bottle culture system for the first time internationally, the yield is far beyond the amplification multiple reported in the world at present, and the yield data obtained by the research provides reliable data support and guarantee for the actual industrial preparation of the red blood cells.
The invention provides an innovative technology for directionally amplifying and differentiating human hematopoietic stem cells into erythrocytes in vitro. Mature red blood cells generated by the technology are used for preparing general blood for clinical treatment and combat readiness blood transfusion, and the problems of shortage of blood sources and safety in the world are solved.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Example 1 in vitro preparation of erythrocytes-method 1
Harvesting and culturing of hematopoietic stem cells (CD34 +): taking fresh full-term healthy newborn umbilical cord blood, separating CD34+ cells by using a MacS magnetic bead sorting system of a company of Maitian and whirlpool, obtaining cells with the number of 1.5 multiplied by 10^6, and identifying the purity of CD34+ cell population by flow type to be about 94%. Adjusting the cell concentration to 5 × 10^4 cells/ml, culturing 1ml per well in a 24-well plate, placing in an incubator (37 ℃, 5% CO)2) And carrying out microscope observation every day, and timely changing the liquid of the cells to keep the cells at a proper density (not more than 2 x 10^6 cells/ml).
Cells were cultured in stages for 21 days. When the medium was prepared, cytokines used were purchased from PEPERO TECH, and compounds were purchased from Sigma, and the medium was changed at the corresponding time. The culture media adopted in different stages are specifically as follows:
1. and (3) an amplification period: hematopoietic stem cell expansion medium (day 0 to 5)
The Stemspan culture medium is used as a basic culture medium, and cytokines and small molecule compounds are added: stem Cell Factor (SCF), Flt 3-ligand (FLT-3L), platelet growth factor (TPO), as shown in Table 4.
TABLE 4
| Final concentration | |
| Stem Cell Factor (SCF) | 100ng/ml |
| Flt 3-ligand (FLT-3L) | 200ng/ml |
| Platelet growth factor (TPO) | 20ng/ml |
2. And (3) differentiation stage: erythrocyte induced differentiation medium (day 6 to 18)
IMDM culture medium as basic culture medium, and adding fetal calf serum, Transferrin (Transferrin), inositol (inositol), folic acid (folic acid), and Insulin (Insulin); addition of cytokines: SCF, FLT-3, interleukin 3(IL-3), granulocyte macrophage stimulating factor (GM-CSF), Erythropoietin (EPO). The details are shown in Table 5.
TABLE 5
| Final concentration | |
| Fetal bovine serum | 15％(v/v) |
| Transferrin (Transferrin) | 100μg/ml |
| Inositol (Insitol) | 40μg/ml |
| Folic acid (Folic acid) | 20μg/ml |
| Insulin (Insulin) | 25μg/ml |
| SCF | 100ng/ml |
| FLT-3 | 100ng/ml |
| Interleukin 3(IL-3) | 25ng/ml |
| Granulocyte macrophage stimulating factor (GM-CSF) | 12.5ng/ml |
| Erythropoietin (EPO) | 6IU/ml |
3. And (3) mature period: erythrocyte enucleation maturation medium (19 th to 21 th day)
The IMDM culture medium is used as a basic culture medium, and human plasma albumin (plasma), D-mannitol (D-mannitol), Adenine (Adenine), Sodium phosphate dibasic dodecahydrate (mifepristone) and mifepristone (mifepristone) are added. The details are shown in Table 6.
TABLE 6
| Final concentration | |
| Human plasma albumin (Plasmanate) | 1％(v/v) |
| D-mannitol (D-mannitol) | 12mg/ml |
| Adenine (Adenine) | 0.1mg/ml |
| Disodium hydrogen phosphate dodecahydrate | 1mg/ml |
| Mifepristone | 1μM |
4. Culture method
In this example, since the number of hematopoietic stem cells (CD34+) initially isolated is small, 25-T flash cell culture bottles (Corning) are used, then the cells are diluted at a reasonable density (not more than 2 × 10^6 cells/mL) with the amplification of culture, the culture volume is gradually enlarged, after the volume exceeds 10mL, the cells are transferred to 75-T flash cell culture bottles (Corning), each 75-Tflash can contain 20-50 mL of culture medium, after the total culture volume exceeds 200mL, the cells are transferred to roller bottle culture boxes (Thermo RACHEELL 240i roller bottles) for in vitro differentiation culture (as shown in FIG. 1), the culture boxes can contain 4 layers of roller bottles at a time, at least 10L of cell culture solution, and each roller bottle can contain 200-650 mL of culture medium.
The culture method in the amplification stage (day 0-5): adding the hematopoietic stem cell amplification culture medium into a culture bottle 25-T flash, wherein the addition amount of the culture medium in each bottle is 10mL, and the inoculation amount of the cells is 1.5 multiplied by 10^ 6. The flasks were incubated at 37 ℃ in a roller bottle incubator with 5% CO 2. For a total of 5 days, cell counts were performed daily, with appropriate addition of medium and flask splitting, keeping the cells at the appropriate density (not more than 2X 10^6 cells/ml). At the end of the fifth day, the culture medium of the next stage is used instead of centrifugation or liquid change or bottle separation.
A differentiation stage culture method (day 6-18): at this time, the culture medium for inducing differentiation of erythrocytes was added to the flask every time the culture medium was changed. The flasks were incubated at 37 ℃ in a roller bottle incubator with 5% CO 2. During this period, cell counts were performed daily, media was added and flasks were split, keeping the cells at the appropriate density (no more than 2X 10^6 cells/ml). At the end of day 18, cells were isolated by centrifugation (300g,6min) for transfer to maturation culture.
Mature period culture method (19 th to 21 th days): adding a culture medium for enucleation maturation of erythrocytes into a culture bottle, wherein the addition amount of the culture medium in each bottle is 600 mL; and adding the cells cultured in the differentiation phase in an amount per flask (the cell density is controlled at 1X 10^6 cells/ml). The flasks were placed in a roller bottle incubator and incubated at 37 ℃ under 5% CO2 for three days, during which time the medium was added and the flasks were counted daily, keeping the cells at the appropriate density (no more than 2X 10^6 cells/ml). At the end of day 21, the cells were separated by centrifugation (300g,6min) to obtain a cell pellet.
4. Culture process monitoring
During 21 days of in vitro culture, cells were counted at different times and fold amplifications were calculated (see FIG. 2); the fold amplification ranged from 20 fold at day 5 to two million fold at day 12. The total cell number of the cells cultured in vitro and expanded and differentiated for 21 days is increased, the expansion times of the cells at 21 days are about 2 hundred million times, namely, late erythroblasts with the total cell number of 2 hundred million as the initial cell number can be obtained at 21 days, and a representative picture of the centrifugal sediment of the red blood cells prepared by culturing is shown in figure 3.
Cells were flow assayed at different time points and all antibodies and isotype controls were purchased from BD. The expression of cell surface markers CD34, CD133, CD45, CD71, CD235a was analyzed by flow cytometry (see fig. 4). The gradually decreased expression level of the cell surface marker CD34 and CD133 (hematopoietic stem progenitor cell marker) indicates that the cells gradually lose the stem and are in the process of differentiation; the expression level of CD45 (common antigen on the surface of leucocytes) is gradually reduced, the expression trend of an early erythrocyte marker CD71 is increased firstly and then reduced, the expression of a late erythrocyte maturation marker CD235a is increased firstly and then maintained at a high level, and the expression conditions of the markers correspond to the process that the erythroid cells are differentiated and gradually matured in vitro.
At different time points of in vitro culture, after staining cell smears with Giemsa riemersa, morphology was observed (FIG. 5A), the staining of nuclei was gradually reduced, the staining of cytoplasm was gradually increased, enucleated erythrocytes were visible on day 18, the number of enucleated cells was significantly increased on day 21, and the enucleation rate was roughly counted by taking multiple visual fields, which was about 50%. The change in cell morphology is consistent with the expression of the cell surface markers described above. Cell clumps were obtained at the bottom of the EP tube after centrifugation (1200rpm/min, 5min) of cell fractions at different time points. The cell mass remained yellowish white at day 9, appeared partly red at day 12 and red at days 15 to 21. As the cells gradually differentiated towards erythroid maturation, more and more cells expressed hemoglobin (fig. 5B).
The content of hemoglobin in cells was measured by OD measurement using a microplate reader using Drabkin's Reagent purchased from Sigma, and a standard curve of the hemoglobin content and OD value was prepared according to the instructions as shown in FIG. 6A) before the measurement. Then, at various stages of differentiation, the cells were taken and tested for hemoglobin using Duchenne's reagent. The HGB content was measured on days 9, 12, 15, 18 and 21 (FIG. 6B), and the increase of the cellular HGB content was gradually close to the HGB content (27-33 pg/cell) in Normal peripheral blood (Normal) of human, corresponding to the deepening of the color of the cytospin (FIG. 5B).
The cells at different stages of differentiation are taken, Real-time PCR is used for detecting the expression conditions of tumor-causing related protooncogenes (c-myc, c-myb, bmi-1, K-ras, cyclin B and hTERT), and compared with the CD34+ cell which is just separated at the 0 th day, the expression of each protooncogene is not obviously increased (figure 7), which shows that the expression level of the erythrocyte protooncogene prepared by the invention is stable and not activated in the in-vitro preparation process, and the cell has no carcinogenic risk.
Mature human erythrocytes are anucleate and negative for the surface marker CD71, and the inventors transplanted human-derived committed differentiated advanced incompletely mature nucleated erythrocytes into NOD/SCID mice (purchased at suzhou university) and observed whether these late erythroblasts could be further enucleated to mature in vivo. Late erythroblasts (day 18 cells) cultured in vitro were labeled with the fluorescent dye CFSE (purchased from Life corporation) and transfused back into NOD/SCID mice, and peripheral blood was taken daily on the third day of detection from the first day after transfusing and maturation of the transfused cells in the mice was monitored by flow. The figure is a flow chart representative of the experiment showing that the nucleated rate (i.e., LDS + fraction, LDS from Life, a nuclear dye) of the reinfused cells in mice gradually decreased (fig. 8A) and essentially all matured to non-nucleated cells on the third day (LDS + fraction less than 1%); CD71+ cells were progressively depleted from the CFSE + cell population (fig. 8B), with the CFSE + cell population being all CD71 "on the third day. The above results show that human umbilical cord blood-derived hematopoietic stem cells can be further matured and enucleated in animals after being efficiently amplified in vitro and directionally differentiated into erythroid cells.
After 6 months of observation after infusion, mice are all kept healthy in the observation period, and no abnormal reaction and tumor formation occur. The animal experiments and in vitro experiments show that the red blood cells prepared in vitro by the technology are safe without side effect and have no tumor or carcinogenic risk in vivo.
Example 2 in vitro preparation of erythrocytes-method 2
The method for obtaining and culturing the hematopoietic stem cells (CD34+) is the same as the previous method.
The culture media adopted in different stages are specifically as follows:
1. and (3) an amplification period: hematopoietic stem cell expansion medium (day 0 to 5)
The Stemspan culture medium is used as a basic culture medium, and cytokines and small molecule compounds are added: stem cell factor, Flt 3-ligand, platelet growth factor, as specified in Table 7.
TABLE 7
| Final concentration | |
| Stem cell factor | 50ng/ml |
| Flt 3-ligand | 100ng/ml |
| Platelet growth factor | 10ng/ml |
2. And (3) differentiation stage: erythrocyte induced differentiation medium (day 9 to 18)
DMEM/F12 culture medium is used as basic culture medium, and fetal calf serum, transferrin, inositol, folic acid and insulin are added; addition of cytokines: SCF, FLT-3, interleukin 3, granulocyte macrophage stimulating factor, erythropoietin. The details are shown in Table 8.
TABLE 8
| Final concentration | |
| Fetal |
10％(v/v) |
| Transferrin | 50μg/ml |
| Inositol | 20μg/ml |
| Folic acid | 10μg/ml |
| Insulin | 15μg/ml |
| SCF | 50ng/ml |
| FLT-3 | 50ng/ml |
| Interleukin 3 | 20ng/ml |
| Granulocyte macrophage stimulating factor | 8ng/ml |
| Erythropoietin | 3IU/ml |
3. And (3) mature period: erythrocyte enucleation maturation medium (19 th to 21 th day)
DMEM/F12 is used as a basic culture medium, and human plasma albumin, D-mannitol, adenine, disodium hydrogen phosphate dodecahydrate and mifepristone are added. The details are shown in Table 9.
TABLE 9
| Final concentration | |
| Human plasma albumin | 0.04％(v/v) |
| D-mannitol | 5mg/ml |
| Adenine | 0.05mg/ml |
| Disodium hydrogen phosphate dodecahydrate | 0.8mg/ml |
| Mifepristone | 0.7μM |
4. Culture method
In this example, 25-T flash cell culture flasks, 75-T flash (Corning Corp.), and roller bottle incubator (Thermo HERACELL 240i roller bottle) were subjected to in vitro differentiation culture, and the parameters of each flask were as in example 1.
The culture method in the amplification stage (day 0-5): adding the hematopoietic stem cell amplification culture medium into a culture bottle 25-T flash, wherein the addition amount of the culture medium in each bottle is 10mL, and the inoculation amount of the cells is 1 multiplied by 10^ 6. The flasks were incubated at 37 ℃ in a roller bottle incubator with 5% CO 2. For a total of 5 days, cell counts were performed daily, with appropriate addition of medium and flask splitting, keeping the cells at the appropriate density (not more than 2X 10^6 cells/ml). At the end of the fifth day, the culture medium of the next stage is used instead of centrifugation or liquid change or bottle separation.
A differentiation stage culture method (day 6-18): at this time, the culture medium for inducing differentiation of erythrocytes was added to the flask every time the culture medium was changed. The flasks were incubated at 37 ℃ in a roller bottle incubator with 5% CO 2. During this period, cell counts were performed daily, media was added and flasks were split, keeping the cells at the appropriate density (no more than 2X 10^6 cells/ml). At the end of day 18, cells were isolated by centrifugation (300g,6min) for transfer to maturation culture.
Mature period culture method (19 th to 21 th days): adding a culture medium for enucleation maturation of erythrocytes into a culture bottle, wherein the addition amount of the culture medium in each bottle is 600 mL; and adding the cells cultured in the differentiation phase in an amount per flask (the cell density is controlled at 1X 10^6 cells/ml). The flasks were placed in a roller bottle incubator and incubated at 37 ℃ under 5% CO2 for three days, during which time the medium was added and the flasks were counted daily, keeping the cells at the appropriate density (no more than 2X 10^6 cells/ml). At the end of day 21, the cells were separated by centrifugation (300g,6min) to obtain a cell pellet.
4. Culture process monitoring
Cells were counted at different times during 21 days of in vitro culture and fold amplifications were calculated. The total cell number of the cells cultured in vitro and expanded and differentiated for 21 days is increased continuously, and the expansion times reach 1.5 hundred million times at 21 days, namely, the late erythroblasts with the total cell number of 1.5 multiplied by 10^14 of the initial cell number can be obtained at 21 days.
Example 3 in vitro preparation of erythrocytes-method 3
The method for obtaining and culturing the hematopoietic stem cells (CD34+) is the same as the previous method.
The culture media adopted in different stages are specifically as follows:
1. and (3) an amplification period: hematopoietic stem cell expansion medium (day 0 to 5)
The Stemspan culture medium is used as a basic culture medium, and cytokines and small molecule compounds are added: stem cell factor, Flt 3-ligand, platelet growth factor, as specified in Table 10.
| Final concentration | |
| Stem cell factor | 200ng/ml |
| Flt 3-ligand | 200ng/ml |
| Platelet growth factor | 50ng/ml |
2. And (3) differentiation stage: erythrocyte induced differentiation medium (day 9 to 18)
The IMDM culture medium is used as a basic culture medium, and fetal calf serum, transferrin, inositol, folic acid and insulin are added; addition of cytokines: SCF, FLT-3, interleukin 3, granulocyte macrophage stimulating factor, erythropoietin. The details are shown in Table 11.
TABLE 11
3. And (3) mature period: erythrocyte enucleation maturation medium (19 th to 21 th day)
The IMDM culture medium is used as a basic culture medium, and human plasma albumin, D-mannitol, adenine, disodium hydrogen phosphate dodecahydrate and mifepristone are added. The details are shown in Table 12.
TABLE 12
| Final concentration | |
| |
1％(v/v) |
| D-mannitol | 12mg/ml |
| Adenine | 0.1mg/ml |
| Disodium hydrogen phosphate dodecahydrate | 1mg/ml |
| Mifepristone | 1μM |
4. Culture method
In this example, in vitro differentiation culture was carried out using 25-T flash cell culture flasks, 75-T flash (Corning Co.), roller bottle incubator (Thermo HERACELL 240i roller bottle), and the parameters of each flask were as in example 1.
The culture method in the amplification stage (day 0-5): adding the hematopoietic stem cell amplification culture medium into a culture bottle 25-T flash, wherein the addition amount of the culture medium in each bottle is 10mL, and the inoculation amount of the cells is 2 multiplied by 10^ 6. The flasks were incubated at 37 ℃ in a roller bottle incubator with 5% CO 2. For a total of 5 days, cell counts were performed daily, with appropriate addition of medium and flask splitting, keeping the cells at the appropriate density (not more than 2X 10^6 cells/ml). At the end of the fifth day, the culture medium of the next stage is used instead of centrifugation or liquid change or bottle separation.
A differentiation stage culture method (day 6-18): at this time, the culture medium for inducing differentiation of erythrocytes was added to the flask every time the culture medium was changed. The flasks were incubated at 37 ℃ in a roller bottle incubator with 5% CO 2. During this period, cell counts were performed daily, media was added and flasks were split, keeping the cells at the appropriate density (no more than 2X 10^6 cells/ml). At the end of day 18, cells were isolated by centrifugation (300g,6min) for transfer to maturation culture.
Mature period culture method (19 th to 21 th days): adding a culture medium for enucleation maturation of erythrocytes into a culture bottle, wherein the addition amount of the culture medium in each bottle is 600 mL; and adding the cells cultured in the differentiation phase in an amount per flask (the cell density is controlled at 1X 10^6 cells/ml). The flasks were placed in a roller bottle incubator and incubated at 37 ℃ under 5% CO2 for three days, during which time the medium was added and the flasks were counted daily, keeping the cells at the appropriate density (no more than 2X 10^6 cells/ml). At the end of day 21, the cells were separated by centrifugation (300g,6min) to obtain a cell pellet.
4. Culture process monitoring
Cells were counted at different times during 21 days of in vitro culture and fold amplifications were calculated. The total cell number of the cells cultured in vitro and expanded and differentiated for 21 days is increased continuously, and the expansion times reach 2.4 hundred million times at 21 days, namely, the late erythroblasts with the total cell number of 4.8 multiplied by 10^14 of the initial cell number can be obtained at 21 days.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (11)
1. A composition for promoting the induction of differentiation of red blood cells, said composition comprising:
2. use of a composition according to claim 1 for promoting induced differentiation of erythrocytes; or used for preparing a culture medium for promoting the induction and differentiation of the red blood cells.
3. A medium for promoting the induction of differentiation of erythrocytes, comprising:
IMDM medium or DMEM/F12 medium; and
the composition of claim 1.
4. The culture medium of claim 3, further comprising fetal bovine serum.
5. A kit, comprising: the composition of claim 1; or the kit comprises: the culture medium of claim 3.
6. The kit of claim 5, further comprising a composition for promoting red blood cell enucleation maturation, said composition comprising:
7. the kit of claim 5, further comprising a culture medium for promoting red blood cell enucleation maturation, said culture medium comprising: IMDM medium or DMEM/F12 medium; and a composition for promoting red blood cell enucleation maturation, the composition comprising:
8. the kit of claim 5, further comprising: a composition comprising or a hematopoietic stem cell expansion medium comprising the following components:
9. a method of targeted preparation of human red blood cells, the method comprising:
(1) culturing CD34+ human hematopoietic stem/progenitor cells in the medium for promoting erythrocyte-induced differentiation according to claim 3; said CD34+ human hematopoietic stem/progenitor cells are expanded CD34+ human hematopoietic stem/progenitor cells, said expansion using a hematopoietic stem cell expansion medium comprising:
(2) culturing the cells obtained in the step (1) by using a culture medium for promoting the enucleation maturation of the erythrocytes to obtain human erythrocytes; wherein the culture medium for promoting the enucleated maturation of the red blood cells comprises: IMDM medium or DMEM/F12 medium and a composition for promoting red blood cell enucleation maturation, the composition comprising:
the culture is roller bottle culture.
10. The method according to claim 9, wherein in the step (1), during the culturing period, the method further comprises the step of replacing 5 to 8 times the fresh medium for promoting the differentiation induction of erythrocytes according to claim 3.
11. The method of claim 9, wherein in step (2), during the culturing period, the method further comprises the step of replacing the fresh culture medium for promoting the enucleated maturation of the red blood cells 1-2 times.
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