WO2022126473A1 - 一种通过物理途径促进干细胞分化的材料及方法 - Google Patents

一种通过物理途径促进干细胞分化的材料及方法 Download PDF

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WO2022126473A1
WO2022126473A1 PCT/CN2020/137150 CN2020137150W WO2022126473A1 WO 2022126473 A1 WO2022126473 A1 WO 2022126473A1 CN 2020137150 W CN2020137150 W CN 2020137150W WO 2022126473 A1 WO2022126473 A1 WO 2022126473A1
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cells
stem cells
microspheres
nanospheres
hematopoietic
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French (fr)
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王鹏元
林姣
陈波
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/16—Apparatus for enzymology or microbiology containing, or adapted to contain, solid media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention belongs to the field of cell biology, and in particular relates to a material and method for promoting stem cell differentiation through a physical approach.
  • Hematopoietic stem cell transplantation for the treatment of hematological diseases is the earliest stem cell technology used in clinic, and it is also the only stem cell therapy technology that has been recognized worldwide.
  • the source of hematopoietic stem cells is limited, and it is difficult to expand in vitro, and it is difficult to meet the requirements of sufficient quantity and high quality in clinical treatment.
  • Human pluripotent stem cells hPSCs
  • hESCs and hiPSCs are a promising source of blood-like cells.
  • the hematopoietic potential of hPSCs has important applications in the treatment of blood-related diseases such as thalassemia [1] or hemophilia [2].
  • AGM-S3 aortic-gonad-mesonephric-derived stromal cells
  • AGM-S3 aortic-gonad-mesonephric-derived stromal cells
  • cSAPs Particle Crystal Film
  • cSAPs Particle Crystal Film
  • Crystalline particles can be pre- or post-modified to finally provide cSAPs with complex surfaces and chemical compositions [10], which result in different surface morphologies, roughness, hydrophilicity, chemical properties, and even stiffness.
  • the behavior of human stem cells and adult cells has been previously studied on cSAPs [11,12], and a method for reprogramming human fibroblasts into human induced pluripotent stem cells (hiPSC) has been successfully established on cSAPs [13], demonstrating that cSAPs Shows the potential to regulate cell adhesion as well as cell fate.
  • hESCs to differentiate into hematopoietic cells in vitro
  • one is embryoid body differentiation induction method
  • the other is co-culture method of ESCs and hematopoietic stromal cells
  • the other company cultured ESCs outside cells Matrix-free cell culture method on matrix proteins.
  • the mouse aortic gonadal mesonephric mechanism (AGM) can well induce ESCs into hematopoietic stem/progenitor cells, and the ratio of induced hematopoietic stem/progenitor cells is higher.
  • AGM mouse aortic gonadal mesonephric mechanism
  • the existing in vitro induction methods are inefficient and costly.
  • the existing methods to improve the efficiency of in vitro differentiation and reduce the cost of differentiation mainly include: optimizing the formulation of the differentiation medium; using a semi-permeable membrane to preserve the medium to find the molecule that needs to be supplemented; using cheaper mimics to replace cytokines; adding specific small molecules Increase the expansion efficiency of pre-erythrocytic difficult cells; establish cell lines of immortalized red progenitor cells; use genetic manipulation to up-regulate the differentiation and expansion efficiency of red blood cells at the gene level.
  • these methods achieve the purpose of improving the differentiation efficiency and reducing the differentiation cost to a certain extent, the method described in this patent is superior to the existing methods in terms of operation technology, cost and differentiation efficiency, and can be obtained in a simple, efficient and low-cost manner to a greater extent. More hematopoietic stem/progenitor cells and erythroid cells.
  • the present invention provides a granular crystal membrane and a cell culture container comprising the granular crystal membrane, and it is verified that the granular crystal membrane can promote the proliferation of stem cells and induce them to differentiate into hematopoietic stem cells and hematopoietic stem cells. Progenitor cells and erythroid cells.
  • One aspect of the present invention provides a granular crystal membrane for promoting the differentiation of stem cells into hematopoietic-related cells, the granular crystal membrane is made of microspheres and nanospheres by self-assembly on the surface of a substrate;
  • microspheres are selected from oxidized inorganic microspheres or polymer organic microspheres;
  • the nanospheres are selected from nanospheres whose surfaces are chemically modified.
  • the particle size of the microspheres is 1 ⁇ m-6 ⁇ m,;
  • the particle size of the nanospheres is selected from 30nm-500nm; and the particle size ratio of the microspheres and the nanospheres is 2:0.03-0.5.
  • the particle size of the nanospheres is 50nm-200nm, preferably 40nm-120nm.
  • the particle size of the microspheres is 1 ⁇ m-3 ⁇ m; preferably 1.5 ⁇ m-2.5 ⁇ m.
  • the oxidized inorganic microspheres are selected from silica microspheres
  • the polymer organic microspheres are selected from polystyrene microspheres, polystyrene microspheres, and polymethyl methacrylate microspheres.
  • the particle size ratio of the microspheres and the nanospheres is 2:0.05-0.1.
  • the chemical modification means that the surface of the nanosphere is modified with chemical groups, and more preferably, the chemical groups are selected from carboxyl groups.
  • the quantity ratio of the microspheres to the nanospheres is 1:10000-1:60000.
  • the quantity ratio of the microspheres and nanospheres is 1:10000-1:15000, or 1:50000-1:60000.
  • the particle size ratio of microspheres and nanospheres in the particle crystal film is 2:0.08-0.12, more preferably 2:0.1.
  • the contact angle of the granular crystal film is less than 30°, preferably less than 25°.
  • the roughness of the granular crystal film is 190-230 nm.
  • Another aspect of the present invention provides a cell culture vessel, the vessel having the surface of the above-mentioned particle crystal film of the present invention.
  • the type of the container is selected from cell culture dishes, cell culture plates, and cell slides.
  • the above-mentioned granular crystal film of the present invention is provided on the surface of the container in contact with the cells.
  • Another aspect of the present invention provides a method for inducing stem cells to differentiate into functional cells, the method comprising the steps of:
  • the functional cells are selected from at least one of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and erythroid cells.
  • the induction method of step 2) includes physical induction, chemical induction or biological induction method, for example, co-culture with aorta-gonad-mesonephric cells to induce stem cells to hematopoietic cells, hematopoietic stem cells, hematopoietic cells Progenitor and erythroid differentiation.
  • Yet another aspect of the present invention provides a method for preparing a surface having a stem cell differentiation promoting surface, the method comprising the step of forming the above-mentioned particle crystal film on a substrate.
  • the above method comprises the following steps:
  • Yet another aspect of the present invention provides the use of the above-mentioned particle crystal membrane as a surface for culturing stem cells for differentiation, or as a surface for culturing stem cells.
  • Another aspect of the present invention provides the use of the above-mentioned granular crystal membrane, which is the use of improving or maintaining the stemness state of stem cells.
  • the use is to promote the differentiation of stem cells into hematopoietic stem cells, hematopoietic progenitor cells and erythroid cells by improving the stemness state of stem cells.
  • Still another aspect of the present invention provides the use of the above-mentioned granular crystal membrane, which is to improve the differentiation of stem cells into hematopoietic stem cells, hematopoietic progenitor cells or erythroid cells.
  • Still another aspect of the present invention provides the use of the above-mentioned granular crystal membrane for increasing the number or ratio of stem cells differentiated into cells expressing C34 and CD43, cells expressing C34 and not expressing CD43 , cells that do not express C34 and express CD43, cells that express C34 and express CD45, cells that express C34 and do not express CD45, cells that do not express C34 and express CD45, or cells that express GPA and express CD71.
  • the stem cells are selected from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
  • Still another aspect of the present invention provides an induced and catalytically treated stem cell, the stem cell being cultured on the granular crystal membrane of the present invention before being induced.
  • having been cultured on the granular crystal film of the present invention means that the stem cells have been in contact with the granular crystal film of the present invention during the culture process,
  • Another aspect of the present invention provides the use of the above induced and catalytically treated stem cells of the present invention in preparing a preparation for treating hematopoietic disorders.
  • the human embryonic stem cells are isolated or obtained from human embryos that have not undergone in vivo development within 14 days of fertilization.
  • the invention uses the granular crystal membrane cSAPs material prepared by self-assembly technology to cultivate human embryonic stem cells (hESCs), and pre-stimulates the hESCs through the surface chemical and physical properties of the cSAPs material, so that the hESCs harvested from the cSAPs are differentiated under the condition of hematopoietic cell differentiation induction To increase the efficiency of hematopoietic stem/progenitor cells and erythroid cells, both in quantity and cell quality. hESCs were cultured on cSAPs for more than 3 passages, then removed, and co-cultured with AGM cells to induce hematopoietic cells.
  • hESCs were cultured on cSAPs for more than 3 passages, then removed, and co-cultured with AGM cells to induce hematopoietic cells.
  • cSAPs themselves have an impact on the stemness maintenance of hESCs, making them in a state of higher stemness and inducible differentiation, so as to differentiate into hematopoietic stem/progenitor cells and erythroid cells under induced conditions. to raise efficiency.
  • the present invention can improve the differentiation efficiency of embryonic stem cells into hematopoietic stem cells, hematopoietic progenitor cells and erythroid blood cells;
  • the method used in the present invention to improve the differentiation efficiency of embryonic stem cells into blood cells does not require the addition of exogenous factors, thereby improving the safety of the obtained hematopoietic cells;
  • the present invention uses granular crystal membranes (cSAPs) to pre-stimulate embryonic stem cells, and cSAPs use physical morphology and chemical properties to affect the stemness maintenance of hESCs, so that they are in a state of higher stemness and higher inducible differentiation, thereby Improve the efficiency of embryonic stem cell differentiation into hematopoietic cells.
  • cSAPs granular crystal membranes
  • the preparation method of the product of the present invention is simple, the preparation raw materials are economical, and it does not require harsh storage conditions, which is more conducive to commercialization.
  • Figure 1 SEM images of the surface topography of different combinations of cSAPs, the scale bar of SEM is 5 ⁇ m.
  • TCPS is a polystyrene cell culture plate
  • #1 represents the 5 ⁇ m silica microspheres and 400nm polystyrene (PS) nanospheres in Example 1
  • 2# represents the 5 ⁇ m silica microspheres and 200nm in Example 1.
  • Polystyrene nanospheres 3# represents the 2 ⁇ m silica microspheres and 65nm polystyrene nanospheres in Example 1
  • 4# represents the 2 ⁇ m silica microspheres and 50nm carboxylated polystyrene (PSC) in Example 1.
  • PSC carboxylated polystyrene
  • the particle crystal membrane material cSAP#4 prepared by nanospheres, 5# represents the particle crystal membrane material cSAP#5 prepared from 2 ⁇ m silica microspheres and 100 nm carboxylated polystyrene (PSC) nanospheres in Example 1.
  • PSC carboxylated polystyrene
  • Figure 2 Cell morphology of H1 hESCs cultured on TCPS plates, plates covered with cSAP#1, cSAP#2, cSAP#3, cSAP#4 and cSAP#5.
  • Figure 3 Immunostaining results of stemness factor OCT4/SOX2/SSEA4 on H1 hESCs after 3 passages on TCPS plates, plates covered with cSAP#4 and cSAP#5.
  • FIG. 4 Co-culture of hESCs with AGM cells in Example 2 induces differentiation into hematopoietic cells, and the expression of cell surface markers was analyzed by flow cytometry on days 8 and 14.
  • Figure 5 The flow chart of the experimental verification of Example 2 of the present invention.
  • Example 6 In Example 2 of the present invention, co-culture of hESCs and AGM cells induces differentiation into hematopoietic cells, and the results of flow cytometry analysis on the 8th and 14th days.
  • Fig. 7 The results of colony formation experiment analysis after culturing for 14 days in Example 2.
  • Figure 8 Sequencing results and analysis in Example 2.
  • Figure 10 Roughness of cSAP#1-5 samples.
  • the invention provides a granular crystal membrane for promoting the differentiation of stem cells into hematopoietic-related cells, the granular crystal membrane is made of microspheres and nanospheres by self-assembly on the surface of a substrate; the microspheres are selected from silica microspheres , polystyrene microspheres, polystyrene microspheres, polymethyl methacrylate microspheres; the nanospheres are selected from nanospheres whose surfaces are chemically modified; the particle size of the microspheres is 1 ⁇ m-6 ⁇ m; The particle size of the nanospheres is selected from 30nm-200nm; and the particle size ratio of the microspheres and the nanospheres is 2:0.03-0.5.
  • the particle size of the nanospheres is selected from 40nm-120nm.
  • the particle size of the microspheres is 1.5 ⁇ m-2.5 ⁇ m.
  • the particle size ratio of the microspheres to the nanospheres is 2:0.05-0.1.
  • the contact angle of the granular crystal film is less than 30°, preferably less than 25°.
  • the chemical modification refers to the modification of chemical groups on the surface of the nanosphere.
  • the chemical groups are selected from carboxyl groups.
  • the chemically modified nanospheres are selected from at least one of carboxylated polystyrene nanospheres, carboxylated polystyrene nanospheres, and carboxylated polymethyl methacrylate nanospheres .
  • the quantity ratio of the microspheres to the nanospheres is 1:10000-1:60000. In some preferred embodiments of the present invention, the quantity ratio of the microspheres to the nanospheres is 1:10000-1:15000, or 1:50000-1:60000.
  • the particle size ratio of microspheres and nanospheres in the particle crystal film is 2:0.08-0.12, more preferably 2:0.1.
  • the particle crystal film is formed on the surface of the substrate by self-assembly of 2 ⁇ m silica microspheres and 100 nm carboxylated polystyrene nanospheres.
  • the quantity ratio of the silica microspheres and nanospheres is 1:10000-1:15000.
  • the particle crystal film is formed on the surface of the substrate by self-assembly of 2 ⁇ m silica microspheres and 50 nm carboxylated polystyrene nanospheres.
  • the quantity ratio of the silica microspheres and nanospheres is 1:50000-1:60000.
  • the present invention also provides a cell culture container having the surface of the above-mentioned particle crystal film of the present invention.
  • the container type is selected from cell culture dishes, cell culture plates, and cell slides.
  • the surface of the container in contact with the cells has the above-mentioned granular crystal film of the present invention.
  • Some embodiments of the present invention also provide a method for promoting the differentiation of stem cells into functional cells, the method comprising the steps of:
  • step 1 Inducing differentiation of stem cells after proliferating in step 1);
  • the functional cells are selected from at least one of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and erythroid cells.
  • the induction method in step 2) includes physical induction, chemical induction or biological induction method, such as induction by co-culturing with aorta-gonad-mesonephros cells, or using mouse bone marrow stromal cells Induce stem cells to differentiate into hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and erythroid cells.
  • Some embodiments of the present invention provide a method of preparing a surface having a stem cell differentiation-promoting surface, the method comprising the step of forming the above-mentioned granular crystal film on a substrate.
  • the above method comprises the following steps:
  • Some specific embodiments of the present invention provide the use of the above-mentioned particle crystal membrane as a surface for culturing stem cells for differentiation, or as a surface for culturing stem cells.
  • Some specific embodiments of the present invention provide the use of the above-mentioned granular crystal membrane for improving or maintaining the stemness state of stem cells.
  • the use is to promote the differentiation of stem cells into hematopoietic stem cells, hematopoietic progenitor cells and erythroid cells by improving the stemness state of stem cells.
  • Some specific embodiments of the present invention provide the use of the above-mentioned granular crystal membrane for enhancing the differentiation of stem cells into hematopoietic stem cells, hematopoietic progenitor cells or erythroid cells.
  • Some specific embodiments of the present invention provide the use of the above-mentioned granular crystal membrane for increasing the number or proportion of stem cells differentiated into cells expressing C34 and CD43, cells expressing C34 and not expressing CD43 Cells, cells that do not express C34 and express CD43, cells that express C34 and express CD45, cells that express C34 and do not express CD45, cells that do not express C34 and express CD45, or cells that express GPA and express CD71.
  • the stem cells are selected from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
  • Some specific embodiments of the present invention provide an induced and catalytically treated stem cell, and the stem cell has been cultured on the granular crystal membrane of the present invention before being induced.
  • having been cultured on the granular crystal film of the present invention means that the stem cells have contacted the granular crystal film of the present invention during the process of proliferation and culture, or The surface of the granular crystal film proliferates.
  • Some specific embodiments of the present invention provide the use of the above induced and catalytically treated stem cells of the present invention in the preparation of preparations for treating hematopoietic disorders.
  • the stem cells are isolated or obtained from human embryos that have not undergone in vivo development within 14 days of fertilization.
  • the hematopoietic-related cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, erythroid cells or blood cells.
  • Silica microspheres and carboxylated polystyrene nanospheres gather on the surface of the substrate under the action of gravity sedimentation, and then the above-mentioned microspheres and nanospheres are arranged on the surface of the substrate through electrostatic interaction to form a multi-level particle crystal film cSAP# 1.
  • Silicon microspheres and polystyrene nanospheres gather on the surface of the substrate under the action of gravity sedimentation, and then the above-mentioned microspheres and nanospheres are arranged on the surface of the substrate through electrostatic interaction to form a multi-level particle crystal film cSAP#2.
  • the silicon microspheres and the polystyrene nanospheres gather on the surface of the substrate under the action of gravity sedimentation, and then the microspheres and the nanospheres are arranged on the surface of the substrate through electrostatic interaction to form a granular crystal film cSAP#3 with a hierarchical structure.
  • silica microspheres and 50nm carboxylated polystyrene nanospheres are aggregated on the surface of the substrate under the action of gravity sedimentation, and then the above-mentioned microspheres and nanospheres are arranged on the surface of the substrate through electrostatic interaction to form a hierarchical structure The granular crystal film cSAP#4.
  • the hydrophilic properties of cSAP#1-5 samples were detected respectively, and the hydrophilic contact angles of each sample are shown in Figure 9.
  • the contact angles of the respective samples were #1: 85.8 ⁇ 3.5, #2: 96.8 ⁇ 4.6, #3: 31.8 ⁇ 1.1, #4: 32.3 ⁇ 3.2, #5: 25.8 ⁇ 2.4.
  • the surface roughness of each sample was #1: 225.91 ⁇ 15.343 nm, #2: 116.371 ⁇ 10.599 nm, #3: 154.873 ⁇ 14.241 nm, #4: 83.03 ⁇ 5.669 nm, #5: 212 ⁇ 11.95 nm.
  • hESCs embryonic stem cells
  • H1hESCs Human embryonic stem cells (hESCs) were cultured on polystyrene tissue culture plates and cSAP#1-5 surfaces: H1hESCs were cultured on polystyrene cell culture plates, covered with cSAP#1, cSAP#2, cSAP#3, cSAP# 4 and cSAP#5 coated cell culture plates. Proliferation culture was performed to observe the morphology of cell clones. The experimental results are shown in Figure 2. Among them, the cell morphology was abnormal on cSAP#1-3, while the cell clone morphology was more uniform on cSAP#4 and cSAP#5, and the cells in the clone were more uniform and dense.
  • carboxyl groups carried in the carboxylated polystyrene (PSC) nanospheres on the surfaces of cSAP#4 and cSAP#5 impart surface properties that are more suitable for cell culture conditions, so the cells grown on it achieve better morphology.
  • cSAP#4 and cSAP#5 were selected for subsequent culture. After 3 passages, the stemness molecular marker OCT4/SSEA4/SOX2 of the cells was identified by immunofluorescence staining.
  • the experimental results are shown in Figure 3.
  • the experimental results show that hESCs can maintain stemness factor expression on cell culture plates coated with cSAP#4 and cSAP#5.
  • hESCs Embryonic Stem Cells
  • AGM aorta-gonad-mesonephros
  • the experimental results are shown in Figure 4 and Figure 6.
  • the experimental results showed that on the 8th day, the number of induced CD34+ cells, that is, the number of hematopoietic stem cells, was significantly higher than that of ordinary polyethylene cells only in the cells obtained by the proliferation culture of the cSAP#4 and cSAP#5-coated cell culture plates.
  • the number of cells cultured in the plate, especially the cSAP#5 group showed that it was about 2-5 times that of the ordinary polyethylene cell culture plate group. This indicates that the coating of granular crystal membrane can significantly increase the proliferation of embryonic stem cells and induce the transformation to hematopoietic stem cell progenitor cells.
  • GPA+CD71+ cells were also sorted, wherein GPA is an important marker on the surface of erythroid cells, and CD71 is also an important molecule on the surface of erythroid cells, and the co-expression of the two shows that the erythroid cells tend to mature. Therefore, the experimental results showed that the granular crystal membrane-coated cSAP#5 group could promote the differentiation of embryonic stem cells into erythroid cells, while the cSAP#4 group showed slightly worse results than the TCPS group.
  • Sorting of more mature blood cells was also performed on day 14, and the cSAP#4 group showed slightly worse results than the TCPS group for the induction of differentiation of the more mature blood cells.
  • the cells differentiated for 14 days were further cultured for 14 days, and the colony formation assay of hematopoietic stem cells was carried out.
  • the experimental results are shown in Figure 7.
  • the results showed that the hematopoietic stem cells cultured on cSAP#4 and cSAP#5 were in granulocyte-monocyte colony forming unit (CFU-GM), erythrocyte colony forming unit (CFU-E), erythrocyte early colony forming unit BFU-E,
  • the colony content of mixed colony forming units (CFU-MIX) was higher than that of TCPS surface.
  • CFU-GM granulocyte-monocyte colony forming unit
  • CFU-E erythrocyte colony forming unit
  • BFU-E erythrocyte early colony forming unit
  • CFU-MIX mixed colony forming units
  • cSAP#5 changed the gene expression pattern of H1hESCs, and the differentially expressed genes were mainly concentrated in signaling pathways such as Mineral absorption, Longevity regulating, Toll-like receptor, HIF1a, Notch, Focal adhesion, TGF-beta, PI3K-Akt, and MAPK. .
  • the experimental results show that the embryonic stem cells cultured on the surface of cSAP#5 and cSAP#4 can induce the transformation into hematopoietic stem cells, and the effect of inducing transformation is much higher than that of the cells cultured on the ordinary culture surface. Moreover, the ability of cSAP#5 to induce embryonic stem cells to hematopoietic stem cells is much higher than that of cSAP#4. At the same time, cSAP#5 also showed a significant increase in the efficiency of inducing hESCs to differentiate into various types of blood cells.
  • hematopoietic stem cells While not wishing to be bound by theory, it is possible that the induction of hematopoietic stem cells by different surfaces is related to their surface roughness, and thus, surfaces composed of particles of different particle sizes show different differentiation-inducing activities.
  • cSAP#5 shows greater roughness than cSAP#4, and it can be considered that the surface under a specific roughness can induce embryonic stem cells and promote their transformation into hematopoietic stem cells and erythroid cells.
  • the present invention finds that when the surface of the nanosphere is modified with chemical groups, it is more suitable for cell culture conditions, and the cells grown on it achieve a better shape. Further, there is a need to achieve a surface with a specific roughness that enables better differentiation-inducing activity of stem cells to hematopoietic-related cells.

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Abstract

本发明涉及一种通过物理途径促进干细胞分化效率的材料及方法，具体公开了一种促进人胚胎干细胞向造血相关细胞亚型分化的颗粒基底材料，所述颗粒基底材料是由微球和纳米球通过自组装在一容器中或基底表面形成；所述的微球与纳米球的粒径尺寸比例为1:0.01-0.3。本发明还公开了上述颗粒基底经由自组装干燥后所形成的二维基底作为人胚胎干细胞扩增的材料，经过材料扩增后的干细胞可提升后续向造血细胞分化的效率，提升造血干组细胞或血细胞数量至少3倍以上。本发明的方法首次利用颗粒材料提高了人胚胎干细胞向造血细胞分化的效率，材料制备工艺简单，并优化了造血细胞的生产过程，降低了生产成本。

Description

一种通过物理途径促进干细胞分化的材料及方法 技术领域
本发明属于细胞生物学领域，具体涉及一种物理途径促进干细胞分化的材料及方法。
背景技术
造血干细胞移植治疗血液系统疾病是最早应用于临床的干细胞技术，也是目前唯一在全球范围均获得认可的干细胞治疗技术。但造血干细胞来源有限，体外扩增难度大，难以满足临床治疗中足数量和高质量的要求。人类多能干细胞(hPSC)，包括hESC和hiPSC，是有希望产生血样细胞的来源。hPSCs的造血潜力在治疗与地中海贫血[1]或血友病[2]等血液相关疾病方面有着重要的应用。已经建立了几种共培养分化系统，例如OP9基质细胞和主动脉-性腺-中肾源性基质细胞(AGM-S3)[3]，用于生成类血细胞。这些系统利用自然界启发的微环境在体外刺激确定的造血作用。这些系统可用于鉴定关键基因在正常或异常造血中的功能。例如，AGM-S3共培养系统可用于检查受关键基因影响的造血作用的详细细胞和分子机制[4-6]。它在筛选促进人类造血功能的化合物方面也具有潜在的实用性，它有可能建立使用AGM-S3共培养系统进行化合物功能筛选的高通量筛选系统[7]。但是，目前的体外血细胞生成方法在质量和数量上都不能满足临床需求[8]。因此，非常需要提高hPS-HCs的造血分化效率。
cSAPs(颗粒晶体膜)为本课题组自主研发的一种由不同尺寸和化学组分胶体颗粒组成的新基质材料家族[9]。晶体颗粒可以进行预修饰或后修饰，最终提供cSAPs复杂表面和化学组分[10]，从而使表面形貌、粗糙度、亲水性、化学性质、甚至刚度不同。在cSAPs上前期已经研究了人类干细胞和成体细胞的行为[11,12]，成功在cSAPs上建立了将人类成纤维细胞重编程为人诱导多能干细胞(hiPSC)的方法[13]，证明了cSAPs显示出调控细胞粘附以及细胞命运的潜力。
目前将hESCs分化在体外诱导分化为造血细胞的常用方法主要有三种:一种是拟胚体分化诱导法，一种是ESCs与造血基质细胞共培养法，另一种公司将ESCs培养在细胞外的基质蛋白上的无基质细胞培养法。其中与小鼠主动脉性腺中肾区机制细胞(AGM)能很好的将ESCs诱导成造血干/祖细胞，并且诱导出的造血干/祖细胞比例更高。但相对于临床应用所需细胞数量而言，现有体外诱导方法的效率较低，并且成本较高。现有提高体外分化效率和降低分化成本的方法主要包括：优化分化培养基配方；使用半渗透膜保存培养基找那个需要补充的分子；采用更便宜的模拟物替代细胞因子；添加特定的小分子增加红细胞前难题细胞的 扩增效率；建立永生化红祖细胞的细胞系；利用基因操作在基因水平上调红细胞的分化及扩增效率等。虽然这些方法在一定程度上达到了提高分化效率、降低分化成本的目的，但本专利所述方法在操作技术和成本、分化效率方面优于现有方法，能更大程度的简单高效低成本获得更多造血干/祖细胞及红系细胞。
参考文献
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发明内容
为解决上述问题，本发明提供了一种颗粒晶体膜，以及包含所述颗粒晶体膜的细胞培养容器，并验证了所述颗粒晶体膜能够促进干细胞的增殖，并诱导其分化成为造血干细胞、造血祖细胞以及红系细胞。
本发明一个方面提供了一种促进干细胞向造血相关细胞分化的颗粒晶体膜，所述颗粒晶体膜由微球和纳米球通过在基底表面自组装制成；
所述的微球选自氧化无机微球或聚合物有机微球；
所述的纳米球选自表面进行化学修饰的纳米球。
所述的微球的粒径为1μm-6μm，；
所述的纳米球的粒径选自30nm-500nm；且微球与纳米球的粒径比例为2:0.03-0.5。
在本发明的技术方案中，所述纳米球的粒径为50nm-200nm，优选为40nm-120nm。
在本发明的技术方案中，所述微球的粒径为1μm-3μm；优选为1.5μm-2.5μm。
在本发明的技术方案中，氧化无机微球选自二氧化硅微球，聚合物有机微球选自聚苯乙烯微球、聚苯乙烯微球、聚甲基丙烯酸甲酯微球。
在本发明的技术方案中，微球与纳米球的粒径比例为2:0.05-0.1。
在本发明的技术方案中，所述化学修饰指纳米球表面进行了化学基团的修饰，更优选地，所述化学基团选自羧基。
在本发明的技术方案中，所述的微球和纳米球的数量比为1:10000～1:60000。
优选地，所述的微球和纳米球的数量比为1:10000-1:15000，或1:50000-1:60000。
在本发明一个优选的实施例中，所述颗粒晶体膜中微球和纳米球的粒径比为2:0.08-0.12，更优选为2：0.1。
在本发明的技术方案中，所述颗粒晶体膜的接触角小于30°，优选小于25°。
在本发明的技术方案中，所述颗粒晶体膜的粗糙度为190-230nm。
本发明另一个方面提供了一种细胞培养的容器，所述容器具有本发明上述的颗粒晶体膜的表面。
在本发明的技术方案中，所述的容器种类选自细胞培养皿、细胞培养板、细胞爬片。
在本发明的技术方案中，所述的容器与细胞接触的表面上具有本发明上述的颗粒晶体膜。
本发明另一个方面提供了诱导干细胞向功能细胞分化的方法，所述方法包括以下步骤：
1)采用本发明所述的颗粒晶体膜表面培养干细胞，进行增殖培养；
2)诱导步骤1)增殖后的干细胞分化。
在本发明的技术方案中，所述的功能细胞选自造血细胞、造血干细胞、造血祖细胞、红系细胞中的至少一种。
在本发明的技术方案中，所述步骤2)诱导的方法包括物理诱导、化学诱导或生物诱导方法，例如采用与主动脉-性腺-中肾细胞共培养诱导干细胞向造血细胞、造血干细胞、造血祖细胞、红系细胞分化。
本发明再一个方面提供了一种制备具有促进干细胞分化的表面的方法，所述方法包括在基底上形成上述颗粒晶体膜的步骤。
进一步地，上述方法包含以下步骤：
1)配制二氧化硅微球与粒径和的纳米球的分散液；
2)将分散液分散在基底表面，并使二氧化硅微球与粒径和纳米球在重力沉降作用下聚集在基板表面并蒸发分散液中的溶剂形成颗粒晶体膜。
本发明再一个方面提供了上述颗粒晶体膜的用途，所述用途为作为培养干细胞进行分化的表面，或者作为进行培养干细胞的表面。
本发明再一个方面提供了上述颗粒晶体膜的用途，所述用途为提高或维持干细胞干性状 态的用途。优选地，所述用途为通过提高干细胞干性状态，促进干细胞向造血干细胞、造血祖细胞、红系细胞分化的用途。
本发明再一个方面提供了上述颗粒晶体膜的用途，所述用途为提高干细胞向造血干细胞、造血祖细胞或红系细胞分化的用途。
本发明再一个方面提供了上述颗粒晶体膜的用途，所述用途为提高干细胞向以下细胞分化的数量或比例的用途，所述细胞为表达C34和CD43的细胞、表达C34且不表达CD43的细胞、不表达C34且表达CD43的细胞、表达C34且表达CD45的细胞、表达C34且不表达CD45的细胞、不表达C34且表达CD45的细胞，或表达GPA且表达CD71的细胞。
在本发明的技术方案中，所述的干细胞选自人胚胎干细胞(hESCs)、人诱导多潜能干细胞(hiPSCs)。
本发明再一个方面提供了一种经诱导催化处理的干细胞，所述的干细胞在被诱导前在本发明所述颗粒晶体膜上进行过培养。
在本发明的技术方案中，在本发明所述颗粒晶体膜上进行过培养为所述干细胞在培养过程中与本发明所述的颗粒晶体膜进行过接触，
本发明再一个方面提供了本发明上述经诱导催化处理的干细胞在制备治疗造血障碍疾病的制剂中的用途。
在本发明的技术方案中，所述的人胚胎干细胞是利用未经过体内发育的受精14天以内的人胚胎分离或者获取干细胞的。
本发明利用自主装技术制备的颗粒晶体膜cSAPs材料培养人胚胎干细胞(hESCs)，通过cSAPs材料表面化学和物理性质对hESCs进行预刺激，使从cSAPs上收获的hESCs在造血细胞分化诱导条件下分化为造血干/祖细胞和红系细胞的效率提高，在数量上和细胞质量上得到提升。hESCs在cSAPs上增殖培养3代以上后取下，与AGM细胞共培养进行造血细胞诱导。cSAPs自身的物理形貌和化学性质对hESCs的干性维持产生影响，使之处于较高干性和较高可诱导分化状态，从而在于诱导条件下分化为造血干/祖细胞和红系细胞的效率提高。
有益效果
1)本专利所述方法在操作技术和成本、分化效率方面优于现有方法，能更大程度的简单高效低成本获得更多造血干/祖细胞及红系细胞。
2)本发明可提高胚胎干细胞向造血干细胞、造血祖细胞以及红系血细胞分化的效率；
3)本发明所用提高胚胎干细胞向血细胞分化效率的方式不需要添加外源因子，提高了所得造血细胞的安全性；
4)本发明用颗粒晶体膜(cSAPs)对胚胎干细胞预刺激，cSAPs利用物理形貌和化学性质对hESCs的干性维持产生影响，使之处于较高干性和较高可诱导分化状态，从而提高胚胎干细胞向造血细胞分化的效率。
5)本发明的产品制备方法简单，制备原料经济且其不需要苛刻的保存条件，更有利于商品化。
附图说明
图1：不同组合cSAPs表面形貌的扫描电镜图，扫描电镜的比例尺为5μm。
其中，TCPS为聚苯乙烯细胞培养板，#1代表实施例1中5μm二氧化硅微球与400nm聚苯乙烯(PS)纳米球，2#代表实施例1中5μm二氧化硅微球与200nm聚苯乙烯纳米球，3#代表实施例1中2μm二氧化硅微球与65nm聚苯乙烯纳米球，4#代表实施例1中2μm二氧化硅微球与50nm羧基化的聚苯乙烯(PSC)纳米球制备的颗粒晶体膜材料cSAP#4，5#代表实施例1中2μm二氧化硅微球与100nm羧基化的聚苯乙烯(PSC)纳米球制备的颗粒晶体膜材料cSAP#5。
图2：在TCPS培养板、覆盖cSAP#1、cSAP#2、cSAP#3、cSAP#4和cSAP#5的培养板上培养H1hESCs的细胞形态。
图3：在TCPS培养板、覆盖cSAP#4和cSAP#5的培养板上传代3次后H1hESCs上干性因子OCT4/SOX2/SSEA4的免疫染色结果。
图4：实施例2中hESCs与AGM细胞共培养诱导向造血细胞分化，第8天和第14天流式分析细胞表面标志物的表达。
图5：本发明实施例2实验验证流程图。
图6：本发明实施例2中hESCs与AGM细胞共培养诱导向造血细胞分化，第8天和第14天流式分析结果。
图7：实施例2中继续培养14天后进行集落形成实验分析结果。
图8：实施例2中测序结果及分析。
图9：cSAP#1-5样品的接触角。
图10：cSAP#1-5样品的粗糙度。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方 式做详细的说明，但不能理解为对本发明的可实施范围的限定。
本发明提供了一种促进干细胞向造血相关细胞分化的颗粒晶体膜，所述颗粒晶体膜由微球和纳米球通过在基底表面自组装制成；所述的微球选自二氧化硅微球、聚苯乙烯微球、聚苯乙烯微球、聚甲基丙烯酸甲酯微球；所述的纳米球选自表面进行化学修饰的纳米球；所述的微球的粒径为1μm-6μm；所述的纳米球的粒径选自30nm-200nm；且微球与纳米球的粒径比例为2:0.03-0.5。
在本发明的一些实施例中，所述的纳米球的粒径选自40nm-120nm。
在本发明的一些实施例中，所述的微球的粒径为1.5μm-2.5μm。
在本发明的一些实施例中，微球与纳米球的粒径比例为2:0.05-0.1。
在本发明的一些实施例中，所述颗粒晶体膜的接触角小于30°，优选小于25°。
在本发明的一些实施例中，所述化学修饰指纳米球表面进行了化学基团的修饰。在本发明的一些优选的实施例，所述化学基团选自羧基。
在一些更优选的实施例中，化学修饰的纳米球选自羧基化的聚苯乙烯纳米球、羧基化的聚苯乙烯纳米球、羧基化的聚甲基丙烯酸甲酯纳米球中的至少一种。
在本发明的一些实施例中，所述的微球和纳米球的数量比为1:10000～1:60000。在本发明的一些优选的实施例中，所述的微球和纳米球的数量比为1:10000-1:15000，或1:50000-1:60000。
在本发明一个优选的实施例中，所述颗粒晶体膜中微球和纳米球的粒径比为2:0.08-0.12，更优选为2：0.1。
在本发明一个优选的实施例中，所述颗粒晶体膜由2μm的二氧化硅微球和100nm羧基化的聚苯乙烯纳米微球通过自组装在基底表面形成。优选地，所述的二氧化硅微球和纳米球的数量比为1:10000～1:15000。
在本发明一个优选的实施例中，所述颗粒晶体膜由2μm的二氧化硅微球和50nm羧基化的聚苯乙烯纳米微球通过自组装在基底表面形成。优选地，所述的二氧化硅微球和纳米球的数量比为1:50000～1：60000。
本发明还提供了一种细胞培养的容器，所述容器具有本发明上述的颗粒晶体膜的表面。
在本发明的一些实施例中，所述的容器种类选自细胞培养皿、细胞培养板、细胞爬片。
在本发明的一些实施例中，所述的容器与细胞接触的表面上具有本发明上述的颗粒晶体膜。
本发明一些实施例还提供了一种促进干细胞向功能细胞分化的方法，所述方法包括以下 步骤：
1)采用本发明所述的颗粒晶体膜表面培养干细胞，进行增殖培养；
2)诱导步骤1)增殖后的干细胞分化；
所述的功能细胞选自造血细胞、造血干细胞、造血祖细胞、红系细胞中的至少一种。
在本发明的一些实施例中，所述步骤2)诱导的方法包括物理诱导、化学诱导或生物诱导方法，例如采用与主动脉-性腺-中肾细胞共培养诱导，或者采用小鼠骨髓基质细胞诱导干细胞向造血细胞、造血干细胞、造血祖细胞、红系细胞分化。
本发明一些具体实施例提供了一种制备具有促进干细胞分化的表面的方法，所述方法包括在基底上形成上述颗粒晶体膜的步骤。
进一步地，上述方法包含以下步骤：
1)配制二氧化硅微球与纳米球的分散液；
2)将分散液分散在基底表面，并使二氧化硅微球与粒径和纳米球在重力沉降作用下聚集在基板表面并蒸发分散液中的溶剂形成颗粒晶体膜。
本发明一些具体实施例提供了上述颗粒晶体膜的用途，所述用途为作为培养干细胞进行分化的表面，或者作为进行培养干细胞的表面。
本发明一些具体实施例提供了上述颗粒晶体膜的用途，所述用途为提高或维持干细胞干性状态的用途。优选地，所述用途为通过提高干细胞干性状态，促进干细胞向造血干细胞、造血祖细胞、红系细胞分化的用途。
本发明一些具体实施例提供了上述颗粒晶体膜的用途，所述用途为提高干细胞向造血干细胞、造血祖细胞或红系细胞分化的用途。
本发明一些具体实施例提供了上述颗粒晶体膜的用途，所述用途为提高干细胞向以下细胞分化的数量或比例的用途，所述细胞为表达C34和CD43的细胞、表达C34且不表达CD43的细胞、不表达C34且表达CD43的细胞、表达C34且表达CD45的细胞、表达C34且不表达CD45的细胞、不表达C34且表达CD45的细胞，或表达GPA且表达CD71的细胞。
在本发明一些技术方案中，所述的干细胞选自人胚胎干细胞(hESCs)、人诱导多潜能干细胞(hiPSCs)。
本发明一些具体实施例提供了一种经诱导催化处理的干细胞，所述的干细胞在被诱导前在本发明所述颗粒晶体膜上进行过培养。
在本发明的技术方案中，在本发明所述颗粒晶体膜上进行过培养为所述干细胞在增殖培养过程中与本发明所述的颗粒晶体膜进行过接触，或者是在本发明所述的颗粒晶体膜表面进 行增殖。
本发明一些具体实施例提供了本发明上述经诱导催化处理的干细胞在制备治疗造血障碍疾病的制剂中的用途。
在本发明中，所述的干细胞是利用未经过体内发育的受精14天以内的人胚胎分离或者获取干细胞的。
在本发明中，所述造血相关细胞选自造血干细胞、造血祖细胞、红系细胞或血细胞。
实施例1颗粒晶体膜cSAPs材料的制备
将粒径为5μm二氧化硅微球与粒径为400nm聚苯乙烯(PS)纳米球分散在溶剂中，颗粒数量比例为1：25000～1：50000，并将分散液分散在基板表面，二氧化硅微球与羧基化的聚苯乙烯纳米球在重力沉降作用下聚集在基板表面，然后上述微球和纳米球之间通过静电作用在基板表面排布形成多级结构的颗粒晶体膜cSAP#1。
将粒径为5μm的二氧化硅微球与粒径为200nm的聚苯乙烯纳米球分散在溶剂中，颗粒数量比例为1：20000～1：40000，并将分散液分散在基板表面，二氧化硅微球与聚苯乙烯纳米球在重力沉降作用下聚集在基板表面，然后上述微球和纳米球之间通过静电作用在基板表面排布形成多级结构的颗粒晶体膜cSAP#2。
将粒径为2μm的二氧化硅微球与粒径为65nm的聚苯乙烯纳米球分散在溶剂中，颗粒数量比例为1：30000～1：60000，并将分散液分散在基板表面，二氧化硅微球与聚苯乙烯纳米球在重力沉降作用下聚集在基板表面，然后上述微球和纳米球之间通过静电作用在基板表面排布形成多级结构的颗粒晶体膜cSAP#3。
将粒径为2μm的二氧化硅微球与粒径为50nm的羧基化的聚苯乙烯(PSC)纳米球分散在溶剂中，颗粒数量比例为1：50000～1：60000，并将分散液分散在基板表面，二氧化硅微球与50nm羧基化的聚苯乙烯纳米球在重力沉降作用下聚集在基板表面，然后上述微球和纳米球之间通过静电作用在基板表面排布形成多级结构的颗粒晶体膜cSAP#4。
将2μm二氧化硅微球与100nm羧基化的聚苯乙烯(PSC)纳米球分散在溶剂中，颗粒数量比例为1：10000～1：15000，并将分散液分散在基板表面，二氧化硅微球与100nm羧基化的聚苯乙烯纳米球在重力沉降作用下聚集在基板表面，然后上述微球和纳米球之间通过静电作用在基板表面排布形成多级结构的颗粒晶体膜cSAP#5。
采用扫描电镜分别观察聚苯乙烯细胞培养板、cSAP#1-5产品的形貌特征，结果参见图1，实验结果显示，cSAP#1-5在基板表面形成了均匀的排列，形成了多级结构。
分别检测cSAP#1-5样品的亲水性能，各样品的亲水接触角如图9所示。各样品的接触角为#1： 85.8±3.5、#2：96.8±4.6、#3：31.8±1.1、#4：32.3±3.2、#5：25.8±2.4。各样品的表面粗糙度为#1：225.91±15.343nm、#2：116.371±10.599nm、#3：154.873±14.241nm、#4：83.03±5.669nm、#5：212±11.95nm。
实施例2胚胎干细胞的培养和诱导分化
对于胚胎干细胞培养以及诱导分化实验流程示意图参见图4。
胚胎干细胞(hESCs)的培养
分别采用聚苯乙烯组织培养板、cSAP#1-5表面对人胚胎干细胞(hESCs)进行培养：将H1hESCs在聚苯乙烯细胞培养板、覆盖cSAP#1、cSAP#2、cSAP#3、cSAP#4和cSAP#5涂层的细胞培养板。进行增殖培养，观察细胞克隆形态。实验结果见图2。其中，在cSAP#1-3上细胞形态异常，而在cSAP#4和cSAP#5上细胞克隆形态更均匀，克隆内细胞更均匀致密。虽然不希望被理论所束缚，但是有可能是由于cSAP#4和cSAP#5表面的羧基化的聚苯乙烯(PSC)纳米球中携带的羧基基团为表面带来表面特性，更适合细胞培养条件，所以在其上生长的细胞实现了更好的形态。
根据细胞克隆形态，选择cSAP#4和cSAP#5进行后续培养。传代3次后对细胞的干性分子标记OCT4/SSEA4/SOX2进行免疫荧光染色鉴定。
实验结果见图3。实验结果显示hESCs在覆盖cSAP#4和cSAP#5涂层的细胞培养板上能够维持干性因子表达。
胚胎干细胞(hESCs)的诱导分化：
分别将聚苯乙烯细胞培养板、覆盖在cSAP#4和cSAP#5涂层的细胞培养板上传代3次后的细胞转移到主动脉-性腺-中肾细胞(AGM)上进行共培养14天，诱导hESCs向造血干/祖细胞和红系细胞分化，分别在第8天和第14天时通过细胞流式分析仪分析CD34/CD43/CD45/CD71/GPA在分化后细胞表面的表达。
实验结果见图4和图6。实验结果显示在第8天时，覆盖在cSAP#4和cSAP#5涂层的细胞培养板增殖培养获得的细胞中经过诱导后CD34+的细胞，即造血干细胞数量显著高于仅采用普通聚乙烯细胞培养板培养的细胞，尤其是cSAP#5组细胞数量显示其是普通聚乙烯细胞培养板组的约2-5倍。说明颗粒晶体膜涂层能够显著增加胚胎干细胞增殖同时诱导向造血干细胞祖细胞的转化。
对于第14天的结果，显示了cSAP#5组细胞数量依然明显高于普通聚乙烯细胞培养板组的细胞数量，且相比于第8天时的结果，也有显著增加，说明cSAP#5组颗粒晶体膜涂层对于胚胎干细胞增殖阶段的影响能够在其分化时持续作用。
在第14天时还进行了GPA+CD71+细胞的分选，其中GPA是红系细胞表面重要的标志 物，同时CD71也是红系细胞表面重要的分子，二者共同表达显示了红系细胞趋向成熟。因此，实验结果显示颗粒晶体膜涂层cSAP#5组能够促进对于胚胎干细胞向红系细胞分化，而cSAP#4组显示出比TCPS组稍差的结果。
在第14天时还进行了较成熟血细胞(CD34-CD43+，或CD34-CD45+)的分选，对于对较成熟的血细胞的诱导分化，cSAP#4组显示出比TCPS组稍差的结果。
对14天分化后的细胞继续进行14天培养，进行造血干细胞集落形成实验分析。实验结果见图7。结果显示在cSAP#4和cSAP#5上培养过的造血干细胞在粒－单核细胞集落形成单位(CFU-GM)、红细胞集落形成单位(CFU-E)、红细胞早期集落形成单位BFU-E、混合集落形成单位(CFU-MIX)的集落含量均高于TCPS表面。与细胞分选结果相同，cSAP#4和cSAP#5相比于TCPS显示了更强的向造血干细胞诱导分化效果。
对在cSAP#5上传代3次后的细胞进行测序分析，实验结果见图8。结果表明，cSAP#5改变了H1hESCs的基因表达模式，差异表达基因主要集中在Mineral absorption、Longevity regulating、Toll-like receptor、HIF1a、Notch、Focal adhesion、TGF-beta、PI3K-Akt、MAPK等信号通路。
综上所述，实验结果显示经过cSAP#5和cSAP#4表面培养的胚胎干细胞，能够诱导其向造血干细胞的转化，其诱导转化的效果远远高于普通培养表面培养后的细胞。而且，cSAP#5诱导胚胎干细胞向造血干细胞的能力又远高于cSAP#4。同时，cSAP#5还显示出了诱导hESCs向各种类型血细胞分化的效率有显著性提高。虽然不希望被理论束缚，但是不同的表面对于造血干细胞的诱导作用有可能和其表面粗糙度相关，因而，在不同粒径颗粒组成的表面上显示了不同的分化诱导活性。cSAP#5比cSAP#4显示了更大的粗糙度，可以认为在特定粗糙度下的表面能够实现对胚胎干细胞的诱导，促进其向造血干细胞和红系细胞转化。综上所述，本发明发现了当纳米球表面进行了化学基团的表面修饰，更适合细胞培养条件，在其上生长的细胞实现了更好的形态。进一步地，还需要实现特定粗糙度的表面，这样的表面能够实现更好的干细胞向造血相关细胞的分化诱导活性。

Claims (17)

  1. 一种促进干细胞向造血相关细胞分化的颗粒晶体膜，其特征在于，所述颗粒晶体膜由微球和纳米球通过自组装在基底表面形成；
    所述的微球选自氧化无机微球或聚合物有机微球；
    所述的纳米球选自表面进行化学修饰的纳米球；
    所述的微球的粒径为1μm-6μm；
    所述的纳米球的粒径选自30nm-500nm；且微球与纳米球的粒径比例为2:0.03-0.5。
  2. 根据权利要求1所述的颗粒晶体膜，其特征在于，氧化无机微球选自二氧化硅微球，聚合物有机微球选自聚苯乙烯微球、聚苯乙烯微球、聚甲基丙烯酸甲酯微球。
  3. 根据权利要求1所述的颗粒晶体膜，其特征在于，表面进行化学修饰的纳米球选自表面进行羧基修饰的纳米球，更优选为表面进行羧基修饰的聚苯乙烯纳米球。
  4. 根据权利要求1所述的颗粒晶体膜，其特征在于，所述纳米球的粒径为50nm-200nm，优选为40nm-120nm。
  5. 根据权利要求1所述的颗粒晶体膜，其特征在于，所述微球的粒径为1μm-3μm；优选为1.5μm-2.5μm。
  6. 根据权利要求1所述的颗粒晶体膜，其特征在于，所述的微球和纳米球的数量比为1:10000～1:60000。
  7. 一种细胞培养的容器，其特征在于，所述的容器与细胞接触的表面上具有如权利要求1-6任一项所述的颗粒晶体膜；优选地，所述的容器的种类选自细胞培养皿、细胞培养板、细胞爬片。
  8. 一种诱导干细胞向功能细胞分化的方法，其特征在于，所述方法包括以下步骤：
    1)采用权利要求1-6任一项所述的颗粒晶体膜表面或者采用权利要求7所述的细胞培养的容器，培养干细胞，进行增殖培养；
    2)诱导步骤1)增殖后的干细胞分化；
    优选地，所述的功能细胞选自造血细胞、造血干细胞、造血祖细胞、红系细胞中的至少 一种。
  9. 一种制备具有诱导干细胞分化的表面的方法，其特征在于，所述方法包括在基底上形成权利要求1-6任一项所述的颗粒晶体膜的步骤。
  10. 如权利要求1-6任一项所述的颗粒晶体膜的用途，其特征在于，所述用途为作为培养干细胞进行分化的表面，或者作为进行培养干细胞的表面。
  11. 如权利要求1-6任一项所述的颗粒晶体膜的用途，其特征在于，所述用途为提高或维持干细胞干性状态的用途；优选地，所述用途为通过提高或维持干细胞干性状态，促进干细胞向造血相关细胞分化的用途。
  12. 如权利要求1-6任一项所述的颗粒晶体膜的用途，其特征在于，所述用途为提高干细胞向造血干细胞、造血祖细胞或红系细胞分化的用途。
  13. 如权利要求1-6任一项所述的颗粒晶体膜的用途，其特征在于，所述用途为提高干细胞向以下细胞分化的数量或比例的用途，所述细胞为表达C34和CD43的细胞、表达C34且不表达CD43的细胞、不表达C34且表达CD43的细胞、表达C34且表达CD45的细胞、表达C34且不表达CD45的细胞、不表达C34且表达CD45的细胞，或表达GPA且表达CD71的细胞。
  14. 一种经诱导催化处理的干细胞，其特征在于，所述的干细胞在被诱导前在权利要求1-6任一项所述的颗粒晶体膜上进行过培养。
  15. 根据权利要求14所述的经诱导催化处理的干细胞在制备治疗造血障碍疾病的制剂中的用途。
  16. 根据权利要求14所述的经诱导催化处理的干细胞在治疗造血障碍疾病中的用途。
  17. 如权利要求1-6任一项所述的颗粒晶体膜，或者如权利要求7所述的容器，或者如权利要求8或9所述的方法，或者如权利要求10-13，15-16任一项所述的用途，或者权利要 求14所述的干细胞，其特征在于，所述干细胞选自人胚胎干细胞、人诱导多潜能干细胞。
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