CN111088213A

CN111088213A - Method for inducing stem cells to gradually differentiate to form keratinocytes and keratinocytes

Info

Publication number: CN111088213A
Application number: CN201811248262.4A
Authority: CN
Inventors: 陈国凯; 钟慧
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2018-10-24
Filing date: 2018-10-24
Publication date: 2020-05-01
Anticipated expiration: 2038-10-24
Also published as: CN111088213B

Abstract

The invention discloses a method for inducing stem cells to gradually differentiate to form keratinocytes and the keratinocytes; the method comprises the steps of differentiating stem cells in a basal differentiation medium to form keratinocyte precursor cells and inducing the keratinocyte precursor cells to differentiate into keratinocytes; the method can gradually induce and differentiate the stem cells to form the keratinocytes, provides a new idea for inducing the stem cells to form the keratinocytes, and has the characteristics of simple operation steps, short period and the like.

Description

Method for inducing stem cells to gradually differentiate to form keratinocytes and keratinocytes

Technical Field

The invention relates to the technical field of stem cell differentiation, in particular to a method for inducing stem cells to gradually differentiate into keratinocytes and the keratinocytes.

Background

The skin is the largest organ of the human body, and keratinocytes are the predominant cell type in the skin, providing a protective barrier function for the body. In animal models, various signaling pathways are involved in keratinocyte differentiation, but many functions and interactions of these pathways remain unclear in pluripotent stem cell models.

At present, methods for inducing differentiation of cells into keratinocytes by using embryonic stem cells are very diverse, but the induction process sometimes does not completely reflect findings in animal models. For example, WNT signaling is essential for epidermal differentiation, but its function still needs to be elucidated in vitro (fuch.e.et al, 2007, pattchey c.et al, 2009). At the same time, it would be interesting to understand how the different pathways synergistically induce the conversion of hescs into keratinocytes. Although WNT and NOTCH pathways are essential in animal models, there are no reports of existing hESC differentiation; at the same time these reports do not discuss how the different pathways cooperate in cell fate determination. The established differentiation program may therefore reflect in vivo signal regulation, which may serve as an ideal model for the study of key molecular mechanisms of keratinocyte differentiation. This differentiation platform can be used for disease models, drug screening and cell therapy. In this report, we will mainly investigate how keratinocytes are induced in a stepwise manner through coordinated signaling pathways.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for inducing stem cells to gradually differentiate into keratinocytes, and the method can be used for gradually inducing the stem cells to differentiate into the keratinocytes.

The invention is realized by the following steps:

in one aspect, the present invention provides a method for inducing stem cells to differentiate stepwise to form keratinocytes, comprising the steps of:

step (a): changing the stem cell culture solution cultured by the E8 culture medium into a basic differentiation culture medium for differentiation culture;

during the period, ALK5 inhibitor is added into the basal differentiation culture medium from the 1 st day to the 6 th day of the culture in the basal differentiation culture medium;

culturing in a basal differentiation medium supplemented with WNT activator and BMP4 from day 2 to day 6;

culturing in a basic differentiation culture medium for 4 days to 6 days, wherein the basic differentiation culture medium is added with a NOTCH inhibitor;

adding only BMP4 and a NOTCH inhibitor to the basal differentiation medium from day 7 to day 8 in the basal differentiation medium;

step (b): culturing the obtained cells in the step (a), and changing the cell culture fluid to a calcium differentiation culture medium containing low calcium to continue differentiation culture so as to obtain keratinocyte precursor cells;

step (c): changing the liquid of the keratinocyte precursor cells obtained in the step (b) to a keratinocyte culture medium for differentiation culture to obtain the keratinocyte.

Further, in some embodiments of the invention, the ALK5 inhibitor is SB 431542.

Further, in some embodiments of the invention, SB431542 is added to the basal differentiation medium at a concentration of 9-11 μm.

Further, in some embodiments of the invention, the WNT activator is CHIR 99021.

Further, in some embodiments of the invention, CHIR99021 is added to the basal differentiation medium at a concentration of 4-6 μm.

Further, in some embodiments of the invention, the NOTCH inhibitor is DAPT.

Further, in some embodiments of the present invention, DAPT is added to the basal differentiation medium at a concentration of 4-6 μm.

Further, in some embodiments of the invention, BMP4 is added to the basal differentiation medium at a concentration of 9-20 ng/ml.

Further, in some embodiments of the invention, the basal differentiation medium is made from E6 medium after removal of TGF β and FGF2 and addition of liposomes.

Further, in some embodiments of the present invention, the calcium differentiation medium contains calcium ions, wherein the calcium ion concentration is 0.05-0.07 mM.

Further, in some embodiments of the invention, the calcium differentiation medium contains the following components: magnesium chloride, magnesium sulfate, sodium bicarbonate, 7.5mg/L L-glutamine, L-leucine, L-lysine monohydrochloride, L-methionine, L-ascorbic acid-2-magnesium phosphate, sodium selenium, insulin, NaHCO₃Transferrin, phenol red and liposomes.

Further, in some embodiments of the invention, the calcium differentiation medium contains the following components in the following concentrations: 28.64mg/L magnesium chloride, 28.84mg/L magnesium sulfate, 1200mg/L sodium bicarbonate, 7.5mg/L L-glutamine, 59.05mg/L L-leucine, 91.25mg/L L-lysine monohydrochloride, 17.24 mg/ion-L L-methionine, 64mg/l L-magnesium ascorbate-2-phosphate, 14 μ g/l sodium selenium, 19.4mg/l insulin, 543mg/l NaHCO₃10.7mg/L transferrin, 15mg/L phenol red, 1xCD liposome and 0.06mM CaCl₂。

Further, in some embodiments of the invention, the keratinocyte culture medium is made by adding to the calcium differentiation medium: BSA, adenine, hydrocortisone, NKH477, and EGF.

Further, in some embodiments of the invention, the keratinocyte culture medium is made by adding to the calcium differentiation medium final concentrations of components: 0.7% BSA, 2.4ng/ml adenine, 0.4ug/ml hydrocortisone, 5ug/ml NKH477 and 10ng/ml EGF.

Further, in some embodiments of the invention, the stem cells are human embryonic stem cells.

In another aspect, the present invention provides a keratinocyte cell prepared by the above method.

The invention has the following beneficial effects:

the method for inducing stem cells to gradually differentiate into keratinocytes provided by the invention comprises the steps of inhibiting and starting ectodermal differentiation through TGF β (namely culturing in a basic differentiation culture medium from day 1 to day 6, adding an ALK5 inhibitor into an E6 culture medium), driving epidermal specifications by utilizing BMP and WNT activation (namely culturing in an E6 culture medium from day 2 to day 6, adding a WNT activator and BMP4 into the basic differentiation culture medium), further promoting keratinocyte maturation through NOTCH activation and calcium regulation conditions (namely culturing in an E6 culture medium from day 4 to day 6, adding a NOTCH inhibitor into the basic differentiation culture medium, culturing in a basic differentiation culture medium from day 7 to day 8, only adding a BMP4 and a NOTCH inhibitor into the basic differentiation culture medium, continuing differentiation culture by changing a cell liquid to the calcium differentiation culture medium to obtain keratinocyte precursor cells, changing the obtained keratinocyte precursor cells to the medium to culture to obtain the keratinocyte precursor cells, and effectively regulating the differentiation of the keratinocytes through a specific differentiation pathway.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 early epidermal fate determination under the influence of multiple signaling pathways in hESCs.

In the figure:

hESCs were treated with TGF β pathway ALK5 inhibitor SB431542(10 μ M) in differentiation medium and cells were harvested at specific time points to analyze gene expression by RT-qPCR.

B. Tp63 appeared under TGF- β inhibitory pulses hescs were treated with SB431542(10 μ M) for specific periods in differentiation media and cells were harvested on day 6 to analyze gene expression by RT-qPCR.

C. hESCs were treated with SB431542 in differentiation medium and different factors were added from day 1 to day 6, at which time cells were harvested for gene expression analysis by RT-qPCR, these factors included BMP4(10ng/ml) and the inhibitor LDN (100. mu.M).

The effect of wnt pathway modulation on cell fate determination between epidermal and extra-embryonic lineages. Hescs were treated with SB431542 and BMP4 in differentiation medium and CHIR99021 (5 μ M) or IWR (1 μ M) was added on days 1 to 6, at which time the cells were harvested by RT-qPCR for gene expression analysis.

FIG. 2 optimization of early epidermal cell fate determination.

In the figure:

A. ectoderm images the cell induction time course. Cells 0d, 1d, 2d or 3d in E6 contained lipid medium treated with SB431542 only, then further treated with SB431542, BMP4 and CHIR99021, and all samples were collected after 6 days and subjected to PCR to analyze the expression of TP63 gene.

B. BMP4 and CHIR99021 were added at specific time points in the presence of TGF β inhibitor SB431542 and gene expression was analyzed on day 6.

Influence of TGF β inhibition time on TP63 Induction SB431542 was added for specific periods of time in the presence of BMP4 and CHIR99021 and gene expression was analyzed on day 6.

Effect of bmp4 timing on TP63 induction. BMP4 was added for a specified period of time in the presence of SB431542 and CHIR99021, and gene expression was analyzed on day 6.

E. Effects of NOTCH inhibition during epidermal differentiation. NOTCH inhibitor DAPT (5 μ M) was added at different time points and gene expression was analyzed by qPCR 6 days after treatment.

Effects of wnt activators on epidermic cell fate assays and NOTCH inhibition. CHIR99021 was added during NOTCH treatment and samples were collected 6 days after treatment.

FIG. 3 keratinocyte maturation in Low calcium culture.

In the figure:

bmp4 and NOTCH inhibitors prolong the efficacy of treatment. Day 6 differentiated cells were treated with or without SB431542 and WNT activators for 2 days under BMP4 and NOTCH inhibitor treatment conditions and examined for gene expression by qPCR (TP63, KRT14, CGB).

B. Immunostaining examined TP63 expression at D0 and D8.

C.a) effect of EGF on keratinocyte marker expression. Additional EGF was treated with BMP4 and DAPT for 3 days 8 days after treatment. PCR measures keratinocyte and placental marker gene expression compared to EGF or not (TP63, Krt14, CGB). b) Effect of calcium concentration on keratinocyte marker expression. After the initial treatment, cells were incubated at different calcium concentrations (1mM versus 0.05mM) between 9 and 11 days and analyzed for gene expression.

D. Immunostaining detected TP63 expression at D8 and D11, TP63 (green), KRT14 (red).

Figure 4 keratinocyte derivation under defined conditions.

In the figure:

A. keratinocyte differentiation scheme;

B. morphological changes in cells during differentiation;

detection of staged keratinocyte gene expression following differentiation of c.24d;

D. immunostaining assay 26 days after differentiation, TP63 (Red), K14 (Green), K1 (Green) and K10 (Green) on day 24

E. Flow cytometry analysis of the percentage of keratinocytes after 28 days of sample;

F. keratinocytes differentiation in other hESC (H9) and hipSC lines (ND1-4, NL-1, NL-4). Gene expression was detected after 20 days of differentiation, compared to HacaT cell gene expression.

FIG. 5. derived keratinocytes and transplantation were further analyzed by global gene expression;

in the figure:

A. global gene expression profile clustering during keratinocyte differentiation;

B. specific gene expression profile in epidermal differentiation process;

hesc-derived keratinocytes (hKC) were transplanted in mouse excision wounds. a) Representative images of mouse excision wound and hKC site; b) representative images of wounds after transplantation; c) represent the figure wound and the wound 7 days after cell transplantation; d) h & E staining of wound healing sites;

immunostaining of KRT14, KRT1, KRT10, Involucrin (IVL) and Filaggrin (FLG);

F. percentage of GFP positive cells expressing a particular maturation marker gene.

FIG. 6.A summary of signaling pathways involved in ectodermal differentiation TGF β inhibits cell fate that induces ectodermal cell fates and induces neurogenesis the WNT signaling pathway is critical for Neural Crest (NC) and mesodermal (Meso) induction, BMP4 and Notch inhibition induce epidermal (Epi) and trophoblast (Troph) profiles, B: summary of factors that require activation and inhibition during epidermal differentiation.

FIG. 7 is a detailed flow chart summarizing keratinocyte differentiation.

FIG. 8. A. Effect of BMP4 concentration on early differentiation; influence of concentration of CHIR on the early differentiation of keratinocytes.

FIG. 9 EGF is capable of inhibiting the expression of CGB.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Experimental example 1

1 Stem cell culture

Cell type: hESCs (H1 and H9) and iPSCs (ND1-1, NL-1, Nd1-4) were used in this study.

H1 is the main cell line used in the research of keratinocyte development, and after a keratinocyte differentiation scheme is obtained, the stem cells hESC (H9) and iPSCs (ND1-1, NL-1 and ND1-4) from different sources are verified to be error-free. All cell lines were maintained on Matrigel coated plates (corning 354230) by E8 medium (Essential 8 medium; Thermo Fisher Scientific Life Sciences). The cells were passaged every 3 to 4 days, washed twice with EDTA/PBS (500ul, 0.5Mm EDTA in PBS, 360g/L NaCl 5ml), then the digested cells were treated with EDTA/PBS for 5 minutes at 37 ℃ and the digested cells were collected with E8 medium containing Rock inhibitor (Y27632, 5. mu.M/ml), reseeded in cell culture plates, and on the next day, the Rock inhibitor contained in the medium was removed by changing the medium with E8, followed by daily replacement of fresh medium E8.

2 monolayer keratinocyte differentiation

On day 1, cells were seeded with E8 medium containing ROCK inhibitor, on day 2 fresh E8 medium (without ROCK inhibitor) was changed, and on day 3 the cell density was approximately 30% for the subsequent differentiation step.

Basal differentiation Medium E6 medium (without TGF β and FGF2) and 1 XCDlipid (100X, Cat. No. 11905-031; Thermo Fisher Scientific Life Sciences).

Differentiation procedure:

(1) performing differential culture on the basic differential culture medium in the following method 8 days before the differential culture;

on day 1, the cells cultured in E8 medium were changed to basal differentiation medium, and ALK5 inhibitor SB431542(10 μm, catalog # S1067; Selleckchem) was added to induce cell differentiation for 24 hours;

day 2 to day 6, during which time ALK5 inhibitor SB431542(10 μm, cat # S1067; Selleckchem), WNT activator CHIR99021 (5 μm, cat # S2924; Selleckchem) and BMP4(10ng/ml, R & D) were added daily to continue induction of differentiation;

in addition, the NOTCH inhibitor DAPT (5 μm, catalog number 2634; Tocris) was additionally added to the medium every day from day 4 to day 6;

in addition, addition of only BMP4(10ng/ml) and DAPT (5 μm) induced cells to continue differentiation from day 7 to day 8.

During this period, the medium was changed every day.

(2) From day 9 to day 11, cells were started to be cultured using the low-calcium differentiation medium, with fresh medium changed daily:

specifically, on day 9, the cells cultured by the above procedure were transferred to a low calcium differentiation medium to continue differentiation culture, and BMP4(10ng/ml), DAPT (5 μm) and EGF (10ng/ml, Cat. AF-100-15; Peprotech) were added to the low calcium differentiation medium every day to gradually induce keratinocyte precursor cells.

The low-calcium differentiation culture medium is prepared by adding 0.06mM CaCl into basic differentiation culture medium₂And (4) preparing.

The calcium concentration in the basal differentiation medium was varied and 0.06mM calcium was added in addition to 0.06mM calcium differentiation medium (calcium-free DMEM/F12 powder, sigma), 1-ascorbic acid, selenium, transferrin and NaHCO3, 1xCD lipid) (Gk. chen. 2011), Bmp4(10ng/ml), DAPT (5 μm), EGF (10ng/ml, Cat. AF-100-15; peprotech) of the group,

wherein the preparation method of the low-calcium differentiation medium (1L) comprises the following steps:

taking 14.8g/L DEME/F12 powder (sigma D9785), dissolving the powder with distilled water (Therm Fisher 15230), and adding the following components in final concentration:

28.64mg/L magnesium chloride (Sigma M4880), 28.84mg/L magnesium sulfate (Sigma M2643), 1200mg/L sodium bicarbonate (Sigma s5761), 7.5mg/L L-glutamine (Sigma G5792), 59.05mg/L L-leucine (Sigma L8912), 91.25mg/L L-lysine monohydrochloride (Sigma L8662), 17.24mg/L L-methionine (Sigma M5308), 64mg/L L-magnesium ascorbate-2-phosphate (Sigma), 14. mu.g/L sodium selenium (Sigma), 19.4mg/L insulin (Sigma), 543mg/L NaHCO₃(sigma), 10.7mg/l transferrin (R)&D) Phenol red 15mg/L (sigma P3532), 1xCD liposome and 0.06mM CaCl₂(ii) a Make up to 1L with distilled water.

(3) On day 12 and later, the keratinocyte precursor cells were maintained in keratinocyte medium (containing 0.06mM Ca)²⁺) To form keratinocytes. During this period, fresh medium was replaced daily.

Keratinocyte medium was prepared by adding the following components to a low calcium differentiation medium at final concentrations: 0.7% BSA, 2.4ng/ml adenine, 0.4ug/ml hydrocortisone, 5ug/ml NKH477 and 10ng/ml EGF.

3 real-time polymerase chain reaction

RNA was purified using RNAiso Plus reagent according to the manufacturer's protocol (Takara, RNAiso Plus Cat. # 99109). Using the recommended protocol (Takara,

taq TM II) for reverse transcription. Using Takara sybr Green Premix (Takara,

taq TM II) was tested in BIO-RAD CFX96(Bio-Rad laboratories, Hercules, CA, http: com) was performed. Quantitative PCR: quantitative PCR (qpcr) was performed on an Applied Biosystems QuantStudio 7Flex real-time PCR system and the data was normalized to GAPDH or TBP expression. The primers used are shown in table 1.

TABLE 1

4 flow cytometry analysis

Cells were dissociated into single cells by treatment with TrypLE Select enzyme (Thermo Fisher Scientific Life Sciences), signal cells were washed in PBS, then fixed with 4% Paraformaldehyde (PFA) for 20 minutes at room temperature, washed with PBS, and permeabilized in 0.5% Triton. X-100/PBS for 10 min. After washing in PBS, blocking was performed in 3% bovine serum albumin (BSA/PBS) for 1 hour and incubated with primary antibody overnight at 4 ℃. Thereafter, the cells were washed 5 min, 3 times in PBS, then the secondary antibody (Alexa 488Thermo Fisher Scientific Life Sciences) was incubated for 30 min, then washed twice, suspended in PBS and analyzed by flow cytometry using BD Accuri TM C6 Cytometer. The following primary antibodies were used: anti-TP 63 antibody (BA1887, Boster); cytokeratin 14(sc-58724, Santa Cruz); cytokeratin 1(sc-65999, Santa Cruz); cytokeratin 10(sc-23877, Santa Cruz).

5 Immunity staining

Cells (PFA) were fixed in 4% paraformaldehyde for 20 min, spun for 5 min at 1000rpm, then the pellet was washed with PBS and permeabilized in 0.5% Triton X-100/PBS for 10 min. Blocking with 3% BSA/PBS for 1h, followed by overnight incubation with primary antibody at 4 ℃. The cells were washed three times with PBS, then the secondary antibody was incubated for 1 hour, washed 5 minutes 3 times, and cell nucleus staining was performed by adding Hochest 33258(Molecular Probes H1398) for 10 minutes.

Results of the experiment

(1) Inhibition of induced ectodermal cells by TGF β

To establish an in vitro platform reflecting in vivo regulation, we summarized the signaling pathways that may exist in keratinocytes, their role (fig. 6A and 6B), and explored the stage-specific role of each factor during keratinocyte differentiation (fig. 7). first, we examined whether TGF β inhibition could induce primitive ectoderm, which is likely to become neuroectoderm and surface ectoderm in hESCs. ALK5 inhibitor SB431542 was reported to inhibit TGF β pathway in the initiation of ectoderm in a mouse model, and used a 6 day time course here, the appearance of lineage markers was significantly reduced within one day with a set of marker genes including NANOG (pluripotency), TP63 (epidermis), PAX3 (neural crest), SIX germ layer 1 (basal plate), PAX6 (neural) and CGB (extraembryonic). under inhibition of TGF β, the pluripotent markers were significantly reduced within one day, while the appearance of all ectoderm and extrablast markers within SIX days (fig. 1. this could indicate that TGF 6A could be used to induce lineage differentiation and all differentiation events were evaluated using TGF β.

We further evaluated whether the time of TGF β inhibition affected ectodermal differentiation, hESCs were first exposed to TGF β inhibition for different times and then differentiated to day 6 without inhibition one day of TGF β inhibition was sufficient to induce epidermal, basal and extra-embryonic gene expression, but neural and neural crest lineage markers required TGF β inhibition for more than 5 days (fig. 1B).

(2) Early epidermal induction through activation of BMP and WNT signaling pathways

BMP4 is widely used for epidermal differentiation, in addition to sustained TGF β inhibition, BMP4 significantly inhibits the neural (PAX6) lineage, while increasing expression in both extra-embryonic (CGB) and epidermal (TP63) lineages (fig. 1C).

To identify factors that can inhibit the differentiation of extra-embryonic cells, we screened various pathway modulators in the presence of BMP4 and SB 431542. We found that WNT activator CHIR99021 significantly inhibited extraembryonic cell formation while increasing epidermal cell differentiation (fig. 1D). In contrast, WNT inhibitor IWR inhibited the epidermal lineage and induced extracellular cell differentiation (fig. 1D). These data suggest that WNT activation is important for epidermal lineage differentiation, a result consistent with findings in animal models.

(3) Treatment time optimization for early epidermal differentiation

We optimized time treatment by SB431542, BMP4 and CHIR99021 during the 6 day time course, by first treating with SB431542 alone for 24 hours, second when BMP4 and CHIR99021 were added, optimal TP63 expression was observed (fig. 2A) when hescs were treated with SB431542 alone for too long, epidermal lineage was inhibited, while we analyzed that prolonged SB431542 exposure did not affect TP63 expression (fig. 2B) when treated with BMP4 and CHIR99021, indicating that early TGF β inhibition can establish a barrier to differentiation of the surface ectodermal lineage with CHIR99021 and BMP 4.

The timing of BMP4 and WNT signaling pathway activation affected TP63 expression. Prolonged exposure to BMP4 resulted in consistent TP63 expression in the presence of SB431542 and CHIR99021, and helped to inhibit CGB and PAX3 expression (figure 2C). Increased doses of BMP4 also inhibited neural crest differentiation based on PAX3 expression (fig. 8A). We also found that prolonged WNT activation increased neural crest PAX3 expression and decreased TP63 when SB431542 and BMP4 were present (fig. 2D). With decreased WNT activation, TP63 increased at the expense of increased extra-embryonic CGB (fig. 8B). These results suggest that BMP and WNT are beneficial for epidermal differentiation, but additional signaling may be required to overcome the WNT effects to drive neural crest cell fate.

(4) NOTCH inhibition improves epidermal progenitor cell differentiation

The NOTCH inhibitor DAPT increased TP63 expression in epidermal differentiation. We demonstrated that DAPT increased TP63 expression in the presence of SB431542, BMP4 and CHIR99021 (FIG. 2E). DAPT is also an inducer of extra-embryonic lineages. When DAPT was added prematurely, extra-embryonic lineages were increased based on CGB expression (fig. 2F). When DAPT was used, the addition of WNT activator CHIR99021 inhibited the extra-embryonic CGB expression, but TP63 was still maintained during differentiation (fig. 2F). These results indicate that NOTCH inhibition favors the transition from the superficial ectoembryonic layer to the keratinocytes in the anterior horn of the epidermis, but WNT activity is also required to inhibit extraembryonic expression.

(5) Keratinocyte maturation in low calcium culture

From pre-epidermal keratinocytes to epidermal keratinocytes, additional treatment is required to promote the transition. After day 6, continuous exposure to BMP4 and DAPT further increased TP63 expression, but SB431542 and CHIR99021 inhibited TP63 expression (fig. 3A). Based on this observation, BMP4 and DAPT were used to drive keratinocyte differentiation after day 6. Immunostaining showed that most cells were TP63 positive after 8 days of differentiation (fig. 3B).

Epidermal Growth Factor (EGF) was used in keratinocyte culture medium and added to the medium, we observed that EGF helped to inhibit CGB while maintaining TP63 expression (fig. 9). Even with elevated TP63 expression, we noted no increase in other key keratinocyte expression markers (fig. 3C). With low calcium applied to the cells, we observed that low calcium at 0.06mM significantly increased the expression of TP63 and KRT14 and resulted in less expression of neural crest PAX3 (FIG. 3D). Immunostaining also confirmed that more than 90% of the cells were TP63 positive (fig. 3E). This indicates that EGF can significantly increase the expression of TP63 at this stage, and low calcium further promotes the expression of marker gene KRT14 in epidermal stem cells.

(6) Keratinocyte derivation under defined conditions

Based on gene expression during differentiation at each stage, we established a protocol for differentiating hescs into pre-epidermal keratinocytes and then epidermal keratinocytes (fig. 4A). During this process, cell morphology changed significantly over time (fig. 4B). Epidermal marker gene expression changes were consistent with the differentiation stage (fig. 4C). After more than 20 days of cell differentiation, most cells showed keratinocyte markers including TP63, KRT14, KRT10, and KRT 1. The experimental results were consistent with the flow cytometry results, i.e., > 95% cells were positive for p63 and K14 (fig. 4D).

The keratinocyte approach works for a variety of hPSC lines, including hESC (H9) and hipSC (NL-1, NL-5, ND-1) lines. Most cell lines showed positive expression of keratinocyte gene expression, and this level was comparable to the immortalized keratinocyte HacaT cell line (fig. 4E). We also noted that different lines had variations in marker gene expression, particularly the ND1 line with reduced K14 expression. This suggests that the protocol may need to be adapted to the differentiation requirements of a particular cell line.

(7) Gene expression in keratinocyte differentiation

Keratinocyte gene expression is programmed in vivo during differentiation (C byrne et al, 1994). We investigated how global gene expression can be altered on an in vitro platform. Clustering analysis of gene expression demonstrated that global gene expression profiles were clustered according to differentiation stage (fig. 5A). The production of skin-specific genes according to BIOGPS (ACAD9, ACPP, ANKRD 9, ANO9, ANXA8L 9, ANXA9, APCDD 9, ARRDC 9, ATP2A 9, BNC 9, BNIPL, C2CD 9, C5orf 9, CACNA 19, CASQ 9, CD109, CERS 9, CFAP 9, CLDN 9, CLIC 9, CNFN, COL7A 9, CPA 9, CST 9, CYSRT 9, DKKL 9, DMKN, DSC 9, DSP, ECHDC 9, EFNB 9, ELAPL 9, ELOVL 9, EPHB 9, EPHX 9, KR 9, EVPL 9, EPGL 9, EPR 9, GALN 9, TFL 363672, TFL 9, TFL 363636363636363636363672, TFL 36363672, TFL 363636363672, TFL 3636363672, TFL 36363672, TFL 36363636363672, TFL 36363672, TFL 363672, TFL 3636363672, TFL 9, TFL 363636363636363636363672, TFL 36363672, TFL 363672, TFL 363636363636363636363636363636363636363636363672, TFL 36363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363672, TFL 363636363636363636363636363636363636363636363636363672, TFL 9, TFL 36363672, TFL 9, TFL 3636363672, RAPGEFL, RDH, RGMB, RNASE, RORA, RYR, S100A, SBSN, SDR16C, SEMA3, SERPINB, SGCG, SH3RF, SLC16A, SLC2A, SLITRK, SMPX, SOSTDC, SOX, SPAG, SPINK, SPTLC, SUSD, TACTD, TANC, TBX, TFAP2, TGM, THEM, TMEM45, TMPRSS11, TNFRSF, TNNT, TP, TRIM, TUFT, ZNF750, NANOG, POU5F, SOX, LIN 28), and the expression of skin-specific genes occurs according to the stage of differentiation (FIG. 5B). GO analysis showed that specific genes reflected function at each specific stage (fig. 5C). Interestingly, some skin-specific genes were not expressed for around 25 days even until the final stage of differentiation (fig. 5D). This suggests that these genes may appear late in keratinocyte maturation. We collected cells that differentiated for 20 days and transplanted them onto the excised wounds of immunodeficient mice by injection and topical application. Wound closure 7 days after transplantation. The H & E staining results confirmed the formation of new keratinized skin (fig. 5C). Immunostaining of serial skin sections showed positive expression of KRT14, KRT1, KRT10, Involucrin and Flaggrin (fig. 5D, red fluorescence). GFP positive cells remained in the skin and green fluorescence co-localized with staining of the keratinocyte maturation marker, indicating survival and continued maturation of the transplanted precursor cells in vivo (fig. 5D-E). These results indicate that hPSC-derived keratinocyte progenitor cells can survive and be incorporated into mouse skin in vivo. These cells are likely to be useful for future therapeutic applications.

In conclusion, keratinocytes form an epithelial barrier against infections and water loss in the skin, and they are also essential in wound healing. Keratinocytes have many applications in disease models and clinical treatments. Among the combination of Induced Pluripotent Stem Cell (iPSC) technology, hpscs have the potential to generate unlimited patient-specific keratinocytes in vitro. To realize the potential of hPSC-derived keratinocytes, there remains a need to improve this technology in a number of ways, including lineage specific differentiation, cell mass control, cell expansion and transplantation.

We focused on how to efficiently produce keratinocytes based on signal regulation and lineage requirements. We have gradually established an in vitro epidermal differentiation procedure. We can demonstrate that effective epidermal cell fate specifications require the cooperation of multiple signaling pathways at specific stages. This study not only provides a new in vitro differentiation protocol to generate keratinocytes, but also provides an in vitro platform to understand the development of keratinocytes.

In conclusion, the present invention explains in detail the growth factors required at various stages during the differentiation of keratinocytes and the activation of the required signals through the differentiation process of keratinocytes under serum-free conditions, and reveals for the first time the process of keratinocyte development in vitro. The invention not only provides a new effective differentiation scheme, but also is simpler and has shorter period compared with the prior differentiation means.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for inducing stem cells to differentiate stepwise to form keratinocytes, comprising the steps of:

step (b): culturing the obtained cells in the step (a), and changing the cell fluid at the stage into a calcium differentiation culture medium containing low calcium to continue differentiation culture so as to obtain keratinocyte precursor cells;

2. The method of claim 1, wherein the ALK5 inhibitor is SB 431542.

3. The method of inducing the stepwise differentiation of stem cells into keratinocytes as claimed in claim 1, wherein the WNT activator is CHIR 99021.

4. The method of inducing the gradual differentiation of stem cells into keratinocytes as claimed in claim 1, wherein the NOTCH inhibitor is DAPT.

5. The method for inducing the gradual differentiation of stem cells into keratinocytes according to any one of claims 1 to 4, wherein said basal differentiation medium is prepared from E6 medium after removing TGF β and FGF2 and adding liposomes.

6. The method for inducing the stepwise differentiation of stem cells into keratinocytes according to any one of claims 1 to 4, wherein said calcium differentiation medium comprises calcium ions, wherein the concentration of calcium ions is 0.05 to 0.07 mM.

7. The method for inducing the stepwise differentiation of stem cells into keratinocytes according to claim 6, wherein said calcium differentiation medium comprises the following components: magnesium chloride, magnesium sulfate, sodium bicarbonate, 7.5mg/L L-glutamine, L-leucine, L-lysine monohydrochloride, L-methionine, L-ascorbic acid-2-magnesium phosphate, sodium selenium, insulin, NaHCO₃Transferrin, phenol red and liposomes.

8. The method for inducing the stepwise differentiation of stem cells into keratinocytes according to claim 7, wherein said keratinocyte medium is prepared by adding the following components to said calcium differentiation medium: BSA, adenine, hydrocortisone, NKH477, and EGF.

9. The method of inducing the stepwise differentiation of stem cells into keratinocytes according to claim 1, wherein said stem cells are human embryonic stem cells.

10. A keratinocyte cell produced by the method of any of claims 1-9.