CN110106144B - Method and kit for inducing hematopoietic stem cells to differentiate into pre-T-lineage cells - Google Patents

Abstract

The invention discloses a method and a kit for inducing hematopoietic stem cells to differentiate into anterior T-lineage cells, and belongs to the technical field of biomedicine. The invention co-cultures fibroblast after over-expressing DLL4 protein and hematopoietic stem cells to induce the differentiation and development of hematopoietic stem cells to anterior T-line cells; or co-culturing cells over-expressing DLL4 protein and hematopoietic stem cells to induce differentiation and development of hematopoietic stem cells in the fibrotic diseases to the front T-line cells. The T cells obtained by induction of the invention are in two important stages of thymus development DN2 or DN 3. Induced allogeneic DN2 or DN3 stage T cells have the thymus engraftment and develop into mature T cells with complete functions, and the cell number is effectively and massively expanded in the allogeneic thymus and becomes T cells with tolerance to a receptor through the development in the allogeneic thymus, thereby becoming the basis of the universal type Car-T cell source.

Description

Method and kit for inducing hematopoietic stem cells to differentiate into pre-T-lineage cells

Technical Field

The invention relates to a method and a kit for inducing hematopoietic stem cells to differentiate into anterior T-lineage cells, belonging to the technical field of biomedicine.

Background

The reconstitution of the immune system, particularly the T cell population, is one of the major clinical problems in the treatment of AIDS, tumors, and aging. Currently, acquired immunodeficiency syndrome (AIDS) caused by HIV-1 infection is a significant public health threat, and AIDS is often accompanied by immune function reduction or suppression, thereby causing a series of clinical symptoms. After complete inhibition of HIV viral propagation and clearance of virus, CD4⁺The gradual recovery and reconstruction of the number and the function of T cells are always the ultimate target of AIDS treatment. Current antiretroviral therapy (ART) improves the quality of life and longevity of patients, reduces the rate of opportunistic infections and early mortality associated with the termination of viral-induced immune degeneration and to some extent the reconstitution of immune function. However, in the short term after the treatment, the immune reconstruction of the body is not quick, and the CD4 is peripheral blood⁺The increase in T lymphocyte count is limited. In addition, clinically, in about 16% of patients, good reconstitution of immunity is not achieved even when the virus is suppressed after long-term treatment with ART, resulting in poor reconstitution of immunity. On the other hand, patients infected with HIV often have concomitant infection with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) due to the decreased immune function, and hepatotoxicity caused by the drugs during ART treatment, which often results in severe cirrhosis and loss of liver function. Therefore, there is also a great need to find effective treatment strategies to meet these AIDS-complicated liver conditionsTreatment of patients with inflammatory virus infections.

It is well known that the thymus is the primary site of T lymphocyte development and differentiation. In essence, it can be viewed as a three-dimensional network of highly specialized thymic epithelial cells, which are filled with T cells at different stages of development. Under the support of various signals provided by epithelial cells, hematopoietic progenitor cells migrating from bone marrow to thymus develop a given developmental program, undergo complex developmental processes such as proliferation, directed differentiation, TCR rearrangement, beta selection, positive selection and negative selection, and finally form a T cell bank which is highly diverse and can effectively distinguish self from non-self. The development of T cells in the thymus is a highly ordered, multi-staged, extremely complex process, with the different stages of development being dependent on the diverse signals provided by the microenvironment of the thymus. FIG. 1 is a schematic diagram showing the process of thymus development, wherein lymphoid precursor (CLP) cells migrate from bone marrow into the thymus and are differentiated into mature T cells following a certain developmental program. Thymic T cell development is divided into three major stages, Double Negative (DN), Double Positive (DP) and Single Positive (SP), depending on whether CD4 and CD8 are expressed. Based on the expression of CD117, CD44 and CD25, DN cells were divided into four groups DN 1-DN 4, which represent different developmental stages.

Different in vitro research systems have been used to mimic the differentiated development of lymphocytes, with the OP9 system being the most mature. OP9 cells were stromal cells from bone marrow of neonatal osteopetrotic (OP9/OP9) mice. This cell line has a gene defect of cytokine M-CSF (cytokine binding-stimulating factor). M-CSF has a role of supporting the differentiation of myeloid (myoloid) cells, i.e., differentiation and development toward cmp (common myoloid prognosticator) cells, and thus OP9 cells lacking M-CSF expression can be differentiated and developed toward clp (common lymphoid prognosticator), i.e., lymphoprecursor cells, by restricting the development of hematopoietic stem cells myeloid cells, thereby achieving the purpose of supporting the development of B-cells and other lymphoid cells, but cannot support the in vitro differentiation of T-cells. In 2002, Schmitt and

-Ptransfecting OP9 cells by using a retrovirus expression vector of a Notch ligand molecule DLL1(Delta-like ligand 1) by using the flechage expression vector of the flechage, and establishing OP9-DL1 cells for stably expressing DLL1 molecules; overexpression of the DLL1 molecule on the surface of OP9 stromal cells provides Notch signals required for directed differentiation of T cells, so that the stromal cells lose the ability to support B cell differentiation, and the T lymphocytes can be efficiently induced to generate in vitro. Subsequent studies also showed that OP9-DL4 (overexpressing DLL4) was also able to exert the same effect. OP9-DL1 cell can make the co-cultured hematopoietic stem cell produce a large amount of T precursor cell (about 1000-4000 times amplification), and differentiate into fully functional mature T lymphocyte (including alpha beta TCR) according to normal development program⁺And gamma delta TCR⁺T cells). OP9-DL1 is easy to culture and expand as an adherently growing stromal cell, and is more convenient and effective for in vitro induction of T cells. In addition, flow sorting of CD45 in the co-culture system was used⁺CD44⁺CD25⁺DN2(Double Negative stage 2) cells, when injected into Rag2 knockout mice by tail vein, CD4 can be detected in peripheral blood of Rag2 knockout mice⁺T and CD8⁺The existence of T shows that DN2 cells obtained by co-culture have the capacity of engrafting thymus and further developing into mature T cells, and simultaneously shows that the in vitro culture system has the potential for in vivo T cell reconstruction. The establishment of the OP9-DL1 induced model not only breaks up one view in the long-term control immunology field, namely: the process of developing T cells from lymphoid progenitors requires an intact thymus, and T cells cannot be differentiated from hematopoietic stem cells with the support of a monolayer of stromal cells; and enables the large-scale induction of T cells in vitro in a non-thymus environment.

Although OP9-DL1 stromal cell induced T cell culture systems have been increasingly adopted and used by laboratories. However, we also see that there are some theoretical and practical problems with this in vitro induction system. Firstly, OP9 cells do not express MHC molecules and CD1d molecules, and positive selection and negative selection of T cells or NKT cells in the development process cannot be realized; in addition, OP9 is a bone marrow stromal cell of murine origin, and therefore, the exact nature of the human T lymphocytes induced by OP9 needs to be further studied, for example: the expression pattern of T cell surface TCR molecule, the in vitro and in vivo functions of T cells, etc. It is therefore also necessary to find cell types which produce the same effect as OP 9.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for inducing differentiation of mouse or human hematopoietic stem cells into thymic T-lineage cells in vitro, wherein primary hepatic fibroblasts can restrict differentiation of hematopoietic stem cells into T/B lymphocyte lineage by a certain mechanism, and can promote differentiation and development of T cells after overexpression of DLL4, and can promote differentiation and development of T cells by co-culturing with hematopoietic stem cells after overexpression of DLL4 using liver-derived primary fibroblasts. Human primary liver fibroblasts also have the same promoting effect after DLL4 is overexpressed, and can promote human umbilical cord-derived hematopoietic stem cells to differentiate into DN2-DN3 cells.

The first purpose of the invention is to provide the application of DLL4 in preparing a medicine for inducing differentiation of hematopoietic stem cells into anterior T cell lines.

Further, the application is specifically that after the DLL4 protein is over-expressed by the fibroblasts, the fibroblasts and the hematopoietic stem cells are co-cultured to obtain the medicine for inducing the hematopoietic stem cells to differentiate into the pre-T-lineage cells.

Further, the application is specifically to co-culture of cells over-expressing DLL4 protein and hematopoietic stem cells to obtain the medicine for inducing differentiation of hematopoietic stem cells to anterior T-lineage cells in fibrotic diseases.

Further, the fibroblasts are primary hepatic fibroblasts.

Further, the cell over expressing the DLL4 protein is one or more of mesenchymal stem cells over expressing the DLL4 protein, thymic epithelial stromal cells over expressing the DLL4 protein and fibroblasts over expressing the DLL4 protein.

Furthermore, the hematopoietic stem cells are flow-sorted human hematopoietic stem cells after the erythrocyte of the umbilical cord blood cells is cracked and then is subjected to negative selection by using a lineage cell depletion kit.

Further, the pre-T line cells are DN2 cells and/or DN3 cells.

Further, the medicine is an injection.

Furthermore, the medicine also comprises an agent for inducing differentiation.

Further, an agent for co-culturing fibroblasts and hematopoietic stem cells is included.

Further, the co-culture comprises the following steps when in vitro culture:

(1) preparing MEM alpha culture medium containing 15% FBS, adding SCF with final concentration of 25-50ng/mL, Flt3L with final concentration of 5-10ng/mL, and IL-7 with final concentration of 1-20ng/mL, subpackaging, and storing at-20 deg.C;

(2) coating the culture dish by using mouse or human DLL4 recombinant protein 24h in advance, wherein the coating concentration is 10 mu g/ml, and the temperature is 24h at 4 ℃ or 4h at 37 ℃; (optional step)

(3) Cell lines over-expressed by DLL4 were recovered and cultured at 1X 10⁵The cell amount is laid on a culture dish, and the culture medium is added to 10 ml;

(4) reviving the cell line at 5X 10⁵Laying a culture dish for cell amount, adding a culture medium to 10ml, and culturing for 24 hours; (implementation of step 2 please select this step)

(5) Will be 1 × 10⁵Adding the hematopoietic stem cells obtained by sorting into culture medium of a culture dish, mixing uniformly, placing at 37 ℃ with 5% CO₂Co-culturing in an incubator;

(6) half liquid change is carried out at intervals of 72 h;

(7) harvesting cells after co-culturing for a certain number of days and blowing the cells away; passing through a 70 μm pore size sieve, and then obtaining DN2 or DN3 cells by adopting CD25+ magnetic bead sorting or CD44+ CD25+ flow antibody labeling post-flow sorting.

The invention has the beneficial effects that:

the method successfully induces the mouse hematopoietic stem cells or the human hematopoietic stem cells to differentiate into thymus T-lineage cells in vitro, and the induced DN2 or DN3 is two important stages of T-cell thymus development. DN2 to DN3 are a relatively continuous process, and the total number of cells increases about 1000 times from DN1 to DN2 through 10 divisions, and the cell differentiation has been completely limited to T-lineage at DN2 stage, while γ σ and α β T cell directed differentiation has also been completed; and DN2 and DN3 stage cells are positioned before beta selection, positive selection and negative selection of T cell development, allogeneic DN2 or DN3 cells induced in vitro have engraftment and develop into mature T cells with complete functions, the cell number is greatly expanded in the allogeneic thymus, and the T cells without tolerance to a receptor are formed by negative selection and positive selection of the allogeneic thymus, thus becoming the basis of universal Car-T.

Drawings

FIG. 1 is a schematic representation of a T cell thymus development procedure;

FIG. 2 shows the expression level of liver tissue DLL4mRNA in mice with different degrees of liver fibrosis;

FIG. 3 is a frozen serial section of different clinical specimens stained with CD31 and DLL4 antibodies, respectively;

FIG. 4 is a frozen serial section of different clinical specimens stained with the α SMA and DLL4 antibodies, respectively;

FIG. 5 is a frozen serial section of different clinical specimens stained with CK18 and DLL4 antibodies, respectively;

FIG. 6 is the mRNA expression levels of DLL4 in different clinical specimens;

FIG. 7 is MSCs mRNA expression levels overexpressing DLL 4;

FIG. 8 shows the results of experiments on the co-culture of OP9-DLL4 with hematopoietic stem cells;

FIG. 9 shows the results of experiments on the co-culture of MSC-DLL4 and 3T3-DLL4 with hematopoietic stem cells;

FIG. 10 shows that MSC-DLL4 promotes differentiation and development of hematopoietic stem cells into CD11b⁺Macrophages;

FIG. 11 is a result of in vitro co-culture of TSC-DLL4 cells in which DLL4 is overexpressed by TSC with HSC for 6 days and 10 days;

FIG. 12 shows the flow analysis results of MSCs as negative controls, primary hepatic fibroblasts, and P5-generation hepatic fibroblasts, which were stained with alpha SMA-FITC after membrane rupture, respectively;

FIG. 13 shows CD45 of P5 generation, primary liver fibroblasts, OP9 cells co-cultured with HSC for 9 days⁺Gate analysis CD19⁺B cell ratio;

FIG. 14 shows DN stage cell ratio by CD45+ gating analysis after 6 days of co-culture with HSC after DLL4 is overexpressed in primary liver fibroblasts of P5 generation;

FIG. 15 shows the results of co-culture with hematopoietic stem cells of primary hepatic fibroblasts mixed with the hepatic parenchymal cell line AML12 overexpressing DLL 4;

FIG. 16 is a qPCR comparison of expression profiles for different cells, different passage numbers;

fig. 17 shows the effect of blockade of CXCL12-CXCR4 signaling pathway to inhibit the promotion of T cell development by DLL4 overexpressing cells;

fig. 18 shows the effect of addition of recombinant CXCL12 protein on the recovery of T cell development by DLL4 overexpressed P5 passaged hepatic fibroblasts.

Detailed Description

The invention will be further described with reference to specific examples, which are intended for illustrative purposes only and are not intended to be limiting. Those skilled in the art can appreciate the features and utilities of the present invention from the description as set forth herein, and that the present invention may be implemented or utilized in various other embodiments. Experiments in which specific conditions are not specified in the examples are generally performed under conventional conditions such as those described in the manufacturer's instructions, experimental guidelines, or the contents of textbooks.

Sorting hematopoietic stem cells: after complete red splitting of mouse bone marrow cells or human umbilical cord blood Cell erythrocyte lysate, negative selection is carried out by a Linear Cell Depletion Kit (Miltenyi Biotec), and then flow-type sorting is adopted for mouse hematopoietic stem cells (Lin)^-Sca1⁺cKit⁺) Or human hematopoietic stem cells (Lin)^-CD34⁺CD38⁺). The linkage cocktail used consisted of labeled B220, CD3e, CD4, CD8 α, CD19, CD11B, Gr1, Ter119, CD11c, and NK1.1 antibodies.

Co-culture operation steps:

(1) preparing MEM alpha culture medium containing 15% FBS, adding SCF with final concentration of 25-50ng/mL, Flt3L with final concentration of 5-10ng/mL, and IL-7 with final concentration of 1-20ng/mL, subpackaging, and storing at-20 deg.C;

(2) coating the culture dish by using mouse or human DLL4 recombinant protein 24h in advance, wherein the coating concentration is 10 mu g/ml, and the temperature is 24h at 4 ℃ or 4h at 37 ℃; (optional step)

(3) ResuscitationCell line over-expressed with DLL4 at 1X 10⁵The cell amount is laid on a culture dish, and the culture medium is added to 10 ml;

(4) reviving the cell line at 5X 10⁵Laying a culture dish for cell amount, adding a culture medium to 10ml, and culturing for 24 hours; (implementation of step 2 please select this step)

(5) Will be 1 × 10⁵Adding the hematopoietic stem cells obtained by sorting into culture medium of a culture dish, mixing uniformly, placing at 37 ℃ with 5% CO₂Co-culturing in an incubator;

(6) half liquid change is carried out at intervals of 72 h;

(7) harvesting cells after co-culturing for a certain number of days and blowing the cells away; passing through a 70 μm pore size sieve, and then obtaining DN2 or DN3 cells by adopting CD25+ magnetic bead sorting or CD44+ CD25+ flow antibody labeling post-flow sorting.

Example 1: the mouse induced liver fibrosis can up-regulate the expression level of liver tissue DLL4

By using CCl₄The liver fibrosis model of C57BL/6 mouse induced for 8 weeks is used as the receptor experimental group, and normal liver mouse is used as the control group. The donor mouse is CD45.1C57BL/6 mouse, and the left and right upper leg humerus and lower leg tibia are collected, and bone marrow cells are obtained by perfusion washing, counted after red splitting by erythrocyte lysate, and counted at 1 × 10⁶Injecting the bone marrow mononuclear cells into the spleen of a receptor mouse by adopting an intrasplenic injection method with the volume of 200 mu l, and introducing the bone marrow mononuclear cells into the liver through the intrasplenic blood vessels via the hepatic portal vein of the receptor mouse; recipient mice were irradiated with a lethal dose of 9.5Gy prior to transplantation.

Considering the important role of notch signal pathways DLL1 and DLL4 in T cell development, we used qPCR for CCl₄The mRNA expression levels of DLL1 and DLL4 in liver tissues of liver fibrosis mice and normal mice are detected and compared with beta-actin to calculate relative values, and the result shows that only DLL4 is expressed in the liver of the mice. And the mRNA expression level of the DLL4 molecule in liver tissue of the liver fibrosis C57BL/6 mouse is found to be obviously higher than that of normal liver tissue, and gradually increases along with the degree of liver fibrosis. The results are shown in FIG. 2.

Example 2: the parenchymal liver cells are the main cells which are up-regulated to DLL4 expression under liver fibrosis

(1) Considering that the notch signal pathway plays an important role in T cell development, DLL1 and DLL4 protein immunohistochemical staining is carried out on liver tissue specimens of AIDS patients with liver cirrhosis, and the results show that: only DLL4 was expressed in the liver. Meanwhile, the parenchymal hepatic cell is the main cell for expressing DLL4, and the liver fibroblast cannot express DLL 4. We then performed DLL4 immunohistochemical staining as well as CD31 (vascular endothelial cell marker), CK18 (liver parenchymal cell marker), α SMA (fibroblast marker) immunohistochemical staining of clinical specimens using frozen serial sections and performed co-localization analysis (fig. 3-5). Indicating that parenchymal hepatocytes are the primary cells expressing DLL4, while neither vascular endothelium nor fibrotic tissue express DLL 4; and with the aggravation of the degree of hepatic fibrosis of the patient, the expression of the liver parenchymal cell DLL4 protein is increased.

FIG. 3 shows that CD31 and DLL4 antibodies are stained after freezing serial sections, and the same position is photographed for co-localization, A1 is a specimen of high degree of hepatic fibrosis of AIDS-complicated liver cirrhosis patients, A2 is a specimen of moderate degree of hepatic fibrosis of AIDS-complicated liver cirrhosis patients, and A3 is a normal liver specimen. It can be seen that CD31 is used as a vascular endothelial cell marker, and DLL4 expression is negative in the positive expression region.

FIG. 4 shows that after freezing serial sections, alpha SMA and DLL4 antibodies were stained respectively and photographed at the same position for co-localization, A1 is a specimen of high degree liver fibrosis in AIDS complicated with liver cirrhosis, A2 is a specimen of moderate degree liver fibrosis in AIDS complicated with liver cirrhosis, and A3 is a normal liver specimen. Alpha SMA is shown as a liver fibroblast marker, and DLL4 expression is negative in the positive expression region. Indicating that hepatic fibroblasts are not cells expressing DLL 4.

FIG. 5 shows that CK18 and DLL4 antibodies are stained respectively after freezing serial sections, and the same position is photographed for co-localization, A1 is a specimen of high degree of hepatic fibrosis of AIDS-complicated liver cirrhosis patients, A2 is a specimen of moderate degree of hepatic fibrosis of AIDS-complicated liver cirrhosis patients, and A3 is a normal liver specimen. CK18 is shown as a hepatocyte marker, and DLL4 expression is positive in the positive expression region. It was shown that the parenchymal hepatocytes are the major cells with high expression of DLL4, not hepatic fibroblasts. Meanwhile, the expression degree of the polypeptide increases along with the increase of the hepatic fibrosis degree of the patient.

(2) The mRNA expression level of DLL4 was measured by qPCR for AIDS-complicated liver cirrhosis patient specimens and normal human liver tissues and compared with beta-actin to calculate relative values, and the results are shown in FIG. 6. A1 is a high-degree liver fibrosis specimen of AIDS-complicated liver cirrhosis patients, A2 is a medium-degree liver fibrosis specimen of AIDS-complicated liver cirrhosis patients, and A3 is a normal liver specimen. The expression level of mRNA of DLL4 molecule in liver tissue of AIDS-complicated liver cirrhosis patients is found to be significantly higher than that of normal liver tissue, and gradually increases with the degree of liver fibrosis.

Example 3: construction of DLL4 lentiviral vector and overexpression cell thereof

Construction of MSCs cells overexpressing DLL4 protein: the expression plasmid pLVX-IRES-Puro-DLL4 is efficiently constructed by adopting the Infusion technology. Primer Forward (5-ATCCCGCGACTCTAGAT GACGCCTGCGTCCCG-3’)Reverse(5’-GGTAGAATTATCTAGTTATACCTCT GTGGCAATCACAC-3', the underlined part indicates 15bp bases complementary to the plasmid insertion position) for amplification of the mature C57BL/6 murine DLL4cDNA gene.

Total RNA of the liver of C57BL/6 mouse was extracted by Trizol method and reverse transcription was performed to obtain cDNA library, AccuPrime^TMPfx SuperMix (Invitrogen) PCR-amplified the cDNA library to obtain the desired fragment, and the PCR product was recovered using 1% agarose gel. pLVX-IRES-Puro plasmid (Invitrogen corporation) was digested with BamHI and XbaI, respectively, overnight, and the PCR product and the plasmid digested product were used

HD Cloning Plus reagent for ligation. Then DH5 alpha competent bacteria were transformed, plated on ampicillin resistant LB plates, and positive clones were selected for sequencing. The upgraded particles were obtained as overexpression plasmid pLVX-IRES-Puro-DLL 4.

Mixing the over-expression plasmid pLVX-IRES-Puro-DLL4 and the packaging plasmid according to a certain proportion, adding lipo2000 to infect 293T cells, collecting supernatant after 24h and 48h, filtering, ultrafiltering, centrifuging and concentrating to obtain virus, infecting MSCs cells with the concentrated virus, and adding puromycin after infecting for 48h for screening. After the cells are screened for two weeks and stabilized, adherent cells are collected to extract RNA, after the RNA is reversely transcribed into cDNA, the qPCR analysis result shows that the over-expressed cell DLL4mRNA transcription level is up to 28 percent relative to the average value of beta-actin, which is obviously higher than transfected empty plasmid MSCs cells and control MSCs cells, and the figure is 7. On the basis, DLL4 overexpression cell strains of OP9, 3T3/NIH, TSC, AML12, primary hepatic fibroblasts, subcultured hepatic fibroblasts and the like are respectively constructed.

Example 4: specific DLL4 overexpression cell has effect of promoting T cell development

Construction of OP9 cell line OP9-DLL4 overexpressing DLL4, and flow sorted hematopoietic Stem cells (Lin)^-Sca1⁺cKit⁺) The cells were co-cultured for 6 days, 10 days, 14 days, and 18 days. As shown in FIG. 8, panels A-D show the development of DN stage by flow analysis after 6, 10, 14 and 18 days of co-culture, respectively, and the results show that DN2(CD 44) appears very clearly after co-culture when analyzed under CD45 gate setting⁺CD25⁺) And DN3(CD 44)^-CD25⁺) The ratio of the two groups is gradually increased along with the increase of the co-culture time. Panel E is an analysis of CD4 and CD8 expression after 18 days of co-culture, indicating the presence of some DP cells.

MSC-DLL4 or 3T3-DLL4 was co-cultured with bone marrow derived hematopoietic stem cells and was found not to exhibit the same T cell development promoting effect as OP9-DLL 4. Then we tried to mix OP9 cells with MSC-DLL4 cells or 3T3-DLL4 cells at a ratio of 1:1 and then repeated the experiments of co-culturing with hematopoietic stem cells, and the results are shown in FIG. 9, and the results of the experiments of co-culturing MSCs cells, MSC-DLL4 cells, MSC-DLL4 cells and HSCs at a ratio of OP 91: 1, respectively, show that MSC-DLL4 has no effect of promoting T cell development, but has the effect of promoting T cell development when mixed with OP9 cells. FIGS. E-G show the results of experiments in which 3T3/NIH cells, 3T3-DLL4 cells, 3T3-DLL4 and OP 91: 1 mixed cells were co-cultured with HSC, respectively, and the results show that 3T3-DLL4 has no effect of promoting T cell development but has an effect of promoting T cell development when mixed with OP9 cells. It was suggested that OP9 cells could provide factors or conditions that would allow MSC-DLL4 or 3T3-DLL4, which did not originally have the effect of promoting T-lineage cell development, to exert this function.

Further analysis showed that MSC-DLL4 cells could not limit hematopoietic stem cell development to T/B cell line, and its high expression of M-SCF promoted its development to CD11B⁺Macrophages (fig. 10). OP9 cells are shown to provide certain factors or conditions to block the action of M-SCF, thereby limiting the differentiation and development of hematopoietic stem cells to T/B lineage.

Thymic epithelial cell lines (TSCs) are given by professor of laughing of health institute of academy of China in Shanghai, and Thymic epithelial-like tissues can be formed in nude mice injected with tail vein and CD4 can be detected in the nude mice⁺And CD8⁺SP T Cells (Established third epitope Progene/Stem Cell-Like Cell Lines differential interaction in the Material third epitope Cells and Support T Cell development. PLOS one,2013.8(9): p.e75222). However, we co-cultured with hematopoietic stem cells in vitro using the same system as OP9-DLL4, but did not show the same effect of promoting T cell differentiation and development; qPCR shows that TSCs are in a DLL4 low expression state, we perform virus transfection on the TSCs, construct TSC-DLL4 cells with DLL4 over-expression, and repeat the co-culture experiment with hematopoietic stem cells again, the results are shown in FIG. 11, A-B are the results of the in vitro co-culture of the TSC-DLL4 cells with TSC over-expression DLL4 and HSC for 6 days and 10 days respectively, and the results show that the TSCs over-expression DLL4 can have the same effect as OP9-DLL 4. TSC has the same effect as OP9 cells in limiting differentiation of hematopoietic stem cells into T/B lineage cells.

Example 5: separating to obtain mouse liver fibroblast

For CCl₄Liver of hepatic fibrosis mouse or normal mouse is treated by Liver Dissociation Kit to separate into single cells, most of parenchymal cells are removed by filtering with a screen, and CD140 alpha is selected by immunomagnetic beads⁺The cells were then labeled with flow-through antibodies CD140 α -APC, CD11b-FITC, CD146-PE, F4/80-PerCP-Cy5.5, and flowedSorting to obtain CD140 alpha by cell analyzer⁺CD11b^-CD146^-F4/80^-A cell; after the DMEM containing 10% FBS is resuspended, slow adherent non-fibroblasts are removed by adopting a differential attachment method, and adherent primary cells with positive alpha SMA expression are obtained after the DMEM is changed; meanwhile, hepatic fibroblasts with high alpha SMA positive expression purity are obtained through continuous passage, as shown in figure 12, and figures A-C are respectively a flow analysis result after MSCs are used as negative controls, primary hepatic fibroblasts and P5-generation hepatic fibroblasts, and alpha SMA-FITC staining is respectively adopted after membrane rupture. It can be seen that the primary hepatic fibroblasts and the passage hepatic fibroblasts have high-proportion alpha SMA positive cells, and the P5 passage hepatic fibroblasts have higher purity.

Example 6: the primary liver fibroblast has the effect of promoting the development of a T line similar to the OP9

Primary hepatic fibroblasts were co-cultured with hematopoietic stem cells, similar to OP9 cells in CD45⁺Under-door analysis can present a population of CDs 19⁺While the passage liver fibroblasts do not. The results are shown in FIG. 13, in which P5 liver fibroblasts were cultured with HSC 9 days later and then treated with CD45⁺Gate analysis CD19⁺B cell ratio; panel B shows CD45 after 9 days of co-culture of primary liver fibroblasts with HSC⁺Gate analysis CD19⁺B cell ratio; panel C shows CD19 being analyzed by CD45+ gating after 9 days of co-culture of OP9 cells and HSC⁺B cell line ratio. As a result, only primary liver fibroblasts and OP9 cells were able to show CD19 in the co-culture system⁺Cells, indicating that primary hepatic fibroblasts, similar to OP9 cells, were able to limit hematopoietic stem cells to differentiation to the T/B cell line, i.e., CLP cells, rather than to CMP cells, and further develop to CD19 in the absence of DLL4⁺B is a cell line. Whereas passaged liver fibroblasts do not have this function.

And (3) carrying out lentivirus transfection on the primary hepatic fibroblasts and the P5 hepatic fibroblasts for generation to obtain primary hepatic fibroblasts over expressing DLL4 and P5 hepatic fibroblasts for generation, and co-culturing the primary hepatic fibroblasts and the P5 hepatic fibroblasts with the separated hematopoietic stem cells. As shown in FIG. 14, Panel A shows the liver fibrosis of P5 generationCell overexpression of DLL4 followed by 6 days of coculture with HSC and CD45⁺Setting a gate to analyze the cell proportion in the DN stage; panel B shows primary liver fibroblasts overexpressing DLL4, co-cultured with HSC for 6 days, and then treated with CD45⁺Setting a gate to analyze the cell proportion in the DN stage; as a result, only primary hepatic fibroblasts over-expressing DLL4 and OP9-DLL4 cells were able to present DN2 cells in the co-culture system, further indicating that primary hepatic fibroblasts, similar to OP9 cells, were able to limit the differentiation of hematopoietic stem cells into T/B cell lines, while further promoting their development into T line cells in the presence of DLL 4.

AML12 is a hepatic parenchymal cell line, and was subjected to lentiviral transfection to obtain AML12-DLL4 cells overexpressing DLL4, which were co-cultured with isolated hematopoietic stem cell cells. Referring to FIG. 15, FIG. A shows primary liver fibroblasts and AML 121: 1, a hepatocyte cell line overexpressing DLL4, mixed at a ratio, co-cultured with HSC for 6 days and then expressed as CD45⁺Setting a gate to analyze the cell proportion in the DN stage; panel B shows that OP9 cells overexpress DLL4 and were co-cultured with HSCs for 6 days before CD45+ gating analysis of DN stage cell proportion as a positive control; panel C shows DN stage cell ratio by CD45+ gating analysis after co-culturing with HSC for 6 days after mouse hepatocyte cell line AML12 overexpresses DLL 4. As a result, AML12-DLL4 did not have the effect of promoting T-lineage cell development. But when mixed with primary hepatic fibroblasts, the same DN2 and DN3 cells as the OP9-DLL4 co-culture system could be present in their co-culture system. It was further shown that primary hepatic fibroblasts, similar to OP9 cells, were able to limit the differentiation of hematopoietic stem cells into T/B cell lines, while further promoting their development into T line cells in the presence of DLL 4. The liver parenchymal cells do not have this effect.

By adopting DN2 and DN3 cells in a flow sorting co-culture system and injecting into a Rag2 knockout mouse by tail vein, CD4 can be detected in peripheral blood of the Rag2 knockout mouse⁺And CD8⁺The existence of the rat 2 mouse is unlikely to generate T cells due to the defect of the rat gene, which shows that DN2 and DN3 cells obtained by the culture system can colonize thymus and further develop into mature CD4+ and CD8+ T, has very wide application prospect and can become a universal Car-T cellAnd (5) planting a foundation.

Example 7: the CXCL12-CXCR4 signal pathway in the hepatic fibrosis microenvironment is possibly involved in the function of DLL4 in promoting the development of T line

Further using qPCR, the expression profiles of different proteins of OP9 cells, TSC cells, primary hepatic fibroblasts, which are capable of promoting T cell development, were compared with the differences between cell lines that did not have such promoting effects, such as NIH/3T3, AML12, MSCs, primary hepatic parenchyma, passaged hepatic fibroblasts, and the like, and the results are shown in fig. 16, in which panel a is the expression profile of different cells, and panel B is the expression level of CXCL12 in hepatic fibroblasts with different passage numbers. Focusing on inflammation-related factors and chemokines, CXCL12 was found to be expressed to a high degree in OP9, TSC and primary hepatic fibroblasts with a promoting effect, whereas in cell lines without this promoting effect: the expression level of NIH/3T3 cells, AML12 cells, MSCs cells, primary liver parenchyma cells and passage liver fibroblasts is very low. And CXCL12 was expressed in primary liver fibroblasts (P0) at a level that decreased proportionally with the increase in the number of passages (P1-P5 passages). CXCL12 was shown to be involved in this process of T cell development in vitro and to play an important role.

To further demonstrate, the use of CXCL12-CXCR4 signaling pathway chemical blocker AMD3100 (whose mechanism of action is to compete preferentially for binding to CXCR4, thus blocking the binding site of CXCL 12.) was added to co-culture systems, see fig. 17, panels a-D: CXCL12-CXCR4 signaling pathway inhibitor AMD3100 varying concentrations from high to low (10 μ M-0 μ M), in co-culture systems of primary hepatic fibroblasts over-expressing DLL4 with bone marrow hematopoietic stem cells. It was seen that the addition of AMD3100 significantly inhibited the differentiation of DN2 and DN3 cells, and DN2 and DN3 gradually decreased with increasing AMD3100 concentration. The results show that CXCL12-CXCR4 signal channel is blocked, the effect of primary hepatic fibroblasts over expressing DLL4 or OP9 cells on promoting T cell development can be effectively inhibited, and further, CXCL12 plays an important role in the process that DLL4 promotes hematopoietic stem cells to develop into T cells.

In another experiment, recombinant CXCL12 protein was added at different concentrations to a co-culture system of DLL4 overexpressed P5 passage hepatic fibroblasts and hematopoietic stem cells, as shown in FIG. 18, and in panels A-D CXCL12 recombinant protein was added at different concentrations from high to low (100ng/ml-0ng/ml) to a co-culture system of DLL4 overexpressed P5 passage hepatic fibroblasts and bone marrow hematopoietic stem cells. It can be seen that the addition of the CXCL12 recombinant protein can promote the appearance of DN2 and DN3 cells which are not possessed originally, and is more remarkable along with the increase of the concentration, but still does not achieve the effect of primary liver fibroblast coculture. The results show that the addition of recombinant protein can only recover the T cell development promoting effect in a limited way, and that DLL4 and CXCL12 play an important role in the process, but do not play a role together, so that the process is much more complicated than imagination, and a plurality of unknown factors and proteins are involved in the liver microenvironment.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (6)

1. An application of DLL4 in preparing medicine for inducing hematopoietic stem cell to differentiate into anterior T cell line is characterized in that human primary hepatic fibroblast is co-cultured with human hematopoietic stem cell after over-expressing DLL4 protein to obtain medicine for inducing human hematopoietic stem cell to differentiate into anterior T cell line,

enhancing the CXCL12-CXCR4 signaling pathway to promote the development of human hematopoietic stem cells into T lineage cells;

when the co-culture is carried out in vitro culture, the method specifically comprises the following steps:

(1) preparing MEM alpha culture medium containing 15% FBS, adding SCF with final concentration of 25-50ng/mL, Flt3L with final concentration of 5-10ng/mL, and IL-7 with final concentration of 1-20ng/mL, subpackaging, and storing at-20 deg.C;

(2) the culture dish is coated with mouse or human DLL4 recombinant protein 24h in advance, and the coating concentration is 10 mug/ml, 24h at 4 ℃ or 4h at 37 ℃, wherein the step (2) is optional;

(3) cell lines over-expressed by DLL4 were recovered and cultured at 1X 10⁵The cell amount is laid on a culture dish, and the culture medium is added to 10 ml;

(4) reviving the cell line at 5X 10⁵Paving a culture dish for the cell amount, adding culture medium to 10ml, and culturing for 24h, wherein the step (4) is required to be carried out when the step (2) is carried out;

(5) will be 1 × 10⁵Adding the hematopoietic stem cells obtained by sorting into culture medium of a culture dish, mixing uniformly, placing at 37 ℃ with 5% CO₂Co-culturing in an incubator;

(6) half liquid change is carried out at intervals of 72 h;

(7) harvesting cells after co-culturing for a certain number of days and blowing the cells away; passing through a 70 μm pore size sieve, and then obtaining DN2 or DN3 cells by adopting CD25+ magnetic bead sorting or CD44+ CD25+ flow antibody labeling post-flow sorting.

2. The use according to claim 1, wherein said hematopoietic stem cells are flow sorted hematopoietic stem cells after lysing umbilical cord blood cells and negative selection with a lineage cell depletion kit.

3. The use of claim 1, wherein said pre-T line cells are DN2 cells and/or DN3 cells.

4. The use of claim 1, wherein the medicament is an injection.

5. The use of claim 1, wherein said medicament further comprises an agent that induces differentiation.

6. A kit for inducing hematopoietic stem cells to differentiate into anterior T-lineage cells is characterized by comprising a reagent for co-culturing human primary hepatic fibroblasts and human hematopoietic stem cells,

the kit further comprises an agent that enhances the CXCL12-CXCR4 signaling pathway to promote the development of human hematopoietic stem cells into T lineage cells;

wherein, when the co-culture is cultured in vitro, the method specifically comprises the following steps:

(1) preparing MEM alpha culture medium containing 15% FBS, adding SCF with final concentration of 25-50ng/mL, Flt3L with final concentration of 5-10ng/mL, and IL-7 with final concentration of 1-20ng/mL, subpackaging, and storing at-20 deg.C;

(2) the culture dish is coated with mouse or human DLL4 recombinant protein 24h in advance, and the coating concentration is 10 mug/ml, 24h at 4 ℃ or 4h at 37 ℃, wherein the step (2) is optional;

(3) cell lines over-expressed by DLL4 were recovered and cultured at 1X 10⁵The cell amount is laid on a culture dish, and the culture medium is added to 10 ml;

(4) reviving the cell line at 5X 10⁵Paving a culture dish for the cell amount, adding culture medium to 10ml, and culturing for 24h, wherein the step (4) is required to be carried out when the step (2) is carried out;

(5) will be 1 × 10⁵Adding the hematopoietic stem cells obtained by sorting into culture medium of a culture dish, mixing uniformly, placing at 37 ℃ with 5% CO₂Co-culturing in an incubator;

(6) half liquid change is carried out at intervals of 72 h;

(7) harvesting cells after co-culturing for a certain number of days and blowing the cells away; passing through a 70 μm pore size sieve, and then obtaining DN2 or DN3 cells by adopting CD25+ magnetic bead sorting or CD44+ CD25+ flow antibody labeling post-flow sorting.

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