WO2016045495A1 - 多能干细胞体外定向分化为心肌细胞的方法 - Google Patents

多能干细胞体外定向分化为心肌细胞的方法 Download PDF

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WO2016045495A1
WO2016045495A1 PCT/CN2015/089046 CN2015089046W WO2016045495A1 WO 2016045495 A1 WO2016045495 A1 WO 2016045495A1 CN 2015089046 W CN2015089046 W CN 2015089046W WO 2016045495 A1 WO2016045495 A1 WO 2016045495A1
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medium
differentiation
concentration
cardiomyocytes
pluripotent stem
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French (fr)
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李天晴
蒋斌
季维智
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云南中科灵长类生物医学重点实验室
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  • the present invention relates to the field of cell biology, and in particular to a method for the directed differentiation of pluripotent stem cells into cardiomyocytes in vitro under three-dimensional suspension conditions.
  • Heart disease is a common disease that threatens human health, especially myocardial infarction caused by myocardial ischemia caused by coronary heart disease, which is 1.5 million in the United States and 2 million in China. After myocardial infarction, local myocardial cells in the ventricle will be necrotic, causing local scarring and not being repaired, causing cardiac arrhythmia, insufficient pumping, light affecting the patient's production and life, severe cases cause shock or death, serious Threats to human health and social stability. But there is currently no good treatment. Stem cells have long-term self-renewal and the ability to differentiate into any cell in the body.
  • cardiomyocytes differentiated from stem cells for replacement therapy provides a possibility for the complete treatment of myocardial infarction.
  • human embryonic stem cells can be differentiated into cardiomyocytes, and can be long-term survival by injection into the heart of a monkey with myocardial infarction, forming a cell connection with the monkey's original cardiomyocytes, producing an ECG activity consistent with the monkey heart. With calcium conduction signals, it significantly improves its cardiac function and has a significant therapeutic effect.
  • the former is to digest the stem cells, suspension culture for 4-5 days to form the embryoid body, and then induced by growth factors and drugs, attached to the bottom of the culture dish to differentiate into cardiomyocytes.
  • EBs EBs
  • This method can only make 50-75% of the cells in EBs.
  • It is a functional cardiomyocyte, and the differentiated cells contain non-myocardial cells of the inner, middle and outer germ layers. Therefore, it is necessary to subject the differentiated cardiomyocytes to complex sorting and purification before being used for drug screening and transplantation.
  • the adherent differentiation method is to grow stem cells in a high-density manner in a culture dish and induce them with a medium in which a combination of expensive cell growth factors and small molecules are combined.
  • the adherent culture method adopts a planar 2D culture method, and stem cells are induced to differentiate by single layer growth, and the production capacity is greatly limited, which is disadvantageous for practical use and industrial production.
  • these methods require special substrates such as martrigel, laminin, etc. on the one hand; and various expensive protein growth factors such as Activin A, BMP4, bFGF, etc., on the other hand.
  • These matrices and growth factors have significant drawbacks: unidentified ingredients, high prices, large differences between batches, and some are animal-derived, resulting in low safety.
  • the present invention first provides a medium for establishing differentiation of cardiomyocytes in vitro under three-dimensional suspension conditions, comprising a differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells for use in The germ layer precursor cells differentiate into a differentiation medium for cardiomyocytes and a long-term maintenance medium for cardiomyocytes.
  • the differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells comprises: a base medium is a mixed medium of DMEM/F12 and IMDM, a KOSR (serum substitute), ⁇ -mercaptoethanol, Glutamine, NEAA (non-essential amino acids), CHIR99021 (agonist of the Wnt signaling pathway) and BIO (agonist of the Wnt signaling pathway).
  • the differentiation medium for differentiating mesodermal precursor cells into cardiomyocytes comprises: a base medium is a mixed medium of DMEM/F12 and IMDM, KOSR, ⁇ -mercapto B Alcohol, glutamine, NEAA, IWP-2 (an inhibitor of the Wnt signaling pathway), XAV939 (an inhibitor of the Wnt signaling pathway), and ascorbic acid.
  • a base medium is a mixed medium of DMEM/F12 and IMDM, KOSR, ⁇ -mercapto B Alcohol, glutamine, NEAA, IWP-2 (an inhibitor of the Wnt signaling pathway), XAV939 (an inhibitor of the Wnt signaling pathway), and ascorbic acid.
  • the long-term maintenance medium of the cardiomyocytes includes: the basic culture medium is a mixed medium of DMEM/F12 and IMDM, KOSR, ⁇ -mercaptoethanol, glutamine, NEAA, and ascorbic acid.
  • the medium for inducing myocardial differentiation comprises mesodermal differentiation medium, myocardial differentiation medium and long-term maintenance medium of cardiomyocytes, and the differentiation medium contains various nutrients for pluripotent stem cells to differentiate into cardiomyocytes. ingredient.
  • CHIR99021 and BIO small molecules act as Wnt signaling activators, which can inhibit pluripotent stem cell self-renewal and promote its differentiation into mesoderm lineage cells
  • IWP-2 and XAV939 small molecules act as inhibitors of Wnt signaling pathway and promote mesoderm Somatic cells differentiate into myocardial precursors and cardiomyocytes
  • ascorbic acid can inhibit cell death during cell differentiation and has a function of promoting cardiomyocyte differentiation.
  • the volume ratio of DMEM/F12 mixed IMDM is 5:1 to 1:5; the concentration of the KOSR is 2 -20%; the concentration of the NEAA is 0.1-2%; the concentration of the ⁇ -mercaptoethanol is 0.1-1%; the concentration of the glutamine is 10-200 mM; the concentration of the CHIR99021 is 1-20 ⁇ M The concentration of the BIO is 1-20 ⁇ M.
  • the volume ratio of DMEM/F12 mixed IMDM is 5:1-1:8; the concentration of the KOSR is 2-20 %; the concentration of the NEAA is 0.1-2%; the concentration of the ⁇ -mercaptoethanol is 0.1-1%; the concentration of the glutamine is 10-200 mM; the concentration of the IWP-2 is 2-30 ⁇ M The concentration of the XAV939 is 1-20 ⁇ M; the concentration of the ascorbic acid is 5-500 ⁇ g/L.
  • the volume ratio of DMEM/F12 mixed IMDM is 5:1-1:8; the concentration of the KOSR is 2-20%; the concentration of the NEAA is 0.1 -2%; the concentration of the ⁇ -mercaptoethanol is 0.1-1%; the concentration of the glutamine is The degree is 10-200 mM; the concentration of the ascorbic acid is 5-500 ⁇ g/L.
  • the invention also provides a method for direct differentiation of pluripotent stem cells into cardiomyocytes in vitro under three-dimensional suspension conditions, comprising the following steps:
  • the cell pellet obtained by cutting is placed in a cell culture incubator for three-dimensional suspension culture, and cultured in a stem cell culture medium for 4-5 days to obtain a pluripotent stem cell sphere having a diameter of 200-300 ⁇ m;
  • the pluripotent stem cell of step 2) is removed from the stem cell culture medium, and the above-mentioned differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells is subjected to differentiation culture under the condition of three-dimensional suspension, and is recorded as D0. Days and later, under the differentiation culture at this time, the different days are represented as D1, D2, D3, etc.; on D2 days of differentiation culture, the medium is replaced with the above-mentioned differentiation for differentiation of mesoderm precursor cells into cardiomyocytes. The medium was further subjected to differentiation culture; on the D12 day of the differentiation culture, the medium was replaced with the long-term maintenance medium of the above cardiomyocytes for culture.
  • human stem cells begin to show significant myocardial beating balls on D6-D8 days; stem cells of non-human primates begin to show significant myocardial beating balls on D4-D6 days.
  • the differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells is capable of producing mesodermal precursor cells of 60-100% purity.
  • the differentiation medium for differentiating mesodermal precursor cells into cardiomyocytes is capable of producing 60-100% pure myocardial precursor cells.
  • the long-term maintenance medium of the cardiomyocytes is capable of producing cardiomyocytes of 60-100% purity.
  • the pluripotent stem cells involved in the present invention include human and non-human primate embryonic stem cells and induced pluripotent stem cells.
  • the formula of the stem cell culture medium is: DMEM/F12 addition KOSR, NEAA, ⁇ -mercaptoethanol, glutamine and bFGF; the concentration of the KOSR is 2-20%; the concentration of the NEAA is 0.1-2%; and the concentration of the ⁇ -mercaptoethanol is 0.1-1%
  • the concentration of the glutamine is 1-5 mM, and the concentration of the bFGF is 1-50 ng/ml.
  • the present invention also provides a differentiated mesodermal precursor cell line, a myocardial precursor cell line, and a cardiomyocyte cell line obtained by the above method.
  • the invention further provides the use of the above cell line in cell replacement therapy, heart disease pathogenesis research and drug screening.
  • the method of the invention can efficiently differentiate pluripotent stem cells into cardiomyocytes in a 3D suspension culture system within 12 days, and the differentiation efficiency reaches 60-95% or more, and the differentiated cardiomyocytes express cTnT, a-Actinin, etc. 60-95% or more.
  • the differentiated cardiomyocytes After transmission electron microscopy analysis, the differentiated cardiomyocytes have the same microscopic structure as the in vivo heart, such as rich myofilament, clear sarcomere, a large number of mitochondria, and abundant intercellular connections, such as sputum.
  • the sinus electrical signal can be detected in a myocardial ball by the method of the invention, and the atrial electrical signal and the ventricular electrical signal have physiological electrical signals similar to those of the inner body.
  • the cardiomyocytes obtained by the method of the present invention can produce a similar reaction to the existing cardiac drugs, and the calcium-wave analysis can be used to accelerate the calcium wave frequency of the myocardial ball produced by the method of the present invention by adding the caffeine for accelerating the heart rate. Drugs such as propranolol which lowers the heart rate can significantly reduce the calcium wave frequency of the myocardial sphere.
  • the method of the present invention does not require expensive and unidentified biological products such as Matrigel, laminin, etc. as a matrix, and the whole differentiation process is cultured and induced to differentiate in a suspension culture system, and the induced differentiation agents used are all inexpensive chemical small molecules.
  • the method of the invention is simple and reliable, low in price, stable and efficient, and high in safety. Due to the suspension culture system, high quality cardiomyocytes can be industrially produced without any subsequent screening and purification steps, and can be directly used for cardiac development research. Cellular treatment of heart disease, transplantation of heart damage, and application requirements for drug screening have immeasurable scientific and socioeconomic benefits.
  • Example 1 is a schematic diagram showing the preparation of uniform pluripotent stem cell globules using human and non-human primate embryonic stem cells in Example 2 of the present invention.
  • Example 2 is a schematic view showing the directional differentiation of pluripotent stem cell beads into autonomous beating myocardial spheres under three-dimensional suspension culture conditions in Example 2 of the present invention.
  • Fig. 3 is a schematic view showing the screening of differentiated cardiomyocytes and drug for myocardial spheres in Example 2 of the present invention.
  • the formulation of the stem cell culture medium used in the following examples was added to DMEM/F12 with KOSR, NEAA, ⁇ -mercaptoethanol, glutamine, bFGF; the concentration of the KOSR was 2%-20%; the concentration of the NEAA was 0.1%. -2%; the concentration of the ⁇ -mercaptoethanol is 0.1-1%, the concentration of the glutamine is 1-5 mM, and the bFGF is 1-50 ng/ml.
  • Example 1 is used to establish a medium for inducing cardiomyocyte differentiation in vitro under three-dimensional suspension conditions
  • the medium for inducing cardiomyocyte differentiation in vitro under the three-dimensional suspension condition provided in the present embodiment includes: a differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells for using mesoderm precursor cells A differentiation medium that differentiates into cardiomyocytes and a long-term maintenance medium for cardiomyocytes.
  • the differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells comprises: a base medium is a mixed medium of DMEM/F12 and IMDM, KOSR (serum substitute), ⁇ -mercaptoethanol, glutamine Amide, NEAA (non-essential amino acid), CHIR99021 (agonist of the Wnt signaling pathway) and BIO (agonist of the Wnt signaling pathway).
  • a base medium is a mixed medium of DMEM/F12 and IMDM, KOSR (serum substitute), ⁇ -mercaptoethanol, glutamine Amide, NEAA (non-essential amino acid), CHIR99021 (agonist of the Wnt signaling pathway) and BIO (agonist of the Wnt signaling pathway).
  • the differentiation medium for differentiating the mesoderm precursor cells into cardiomyocytes comprises: the basic culture medium is a mixed medium of DMEM/F12 and IMDM, KOSR, ⁇ -mercaptoethanol, glutamine, NEAA, IWP-2 (Inhibitor of Wnt signaling pathway), XAV939 (inhibitor of Wnt signaling pathway) and ascorbic acid.
  • the basic culture medium is a mixed medium of DMEM/F12 and IMDM, KOSR, ⁇ -mercaptoethanol, glutamine, NEAA, IWP-2 (Inhibitor of Wnt signaling pathway), XAV939 (inhibitor of Wnt signaling pathway) and ascorbic acid.
  • the long-term maintenance medium of the cardiomyocytes includes: the basic culture medium is a mixed medium of DMEM/F12 and IMDM, KOSR, ⁇ -mercaptoethanol, glutamine, NEAA, and ascorbic acid.
  • the following is a differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells of the present embodiment, a differentiation medium for differentiating mesoderm precursor cells into cardiomyocytes, and a long-term maintenance medium for cardiomyocytes. Specific examples.
  • Differentiation medium for inducing differentiation of pluripotent stem cells into mesodermal precursor cells is a substance for inducing differentiation of pluripotent stem cells into mesodermal precursor cells
  • Example 1 DMEM/F12 and IMDM were mixed in a 5:1 volume ratio, 5% KOSR, 1% NEAA, 0.025% ⁇ -mercaptoethanol, 100 mM glutamine, 5 ⁇ M CHIR99021 and 5 ⁇ M BIO.
  • Example 2 DMEM/F12 was mixed with IMDM in a 1:1 volume ratio, 10% KOSR, 1% NEAA, 0.025% ⁇ -mercaptoethanol, 50 mM glutamine, 10 ⁇ M CHIR99021 and 10 ⁇ M BIO.
  • Example 3 DMEM/F12 was mixed with IMDM in a 1:5 volume ratio, 10% KOSR, 1% NEAA, 0.025% ⁇ -mercaptoethanol, 50 mM glutamine, 3 ⁇ M CHIR99021 and 3 ⁇ MIO.
  • Example 1 The basal medium was mixed with DMEM/F12 and IMDM in a volume ratio of 5:1, 5% KOSR, 0.025% ⁇ -mercaptoethanol, 100 mM glutamine, 1% NEAA, 10 ⁇ MIWP-2, 5 ⁇ M XAV939 (Wnt signaling pathway) Inhibitor) and 50 ⁇ g/L ascorbic acid.
  • Example 2 The basal medium was mixed with DMEM/F12 and IMDM in a volume ratio of 1:2, 10% KOSR, 0.025% ⁇ -mercaptoethanol, 50 mM glutamine, 1% NEAA, 5 ⁇ M IWP-2, 2 ⁇ M XAV939 (Wnt signal Inhibitor of the pathway) and 25 ⁇ g/L ascorbic acid.
  • Example 3 The basal medium was mixed with DMEM/F12 and IMDM in a volume ratio of 1:8, 10% KOSR, 0.025% ⁇ -mercaptoethanol, 100 mM glutamine, 1% NEAA, 20 ⁇ MIWP-2, 10 ⁇ M XAV939 (Wnt signaling pathway) Inhibitor) and 100 ⁇ g/L ascorbic acid.
  • Example 1 The base medium was DMEM/F12 mixed with IMDM in a volume ratio of 5:1, 5% KOSR, 0.025% ⁇ -mercaptoethanol, 100 mM glutamine, 1% NEAA and 50 ⁇ g/L ascorbic acid.
  • Example 2 The base medium was DMEM/F12 mixed with IMDM in a volume ratio of 1:2, 10% KOSR, 0.025% ⁇ -mercaptoethanol, 50 mM glutamine, 1% NEAA and 25 ⁇ g/L ascorbic acid.
  • Example 3 The base medium was DMEM/F12 mixed with IMDM in a volume ratio of 1:8, 10% KOSR, 0.025% ⁇ -mercaptoethanol, 100 mM glutamine, 1% NEAA and 100 ⁇ g/L ascorbic acid.
  • Example 2 Method for directional differentiation of pluripotent stem cells into cardiomyocytes in vitro under three-dimensional suspension conditions
  • Example 1 Using the differentiation medium corresponding to Example 1 in Example 1 for inducing differentiation of pluripotent stem cells into mesodermal precursor cells, for differentiating medium for differentiation of mesoderm precursor cells into cardiomyocytes and long-term maintenance of cardiomyocytes
  • the medium is used to establish a method for differentiating pluripotent stem cells into cardiomyocytes in vitro under three-dimensional suspension conditions. Includes the following steps:
  • pluripotent stem cell spheroids of uniform size using pluripotent stem cells of human and non-human primates prepared pluripotent stem cells of human and non-human primates. Specifically, in this step, pluripotent stem cells under two-dimensional conditions are used for experiments, and the pluripotent stem cells are involved in feeder layer co-culture and feeder-free culture of pluripotent stem cells, and the stem cell culture medium is removed, and 1 ⁇ PBS is washed 2 Add 1mg/ml dispase 2-4ml to cover pluripotent stem cells, incubate for 4-8mins at 37°C; when the edge of the stem cell clone is rolled up, remove dispase, wash 2 times with 1 ⁇ PBS; add 3ml stem cell culture medium, dry cells The clones were gently washed off; the stem cells were cloned, 3 ml of stem cell medium and 10 ⁇ M Y-27632 were added to mix; the resuspended stem cells were passed through a 50
  • Figure 1 shows the process of three-dimensional suspension culture of pluripotent stem cells to produce sized pluripotent stem cell spheres.
  • A-B is used to cut cell sieves of pluripotent stem cells
  • C The pluripotent stem cells after cell sieve cutting are fast
  • D is a schematic diagram of the method for forming pluripotent stem cell spheres after dicing pluripotent stem cells in three-dimensional suspension condition
  • EG pluripotent stem cell mass in three-dimensional suspension culture In the process, pluripotent stem cell spheres produced by different days
  • HI is a representative pluripotent stem cell sphere within five days, the stem cell size and cell number increase
  • J three-dimensional suspension long-term culture The stem cells still maintain a stable karyotype
  • (KO) three-dimensional suspension cultured pluripotent stem cells still maintain the pluripotency of stem cells, expressing stem cell marker proteins such as Oct4, SSEA-4, TRA-1-60 and TRA-1 -80, but does not
  • the pluripotent stem cell globules were differentiated into self-pulsating myocardial spheres under suspension culture conditions. Specifically, in this step, the pluripotent stem cell pellet is collected into a centrifuge tube, centrifuged at a speed of 600 rpm for 3 minutes; the stem cell culture medium is removed, the mesoderm precursor cell differentiation medium is added, and the cells are resuspended, the time period. Recorded as D0 days; cells were cultured in Petri dishes and in a 37 ° C, 5% CO 2 incubator; on day D2, the mesoderm precursor cell differentiation medium was completely replaced with cardiomyocyte differentiation medium, changed every 1-2 days.
  • the fluid was once; on the D6 day, the self-pulsating myocardial sphere was observed; from D12 days, the myocardial differentiation medium was changed into a long-term maintenance medium of cardiomyocytes, and the medium was changed once every 1-2 days (Fig. 2).
  • FIG. 2 shows the process of differentiation of pluripotent stem cell spheres produced by three-dimensional suspension into cardiomyocyte spheres.
  • A is a schematic diagram of the whole differentiation process;
  • BD is a pluripotent stem cell that differentiates into mesodermal precursor cells after two days of action in the mesoderm precursor cell differentiation medium, and 99.6% of the cells express Brachyury;
  • EG differentiated mesenchymal precursor cells into myocardial precursor cells after two days of differentiation, and 99.3% of cells express NKX2.5;
  • HM differentiated myocardial precursor cells in cardiac differentiation medium After 12-14 days of continuous action, differentiated into mature cardiomyocytes, 95.7% of cells express ⁇ -actinin and cTnT;
  • N is the ratio of spontaneously beating cardiomyocytes to total stem cell spheres during different days of differentiation; a differentiated cardiomyocyte that is digested into a single cardiomyocyte, Re-single-layer culture and staining showed that the differentiated cardiomy
  • pluripotent stem cell pellet and its subsequently differentiated cells were fixed with 4% PFA (paraformaldehyde); embedded in OCT, frozen section, slice thickness 10 ⁇ m; soaked in 1 ⁇ PBS for 10 min, removed Embedding agent OCT; 1 ⁇ PBS soaked section, 5 min; 0.2% Triton X100 permeable membrane for 10 min; 5% serum blocked for 2 h; add primary antibody at 4 ° C overnight, antibody ratio was SSEA4 1:400, Brachyury 1:300, Nkx2.51 :300, CTnT 1:400, a-Actinin 1:400; 1 ⁇ PBS, 3 min, 4 times addition of secondary antibody, normal temperature for 1 hour, antibody ratio is 1:1000; 1 ⁇ PBS, 3 min, 4 times; add DAPI Staining, 5 min; 1 x PBS, 3 min, 2 times,
  • Differentiated cardiomyocytes exhibit the function of cardiomyocytes and show potential for response to cardiac drugs.
  • the differentiated myocardial sphere is placed on a cardiomyocyte function tester to detect its action potential; for the detection of calcium waves, the fluorescent calcium indicator of Furo 4 is mixed with the medium for 1:1 for 20 minutes.
  • a continuous rapid sweep was performed using a Leica co-microscopy microscope to capture continuous calcium waves.
  • caffeine and propranolol were added to the long-term maintenance solution of cardiomyocytes according to a certain concentration, and the calcium waves of the cardiomyocytes were detected.
  • the role of drugs in myocardial globules establishes a drug screening platform (Figure 3).
  • FIG. 3 shows the process of drug screening using three-dimensional suspension-differentiated cardiomyocytes.
  • A is a differentiated cardiomyocyte showing three typical action potentials of cardiomyocytes, which are atrial, ventricular and sinus nodes respectively;
  • BC differentiated myocardial spheres have typical myocardial calcium waves;
  • DE Caffeine significantly promotes the contraction of cardiomyocytes, while propranolol significantly inhibits the contraction of cardiomyocytes.
  • mesoderm precursor cells, myocardial precursor cells, cardiomyocytes, and cell lines produced by the three types of cell cultures established by the method of the present embodiment; and cell lines produced by these three types of cell culture are in cell replacement therapy, Cardiac disease pathogenesis and drug screening
  • the selected application is also within the scope of the invention.
  • the invention provides a method for direct differentiation of pluripotent stem cells into cardiomyocytes in vitro under three-dimensional suspension conditions, the method is simple and reliable, low in price, stable and efficient, and high in safety, and can be industrially produced with high quality due to the use of a suspension culture system.
  • Cardiomyocytes without any subsequent screening and purification steps, can be directly used for cardiac developmental research, cell therapy for heart disease, transplantation of cardiac lesions, and application requirements for drug screening, with immeasurable scientific and socioeconomic benefits.

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Abstract

提供了一种在三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法，还提供了用于建立三维悬浮条件下体外诱导心肌细胞分化的培养基。该培养基包括用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基，用于将中胚层前体细胞分化为心肌细胞的分化培养基以及心肌细胞的长期维持培养基。

Description

多能干细胞体外定向分化为心肌细胞的方法 技术领域
本发明涉及细胞生物学领域，具体地说，涉及在三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法。
背景技术
心脏疾病是威胁人类健康的常见疾病，尤其是冠心病等造成的心肌缺血导致的心肌梗死病例，每年在美国有150万，在中国有200万。发生心肌梗死后，心室的局部心肌细胞会发生坏死，引起局部结疤而得不到修复，引发心脏心律不全，泵血不足，轻者影响病人的生产和生活，重者引起休克或死亡，严重威胁人类健康和社会稳定。但目前没有很好的治疗方法。干细胞具有长期自我更新以及分化为体内任何细胞的能力，因此使用干细胞分化来的心肌细胞进行替代治疗，为彻底治疗心肌梗死提供了可能。有研究表明，将人类胚胎干细胞定向分化成为心肌细胞，通过注射进入患有心肌梗死的猴子心脏中可以长期存活，形成和猴子原有心肌细胞的细胞连接，产生具有跟猴子心脏一致的心电活动与钙传导信号，明显改善其心脏功能，具有显著的治疗效果。
安全、均质、规模化产生足够量的功能心肌细胞是其应用的前提和关键。另外，心肌药物的筛选同样也需要大量具有功能的心肌细胞。虽然，目前有很多方法可以将干细胞定向分化为心肌细胞，但是心肌细胞的分化效率低，价格高昂，细胞系之间的差异很大，不利于规模化生产和应用。目前将干细胞定向分化为心肌细胞的方法主要包括两种：1)拟胚体(Embryoid bodies,EBs)诱导分化法；2)贴壁诱导分化法。前者是将干细胞消化下来，悬浮培养4-5天形成拟胚体，然后用生长因子和药物诱导后，贴附在培养皿底分化为心肌细胞。但仅20-50％EBs能自发搏动，并且这种方法只能使EBs中50-75％的细胞分 化成具有功能的心肌细胞，另外分化的细胞中含有内、中、外三个胚层的非心肌细胞，所以在用于药物筛选和移植前，需要对分化的心肌细胞经过复杂的分选纯化。贴壁分化法是将干细胞以高密度的方式培养在培养皿中，用昂贵的细胞生长因子和小分子组合的培养基进行诱导。贴壁培养方法采用的是平面2D的培养方式，干细胞以单层生长的方式诱导分化，生产能力受到极大的限制，不利于实用化和工业化生产。另外，这些方法一方面需要特殊的基质如martrigel、laminin等；另一方面需要各种昂贵的蛋白生长因子例如ActivinA、BMP4、bFGF等。这些基质和生长因子具有明显的缺陷：成分不明、价格昂贵、不同批次之间存在巨大差异；部分是动物源性的，导致安全性低等。
因此，开发出一种完全是由小分子物质组成的成分限定、高效以及廉价的心肌分化培养基，并利用此培养基，建立一种三维悬浮条件下诱导多能干细胞定向分化为心肌细胞的方法，是本领域亟待解决的一个技术问题。
发明内容
本发明的目的是提供一种在三维(3D)悬浮培养环境中，将多能干细胞体外定向分化为心肌细胞的方法。
为了实现本发明目的，本发明首先提供用于建立三维悬浮条件下体外诱导心肌细胞分化的培养基，包括用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基，用于将中胚层前体细胞分化为心肌细胞的分化培养基以及心肌细胞的长期维持培养基。
其中，所述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR(血清替代品)、β-巯基乙醇、谷氨酰胺、NEAA(非必需氨基酸)、CHIR99021(Wnt信号通路的激动剂)和BIO(Wnt信号通路的激动剂)。
所述用于将中胚层前体细胞分化为心肌细胞的分化培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙 醇、谷氨酰胺、NEAA、IWP-2(Wnt信号通路的抑制剂)、XAV939(Wnt信号通路的抑制剂)和抗坏血酸。
所述心肌细胞的长期维持培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙醇、谷氨酰胺、NEAA和抗坏血酸。
本发明提供的诱导心肌分化的培养基，包括中胚层分化培养基、心肌分化培养基和心肌细胞长期维持培养基，分化培养基中含有多种可供多能干细胞分化为心肌细胞所需的营养成分。其中，CHIR99021和BIO小分子作为Wnt信号激活剂，可以抑制多能干细胞自我更新，促进其往中胚层谱系细胞的分化；IWP-2和XAV939小分子作为Wnt信号通路的抑制剂，促进中胚层前体细胞往心肌前体和心肌细胞分化；抗坏血酸可以抑制细胞分化过程中细胞的死亡，并且具有促进心肌细胞分化的功能。
优选地，在所述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基中，DMEM/F12混合IMDM的体积比为5:1-1:5；所述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％；所述谷氨酰胺的浓度为10-200mM；所述CHIR99021的浓度为1-20μM；所述BIO的浓度为1-20μM。
优选地，在所述用于将中胚层前体细胞分化为心肌细胞的分化培养基中，DMEM/F12混合IMDM的体积比为5:1-1:8；所述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％；所述谷氨酰胺的浓度为10-200mM；所述IWP-2的浓度为2-30μM；所述XAV939的浓度为1-20μM；所述抗坏血酸的浓度为5-500μg/L。
优选地，在所述心肌细胞的长期维持培养基中，DMEM/F12混合IMDM的体积比为5:1-1:8；所述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％；所述谷氨酰胺的浓 度为10-200mM；所述抗坏血酸的浓度为5-500μg/L。
本发明还提供在三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法，包括以下步骤：
1)将贴壁生长的多能干细胞消化下来，通过直径为20-70μm的细胞筛切割成大小均一的细胞团块；
2)切割得到的细胞团块放入细胞培养箱中进行三维悬浮培养，在干细胞培养基中培养4-5天获得直径为200-300μm的多能干细胞球；
3)将步骤2)的多能干细胞球除去干细胞培养基，在三维悬浮的条件下，添加上述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基进行分化培养，记为D0天，以后在此时间的分化培养下，不同天数分别表示为D1、D2、D3等；在分化培养的D2天，将培养基替换成上述用于将中胚层前体细胞分化为心肌细胞的分化培养基继续进行分化培养；在分化培养的D12天，将培养基替换成上述心肌细胞的长期维持培养基进行培养。
在本发明所述的心肌分化过程中，对人的干细胞在D6-D8天开始出现明显的心肌跳动球；对非人灵长类动物的干细胞在D4-D6天开始出现明显的心肌跳动球。
优选地，利用所述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基能产生60-100％纯度的中胚层前体细胞。
优选地，利用所述用于将中胚层前体细胞分化为心肌细胞的分化培养基能产生60-100％纯度的心肌前体细胞。
优选地，利用所述心肌细胞的长期维持培养基能产生60-100％纯度的心肌细胞。
本发明中涉及的多能干细胞包括人和非人灵长类动物胚胎干细胞(embryonic stem cell)以及诱导多能性干细胞(induced pluripotent stem cell)。
前述方法中，所述干细胞培养基的配方为：DMEM/F12添加 KOSR、NEAA、β-巯基乙醇、谷氨酰胺和bFGF；所述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％，所述谷氨酰胺的浓度为1-5mM，所述bFGF的浓度为1-50ng/ml。本发明还提供利用上述方法获得的分化的中胚层前体细胞系、心肌前体细胞系和心肌细胞系。
本发明进一步提供上述细胞系在细胞替代治疗，心脏疾病致病机制研究及药物筛选中的应用。
采用本发明方法可以在12天内将多能干细胞在3D悬浮培养系统中高效的分化为心肌细胞，分化效率达到60-95％以上，分化的心肌细胞60-95％以上表达cTnT、a-Actinin等心肌细胞特有的标记物。经过透射电镜分析，分化的心肌细胞具有跟体内心脏一致的微观组织结构如丰富的肌丝，清晰的肌节，大量的线粒体，丰富的细胞间连接，如闰盘等。采用本发明方法可以在一个心肌球中检测到窦性电信号，心房电信号和心室电信号三种具有跟体内心脏相似的生理电信号。另外，采用本发明方法获得的心肌细胞可以对现有的心脏病药物产生相似的反应，通过钙波分析，添加加速心率的药物咖啡因可加速本发明方法所产生心肌球的钙波频率，添加心得安等降低心率的药物可以明显的减低心肌球的钙波频率，这些证据充分表明本发明所生产的心肌细胞是具有与体内心肌细胞一致的有功能的细胞。此外，本发明方法无需昂贵且成分不明的生物产物如Matrigel、laminin等作为基质，整个分化过程都是在悬浮培养系统中培养诱导分化，所用的诱导分化剂均为价格低廉的化学小分子。本发明方法简单可靠，价格低廉，稳定高效，安全性高，由于采用了悬浮培养系统，可以工业化生产高品质的心肌细胞，无需任何后续的筛选和纯化步骤，可以直接用于心脏发育科学研究，心脏疾病的细胞治疗，心脏损伤的移植以及药物筛选的应用需求，具有不可估量的科学和社会经济效益。
附图说明
图1为本发明实施例2中利用人和非人灵长类动物的胚胎干细胞制备大小均一的多能干细胞小球的示意图。
图2为本发明实施例2中多能干细胞小球在三维悬浮培养条件下定向分化为自主搏动心肌球的示意图。
图3为本发明实施例2中分化的心肌细胞以及心肌球用药物筛选的示意图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中使用的干细胞培养基的配方为DMEM/F12添加KOSR，NEAA，β-巯基乙醇，谷氨酰胺，bFGF；所述KOSR的浓度为2％-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％，所述谷氨酰胺的浓度为1-5mM，所述bFGF为1-50ng/ml。
实施例1用于建立三维悬浮条件下体外诱导心肌细胞分化的培养基
本实施例中提供的用于建立三维悬浮条件下体外诱导心肌细胞分化的培养基包括：用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基，用于将中胚层前体细胞分化为心肌细胞的分化培养基以及心肌细胞的长期维持培养基。
所述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR(血清替代品)、β-巯基乙醇、谷氨酰胺、NEAA(非必需氨基酸)、CHIR99021(Wnt信号通路的激动剂)和BIO(Wnt信号通路的激动剂)。
所述用于将中胚层前体细胞分化为心肌细胞的分化培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙醇、谷氨酰胺、NEAA、IWP-2(Wnt信号通路的抑制剂)、XAV939(Wnt信号通路的抑制剂)和抗坏血酸。
所述心肌细胞的长期维持培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙醇、谷氨酰胺、NEAA和抗坏血酸。
以下为本实施例的用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基，用于将中胚层前体细胞分化为心肌细胞的分化培养基以及心肌细胞的长期维持培养基的具体实例。
用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基：
实例1：DMEM/F12与IMDM按5:1体积比混合、5％KOSR、1％NEAA、0.025％β-巯基乙醇、100mM谷氨酰胺、5μM CHIR99021和5μM BIO。
实例2：DMEM/F12与IMDM按1:1体积比混合、10％KOSR、1％NEAA、0.025％β-巯基乙醇、50mM谷氨酰胺、10μM CHIR99021和10μM BIO。
实例3：DMEM/F12与IMDM按1:5体积比混合、10％KOSR、1％NEAA、0.025％β-巯基乙醇、50mM谷氨酰胺、3μM CHIR99021和3μMBIO。
用于将中胚层前体细胞分化为心肌细胞的分化培养基：
实例1：基础培养液为DMEM/F12与IMDM按5:1体积比混合、5％KOSR、0.025％β-巯基乙醇、100mM谷氨酰胺、1％NEAA、10μMIWP-2、5μM XAV939(Wnt信号通路的抑制剂)和50μg/L抗坏血酸。
实例2：基础培养液为DMEM/F12与IMDM按1:2体积比混合、10％KOSR、0.025％β-巯基乙醇、50mM谷氨酰胺、1％NEAA、5μM IWP-2、2μM XAV939(Wnt信号通路的抑制剂)和25μg/L抗坏血酸。
实例3：基础培养液为DMEM/F12与IMDM按1:8体积比混合、10％KOSR、0.025％β-巯基乙醇、100mM谷氨酰胺、1％NEAA、20μMIWP-2、10μM XAV939(Wnt信号通路的抑制剂)和100μg/L抗坏血酸。
心肌细胞的长期维持培养基：
实例1：基础培养液为DMEM/F12与IMDM按5:1体积比混合、5％KOSR、0.025％β-巯基乙醇、100mM谷氨酰胺、1％NEAA和50μg/L抗坏血酸。
实例2：基础培养液为DMEM/F12与IMDM按1:2体积比混合、10％KOSR、0.025％β-巯基乙醇、50mM谷氨酰胺、1％NEAA和25μg/L抗坏血酸。
实例3：基础培养液为DMEM/F12与IMDM按1:8体积比混合、10％KOSR、0.025％β-巯基乙醇、100mM谷氨酰胺、1％NEAA和100μg/L抗坏血酸。
实施例2在三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法
利用实施例1中实例1对应的用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基，用于将中胚层前体细胞分化为心肌细胞的分化培养基以及心肌细胞的长期维持培养基，建立三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法。包括以下步骤：
1、利用人和非人灵长类动物的多能干细胞制备大小均一的多能干细胞小球。具体地，在该步骤中，二维条件下的多能干细胞用于实验，所述多能干细胞涉及饲养层共培养和无饲养层培养的多能干细胞，除去干细胞培养基，1×PBS清洗2遍；添加1mg/ml的dispase2-4ml覆盖多能干细胞，37℃条件下孵育4-8mins；待干细胞克隆边缘卷起时，除去dispase，1×PBS清洗2遍；添加3ml干细胞培养基，将干细胞克隆轻轻吹洗下来；将干细胞克隆收集起来，添加3ml干细胞培养基和10μM Y-27632混匀；将重悬的干细胞通过50μm网径的细胞筛；将干细胞悬液铺在Petri皿中，在37℃，5％CO2培养箱中培养过夜；4-5天后可以收获大小均质的多能干细胞小球(图1)。
图1示出了多能干细胞三维悬浮培养产生大小均质的多能干细胞球的过程。其中，(A-B)用于切割多能干细胞的细胞筛；(C)经 过细胞筛切割后的多能干细胞小团快；(D)为切割后的多能干细胞在三维悬浮条件下，形成多能干细胞球的方法示意图；(E-G)多能干细胞团块在三维悬浮培养过程中，不同天数产生的多能干细胞球；(H-I)为一个具有代表性的多能干细胞球在五天之内，干细胞球大小和细胞数量的增值情况；(J)三维悬浮长期培养的多能干细胞仍然保持了稳定的核型；(K-O)三维悬浮培养的多能干细胞仍然保持干细胞的多能性，表达干细胞的标志蛋白，如Oct4、SSEA-4、TRA-1-60和TRA-1-80，但不表达分化细胞的标志蛋白SSEA-1和Nestin；(P-Q)三维悬浮培养的多能干细胞注射到免疫缺陷的小鼠里能够分化产生三种不同胚层的细胞，显示分化的多能性。
2、将多能干细胞小球在悬浮培养条件下定向分化为自主搏动的心肌球。具体地，在该步骤中，将多能干细胞小球收集到离心管里面，离心，速度600rpm，时间3min；除去干细胞培养基，添加中胚层前体细胞分化培养基，重新悬浮细胞，该时间段记为D0天；细胞培养在Petri皿以及在37℃，5％CO2培养箱中；在D2天，将中胚层前体细胞分化培养基完全换成心肌分化培养基，每1-2天换液一次；在D6天可见自主搏动的心肌球；从D12天，将心肌分化培养基换成心肌细胞长期维持培养基，每1-2天换液一次(图2)。
图2示出了三维悬浮产生的多能干细胞球分化为心肌细胞球的过程。其中，(A)为整个分化过程的示意图；(B-D)为多能干细胞球在中胚层前体细胞分化培养基中作用两天后，分化为中胚层前体细胞，99.6％的细胞表达Brachyury；(E-G)为分化的中胚层前体细胞在心肌分化培养基作用两天后，分化为心肌前体细胞，99.3％的细胞表达NKX2.5；(H-M)为分化的心肌前体细胞在心肌分化培养基继续作用12-14天后，分化为成熟的心肌细胞，95.7％的细胞表达α-actinin和cTnT；(N)为分化过程中，不同天数产生自发跳动的心肌球占整个干细胞球的比率；(O)分化的心肌球，被消化为单个心肌细胞， 重新进行单层培养，进行染色显示，分化的心肌细胞具有多核以及表达丰富的肌节，显示心肌细胞的成熟。
3、通过荧光染色鉴定多能干细胞小球的多能性及其心肌分化标记物。在该步骤中，将多能干细胞小球及其后续分化的细胞采用4％PFA(多聚甲醛)固定；使用OCT包埋，进行冰冻切片，切片厚度10μm；使用1×PBS浸泡切片10min，去除包埋剂OCT；1×PBS浸泡切片，5min；0.2％Triton X100透膜10min；5％血清封闭2h；添加一抗4℃过夜，抗体比例为SSEA4 1:400，Brachyury 1:300，Nkx2.51:300，CTnT 1:400，a-Actinin 1:400；1×PBS，3min，4遍添加二抗，常温1小时，抗体比例均为1:1000；1×PBS，3min，4遍；添加DAPI染色，5min；1×PBS，3min，2遍，封片，镜检。
4、分化的心肌细胞表现心肌细胞的功能，显示出对心脏药物的反应潜力。具体地，在该步骤中，分化的心肌球放在心肌细胞功能检测仪上，检测其动作电位；对于钙波的检测，将Furo 4的荧光钙指示剂与培养基1：1混合作用20分钟后，利用Leica共聚交显微镜进行连续的快扫，拍摄出连续的钙波；在此基础上将咖啡因和propranolol按照一定浓度加入心肌细胞长期维持液中，检测心肌细胞的钙波，测试这两种药物对心肌球的作用，建立药物筛选平台(图3)。
图3示出了利用三维悬浮分化的心肌球进行药物筛选的过程。其中，(A)为分化的心肌球表现出心肌细胞三种典型动作电位，分别为心房的、心室的以及窦房节的；(B-C)分化的心肌球具有典型的心肌钙波；(D-E)咖啡因显著地促进心肌细胞的收缩，而propranolol则显著地抑制心肌细胞的收缩。这些结果显示，分化的心肌球可以作为一个药物筛选的细胞模型。
此外，通过本实施例的方法建立的中胚层前体细胞、心肌前体细胞、心肌细胞以及利用该三类细胞培养产生的细胞系；以及这三类细胞培养产生的细胞系在细胞替代治疗、心脏疾病致病机制以及药物筛 选中的应用也属于本发明的保护范围。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种在三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法，该方法简单可靠，价格低廉，稳定高效，安全性高，由于采用了悬浮培养系统，可以工业化生产高品质的心肌细胞，无需任何后续的筛选和纯化步骤，可以直接用于心脏发育科学研究，心脏疾病的细胞治疗，心脏损伤的移植以及药物筛选的应用需求，具有不可估量的科学和社会经济效益。

Claims (8)

  1. 用于建立三维悬浮条件下体外诱导心肌细胞分化的培养基，其特征在于，所述培养基包括用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基，用于将中胚层前体细胞分化为心肌细胞的分化培养基以及心肌细胞的长期维持培养基；
    所述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙醇、谷氨酰胺、NEAA、CHIR99021和BIO；
    所述用于将中胚层前体细胞分化为心肌细胞的分化培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙醇、谷氨酰胺、NEAA、IWP-2、XAV939和抗坏血酸；
    所述心肌细胞的长期维持培养基包括：基础培养液为DMEM/F12与IMDM的混合培养基、KOSR、β-巯基乙醇、谷氨酰胺、NEAA和抗坏血酸。
  2. 根据权利要求1所述的培养基，其特征在于，在所述用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基中，DMEM/F12混合IMDM的体积比为5:1-1:5；所述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％；所述谷氨酰胺的浓度为10-200mM；所述CHIR 99021的浓度为1-20μM；所述BIO的浓度为1-20μM。
  3. 根据权利要求1所述的培养基，其特征在于，在所述用于将中胚层前体细胞分化为心肌细胞的分化培养基中，DMEM/F12混合IMDM的体积比为5:1-1:8；所述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％；所述谷氨酰胺的浓度为10-200mM；所述IWP-2的浓度为2-30μM；所述XAV939的浓度为1-20μM；所述抗坏血酸的浓度为5-500μg/L。
  4. 根据权利要求1所述的培养基，其特征在于，在所述心肌细胞的长期维持培养基中，DMEM/F12混合IMDM的体积比为5:1-1:8；所 述KOSR的浓度为2-20％；所述NEAA的浓度为0.1-2％；所述β-巯基乙醇的浓度为0.1-1％；所述谷氨酰胺的浓度为10-200mM；所述抗坏血酸的浓度为5-500μg/L。
  5. 在三维悬浮条件下将多能干细胞体外定向分化为心肌细胞的方法，其特征在于，包括以下步骤：
    1)将贴壁生长的多能干细胞消化下来，通过直径为20-70μm的细胞筛切割成大小均一的细胞团块；
    2)切割得到的细胞团块放入细胞培养箱中进行三维悬浮培养，在干细胞培养基中培养4-5天获得直径为200-300μm的多能干细胞球；
    3)将步骤2)的多能干细胞球除去干细胞培养基，在三维悬浮的条件下，添加权利要求1-4任一项所述的用于将多能干细胞诱导分化为中胚层前体细胞的分化培养基进行分化培养，记为D0天；在分化培养的D2天，将培养基替换成权利要求1-4任一项所述的用于将中胚层前体细胞分化为心肌细胞的分化培养基继续进行分化培养；在分化培养的D12天，将培养基替换成权利要求1-4任一项所述的心肌细胞的长期维持培养基进行培养。
  6. 根据权利要求5所述的方法，其特征在于，所述多能干细胞包括人和非人灵长类动物胚胎干细胞以及诱导多能性干细胞。
  7. 根据权利要求5或6所述方法获得的分化的中胚层前体细胞系、心肌前体细胞系和心肌细胞系。
  8. 权利要求7所述的细胞系在心脏疾病致病机制研究及药物筛选中的应用。
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