Kidney epithelial cell and preparation method and application thereof

An epithelial cell and renal epithelium technology, applied in the field of stem cell biology, can solve the problems of low differentiation efficiency, inability to obtain renal epithelial cells, complex sources of medium components, and high differentiation cost, and achieve the effects of clear components, simple components and simple operation.

Pending Publication Date: 2019-11-19
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, the existing renal epithelial cell differentiation schemes are different in the process and principle of the differentiation schemes. These differentiation schemes have the following disadvantages: 1)

Method used

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  • Kidney epithelial cell and preparation method and application thereof
  • Kidney epithelial cell and preparation method and application thereof
  • Kidney epithelial cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] This embodiment discloses a method for preparing renal epithelial cells, comprising the following steps:

[0157] S1: Pluripotent stem cells differentiate into posterior primitive streak cells;

[0158] S2: Posterior primitive streak cells differentiate to posterior intermediate mesoderm;

[0159] S3: Posterior intermediate mesoderm differentiates into renal progenitor cells;

[0160] S4: Renal progenitor cells differentiated into renal tubular aggregate precursors;

[0161] S5: Renal tubular aggregate precursors differentiate into renal vesicles;

[0162] S6: Renal vesicles differentiate into renal epithelial cells.

[0163] Specifically, the flow chart is as figure 1 As shown, the steps are as follows:

[0164] (1) Culture of pluripotent stem cells (D-3-D0)

[0165] The pluripotent stem cells used in the experiment have undergone strict pluripotency verification (expressing various pluripotency markers, and can form teratomas including inner, middle and outer ge...

Embodiment 2

[0209] This example discloses a method for preparing renal epithelial cells. The only difference from Example 1 is that step (5) "differentiation of metanephric mesenchymal cells to renal tubular aggregate precursors (Day8-10)" uses a 3D method nourish. Include the following steps:

[0210] The obtained renal progenitor cells were subjected to an embryoid body (EB) formation experiment, and the specific operation was: add 0.2-0.8 mL / cm2 to the differentiated renal progenitor cells 2 After incubating at 37°C for 3-6 minutes, pipette gently with a gun, transfer the cell suspension to a 1.5mL centrifuge tube, centrifuge briefly for 5-10s, and discard the supernatant. Add 0.2-0.8mL / cm 2 Resuspend the cells in complete differentiation medium D containing a certain concentration of Rock inhibitor and transfer to a low-adsorption culture flask, place on a three-dimensional shaker, 37°C, 5% CO 2 Concentration, cultivated in an incubator with saturated humidity. Cell density can be...

Embodiment 3

[0215] This example discloses a method for preparing renal epithelial cells. The only difference from Example 1 is that the sources of pluripotent stem cells are different, namely EGFP-iPSC, iPSC from polycystic kidney disease patients and iPSC from healthy people. The typical renal epithelial cell structure that can be obtained by the three cell lines (see image 3 ), indicating that the differentiation method disclosed in the present invention has universal applicability to various hPSCs.

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Abstract

The invention relates to the field of stem cell biology, in particular to a kidney epithelial cell and a preparation method and application thereof. The first disclosed kidney epithelial cell expresses CDH1<+> and PODXL<+>, and at least expresses one of CDH2<+> and LTL<+>. The second disclosed preparation method of the kidney epithelial cell includes the following steps that pluripotent stem cellsare differentiated into posterior streak cells; the posterior streak cells are differentiated into posterior intermediate mesoderms; the posterior intermediate mesoderms are differentiated into kidney progenitor cells; the kidney progenitor cells are differentiated into kidney tubular aggregated precursors; the kidney tubular aggregated precursors are differentiated into kidney vesicles; and thekidney vesicles are differentiated into kidney epithelial cells. The provided preparation method has high differentiation efficiency and stable differentiation effect, and is applied to a variety of hPSC cell lines; and the differentiated nephron progenitor cells can meet the requirements of kidney disease model building, kidney drug toxicity testing, kidney disease related drug screening and other scientific research experiment.

Description

technical field [0001] The invention relates to the field of stem cell biology, in particular to a kidney epithelial cell and its preparation method and application. Background technique [0002] The kidney is an important organ in the human body with a complex spatial structure. As a component of the urinary system, the main function of the kidneys is to regulate the composition and volume of body fluids and to remove metabolic waste. The mature kidney will filter the blood through the nephron and recycle the substances needed by the human body many times, and finally produce urine to be excreted from the body. In addition, the kidney also has functions such as regulating blood pressure and maintaining acid-base balance in the body. [0003] Differentiation of human pluripotent stem cells (hPSCs, including human embryonic stem cells hESCs and human induced pluripotent stem cells hiPSCs) into renal progenitor cells and renal organoids for the study of human kidney developm...

Claims

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Application Information

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IPC IPC(8): C12N5/071A01N1/02A61K35/22A61P13/12
CPCA01N1/0221A61K35/22A61P13/12C12N5/0625C12N5/0686C12N2500/25C12N2500/32C12N2501/115C12N2501/15C12N2501/155C12N2501/415C12N2506/02C12N2506/45
Inventor 高雅丽胡青松俞君英张颖
Owner 安徽中盛溯源生物科技有限公司
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