JP7080647B2 - Method for producing highly differentiated pluripotent stem cell-derived cells - Google Patents

Description

The present invention relates to a technique for inducing differentiation of pluripotent stem cell-derived cells. More specifically, when cells derived from pluripotent stem cells differentiate into specific cell lines, there are relatively low-differentiation stages and high-differentiation stages, and the differentiation of poorly differentiated cells The present invention relates to a method for producing a highly differentiated pluripotent stem cell-derived cell, which comprises a step of promoting the above-mentioned.

Pluripotent stem cells, including induced pluripotent stem cells (iPS cells) and embryonic stem cells, have the potential to induce differentiation into all cells, tissues, and organs that make up the body, and have the potential for unlimited proliferation. Therefore, various clinical applications including regenerative medicine are expected.
One such application field is a disease related to skin pigment.
Human epidermal melanocytes (pigment cells) are involved in the development of pigment cell damage. Dyschromatosis dermatitis, such as vitiligo and hereditary contralateral dyschromatosis, leads to loss of melanocytes at the lesion site, but the mechanism has not yet been clarified.
For these diseases, the usual autologous transplantation of epidermal melanocytes to the decolorized skin lesion site is often performed, but the effect at the decolorized skin lesion site is not always recognized (Non-Patent Document 1).

Although vitiligo is thought to be caused by the loss of melanocytes, several factors have been identified for human epidermal melanocytes that play important roles in controlling proliferation and differentiation.
In controlling the proliferation and differentiation of human epidermal melanocytes, stem cell factor (SCF), Wnt3a, bone morphogenetic protein 4 (BMP-4), ascorbic acid, 1,25-dihydroxyvitamin D3 ₍ active vitamin _D3 ), α-human melanocyte stimulating hormone (αMSH), N6,2'-O-dibutyryl adenosine 3', 5'-cyclic monophosphate sodium salt (DBcAMP), L-tyrosine, transferase, keratinocyte growth factor (KGF), granulocytes -Macrophage colony stimulating factor (GM-CSF), hepatocellular growth factor (HGF), basic fibroblast growth factor (bFGF), hydrocortisone, and endoserin 1 have been found to be important (Non-Patent Document 2). -7, Patent Document 1). In the present specification, the notation in parentheses is also used for these factors.
Although there have already been reports of differentiation of human iPS cells into melanocytes (Non-Patent Documents 8 and 9), none of them is a technique capable of mass-culturing epidermal melanocytes.

Further, Patent Document 2 describes a growth factor that induces differentiation of stem cells into melanocytes and stimulates cell proliferation, and Patent Document 3 is pluripotent using a specific drug and a specific feeder cell. A method for obtaining a population of melanocytes from sexual stem cells is described.
However, in the treatment of melanocyte transplantation for a wide range of vitiligo and depigmented spot lesions, there is a demand for a production method capable of more easily producing a large amount of melanocytes.
In order to achieve this, when cells derived from pluripotent stem cells differentiate into specific cell lines, there are relatively low-differentiation stages and high-differentiation stages, and the degree of differentiation is low. Technology that promotes cell differentiation is required.

In Patent Document 4, cells at any stage from pancreatic progenitor cells to endocrine progenitor cells are used as feeder cells, and then cells differentiated to endocrine progenitor cells are dispersed in a single cell and then cultured. , It is described that a cell mass having a three-dimensional structure similar to that of a pancreatic island in a living body can be obtained. This is to further advance the differentiation stage of cells with a high differentiation stage by co-culturing cells with a low differentiation stage and cells with a high differentiation stage. However, this technique cannot promote the differentiation of cells with a low differentiation stage and catch up with that of cells with a high differentiation stage.

Further, Patent Document 5 describes a method for inducing differentiation of human-derived pluripotent stem cells in co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. Stem cells only function as feeder cells and do not progress in their own differentiation stage.
Patent Document 6 also discloses a technique for producing organ buds by culturing organ cells together with vascular endothelial cells and mesenchymal cells, but this also promotes the differentiation of cells having a low differentiation stage and is in the differentiation stage. It is not a technology that can catch up with that of high-quality cells.

Japanese Unexamined Patent Publication No. 2015-146803 Special Table 2014-534815 Gazette Japanese Patent Publication No. 2013-520163 Japanese Unexamined Patent Publication No. 2014-161257 International release WO2012 / 043814 International release WO2013 / 047639

J Dermatol. 2013 Sep; 40 (9): 771-2 J Dermatol Sci. 2015 Dec; 80 (3): 203-11 J Dermatol Sci. 2014 Aug; 75 (2): 100-8 J Dermatol Sci. 2013 Jul; 71 (1): 45-57 Pigment Cell Melanoma Res. 2011 Jun; 24 (3): 462-78 J Invest Dermatol. 2008 May; 128 (5): 1220-6 J Dermatol Sci. 2014 Oct; 76 (1): 72-4 PLoS One. 2011; 6 (1): e16182 Proc Natl Acad Sci U S A. 2011; 108 (36): 14861-6

An object of the present invention is to provide a cell having a relatively low degree of differentiation when a cell derived from a pluripotent stem cell has a relatively low degree of differentiation stage and a high degree of differentiation stage in the process of differentiating into a specific cell lineage. It is an object of the present invention to provide a method for producing a highly differentiated pluripotent stem cell-derived cell, which comprises a step of promoting differentiation.

In the present invention, when a pluripotent stem cell-derived cell differentiates into a specific cell lineage, there are a relatively low degree of differentiation stage and a relatively high degree of differentiation stage, and the present invention differentiates into a specific cell lineage. By co-culturing pluripotent stem cell-derived cells with a low degree of differentiation and pluripotent stem cell-derived cells with a high degree of differentiation into the same cell lineage, pluripotent stem cells with a low degree of differentiation It is a method for producing a highly differentiated pluripotent stem cell-derived cell, which comprises a step of differentiating the derived cell into a highly differentiated pluripotent stem cell-derived cell.

The invention also contains SCF, Wnt3a, BMP-4, ascorbic acid, active vitamin D ₃ , αMSH, DBcAMP, L-tyrosine, transferrin, KGF, GM-CSF, HGF, bFGF, hydrocortisone, and endoserin 1. It is a medium for culturing animal cells.
Furthermore, the present invention is a method for culturing animal cells using such an animal cell culture medium.

According to the present invention, a highly differentiated pluripotent stem cell-derived cell differentiated from a pluripotent stem cell-derived cell into a target cell lineage efficiently, in a short period of time, and / or without using a feeder cell. Can be manufactured.

The figure which shows the epidermal melanocyte derived from the human iPS cell on the culture dish coated in the form of a gel with Matrigel ™. It can be recognized as a black spot or a blackish area. The figure which shows the epidermal melanocyte derived from the high density human iPS cell which is seen at the boundary of the iPS cell nest. The figure which shows the melanocyte derived from the human iPS cell in which a large number of melanin granules are observed in the cytoplasm. From left to right, the mRNA expression levels of NANOG, which is a pluripotent gene, in human iPS cells, melanocytes derived from human iPS cells, and HEMa-LP cells (normal human adult epidermal melanocytes, the same applies hereinafter) are compared by the RT-PCR method. Figure. The vertical axis shows the absorbance at 405 nm. From the left, the mRNA expression levels of melanocytes-specific MITF in human iPS cells, melanocytes derived from human iPS cells, and HEMa-LP cells are compared by the RT-PCR method. The vertical axis shows the absorbance at 405 nm. From the left, the mRNA expression levels of TRP1 peculiar to melanocytes in human iPS cells, melanocytes derived from human iPS cells, and HEMA-LP cells are compared by the RT-PCR method. The vertical axis shows the absorbance at 405 nm. From the left, the mRNA expression levels of melanocytes peculiar to melanocytes in human iPS cells, melanocytes derived from human iPS cells, and HEMa-LP cells are compared by the RT-PCR method. The vertical axis shows the absorbance at 405 nm. The figure which shows the epidermal melanocyte derived from the human iPS cell which reached confluence by culture | culture | culture | culture dish coated with Matrigel gel form for 8 weeks. The figure which shows the situation that two black colonies from a plate which reached confluence are engrafted and proliferated in a new culture system on a matrigel of human iPS cell-derived melanocytes. A partially enlarged view of the two colonies is also shown in the figure. A diagram showing a situation in which colored cells obtained from a culture medium of human iPS cell-derived melanocytes are engrafted and proliferated as blackish parts in a new culture system on a matrigel of human iPS cell-derived melanocytes (left). , And the cells obtained from the culture medium and the colony cells obtained by the pipette showed that the induction and proliferation of new human iPS cell-derived melanocytes were brought about on the culture dish coated with Matrigel in a gel form (. right). A partially enlarged view of the colored cells is also shown in the figure. The figure which shows a large number of human iPS cell-derived melanocytes observed in the culture dish 12 days after culture on the culture dish coated with Matrigel in the form of a gel. The figure which shows the black human iPS cell-derived melanocyte mass (left) and the black solution (right) which gathered in the bottom of the centrifuge tube by the centrifuge operation. The figure which shows that the skin of the injection site on the back of a nude mouse was colored blue or black. The figure which shows that the skin of the injection site on the back of a nude mouse was colored blue or black. An anatomical chart showing that a black mass was found at the injection site on the back of a nude mouse. An anatomical chart showing that a black mass was found at the injection site on the back of a nude mouse. 100-fold magnified view showing melanocyte cell mass by Fontana Masson staining of colored site sections. 400x magnified view showing melanocyte cell mass by Fontana Masson staining of colored site sections. 400-fold magnified view showing that the melanocyte cell mass in the dermis at the injection site on the back of a nude mouse was positive for immunostaining with an anti-Melan-A antibody. A 1000-fold magnified view showing that the melanocyte cell mass in the dermis at the injection site on the back of a nude mouse was positive for immunostaining with an anti-Melan-A antibody. The figure which compared the ratio of the area of the black part in the culture of the human iPS cell-derived melanocyte obtained by the manufacturing method of this invention and the manufacturing method described in Patent Document 1. FIG.

In the present invention, when a pluripotent stem cell-derived cell differentiates into a specific cell lineage, there are a relatively low degree of differentiation stage and a relatively high degree of differentiation stage, and the present invention differentiates into a specific cell lineage. By co-culturing pluripotent stem cell-derived cells with a low degree of differentiation and pluripotent stem cell-derived cells with a high degree of differentiation into the same cell lineage, pluripotent stem cells with a low degree of differentiation It is a method for producing a highly differentiated pluripotent stem cell-derived cell, which comprises a step of differentiating the derived cell into a highly differentiated pluripotent stem cell-derived cell.

As for the pluripotent stem cell-derived cells having a high degree of differentiation into the same cell lineage used for co-culture, the culture system of the pluripotent stem cell-derived cells having a low degree of differentiation into the cell lineage is used. It is preferable to use cells obtained by separating cells having a high degree of differentiation based on an index of the degree of differentiation into the cell lineage.

In addition, as a pluripotent stem cell-derived cell having a high degree of differentiation into the same cell lineage used for co-culture, a culture supernatant of a pluripotent stem cell-derived cell having a low degree of differentiation into the cell lineage. The cells present in it are also used.
For example, when differentiation into a specific cell line is differentiation into melanosite, poorly differentiated pluripotent stem cell-derived cells are uncolored melanosite, and highly differentiated pluripotent stem cell-derived cells are colored melanosite. As such, it is possible to produce melanosites derived from highly differentiated pluripotent stem cells.
In this case, the index of the degree of differentiation into the melanocyte line is the presence or absence of coloring.

Further, as a cell obtained by separating a cell having a high degree of differentiation from a culture system of a pluripotent stem cell-derived cell having a low degree of differentiation in this case based on an index of the degree of differentiation into the cell lineage, for example, iPS cell-derived. Black cell clusters found in confluent melanosite (usually after 5-6 weeks) can be used.
Alternatively, as cells whose differentiation stage into the cell lineage is present in the culture supernatant of pluripotent stem cell-derived cells having a low degree of differentiation, for example, cell culture of iPS cell-derived melanosite is performed almost every day for about 3-4 weeks. When culturing while exchanging the liquid, it is also possible to collect the culture liquid and use the cells obtained by centrifuging it.
By co-culturing with such cells, iPS cells in the new culture system are also stimulated, and their differentiation into melanocyte pigment cells is accelerated.

In addition, the co-culture step in the production method of the present invention is preferably carried out in a medium containing a component that stimulates epidermal induction, a component that stimulates final differentiation into keratinocytes, and / or a growth factor or a culture agent.
As a component that stimulates such epidermal induction, one or more selected from the group consisting of SCF, BMP-2, BMP-4, BMP-7, Smad1, Smad5, Smad7, GDF-6, EGF, Wnt3a, and bFGF. It is preferable that it is a component of.

Further, ascorbic acid and / or active vitamin _D3 is preferably used as a component that stimulates the final differentiation into such keratinocytes.
Further, as the growth factor or culture agent, one or more components selected from the group consisting of Ca salt, KGF, GM-CSF, HGF, α-MSH, DBcAMP, L-tyrosine, and transferrin can be used. preferable.

In the co-culture step in the production method of the present invention, SCF, Wnt3a, BMP-4, ascorbic acid, active vitamin D ₃ , αMSH, DBcAMP, L-tyrosine, transferrin, KGF, GM-CSF, HGF, bFGF, hydrocortisone. , And a medium containing transferrin 1 is particularly preferred.
In addition, such a medium can also be used for animal cell culture other than the production method of the present invention.

As the pluripotent stem cells used in the production method of the present invention, it is preferable to use human pluripotent stem cells, and it is particularly preferable to use human iPS cells.
Moreover, it is preferable that the co-culture step in the production method of the present invention is a step in which neither other cells nor the culture supernatant of other cells are used.

1. 1. Generation and analysis of human iPS cells T cells grown from the blood of a 40-year-old healthy Japanese man were subjected to four reprogramming factors OCT3 / 4, SOX2, cMYC, and K by Sendai virus vector (SeVdp).
LF4 was expressed and initialized (infection multiplicity = 2, 37 ° C, 2 hours). See J Biol Chem. 2011 Feb 11; 286 (6): 4760-71. SeVdp was acquired from Dr. Masato Nakanishi of the Stem Cell Engineering Research Center, National Institute of Advanced Industrial Science and Technology.
These transformed cells were subjected to human T cell medium (RPMI1640, 10% fetal bovine serum, 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, Massachusetts), interleukin 2 (PeproTech, Rocky Hill, NJ)).
), The cells were seeded on X-ray-irradiated mouse fetal fibroblasts (MEF).

Two days later, bFGF (5 ng) was added to the medium for primate ES cell culture (ReproCELL, Yokohama).
/ Ml, R & D Systems, Minneapolis, Minnesota) was replaced and cultured until colonies were observed. Colonies of iPS cells were mechanically aspirated and placed on X-ray irradiated MEFs for growth and analysis. The established iPS cell line, WT-iPSC1, was maintained in human iPS cell medium (hiPSCM). This medium is DMEM / F12 (Sigma-Aldrich, St. Louis, Missouri) with 20% KnockOut ™ serum supplement (Invitrogen), 1% non-essential amino acids (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol. 1% penicillin-streptomycin, and 5 ng / ml bFGF added.

To completely remove SeVdp, a siRNA mixture (40 nM L527 siRNA-1 and 2.5 μl RNAiMAX (Thermo Fisher Scientific) in 500 μl OptiMEM®) was added to the medium several times.
L527siRNA-1: 5'-GGUUCAGCAUCAAAUAUGAAG-3'(SEQ ID NO: 1)
L527siRNA-2: 5'-UCAUAUUUGAUGCUGAACCAU-3'(SEQ ID NO: 2)
Then, the authenticity of this iPS cell line was confirmed according to the known literature. Stem Cell Research, Volume 17, Issue 1, July 2016, Pages 22-24, Stem Cell Research, Volume 17, Issue 1, July 2016, Pages 16-18, and Stem Cell Research, 16 (3) (2016), pp ..
See 611-613.

2. 2. Establishment of human iPS cell line, WT-iPSC1 These human iPS cells were seeded in hiPSCM on MEF treated with mitomycin C. Y-27632, a selective inhibitor of p160-Rho-binding kinase (ROCK), was added to the medium for effective inhibition of apoptosis and enhancement of survival of human iPS cells.
Before differentiation into enterocytes begins, human iPS cells were dispersed as cell clumps using dispase (Sigma-Aldrich) and placed on a Matrigel basement membrane matrix (BD Biosciences, Lexington, Kentucky) with reduced growth factors. The cells were cultured in MEF conditioned medium for 2 to 3 days.

3. 3. Cell Culture Normal human adult epidermal melanocytes (HEMa-LP cells) were purchased from Invitrogen Gibco (Carlsbad, CA). This melanocyte was maintained in Gibco's melanocyte medium 254CF with the addition of human melanocyte growth supplement (HMGS-2) and 0.2 mM calcium chloride. HMGS-2 contains 3 ng / ml bFGF, 0.18 μg / ml hydrocortisone, and 10 nM endothelin 1.
Further, as the medium in the production method of the present invention, for example, a medium having the following composition (also referred to as “human iPS cell-derived melanocyte medium”) is used, but those skilled in the art may be skilled in the art regarding the types of factors to be added. For example, it will be possible to make appropriate selections with reference to this, and it will be possible to optimize the concentrations of these factors as appropriate.

Melanosite Medium 254CF (Gibco), HMGS-2 (Gibco), 0.2 mM Calcium Chloride, 50 ng / ml SCF (R & D Systems), 50 ng / ml Wnt3a (R & D Systems), 20 nM Human Recombinant BMP-4 (R & D) Systems), 0.3 mM ascorbic acid (Sigma-Aldrich), ^10-7 M 1,25-dihydroxyvitamin D ₃ (Enzo Life Sciences, Plymouth Meeting, Pennsylvania), 100 μM αMSH (Sigma-Aldrich), 0 .2 mM DBcAMP (Sigma-Aldrich), 0.1 mM L-tyrosine (Sigma-Aldrich), 100 μg / ml transferase (Sigma-Aldrich), 10 ng / ml KGF (Sigma-Aldrich), 0.1 ng / ml GM-CSF (Sigma-Aldrich), 0.1 ng / ml HGF (Sigma-Aldrich).

4. Nude Mouse A 5-week-old male KSN / Slc nude mouse was purchased from SLC (Nagoya). All of these mice were kept and fed at the facility of the Japan Experimental Animal Care Evaluation and Certification Association. Melanocytes derived from human iPS cells were injected intradermally into these mice.

5. Statistical analysis All data are expressed as mean ± standard deviation. A p-value less than 0.05 was considered statistically significant. Quantitative parameter differences between populations were assessed by the Mann-Whitney U test.

[Reference Example 1] Differentiation from human iPS cells to melanocytes
In order to induce differentiation of human iPS cells into melanosite, they were placed in a 60 mm culture dish gelled with Matrigel (BD Biosciences) in DMEM / F12.
Human iPS cells were subcultured on Matrigel in MEF conditioned medium for 1 day, and then these cells were subjected to the above-mentioned medium, HMGS-2, 0.2 mM calcium chloride, 50 ng / ml SCF, 50 ng /. ml Wnt3a, 20 nM human recombinant BMP-4, 0.3 mM ascorbic acid, ^10-7 M 1,25-dihydroxyvitamin D ₃ , 100 μg / ml αMSH, 0.2 mM DBcAMP, 0.1 mM L-tyrosine, 100 μg / m
l Transferrin, 10 ng / ml KGF, 0.1 ng / ml GM-CSF, 0.
The cells were cultured in 254CF of melanocyte medium containing 1 ng / ml HGF.

[Reference Example 2] RT-PCR analysis of melanocytes differentiated from human iPS cells
RNA was extracted from cells using the RNeasy minikit (Qiagen, Hilden, Germany), of which 2 μg RNA was used to extract SuperScript ™ III reverse transcriptase and oligo-dT.
The cDNA was synthesized using primers (Thermo Fisher Scientific) according to the manufacturer's instructions, PowerSYBR ™ Green PCR Master Mix (Applied Biosystis, Foster City, CA) and StepOne ™ Real-Time PCR System. Quantitative analysis was performed by real-time PCR using (Applied Biosystis).
The PCR primers used are as follows.
NANOG: 5'-CAAAGGCAAACAACCCACTT-3' (SEQ ID NO: 3)
And 5'-TCTGCTGGAGGCTGAGGTAT-3'(SEQ ID NO: 4)
PAX3: 5'-CTGGAACATTTGCCCAGACT -3'(SEQ ID NO: 5)
And 5'-GCTGTCGGTTCCTAGTCCAG-3'(SEQ ID NO: 6)
microphthalmia-associated transcription factor (MITF): 5'-CCGGGTGCAGAATTGTAACT-3' (SEQ ID NO: 7)
And 5'-GGACAATTTTGGCATTTTGG-3'(SEQ ID NO: 8)
tyrosinase-related protein 1 (TRP1): 5'-AGCAGTAGTTGGCGCTTTGT-3'(SEQ ID NO: 9)
And 5'-TCAGTGAGGAGAGGCTGGTT-3'(SEQ ID NO: 10), and
tyrosinase: 5'-TTGTACTGCCTGCTGTGGAG-3'(SEQ ID NO: 11)
And 5'-CAGGAACCTCTGCCTGAAAG-3' (SEQ ID NO: 12)

[Reference Example 3] Intradermal injection of human iPS cell-derived melanocytes into nude mice
All experimental procedures have been approved by the Animal Care and Use Committee of St. Marianna University School of Medicine.
Human iPS cell-derived melanocytes on the plate were stripped using StemPro® Accutase® (Gibco), washed and centrifuged to form a cell mass. The melanocytes were suspended in an isotonic sodium chloride solution at a density of 3 × ¹⁰⁶ cells / ml.
Nude mice (n = 5) were anesthetized with diethyl ether and 1 ml of cell suspension was injected intradermally into the dorsal aspect of the mice. Skin specimens of the nude mice were fixed with PBS containing 4% formaldehyde and embedded in paraffin.

[Reference Example 4] Melanocyte-related staining
Tissue specimens fixed with this formalin and embedded in paraffin were used as 4 μm sections. This was left at room temperature for 10 hours in a silver ammonia solution of Fontana for Fontana Masson staining according to the manufacturer's instructions. Hematoxylin and eosin staining of this tissue specimen was also performed for morphological analysis. Further, this formalin-fixed, paraffin-embedded tissue specimen was left overnight at 4 ° C. in a 50-fold diluted solution of an anti-Melan-A monoclonal antibody (Dako, Santa Clara, CA). The tissue section slides were then treated with biotinylated secondary antibody and peroxidase-labeled avidin (Dako), as well as the chromogenic substrate diaminobenzidine. The one using Tris-buffered saline instead of the primary antibody was used as a negative control.

[Example 1] Induction of differentiation of low-differentiation human iPS cell-derived melanocytes by the method of the present invention
Human iPS cell-derived melanocytes with SCF, Wnt3a, BMP-4, ascorbic acid, active vitamin D ₃ , αMSH, DBcAMP, L-tyrosine, transferrin, KGF, GM-CSF, HGF, bFGF, hydrocortisone, and endoserin 1. After culturing in the medium containing the cells for 20 days, the formation of a large number of colored cells with black granules was observed on the edge of the human iPS cell nest on the culture dish coated with Matrigel in the form of gel (Fig. 1-3).

The expression of NANOG, MITF, TRP-1, and tyrosinase in the colored cells derived from human iPS cells was examined by the RT-PCR method (Fig. 4-7). The mRNA expression of NANOG, a marker of pluripotency, in these colored cells was significantly lower than that in human iPS cells (p <0.001). On the other hand, the mRNA expression of melanocyte-specific markers MITF, TRP-1, and tyrosinase in these colored cells was significantly higher than that of human iPS cells (p <0.001). The mRNA expression of NANOG, MITF, TRP-1, and tyrosinase in these colored cells was similar to that of HEMA-LP cells, which are normal human melanocytes. From these facts, it can be said that these colored cells are melanocytes derived from human iPS cells.

After approximately 5-6 weeks, human iPS cell-derived melanocytes reached confluence on a gel-coated culture dish with Matrigel (FIG. 8). Colonies of human iPS cell-derived melanocytes were sucked up by gentle pipetting in the culture dish. This colony was directly added to a culture system of fresh human iPS cell-derived melanocytes cultured for 2 days in human iPS cell-derived melanocyte medium on a culture dish coated with Matrigel in a gel form. After 3 days, these melanocyte colonies were engrafted on Matrigel and differentiated and proliferated together with other human iPS cell-derived melanocytes (Fig. 9).

On the other hand, an operation different from the cell acquisition by pipetting was also performed. That is, three weeks after the start of culture of human iPS cell-derived cells, the medium was collected and centrifuged, and the black cell mass observed at the bottom of the centrifuge tube was directly added to a new human iPS cell-derived melanosite medium and gently added. Stirred. The medium containing the cells was directly added to a new culture system of human iPS cell-derived melanocytes cultured for 2 days in a human iPS cell-derived melanocyte medium on a culture dish coated with Matrigel in the form of a gel. After a few days, these melanocytes contained a small number of small black particles, engrafted on Matrigel, differentiated and proliferated with other human iPS cell-derived melanocytes (Fig. 10).

In this way, the presence of "colony sucked up by pipetting" and "human iPS cell-derived melanosite recovered from the medium" is present in the differentiation and proliferation of new human iPS cell-derived melanosite on a culture dish coated in a gel form with Matrigel. Was induced. Twelve days later, a large number of high-quality human iPS cell-derived melanocytes were obtained on a gel-like culture dish coated with Matrigel by these two methods (FIG. 11). With the conventional technique, melanocytes can be confirmed only 8 weeks after the induction of differentiation.

[Example 2] Infiltration of human iPS cell-derived melanocytes into the skin tissue of nude mice.
When human iPS cell-derived melanocytes were centrifuged, both the centrifugal pellet and the supernatant were black (Fig. 12). The cells were injected intradermally into the back of a nude mouse. Three days after the injection, a blue or black colored skin lesion was observed at the injection site on the back of the mouse (FIGS. 13 and 14). Then, on the 7th day, an autopsy revealed a black mass on the back of the injected nude mouse (FIGS. 15 and 16). When the colored lesion site section was microscopically stained with Fontana Masson histochemical staining, a positive black color was observed, confirming that it was melanocyte (FIGS. 17 and 18). Immunohistochemical staining with an anti-Melan-A monoclonal antibody revealed brown or black cytoplasmic cells corresponding to the injection site compared to the stained section of the negative control (FIG. 19, FIG. 20). In all four nude mice injected with human iPS cell-derived melanocytes, the transplanted melanocytes were mainly present between the dermis and the subcutaneous fascia, but some were also present in the epithelium.

[Example 3] Long-term observation of human iPS cell-derived melanocytes after transplantation into SCID mice
Cultured cells of human iPS cell-derived melanosite obtained by the production method of the present invention were subcutaneously injected into SCID mice. As a result of observation for 12 months after administration, no canceration was observed. This proved that the human iPS cell-derived melanocytes obtained by the production method of the present invention have no risk of canceration even after being transplanted into a living body and are highly safe.

[Example 4] Comparison between the production method of the present invention and the production method described in Patent Document 1.
Since black melanin is produced from melanocytes, measuring the ratio of the area of the black part generated when melanocytes are cultured in a cell culture dish is an index of the degree of differentiation of melanocytes.

Therefore, human iPS cell-derived melanocytes according to the production method of the present invention and human iPS cell-derived melanocytes according to the production method described in Patent Document 1 are cultured, and the ratio of the area of the black portion in the cell culture dish having a diameter of 60 mm is image-processed. Measurements were made using software ImageJ. As a result, as shown in FIG. 21, the ratio of the area of the black portion of the human iPS cell-derived melanocyte according to the production method of the present invention is 2.3% on the 21st day, whereas it is described in Patent Document 1. The ratio of the area of the black portion of the human iPS cell-derived melanocytes according to the production method was 0.1%. Therefore, it was clarified that a highly differentiated melanocyte can be obtained by the production method of the present invention (p <0.01).

When the production method of the present invention was applied to the differentiation into melanocytes, it became possible to prepare a large amount of high-quality melanocytes having a high degree of differentiation from human iPS cell-derived melanocytes having a low degree of differentiation. In addition, these melanocytes could be used to pigment the skin of nude mice. The melanocytes thus obtained are used for the treatment of diseases in which skin pigment cells are deficient, such as vitiligo and depigmented spot diseases.

In addition, when a tattooed patient makes an MRI diagnosis, there is a problem of skin burns caused by the ferromagnetic metal compound in the tattoo pigment, and there is a risk of distorting the MRI image. Using cell-derived melanosite eliminates these problems and concerns.
Furthermore, although areola reconstruction is important in breast reconstruction after mastectomy, intradermal injection with human iPS cell-derived melanocytes will be a safe areola reconstruction.
In addition, it is expected to be applied to the blackening of white hair and the treatment of malignant melanoma.

The production method of the present invention enables efficient production of highly differentiated pluripotent stem cell-derived cells that can be used for autologous transplantation as a cell medicine, and contributes to improvement of various quality of life. ..

According to the method for producing pluripotent stem cell-derived cells having a high degree of differentiation of the present invention, pluripotent stem cell-derived cells that are highly differentiated in a intended direction can be prepared, and are therefore used, for example, in the cell pharmaceutical manufacturing industry.

Claims (9)

In the process of differentiating human pluripotent stem cells into melanocytes , there are relatively low-differentiation stages and relatively high-differentiation stages.
By co-culturing human pluripotent stem cell-derived melanosite with a low degree of differentiation into melanosite and human pluripotent stem cell-derived melanosite with a high degree of differentiation into melanosite , the degree of low differentiation can be achieved. Including the step of differentiating human pluripotent stem cell-derived melanosite into highly differentiated human pluripotent stem cell-derived melanosite .
Poorly differentiated human pluripotent stem cell-derived melanosites are uncolored melanosites, and highly differentiated human pluripotent stem cell-derived melanosites are colored melanosites.
Human pluripotent stem cells are human iPS cells,
Highly differentiated human pluripotent stem cell-derived melanosite co-cultured from a culture system of human pluripotent stem cell-derived melanosite with a low degree of differentiation to melanosite based on an index of the degree of differentiation into melanosite. It was obtained by separating the cells of the degree.
The co-culture step is performed in a medium containing at least SCF, Wnt3a, BMP-4, α-MSH, DBcAMP, L-tyrosine, transferrin, KGF, GM-CSF, HGF, bFGF, hydrocortisone, and endothelin 1. , A method for producing highly differentiated human pluripotent stem cell-derived melanocytes . Claimed that the co-cultured highly differentiated human pluripotent stem cell-derived melanosite is a cell in which the stage of differentiation into the melanosite was present in the culture supernatant of the poorly differentiated human pluripotent stem cell-derived melanosite . The manufacturing method according to 1. The production method according to claim 1 or 2 , wherein the co-culture step is carried out in a medium further containing a component that stimulates epidermal induction, a component that stimulates final differentiation into keratinocytes, and a growth factor or a culture agent. 3. The component that stimulates epidermal induction is one or more components selected from the group consisting of B MP-2 , B MP-7, Smad1, Smad5, Smad7 , GDF-6, and EGF. The manufacturing method described in. The production method according to claim 3 or 4 , wherein the component that stimulates the final differentiation into keratinocytes is ascorbic acid and / or active vitamin D3. The production method according to any one of claims 3 to 5 , wherein the growth factor or the culture agent is a Ca salt . The co-culture step is in a medium containing SCF, Wnt3a, BMP4, ascorbic acid, active vitamin D3, αMSH, DBcAMP, L-tyrosine, transferrin, KGF, GM-CSF, HGF, bFGF, hydrocortisone, and endoserin 1. The manufacturing method according to claim 1 , which is carried out in 1. The production method according to any one of claims 1 to 7 , wherein the co-culture step is a step in which neither other cells nor culture supernatants of other cells are used. In the process of differentiating human pluripotent stem cells into melanocytes , there are relatively low-differentiation stages and relatively high-differentiation stages.
By co-culturing human pluripotent stem cell-derived melanosite with a low degree of differentiation into melanosite and human pluripotent stem cell-derived melanosite with a high degree of differentiation into melanosite , the degree of low differentiation can be achieved. Including the step of differentiating human pluripotent stem cell-derived melanosite into highly differentiated human pluripotent stem cell-derived melanosite .
Poorly differentiated human pluripotent stem cell-derived melanosites are uncolored melanosites, and highly differentiated human pluripotent stem cell-derived melanosites are colored melanosites.
Human pluripotent stem cells are human iPS cells,
Highly differentiated human pluripotent stem cell-derived melanosite co-cultured from a culture system of human pluripotent stem cell-derived melanosite with a low degree of differentiation to melanosite based on an index of the degree of differentiation into melanosite. A medium composition for use in co-culture of a method for producing highly differentiated human pluripotent stem cell-derived melanosite, which is obtained by separating cells of different degrees.
Medium composition containing SCF, Wnt3a, BMP-4, ascorbic acid, active vitamin D3, αMSH, DBcAMP, L-tyrosine, transferrin, KGF, GM-CSF, HGF, bFGF, hydrocortisone, and endothelin 1. ..

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