A method for inducing stem cells to differentiate into keratinocytes

Abstract

The present invention discloses a method for inducing stem cells to differentiate into keratinocytes, and relates to the technical field of stem cell differentiation. The present invention proposes that by adding an ERK pathway activator at any time between the 0th and 4th days of inducing stem cells to differentiate into keratinocytes, keratinocytes derived from extraembryonic trophoblast cells can be avoided or reduced, so that the source of keratinocytes differentiated from stem cells is clear, the physiology and function of the cells are stable, the differentiation efficiency and yield of keratinocytes derived from the cells can be optimized, and a new idea for the stable and effective acquisition of keratinocytes is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell differentiation, and in particular to a method for inducing stem cells to differentiate into keratinocytes. Background Art

[0002] Skin keratinocytes obtained from the human body have poor expansion ability and cannot be mass-produced. Therefore, skin keratinocytes differentiated from hPSCs provide another platform for future skin transplantation and safety and efficacy evaluation of beauty care products. However, the origin of most hPSC-differentiated skin keratinocytes is unknown. They are mixed with keratinocytes derived from extraembryonic trophoblast cells, which may differ from normal skin keratinocytes in physiology and function. Therefore, clarifying the cell source is a problem that needs to be solved when using skin keratinocytes differentiated from hPSCs.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The object of the present invention is to provide a method for inducing stem cells to differentiate into keratinocytes.

[0005] The present invention is achieved in that:

[0006] In a first aspect, an embodiment of the present invention provides a method for inducing stem cells to differentiate into keratinocytes, comprising inducing stem cells to differentiate into keratinocytes, wherein at any time between days 0 and 4 of the induction culture, the differentiation medium used contains an ERK pathway activator.

[0007] In a second aspect, an embodiment of the present invention provides the use of a target agent in preparing a kit for reducing or inhibiting the differentiation of stem cells into skin keratinocytes mixed with keratinocytes derived from extraembryonic trophoblast cells, wherein the target agent includes an ERK pathway activator.

[0008] The present invention has the following beneficial effects:

[0009] The present invention proposes that the addition of an ERK pathway activator during the process of inducing stem cells to differentiate into keratinocytes can avoid or reduce keratinocytes derived from extraembryonic trophoblast cells, thereby making the source of keratinocytes differentiated from stem cells clear, the physiology and function of the cells stable, and the differentiation efficiency and yield of cell-derived keratinocytes can be optimized, providing a new idea for the stable and effective acquisition of keratinocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0011] Figure 1 This is the timeline of the differentiation method. From day 0 to day 8, differentiation medium was used for differentiation and different treatments were used to guide H1 cells to skin keratinocytes.

[0012] Figure 2 The cell morphology of H1 cells during differentiation with or without FGF2 from day 0 to day 2, scale bar 400 μm;

[0013] Figure 3 To investigate the effect of adding FGF2 at different times on the expression of genes related to extraembryonic trophoblast cells and skin keratinocytes; extract mRNA on the 8th day of differentiation and measure the expression of related genes by real-time nucleic acid quantification, using TBP as a housekeeping gene to quantify gene expression;

[0014] Figure 4 To investigate the effects of adding various ERK pathway activators on the expression of genes related to extraembryonic trophoblast cells and skin keratinocytes; extract mRNA on the 8th day of differentiation and measure the expression of related genes by real-time nucleic acid quantification, using TBP as a housekeeping gene to quantify gene expression;

[0015] Figure 5 To investigate the effects of CHIR and / or FGF2 addition on the expression of genes related to extraembryonic trophoblast cells and skin keratinocytes during differentiation, mRNA was extracted on day 8 of differentiation and the expression of related genes was measured by real-time nucleic acid quantification. TBP was used as a housekeeping gene to quantify gene expression.

[0016] Figure 6 To investigate the effect of adding or not adding a BMP pathway inhibitor (LDN193189) during FGF2 differentiation on the expression of genes related to extraembryonic trophoblast cells and skin keratinocytes, mRNA was extracted on the 8th day of differentiation and the expression of related genes was measured by real-time nucleic acid quantification. TBP was used as a housekeeping gene to quantify gene expression. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0018] An embodiment of the present invention provides a method for inducing stem cells to differentiate into keratinocytes, comprising inducing stem cells to differentiate into keratinocytes, wherein at any time between days 0 and 4 of the induction culture, the differentiation medium used contains an ERK pathway activator.

[0019] Corneocytes, also known as keratinocytes or keratin-forming cells, are the main cellular components of the epidermis.

[0020] The step of "inducing stem cells to differentiate into keratinocytes" herein can be performed based on the prior disclosed method of inducing stem cells to differentiate into keratinocytes.

[0021] In the existing skin keratinocyte differentiation, many reagents can induce stem cells to differentiate into keratinocytes, but at the same time they will also cause stem cells to differentiate into extraembryonic trophoblast cells. For example, BMP4 is a key factor in guiding skin keratinocytes. Extraembryonic trophoblast cells will further differentiate into keratinocytes, making the source of the obtained keratinocytes unclear, and the keratinocytes obtained by differentiation of extraembryonic trophoblast cells are also physiologically and functionally different from normal skin keratinocytes. ERK pathway activators, especially FGF2, can solve, improve or avoid this technical problem. They can inhibit or reduce the differentiation of stem cells into extraembryonic trophoblast cells, thereby inhibiting or reducing keratinocytes derived from extraembryonic trophoblast cells, so that the final keratinocytes have a clear source and stable physiological functions, and can optimize the differentiation efficiency and yield of cell-derived keratinocytes.

[0022] The "day 0" in this article refers to the starting point of inducing differentiation, and day 0 to 1 can be understood as 0 h to 24 h, and "any time between days 0 to 4" can refer to any time point or any time period between days 0 to 4. Subsequent similar expressions are understood in the same way and will not be repeated.

[0023] The basic components of the "differentiation medium" herein are the same as the components of the existing conventional culture medium for inducing stem cells to differentiate into keratinocytes, and "the differentiation medium used contains an ERK pathway activator" can be understood as adding an ERK pathway activator to the basic components of the existing differentiation medium.

[0024] Preferably, the basic components of the differentiation medium include: at least one of: DMEM / F12, L-ascorbic acid 2-phosphate (effective concentration range: 10~100 mg / L), sodium selenite (effective concentration range ~ 1~20 µg / mL), transferrin (effective concentration range: 1~20μg / mL) and Chemical defined lipid concentrate (chemically defined concentrated lipid, effective concentration range: 0.5%~3%).

[0025] Specifically, in this embodiment, the time when the differentiation medium contains the ERK pathway activator can be any time from 0 to 4 days, specifically any time point or any time period from 0 to 4 days, 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, and 4 days. It should be noted that when the time for culturing with a differentiation medium containing a specific agent is a time period, the culture medium can be selectively replaced several times in the time period so that the differentiation medium contains a specific agent. For example, in the time period of 0 to 4 days, a new culture medium can be selected every day, every 10 hours or 20 hours to make the process of inducing differentiation more stable and effective. Subsequent similar situations are understood in the same way and will not be repeated.

[0026] Preferably, the time when the ERK pathway activator is contained in the differentiation medium is any time between day 0 and day 2 of the induction culture.

[0027] Preferably, the differentiation medium contains the ERK pathway activator on day 0 to 2 of the induction culture. Adding the ERK pathway activator at different time periods has an effect on cell differentiation. The best effect is achieved by using the medium containing the ERK pathway activator for induction culture on day 0 to 2, which can effectively inhibit the differentiation of extraembryonic trophoblast cells while promoting or not affecting the acquisition of keratinocytes.

[0028] Preferably, the concentration of the ERK pathway activator in the differentiation medium is 1-40 ng / mL, specifically 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL and 40 ng / mL, or any two of them. In the case where the concentration of the ERK pathway activator is limited, the amount of addition can be selected according to the limited concentration, or the culture medium can be replaced with a differentiation medium containing a specific concentration of the ERK pathway activator for induction culture.

[0029] The ERK pathway activator is selected from at least one of FGF1, FGF2, FGF4, FGF9, EGF and Heregulin β.

[0030] Preferably, the ERK pathway activator is FGF2.

[0031] FGF2, whose English name is Fibroblast growth factor 2, and whose Chinese full name is fibroblast growth factor 2, has the function of maintaining the pluripotency of hPSC (human pluripotent stem cells).

[0032] In the present invention, the effect of FGF2 is more effective and prominent than other ERK pathway activators.

[0033] Preferably, the differentiation medium further contains a WNT pathway agonist at any time between day 1 and day 6 of the induction culture.

[0034] It has been verified that when the differentiation medium for inducing stem cells to differentiate into keratinocytes contains a WNT pathway agonist, the addition of FGF2 can more effectively exert its effect of inhibiting extraembryonic trophoblast cells.

[0035] Specifically, in this embodiment, the differentiation medium also contains a WNT pathway agonist on days 1 to 6 of the induction culture, specifically any time point on days 1, 2, 3, 4, 5 and 6 or a time period between any two of them.

[0036] Preferably, the differentiation medium also contains a WNT pathway agonist on day 1 to day 6 of the induction culture.

[0037] In an optional embodiment, the present invention does not limit the type of WNT pathway agonist, and it can be selected from any currently disclosed or commercially available WNT pathway agonist, as long as it has the function of activating the WNT pathway.

[0038] Preferably, the WNT pathway agonist is CHIR99021.

[0039] Preferably, at any time between day 0 and day 1 of the induction culture, the differentiation medium used further contains a BMP pathway inhibitor.

[0040] Specifically, in this embodiment, the differentiation medium used also contains the BMP pathway inhibitor on day 0 to day 1 of the induction culture.

[0041] Preferably, the concentration of the BMP pathway inhibitor in the differentiation medium is 50-150 nM, specifically, any one or any two of 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, and 150 nM. Within the limited action concentration and the limited action time range, the BMP pathway inhibitor combined with FGF2 can more effectively inhibit the differentiation of extraembryonic trophoblast cells and maintain or promote the stability of the source of keratinocytes.

[0042] Preferably, the BMP pathway inhibitor is LDN193189.

[0043] Preferably, in the process of inducing stem cells to differentiate into keratinocytes, the differentiation medium used further contains at least one of the following agents: a TGFβ pathway inhibitor, BMP-4 and a NOTCH pathway inhibitor.

[0044] TGFβ pathway inhibitors can induce stem cells to differentiate into ectoderm cells. Preferably, the differentiation medium contains TGFβ pathway inhibitors at any time between days 0 and 6 of inducing differentiation, preferably days 0 to 6.

[0045] Preferably, the effective concentration of the TGFβ pathway inhibitor is 1 to 40 µM, specifically, it can be the range of any one or between any two of 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL and 40 ng / mL.

[0046] The TGFβ pathway inhibitor may be SB431542.

[0047] BMP-4 induces stem cells to differentiate into ectoderm cells. Preferably, the differentiation medium contains BMP-4 at any time between the first and eighth days of inducing differentiation, preferably between the first and eighth days.

[0048] Preferably, the effective concentration of BMP-4 is 1-20 ng / mL, specifically, it can be any one of 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL and 20 ng / mL, or a range between any two of them.

[0049] NOTCH pathway inhibitors can enhance the differentiation of skin keratinocytes. Preferably, the differentiation medium contains the NOTCH pathway inhibitor at any time between the 4th and 8th days of inducing differentiation, preferably the 4th and 8th days.

[0050] Preferably, the effective concentration of the NOTCH pathway inhibitor is 1-40 µM, specifically, it can be the range of any one or between any two of 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL and 40 ng / mL.

[0051] Preferably, the NOTCH pathway inhibitor may be DAPT.

[0052] Preferably, the stem cells are human pluripotent stem cells, which include human embryonic stem cells and human induced pluripotent stem cells. Human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0053] The embodiment of the present invention also provides the use of a target agent in preparing a kit for reducing or inhibiting the differentiation of stem cells into skin keratinocytes mixed with keratinocytes derived from extraembryonic trophoblast cells, wherein the target agent includes FGF2.

[0054] Preferably, the target agent comprises a BMP pathway inhibitor.

[0055] Preferably, the target agent further comprises at least one of BMP-4, a WNT pathway agonist, a TGFβ pathway inhibitor and a NOTCH pathway inhibitor.

[0056] The present invention also provides a target agent for improving or reducing the differentiation of stem cells into skin keratinocytes mixed with keratinocytes derived from extraembryonic trophoblast cells. The target agent is the same as that described in any of the above embodiments and will not be described in detail.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] In order to study the role of FGF2 in early skin differentiation, this example selected a staged skin differentiation method ( Figure 1 ). Under this differentiation method, the stem cells were induced and cultured using a differentiation medium containing FGF2 in the early stages of differentiation, and the effects on skin and extraembryonic differentiation were detected on the eighth day of differentiation.

[0060] The specific implementation plan is as follows: Human embryonic stem cells H1 are cultured in E8 medium, and fresh medium is replaced every day. When the cell density reaches 70%-80%, the cells are passaged. First, wash twice with dPBS-EDTA, incubate at room temperature for 8 minutes for the third time, aspirate DPBS-EDTA, add E8 medium containing 5 μM ROCK inhibitor, resuspend the cells, and spread them into a 12-well plate pre-coated with matrix gel at a density of 1:12-1:15. After one day, when the cells are attached to the plate and stabilized, differentiation medium (E8 medium without TGFβ and FGF2, and 1% Chemically Defined Lipid Concentrate) is used according to the staged skin differentiation method ( Figure 1 ), FGF2 was added from day 0 to day 2 / 4 at a concentration of 20 ng / mL, the culture medium was changed every day, and the levels of indicator genes of skin keratinocytes and extraembryonic trophoblast cells of differentiated cells were detected on day 8.

[0061] The results show that ( Figure 2~3 ), adding FGF2 on days 0-2 can significantly inhibit the expression of extraembryonic trophoblast cells (CGA, CGB), and has little effect on the early indicator gene of skin keratinocytes (p63), but treatment on days 0 to 4 was found to affect the early indicator gene of skin keratinocytes (p63). This shows that FGF2 treatment can inhibit the differentiation of extraembryonic trophoblast cells and the optimal treatment time is day 0-2.

[0062] Example 2

[0063] ERK pathway activators (fibroblast growth factor family, EGF and Heregulin β) also have the ability to inhibit the differentiation of extraembryonic trophoblast cells.

[0064] The specific implementation plan is as follows: Human embryonic stem cells H1 are cultured in E8 medium, and fresh medium is replaced every day. When the cell density reaches 70%-80%, the cells are passaged. First, wash twice with dPBS-EDTA, incubate at room temperature for 8 minutes for the third time, aspirate dPBS-EDTA, add E8 medium containing 5 μM ROCK inhibitor (Y-27632), resuspend the cells, and spread them into a 12-well plate pre-coated with matrix gel at a density of 1:12-1:15. After one day for the cells to adhere to the plate and stabilize, use differentiation medium (E8 medium without TGFβ and FGF2, and add Chemically Defined LipidConcentrate), and set up 9 groups of experiments at the same time. The differentiation methods of the 9 experimental groups are roughly the same. Figure 1 As shown, the difference is that no or various ERK pathway activators are added, the culture medium is changed every day, and the levels of indicator genes of skin and extraembryonic trophoblast cells of differentiated cells are finally detected on the eighth day. The results show ( Figure 4 ), the expression of extraembryonic trophoblast cell marker genes (CGA, CGB) with the addition of different ERK pathway activators (FGF1, FGF2, FGF4, FGF9, EGF or heregulin β) was significantly lower than that without the addition of fibroblast growth factor, and had little effect on the early marker gene of skin keratinocytes (p63).

[0065] This indicates that activation of the ERK pathway can inhibit the differentiation of extraembryonic trophoblast cells.

[0066] Example 3

[0067] FGF2 and WNT work synergistically to regulate the differentiation of extraembryonic trophoblast cells.

[0068] The specific implementation plan is as follows: Human embryonic stem cells H1 are cultured in E8 medium, and fresh medium is replaced every day. When the cell density reaches 70%-80%, the cells are passaged. First, wash twice with dPBS-EDTA, incubate at room temperature for 8 minutes for the third time, aspirate dPBS-EDTA, add E8 medium containing 5 μM ROCK inhibitor (Y-27632), resuspend the cells, and spread them into a 12-well plate pre-coated with matrix gel at a density of 1:12-1:15. After one day for the cells to adhere to the plate and stabilize, use differentiation medium (E8 medium without TGFβ and FGF2, and add Chemically Defined LipidConcentrate), and set up 4 experimental groups at the same time. The differentiation methods of the 4 experimental groups are roughly the same. Figure 1 As shown, the difference lies in the addition or not of WNT pathway agonist (CHIR99021) and the addition or not of FGF2 on days 0-2. The culture medium was changed every day during this period, and the levels of indicator genes of skin and extraembryonic trophoblast cells of differentiated cells were finally detected on the eighth day.

[0069] The results show that ( Figure 5 ), in the differentiation condition without adding WNT pathway agonist (CHIR99021), the expression of early skin keratinocyte indicator gene (p63) decreased, and the expression of extraembryonic trophoblast cell indicator gene (CGA, CGB) with the addition of FGF2 increased significantly compared with that without the addition of FGF2. This indicates that FGF2 can only inhibit extraembryonic trophoblast cells when the WNT pathway is activated.

[0070] Example 4

[0071] Controlling the timing of BMP4 initiation can regulate the differentiation of extraembryonic trophoblast cells.

[0072] The specific implementation plan is as follows: Human embryonic stem cells H1 were cultured in E8 medium, and fresh medium was replaced every day. When the cell density reached 70%-80%, the cells were passaged. First, they were washed twice with dPBS-EDTA, and incubated at room temperature for 8 minutes for the third time. The dPBS-EDTA was aspirated, and E8 medium containing 5 μM ROCK inhibitor (Y-27632) was added. After resuspending the cells, they were spread into a 12-well plate pre-coated with matrix gel at a density of 1:12-1:15. After one day for the cells to adhere to the plate and stabilize, differentiation medium (E8 medium removed TGFβ and FGF2, and added Chemically Defined LipidConcentrate) was used, and 2 experimental groups were set up at the same time. The differentiation methods of the 2 experimental groups were as follows: Figure 1The two groups were shown in Figure 1 and 20 ng / mL FGF2 was added on days 0-2. The difference between the two groups was that LDN193189 was added on days 0-1, while the other group did not. The culture medium was changed every day during the period, and the levels of the marker genes of the skin keratinocytes and extraembryonic trophoblast cells of the differentiated cells were finally detected on the eighth day.

[0073] The results show that ( Figure 6 ), when the BMP pathway inhibitor (LDN193189) was added, the expression of extraembryonic trophoblast cell index genes (CGA, CGB) decreased significantly, while the skin keratinocyte index (p63) did not change significantly. This indicates that the inhibitor of BMP pathway can further inhibit the function of extraembryonic trophoblast cells at the beginning.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing stem cells to differentiate into keratinocytes, characterized in that: It includes: The stem cells were induced to differentiate into keratinocytes. On days 0 to 2 of the induction culture, the differentiation medium used contained the ERK pathway activator FGF2; The differentiation medium used also contained: TGFβ pathway inhibitor SB431542, WNT pathway agonist CHIR99021, BMP-4, and NOTCH pathway inhibitor DAPT; The differentiation medium used contained the TGFβ pathway inhibitor SB431542 from day 0 to day 6 of differentiation induction; The differentiation medium used also contained the WNT pathway agonist CHIR99021 from day 1 to day 6 of induction culture; The differentiation medium used contained BMP-4 from day 1 to day 8 of differentiation induction; The differentiation medium used contained the NOTCH pathway inhibitor DAPT from day 4 to day 8 of differentiation induction; The stem cells are human pluripotent stem cells, which include human embryonic stem cells and human induced pluripotent stem cells. Human embryonic stem cells are stem cells separated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

2. The method for inducing stem cells to differentiate into keratinocytes according to claim 1, characterized in that: The concentration of the ERK pathway activator in the differentiation medium is 1-40 ng / mL.

3. The method for inducing stem cells to differentiate into keratinocytes according to claim 1, characterized in that: On day 0-1 of induction culture, the differentiation medium used also contains a BMP pathway inhibitor, and the BMP pathway inhibitor is LDN193189.

4. The method for inducing stem cells to differentiate into keratinocytes according to claim 3, characterized in that: The BMP pathway inhibitor has an effective concentration of 50-150 nM in the differentiation medium.

5. The method for inducing stem cells to differentiate into keratinocytes according to claim 1, characterized in that: The basic components of the differentiation medium include: at least one of DMEM / F12, L-ascorbic acid 2-phosphate, sodium selenite, transferrin and Chemically defined lipid concentrate.

6. Use of a target agent in the preparation of a kit for reducing or inhibiting the differentiation of stem cells into skin keratinocytes mixed with keratinocytes derived from extraembryonic trophoblast cells, characterized in that: The target reagents include ERK pathway activator FGF2, TGFβ pathway inhibitor SB431542, WNT pathway agonist CHIR99021, BMP-4 and NOTCH pathway inhibitor DAPT.

Images