Method for differentiating iPSC cell strain into melanocytes and application thereof

Abstract

The invention belongs to the technical field of regenerative medicine, and discloses a method for differentiating an i PSC cell strain into melanocytes and application of the method, and the method comprises the following steps: firstly establishing an HLA monomer high-matching i PSC cell strain, and then establishing a method for effectively directionally differentiating into the melanocytes, so as to prepare the melanocytes on a large scale; the application is the application of the method for differentiating the i PSC cell strain into the melanocytes in the aspect of clinical white hair blackening by allograft transplantation. The melanocyte preparation method has the advantages of strong in-vitro amplification capability, high success rate and low preparation cost, and is beneficial to clinical popularization and application in the future.

Description

Technical Field

[0001] The present invention belongs to the technical field of regenerative medicine, and specifically relates to a method for differentiating an iPSC cell line into melanocytes and an application thereof. Background Art

[0002] Hair is a symbol of personal beauty and self-esteem, and plays a vital role in daily life. However, gray hair, caused by many factors such as genetics, disease, stress and aging, is troubling more and more people. Although gray hair is known to greatly affect people's mental health and social life, there is a lack of effective countermeasures except for hair dyes (the use of hair dyes is positively correlated with the risk of cancer).

[0003] The human hair growth cycle is 3-5 years, and the hair begins to turn white after about 10 cycles. White hair can be divided into physiological white hair and pathological white hair. The hair does not mutate to white by itself, but is caused by the reduction or absence of melanin synthesized by melanocytes in the newly generated hair in this cycle. For example, stress causes the sympathetic nerves to release norepinephrine, which causes excessive division and exhaustion of melanocytes. Aging white hair is caused by melanocyte stem cells being stuck and unable to move up and down the hair correctly to receive the correct signals and then differentiate into melanocytes. Although there are many hypotheses about the formation mechanism of white hair, they are fundamentally caused by the imbalance of melanocyte stem cells (McSCs) and their lineages in the hair follicles. Such as the reduction of McSCs and the destruction of their homeostasis, the loss of melanocyte migration ability, the loss of the growth phase in the hair cycle, the apoptosis of melanocytes, and the transport and synthesis of melanosomes. However, as the core of melanocyte regeneration and the source of the entire pigment cycle, once the ability of McSCs to maintain and renew itself is destroyed, the subsequent melanocyte lineage will eventually lose its function and cannot be restored. Therefore, the destruction of the homeostasis of McSCs is the key to the formation of gray hair.

[0004] The hair follicle is a skin appendage composed of multiple cell types that controls hair growth and is the "birthplace" of the hair shaft. The bulge and hair germ area of ​​the hair follicle contain two groups of stem cells: epithelial-derived hair follicle stem cells (HFSCs) and neural crest-derived melanocyte stem cells (MeSCs). As a "supply station" for the pigment cell cycle, melanocyte stem cells (McSCs) located in the hair follicle play an important role in maintaining homeostasis. Once destroyed, other subsequent daughter melanocytes will eventually lose their function, such as being unable to produce melanin granules to color the hair. Therefore, how to maintain the homeostasis of endogenous McSCs and activate and regenerate them, or supplement exogenous melanocyte stem cells has become an important breakthrough in the treatment of gray hair.

[0005] Although primary melanocytes can be isolated and cultured from individual skin or hair follicle tissue, firstly, their in vitro proliferation capacity is limited; secondly, they are easily contaminated by fibroblasts and keratinocytes, resulting in a low culture success rate; and thirdly, the cost of individualized preparation is too high, which is not conducive to future clinical promotion and application.

[0006] Induced pluripotent stem cells (iPSCs) are a Nobel Prize-winning technology developed by Professor Shinya Yamanaka of Kyoto University in Japan in 2006. Four transcription factors, Oct3 / 4, Sox2, c-Myc and Klf4, are introduced into somatic cells (such as skin fibroblasts) to induce their reprogramming. The cells produced are very similar to embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cell multiplication ability, embryoid body and teratoma generation ability, and three-germ layer differentiation ability, so they are called induced pluripotent stem cells. It is precisely because of the powerful in vitro expansion ability and multipotential differentiation ability of iPSC that it has been used as seed cells to produce a variety of functional cells, such as macrophages, immune cells, dopaminergic neurons, hepatocytes, platelets, pancreatic islet cells and retinal pigment epithelial cells. Due to the affirmation of the efficacy, the development of a number of new cell biological drugs derived from iPSC cells has entered the clinical trial stage, such as for the treatment of Parkinson's disease, diabetes and age-related macular degeneration.

[0007] The purpose of iPSC cell HLA haplotype library construction (Haplobanking) is to match a higher proportion of the population with the least cell lines, thereby avoiding rejection reactions during allogeneic stem cell transplantation in the future or reducing the use of anti-immune rejection drugs. For example, British scholar Taylor's research shows that 10 HLA-A, HLA-B and HLA-DR homozygous donors screened from 10,000 people, using their somatic cells to reprogram iPSC cells to build a cell bank, can provide HLA phenotype-matched stem cells for at least 37.7% of the British. If matching stem cells are to be provided for 93% of the British, 150 HLA phenotype high-matching cell lines need to be built. Nakatsuji's research shows that 30 homozygous donors were screened from 15,000 people to build a line, which can match 82.2% of Japanese people. 50 homozygous donors screened from 24,000 people can increase this proportion to 90.7%. The above international studies only focus on the three loci of HLA-A, HLA-B and HLA-DR. Our design is based on the same principle but increases HLA to 5 loci of AB-CW-DQ-DR to further improve the accuracy of matching and reduce potential rejection reactions.

[0008] In view of the above reasons, the purpose of the present invention is to use HLA monomer high-matching iPSC cells for in vitro directed differentiation to prepare melanocytes on a large scale for use in allogeneic transplantation to convert white hair to black. Summary of the invention

[0009] To overcome the above technical problems, the present invention provides a method for differentiating iPSC cell lines into melanocytes and its application, firstly establishing an HLA monomer high-matching iPSC cell line, and then establishing an effective method for directed differentiation into melanocytes, thereby preparing melanocytes on a large scale.

[0010] The present invention adopts the following technical solutions:

[0011] The method of differentiating an iPSC cell line into a melanocyte comprises the following:

[0012] S1. Digest iPSC cell lines with Stempro Accutase cell dissociation buffer (ThermoFisher) for 1–2 min and collect cell clumps.

[0013] S2. Centrifuge the cells at room temperature and gently resuspend the cells in neurobasal / Ham's F12 (ratio 1:1) medium (supplemented with 1% of N-2 and 2% B-27 without vitamin A, ThermoFisher) to keep the cells in pellets;

[0014] S3. Transfer the cell clumps to low-adhesion well plates to form embryoid bodies, and add CHIR-99021, LDN-193189, and SB 431542 to the culture medium for 4 days to induce the embryoid bodies to differentiate into the neural crest lineage.

[0015] S4. Add CHIR99021, EDN3, SCF, human recombinant BMP4 and ascorbic acid to the neural basal medium and continue to induce differentiation for 3 days;

[0016] S5. From day 7 to day 30, embryoid bodies were transferred to gelatine-coated plates and cultured in melanocyte medium MGM-4 supplemented with CHIR99021, EDN3, SCF, recombinant human BMP4, and ascorbic acid to induce differentiation into melanocytes.

[0017] S6. When the cells are confluent, they are passaged with 0.05% trypsin and cultured for two generations in melanocyte differentiation medium supplemented with 20 μM forskolin.

[0018] S7. Starting from the third generation, the differentiated melanocytes were continuously passaged in MGM-4 medium without any additives until the 20th generation;

[0019] S8. After melanocytes are identified, the black precipitate is collected by centrifugation to obtain the melanocytes.

[0020] Preferably, the preparation process of the iPSC cell line used is:

[0021] S01. Screening of mononuclear cells isolated from cord blood with homozygous haplotype combinations of 10 loci of 5 tandem loci A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0202 for reprogramming to prepare iPSC cells;

[0022] S02. Culture monocytes with StemPro-34 serum-free complete medium;

[0023] S03. Transduce cells using the Sendai virus reprogramming kit, add Klf4, Oct4 and Sox2 (KOS) viruses and c-Myc virus, and incubate at 37°C;

[0024] S04. After 1 day, the Sendai virus was washed away and the cells continued to be cultured in StemPro-34;

[0025] S05. Plate cells on Geltrex matrix-coated culture dishes using StemPro-34 complete medium and culture continuously;

[0026] S06. Transition the cells to Essential 8 medium and continue culturing;

[0027] S07. On days 15-21 after transduction, when the colonies grow to a size suitable for transfer, pick the colonies and transfer them to the prepared Geltrex matrix-coated culture plates for clonal expansion;

[0028] S08. After the iPSC cell surface markers and three-germ layer differentiation ability are identified and meet the standards, the iPSC cell line is obtained.

[0029] Preferably, in S02, mononuclear cells from cord blood are cultured in one well of a 24-well plate using 1 ml of StemPro-34 serum-free complete medium at a cell concentration of 2.5×10 5 – 5×10 5 cells / mL and cultured for 1 day.

[0030] Preferably, S03 is: taking out a set of CytoTune-iPS2.0 Sendai virus reprogramming kit from a storage device at -80°C, adding 1 ml of Klf4, Oct4 and Sox2 (KOS) viruses with MOI = 5 and MOI = 5 c-Myc virus, plating the cells in a 12-well tissue culture plate, and incubating overnight at 37°C to perform cell transduction.

[0031] Preferably, in S04, the cells are cultured in StemPro-34 for 2 days; and in S05, the cells are cultured continuously for 7 days with half of the medium changed every day.

[0032] Preferably, S5 is: from day 7 to day 30, embryoid bodies are transferred to a 0.1% gelatine (Sigma-Aldrich) coated well plate, cultured with melanocyte medium MGM-4 supplemented with 0.5 μM CHIR99021, 100 nM EDN3, 50 ng / mL SCF, 0.02 nM of human recombinant BMP4, and 0.3 mM ascorbic acid, and the medium is changed every 2-3 days to induce differentiation into melanocytes.

[0033] Preferably, in S8, melanocyte identification is performed by using immunofluorescence staining to prove that the differentiated melanocytes express typical markers MITF and TYRP1.

[0034] The present invention also includes the application of the above-mentioned method of differentiating iPSC cell lines into melanocytes in clinical conversion of white hair to black in allogeneic transplantation.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The preparation method of the present invention has a strong in vitro amplification ability and a high success rate, and can prepare off-the-shelf universal melanocytes in large quantities. Compared with individualized preparation, it has low cost and is conducive to future clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is an image of iPSC cell clones obtained by reprogramming HLA-high-matched cord blood mononuclear cells;

[0038] Figure 2 This is an image of the identification of iPSC cell stemness surface markers;

[0039] Figure 3 It is the result of the identification of iPSC cell stemness surface markers;

[0040] Figure 4 This is an image of the identification of the three germ layers of iPSC cells;

[0041] Figure 5This is an image of embryoid bodies obtained from iPSC suspension culture being induced into melanocytes in vitro. DETAILED DESCRIPTION

[0042] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. Unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are conventional methods in the art. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0043] 1. Establishment of HLA-high iPSC cell lines:

[0044] S01. Screening of mononuclear cells isolated from cord blood with homozygous haplotype combinations of 10 loci of 5 tandem loci A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0202 for reprogramming to prepare iPSC cells;

[0045] According to bioinformatics analysis, when 10 sites of the five tandem loci A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0202 are homozygous, the haplotype can be matched with the Chinese population (providing matching stem cells) with the highest probability during haplotype transplantation. The cord blood mononuclear cells isolated by Ficoll-paue density gradient centrifugation were screened for this haplotype combination and used for reprogramming to prepare iPSC cells.

[0046] S02. On day 1, culture mononuclear cells from cord blood in one well of a 24-well plate with 1 ml of StemPro-34 serum-free complete medium at a cell concentration of 2.5 × 10 5 – 5×10 5 cells / mL, cultured for 1 day;

[0047] S03. On the second day, take out a set of CytoTune-iPS2.0 Sendai virus reprogramming kit from the storage device at –80°C, add 1 ml of Klf4, Oct4 and Sox2 (KOS) virus with MOI=5 and c-Myc virus with MOI=5, plate the cells in a 12-well tissue culture plate, and incubate at 37°C overnight to perform cell transduction and mediate reprogramming of the four transcription factors;

[0048] S04. After 1 day, the Sendai virus was washed away and the cells were cultured in StemPro-34 for another 2 days;

[0049] S05. On day 5, cells were plated on Geltrex matrix-coated culture dishes using StemPro-34 complete medium and cultured for 7 consecutive days, with half of the medium changed every day;

[0050] S06. On day 12, the cells were transitioned to Essential 8 medium and cultured for 20 consecutive days, during which time the cells were fed and monitored.

[0051] S07. On the 15th to 21st day after transduction, when the colonies grow to a size suitable for transfer, select the clones with compact cells, clear edges, and independent existence under the microscope, gently scrape them with a 27-gauge needle, transfer them to new wells of a 24-well plate pre-coated with Geltrex, and expand and culture them in E8 medium. Figure 1 This is the clone morphology of the expanded iPSC cells. Under a 40X microscope, single cells with a high nuclear-cytoplasmic ratio can be seen.

[0052] S08. After the iPSC cell surface markers and three-germ layer differentiation ability are identified and meet the standards, the iPSC cell line is obtained.

[0053] 2. iPSCs are induced to differentiate into melanocytes:

[0054] S1. Digest iPSC cell lines with Stempro Accutase cell dissociation buffer (ThermoFisher) for 1–2 min and collect cell clumps.

[0055] S2. Centrifuge the cells at 110 × g for 3 min at room temperature and gently resuspend the cells in neurobasal / Ham's F12 (ratio 1:1) medium (supplemented with 1% of N-2 and 2% B-27 without vitamin A, ThermoFisher) to maintain the pellet;

[0056] S3. The cell clumps were transferred to low-adhesion well plates to form embryoid bodies, and CHIR-99021 (3 μM) (TOCRIS), LDN-193189 (0.2 μM) (Miltenyi Biotec, Bergisch Gladbach, Germany), and SB431542 (40 μM) (TOCRIS) were added to the culture medium for 4 days to induce the embryoid bodies to differentiate into the neural crest lineage.

[0057] S4. Starting from day 4, 3 μM CHIR99021, 100 nM EDN3 (American peptide, Albuquerque, NM, USA), 50 ng / mL SCF (Peprotech, NJ, USA), 0.02 nM of human recombinant BMP4 (Peprotech, Cranbury, NJ, USA) and 0.3 mM ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) were added to the neural basal medium and differentiation was induced for another 3 days.

[0058] S5. From day 7 to day 30, embryoid bodies were transferred to 0.1% gelatine (Sigma-Aldrich)-coated plates and cultured with melanocyte medium MGM-4 (MGM-4 Bullet Kit Lonza, Allendale, NJ, USA) supplemented with 0.5 μM CHIR99021, 100 nM EDN3, 50 ng / mL SCF, 0.02 nM of human recombinant BMP4, and 0.3 mM Mascorbic acid. The medium was changed every 2–3 days to induce differentiation into melanocytes.

[0059] S6. When the cells were confluent, they were passaged with 0.05% trypsin and cultured for two generations in melanocyte differentiation medium supplemented with 20 μM forskolin (Sigma-Aldrich, St. Louis, MO, USA);

[0060] S7. Starting from the third generation, the differentiated melanocytes were continuously passaged in MGM-4 medium without any additives until the 20th generation;

[0061] S8. Identification of mature and functional melanocytes was performed using immunofluorescence staining (1:200 mouse anti-MITF (DAKO, Santa Clara, CA, USA; M3621), 1:100 mouse anti-TYRP1 (Lsbio, Seattle, WA, USA; LSC39939)) to demonstrate that differentiated melanocytes expressed typical markers MITF and TYRP1. After cell centrifugation, black precipitates (melanin granules) were visible to the naked eye, and the precipitates were used to obtain melanocytes.

[0062] From the results of immunofluorescence staining, NANOG and OCT4 were highly expressed in both morphological cell lines, proving that the cells obtained by reprogramming in this study were iPSCs. It also proved that HLA homozygous umbilical cord blood mononuclear cells can also be reprogrammed into iPSCs. The purity of iPSC cells was detected by flow cytometry, and the expression of surface marker SSEA4 and nuclear marker Oct3 / 4 could reach 94%, proving that the purity of iPSCs was high, reaching more than 90%. Figure 2 .

[0063] Another indicator for identifying iPSC cells is "pluripotency". True iPSC cells have the potential to differentiate into all cells of any of the three germ layers: endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract) or ectoderm (epidermal tissue and nervous system). According to the instructions for use of the three-germ layer in vitro differentiation kit (Nanjing ALP Regenerative Medicine Technology Co., Ltd.), in vitro differentiation was carried out for 14 days, and then the expression of special markers of the three germ layers was identified by immunofluorescence, such as Figure 5 As shown, it can be seen that the obtained i PSC cell line can differentiate into AFP+ (inside), Brachyury+ (middle) and PAX6+ (outside) cells at a high proportion, indicating that the obtained i PSC clones have the "pluripotency" of three-germ layer differentiation, which functionally proves that the cells obtained by reprogramming in this study are i PSCs.

[0064] The above-mentioned method of differentiating iPSC cell lines into melanocytes is used in the clinical application of allogeneic transplantation to convert white hair into black hair, and is used to provide melanocytes.

[0065] Although the embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the above embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for differentiating an iPSC cell line into a melanocyte, characterized in that: Includes the following: S1. Digest iPSC cell lines with Stempro Accutase cell dissociation buffer (ThermoFisher) for 1–2 min and collect cell clumps. S2. Centrifuge the cells at room temperature and gently resuspend the cells in neurobasal / Ham's F12 (ratio 1:1) medium (supplemented with 1% of N-2 and 2% B-27 without vitamin A, ThermoFisher) to keep the cells in pellets; S3. Transfer the cell clumps to low-adhesion well plates to form embryoid bodies, and add CHIR-99021, LDN-193189, and SB 431542 to the culture medium for 4 days to induce the embryoid bodies to differentiate into the neural crest lineage. S4. Add CHIR99021, EDN3, SCF, human recombinant BMP4 and ascorbic acid to the neural basal medium and continue to induce differentiation for 3 days; S5. From day 7 to day 30, embryoid bodies were transferred to gelatine-coated plates and cultured in melanocyte medium MGM-4 supplemented with CHIR99021, EDN3, SCF, recombinant human BMP4, and ascorbic acid to induce differentiation into melanocytes. S6. When the cells are confluent, they are passaged with 0.05% trypsin and cultured for two generations in melanocyte differentiation medium supplemented with 20 μM forskolin. S7. Starting from the third generation, the differentiated melanocytes were continuously passaged in MGM-4 medium without any additives until the 20th generation; S8. After melanocytes are identified, the black precipitate is collected by centrifugation to obtain the melanocytes.

2. The method for differentiating an iPSC cell line into melanocytes according to claim 1, characterized in that: The preparation process of the iPSC cell line used is as follows: S01. Screening of mononuclear cells isolated from cord blood with homozygous haplotype combinations of 10 loci of 5 tandem loci A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0202 for reprogramming to prepare iPSC cells; S02. Culture monocytes with StemPro-34 serum-free complete medium; S03. Transduce cells using the Sendai virus reprogramming kit, add Klf4, Oct4 and Sox2 (KOS) viruses and c-Myc virus, and incubate at 37°C; S04. After 1 day, the Sendai virus was washed away and the cells continued to be cultured in StemPro-34; S05. Plate cells on Geltrex matrix-coated culture dishes using StemPro-34 complete medium and culture continuously; S06. Transition the cells to Essential 8 medium and continue culturing; S07. On days 15-21 after transduction, when the colonies grow to a size suitable for transfer, pick the colonies and transfer them to the prepared Geltrex matrix-coated culture plates for clonal expansion; S08. After the iPSC cell surface markers and three-germ layer differentiation ability are identified and meet the standards, the iPSC cell line is obtained.

3. The method for differentiating an iPSC cell line into melanocytes according to claim 2, characterized in that: In S02, mononuclear cells from cord blood were cultured in 1 well of a 24-well plate with 1 ml of StemPro-34 serum-free complete medium at a cell concentration of 2.5 × 10 5 – 5×10 5 cells / mL and cultured for 1 day.

4. The method for differentiating an iPSC cell line into melanocytes according to claim 2, characterized in that: S03 is: take out a set of CytoTune-iPS2.0 Sendai virus reprogramming kit from the storage device at -80°C, add 1ml of Klf4, Oct4 and Sox2 (KOS) viruses with MOI = 5 and c-Myc virus with MOI = 5, plate the cells in a 12-well tissue culture plate, and incubate them at 37°C overnight to perform cell transduction.

5. The method for differentiating an iPSC cell line into melanocytes according to claim 2, characterized in that: In S04, the cells were cultured in StemPro-34 for 2 days; in S05, the cells were cultured for 7 consecutive days with half medium change every day.

6. The method for differentiating an iPSC cell line into melanocytes according to claim 1, characterized in that: S5: From day 7 to day 30, embryoid bodies were transferred to 0.1% gelatine (Sigma-Aldrich)-coated well plates and cultured with melanocyte culture medium MGM-4 supplemented with 0.5 μM CHIR99021, 100 nM EDN3, 50 ng / mL SCF, 0.02 nM of human recombinant BMP4, and 0.3 mM ascorbic acid. The culture medium was changed every 2–3 days to induce differentiation into melanocytes.

7. The method for differentiating an iPSC cell line into melanocytes according to claim 1, characterized in that: In S8, melanocytes were identified by immunofluorescence staining to demonstrate that differentiated melanocytes expressed typical markers MITF and TYRP1.

8. Use of the method for differentiating the iPSC cell line into melanocytes according to any one of claims 1 to 7 in converting white hair to black in clinical allogeneic transplantation.

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