Method and culture media for inducing osteogenic differentiation of induced pluripotent stem cell of mouse

A technology of differentiation medium and pluripotent stem cells, which is applied in the field of induction of mouse induced pluripotent stem cells to differentiate into bone, can solve the problems of unclear medium composition and low purity of differentiated cells, and achieve broad application prospects and practical value. High differentiation rate and stable effect

Inactive Publication Date: 2014-04-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing directional induction technology generally has the problems of unclear medium composition and low purity of differentiated cells
In addition, in order to meet clinical sta

Method used

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  • Method and culture media for inducing osteogenic differentiation of induced pluripotent stem cell of mouse
  • Method and culture media for inducing osteogenic differentiation of induced pluripotent stem cell of mouse
  • Method and culture media for inducing osteogenic differentiation of induced pluripotent stem cell of mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Culture and Identification of Mouse iPS Cells

[0036] Obtaining and maintaining mouse iPS cell lines with good pluripotency and differentiation ability is the basis for cell-directed differentiation in the later stage. This example is a description of the operation process of culturing and identifying mouse iPS cells used in the present invention.

[0037] 1. Culture of mouse iPS cells

[0038] The mouse iPS cell line p14-2-2 used in this example was provided by the Chinese Academy of Sciences Stem Cell Bank.

[0039] Mitomycin C working solution: Dissolve 2mg of Mitomycin C in 200mL high-glucose DMEM containing 10% fetal bovine serum, and filter to sterilize.

[0040] 0.1% gelatin: Dissolve 0.1g gelatin powder in 100mL double distilled water and autoclave.

[0041] Fibroblast culture medium: high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0042] LN culture medium: containing 48%DMEM / F12, 48%Neurobasal, 1000U / mL LIF, ...

Embodiment 2

[0055] Example 2 Osteotropic differentiation of mouse iPS cells

[0056] In the present invention, the osteotropic differentiation of mouse iPS cells is accomplished through two steps: the first step is to obtain embryoid bodies enriched in mesenchymal cells under appropriate culture conditions; and the second step is to induce differentiation Obtain osteoblasts. Operation process see figure 1 .

[0057] 1. Preparation of embryoid bodies

[0058] Differentiation medium I: add 5×10 to the fibroblast culture medium -8 mol / L all-trans retinoic acid and 10ng / mL bFGF, pH 7.0-7.4. Wherein, the fibroblast culture medium is high-sugar DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0059] The operation process of this step includes: referring to Example 1, removing the feeder layer cells and collecting the suspension cells by gelatin attachment method; adjusting the cell density to 2×10 5 cells / mL, inoculate cells into ultra-low adhesion culture dishes;...

Embodiment 3

[0066] Example 3 Identification of osteogenic differentiated cells

[0067] 1. Calcium deposition level detection

[0068] 1% Alizarin Red S: 0.1M tris-Hcl 100mL (pH8.3), add 0.1g Alizarin Red S, store at 4°C.

[0069] In this example, the Alizarin red staining method was used to detect the calcium deposition level of differentiated cells, and the steps included: induction of differentiation medium I for 5 days (step 1 of Example 2) and induction of differentiation medium II for 14 days (Example 2 Remove the culture medium from the cells in step 2), wash with PBS 3 times; fix the cells with 95% ethanol at room temperature for 10 minutes, wash 3 times with PBS, add 1% Alizarin Red S staining solution, incubate at 37°C for 30 minutes; wash with PBS Cells were washed 3 times, observed and photographed under an inverted microscope.

[0070] The staining result is as Figure 4 As shown (where, A is the control group; B is the osteoblast medium group; C is the differentiation mediu...

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Abstract

The invention discloses a method and culture media for inducing osteogenic differentiation of an induced pluripotent stem cell of a mouse. The method provided by the invention comprises the steps of inoculating the pluripotent stem cell of the mouse into a differentiation culture medium I to form an embryoid body with enriched mesenchymal cells, then separating the embryoid body and culturing in a differentiation culture medium II to obtain an osteoblast, wherein the differentiation culture medium I disclosed by the invention is formed by adding all-transretinoic acid and basic fibroblast growth factor on the basis of a fibroblast culture medium, and the differentiation culture medium II is formed by reducing the D-glucose content and adding BMP4 or BMP7 on the basis of an osteoblast culture medium. The method and the culture media provided by the invention can significantly promote the osteogenic differentiation of the pluripotent stem cell of the mouse, have the characteristics of short cycle, high differentiation rate, stable effect and the like, and have broad application prospect and practical value.

Description

technical field [0001] The invention relates to a method for inducing osteogenic differentiation of mouse induced pluripotent stem cells and a culture medium thereof, belonging to the field of biotechnology. technical background [0002] Pluripotent stem cells refer to a type of cells that have the potential to form all types of cells in an individual animal and are capable of continuous self-renewal. On the one hand, they maintain the size or expansion of their own population through cell division, and on the other hand, they can further differentiate into specific cell types. Due to the above-mentioned characteristics of pluripotent stem cells, they play a unique role and advantages in the fields of cell replacement therapy, gene therapy, developmental biology, and pharmacology. At present, pluripotent stem cells are divided into three categories according to their sources: embryonic stem cells (embryonic stem cells, ES) derived from the inner cell mass of embryonic blast...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/077
Inventor 张宇刘忠华史久慧邢舰誉
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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