WO2019033482A1 - Method for directional differentiation of human pluripotent stem cells - Google Patents

Method for directional differentiation of human pluripotent stem cells Download PDF

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WO2019033482A1
WO2019033482A1 PCT/CN2017/101430 CN2017101430W WO2019033482A1 WO 2019033482 A1 WO2019033482 A1 WO 2019033482A1 CN 2017101430 W CN2017101430 W CN 2017101430W WO 2019033482 A1 WO2019033482 A1 WO 2019033482A1
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stem cells
pluripotent stem
differentiation
cells
human
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萨莫赫瓦洛夫伊戈尔•M
尤昊
谭颖
樊琛语
菲洛年科埃琳娜•S
王翠华
沙阿查希尔
张建光
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中国科学院广州生物医药与健康研究院
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Definitions

  • the present invention belongs to the technical field of cell culture differentiation methods, and more particularly, the present invention relates to the use of human pluripotent stem cells for directional differentiation in the absence of exogenous hematopoietic cytokines, serum-free, stromal-free cells, and well-defined culture conditions. method.
  • hPSCs human pluripotent stem cells
  • culture systems using hPSCs for directed hematopoietic development mostly use mouse cell lines OP9, mAGM-S3, S17, and MS-5 trophoblast cells for co-culture. These co-culture systems require fetal bovine serum to maintain mouse stromal cells. Growth and differentiation of hPSCs.
  • the stromal cell co-culture system is very sensitive to changes in serum mass, stromal cell culture quality, and hPSCs colony size for differentiation.
  • Both mouse trophoblast/stromal cells and fetal bovine serum are heterogeneous components of unknown composition, the existence of which fundamentally hinders the study of the role of the microenvironment in hematopoietic development.
  • serum as a complex mixture, may contain a variety of cytotoxins that are harmful to primary cells and cell lines, and its components vary greatly from batch to batch, easily leading to difficult cell culture conditions. Different batches of serum can produce different cell differentiation and analysis results. If the best batch is screened before using the serum, there are problems such as time-consuming and costly.
  • commercial serum may contain a variety of harmful contaminants such as viruses, prions and mycoplasmas.
  • EBs embryoid bodies
  • hematopoietic differentiation is usually induced by the addition of a series of growth factors to induce mesoderm differentiation, and then using high, often beyond, physiological concentrations of cytokines.
  • this mode of culture significantly reduces the number of hematopoietic progenitor cells and limits the range of applications of this protocol.
  • the experimental method is based on the directed differentiation of exogenous cytokines in hPSCs, and it is not a good model for functional studies of human blood development. This is because exogenous cytokine-activated cellular pathways and endogenous signal transduction interfere with and influence molecular action, making the molecular mechanism of studying blood development complicated.
  • cell differentiation using cytokines results in increased costs, and experimental reproducibility is also affected by a range of factors including cytokine production processes, cytokine stability, use errors, and potential impurities in cytokines.
  • human pluripotent stem cell hematopoietic differentiation systems often incorporate high concentrations of different blood cytokine mixtures to drive the development of hematopoietic progenitor cells.
  • hematopoietic-associated cytokines adversely affects the maintenance of hematopoietic progenitor cells produced by hPSCs, significantly reducing the yield and proliferative capacity of hematopoietic progenitor cells, thereby reducing the production of functional blood cells that can be used in the biomedical field.
  • the addition of cytokine differentiation means interferes with normal blood development processes and is not suitable as a model for studying human hematopoietic development processes and for testing potential drugs.
  • the object of the present invention is to overcome a series of problems and deficiencies in the prior methods for utilizing human pluripotent stem cells for hematopoietic differentiation, and to provide a directional differentiation of human pluripotent stem cells in a well-defined and cytokine-free culture condition. method.
  • the present invention provides a method for the directed differentiation of human pluripotent stem cells (hPSCs), which is to first polymerize human pluripotent stem cells to form embryoid bodies, and then to explant the embryoid bodies in the absence of exogenous hematopoietic cytokines.
  • hPSCs human pluripotent stem cells
  • Two-dimensional culture was carried out in serum-free, stromal-free, and well-defined cell culture environments, and only vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP4) were added to induce mesoderm development.
  • VEGF vascular endothelial growth factor
  • BMP4 bone morphogenetic protein 4
  • the entire differentiation process is carried out on the surface coated with collagen IV to produce hematopoietic progenitor cells and blood cells.
  • blood development begins without using any exogenous hematopoietic cytokines, and the blood progenitor cells are maintained by endogenous factors produced by
  • the method for directed differentiation of pluripotent stem cells of the present invention includes the following steps:
  • the human pluripotent stem cells cultured in the step (1) are made into a single cell suspension, and after centrifugation and resuspension, they are added to the culture plate to be cultured to form an embryoid body;
  • the embryoid body was suspended in the mTeSR1 culture medium supplemented with human vascular endothelial growth factor, human bone morphogenetic protein 4 and Thiazovivin, and then the embryoid body was attached to the plate.
  • the surface of collagen IV is attached and cultured;
  • the StemLine II medium supplemented with human vascular endothelial growth factor was added to continue culture to produce hematopoietic progenitor cells and blood cells.
  • the human pluripotent stem cells of the present invention may be human embryonic stem cells (hESCs), human nuclear transfer embryonic stem cells (hNT-ESCs) or human induced multifunctionality. Stem cells (hiPSCs).
  • the single cell suspension in the step (2), can be added to the AggreWell400 culture plate to form an embryoid body, and each culture hole is added.
  • the amount is 4 ⁇ 10 5 to 2 ⁇ 10 6 cells.
  • each of the above culture wells is added in an amount of 1 ⁇ 10 6 cells to form an optimal number of embryoid bodies.
  • the single cell suspension can also be cultured in other brands and models of cell culture plates other than the AggreWell 400 culture plate, and the amount of each culture hole will also change. .
  • each of the embryoid bodies comprises 200 to 500 cells.
  • the human vascular endothelial growth factor is added in an amount of 2 to 100 ng/mL, and when the addition amount is 50 ng/mL. When it comes to cost efficiency.
  • the human bone morphogenetic protein 4 is added in an amount of 1 to 40 ng/mL, and when the addition amount is 1 to 2 ng. At /mL, the optimal concentration for promoting hematopoietic differentiation.
  • the concentration of the Thiazovivin is 1 to 20 ⁇ M, and when the concentration is 10 ⁇ M, the attachment to the embryoid body and the cells Differentiation is most beneficial.
  • step (3) when the embryoid body is attached to the surface coated with collagen IV for attachment culture, the seedling density of the embryoid body It is 15-20 embryoid bodies/cm 2 .
  • the collagen IV is mouse collagen IV or human collagen IV.
  • the amount of the collagen IV laid is 0.2 to 10 ⁇ g/cm 2 ; it has been found that laying a high concentration of collagen IV has a negative effect on hematopoietic differentiation when the amount of collagen IV laid is 0.5 ⁇ g/ At cm 2 , the best differentiation effect can be achieved.
  • the culture environment in which the StemLine II culture solution is continuously cultured is a 5% carbon dioxide, 21% oxygen, and 37 ° C humidified culture environment.
  • the present invention finds in a hypoxic environment (5% oxygen) commonly used in traditional cell differentiation cultures between different cell lines (hESC cell lines, such as H1, H9, Mel1, HN14; and hiPSC cell lines, such as IPS9, IPS12). , 5% carbon dioxide) used in the method of the invention does not increase blood Cell yield.
  • the StemLine II culture solution is further added with a non-essential amino acid, GlutaMAX and/or ⁇ -mercaptoethanol to achieve better. Differentiation effect.
  • the StemLine II culture solution can be replaced with other well-defined, serum-free culture solutions, such as StemSpan SFEM culture solution or STEMdiff APEL culture solution.
  • StemSpan SFEM culture solution has a slightly lower yield of human blood cells and hematopoietic cells
  • STEMdiff APEL medium can produce equal or more multipotential progenitor blood progenitors compared with StemLine II medium. .
  • the directed differentiation method of the pluripotent stem cells of the present invention can be used not only for the production of hematopoietic progenitor cells and blood cells, but also for efficiently producing endothelial cells of CD31 + CD146 + VE-cadherin + CD34 + and mesenchymal cells of CD31 - CD146 + .
  • Activin A (0.5-2 ng/mL) can be added on the 0th to 2nd day of differentiation to slightly promote the development of CD34 + CD43 - and CD34 + CD43 + original blood cells.
  • basic fibroblast growth factor (FGF2) is selectively added to the culture solution.
  • the directed differentiation method of the pluripotent stem cells of the present invention has the following beneficial effects:
  • the present invention realizes hematopoietic differentiation under the culture condition with clear components and no hematopoietic cytokine addition, can improve the blood cell yield and quality obtained by differentiation, and can more accurately reduce the human embryo development process, and is suitable for studying human blood development molecules. Model of the mechanism.
  • the method of directed differentiation of the present invention does not use serum and stromal cells, and uses the least amount of external induction factor, and has the advantages of low cost, simple operation steps, easy expansion of culture, easy control of culture conditions, and orderly blood differentiation process, and avoiding serum.
  • the negative effects of the unidentified ingredients have significantly improved the repeatability of the method.
  • the directed differentiation method of the present invention can produce a large number of hematopoietic progenitor cells and CD45 antigen high expressing cells, macrophages and dendritic cells which can differentiate into mature blood cells.
  • FIG. 1 is a schematic flow chart showing a method for directing differentiation of human pluripotent stem cells.
  • A is The embryoid bodies attached to the next day of differentiation
  • B is the embryoid body attached to the fourth day of differentiation
  • C is the vascular plexus formed on the sixth day of differentiation
  • D is a large number of non-adherent blood cells produced on the twelfth day of differentiation.
  • Figure 3 shows the apparent characteristics of the directed differentiation method of the present invention - "in vitro blood island", that is, the formed vascular plexus structure and the VE-CADHERIN + blood cell mass producing the earliest CD43 + blood cells; wherein A is human embryonic stem cell H1 The sixth day of cell line differentiation; B is the eighth day of differentiation of human embryonic stem cell H1 cell line; the upper row is the field of view of the microscope, and the lower row is the result of immunocytochemical staining.
  • Figure 4 is a graph showing the expression of blood and endothelial-associated cell surface antigens in different stages of differentiation of human pluripotent stem cells in the directed differentiation method of the present invention; wherein A shows that early CD43 + blood cells (grey markers) co-express CD146 (mesenchymal) and CD31 (endothelium) antigen; in the late stage of differentiation, the cell populations expressing these three antigens are gradually separated; B shows that CD235a antigen (glycophorin A) expression is continuously decreased, and CD41a + cells are increased, CD33 + cells are marked with gray; C shows CD43 + Early blood cells are CD31 + (grey label) and express low levels of VE-CADHERIN.
  • A shows that early CD43 + blood cells (grey markers) co-express CD146 (mesenchymal) and CD31 (endothelium) antigen; in the late stage of differentiation, the cell populations expressing these three antigens are gradually separated; B shows that CD235a antigen (glycophorin A
  • FIG. 5 shows that in the directed differentiation method of the present invention, human pluripotent stem cells gradually lose their pluripotency with differentiation and differentiate into hematopoietic progenitor cells; wherein A shows the initial stage of hematopoietic differentiation, and the expression of human pluripotent stem cell pluripotency antigen is down-regulated; Six to eight days of differentiation, E-cadherin (CDH1, CD324) was expressed on a group of immature erythroid cells; B showed different time points, CD43 + CD45 + blood progenitor cells differentiated without cytokines, and CD45 + After the tenth day of differentiation, the cells accounted for 64% of all living cells.
  • A shows the initial stage of hematopoietic differentiation, and the expression of human pluripotent stem cell pluripotency antigen is down-regulated
  • E-cadherin CDH1, CD324
  • B showed different time points, CD43 + CD45 + blood progenitor cells differentiated without cytokines, and
  • Fig. 6 shows the results of blood cell colony analysis of cells obtained after directed differentiation of the method of the present invention, and the differentiated whole cells are cultured in a serum-free methylcellulose medium after digestion, and the colonies are counted after 14 to 18 days; wherein A is utilized.
  • CFU-mix erythroid myeloid
  • CFU-G, M GM (myeloid) hematopoietic progenitor cells with differentiation It can be maintained or increased
  • B shows differentiation using iPS12 cell line, and blood progenitor cells are all present in CD43 + cell population on the 16th day
  • C is a typical blood cell colony morphology formed by differentiation of human pluripotent stem cells
  • a and B They are all a histogram comparing four columns. In each of the four columns, from left to right are: CFU-E, BFU-E, CFU-G/M/GM and CFU-Mix.
  • Figure 7 shows the expression levels of transcription factors (TFs), cytokine receptors and other blood progenitor-related genes in CD43 + and CD43 - cells; RNaseq transcriptome analysis derived from cells on the twelfth day of the directed differentiation method of the present invention; CD43 Three replicates of + and CD43 - cell populations were used for analysis.
  • TFs transcription factors
  • cytokine receptors cytokine receptors
  • CD43 Three replicates of + and CD43 - cell populations were used for analysis.
  • hPSCs Human pluripotent stem cells
  • Matrigel-free serum-free mTeSR1 medium After 3 to 5 short-interval passages of hPSCs, cells in the exponential growth phase were trypsinized into single cell suspensions. After centrifugation, the cells were resuspended in mTeSR1 medium containing 1 ⁇ M Thiazovivin, and the single cell suspension was added to AggreWell plates to form EBs, and each AgreeWell 400 model culture well was one million cells. Each EB contains 200 to 500 cells.
  • EBs were cultured for 24 h under standard hPSCs culture conditions (5% carbon dioxide, 21% oxygen, 37 °C, humidified environment), then carefully pipetted out EBs with a pipette containing 50 ng/mL of hVEGF 165 , 2 ng/ mL hBMP4 and 10 ⁇ M Thiazovivin in mTeSR1 medium were suspended and cultured on plastic plates plated with mouse or human collagen IV (0.5 ⁇ g/cm 2 ). The culture density of EBs is 15-20 per square centimeter. Addition of Activin A (0.5-2 ng/mL) on days 0 to 2 of differentiation slightly promoted the development of CD34 + CD43 - and CD34 + CD43 + primordial blood cells.
  • FIG. 3 shows the apparent characteristics during the directed differentiation of the present embodiment - "in vitro blood island", that is, the formed vascular plexus structure and the VE-CADHERIN + blood cell mass which produces the earliest CD43 + blood cells;
  • CD43 + blood cells appeared around the "in vitro blood island”;
  • human embryonic stem cell H1 cell line differentiated on the eighth day, blood island hematopoiesis and blood cell dispersion in vitro, CD43 + blood cells began Dispersed from VE-CADHERIN + "in vitro blood island”.
  • the "in vitro blood island” is located on a single layer of endothelial cells of VE-CADHERIN + .
  • the hESC cell line H1 When the hESC cell line H1 was used for differentiation, the earliest CD43 + blood cells appeared on the fourth day of differentiation, and CD43 + CD45 + blood progenitor cells were first detected on the eighth day of differentiation, after which the CD43 + CD45 + cell population differentiated and expanded. Overtakes more than 50% of the cells in the entire culture system (see Figure 5).
  • the CD45 high expression cell population mainly includes mature macrophages and dendritic cells. Endothelial cells express high levels of VE-CADHERIN, CD34 and CD31 during differentiation, whereas these surface antigens are in low to medium expression levels in blood cells. The level of endothelial antigen expression on CD43 + blood cells steadily decreased as differentiation progressed.
  • CD43 + blood cells co-expressed CD146 (mesenchymal) and CD31 (endothelial) antigens; in the late stage of differentiation, the cell populations expressing these three antigens gradually separated; the expression of CD235a antigen (glycophorin A) continued to decrease. And CD41a + cells increased; CD43 + early blood cells were CD31 + and expressed low levels of VE-CADHERIN.
  • This example provides a method for expanding the scale of cell culture, the same steps as in Example 1, except that the cells are treated with trypsin at the second to fourth days of differentiation, and then the plate coated with collagen IV is re-plated or Incubate on a culture dish and incubate for 1 h in a standard culture environment.
  • the cells in the still suspended or semi-adsorbed state are then gently aspirated to remove residual undifferentiated hPSCs.
  • the adsorbed cell fraction can be cultured in the original culture dish under the above conditions, or seeded in a new collagen IV-coated petri dish at a density of 10,000 cells per square centimeter.
  • the method of this example can expand the entire cell culture scale by about ten times.
  • erythroid progenitor cells and multipotential progenitor cells are the predominantly hematopoietic progenitors.
  • multi-directional progenitor cells were well maintained due to the absence of exogenous cytokines.
  • Myeloid progenitor cells CFU-M, CFU-G, CFU-GM
  • erythroid CFU-Es decreased significantly (see Figure 6).

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Abstract

Provided is a method for directional differentiation of human pluripotent stem cells. The method comprises cultivating human pluripotent stem cells in a clearly-composed cell culture environment without exogenous hematopoietic cytokines, serum or stromal cells, and after the formation of the embryoid body, adding vascular endothelial growth factors and bone morphogenetic protein 4 and attaching the embryoid body to the surface covered with collagen IV to induce mesoderm differentiation, thereby generating hematopoietic progenitor cells and blood cells.

Description

一种人多能干细胞的定向分化方法Directional differentiation method of human pluripotent stem cells 技术领域Technical field
本发明属细胞培养分化方法技术领域，更具体地说，本发明涉及一种利用人多能干细胞在无外源造血细胞因子、无血清、无基质细胞、成分明确的培养条件下进行定向分化的方法。The present invention belongs to the technical field of cell culture differentiation methods, and more particularly, the present invention relates to the use of human pluripotent stem cells for directional differentiation in the absence of exogenous hematopoietic cytokines, serum-free, stromal-free cells, and well-defined culture conditions. method.
背景技术Background technique
在体外利用人多能干细胞(hPSCs)进行定向分化，为规模化获得临床级别治疗用的造血祖细胞和成熟血细胞提供了机会，并为人体早期造血发育中的功能研究提供条件。Directed differentiation using human pluripotent stem cells (hPSCs) in vitro provides opportunities for large-scale access to hematopoietic progenitor cells and mature blood cells for clinical grade therapy, and provides conditions for functional studies in early human hematopoietic development.
通常，利用hPSCs进行定向造血发育的培养体系大多利用小鼠细胞系OP9、mAGM-S3、S17、MS-5滋养层细胞进行共培养，这些共培养体系需要使用胎牛血清以维持小鼠基质细胞生长和实现hPSCs分化。基质细胞共培养体系对不同批次间血清质量变化、基质细胞培养质量和用于分化的hPSCs集落大小非常敏感。小鼠滋养层/基质细胞和胎牛血清均属于成分不明的异源性成分，其存在从根本上阻碍了对微环境在造血发育中的作用的研究。并且，上述培养体系也不能用于获得临床级别治疗用的血细胞。这是因为血清作为一种复杂的混合物，可能含有包括各种对原代细胞和细胞系有害的细胞毒素，并且其组分在不同批次产品之间差异巨大，容易导致细胞培养条件难以控制、且不同批次的血清可以产生不同的细胞分化和分析结果。若在使用血清之前筛选出最佳批次，又存在耗时费钱等问题。此外，商业血清可能含有各种有害污染物，比如病毒，朊病毒和支原体等。In general, culture systems using hPSCs for directed hematopoietic development mostly use mouse cell lines OP9, mAGM-S3, S17, and MS-5 trophoblast cells for co-culture. These co-culture systems require fetal bovine serum to maintain mouse stromal cells. Growth and differentiation of hPSCs. The stromal cell co-culture system is very sensitive to changes in serum mass, stromal cell culture quality, and hPSCs colony size for differentiation. Both mouse trophoblast/stromal cells and fetal bovine serum are heterogeneous components of unknown composition, the existence of which fundamentally hinders the study of the role of the microenvironment in hematopoietic development. Moreover, the above culture system cannot be used to obtain blood cells for clinical grade treatment. This is because serum, as a complex mixture, may contain a variety of cytotoxins that are harmful to primary cells and cell lines, and its components vary greatly from batch to batch, easily leading to difficult cell culture conditions. Different batches of serum can produce different cell differentiation and analysis results. If the best batch is screened before using the serum, there are problems such as time-consuming and costly. In addition, commercial serum may contain a variety of harmful contaminants such as viruses, prions and mycoplasmas.
另一种不依靠基质细胞进行hPSCs定向造血分化的方法则需要形成类胚体(EBs)。然而，这种途径经常需要使用异源性血清，具有高度变化性和造血发育不同步的缺点。起始细胞团的大小和EBs的大小对hPSCs的分化效率影响巨大，由于分化过程中EBs间经常互相粘附，并吸附在培养器皿表面，使得其大小很难控制。在基于EBs的培养体系中，通常先通过添加一系列生长因子以诱导中胚层分化，然后利用高的、往往超出生理浓度范围的细胞因子诱导造血分化， 促进血祖细胞扩增，并影响它们的生存和分化潜能。但是，在血液早期发育过程中，初生血液祖细胞的过早扩增可能不利于建立祖细胞的潜能；此外，添加的细胞因子可能导致过早分化、成熟，因此消耗造血祖细胞对多向潜能血祖细胞负面影响可能最大。Another method that does not rely on stromal cells for hematopoietic differentiation of hPSCs requires the formation of embryoid bodies (EBs). However, this approach often requires the use of heterologous serum with the disadvantage of high variability and unsynchronized hematopoietic development. The size of the initial cell mass and the size of EBs have a great influence on the differentiation efficiency of hPSCs. Because the EBs often adhere to each other during the differentiation process and adsorb on the surface of the culture vessel, the size is difficult to control. In EBs-based culture systems, hematopoietic differentiation is usually induced by the addition of a series of growth factors to induce mesoderm differentiation, and then using high, often beyond, physiological concentrations of cytokines. Promotes the expansion of blood progenitor cells and affects their survival and differentiation potential. However, premature expansion of primary blood progenitor cells may be detrimental to the potential of progenitor cells during early blood development; in addition, added cytokines may lead to premature differentiation and maturation, thus consuming hematopoietic progenitor cells for multipotential potential The negative effects of blood progenitors may be greatest.
有人报道了利用人多能干细胞在成分明确的培养条件下定向分化血细胞的实验方案；此后，还有人对其进行了进一步的优化。优化后的分化体系是利用化学成分明确的培养基，并采用纯化后的细胞外基质蛋白质取代基质细胞。后者实验方案的必备步骤包括在形成中胚层后，向细胞添加外源性细胞因子，并且选择的细胞因子种类和工作浓度是不符合生理条件的，大多数使用的细胞因子并不表达于人类早期发育孕体中。外源性的细胞因子刺激初生血细胞过度增殖并诱导它们进一步成熟为往往只能短暂存活的成熟血细胞。因此，这种培养方式显著地减少了造血祖细胞的数量，并限制了该实验方案的应用范围。另外，该实验方法是基于外源性细胞因子的hPSCs定向分化，无法为人体血液发育的功能性研究提供好的模型。这是因为外源性细胞因子激活的细胞通路和内源性信号转导相干扰并影响分子作用，使得研究血液发育的分子机制变得复杂。此外，使用细胞因子进行细胞分化使得费用升高，并且实验可重复性还受到包括源自细胞因子生产过程、细胞因子稳定性、使用失误和细胞因子中的潜在杂质等一系列因素的影响。Experiments have been reported on the use of human pluripotent stem cells to differentiate blood cells under well-defined culture conditions; since then, others have further optimized them. The optimized differentiation system utilizes a chemically defined medium and replaces the stromal cells with purified extracellular matrix proteins. The necessary steps in the latter experimental protocol include the addition of exogenous cytokines to the cells after the formation of mesoderm, and the selected cytokine species and working concentration are not in accordance with physiological conditions, and most of the cytokines used are not expressed in Early human development in the conception. Exogenous cytokines stimulate the proliferating of primary blood cells and induce their further maturation into mature blood cells that tend to survive only transiently. Therefore, this mode of culture significantly reduces the number of hematopoietic progenitor cells and limits the range of applications of this protocol. In addition, the experimental method is based on the directed differentiation of exogenous cytokines in hPSCs, and it is not a good model for functional studies of human blood development. This is because exogenous cytokine-activated cellular pathways and endogenous signal transduction interfere with and influence molecular action, making the molecular mechanism of studying blood development complicated. In addition, cell differentiation using cytokines results in increased costs, and experimental reproducibility is also affected by a range of factors including cytokine production processes, cytokine stability, use errors, and potential impurities in cytokines.
综合以上，目前利用hPSCs进行造血分化的方法存在一系列问题，包括：培养液中使用动物源性和成分不明的物质、使用滋养层细胞或基质细胞系共培养、添加过高浓度的生长因子诱导中胚层分化，以及使用造血相关细胞因子以扩增新生成的血液细胞。这些问题导致目前造血分化实验的低可重复性，限制了以规模化生产人血细胞为目的的技术应用和针对血细胞功能和机制的研究。而在现有的使用成分明确的培养基进行造血分化的方案中，人多能干细胞造血分化体系中往往添加有高浓度不同种血液细胞因子混合物以驱动造血祖细胞的发育。鉴于添加造血相关细胞因子会负面影响由hPSCs产生的造血祖细胞的维持性，显著降低血液祖细胞的得率和增殖能力，因此降低了可用于生物医学领域的功能性血细胞的产量。添加细胞因子的分化手段干扰正常血液发育过程，不适合作为研究人类造血发育过程的模型和用于测试潜在药物。In summary, there are a number of problems in the current method of hematopoietic differentiation using hPSCs, including the use of animal-derived and unidentified substances in culture fluids, co-culture with trophoblast cells or stromal cell lines, and induction of excessive concentrations of growth factors. Mesoderm differentiation, and the use of hematopoietic-associated cytokines to amplify newly generated blood cells. These problems lead to the low reproducibility of current hematopoietic differentiation experiments, limiting the technical application for the production of human blood cells on a large scale and the study of blood cell functions and mechanisms. In existing protocols for hematopoietic differentiation using well-defined medium, human pluripotent stem cell hematopoietic differentiation systems often incorporate high concentrations of different blood cytokine mixtures to drive the development of hematopoietic progenitor cells. In view of the fact that the addition of hematopoietic-associated cytokines adversely affects the maintenance of hematopoietic progenitor cells produced by hPSCs, significantly reducing the yield and proliferative capacity of hematopoietic progenitor cells, thereby reducing the production of functional blood cells that can be used in the biomedical field. The addition of cytokine differentiation means interferes with normal blood development processes and is not suitable as a model for studying human hematopoietic development processes and for testing potential drugs.
因此，迫切需要一种使用成分明确的分化培养基、无细胞因子添加的hPSC 诱导分化体系以解决上述问题，但此前从未报道过这种策略的成功案例。Therefore, there is an urgent need for a well-defined differentiation medium, hPSC without cytokine addition. The differentiation system was induced to solve the above problems, but the success of this strategy has never been reported before.
发明内容Summary of the invention
本发明的目的在于：克服现有利用人多能干细胞进行造血分化的方法中存在的一系列问题和不足，提供一种利用人多能干细胞在成分明确、无细胞因子培养条件下进行定向分化的方法。The object of the present invention is to overcome a series of problems and deficiencies in the prior methods for utilizing human pluripotent stem cells for hematopoietic differentiation, and to provide a directional differentiation of human pluripotent stem cells in a well-defined and cytokine-free culture condition. method.
为达到上述发明目的，本发明提供了一种人多能干细胞(hPSCs)的定向分化方法，其是将人多能干细胞先聚合形成类胚体，然后将类胚体在无外源造血细胞因子、无血清、无基质细胞、成分明确的细胞培养环境下进行二维培养，只添加血管内皮生长因子(VEGF)和骨形态发生蛋白4(BMP4)以诱导中胚层发育。整个分化过程在铺有胶原蛋白IV的表面进行，生成造血祖细胞和血细胞。本发明方法中，血液发育在不使用任何外源性造血细胞因子的条件下开始，血液祖细胞依靠人多能干细胞分化产生的内源性因子维持达16～20天。In order to achieve the above object, the present invention provides a method for the directed differentiation of human pluripotent stem cells (hPSCs), which is to first polymerize human pluripotent stem cells to form embryoid bodies, and then to explant the embryoid bodies in the absence of exogenous hematopoietic cytokines. Two-dimensional culture was carried out in serum-free, stromal-free, and well-defined cell culture environments, and only vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP4) were added to induce mesoderm development. The entire differentiation process is carried out on the surface coated with collagen IV to produce hematopoietic progenitor cells and blood cells. In the method of the present invention, blood development begins without using any exogenous hematopoietic cytokines, and the blood progenitor cells are maintained by endogenous factors produced by differentiation of human pluripotent stem cells for 16 to 20 days.
具体地，本发明人多能干细胞的定向分化方法包括如下步骤：Specifically, the method for directed differentiation of pluripotent stem cells of the present invention includes the following steps:
(1)将人多能干细胞在铺有Matrigel的无血清mTeSR1培养液中进行常规培养；(1) routinely culturing human pluripotent stem cells in Matrigel-free serum-free mTeSR1 medium;
(2)将步骤(1)培养的人多能干细胞制成单细胞悬液，经离心、重悬后，加入培养板中培养，形成类胚体；(2) The human pluripotent stem cells cultured in the step (1) are made into a single cell suspension, and after centrifugation and resuspension, they are added to the culture plate to be cultured to form an embryoid body;
(3)将步骤(2)所得类胚体培养24h后，用添加了人血管内皮生长因子、人骨形态发生蛋白4和Thiazovivin的mTeSR1培养液悬浮类胚体，然后使类胚体贴附于铺有胶原蛋白IV的表面进行贴附培养；(3) After culturing the embryoid body obtained in the step (2) for 24 hours, the embryoid body was suspended in the mTeSR1 culture medium supplemented with human vascular endothelial growth factor, human bone morphogenetic protein 4 and Thiazovivin, and then the embryoid body was attached to the plate. The surface of collagen IV is attached and cultured;
(4)在类胚体贴附培养48h后，换成添加有人血管内皮生长因子的StemLineII培养液继续培养，生成造血祖细胞和血细胞。(4) After 48 hours of attachment to the embryoid body, the StemLine II medium supplemented with human vascular endothelial growth factor was added to continue culture to produce hematopoietic progenitor cells and blood cells.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，本发明所述人多能干细胞可以是人胚胎干细胞(hESCs)、人细胞核移植胚胎干细胞(hNT-ESCs)或人诱导多功能干细胞(hiPSCs)。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, the human pluripotent stem cells of the present invention may be human embryonic stem cells (hESCs), human nuclear transfer embryonic stem cells (hNT-ESCs) or human induced multifunctionality. Stem cells (hiPSCs).
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(2)中，所述单细胞悬液可加入到AggreWell400培养板中进行培养形成类胚体，每个培养孔的加入量为4×105～2×106个细胞。 As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (2), the single cell suspension can be added to the AggreWell400 culture plate to form an embryoid body, and each culture hole is added. The amount is 4 × 10 5 to 2 × 10 6 cells.
更优选地，上述每个培养孔的加入量为1×106个细胞，以形成最佳数量的类胚体。More preferably, each of the above culture wells is added in an amount of 1 × 10 6 cells to form an optimal number of embryoid bodies.
可以理解的是，步骤(2)中，所述单细胞悬液也可以加入AggreWell400培养板以外的其它品牌和型号的细胞培养板中进行培养，每个培养孔的加入量也会随之发生变化。It can be understood that, in the step (2), the single cell suspension can also be cultured in other brands and models of cell culture plates other than the AggreWell 400 culture plate, and the amount of each culture hole will also change. .
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(2)中，每个所述类胚体包含200～500个细胞。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (2), each of the embryoid bodies comprises 200 to 500 cells.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(3)中，所述人血管内皮生长因子的添加量为2～100ng/mL，且当其添加量为50ng/mL时，成本效率最佳。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (3), the human vascular endothelial growth factor is added in an amount of 2 to 100 ng/mL, and when the addition amount is 50 ng/mL. When it comes to cost efficiency.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(3)中，所述人骨形态发生蛋白4的添加量为1～40ng/mL，且当其添加量为1～2ng/mL时，为促进造血分化的最佳浓度。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (3), the human bone morphogenetic protein 4 is added in an amount of 1 to 40 ng/mL, and when the addition amount is 1 to 2 ng. At /mL, the optimal concentration for promoting hematopoietic differentiation.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(3)中，所述Thiazovivin的浓度为1～20μM，且当其浓度为10μM时，对类胚体的贴附和细胞分化最为有利。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (3), the concentration of the Thiazovivin is 1 to 20 μM, and when the concentration is 10 μM, the attachment to the embryoid body and the cells Differentiation is most beneficial.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(3)中，类胚体贴附于铺有胶原蛋白IV的表面进行贴附培养时，所述类胚体的接种密度为15～20个类胚体/cm2。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in step (3), when the embryoid body is attached to the surface coated with collagen IV for attachment culture, the seedling density of the embryoid body It is 15-20 embryoid bodies/cm 2 .
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(3)中，所述胶原蛋白IV为小鼠胶原蛋白IV或人胶原蛋白IV。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (3), the collagen IV is mouse collagen IV or human collagen IV.
更进一步地，所述胶原蛋白IV的铺设量为0.2～10μg/cm2；实验发现，铺设过高浓度的胶原蛋白IV会对造血分化产生消极影响，当胶原蛋白IV的铺设量为0.5μg/cm2时，可达到最佳分化效果。Further, the amount of the collagen IV laid is 0.2 to 10 μg/cm 2 ; it has been found that laying a high concentration of collagen IV has a negative effect on hematopoietic differentiation when the amount of collagen IV laid is 0.5 μg/ At cm 2 , the best differentiation effect can be achieved.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(4)中，以StemLine II培养液继续培养的培养环境为5％二氧化碳、21％氧气、37℃加湿培养环境。本发明在不同种细胞系之间(hESC细胞系，如H1、H9、Mel1、HN14；以及hiPSC细胞系，如IPS9、IPS12)试验发现，传统细胞分化培养所常用的低氧环境(5％氧气，5％二氧化碳)应用于本发明方法中，并不能提高血 细胞的得率。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (4), the culture environment in which the StemLine II culture solution is continuously cultured is a 5% carbon dioxide, 21% oxygen, and 37 ° C humidified culture environment. The present invention finds in a hypoxic environment (5% oxygen) commonly used in traditional cell differentiation cultures between different cell lines (hESC cell lines, such as H1, H9, Mel1, HN14; and hiPSC cell lines, such as IPS9, IPS12). , 5% carbon dioxide) used in the method of the invention does not increase blood Cell yield.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，步骤(4)中，所述StemLine II培养液还添加有非必需氨基酸、GlutaMAX和/或β-巯基乙醇，以达到更好的分化效果。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, in the step (4), the StemLine II culture solution is further added with a non-essential amino acid, GlutaMAX and/or β-mercaptoethanol to achieve better. Differentiation effect.
作为本发明人多能干细胞的定向分化方法的一种优选技术方案，所述StemLine II培养液可替换为其它成分明确、无血清的培养液，如可替换为StemSpan SFEM培养液或STEMdiff APEL培养液。StemSpan SFEM培养液与StemLine II培养液相比，生成人血细胞和血祖细胞得率稍低；STEMdiff APEL培养液与StemLine II培养液相比，能生成等数目或更多的多向潜能血祖细胞。As a preferred technical solution of the directed differentiation method of the pluripotent stem cells of the present invention, the StemLine II culture solution can be replaced with other well-defined, serum-free culture solutions, such as StemSpan SFEM culture solution or STEMdiff APEL culture solution. . Compared with StemLine II medium, StemSpan SFEM medium has a slightly lower yield of human blood cells and hematopoietic cells; STEMdiff APEL medium can produce equal or more multipotential progenitor blood progenitors compared with StemLine II medium. .
本发明人多能干细胞的定向分化方法不仅可以用于生成造血祖细胞和血细胞，还可用于有效生成CD31+CD146+VE-cadherin+CD34+的内皮细胞和CD31-CD146+的间充质细胞。当期望定向分化为内皮细胞和/或间充质细胞时，可在分化第0～2天添加Activin A(0.5～2ng/mL)微微促进CD34+CD43-和CD34+CD43+原始血液细胞的发育，并在此阶段在培养液中选择性添加碱性成纤维细胞生长因子(FGF2)。The directed differentiation method of the pluripotent stem cells of the present invention can be used not only for the production of hematopoietic progenitor cells and blood cells, but also for efficiently producing endothelial cells of CD31 + CD146 + VE-cadherin + CD34 + and mesenchymal cells of CD31 - CD146 + . When it is desired to differentiate into endothelial cells and/or mesenchymal cells, Activin A (0.5-2 ng/mL) can be added on the 0th to 2nd day of differentiation to slightly promote the development of CD34 + CD43 - and CD34 + CD43 + original blood cells. At this stage, basic fibroblast growth factor (FGF2) is selectively added to the culture solution.
相对于现有技术，本发明人多能干细胞的定向分化方法具有如下有益效果：Compared with the prior art, the directed differentiation method of the pluripotent stem cells of the present invention has the following beneficial effects:
(1)本发明使用成分明确且不添加造血细胞因子的培养条件下实现造血分化，可提高分化获得的血细胞产量和质量，还可更准确地还原人体胚胎发育过程，适合作为研究人类血液发育分子机制的模型。(1) The present invention realizes hematopoietic differentiation under the culture condition with clear components and no hematopoietic cytokine addition, can improve the blood cell yield and quality obtained by differentiation, and can more accurately reduce the human embryo development process, and is suitable for studying human blood development molecules. Model of the mechanism.
(2)本发明定向分化方法不使用血清和基质细胞，最少量地使用外部诱导因子，具有成本低，操作步骤简单、容易扩大培养、培养条件易于控制、血液分化过程有序的优点，避免血清中不明成分造成负面影响，显著提高了该方法的可重复性。(2) The method of directed differentiation of the present invention does not use serum and stromal cells, and uses the least amount of external induction factor, and has the advantages of low cost, simple operation steps, easy expansion of culture, easy control of culture conditions, and orderly blood differentiation process, and avoiding serum. The negative effects of the unidentified ingredients have significantly improved the repeatability of the method.
(3)本发明定向分化方法能大量产生可分化为成熟血细胞的造血祖细胞和CD45抗原高表达细胞、巨噬细胞和树突状细胞。(3) The directed differentiation method of the present invention can produce a large number of hematopoietic progenitor cells and CD45 antigen high expressing cells, macrophages and dendritic cells which can differentiate into mature blood cells.
(4)利用本发明定向分化方法，还可通过敲除关键造血基因高度重演人体血液发育过程，在人体血液发育过程的机制研究中具有重要意义。(4) By using the directed differentiation method of the present invention, it is also important to recapture the blood development process of the human body by knocking out key hematopoietic genes, which is of great significance in the study of the mechanism of human blood development.
附图说明 DRAWINGS
下面结合附图和具体实施方式，对本发明人多能干细胞的定向分化方法和有益效果进行详细说明。The method and the beneficial effects of the directed differentiation of the pluripotent stem cells of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
图1为本发明人多能干细胞的定向分化方法的流程示意图。1 is a schematic flow chart showing a method for directing differentiation of human pluripotent stem cells.
图2为本发明实施例中类胚体在培养皿底部培养的电子显微镜图像，将第零天定义为类胚体被冲出AggreWell培养板并铺于胶原蛋白IV表面的当天；其中，A为分化第二天附着的类胚体；B为分化第四天附着的类胚体；C为分化第六天形成的血管状丛；D为分化第十二天时产生的大量非附着血细胞。2 is an electron microscope image of the embryoid body cultured at the bottom of the culture dish in the embodiment of the present invention, and the zeroth day is defined as the day when the embryoid body is punched out of the AggreWell culture plate and laid on the surface of the collagen IV; wherein A is The embryoid bodies attached to the next day of differentiation; B is the embryoid body attached to the fourth day of differentiation; C is the vascular plexus formed on the sixth day of differentiation; and D is a large number of non-adherent blood cells produced on the twelfth day of differentiation.
图3显示本发明定向分化方法过程中的表观特征——“体外血岛”，即形成的血管丛状结构和产生最早CD43+血细胞的VE-CADHERIN+血细胞团；其中，A为人胚胎干细胞H1细胞系分化第六天的情况；B为人胚胎干细胞H1细胞系分化第八天的情况；上排为显微镜明场视野，下排为免疫细胞化学染色结果。Figure 3 shows the apparent characteristics of the directed differentiation method of the present invention - "in vitro blood island", that is, the formed vascular plexus structure and the VE-CADHERIN + blood cell mass producing the earliest CD43 + blood cells; wherein A is human embryonic stem cell H1 The sixth day of cell line differentiation; B is the eighth day of differentiation of human embryonic stem cell H1 cell line; the upper row is the field of view of the microscope, and the lower row is the result of immunocytochemical staining.
图4显示本发明定向分化方法中，人多能干细胞不同分化时期血液和内皮相关细胞表面抗原的表达情况；其中，A显示早期CD43+血细胞(灰色标示)共表达CD146(间充质性)和CD31(内皮性)抗原；分化晚期，表达这三个抗原的细胞群逐渐分开；B显示CD235a抗原(glycophorin A)表达持续降低，以及CD41a+细胞增多，CD33+细胞用灰色标记；C显示CD43+早期血细胞为CD31+(灰色标示)，并表达低水平VE-CADHERIN。Figure 4 is a graph showing the expression of blood and endothelial-associated cell surface antigens in different stages of differentiation of human pluripotent stem cells in the directed differentiation method of the present invention; wherein A shows that early CD43 + blood cells (grey markers) co-express CD146 (mesenchymal) and CD31 (endothelium) antigen; in the late stage of differentiation, the cell populations expressing these three antigens are gradually separated; B shows that CD235a antigen (glycophorin A) expression is continuously decreased, and CD41a + cells are increased, CD33 + cells are marked with gray; C shows CD43 + Early blood cells are CD31 + (grey label) and express low levels of VE-CADHERIN.
图5显示本发明定向分化方法中，人多能干细胞随着分化逐渐丧失其全能性，并分化为造血祖细胞；其中，A显示造血分化起始阶段，人多能干细胞全能性抗原表达下调；分化第六至八天，E-cadherin(CDH1,CD324)表达于一群未成熟红系细胞上；B显示不同时间点，CD43+CD45+血祖细胞在无细胞因子条件下分化情况，且CD45+细胞在分化第十天后，占所有活细胞数64％之多。5 shows that in the directed differentiation method of the present invention, human pluripotent stem cells gradually lose their pluripotency with differentiation and differentiate into hematopoietic progenitor cells; wherein A shows the initial stage of hematopoietic differentiation, and the expression of human pluripotent stem cell pluripotency antigen is down-regulated; Six to eight days of differentiation, E-cadherin (CDH1, CD324) was expressed on a group of immature erythroid cells; B showed different time points, CD43 + CD45 + blood progenitor cells differentiated without cytokines, and CD45 + After the tenth day of differentiation, the cells accounted for 64% of all living cells.
图6为本发明方法定向分化后所得细胞进行血细胞集落数分析结果，分化的全细胞在消化后加入无血清甲基纤维素培养基培养，14～18天后对集落进行计数；其中，A为利用人多能干细胞分化第12天和16天细胞进行集落分析的血细胞集落数，注意产生CFU-mix(红系髓系)和CFU-G,M,GM(髓系)的造血祖细胞随着分化得以保持或增加；B显示利用iPS12细胞系分化，第十六天血祖细胞全存在于CD43+细胞群中；C为几种利用人多能干细胞分化形成的典型血细胞集落形态；A和B中均是以四个柱为一组进行比较的柱状图，每四个柱中，从左至右 依次为：CFU-E，BFU-E、CFU-G/M/GM和CFU-Mix。Fig. 6 shows the results of blood cell colony analysis of cells obtained after directed differentiation of the method of the present invention, and the differentiated whole cells are cultured in a serum-free methylcellulose medium after digestion, and the colonies are counted after 14 to 18 days; wherein A is utilized. Human pluripotent stem cell differentiation on the 12th and 16th day cells for colony analysis of the number of blood cell colonies, pay attention to the production of CFU-mix (erythroid myeloid) and CFU-G, M, GM (myeloid) hematopoietic progenitor cells with differentiation It can be maintained or increased; B shows differentiation using iPS12 cell line, and blood progenitor cells are all present in CD43 + cell population on the 16th day; C is a typical blood cell colony morphology formed by differentiation of human pluripotent stem cells; A and B They are all a histogram comparing four columns. In each of the four columns, from left to right are: CFU-E, BFU-E, CFU-G/M/GM and CFU-Mix.
图7为CD43+和CD43-细胞中转录因子(TFs)、细胞因子受体和其它血祖细胞相关基因表达水平情况；RNaseq转录组分析源于本发明定向分化方法第十二天的细胞；CD43+和CD43-细胞群各三个重复被用于分析。Figure 7 shows the expression levels of transcription factors (TFs), cytokine receptors and other blood progenitor-related genes in CD43 + and CD43 - cells; RNaseq transcriptome analysis derived from cells on the twelfth day of the directed differentiation method of the present invention; CD43 Three replicates of + and CD43 - cell populations were used for analysis.
具体实施方式Detailed ways
为了使本发明的目的、技术方案和有益技术效果更加清晰，以下结合实施例，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的实施例仅仅是为了解释本发明，并非为了限定本发明，实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。In order to make the objects, technical solutions and beneficial technical effects of the present invention more clear, the present invention will be further described in detail below with reference to the embodiments. It is to be understood that the embodiments described in the specification are merely illustrative of the invention and are not intended to limit the invention. The parameters, proportions, and the like of the embodiments may be selected in accordance with the present invention without substantially affecting the results.
实施例1Example 1
(1)人多能干细胞(hPSCs)在铺有Matrigel的无血清mTeSR1培养液中进行常规培养。在对hPSCs进行3～5次短间隔的传代后，对处于指数生长阶段的细胞用胰蛋白酶消化成单细胞悬液。离心后接着用含有1μM Thiazovivin的mTeSR1培养液重悬浮细胞，并将单细胞悬液加入AggreWell培养板中形成EBs，每个AgreeWell400型号培养孔一百万细胞。每个EB含有200～500个细胞。(1) Human pluripotent stem cells (hPSCs) were routinely cultured in Matrigel-free serum-free mTeSR1 medium. After 3 to 5 short-interval passages of hPSCs, cells in the exponential growth phase were trypsinized into single cell suspensions. After centrifugation, the cells were resuspended in mTeSR1 medium containing 1 μM Thiazovivin, and the single cell suspension was added to AggreWell plates to form EBs, and each AgreeWell 400 model culture well was one million cells. Each EB contains 200 to 500 cells.
(2)EBs在标准hPSCs培养条件下(5％二氧化碳，21％氧气，37℃，加湿环境)培养24h，之后小心将EBs用移液器吹打出来，用含有50ng/mL的hVEGF165，2ng/mL hBMP4和10μM Thiazovivin的mTeSR1培养液悬浮后，加在铺有小鼠或人胶原蛋白IV(0.5μg/cm2)的塑料培养板上培养。EBs的培养密度为每平方厘米15～20个。在分化第0到第2天添加Activin A(0.5～2ng/mL)微微促进CD34+CD43-和CD34+CD43+原始血液细胞的发育。(2) EBs were cultured for 24 h under standard hPSCs culture conditions (5% carbon dioxide, 21% oxygen, 37 °C, humidified environment), then carefully pipetted out EBs with a pipette containing 50 ng/mL of hVEGF 165 , 2 ng/ mL hBMP4 and 10 μM Thiazovivin in mTeSR1 medium were suspended and cultured on plastic plates plated with mouse or human collagen IV (0.5 μg/cm 2 ). The culture density of EBs is 15-20 per square centimeter. Addition of Activin A (0.5-2 ng/mL) on days 0 to 2 of differentiation slightly promoted the development of CD34 + CD43 - and CD34 + CD43 + primordial blood cells.
(3)EBs在培养皿底部附着培养48小时(请参见图2)后，将培养液换成添加有非必须氨基酸，GlutaMAX，2-mercaptoethano和50ng/mL hVEGF165的StemLine II(5％二氧化碳，21％氧气，37℃，加湿环境)。从此阶段起，每隔一天换一半培养液，换液时应小心以避免吸走沉积在底部的非粘附性血细胞(本发明定向分化流程请参见图1)。(3) After EBs were incubated at the bottom of the culture dish for 48 hours (see Figure 2), the culture medium was replaced with StemLine II (5% carbon dioxide, supplemented with non-essential amino acids, GlutaMAX, 2-mercaptoethano and 50 ng/mL hVEGF 165 . 21% oxygen, 37 ° C, humidified environment). From this stage, half of the culture medium should be changed every other day. Care should be taken to avoid aspiration of non-adherent blood cells deposited at the bottom (see Figure 1 for the directed differentiation process of the present invention).
请参见图3，图3显示本实施例定向分化过程中的表观特征——“体外血岛”，即形成的血管丛状结构和产生最早CD43+血细胞的VE-CADHERIN+血细胞团； 可以看出，在人胚胎干细胞H1细胞系分化第六天，CD43+血细胞出现在“体外血岛”的周围；人胚胎干细胞H1细胞系分化第八天，体外血岛造血和血细胞分散，CD43+血细胞开始从VE-CADHERIN+“体外血岛”向外分散。注意“体外血岛”坐落于VE-CADHERIN+的单层内皮细胞上。Referring to FIG. 3, FIG. 3 shows the apparent characteristics during the directed differentiation of the present embodiment - "in vitro blood island", that is, the formed vascular plexus structure and the VE-CADHERIN + blood cell mass which produces the earliest CD43 + blood cells; On the sixth day of differentiation of human embryonic stem cell H1 cell line, CD43 + blood cells appeared around the "in vitro blood island"; human embryonic stem cell H1 cell line differentiated on the eighth day, blood island hematopoiesis and blood cell dispersion in vitro, CD43 + blood cells began Dispersed from VE-CADHERIN + "in vitro blood island". Note that the "in vitro blood island" is located on a single layer of endothelial cells of VE-CADHERIN + .
在利用hESC细胞系H1进行分化时，最早的CD43+血细胞出现于分化第四天，CD43+CD45+血祖细胞最先在分化第八天被检测到，之后CD43+CD45+细胞群分化并扩增占到整个培养体系中超过50％的细胞(请参见图5)。分化第十天后，CD45高表达细胞群主要包括成熟的巨噬细胞和树突状细胞。在分化过程中内皮细胞表达高水平的VE-CADHERIN、CD34和CD31，而血细胞中这些表面抗原处于低至中表达水平。CD43+血细胞上的内皮抗原表达水平随着分化的持续而稳步降低。由图4可见，早期CD43+血细胞共表达CD146(间充质性)和CD31(内皮性)抗原；分化晚期，表达这三个抗原的细胞群逐渐分开；CD235a抗原(glycophorin A)表达持续降低，以及CD41a+细胞增多；CD43+早期血细胞为CD31+，并表达低水平VE-CADHERIN。When the hESC cell line H1 was used for differentiation, the earliest CD43 + blood cells appeared on the fourth day of differentiation, and CD43 + CD45 + blood progenitor cells were first detected on the eighth day of differentiation, after which the CD43 + CD45 + cell population differentiated and expanded. Overtakes more than 50% of the cells in the entire culture system (see Figure 5). After the tenth day of differentiation, the CD45 high expression cell population mainly includes mature macrophages and dendritic cells. Endothelial cells express high levels of VE-CADHERIN, CD34 and CD31 during differentiation, whereas these surface antigens are in low to medium expression levels in blood cells. The level of endothelial antigen expression on CD43 + blood cells steadily decreased as differentiation progressed. As can be seen from Fig. 4, early CD43 + blood cells co-expressed CD146 (mesenchymal) and CD31 (endothelial) antigens; in the late stage of differentiation, the cell populations expressing these three antigens gradually separated; the expression of CD235a antigen (glycophorin A) continued to decrease. And CD41a + cells increased; CD43 + early blood cells were CD31 + and expressed low levels of VE-CADHERIN.
实施例2Example 2
本实施例提供了扩大细胞培养规模的方法，步骤同实施例1，不同之处是在分化第二至四天时，用胰酶处理细胞，然后重新铺回包被有胶原蛋白IV的培养板或培养皿上，再在标准培养环境下孵育1h。接着轻轻吹打吸除仍悬浮或半吸附状态的细胞，以除去残存的非分化hPSCs。吸附的细胞部分可以在原来的培养皿内继续按上述条件培养，或按照每平方厘米一万细胞数的密度播种在新的胶原蛋白IV包被过的培养皿内培养。本实施例方法可以扩大整个细胞培养规模约十倍。This example provides a method for expanding the scale of cell culture, the same steps as in Example 1, except that the cells are treated with trypsin at the second to fourth days of differentiation, and then the plate coated with collagen IV is re-plated or Incubate on a culture dish and incubate for 1 h in a standard culture environment. The cells in the still suspended or semi-adsorbed state are then gently aspirated to remove residual undifferentiated hPSCs. The adsorbed cell fraction can be cultured in the original culture dish under the above conditions, or seeded in a new collagen IV-coated petri dish at a density of 10,000 cells per square centimeter. The method of this example can expand the entire cell culture scale by about ten times.
实验例1Experimental example 1
为了分析分化过程中生成的血祖细胞，处于不同分化阶段的全细胞或分选后细胞被添加到含有人细胞因子SCF、G-CSF、GM-CSF、IL-3、IL-6和EPO的无血清甲基纤维素培养基中进行集落分析。最早的血祖细胞出现于分化第四天。大部分具有血液集落生成能力的血细胞存在于悬浮细胞部分。基本上所有血祖细胞都存在于CD43+细胞群中(请参见图6)。CD43+细胞与CD43-细胞相比，高表达很多血细胞相关基因(请参见图7)。在分化的早期，红系祖细胞和多向潜能祖 细胞(CFU-E,BFU-E and CFU-Mix)是主要产生的造血祖细胞。在分化后期(分化第十四至十六天)，由于无添加外源性细胞因子，多向潜能祖细胞得以很好维持。髓系祖细胞(CFU-M，CFU-G，CFU-GM)显著增长，而红系CFU-Es大量减少(请参见图6)。In order to analyze the progenitor cells generated during differentiation, whole cells or sorted cells at different stages of differentiation were added to human cytokines SCF, G-CSF, GM-CSF, IL-3, IL-6 and EPO. Colony analysis was performed in serum-free methylcellulose medium. The earliest blood progenitor cells appeared on the fourth day of differentiation. Most blood cells with blood colony forming ability are present in the suspended cell fraction. Essentially all blood progenitor cells are present in the CD43 + cell population (see Figure 6). CD43 + cells express a high number of blood cell-associated genes compared to CD43 - cells (see Figure 7). In the early stages of differentiation, erythroid progenitor cells and multipotential progenitor cells (CFU-E, BFU-E and CFU-Mix) are the predominantly hematopoietic progenitors. In the late stage of differentiation (fourteenth to sixteenth day of differentiation), multi-directional progenitor cells were well maintained due to the absence of exogenous cytokines. Myeloid progenitor cells (CFU-M, CFU-G, CFU-GM) increased significantly, while erythroid CFU-Es decreased significantly (see Figure 6).
根据上述说明书的揭示和教导，本发明所属领域的技术人员还可以对上述实施方式进行适当的变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对本发明的一些修改和变更也应当落入本发明的权利要求的保护范围内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制。 The above embodiments may be modified and modified as appropriate by those skilled in the art in light of the above disclosure. Therefore, the invention is not limited to the specific embodiments disclosed and described herein, and the modifications and variations of the invention are intended to fall within the scope of the appended claims. In addition, although specific terms are used in the specification, these terms are merely for convenience of description and do not limit the invention.

Claims (10)

  1. 一种人多能干细胞的定向分化方法，其特征在于，该方法是将人多能干细胞在无外源造血细胞因子、无血清、无基质细胞、成分明确的细胞培养环境下培养，形成类胚体后，添加血管内皮生长因子和骨形态发生蛋白4并使类胚体贴附于铺有胶原蛋白IV的表面，以诱导中胚层分化，生成造血祖细胞和血细胞。The invention relates to a method for directed differentiation of human pluripotent stem cells, which is characterized in that the human pluripotent stem cells are cultured in a cell culture environment without exogenous hematopoietic cytokines, serum-free, stromal cells and components, to form embryos. After the body, vascular endothelial growth factor and bone morphogenetic protein 4 are added and the embryoid body is attached to the surface coated with collagen IV to induce mesoderm differentiation to produce hematopoietic progenitor cells and blood cells.
  2. 根据权利要求1所述的人多能干细胞的定向分化方法，其特征在于，包括如下步骤：The method for directed differentiation of human pluripotent stem cells according to claim 1, comprising the steps of:
    (1)将人多能干细胞在铺有Matrigel的无血清mTeSR1培养液中进行常规培养；(1) routinely culturing human pluripotent stem cells in Matrigel-free serum-free mTeSR1 medium;
    (2)将步骤(1)培养的人多能干细胞制成单细胞悬液，经离心、重悬后，加入培养板中培养，形成类胚体；(2) The human pluripotent stem cells cultured in the step (1) are made into a single cell suspension, and after centrifugation and resuspension, they are added to the culture plate to be cultured to form an embryoid body;
    (3)将步骤(2)所得类胚体培养24h后，用添加了人血管内皮生长因子、人骨形态发生蛋白4和Thiazovivin的mTeSR1培养液悬浮类胚体，然后使类胚体贴附于铺有胶原蛋白IV的表面进行贴附培养；(3) After culturing the embryoid body obtained in the step (2) for 24 hours, the embryoid body was suspended in the mTeSR1 culture medium supplemented with human vascular endothelial growth factor, human bone morphogenetic protein 4 and Thiazovivin, and then the embryoid body was attached to the plate. The surface of collagen IV is attached and cultured;
    (4)在类胚体贴附培养48h后，换成添加有人血管内皮生长因子的StemLine II培养液继续培养，生成造血祖细胞和血细胞。(4) After 48 hours of attachment to the embryoid body, the StemLine II medium supplemented with human vascular endothelial growth factor was added to continue culture to produce hematopoietic progenitor cells and blood cells.
  3. 根据权利要求2所述的人多能干细胞的定向分化方法，其特征在于，所述人多能干细胞包括人胚胎干细胞、人细胞核移植胚胎干细胞和人诱导多功能干细胞。The method for directed differentiation of human pluripotent stem cells according to claim 2, wherein the human pluripotent stem cells include human embryonic stem cells, human nuclear transfer embryonic stem cells, and human induced pluripotent stem cells.
  4. 根据权利要求2所述的人多能干细胞的定向分化方法，其特征在于，步骤(2)中，每个所述类胚体包含200～500个细胞。The method for directed differentiation of human pluripotent stem cells according to claim 2, wherein in the step (2), each of the embryoid bodies comprises 200 to 500 cells.
  5. 根据权利要求2所述的人多能干细胞的定向分化方法，其特征在于，步骤(3)中，所述胶原蛋白IV为小鼠胶原蛋白IV或人胶原蛋白IV。The method for directed differentiation of human pluripotent stem cells according to claim 2, wherein in the step (3), the collagen IV is mouse collagen IV or human collagen IV.
  6. 根据权利要求5所述的人多能干细胞的定向分化方法，其特征在于，所述胶原蛋白IV的铺设量为0.2～10μg/cm2。The method for directed differentiation of human pluripotent stem cells according to claim 5, wherein the amount of the collagen IV laid is 0.2 to 10 μg/cm 2 .
  7. 根据权利要求2所述的人多能干细胞的定向分化方法，其特征在于，步骤(4)中，所述StemLine II培养液继续培养的培养环境为5％二氧化碳、21％氧气、37℃加湿培养环境。 The method for directional differentiation of human pluripotent stem cells according to claim 2, wherein in the step (4), the culture environment in which the StemLine II culture solution is continuously cultured is 5% carbon dioxide, 21% oxygen, and humidified at 37 ° C. surroundings.
  8. 根据权利要求2所述的人多能干细胞的定向分化方法，其特征在于，步骤(4)中，所述StemLine II培养液还添加有非必需氨基酸、GlutaMAX和/或β-巯基乙醇。The method for directed differentiation of human pluripotent stem cells according to claim 2, wherein in the step (4), the StemLine II culture solution is further supplemented with a non-essential amino acid, GlutaMAX and/or β-mercaptoethanol.
  9. 根据权利要求2所述的人多能干细胞的定向分化方法，其特征在于，步骤(4)中，所述StemLine II培养液可替换为StemSpan SFEM培养液、STEMdiff APEL培养液或其它成分明确、无血清的培养液。The method for direct differentiation of human pluripotent stem cells according to claim 2, wherein in the step (4), the StemLine II culture solution can be replaced with StemSpan SFEM culture solution, STEMdiff APEL culture solution or other components, and no Serum broth.
  10. 根据权利要求1～9中任意一条权利要求所述的人多能干细胞的定向分化方法，其特征在于，所述定性分化方法还用于生成内皮细胞和间充质细胞。 The method for directed differentiation of human pluripotent stem cells according to any one of claims 1 to 9, characterized in that the qualitative differentiation method is further used for producing endothelial cells and mesenchymal cells.
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