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Culture medium and method for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells

A technology for stem cell differentiation and induction differentiation, which is applied in the field of induction medium for the differentiation of airway basal stem cells into bronchoalveolar stem cells, which can solve the problem of high induction transformation rate, and achieve the effects of high differentiation efficiency, good replacement and short time.

Active Publication Date: 2021-06-25
中山大学深圳
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Problems solved by technology

[0006] Based on this, it is necessary to provide a medium for inducing the differentiation of airway basal stem cells into bronchoalveolar stem cells in order to solve the problem that the patient's own bronchoalveolar stem cells cannot be directly obtained clinically. Successfully differentiated into bronchoalveolar stem cells with a high induction transformation rate, providing transplanted cell samples for transplantation treatment of pulmonary fibrosis and other diseases

Method used

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  • Culture medium and method for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells
  • Culture medium and method for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells
  • Culture medium and method for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells

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Embodiment 1

[0034] A medium for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells, comprising a primary inducing differentiation medium and a secondary inducing differentiation medium.

[0035] The primary induced differentiation medium was prepared by the following method: FGF10, KGF and CHIR99021 were added to SAGM medium (Lonza), and the mixture was uniformly mixed. In the medium, the concentration of FGF10 was 10 μg / L, the concentration of KGF was 10 μg / L, and the concentration of CHIR99021 was 3 μM.

[0036] The secondary induced differentiation medium is prepared by the following method: FGF10, KGF, CHIR99021, DAPT and IBMX are added to SAGM medium, and the mixture is uniformly mixed. In the medium, the concentration of FGF10 was 10 μg / L, the concentration of KGF was 10 μg / L, the concentration of CHIR99021 was 3 μM, the concentration of DAPT was 45 μM, and the concentration of IBMX was 90 μM.

Embodiment 2

[0038] A method for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells, comprising the following steps (the steps of inducing BC to differentiate into BASC are as follows: figure 1 shown):

[0039] (1) Cell expansion: use SAGM basal medium (Lonza) to culture BC in a T25 culture flask, change the culture medium every 3 days, suck up the original medium before changing the medium, add 2 mL of DPBS to rinse for 2 minutes, and then aspirate again. Using DPBS, add 4 mL of stabilizing medium. Passage once in about 6 days. When passage, drain the medium, wash the cells twice with 2 mL of DPBS, add 2 mL of TrypLEExpress to digest for 5 min, pat and shake by hand, and observe that most of the cells float up. Add 2 mL of serum containing serum to the culture flask. The medium terminates the digestion. After the cells were pipetted several times, they were transferred to a centrifuge tube and centrifuged at 500 g for 5 min. Remove the supernatant, add 1 ...

experiment example 1

[0058] Phenotypic identification.

[0059] Immunofluorescence and q-PCR were used to detect the expression of SPC in the BASC cells induced and differentiated in Example 2. The results are as follows figure 2 and image 3 As indicated, the induction of differentiation efficiency was analyzed. image 3 The circle or ellipse with bright color in the nucleus (the original color of the picture is blue) is the staining of the nucleus by DAPI, and the irregular shape of the cytoplasmic part of the BASC with a light color (the original color of the picture is red) is the positive staining of SPC antibody.

[0060] Immunofluorescence staining and q-PCR experiments showed that the transformation rate of BC cells into BASC cells was about 95%. It can be seen that the method of using autologous BC cells to induce and differentiate BASC cells in the embodiment of the present invention has high induction efficiency and short time.

[0061] The induction results of Comparative Examples ...

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Abstract

The invention provides a culture medium and a method for inducing airway basal stem cells to differentiate into bronchoalveolar stem cells, and relates to the technical field of stem cell induced differentiation. The culture medium comprises a primary induced differentiation culture medium and a secondary induced differentiation culture medium; the primary induced differentiation culture medium comprises the following components: a fibroblast growth factor 10, a keratinocyte growth factor and a glycogen synthase kinase-3 inhibitor; the secondary induced differentiation culture medium is prepared from the following components: a fibroblast growth factor 10, a keratinocyte growth factor, a keratinocyte growth factor, a gamma secretase inhibitor and a phosphodiesterase inhibitor. According to the method disclosed by the invention, the BC is subjected to induction culture by adopting the induction culture medium and can be efficiently converted into the BASC. According to the culture medium and the culture method, autologous airway basal stem cells can be successfully induced into bronchoalveolar stem cells, and the induction conversion rate is high.

Description

technical field [0001] The invention relates to the technical field of stem cell induction and differentiation, in particular to an induction medium and method for differentiating airway basal stem cells into bronchoalveolar stem cells. Background technique [0002] Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by diffuse alveolitis and alveolar structural disturbances, ultimately leading to interstitial fibrosis. According to statistics, the prevalence of IPF in the overall population is about 2 to 29 / 100,000 per year, and it is more common in the elderly. As an aging country, my country is conservatively estimated to have at least 500,000 IPF patients, and the trend is increasing year by year. In recent years, IPF has become a common disease, and its prognosis is poor. The survival period after symptoms is generally 3 to 8 years, which seriously affects people's life and health. [0003] Currently, there are only two FDA-approved trea...

Claims

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Application Information

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IPC IPC(8): C12N5/074
CPCC12N5/0689C12N2501/119C12N2501/117C12N2501/727C12N2501/73C12N2501/734C12N2506/27
Inventor 聂怡初蒋近军邓文斌麦扬徐健赵景新萧倩谢芫
Owner 中山大学深圳
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