CN114181968A - Preparation method and application of artificial blood progenitor cells with B lineage differentiation potential - Google Patents
Preparation method and application of artificial blood progenitor cells with B lineage differentiation potential Download PDFInfo
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- CN114181968A CN114181968A CN202210131437.3A CN202210131437A CN114181968A CN 114181968 A CN114181968 A CN 114181968A CN 202210131437 A CN202210131437 A CN 202210131437A CN 114181968 A CN114181968 A CN 114181968A
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Abstract
The invention provides a preparation method and application of an artificial blood progenitor cell with B lineage differentiation potential, wherein the preparation method comprises the following steps: simultaneously or respectively introducing three transcription factors of RUNX1, HOXA9 and LHX2 into cells of a human pluripotent stem cell induced differentiation system, and performing induced expression and induced differentiation to obtain the artificial blood progenitor cells with the B lineage differentiation potential; the cells of the human pluripotent stem cell induced differentiation system comprise any one or the combination of at least two of human pluripotent stem cells, mesodermal cells, hematopoietic endothelial cells or hematopoietic progenitor cells; the method for inducing differentiation comprises a monolayer culture differentiation induction method or an organoid three-dimensional culture differentiation induction method based on stromal cell co-culture. The preparation method has good induction and differentiation efficiency, and the prepared cells have good activity and high differentiation capability and have important application value.
Description
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to a preparation method and application of an artificial blood progenitor cell with B lineage differentiation potential.
Background
B cells are a key cellular component of the human humoral immune system, and patients with poor or defective B cell function, such as the elderly or patients with underlying diseases, will have impaired humoral immunity and even severe pathogen infection. In addition, genetically engineered B cells (engineering B cells) have very considerable application prospects in the fields of clinical infection resistance, tumor resistance and disease treatment.
However, clinically, drugs such as B cells and B cell-derived gamma globulin preparations are derived from human peripheral blood or bone marrow banks, so that resources are limited, preparation time is long, and cost is high. Pluripotent Stem Cells (PSCs) are cells that have unlimited proliferation potential, multilineage differentiation potential, and are convenient for genetic editing and modification, and are hot spots for regenerative medicine research of cell therapy. Therefore, the realization of the pluripotent stem cells to induce the regeneration of the human B cell seeds is expected to benefit a plurality of patients with abnormal humoral immune system, and the application of the genetically engineered B cell therapy is greatly promoted. However, the current research on the induction of human B cell regeneration by pluripotent stem cells has few reports, and no substantial progress and breakthrough and no clinical transformation cases exist so far. This suggests that there is a high barrier to overcome in the field of B cell regeneration.
Research shows that the in vitro co-culture of human pluripotent stem cells and stromal cells can sequentially induce hematopoietic progenitor cells, B progenitor cells and CD19+sIgM+B cells (French, Anna et al, Human induced plotter Stem cell-derived B lymphocytes expressed sIgM and can be generated via a genetic endogenous endobacterium intermediate. Stem cells and genetic antagonist vol. 24,9 (2015): 1082-95.). However, mature B cells cannot be induced in vitro, and the in vitro induction mode is long in time and low in efficiency. Since there is a great difficulty in the art of directly inducing functional, mature human B cells in vitro, researchers have begun to consider the realization of human B cell regeneration by means of in vivo transplantation.
Currently, only a few studies on the regeneration of B cells in vivo have been conducted, mainly by inducing pluripotent stem cells in vitro to induce B progenitor cells, and then transplanting them into animal models to achieve B cell regeneration. However, there are the following problems: first, B cells are present in vivo for a short period of time, and no secreted antibody is detectable 6-8 weeks after transplantation (Potocnik, A J et al Reconstation of B cells in rat specific minute by transplantation of in vitro differentiated organizing cells. Immunology letters. vol. 57,1-3 (1997): 131-7); second, B2 Cells that are more important to adaptive humoral immune responses are not available (Lin, Yang et al Long-Term Engraftment of ESC-Derived B-1 promoter Cells Supports HSC-Independent lymphopeptides. Stem cell reports vol. 12,3 (2019): 572-583). The above studies indicate that there are certain deficiencies in the systems for the induction of B progenitor cells in vitro and the regeneration of B cells in vivo after B progenitor cell transplantation, presumably due to the lack of specific transcription factors resulting in a certain deficiency in the regenerated B lymphocyte lineage.
In previous studies, it was found that the key hematopoietic regulatory transcription factors RUNX1 and HOXA9, which play important roles in endothelial hematopoiesis and lymphopoiesis, and LHX2, which plays an important role in immortalization of hematopoietic stem progenitor cells and is highly expressed in multiple pre-B Cell lines, are synergistically expressed to promote the in vitro induction of mouse pluripotent stem cells to generate B lineage seed cells, which can generate all types of mature B cells after transplantation, and can perform adaptive immune responses (Zhang, Qi et al Regeneration of immune complex B lymphoid stem cells regulated by transformation factors Cell Mol Immunol). However, there is no report on inducing differentiation of human pluripotent stem cells in vitro to generate hematopoietic progenitor cells (i.e., B lineage seed cells) with B lineage differentiation potential and realizing regeneration of human B cells after transplantation.
Therefore, how to provide a method for inducing human pluripotent stem cells to generate human B lineage cells with high efficiency has become a problem to be solved.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a preparation method and application of an artificial blood progenitor cell with B lineage differentiation potential.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing an artificial blood progenitor cell having differentiation potential of B lineage, the method comprising:
simultaneously or respectively introducing three transcription factors of RUNX1, HOXA9 and LHX2 into cells of a human pluripotent stem cell induced differentiation system, and performing induced expression and induced differentiation to obtain the artificial blood progenitor cells with the B lineage differentiation potential;
the cells of the human pluripotent stem cell induced differentiation system comprise any one or the combination of at least two of human pluripotent stem cells, mesodermal cells, hematopoietic endothelial cells or hematopoietic progenitor cells;
the method for introducing the transcription factor comprises any one or a combination of at least two of gene overexpression, endogenous gene activation or gene editing;
the method for inducing differentiation comprises the step of inducing differentiation by monolayer culture or organoid three-dimensional culture based on stromal cell co-culture.
In the invention, three transcription factors of RUNX1, HOXA9 and LHX2 are introduced into cells of a human pluripotent stem cell induced differentiation system for induced expression and induced differentiation to obtain artificial blood progenitor cells with B lineage differentiation potential, and the artificial blood progenitor cells have high induction efficiency and good induction effect; the prepared artificial blood progenitor cells with the B lineage differentiation potential can be further differentiated into human B cells, and conditions are created for regeneration of the B cells and reconstruction of a humoral immune system; the prepared B cells can also secrete related therapeutic proteins, antibodies and the like, can be used for preparing related cell preparations, and creates conditions for clinical application and transformation of B cell therapy.
Preferably, said gene overexpression comprises DNA transfection and/or mRNA transfection.
Preferably, the endogenous gene activation comprises RNA activation and/or CRISPR-dCas 9.
Preferably, the gene editing comprises any one or a combination of at least two of homologous recombination, CRISPR/Cas9, TALEN, ZFN, lentiviral infection, or retroviral infection.
In the present invention, the three transcription factors RUNX1, HOXA9 and LHX2 may be simultaneously introduced into cells of a human pluripotent stem cell-induced differentiation system including any one or a combination of at least two of human pluripotent stem cells, mesodermal cells, Hematopoietic Endothelial Cells (HEC) or Hematopoietic Progenitor Cells (HPC).
In the present invention, the three transcription factors RUNX1, HOXA9 and LHX2 may be introduced into cells of a human pluripotent stem cell-induced differentiation system, respectively, and as a preferred embodiment of the present invention, RUNX1 and HOXA9 are introduced together, and at a later stage, LHX2 gene is introduced.
Preferably, RUNX1 and HOXA9 are first introduced into human pluripotent stem cells together, LHX2 gene is introduced at the stage of subsequent mesodermal cells or Hematogenic Endothelial Cells (HEC), and three transcription factors RUNX1, HOXA9 and LHX2 are co-induced to generate Hematopoietic Progenitor Cells (HPC), followed by transplantation.
Preferably, RUNX1 and HOXA9 are firstly introduced into the human pluripotent stem cells together, and then mesodermal cell induction, hematogenic endothelial cell induction and hematopoietic progenitor cell generation are carried out, and then LHX2 gene is introduced at the hematopoietic progenitor stage, and the three transcription factors RUNX1, HOXA9 and LHX2 are cultured for a period of time, optionally 1-20 days, for example, 1 day, 5 days, 10 days, 15 days or 20 days, preferably 4-15 days, and then transplanted.
Preferably, RUNX1 and HOXA9 are first introduced into mesodermal cells, followed by subsequent induction of hematopoietic endothelial cells and hematopoietic progenitor cell generation, and then LHX2 gene is introduced into hematopoietic progenitor cells, and the culture is continued for a period of time, optionally 1-20 days, for example, 1 day, 5 days, 10 days, 15 days, or 20 days, preferably 4-15 days, in the presence of the three transcription factors RUNX1, HOXA9 and LHX2, before transplantation.
As a preferred technical scheme of the invention, the human pluripotent stem cell line with three transcription factors expressed or co-expressed is constructed by transfecting an expression vector containing three transcription factors of RUNX1, HOXA9 and LHX2 into human pluripotent stem cells, inserting RUNX1, HOXA9 and LHX2 into a safe site of a genome;
the expression vector contains any one or a combination of at least two of RUNX1, HOXA9 or LHX 2;
the expression vector also comprises a fluorescent marker gene, a resistance gene and a gene expression regulation system.
Preferably, the fluorescent marker gene includes but is not limited to GFP, YFP, RFP or td Tomato, etc., preferably GFP.
Preferably, the resistance gene includes, but is not limited to, puromycin resistance gene (PuroR), hygromycin resistance gene (HygroR), ampicillin resistance gene (AmpR), and the like, preferably PuroR.
Preferably, the gene expression regulation system includes, but is not limited to, a tetracycline-induced expression Tet-On system or a steroid hormone-induced Cre-ER system, and the like, and preferably a tetracycline derivative Doxycycline (dox) induced expression Tet-On system.
Preferably, the three transcription factors RUNX1, HOXA9 and LHX2 are induced to be expressed simultaneously or separately, said induced expression starting from mesodermal cells, hematogenic endothelial cells or hematopoietic progenitor cells, preferably starting from mesodermal cells.
For the cells for respectively introducing the three transcription factors of RUNX1, HOXA9 and LHX2 into a human pluripotent stem cell induced differentiation system, the RUNX1 and HOXA9 can be used for inducing expression at first and then LHX2 genes can be used for expressing.
Preferably, expression of RUNX1 and HOXA9 is first induced in mesodermal cells and LHX2 gene is then expressed in hematopoietic endothelial cells or hematopoietic progenitor cells.
Preferably, the transfection method of the expression vector includes, but is not limited to, electroporation, lipofection, calcium phosphate precipitation, or the like, and preferably, electroporation.
Preferably, the safe site includes, but is not limited to, AAVS1 or HIPP11 and the like, preferably AAVS1 site.
Preferably, the human pluripotent stem cells comprise human induced pluripotent stem cells and/or human embryonic pluripotent stem cells.
In the invention, based on CRISPR/Cas9 system combined homologous recombination, sgRNA and a targeting vector are electrotransferred into hPSCs for 18-24 h, and then hPSCs culture medium containing puromycin (with final concentration of 0.25-1 mu M) is added for drug screening and positive clone enrichment. And after 8-12 days of drug screening, adopting a puromycin-free culture medium for culture amplification and passage. A plurality of fluorescent single cells are sorted out by a flow sorter AriaII Sorp (BD Bioscience) for cloning culture, finally, cell genome extraction is carried out on each cloned cell, and positive clones are identified by PCR. Passage, expansion and cryopreservation are carried out according to the cell state and growth density.
In a preferred embodiment of the present invention, the method for inducing differentiation is monolayer culture induced differentiation based on stromal cell co-culture, which comprises three induction stages: the method comprises the steps of inducing human pluripotent stem cells to generate mesodermal cells (D0-D2), inducing differentiation of hematogenic endothelial cells (D2-D9) and inducing differentiation and maturation of hematopoietic progenitor cells (D9-D16 or D9-D24).
Preferably, the D0 medium and D1 medium are used in the induction of human pluripotent stem cells to produce mesodermal cells;
d2 culture medium is used in the induction of the differentiation of the hematogenous endothelial cells;
the D9 medium and/or D16 medium is used in the induction of differentiation and maturation of hematopoietic progenitor cells.
In the invention, factors rhIL-6, rhIL-7 and Thymic Stromal Lymphopoietin (TSLP) for promoting the proliferation and differentiation of B cells are added in the process of inducing the differentiation and maturation of the hematopoietic progenitor cells, so that the regeneration efficiency of the B cells can be improved.
Preferably, the D0 medium includes human basic fibroblast growth factor (hbFGF), recombinant human activin (rhActivin a), recombinant human bone morphogenetic protein (rhBMP 4), GSK-3 α/β inhibitor CHIR99021, PI3K inhibitor LY294002, and TeSR-E6 medium.
Preferably, the D1 culture medium comprises recombinant human bone morphogenetic protein (rhBMP 4), ALK5 inhibitor A83-01, Wnt pathway inhibitor IWR-1-endo and TeSR-E6 culture medium.
Preferably, the D2 medium includes vitamin C, recombinant human FMS-like tyrosine kinase 3 ligand (rhFlt 3L), human basic fibroblast growth factor (hbFGF), human vascular endothelial growth factor (hVEGF), recombinant human stem cell factor (rhSCF), recombinant human interleukin 3 (rhIL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), sodium butyrate, and StemPro-34 medium.
Preferably, the D9 medium includes recombinant human thrombopoietin (rhTPO), recombinant human interleukin 3 (rhIL-3), recombinant human stem cell factor (rhSCF), recombinant human interleukin 11 (rhIL-11), human insulin-like growth factor 1 (hIGF-1), human vascular endothelial growth factor (hVEGF), human basic fibroblast growth factor (hbFGF), recombinant human bone morphogenetic protein (rhBMP 4), recombinant human FMS-like tyrosine kinase 3 ligand (rhFlt 3L), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), Doxycycline (DOX), and StemPro-34 medium.
Preferably, the D16 medium includes serum replacement B27, ROCK inhibitor Y-27632, recombinant human thrombopoietin (rhTPO), recombinant human stem cell factor (rhSCF), recombinant human interleukin 3 (rhIL-3), recombinant human FMS-like tyrosine kinase 3 ligand (rhFlt 3L), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), Doxycycline (DOX), and StemPro-34 medium.
Preferably, the D0 culture medium comprises 5-40 ng/mL of hbFGF, 10-50 ng/mL of rhActivin A, 10-60 ng/mL of rhBMP4, 2-10 μ M of CHIR99021 and 5-20 μ M of LY294002, wherein the concentration of hbFGF can be 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL or 40 ng/mL, etc., the concentration of rhActivin A can be 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL or 50 ng/mL, etc., and the concentration of rhBMP4 can be 10 ng/mL, 15 ng/mL, 20 ng/mL, or 50 ng/mL, etc, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, or 60 ng/mL, etc., the concentration of CHIR99021 may be, for example, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, or 10. mu.M, etc., and the concentration of LY294002 may be, for example, 5. mu.M, 10. mu.M, 15. mu.M, or 20. mu.M, etc., and other specific points in the numerical range may be selected, and will not be described in detail herein.
Preferably, the D1 culture medium comprises 10-60 ng/mLrhBMP4, 0.5-3 μ M A83-01 and 0.5-5 μ M IWR-1-endo, wherein the concentration of rhBMP4 can be, for example, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL or 60 ng/mL, etc., the concentration of A83-01 can be, for example, 0.5 μ M, 1 μ M, 1.5 μ M, 2 μ M, 2.5 μ M or 3 μ M, etc., the concentration of R-1-endo can be, for example, 0.5 μ M, 1 μ M, 1.5 μ M, 2 μ M, 2.5 μ M, 3 μ M, 3.5 μ M, 4 μ M, 4.5 μ M or 5 μ M, etc., other specific point values in the numerical range can be selected, and are not described in detail herein.
Preferably, the D2 medium comprises 1-3 mM GlutaMAXTM-I, 0.5-3 mM NEAA, 0.05-0.2 mM beta-mercaptoethanol, 25-75 ng/mL vitamin C, 10-40 ng/mL rhFLT3L, 1-10 ng/mL hbFGF, 25-75 ng/mL hVEGF, 25-75 ng/mL rhSCF, 5-40 ng/mL rhIL-3, 5-25 ng/mL GM-CSF, 5-25 ng/mL M-CSF, 0.1-1 mM sodium butyrate and 0.5-2 μ g/mL DOX, wherein GlutaMAX is a compound of formula I, formula IITMThe concentration of-I may be, for example, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, etc., the concentration of NEAA may be, for example, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, etc., the concentration of β -mercaptoethanol may be, for example, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, etc., the concentration of vitamin C may be, for example, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, etc., the concentration of rhFLT3L may be, for example, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, etc., the concentration of hbFGF can be, for example, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, or 10 ng/mL, etc., the concentration of hVEGF can be, for example, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, or 75 ng/mL, etc., the concentration of rhSCF can be, for example, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, etc, 65 ng/mL, 70 ng/mL, 75 ng/mL, etc., the rhIL-3 concentration may be, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, etc., the GM-CSF concentration may be, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, etc., and the M-CSF concentration may be, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, etcThe concentration of sodium butyrate can be 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, and the concentration of DOX can be 0.5 mu g/mL, 1 mu g/mL, 1.5 mu g/mL or 2 mu g/mL, and other specific points in the numerical range can be selected, which is not described in detail herein.
Preferably, the D9 culture medium comprises 10-60 ng/mL rhTPO, 1-10 ng/mL rhIL3, 25-75 ng/mL rhSCF, 2-20 ng/mL rhIL-11, 10-50 ng/mL hIGF-1, 2-20 ng/mL hVEGF, 1-20 ng/mL hbFGF, 5-25 ng/mL rhBMP4, 5-25 ng/mL rhFLT3L, 5-25 ng/mL GM-CSF, 5-25 ng/mL M-CSF and 0.5-2 μ g/mL DOX, wherein the concentration of rhTPO may be, for example, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, or, 55 ng/mL or 60 ng/mL, etc., the concentration of rhIL3 can be, for example, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, or 10 ng/mL, etc., the concentration of rhSCF can be, for example, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, or 75 ng/mL, etc., the concentration of rhIL-11 can be, for example, 2 ng/mL, 4 ng/mL, 6 ng/mL, 8 ng/mL, 10 ng/mL, etc, 12 ng/mL, 14 ng/mL, 16 ng/mL, 18 ng/mL, or 20 ng/mL, etc., for example, the concentration of hIGF-1 may be 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, or 50 ng/mL, etc., the concentration of hVEGF may be 2 ng/mL, 4 ng/mL, 6 ng/mL, 8 ng/mL, 10 ng/mL, 12 ng/mL, 14 ng/mL, 16 ng/mL, 18 ng/mL, or 20 ng/mL, etc., the concentration of hbFGF may be 1 ng/mL, 2 ng/mL, 4 ng/mL, 6 ng/mL, etc, 8 ng/mL, 10 ng/mL, 12 ng/mL, 14 ng/mL, 16 ng/mL, 18 ng/mL, or 20 ng/mL, etc., rhBMP4 may have a concentration of, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, or 25 ng/mL, rhFLT3L may have a concentration of, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, or 25 ng/mL, etc., GM-CSF may have a concentration of, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, or 25 ng/mL, M-CSF may have a concentration of, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, or 25 ng/mL, etc., the concentration of DOX can be, for example, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, or 2. mu.g/mL, etc., and other specific values within the numerical range can be selected, which is not described herein again.
Preferably, the D16 culture medium comprises 2% -10% serum substitute B27, 5% -20 μ M Y-27632, 10% -50 ng/mL rhTPO, 20% -70 ng/mL rhSCF, 5% -20 ng/mL rhIL-3, 1% -10 ng/mL rhFLT3L, 5% -25 ng/mL GM-CSF, 5% -25 ng/mL M-CSF and 0.5% -2 μ g/mL DOX, wherein the volume fraction of B27 can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% and the like, the concentration of Y-27632 can be 5 μ M, 10 μ M, 15 μ M or 20 μ M and the like, and the concentration of rhTPO can be 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, or 50 ng/mL, etc., for example, the concentration of rhSCF may be 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, or 70 ng/mL, etc., the concentration of rhIL-3 may be 5ng/mL, 10 ng/mL, 15 ng/mL, or 20 ng/mL, etc., the concentration of rhFLT3L may be 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, or 10 ng/mL, etc., the concentration of GM-CSF may be 5ng/mL, 10 ng/mL, 15 ng/mL, etc., for example, 20 ng/mL or 25 ng/mL, etc., the concentration of M-CSF can be, for example, 5ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL or 25 ng/mL, etc., the concentration of DOX can be, for example, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL or 2. mu.g/mL, etc., and other specific points in the numerical range can be selected, which is not repeated herein.
As a preferred technical scheme, the components of the culture medium are as follows:
the D0 culture medium is 15 ng/mL human basic fibroblast growth factor, 20 ng/mL recombinant human activin, 30 ng/mL recombinant human bone morphogenetic protein, 4 mu M GSK-3 alpha/beta inhibitor CHIR99021, 6 mu M PI3K inhibitor LY294002, and the balance is TeSR-E6 culture medium.
The D1 culture medium is 20 ng/mL recombinant human bone morphogenetic protein, 0.5 mu M ALK5 inhibitor A83-01 and 1 mu M Wnt pathway inhibitor IWR-1-endo, and the balance is TeSR-E6 culture medium.
The D2 culture medium is 2 mM GlutaMAXTM-I, 1 mM NEAA, 0.1 mM beta-mercaptoethanol, 50 ng/mL vitamin C, 10 ng/mL recombinant human FMS-like tyrosine kinase 3 ligand, 2 ng/mL human basic compositionFibroblast growth factor, 40 ng/mL human vascular endothelial growth factor, 40 ng/mL recombinant human stem cell factor, 10 ng/mL recombinant human interleukin 3, 5ng/mL granulocyte-macrophage colony stimulating factor, 10 ng/mL macrophage colony stimulating factor, 0.15 mM sodium butyrate and 1 mu g/mL doxycycline, and the balance being StemPro-34 culture medium.
The D9 culture medium is 20 ng/mL recombinant human thrombopoietin, 5ng/mL recombinant human interleukin 3, 40 ng/mL recombinant human stem cell factor, 2 ng/mL recombinant human interleukin 11, 20 ng/mL human insulin-like growth factor 1, 2.5 ng/mL human vascular endothelial growth factor, 2.5 ng/mL human basic fibroblast growth factor, 5ng/mL recombinant human bone morphogenetic protein, 5ng/mL recombinant human FMS-like tyrosine kinase 3 ligand, 5ng/mL granulocyte-macrophage colony stimulating factor, 10 ng/mL macrophage colony stimulating factor and 1 mu g/mL doxycycline, and the balance is StemPro-34 culture medium.
The culture medium of D16 is 2% serum substitute B27, 5 mu M ROCK inhibitor Y-27632, 20 ng/mL recombinant human thrombopoietin, 40 ng/mL recombinant human stem cell factor, 5ng/mL recombinant human interleukin 3, 2.5 ng/mL recombinant human FMS-like tyrosine kinase 3 ligand, 5ng/mL granulocyte-macrophage colony stimulating factor, 10 ng/mL macrophage colony stimulating factor and 1 mu g/mL doxycycline, and the balance is StemPro-34 culture medium.
As a preferred technical scheme, the preparation method of the artificial blood progenitor cells with the B lineage differentiation potential comprises the following steps:
introducing an expression vector containing three transcription factors of RUNX1, HOXA9 and LHX2 into a safe site of the genome of the human pluripotent stem cell by a gene editing technology;
after screening positive clones, carrying out three-stage induced differentiation by a monolayer culture induced differentiation method based on stromal cell co-culture to obtain the artificial blood progenitor cells with the B lineage differentiation potential,
the first stage is the induction of human pluripotent stem cells to produce mesodermal cells (D0 to D2):
culturing with D0 culture medium and D1 culture medium, and cleaning with DPBS 2 times during D0 and D1 cell changing;
the second stage is the induction of Hematogenic Endothelial Cell (HEC) differentiation (D2-D9):
culturing by using a D2 culture medium;
d2, digesting the induced mesoderm cells into single cells using a digestion solution, wherein the digestion solution comprises, but is not limited to, any one or a combination of at least two of Accutase enzyme, 0.5 mM EDTA or 0.05% pancreatin, preferably an equal volume of a mixture of Accutase enzyme and 0.5 mM EDTA;
during the culture process, fluid supplementation is carried out every day or every two days, when D9 is carried out, digestion solution is used for digesting induced HEC, and the HEC can be directly paved into a new culture dish for subsequent HPC induced culture; optionally, the subsequent HPC can be induced by co-culturing the cells in a culture dish containing matrix cells laid in advance, preferably by co-culturing the cells with the matrix cells;
the digestive fluid may include, but is not limited to, TrypLETM Express, 0.05% pancreatin, Accutase, etc., preferably TrypLETM Express (Thermo fisher scientific), and the stromal cells may include, but are not limited to, OP9, OP9-DL1, OP9-DL4, OP9-DL1-APLN, OP9-DL4-APLN, modified OP9 cells, AFT024-DL1, AFT024-LHX2 or modified AFT024 cells, or a combination of at least two thereof, preferably OP9-DL 1;
the third stage is to induce differentiation and maturation of Hematopoietic Progenitor Cells (HPCs) (D9-D16 or D9-D24):
the method for selecting the D9-D16 mode comprises the following steps: culturing to D16 using D9 medium;
during the culture process, fluid replacement is carried out every day or every two days. D13-D16, transferring supernatant, digesting adherent cells with digestive juice for 2-3 min, analyzing induced HPC phenotype by a flow sorter, and sorting CD34+HPC. Staining protocols include, but are not limited to: GFP, CD45-APC/cy7, linkage (CD3/4/8/14/19/20) -Biotin-Percp/cy5.5, CD11b-PE/cy7, CD201-APC, CD34-PE and DAPI, data were analyzed using Flowjo software; the digestive juices may include, but are not limited to, 0.25% pancreatin and tryple Express, and the like;
wherein, when the D9-D24 mode is selected, the method further comprises the step of co-culturing the strain to D24 by using a D16 culture medium:
d16 sorted CD34+HPC is co-cultured with preirradiation-treated stromal cells for expansion and maintenance of a dry state, and the stromal cells used may include, but are not limited to, any one or a combination of at least two of OP9, OP9-DL1, OP9-DL4, OP9-DL1-APLN, OP9-DL4-APLN, modified OP9 cells, AFT024-DL1, AFT024-LHX2, or modified AFT024 cells.
In a second aspect, the present invention provides an artificial blood progenitor cell with B-lineage differentiation potential, which is prepared by the method for preparing an artificial blood progenitor cell with B-lineage differentiation potential according to the first aspect;
the artificial blood progenitor cells with the B lineage differentiation potential realize human B cell regeneration after transplantation.
Preferably, the B cell type differentiated from the artificial blood progenitor cells having B lineage differentiation potential includes any one or a combination of at least two of B progenitor cells (B progenitors), non-mature B cells (immatur B cells), mature B cells (mature B cells), or plasma cells, without limitation.
Preferably, the B progenitor cells (B progenitor cells) include, but are not limited to, pro-B cells and/or pre-B cells.
Preferably, the mature B cells include, but are not limited to, B1 cells and/or B2 cells.
Preferably, the B1 cells include, and are not limited to, B1a and/or B1B cells.
Preferably, the B2 cells include, but are not limited to, Follicular B cells (FO B) and/or Marginal zone B cells (MZ B).
In a third aspect, the present invention provides a cell product, wherein the cell product comprises the cells of the human pluripotent stem cell-induced differentiation system of the first aspect and the genetically engineered cells thereof, the artificial blood progenitor cells with B-lineage differentiation potential of the second aspect and the genetically engineered cells thereof, the B cells formed by the differentiation of the artificial blood progenitor cells with B-lineage differentiation potential of the second aspect and the genetically engineered cells thereof, or the B cells formed by the differentiation of the artificial blood progenitor cells with B-lineage differentiation potential of the second aspect and the secretion products of the genetically engineered cells thereof.
Preferably, the cells of the human pluripotent stem cell-induced differentiation system include any one of or a combination of at least two of human pluripotent stem cells, mesodermal cells, hematopoietic endothelial cells or hematopoietic progenitor cells.
Preferably, the genetically engineered cell comprises any one of or a combination of at least two of cells expressing a protein of interest, a cytokine of interest, an antibody of interest, or a chimeric antigen receptor, constructed by genetic engineering techniques.
Preferably, the secretion products of the B cells and their genetically engineered cells include any one or a combination of at least two of antibodies, cytokines, or proteins.
In a fourth aspect, the present invention provides a method of in vivo B lymphocyte lineage regeneration comprising:
transplanting the artificial blood progenitor cells having B-lineage differentiation potential according to the second aspect into a body.
Preferably, the method for in vivo regeneration of B lymphocyte lineage further comprises the step of detecting human hematopoietic cells in vivo after transplantation.
In the present invention, HPCs generated by induced differentiation can be used as B lineage seed cells, either directly or sorted for CD34+The cells are transplanted into immunodeficient mice (the dose is 0.5-5 millions/mouse) which are treated by sublethal irradiation, and the in-vivo B lymphocyte lineage regeneration is carried out. The transplantation method includes but is not limited to any one of angular intravenous injection, medullary cavity injection or tail vein injection, and preferably angular intravenous injection.
After 6 weeks of transplantation, peripheral blood, bone marrow, spleen and lymph node of recipient mice were collected, and the proportion of hPSCs-derived blood cells and CD19 were analyzed by flow cytometer LSR Fortessa (BD Bioscience)+B cell ratio to confirm that hPSCs induced differentiation of artificial blood progenitor cells can achieve B lymphopoiesis in vivoCell lineage reconstitution.
Sorting GFP from recipient mouse bone marrow, lymph nodes and spleen+hCD45+Cells, genome extraction using a cell genome extraction kit (Tiangen), PCR identification using primers specific for the knock-in gene sequence, sequencing of the PCR-amplified sequence, and confirmation of GFP in recipient mice at the genome level+hCD45+Hematopoietic cells (primarily B cells) are derived from transplanted artificial blood progenitor cells.
Meanwhile, after 4-8 weeks of transplantation, the type content of immunoglobulin in the serum of the nonimmune receptor mouse is detected by ELISA so as to verify the antibody secretion function of the B cell derived from hPSCs.
Finally, after the receptor mouse is immunized by the specific antigen, whether the receptor mouse generates the specific antibody or not is detected, so that the fact that the B cell from the hPSCs can generate the specific antibody and has the function of secreting the specific antibody is verified. After a period of primary antigen stimulation, antigen stimulation was again used to demonstrate that hPSCs-derived B cells can be efficiently involved in adaptive immune responses by flow-testing for the production of plasma cells and memory B cells.
In the present invention, the success rate of HPC transplantation can be improved by injecting factors promoting B cell development, such as rhIL-6, rhIL-7, and Thymic Stromal Lymphopoietin (TSLP), into the recipient mouse during HPC transplantation.
In a fifth aspect, the present invention provides the use of any one or a combination of at least two of the method for producing an artificial blood progenitor cell with B-lineage differentiation potential according to the first aspect, the artificial blood progenitor cell with B-lineage differentiation potential according to the second aspect, or the cell product according to the third aspect, for the preparation of a pharmaceutical composition.
In a sixth aspect, the present invention provides a pharmaceutical composition comprising the artificial blood progenitor cells of the second aspect having B-lineage differentiation potential and/or the cell products of the third aspect.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
Preferably, the pharmaceutically acceptable excipients include any one of a carrier, an excipient or a diluent or a combination of at least two thereof.
In a seventh aspect, the present invention provides a use of the pharmaceutical composition of the sixth aspect in the preparation of a disease prevention medicament, a disease treatment medicament, or a vaccine.
Preferably, the disease prevention drug comprises a drug that enhances the immune response of the body.
Preferably, the immune response comprises a humoral and/or cellular immunity.
Preferably, the disease treatment drug includes any one of or a combination of at least two of a B cell immunodeficiency treatment drug, an anti-pathogen infection drug, an anti-tumor drug, an autoimmune disease treatment drug, or a genetic disease treatment drug.
Preferably, the genetic disease comprises any one of hemophilia, lysosomal storage disease, hypophosphatasia, or phenylketonuria, or a combination of at least two of them.
Preferably, the drug comprises an antibody drug.
In the present invention, the pharmaceutical composition may also be used as a cell carrier to secrete and/or deliver therapeutic drugs for the treatment of related diseases, or as a protein or enzyme replacement therapy.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention obtains the artificial blood progenitor cells with the B lineage differentiation potential by introducing three transcription factors of RUNX1, HOXA9 and LHX2 into cells of a human pluripotent stem cell induced differentiation system and performing induced expression and induced differentiation; by optimizing the induction process and the culture medium formula, the induction efficiency of the cells is obviously improved, and the obtained artificial blood progenitor cells have more quantity and high activity;
(2) the artificial blood progenitor cells prepared by the invention can regenerate human B cells in an animal model after transplantation, the B cells can mature in vivo and secrete corresponding antibodies, the regeneration of a humoral immune system of a humanized animal is realized, the regeneration of the B cells in a human body is finally expected, and the clinical application and transformation of a B cell therapy are promoted;
(3) the regenerated B cell can secrete a large amount of cell products such as target antibodies, target cell factors, functional proteins and the like after being modified by genetic engineering, can be applied to preparation of related medicines, and has extremely high application value.
Drawings
FIG. 1A is a schematic diagram of the structure of a knockin inducible expression sequence of example 1;
FIG. 1B is a photograph showing the result of electrophoresis of the amplification product of primer F2/R2 in example 1, wherein lane M: standard DNA molecular weight Marker, lane 1: negative control, lanes 2-4: amplification products of 3 positive clones;
FIG. 1C is a photograph showing the result of electrophoresis of the amplification product of primer F1/R1 in example 1, in lane M: standard DNA molecular weight Marker, lane 1: negative control, lanes 2-4: amplification products of 3 positive clones;
FIG. 2A is a schematic diagram showing the procedure of example 2 in which human pluripotent stem cells are induced to differentiate to generate hematopoietic progenitor cells;
fig. 2B is a bright field diagram (scale bar =200 μm) of cells in the process of inducing human pluripotent stem cells to generate mesodermal cells in example 2;
figure 2C is a picture of the results of streaming sorting of HPC in example 2;
FIG. 3 is the CD34 transplanted in example 3+A picture of the results of flow analysis of peripheral blood of a recipient mouse of cells;
FIG. 4A is a schematic diagram of the structure of the knockin inducible expression sequence of example 4;
FIG. 4B is a picture of the results of streaming sorting of HPC in example 4;
FIG. 5A is a plasmid map of the lentiviral expression plasmid pRRLsin-LHX2-EGFP of example 5;
FIG. 5B is a schematic diagram showing the procedure for viral infection and co-cultivation in example 5;
FIG. 5C is a photograph showing the results of flow measurement of the infection efficiency in example 5;
fig. 5D is a picture showing the results of flow analysis of peripheral blood of a recipient mouse into which human HPCs were transplanted in example 5, wherein #1, # 2: two transplant recipient mice.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 preparation of human pluripotent Stem cells inducibly expressing exogenous RUNX1, HOXA9 and LHX2 genes
In this example, based on CRISPR/Cas9 system, an inducible expression sequence is knocked in at AAVS1 site of human pluripotent stem cells (hpscs) by an electrotransformation method, and the knocked-in sequence includes RUNX1-p2a-HOXA9-t2a-LHX2 tandem sequence, fluorescent protein gene GFP and puromycin resistance gene sequence for resistance selection, and the structural schematic diagram is shown in fig. 1A.
The sgRNA and targeting vector were electroporated into hPSC, and 20 h later, hPSC medium containing puromycin (1. mu.M) was added and the medium was changed daily. Wherein, the targeting sequence corresponding to the sgRNA is shown in SEQ ID No. 1.
SEQ ID No.1：5’-gtcaccaatcctgtccctagtgg-3’（hAAVS1-sgRNA）。
After 10 days of selection with puromycin, culture, expansion and passage were performed using puromycin-free medium. Sorting multiple GFP s by flow sorter+And (3) culturing the single cells in a 96-well plate, after the single cells form clones, digesting the clones by using 0.5 mM EDTA, and carrying out amplification culture after passage to a 12-well plate.
Finally, collecting corresponding cells from each clone, extracting cell genome (Tiangen cell gene extraction kit), designing PCR primers, identifying positive clones by PCR, wherein the corresponding relation between the PCR primers and the sites is shown in figure 1A, and the sequence is as follows:
primer F1 (SEQ ID No. 2): 5'-AGGCCCGTTCCAAGCCA-3', respectively;
primer R1 (SEQ ID No. 3): 5'-AGATGCCCAGGTGGCAG-3', respectively;
primer F2 (SEQ ID No. 4): 5'-GTAGCGTGGGCATGGATCC-3', respectively;
primer R2 (SEQ ID No. 5): 5'-CTAAGAACTTGGGAACAGCCACAGC-3' are provided.
Wherein, the size of the F1/R1 amplification product is 1541 bp, and the size of the F2/R2 amplification product is 2560 bp.
The electrophoresis results of the amplification products are shown in FIGS. 1B and 1C. Finally obtaining 3 positive clones, and carrying out passage, amplification and cryopreservation according to the cell state and the growth density.
Example 2 three-Gene modification of human pluripotent Stem cells with RUNX1, HOXA9 and LHX2 induced differentiation to give hematopoietic progenitor cells
A schematic of the process for the generation of Hematopoietic Progenitor Cells (HPCs) by induced differentiation of human pluripotent stem cells is shown in FIG. 2A.
A monolayer culture induced differentiation system based on stromal cell co-culture is adopted, and the induced differentiation process is divided into three stages:
(1) the first stage is the induction of human pluripotent stem cells to produce mesodermal cells (D0 to D2), and fig. 2B shows the bright field pattern of the cells during induction of mesodermal cells:
culturing with D0 medium and D1 medium, respectively;
the formula of the D0 culture medium is as follows: 15 ng/mL hbFGF (Peprotech), 20 ng/mL rhActivin A (Peprotech), 30 ng/mL rhBMP4 (R & D), 4. mu.M CHIR99021 (Selleck), and 6. mu.M LY294002 (Selleck), with the balance being TeSR-E6 basal medium (STEMCELL Technologies);
the formula of the D1 culture medium is as follows: 20 ng/mL rhBMP4, 0.5 mu M A83-01 (Selleck), 1 mu M IWR-1-endo (Selleck), and the balance of TeSR-E6 basal medium;
d2, induced mesodermal cells were digested into single cells using a digest (an equal volume of mixture of Accutase enzyme < Sigma-Aldrich > and 0.5 mM EDTA < Gibco >).
(2) The second stage is the induction of Hematogenic Endothelial Cell (HEC) differentiation (D2-D9):
culturing by using a D2 culture medium;
the formula of the D2 culture medium is as follows: 2 mM GlutaMAXTM-I (Thermo fisher scientific), 1 mM NEAA (Thermo fisher scientific), 0.1 mM beta-mercaptoethanol (Sigma-Aldrich), 50 ng/mL vitamin C (Sigma-Aldrich), 10 ng/mL rhFLT3L (Peprotech), 2 ng/mL hbFGF (Peprotech), 40 ng/mL hVEGF (Peprotech), 40 ng/mL rhSCF (Peprotech), 10 ng/mL rhIL-3 (Peprotech), 5ng/mL LGM-CSF (Peprotech), 10 ng/mLM-CSF (Peprotech), 0.15 mM NaB (Selleck), and 1. mu.g/mL doxycycline (blue-Hakko-34 medium, the balance being StemPro-34 medium;
supplementing liquid every 1-2 days during culture, and using TrypLE as digestive juice in D9TMExpress (thermo fisher scientific) digestion induced HEC was replated to a petri dish previously plated with stromal cells OP9-DL1 for co-culture.
(3) The third stage is the induction of hematopoietic progenitor differentiation and maturation (D9-D16):
co-culturing with D9 medium;
the formula of the D9 culture medium is as follows: 20 ng/mL rhTPO, 5ng/mL rhIL3, 40 ng/mL rhSCF, 2 ng/mL rhIL-11, 20 ng/mL hIGF-1, 2.5 ng/mL hVEGF, 2.5 ng/mL hbFGF, 5ng/mL rhBMP4, 5ng/mL rhFLT3L, 10 ng/mL GM-CSF, 10 ng/mL M-CSF and 1 μ g/mL DOX, with the balance being StemPro-34 medium;
and supplementing the culture solution every 1-2 days in the culture process. D16, after transfer of supernatant, adherent cells were digested with 0.25% pancreatin (Thermo fisher scientific), HPC phenotype was analyzed by flow sorter (as shown in FIG. 2C) and CD34 was sorted+A cell. The staining protocol was as follows: GFP, CD45-APC/cy7, linkage (CD3/4/8/14/19/20) -Biotin-Percp/cy5.5, CD11b-PE/cy7, CD201-APC, CD34-PE and DAPI (all antibodies from Biolegend), data were analyzed using Flowjo software.
Example 3 iRUNX1-p2a-HOXA9-t2a-LHX 2-hPSC-derived hematopoietic progenitor cells (B lineage seed cells) were transplanted into immunodeficient mice and B cell regeneration was examined
Sorting D16 into CD34+The cells are transplanted into immunodeficient NCG mice (Jiejiankang) irradiated sublethally (2-3 Gy) by means of angular intravenous injection, and the dosage is 1 million/mouse.
Peripheral blood from recipient mice was obtained by tail vein blood collection for flow analysis at 6, 8 and 10 weeks of transplantation, and the staining protocol was as follows: GFP, mTER119-PerCP/Cy5.5, hCD45-PE/Cy7, hCD19-BV785, hCD33-PE, hCD3/4/8-APC and DAPI (all antibodies described above were purchased from Biolegend). Data were analyzed using Flowjo software.
As shown in FIG. 3, it is clear that iRUNX1-p2a-HOXA9-t2a-LHX2-HPC can be used as a seed cell of B lineage to regenerate CD19 in recipient mice+B cells.
Example 4 preparation of human pluripotent Stem cells inducibly expressing exogenous RUNX1 and HOXA9 genes and Induction of differentiation to give hematopoietic progenitor cells
In the embodiment, based on a CRISPR/Cas9 system, an inducible expression sequence is knocked in at the AAVS1 site of hPSC by an electrotransformation method, wherein the knocked-in sequence comprises a RUNX1-p2a-HOXA9 tandem sequence, a fluorescent protein gene GFP and a puromycin resistance gene sequence for resistance screening, and a structural schematic diagram is shown in FIG. 4A.
The expression sequences were knocked in by the same method as in example 1, and positive clones were identified by drug screening and genomic PCR, to finally obtain 2 positive clones.
HPCs expressing exogenous RUNX1c and HOXA9 genes were induced to differentiate to produce HPCs using the same method as in example 2.
Upon induction of differentiation D16, HPC phenotypes were analyzed by flow analysis, and the staining protocol was as follows: GFP, hCD45-APC/Cy7, CD43-PE/Cy7, CD34-PE and DAPI, the data were analyzed using Flowjo software.
As shown in FIG. 4B, RUNX1-p2a-HOXA9-hPSC induced differentiation to produce CD34+Hematopoietic progenitor cells.
Example 5 RUNX1-p2a-HOXA9-hPSC Induction derived CD34+Hematopoietic progenitor cells infected with LHX2 virus to prepare hematopoietic progenitor cells, which are transplanted into immunodeficient mice for detection of B cell regeneration
In this example, a lentiviral expression plasmid pRRLsin-LHX2-EGFP expressing LHX2 gene was constructed, and 293T cells (ATCC) were transfected with lentiviral packaging plasmids psPAX2 and pMD2.G, or VSVG, pLP1 and pLP2 (Addgene) under the action of PEI, and virus-coated. Wherein, the map of the pRRLsin-LHX2-EGFP plasmid is shown in figure 5A.
Virus liquid was collected at 48 h and 72 h of transfection, virus was concentrated by ultracentrifugation (50000 g, 2 h), virus titer was determined by gradient dilution of concentrated virus liquid to infect Jarkat cells (university of river-south), and infection efficiency was assayed by flow-assay 48 h after infection (i.e., GFP)+Cell ratio) according to GFP+Ratio calculation of viral titer.
D16, changing into D16 culture medium, culturing and sorting to obtain RUNX1-p2a-HOXA 9-hPSC-derived CD34+Hematopoietic progenitor cells, followed by viral infection and co-culture, are schematically depicted in FIG. 5B. The concentrated LHX2 virus was infected with the induced D16 HPC described above, at a virus multiplicity MOI of 20.0. After the virus is infected for 12-24 h, the virus is washed away by using DPBS, and the virus and the stroma cell AFT024-LHX2 (AFT 024 cell is from ATCC and is constructed by gene targeting AFT024-LHX 2) which is subjected to radiation treatment (the total amount is 10-20 Gy and is 1 Gy/min) are co-cultured for about 1 week in a low-temperature incubator at 33 ℃ and the dryness is maintained.
The formula of the culture medium of D16 is as follows: 2% serum replacement B27 (Gibco), 5. mu.M ROCK inhibitor Y-27632, 20 ng/mL rhTPO, 40 ng/mL rhSCF, 5ng/mL rhIL-3, 2.5 ng/mL rhFLT3L, 5ng/mL GM-CSF, 10 ng/mL M-CSF and 1. mu.g/mL DOX, the balance being StemPro-34 medium.
After 48 h of virus infection, a small amount of cells were taken to measure the infection efficiency by flow, and the uninfected HPC was used as a blank control, and the results of flow measurement of the infection efficiency are shown in FIG. 5C, which shows that the LHX2 virus had an infection efficiency of HPC of 90% or more.
The artificial blood progenitor cells obtained after 1 week of co-culture are transplanted into the immunodeficient NCG mice under sublethal irradiation (2-3 Gy) in a dose of 0.7 million/mouse by means of ocular intravenous injection.
At 6 weeks of transplantation, peripheral blood of the recipient mice was obtained by tail vein blood collection for flow analysis, and the staining protocol was as follows: GFP, mTER119-PerCP/Cy5.5, hCD45-PE/Cy7, hCD19-BV785, hCD33-PE, hCD3/4/8-APC and DAPI. Data were analyzed using Flowjo software.
The results are shown in FIG. 5D, which shows that LHX2 virus-infected iRUNX1c-p2a-HOXA9-HPC can regenerate B cells in recipient mice as B lineage seed cells.
In conclusion, the invention prepares the artificial blood progenitor cells with the B lineage differentiation potential by transferring three factors of RUNX1, HOXA9 and LHX2 into human pluripotent stem cells; by optimizing the conditions of induction and differentiation, the induction efficiency and the activity of the induced artificial blood progenitor cells are improved; the artificial blood progenitor cells have the differentiation potential of B lineage, and CD19 can be detected after transplantation+B cells, thereby realizing the lineage reconstruction of B lymphocytes in vivo and providing a theoretical basis for related treatment; the B cells derived from the induced differentiation and formed by the final differentiation of the artificial blood progenitor cells have the capacity of secreting various cell products including cell factors and functional proteins, can be applied to the preparation of various medicaments, and have the value of practical clinical application.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
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Claims (10)
1. A method for producing an artificial blood progenitor cell having a differentiation potential of B lineage, comprising:
simultaneously or respectively introducing three transcription factors of RUNX1, HOXA9 and LHX2 into cells of a human pluripotent stem cell induced differentiation system, and performing induced expression and induced differentiation to obtain the artificial blood progenitor cells with the B lineage differentiation potential;
the cells of the human pluripotent stem cell induced differentiation system comprise any one or the combination of at least two of human pluripotent stem cells, mesodermal cells, hematopoietic endothelial cells or hematopoietic progenitor cells;
the method for introducing the transcription factor comprises any one or a combination of at least two of gene overexpression, endogenous gene activation or gene editing;
the method for inducing differentiation comprises the step of inducing differentiation by monolayer culture or organoid three-dimensional culture based on stromal cell co-culture.
2. The method for preparing artificial blood progenitor cells having B-lineage differentiation potential according to claim 1, wherein three transcription factors RUNX1, HOXA9 and LHX2 are induced to express simultaneously or separately, wherein the induced expression starts from mesodermal cells, hematopoietic endothelial cells or hematopoietic progenitor cells;
the human pluripotent stem cells include human induced pluripotent stem cells and/or human embryonic pluripotent stem cells.
3. The method for producing artificial blood progenitor cells having B-lineage differentiation potential according to claim 1 or 2, wherein the differentiation induction method is a monolayer culture-induced differentiation based on stromal cell co-culture, and comprises three stages: inducing human pluripotent stem cells to produce mesodermal cells, inducing hematopoietic endothelial cells to differentiate and inducing hematopoietic progenitor cells to differentiate and mature.
4. The method for producing artificial blood progenitor cells having B-lineage differentiation potential according to claim 3, wherein the inducing human pluripotent stem cells to produce mesodermal cells uses D0 medium and D1 medium;
d2 culture medium is used in the induction of the differentiation of the hematogenous endothelial cells;
d9 medium and/or D16 medium are used in the induction of differentiation and maturation of hematopoietic progenitor cells;
the D0 culture medium comprises human basic fibroblast growth factor, recombinant human activin, recombinant human bone morphogenetic protein, GSK-3 alpha/beta inhibitor CHIR99021, PI3K inhibitor LY294002 and TeSR-E6 culture medium;
the D1 culture medium comprises recombinant human bone morphogenetic protein, ALK5 inhibitor A83-01, Wnt pathway inhibitor IWR-1-endo and TeSR-E6 culture medium;
the D2 culture medium comprises vitamin C, recombinant human FMS-like tyrosine kinase 3 ligand, human basic fibroblast growth factor, human vascular endothelial growth factor, recombinant human stem cell factor, recombinant human interleukin 3, granulocyte-macrophage colony stimulating factor, sodium butyrate and StemPro-34 culture medium;
the D9 culture medium comprises a recombinant human thrombopoietin, a recombinant human interleukin 3, a recombinant human stem cell factor, a recombinant human interleukin 11, a human insulin-like growth factor 1, a human vascular endothelial growth factor, a human basic fibroblast growth factor, a recombinant human bone morphogenetic protein, a recombinant human FMS-like tyrosine kinase 3 ligand, a granulocyte-macrophage colony stimulating factor, a macrophage colony stimulating factor, doxycycline and StemPro-34 culture medium;
the D16 culture medium comprises a serum substitute B27, ROCK inhibitor Y-27632, recombinant human thrombopoietin, recombinant human stem cell factor, recombinant human interleukin 3, recombinant human FMS-like tyrosine kinase 3 ligand, granulocyte-macrophage colony stimulating factor, doxycycline and StemPro-34 culture medium.
5. An artificial blood progenitor cell having a B-lineage differentiation potential, which is prepared by the method for preparing an artificial blood progenitor cell having a B-lineage differentiation potential according to any one of claims 1 to 4;
the artificial blood progenitor cells with the B lineage differentiation potential are transplanted to realize human B cell regeneration;
the B cell type differentiated from the artificial blood progenitor cells with the B lineage differentiation potential comprises any one or a combination of at least two of B progenitor cells, non-mature B cells, mature B cells or plasma cells.
6. A cell product comprising the cells of the induced differentiation system of human pluripotent stem cells according to any one of claims 1 to 4 and genetically engineered cells thereof, the artificial blood progenitor cells with B-lineage differentiation potential according to claim 5 and genetically engineered cells thereof, the B-cells differentiated from the artificial blood progenitor cells with B-lineage differentiation potential according to claim 5 and genetically engineered cells thereof, or the secretory products of the B-cells differentiated from the artificial blood progenitor cells with B-lineage differentiation potential according to claim 5 and genetically engineered cells thereof;
the cells of the human pluripotent stem cell induced differentiation system comprise any one or the combination of at least two of human pluripotent stem cells, mesodermal cells, hematopoietic endothelial cells or hematopoietic progenitor cells;
the genetically engineered cells comprise any one or combination of at least two of cells which are constructed by genetic engineering technology and express target proteins, target cytokines, target antibodies or chimeric antigen receptors;
the secretion products of the B cell and the genetically engineered cell thereof comprise any one or a combination of at least two of antibodies, cytokines or proteins.
7. A method of in vivo B lymphocyte lineage regeneration, comprising:
transplanting the artificial blood progenitor cells having B-lineage differentiation potential according to claim 5 into a body;
the method for the in vivo regeneration of B lymphocyte lineage further comprises the step of detecting the human hematopoietic cells in vivo after transplantation.
8. Use of any one or a combination of at least two of the method for producing an artificial blood progenitor cell with B-lineage differentiation potential according to any one of claims 1 to 4, the artificial blood progenitor cell with B-lineage differentiation potential according to claim 5, or the cell product according to claim 6 for the preparation of a pharmaceutical composition.
9. A pharmaceutical composition comprising the artificial blood progenitor cells having B lineage differentiation potential according to claim 5 and/or the cell product according to claim 6;
the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials;
the pharmaceutically acceptable auxiliary materials comprise any one or the combination of at least two of a carrier, an excipient or a diluent.
10. Use of the pharmaceutical composition of claim 9 for the preparation of a prophylactic agent, a therapeutic agent or a vaccine for a disease;
the disease prevention drug comprises a drug for improving the immune response of the organism;
the immune response comprises a humoral immunity and/or a cellular immunity;
the disease treatment medicine comprises any one or the combination of at least two of B cell immunodeficiency treatment medicine, anti-pathogen infection medicine, anti-tumor medicine, autoimmune disease treatment medicine or hereditary disease treatment medicine;
the genetic disease comprises any one of hemophilia, lysosomal storage disease, hypophosphatasia, or phenylketonuria, or a combination of at least two thereof;
the drug includes an antibody drug.
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