CN108676777A - Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation - Google Patents

Abstract

The present invention proposes purposes of the tire sinusoidal endothelial cell in building candidate stem cell microenvironment.Tire sinusoidal endothelial cell can be that candidate stem cell builds microenvironment, the amplification in vitro of hematopoiesis support stem cell, and promotes pluripotent stem cell to break up to HSCs or directly reprogram body cell and obtain HSCs.

Description

Application of Fetal Liver Sinusoidal Endothelial Cells in Expansion and Differentiation of Hematopoietic Stem Cells

technical field

The present invention relates to the field of biology. Specifically, the present invention relates to the application of fetal liver sinusoidal endothelial cell line in the expansion of hematopoietic stem cells.

Background technique

Hematopoietic stem cells (HSC) have high self-renewal ability and multi-directional differentiation potential, and can produce all types of blood cells, such as red blood cells, white blood cells, platelets and lymphocytes. It can not only rebuild the whole hematopoietic system in the course of life, but also has the function of maintaining long-term hematopoiesis. In recent years, HSC transplantation has been increasingly used in the clinical treatment of hematological or non-hematological malignancies, showing broad application prospects. Among them, umbilical cord blood transplantation (umbilical cord blood transplantation, UCBT) is favored in particular, it has a wide range of sources, easy to collect, no harm to the donor, low requirements for HLA matching, recurrence rate after transplantation and graft-versus-host disease ( Graft-versus-host disease, GVHD) lower incidence rate and other advantages. However, the absolute number of HSCs in a single cord blood is small, which can easily lead to delayed recovery of neutrophils and increase the risk of bacterial and viral infections after transfusion, while double cord blood transplants will bring the incidence of graft-versus-host disease A series of problems such as increase and prolongation of platelet recovery time. On the whole, the in vitro expansion and culture of HSCs is the most direct and convenient solution, but so far, the construction of a suitable microenvironment for HSC in vitro expansion is still a bottleneck problem that needs to be solved urgently.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent.

It should be noted that the present invention is based on the inventor's following findings:

In 1978, Schofield first proposed the concept of stem cell niche (Niche), pointing out that a specific microenvironment (Microenvironment) would have an effect on stem cell function, and a large number of literatures subsequently confirmed the existence of stem cell niche in various tissues. People have tried many ways to simulate the hematopoietic microenvironment in vivo, in order to construct an in vitro culture system that maintains the self-renewal and expansion of HSCs, such as hematopoietic-related cytokines, small molecules, stromal cell co-culture, Notch ligands, etc., but the effect Still passable.

The hematopoietic microenvironment is composed of a variety of supporting cells adjacent to HSCs, including osteoblasts, endothelial cells, adipocytes, stromal cells, and immune cells. Transcription factors such as CD34, CD31, Runx1, GATA-2, etc., can affect the proliferation and self-renewal of HSCs by secreting a variety of factors, and become the core components of the hematopoietic microenvironment.

In embryonic development, the liver is an important hematopoietic organ, and it is also the most vigorous place for the self-renewal and expansion of HSCs. Among them, human fetal liver sinusoid endothelial cells (HFLSECs) are a kind of sinusoid with unique structure Endothelium, accounting for about 70% of the total number of liver non-parenchymal cells, has functions such as phagocytosis, antigen presentation, blood flow regulation, etc., and can secrete ET-1, NO, vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1 ), tumor necrosis factor (TNF) and other cytokines, also involved in extramedullary hematopoiesis and the selective implantation of HSCs in the hepatic lobules.

In view of this, the inventors used fetal liver sinusoidal endothelial cells as feeder cells to construct a microenvironment for hematopoietic stem cells and promote the expansion of hematopoietic stem cells. Further, the inventors found that since the survival of fetal liver sinusoidal endothelial cells depends on serum and endothelial cell growth factors, but the expansion environment of hematopoietic stem cells requires no serum and no endothelial cell growth factors, resulting in fetal liver sinusoidal endothelial cells and hematopoietic growth factors Apoptosis of fetal liver sinusoidal endothelial cells occurred after stem cells co-cultured for a short period of time.

E4orf1 is the gene product of adenovirus E4 coding region, which can regulate cell signaling pathways and affect cell cycle and apoptosis. The inventors found that introducing the E4orf1 gene into fetal liver sinusoidal endothelial cells can enable fetal liver sinusoidal endothelial cells to survive and maintain their own characteristics without the need for mitomycin C or radiation. Pretreatment can maintain the survival state without proliferation. Thus, constructing a microenvironment for hematopoietic stem cells is conducive to the expansion of hematopoietic stem cells, and promoting the differentiation of pluripotent stem cells into HSCs or directly reprogramming somatic cells to obtain HSCs.

Therefore, in one aspect of the present invention, the present invention proposes the use of fetal liver sinusoidal endothelial cells in constructing a microenvironment for hematopoietic stem cells. Fetal liver sinusoidal endothelial cells have functions such as phagocytosis, antigen presentation, and blood flow regulation, and can secrete ET-1, NO, vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), and tumor necrosis factor (TNF) It is also involved in extramedullary hematopoiesis and the selective implantation of HSCs in the hepatic lobules. Furthermore, the inventors found that fetal liver sinusoidal endothelial cells can construct a microenvironment for hematopoietic stem cells, support the in vitro expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the use may also have the following additional technical features:

According to an embodiment of the present invention, the fetal liver sinusoidal endothelial cells are provided in the form of recombinant cells, and the recombinant cells carry E4orf1 gene and optional GFP gene.

According to an embodiment of the present invention, the fetal liver sinusoidal endothelial cells are used as feeder cells.

According to an embodiment of the present invention, the fetal liver sinusoidal endothelial cells are used to maintain the expansion of hematopoietic stem cells.

According to an embodiment of the present invention, the expression rates of endothelial progenitor cell marker CD133, stemness marker CD117 and hematopoietic cell marker CD45 on the recombinant cells are all lower than 0.4%, and the expression rate of vascular endothelial growth factor receptor KDR is 55-80%. , the expression rates of endothelial cell markers CD144 and CD31 were greater than 99%.

In yet another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cells are fetal liver sinusoidal endothelial cells carrying the E4orf1 gene. The inventors found that since the survival of fetal liver sinusoidal endothelial cells depends on serum and endothelial cell growth factors, but the expansion environment of hematopoietic stem cells requires no serum and no endothelial cell growth factors, resulting in co-culture of fetal liver sinusoidal endothelial cells and hematopoietic stem cells Apoptosis of fetal liver sinusoidal endothelial cells will appear after a short period of time. Furthermore, the inventors have found through in-depth research that introducing the E4orf1 gene into fetal liver sinusoidal endothelial cells allows fetal liver sinusoidal endothelial cells to survive and maintain their own characteristics without the need for mitomycin Pretreatment with C or radiation can maintain a non-proliferative survival state, thereby constructing a microenvironment that promotes the expansion of hematopoietic stem cells in vitro, is conducive to the expansion of hematopoietic stem cells, and promotes the differentiation of pluripotent stem cells into HSCs or direct reprogramming of somatic cells Obtain HSCs.

According to an embodiment of the present invention, the fetal liver sinusoidal endothelial cells carry the GFP gene.

According to an embodiment of the present invention, the recombinant cells are used as feeder cells.

In yet another aspect of the present invention, the present invention provides a method for preparing the aforementioned recombinant cells. According to an embodiment of the present invention, the method includes: adding the first vector carrying the E4orf1 gene into the first culture medium containing the fetal liver sinusoidal endothelial cells, and culturing, so as to obtain the recombinant cells. Thus, the recombinant cells according to the embodiments of the present invention can construct a microenvironment for hematopoietic stem cells, facilitate the expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the method further includes: separately adding the first vector and the second vector carrying the GFP gene into the culture medium of the fetal liver sinusoidal endothelial cells.

According to an embodiment of the present invention, the first vector is a retrovirus packaged with a plasmid MSCV-N E4orf1, and the plasmid carries a puromycin resistance gene.

According to an embodiment of the present invention, the second vector is a retrovirus packaged with a plasmid pMX-GFP.

According to an embodiment of the present invention, the volume ratio of the first carrier and the second carrier is 1:1.

According to an embodiment of the present invention, the first culture medium contains 0.5 μg/ml of puromycin, and does not contain serum and endothelial cell growth factor.

According to an embodiment of the present invention, the first medium is EGM-2 basal medium.

In yet another aspect of the present invention, the present invention provides a method for expanding hematopoietic stem cells. According to an embodiment of the present invention, the method includes: obtaining recombinant cells according to the aforementioned method for preparing recombinant cells; Hematopoietic stem cell expansion.

According to an embodiment of the present invention, the hematopoietic stem cells are CD34 ⁺ cells.

According to an embodiment of the present invention, the second medium does not contain serum and endothelial cell growth factors, and the second medium is StemSpan medium containing 50ng/ml SCF, 50ng/ml Flt-3L and 50ng/ml TPO.

In yet another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes: fetal liver sinusoidal endothelial cells or the aforementioned recombinant cells. Therefore, using the kit according to the embodiment of the present invention can construct a hematopoietic stem cell microenvironment, facilitate the expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the kit is used to construct a microenvironment for hematopoietic stem cells.

According to an embodiment of the present invention, the kit is used for expanding the hematopoietic stem cells.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Figure 1 shows a schematic diagram of immunofluorescence identification of the expression of vWF in HFLSECs according to an embodiment of the present invention;

Figure 2 shows the light micrographs of HFLSECs under the culture condition containing 0.5 μg/ml puromycin according to an embodiment of the present invention, wherein, the left picture shows puromycin pressurized selection for 2 days, and the right picture shows puromycin pressurized selection for 3 days;

Fig. 3 has shown the light micrograph of the retrovirus packaged with plasmid pMX-GFP according to one embodiment of the present invention;

Fig. 4 shows the flow cytometric analysis schematic diagram and the light microscope image of E4orf1-GFP/HFLSECs under the condition of serum-free and endothelial cell growth factor culture according to one embodiment of the present invention;

Figure 5 shows a schematic diagram of flow cytometric analysis of surface marker proteins of E4orf1-GFP/HFLSECs according to an embodiment of the present invention;

Figure 6 shows a schematic diagram of in vitro expansion analysis of E4orf1-GFP/HFLSECs as a feeder layer supporting CD34 ⁺ cells derived from human umbilical cord blood according to an embodiment of the present invention;

Fig. 7 shows a schematic diagram of hematopoietic colony formation ability of CD34+ cells derived from human umbilical cord blood after in vitro expansion according to an embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

The invention proposes the use of fetal liver sinusoidal endothelial cells in constructing the microenvironment of hematopoietic stem cells, recombinant cells, a method for preparing recombinant cells, a method for expanding hematopoietic stem cells and a kit. Each of them will be described in detail below.

Application of fetal liver sinusoidal endothelial cells in constructing hematopoietic stem cell microenvironment

In one aspect of the present invention, the present invention proposes the use of fetal liver sinusoidal endothelial cells in constructing a microenvironment for hematopoietic stem cells. Fetal liver sinusoidal endothelial cells have functions such as phagocytosis, antigen presentation, and blood flow regulation, and can secrete ET-1, NO, vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), and tumor necrosis factor (TNF) It is also involved in extramedullary hematopoiesis and the selective implantation of HSCs in the hepatic lobules. Furthermore, the inventors found that fetal liver sinusoidal endothelial cells can construct a microenvironment for hematopoietic stem cells, support the in vitro expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the fetal liver sinusoidal endothelial cells are provided in the form of recombinant cells, and the recombinant cells carry the E4orf1 gene and optionally the GFP gene. According to a specific embodiment of the present invention, the recombinant cells are obtained by introducing E4orf1 gene and optional GFP gene into fetal liver sinusoidal endothelial cells.

The inventors found that since the survival of fetal liver sinusoidal endothelial cells depends on serum and endothelial cell growth factors, but the expansion environment of hematopoietic stem cells requires no serum and no endothelial cell growth factors, resulting in co-culture of fetal liver sinusoidal endothelial cells and hematopoietic stem cells Apoptosis of fetal liver sinusoidal endothelial cells will appear after a short period of time. To this end, the inventors introduced the E4orf1 gene into the fetal liver sinusoidal endothelial cells, so that the fetal liver sinusoidal endothelial cells can survive and maintain their own characteristics without mitomycin C or radiation. Pretreatment can maintain the survival state without proliferation. Therefore, in order to construct the hematopoietic stem cell microenvironment, support the in vitro expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

In addition, in order to observe the transformed cells and distinguish them from cells without the target gene, the inventors tried to introduce the tracer protein gene into the fetal liver sinusoidal endothelial cells. After a large number of experiments, it was found that the introduction of the GFP gene encoding green fluorescent protein as a tracer gene into fetal liver sinusoidal endothelial cells can not only play a tracer role, but also not affect the expression of E4orf1 gene. It is especially suitable for distinguishing from the initial cells when pluripotent stem cells are differentiated into HSCs or directly reprogrammed somatic cells to obtain HSCs.

According to an embodiment of the present invention, the expression rates of endothelial progenitor cell markers CD133, stemness marker CD117, and hematopoietic cell marker CD45 on recombinant cells are all lower than 0.4%, and the expression rate of vascular endothelial growth factor receptor KDR is 55-80%. The expression rates of cell markers CD144 and CD31 were both greater than 99%. The recombinant cells themselves are the primary isolated fetal liver sinusoidal endothelial cells. The inventors performed flow cytometry experiments to verify the surface markers of the cells, which proved that the recombinant cells of the present invention highly express the markers of mature endothelial cells, but hardly express endothelial progenitor cells and Markers of stemness, nor expression of markers of hematopoietic cells. Thus, it shows that the recombinant cells of the present invention have higher purity.

According to an embodiment of the present invention, fetal liver sinusoidal endothelial cells are used as feeder cells. The inventors found that using fetal liver sinusoidal endothelial cells as feeder cells to co-culture with hematopoietic stem cells can construct a microenvironment for hematopoietic stem cells, which is conducive to promoting their expansion and maintaining self-renewal.

According to an embodiment of the present invention, fetal liver sinusoidal endothelial cells are used to promote the expansion of hematopoietic stem cells. The inventors found that fetal liver sinusoidal endothelial cells can build a microenvironment for hematopoietic stem cells, which is conducive to promoting their expansion and maintaining self-renewal.

recombinant cells

In another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cells are fetal liver sinusoidal endothelial cells carrying the E4orf1 gene. The inventors found that since the survival of fetal liver sinusoidal endothelial cells depends on serum and endothelial cell growth factors, but the expansion environment of hematopoietic stem cells requires no serum and no endothelial cell growth factors, resulting in co-culture of fetal liver sinusoidal endothelial cells and hematopoietic stem cells Apoptosis of fetal liver sinusoidal endothelial cells will appear after a short period of time. Furthermore, the inventors have found through in-depth research that introducing the E4orf1 gene into fetal liver sinusoidal endothelial cells allows fetal liver sinusoidal endothelial cells to survive and maintain their own characteristics without the need for mitomycin The pretreatment of protein C or radiation can maintain the survival state without proliferation. Therefore, constructing a microenvironment that promotes the expansion of hematopoietic stem cells in vitro is conducive to the expansion of hematopoietic stem cells, and promotes the differentiation of pluripotent stem cells into HSCs or directly reprograms somatic cells to obtain HSCs.

According to an embodiment of the present invention, the fetal liver sinusoidal endothelial cells carry the GFP gene. In order to observe the transformed cells and distinguish them from the cells without the target gene, the inventors tried to introduce the tracer protein gene into the fetal liver sinusoidal endothelial cells. After a large number of experiments, it was found that the introduction of the GFP gene encoding green fluorescent protein as a tracer gene into fetal liver sinusoidal endothelial cells can not only play a tracer role, but also not affect the expression of E4orf1 gene. It is especially suitable for distinguishing from initial cells when promoting the differentiation of pluripotent stem cells into HSCs or directly reprogramming somatic cells to obtain HSCs.

According to an embodiment of the present invention, recombinant cells are used as feeder cells. The inventors found that recombinant cells were used as feeder cells for co-culture with hematopoietic stem cells. Recombinant cells can survive without serum and endothelial cell growth factor and maintain their own characteristics, and can maintain a non-proliferative state without pretreatment with mitomycin C or radiation. Thus, constructing a microenvironment for hematopoietic stem cells is conducive to the expansion of hematopoietic stem cells, and promoting the differentiation of pluripotent stem cells into HSCs or directly reprogramming somatic cells to obtain HSCs.

Method for preparing recombinant cells

In yet another aspect of the present invention, the present invention provides a method for preparing the aforementioned recombinant cells. According to an embodiment of the present invention, the method includes: adding the first vector carrying the E4orf1 gene into the first culture medium containing fetal liver sinusoidal endothelial cells, and culturing, so as to obtain recombinant cells. The inventors found that since the survival of fetal liver sinusoidal endothelial cells depends on serum and endothelial cell growth factors, but the expansion environment of hematopoietic stem cells requires no serum and no endothelial cell growth factors, resulting in co-culture of fetal liver sinusoidal endothelial cells and hematopoietic stem cells Apoptosis of fetal liver sinusoidal endothelial cells will appear after a short period of time. To this end, the inventors introduced the E4orf1 gene into the fetal liver sinusoidal endothelial cells, so that the fetal liver sinusoidal endothelial cells can survive and maintain their own characteristics without mitomycin C or radiation. Pretreatment can maintain the survival state without proliferation. Thus, construct the hematopoietic stem cell microenvironment, support the expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the method further includes: separately adding the first vector and the second vector carrying the GFP gene into the culture medium of fetal liver sinusoidal endothelial cells. In order to observe the transformed cells and distinguish them from the cells without the target gene, the inventors tried to introduce the tracer protein gene into the fetal liver sinusoidal endothelial cells. After a large number of experiments, it was found that the introduction of the GFP gene encoding green fluorescent protein as a tracer gene into fetal liver sinusoidal endothelial cells can not only play a tracer role, but also not affect the expression of E4orf1 gene. It is especially suitable for distinguishing from initial cells when promoting the differentiation of pluripotent stem cells into HSCs or directly reprogramming somatic cells to obtain HSCs.

According to an embodiment of the present invention, the first vector is a retrovirus packaged with a plasmid MSCV-N E4orf1, and the plasmid carries a puromycin resistance gene. The inventors used retrovirus to transfect host cells (fetal liver sinusoidal endothelial cells), so that the target gene (E4orf1 gene) entered the host cells and integrated into the cell genome, and the inserted sequence was expressed in the host cells to produce the target protein. Wherein, since the host cell does not contain a puromycin-resistant gene, it cannot tolerate a certain concentration of puromycin. Furthermore, when the first vector carrying the puromycin-resistant gene is introduced into the host cell, it can cause the host cell to develop resistance and grow in a medium containing a certain concentration of puromycin, thereby determining whether the first vector is successfully introduced . Thus, the fetal liver sinusoidal endothelial cells can survive and maintain their own characteristics without serum and endothelial cell growth factors, and can maintain a non-proliferative survival state without pretreatment with mitomycin C or radiation. Thus, construct the hematopoietic stem cell microenvironment, support the expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the second vector is a retrovirus packaged with plasmid pMX-GFP. The inventors transfect host cells with a retrovirus, so that the target gene (GFP gene) enters the host cell and integrates into the cell genome, and the inserted sequence is expressed in the host cell to produce the target protein. Thus, it is convenient to observe the transformed cells and distinguish them from the cells not introduced with the target gene without affecting the expression of the E4orf1 gene.

According to an embodiment of the present invention, the volume ratio of the first carrier and the second carrier is 1:1. The inventor obtained the above-mentioned optimal ratio through a large number of experiments, and the efficiency of co-transfection is the best under this condition.

According to an embodiment of the present invention, the first culture medium contains 0.5 μg/ml of puromycin, and does not contain serum and endothelial cell growth factor. The inventors found that normal fetal liver sinusoidal endothelial cells do not contain a puromycin-resistant gene, so they cannot grow in a medium containing 0.5 μg/ml puromycin, and gradually undergo apoptosis from the second day of culture. Furthermore, introducing the first vector and the second vector carrying the anti-puromycin into the fetal liver sinusoidal endothelial cells can make the fetal liver sinusoidal endothelial cells grow normally. Therefore, 0.5 μg/ml of puromycin was added to the first medium to select transformed cells.

According to an embodiment of the present invention, the first medium is EGM-2 basal medium. Thus, the fetal liver sinusoidal endothelial cells survive and maintain their own characteristics, thereby constructing a microenvironment for hematopoietic stem cells, facilitating the expansion of hematopoietic stem cells, and promoting the differentiation of pluripotent stem cells into HSCs or directly reprogramming somatic cells to obtain HSCs.

Those skilled in the art can understand that the features and advantages described above for recombinant cells are also applicable to the method for preparing recombinant cells, and will not be repeated here.

Methods for the expansion of hematopoietic stem cells

In yet another aspect of the present invention, the present invention provides a method for expanding hematopoietic stem cells. According to an embodiment of the present invention, the method includes: obtaining recombinant cells according to the aforementioned method for preparing recombinant cells; and inoculating hematopoietic stem cells in a second medium containing recombinant cells and culturing them so as to expand the hematopoietic stem cells. The recombinant cells of the present invention carry the E4orf1 gene, so that the fetal liver sinusoidal endothelial cells can survive and maintain their own characteristics in the absence of serum and endothelial cell growth factors, and can be cured without pretreatment with mitomycin C or radiation. maintain a non-proliferative state of survival. In this way, the hematopoietic stem cell microenvironment is constructed, which is conducive to the expansion of hematopoietic stem cells, and promotes the differentiation of pluripotent stem cells into HSCs or directly reprograms somatic cells to obtain HSCs.

According to an embodiment of the present invention, the hematopoietic stem cells are CD34 ⁺ cells. CD34 is an important marker of hematopoietic stem cells. Furthermore, the inventors purified and isolated CD34 ⁺ hematopoietic stem cells from mononuclear cells of umbilical cord blood.

According to an embodiment of the present invention, the second culture medium does not contain serum and endothelial cell growth factor, the second culture medium is StemSpan medium, contains 50ng/ml SCF (Stem cell factor), 50ng/ml Flt-3L (Fms- liketyrosine kinase 3ligand) and 50ng/ml TPO (Thrombopoietin). In this way, the expansion and self-renewal of the hematopoietic stem cells can be realized, and at the same time, the survival state of the fetal liver sinusoidal endothelial cells without proliferation can be maintained.

Those skilled in the art can understand that the features and advantages described above for the method for preparing recombinant cells are also applicable to the method for expanding hematopoietic stem cells, and will not be repeated here.

Reagent test kit

In yet another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes: fetal liver sinusoidal endothelial cells or the aforementioned recombinant cells. Therefore, using the kit according to the embodiment of the present invention can construct a hematopoietic stem cell microenvironment, facilitate the expansion of hematopoietic stem cells, and promote the differentiation of pluripotent stem cells into HSCs or directly reprogram somatic cells to obtain HSCs.

According to an embodiment of the present invention, the kit is used to construct a microenvironment for hematopoietic stem cells.

According to an embodiment of the present invention, the kit is used for expanding hematopoietic stem cells and maintaining their self-renewal.

Those skilled in the art can understand that the features and advantages described above for the hematopoietic stem cell expansion method are also applicable to the kit, and will not be repeated here.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1

1. Isolation of human umbilical cord blood (Umbilical cord blood, UCB) CD34 ⁺ cells

Transfer the umbilical cord blood into a sterile T75 cell culture bottle, add erythrocyte sedimentation solution according to one-third of the total volume, mix well, let it settle naturally for 20-40 minutes, collect the upper plasma layer with a dropper into a 50ml centrifuge tube, and store at room temperature After centrifuging at 2000rpm for 5min, wash once with 10ml PBS, and resuspend the cells with 5ml PBS; obtain mononuclear cells (Mononuclear cells, MNCs) by density gradient centrifugation, add 5ml peripheral blood lymphocyte separation solution to a 15ml centrifuge tube, and use a dropper Slowly add 5ml of cell suspension at 1cm above the liquid surface of the separation liquid along the wall of the centrifuge tube to above the liquid level of the separation liquid, place the centrifuge tube in a horizontal centrifuge, and centrifuge at room temperature at 2000rpm at the lowest speed for 20min. The cell suspension is divided into four layers. Use a dropper to absorb the gray-white cloudy cell layer and place it in a 50ml centrifuge tube. Add PBS to wash once, resuspend the cells into a 1.5ml EP tube, and add CD34 sorting magnetic beads according to the instructions of the CD34Microbeads kit. After rotating and incubating at 4°C for 40 min in the dark, washed once with PBS, and sorted CD34 ⁺ cells in a magnetic field.

2. Preparation of E4orf1-GFP/HFLSECs feeder layer

(1) Screening the optimal drug concentration of HFLSECs stably transfected with MSCV-N E4orf1

vWF, also known as factor VIII-related antigen, is a macromolecular protein polymer synthesized and secreted by vascular endothelial cells. It participates in blood coagulation and thrombus formation in vivo, so it is often used as a characteristic factor to identify endothelial cells cultured in vitro. Immunofluorescence results (Figure 1) showed that the primary cultured HFLSECs expressed positive vWF.

Digest HFLSECs with 0.25% trypsin, inoculate 1×10 ⁵ /well into a 24-well plate for culture, and replace with 0.5μg/ml, 1μg/ml, 2μg/ml, 4μg/ml Puromycin after 16-24h EGM-2 basal medium, the last well of HFLSECs was still cultured with EGM-2 basal medium without Puromycin, as a control group, and the cell survival was observed under a microscope. The results showed that under the culture conditions containing 0.5 μg/ml puromycin, the primary cultured HFLSECs that originally adhered to the wall began to gradually become round from the 2nd day, and gradually floated into the medium from the 3rd day (Figure 2), Within 5 days, almost no adherent growth cells were observed, and under the culture conditions of 1 μg/ml, 2 μg/ml, and 4 μg/ml puromycin, the primary cultured HFLSECs all floated from the second day, and the drug effect was too strong, so , choose 0.5μg/ml puromycin as the optimum drug screening concentration.

(2) Retrovirus packaging, cell transfection, drug screening and flow cytometric sorting of HFLSECs after transfection

Retroviral packaging plasmids MSCV-N E4orf1 and pMX-GFP were made respectively, and the two venoms were collected. Among them, the expression of GFP in the retroviral cells can be seen after 20 hours of packaging, and the expression rate exceeds 90% ( FIG. 3 ). The two venoms were simultaneously transfected into HFLSECs at a ratio of 1:1, and screened with 0.5 μg/ml Puromycin for a week under pressure, and then analyzed by flow cytometry, the positive rate of GFP was 90.5%, and GFP ⁺ was obtained by flow sorting Cells (Figure 4A), the sorted E4orf1-GFP/HFLSECs can be stably expanded and passaged in the medium containing 0.5 μg/ml Puromycin (Figure 4B), and then transferred to serum-free EGM-2 for culture, Still able to maintain survival and not proliferate ( FIG. 4C ), thus obtaining E4orf1-GFP/HFLSECs feeder cells.

Flow cytometric detection of E4orf1-GFP/HFLSECs cells. The results are shown in Figure 5. The positive rates of the endothelial cell surface markers CD144 and CD31 in E4orf1-GFP/HFLSECs were 99% and 99.5%, respectively, while the stem cell surface marker CD117, endothelial progenitor cell surface marker CD133 and blood cell surface marker CD45 were almost positive. No expression, the expression rate of vascular endothelial growth factor receptor KDR was 69%.

3. E4orf1-GFP/HFLSECs as a feeder layer for culturing CD34 ⁺ cells derived from human umbilical cord blood

CD34 ⁺ cells were isolated from UCB, seeded into E4orf1-GFP/HFLSECs at 5×10 ⁴ /well, suspension culture without feeder layer was used as control group, and StemSpan medium (containing 50ng/ml SCF, 50ng/ml Flt-3L, 50ng/ml TPO) were continuously cultured for 15 days, when the total number of cells exceeded 1×10 ⁶ , the cells needed to be subcultured to a new feeder layer for further culture. Figure 6 shows that in the in vitro expansion system of E4orf1-GFP/HFLSECs as a feeder layer, the total number of nucleated cells of CD34 ⁺ cells derived from human cord blood was expanded by 360 times at 15 days, while the suspension culture group with simple factors as a control only The amplification was 165 times, and the amplification efficiency of the experimental group was 2.2 times that of the control group.

In the in vitro expansion system using E4orf1-GFP/HFLSECs as the feeder layer, human cord blood-derived CD34 ⁺ cells still have the ability to differentiate into different CFUs in vitro after expansion (Figure 7), including erythroid blasts Colony forming unit (BFU-E), colony forming unit erythroid (CFU-E), colony forming unit granulocyte (CFU-G), colony forming unit megakaryote (CFU-M), colony formation of granulocyte and macrophage Unit (CFU-GM) and erythroid, granulocyte, megakaryotic, macrophage mixed colony forming unit (CFU-EGMM).

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (10)

1. purposes of the tire sinusoidal endothelial cell in building candidate stem cell microenvironment.

2. purposes according to claim 1, which is characterized in that the tire sinusoidal endothelial cell is in the form of recombinant cell It provides, the recombinant cell carries E4orf1 genes and optional GFP genes；

Optionally, the tire sinusoidal endothelial cell is used as feeder cells；

Optionally, the tire sinusoidal endothelial cell is for maintaining hematopoietic stem cell expansion；

Optionally, endothelial progenitor cells mark CD133, dryness mark CD117 and hematopoietic cell mark on the recombinant cell The expression rate of CD45 is below 0.4%, and vascular endothelial growth factor receptor KDR expression rates are 55~80%, the mark of endothelial cell Will CD144 and CD31 expression rate is all higher than 99%.

3. a kind of recombinant cell, which is characterized in that the recombinant cell is the tire sinusoidal endothelial cell for carrying E4orf1 genes.

4. recombinant cell according to claim 3, which is characterized in that the tire sinusoidal endothelial cell carries GFP genes；

Optionally, the recombinant cell is used as feeder cells.

5. a kind of method preparing the recombinant cell of claim 3 or 4, which is characterized in that including：

The first vector for carrying E4orf1 genes is added in the first culture medium containing the tire sinusoidal endothelial cell, is carried out Culture, to obtain the recombinant cell.

6. according to the method described in claim 5, it is characterized in that, further comprising：

The first vector is added to the training of the tire sinusoidal endothelial cell jointly with the Second support for carrying GFP genes respectively It supports in base,

Optionally, the first vector is to be packaged with the retrovirus of plasmid MSCV-N E4orf1, and taken on the plasmid With anti-puromycin gene,

Optionally, the Second support is the retrovirus for being packaged with plasmid pMX-GFP；

Optionally, the first vector and the volume ratio of Second support are 1:1；

Optionally, the puromycin containing 0.5 μ g/ml in first culture medium, without containing serum and endothelial cell growth because Son；

Optionally, first culture medium is EGM-2 basal mediums.

7. a kind of method of hematopoietic stem cell expansion, which is characterized in that including：

Recombinant cell is obtained according to the method for the Prepare restructuring cell of embodiment 5 or 6；And

Candidate stem cell is inoculated in the second culture medium containing the recombinant cell, is cultivated, to make the hematopoiesis Expansion of stem cells.

8. the method according to the description of claim 7 is characterized in that the candidate stem cell is CD34⁺Cell；

Optionally, serum and endothelial growth factor are not contained in second culture medium, second culture medium is StemSpan culture mediums contain 50ng/ml SCF, 50ng/ml Flt-3L and 50ng/ml TPO.

9. a kind of kit, which is characterized in that including：Tire sinusoidal endothelial cell or the recombinant cell of claim 3 or 4.

10. kit according to claim 9, which is characterized in that the kit is used to build the micro-loop of candidate stem cell In border；

Optionally, the kit is for expanding the candidate stem cell.