Method for inducing pluripotent stem cells to differentiate into melanocytes in serum-free culture solution

A serum-free culture medium, pluripotent stem cell technology, applied in the field of cell engineering and cell biology, can solve the problem of low differentiation efficiency, and achieve the effect of high differentiation efficiency and short time-consuming

Pending Publication Date: 2022-03-01
SHANGHAI SHANGRUI BIOLOGICAL PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is that it is easy to operate, but at present, the differentiation efficiency of this method is low

Method used

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  • Method for inducing pluripotent stem cells to differentiate into melanocytes in serum-free culture solution
  • Method for inducing pluripotent stem cells to differentiate into melanocytes in serum-free culture solution
  • Method for inducing pluripotent stem cells to differentiate into melanocytes in serum-free culture solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Differentiation of the H9 human embryonic stem cell line into epidermal melanocytes:

[0066] 1. Melanocyte differentiation:

[0067] 1. The configuration of culture medium:

[0068] Medium No. 1: Add 10-50% KnockOut produced by Gibco to 500mL DMEM / F-12 (1:1) medium TM Serum Replacement, then add 4-20ng / mL recombinant human fibroblast growth factor 2 (FGF-2) and 2-50mM high-purity β-mercaptoethanol.

[0069] Medium No. 2: Add 1-10g / L human transferrin, 50-200mg / L recombinant human insulin, and 0.063-0.63mg / L corpus luteum to 500mL DMEM / F-12 (1:1) medium Ketones, 100-1600mg / L putrescine and 0.1-1mg / L sodium selenite.

[0070] DiffMel medium: based on Ham's F12 Medium, add 4-20ng / mL recombinant human FGF-2, 0.1-10ng / ml recombinant human stem cell factor (SCF), 0.1-200nM human endothelin 3 (EDN3), 0.1-10 ng / mL bone morphogenetic protein 4 (BMP4), 1-10% bovine serum albumin (BSA), 1-200 uM ascorbic acid, 1-10 uM CHIR99021, 0.1-1 mM dibutyryl cyclic adenosine (dbcAMP),...

Embodiment 2

[0115] Differentiation of human induced pluripotent stem cells into epidermal melanocytes:

[0116] Human induced pluripotent stem cells were induced to differentiate into epidermal melanocytes according to the melanocyte differentiation method described in Example 1.

[0117] Characterization of differentiated melanocytes:

[0118] 1. Cell morphology:

[0119] Such as Figure 11 As shown, the changes in cell morphology throughout the differentiation process are demonstrated.

[0120] Changes in cell morphology during differentiation of human induced pluripotent stem cells into melanocytes. The first row of pictures from left to right shows the cell morphology on day 1, day 3, and day 8 of differentiation, and the second row of pictures shows from left to right day 11 of differentiation, day 3 after passage, and passage The cell morphology on the 7th day after subculture shows that obvious melanocytes have appeared on the 7th day after subculture.

[0121] Such as Figur...

Embodiment 3

[0126] Comparison to Other Melanocyte Cell Differentiation Methods

[0127] using equivalent Basement Membrane Matrix, 3x10 in LDEV-free coated cell culture dishes 6 H9 embryonic stem cells and Essential 8 produced by Gibco TM Medium cultured to day 3. Subsequently, the human embryonic stem cells were differentiated into melanocytes by following the melanocyte differentiation method provided in Example 1, and the embryonic stem cells were differentiated into melanocytes by using other two reported differentiation methods at the same time. On the 11th day, the difference in the expression of melanocyte-related genes among the three groups of cells was detected by Q-PCR, and embryonic stem cells and primary human melanocytes were used as controls. The results are as follows Figure 15 shown. From Figure 15 It can be seen from the figure that the levels of melanocyte-related PAX3, SOX10, MITF, and TYR genes expressed by the cells differentiated by the method in Example 1...

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Abstract

The invention relates to a method for inducing pluripotent stem cells to be differentiated into melanocytes in a serum-free culture solution, which comprises the following steps: preparing a serum-free matrix culture solution of differentiated cells, including a No.1 culture medium, a No.2 culture medium and a DiffMel culture medium; culturing the pluripotent stem cells in the cell culture plate until the pluripotent stem cells overgrow 80% of the cell culture plate; inducing the pluripotent stem cells to be differentiated into precursor cells of melanocytes in a matrix culture solution; a culture dish for treatment and passage is coated, and precursor cells of the melanocytes are continuously induced to be differentiated into the melanocytes in a DiffMel culture medium; and culturing the differentiated melanocytes. The differentiated melanocytes have cell characteristics similar to those of human primary melanocytes, and have great difference from malignant melanoma cells. Compared with the prior art, the melanocyte differentiation method provided by the invention is short in time consumption and high in differentiation efficiency, and serum or other animal-derived preparations are not needed, so that a basis is provided for future clinical application.

Description

technical field [0001] The invention relates to the fields of cell engineering and cell biology, in particular to a method for inducing pluripotent stem cells to differentiate into melanocytes in serum-free culture fluid. Background technique [0002] Melanocytes are pigment cells that exist in the basal layer of human skin, hair follicles, and sweat glands. They can secrete melanin and transfer melanin to keratinocytes in the epidermis to help the body resist ultraviolet rays and various light radiations. Damage to the skin. At the same time, the melanin secreted by melanocytes is also one of the important factors that determine the skin color of the human body. [0003] The hyperfunction of melanocytes will lead to abnormal melanin deposition in the skin, which will lead to hyperpigmentation skin diseases such as freckles; the decline or reduction of melanocyte function will lead to insufficient synthesis of melanin, which will cause pigmentation deficiency diseases such ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0626C12N2506/45C12N2500/90C12N2501/115C12N2500/44C12N2500/25C12N2500/46C12N2501/30C12N2501/125C12N2501/365C12N2501/155C12N2500/38C12N2500/40C12N2501/33C12N2500/32
Inventor 曾炫浩徐金华吴复跃何振东
Owner SHANGHAI SHANGRUI BIOLOGICAL PHARMA
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