JPWO2009110215A1 - Ciliary cell differentiation induction method - Google Patents

Ciliary cell differentiation induction method Download PDF

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JPWO2009110215A1
JPWO2009110215A1 JP2009529336A JP2009529336A JPWO2009110215A1 JP WO2009110215 A1 JPWO2009110215 A1 JP WO2009110215A1 JP 2009529336 A JP2009529336 A JP 2009529336A JP 2009529336 A JP2009529336 A JP 2009529336A JP WO2009110215 A1 JPWO2009110215 A1 JP WO2009110215A1
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cells
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浅島 誠
誠 浅島
佑介 西村
佑介 西村
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独立行政法人科学技術振興機構
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/10Screening for compounds of potential therapeutic value involving cells

Abstract

The purpose of the present invention is to identify a substance that is considered to be contained in FBS that inhibits differentiation from differentiated cells to ciliated cells, to suppress the signal, and to forcibly express genes involved in differentiation of ciliated cells. Thus, to provide a method capable of inducing differentiation of cilia cells even in a general culture system using serum such as FBS used in a normal culture system. The present invention relates to the induction of differentiation of ciliated cells in a general culture system using serum by suppressing the BMP signal or expressing the cilia-related gene Foxj1 or Foxa2, which is important for respiratory differentiation.

Description

The present invention includes a method for inducing differentiation from an undifferentiated cell to a cilia cell, comprising a step of culturing an embryoid body formed from an undifferentiated cell in a medium containing serum, and a ciliated cell induced to differentiate by the method, and Further, the present invention relates to a method for screening a drug that regenerates or inhibits cilia using the ciliated cells.

Prior to 2004, a method for inducing ciliated cells from undifferentiated cells such as embryonic stem cells (ES cells) was not known. In order to solve this situation, the present inventors induced ciliated cells from undifferentiated cells such as ES cells by a special culture system using Knockout Serum Replacement (KSR), a commercially available cell culture solution. (Patent Document 1). However, fetal bovine serum (FBS), which is generally used for cell culture, has the effect of suppressing differentiation into ciliated cells. In general culture systems using FBS, Ciliated cells could not be differentiated from undifferentiated cells.
JP 2006-149329 A

Therefore, the main object of the present invention is to identify a substance that appears to be contained in FBS that inhibits differentiation of undifferentiated cells into ciliated cells, to suppress the signal thereof, and to a gene involved in differentiation of ciliated cells. It is to provide a method capable of inducing differentiation of cilia cells even in a general culture system using serum such as FBS used in a normal culture system.

Bone morphogenetic protein (BMP) is a secreted growth factor and belongs to the spar family of transforming growth factors. In addition to bone formation, BMP has morphogenesis and various other actions. BMP signal regulates downstream gene expression by receptor activated by BMP phosphorylates intracellular signaling molecules Smad1, Smad5, and Smad8, forms heterodimer with Smad4, and moves into the nucleus . On the other hand, Smad6 or Smad7 is known as inhibitory Smad and suppresses BMP signal.

The present inventor has found that by inhibiting BMP signal using Smad6 or Smad7, ciliary cells can be induced to differentiate even in a medium containing serum such as FBS that has not been differentiated until now.

Furthermore, the present inventor is able to induce differentiation of cilia cells even in a medium containing serum such as FBS by expressing Foxj1 which is an essential gene for cilia formation or Foxa2 which has an important function in the differentiation of respiratory epithelium. I found it. The present invention has been completed based on the above findings.

That is, the present invention relates to the following aspects.
[Aspect 1] A method of inducing differentiation from undifferentiated cells to ciliated cells by suppressing BMP signals in undifferentiated cells in the step of culturing embryoid bodies formed from undifferentiated cells in a medium containing serum.
[Aspect 2] The method according to Aspect 1, wherein the BMP signal is suppressed by introducing Smad6 and / or Smad7 genes into undifferentiated cells and forcibly expressing the genes.
[Aspect 3] The method according to Aspect 1, wherein the BMP signal is suppressed by enhancing the expression of Smad6 and / or Smad7 genes in undifferentiated cells.
[Aspect 4] The method according to Aspect 3, wherein the Smad6 and / or Smad7 gene is derived from a mammal.
[Aspect 5] In the step of culturing embryoid bodies formed from undifferentiated cells in a medium containing serum, differentiation is induced from the undifferentiated cells to cilia cells by enhancing the expression of genes involved in the differentiation of cilia cells. how to.
[Aspect 6] The method according to Aspect 5, wherein the gene involved in ciliary cell differentiation is Foxj1 and / or Foxa2 gene.
[Aspect 7] The method according to Aspect 6, comprising introducing Foxj1 and / or Foxa2 genes into undifferentiated cells and forcibly expressing the genes.
[Aspect 8] The method according to Aspect 7, wherein Foxj1 and / or Foxa2 gene is derived from a mammal.
[Aspect 9] The method according to any one of Aspects 1 to 8, wherein the undifferentiated cells are embryonic stem cells.
[Aspect 10] The method according to any one of Aspects 1 to 9, wherein the undifferentiated cells are derived from a human.
[Aspect 11] The method according to any one of Aspects 1 to 10, wherein the serum is fetal calf serum.
[Aspect 12] Ciliated cells induced to differentiate by the method according to any one of Aspects 1 to 11.
[Aspect 13] The ciliated cell according to aspect 12, which is a ciliary cell of the ventricle.
[Aspect 14] A screening method for a drug that regenerates or inhibits cilia using the ciliary cells according to Aspect 12 or 13.

A special culture system using KSR has been developed so far to induce differentiation of cilia cells, but according to the present invention, in a general medium using serum such as FBS used in normal cell culture. However, it became possible to induce differentiation of ciliated cells by suppressing BMP signal and expressing cilia-related gene Foxj1 and Foxa2, which is important for respiratory differentiation.

Furthermore, cilia cells obtained by differentiation induction according to the present invention can be used for screening for drugs that regenerate or inhibit cilia.

Immunostaining of beta-tubulin IV of ciliated cells differentiated by Smad7. Immunostaining of beta-tubulin IV and Musashi1 of ciliated cells differentiated by Smad7. The graph which shows the expression of Foxj1, beta-tubulin IV, and Centrin4 by Real-time PCR (* P <0.05). Immunostaining of beta-tubulin IV of ciliated cells differentiated by Foxj1. Immunostaining of beta-tubulin IV of ciliated cells differentiated by Foxa2. Graph showing the expression of Foxj1, beta-tubulin IV, and Centrin4 by Real-time PCR method when Foxj1 is expressed (* P <0.05). Graph showing the expression of Foxj1, beta-tubulin IV and Centrin4 by Real-time PCR method when Foxa2 is expressed (* P <0.05).

In the first aspect of the present invention, in the step of culturing embryoid bodies formed from undifferentiated cells in a medium containing serum, the BMP signal in the undifferentiated cells is suppressed, whereby the undifferentiated cells are converted into ciliated cells. It relates to a method of inducing differentiation.

As a specific means for suppressing BMP signal in undifferentiated cells, for example, by introducing Smad6 and / or Smad7 gene into undifferentiated cells and forcibly expressing the gene, or suppressing BMP signal, or It can be achieved by a method such as enhancing the expression of endogenous Smad6 and / or Smad7 genes in undifferentiated cells. This inhibits the action of serum that suppresses differentiation from undifferentiated cells to cilia cells, and promotes differentiation induction from the undifferentiated cells to cilia cells.

Furthermore, in the second aspect of the present invention, in the step of culturing embryoid bodies formed from undifferentiated cells in a medium containing serum, the undifferentiated state is enhanced by enhancing the expression of genes involved in ciliary cell differentiation. The present invention relates to a method for inducing differentiation from a cell to a ciliated cell. Examples of genes involved in ciliary cell differentiation include arbitrary genes known to those skilled in the art, for example, Foxj1 and Foxa2 genes.

Specific means for enhancing the expression of such a gene includes, for example, introducing Foxj1 and / or Foxa2 genes into undifferentiated cells and forcibly expressing the genes.

  In this specification, “undifferentiated cells” broadly mean cells in an undifferentiated state. For example, in addition to embryonic stem cells (ES cells), various tissue stem cells such as hematopoietic stem cells, neural stem cells, skin tissue stem cells, etc. It is a concept that includes the like. Furthermore, genes such as Oct3 / 4, Klf4, c-Myc and Sox2 are introduced into differentiated cells such as various somatic cells derived from mice and humans, or various stem cells or progenitor cells, or other drugs. Induced Pluripotent Stem Cells induced by treatment and the like are also included in the “undifferentiated cells” in the present specification (Japanese Patent No. 4183742, JP 2008-307007 A, Takahashi K. et al ., Cell, 2007 Nov. 30: 131 (5): 861-72)

One of the processes of growing a fertilized egg into a fetus is a state called a blastocyst. The blastocyst has a spherical shape and is composed of a trophectoderm, which is an outer cell layer, and a blastocoel containing an inner cell mass that will form the body in the future. When undifferentiated cells such as ES cells are cultured in a floating state, a large cell mass similar to the blastocoel whose peripheral portion is differentiated like an endothelium is formed. In the present invention, this cell cluster is referred to as embryoid bodies.

The embryoid body can be prepared according to a conventionally known technique. The cells constituting the embryoid body are not particularly limited as long as they are undifferentiated cells that can form an embryoid body, but are preferably ES cells or somatic stem cells, particularly preferably. Are ES cells. For example, ES cells can be used as a starting material, and embryoid bodies that are aggregates of ES cells can be prepared by suspension culture in a serum medium or fetal serum-free medium containing fetal calf serum or the like.

The undifferentiated cells that can be used in the present invention are not particularly limited as long as they are cells derived from mammals, but are preferably cells derived from rodents such as mice or primates, particularly preferably. Are cells derived from humans.

Cultivation of undifferentiated cells and transformants into which the nuclear gene has been introduced can be performed by any method known to those skilled in the art. For example, undifferentiated cells can be cultured by seeding on feeder cells as necessary. As the feeder cells, cells that have survived but not proliferated by treatment with X-rays, γ-rays, mitomycin C, and the like can be used. For example, mouse fetal fibroblasts treated with mitomycin C can be used as feeder cells for mouse-derived ES cells. Typically, the medium is changed every day, and subculture is performed every 3 days.

The cultured undifferentiated cells are made into a single cell suspension by trypsin treatment, and the obtained suspension is seeded in a culture vessel and cultured in a CO 2 incubator. By this operation, feeder cells adhere to the culture surface, but undifferentiated cells do not adhere to the culture surface and remain floating. Therefore, undifferentiated cells can be separated from feeder cells by collecting the culture supernatant and centrifuging.

Next, the obtained undifferentiated cells are suspended and cultured overnight in a tilted, low-adhesive culture container, and then the container is placed in a horizontal state and further cultured for 2 to 3 days. To prepare. An embryoid body can also be prepared by culturing undifferentiated cells in a suspended state in droplets formed on mineral oil. As this embryoid body preparation medium, a medium containing fetal calf serum or a serum-free medium can be used.

The obtained embryoid body can be attached to the bottom surface of the culture container by culturing it in a serum medium or serum-free medium using an adhesive container.

In the present invention, the embryoid body thus obtained is cultured in a normal serum-containing medium without using a conventional special serum-free medium, so that cells having countless cilia-like structures can be obtained. Differentiation can be induced. Here, there is no restriction | limiting in particular in kind, origin, etc. of "serum", What is normally used for the culture medium of various cells, especially an undifferentiated cell, and various differentiated cells from it is contained. Examples thereof include serum derived from various animals such as cows, particularly fetal bovine serum.

A ciliated epithelial cell is a cell having a cilia-like structure, which has a “climbing movement” peculiar to cilia and has a 9 + 2 structure that is an arrangement structure of axial yarns peculiar to cilia such as the ventricle, tracheal epithelium and oviduct. Furthermore, it expresses tubulin (β-tubulin IV), which is a protein constituting the axial thread, and Foxj1, which is also a gene specific to cilia (marker protein). Therefore, for example, by detecting β-tubulin IV which is a constituent protein of the axial thread using an antigen staining method, and identifying the expression of Foxj1 using an RT-PCR method, ciliated epithelial cells Can be confirmed. The ciliated epithelial cells are present in the tracheal epithelium, oviduct, and the like in vivo.

Smad6, Smad7, and genes involved in ciliary cell differentiation such as Foxj1 and Foxa2 are preferably derived from mammals, particularly primates including rodents and humans. For example, the amino acid sequences and the like of these genes derived from humans are known, and the databases of each public institution can be referred to based on the following ID numbers:
Smad6: NM_005585, Smad7: NM_005904, Foxj1: NM_001454, Foxa2: BC011780.

Furthermore, as each of the above genes,
(a) a known amino acid sequence encoded by each gene, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added, and the biological activity represented by the known amino acid sequence A nucleic acid comprising a nucleotide sequence encoding a protein;
(b) It consists of an amino acid sequence having 90% or more, preferably 93% or more, more preferably 95% or more, more preferably 99% or more homology with the known amino acid sequence, and the known amino acid sequence is A nucleic acid comprising a nucleotide sequence encoding a protein that exhibits a biological activity; or
(c) hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to a nucleic acid consisting of a base sequence encoding the known amino acid sequence, and has a biological activity exhibited by the known amino acid sequence A nucleic acid comprising a nucleotide sequence encoding a protein;
Can be used in the present invention as well as each gene.

In the method of the present invention, each of the genes may be the same or different species from the stem cell to be introduced, but it is preferable that the genes are closely related to each other, and particularly from the same species. It is preferable.

Here, “homology” refers to each amino acid residue or each base constituting the chain between two chains in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence). It means the amount (number) of things that can be determined to be identical in the mutual relationship of each other, and means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. Homology can be easily calculated. Many methods for measuring homology between two polynucleotide or polypeptide sequences are known, and the term “homology” (also referred to as “identity”) is well known to those skilled in the art (eg, , Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin , AM & Grifin, HG (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987 Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.). Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman , D., SIAM J. Applied Math., 48: 1073 (1988), etc., but are not limited thereto.

The nucleic acid (gene) of the present invention encodes the above protein. Here, “encode” means that the protein of the present invention is expressed in a state having the activity. The term “encode” includes both encoding the protein of the present invention as a continuous structural sequence (exon) or encoding the protein of the present invention via an appropriate intervening sequence (intron). Yes.

"Nucleic acid" includes ribonucleic acid, deoxyribonucleic acid, or any modified nucleic acid. The nucleic acid includes single-stranded or double-stranded DNA.

As used herein, “stringent conditions” are conditions that enable selective and detectable specific binding between the polynucleotide or oligonucleotide and genomic DNA. Stringent conditions are defined by a suitable combination of salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, stringency increases depending on whether the salt concentration is reduced, the organic solvent concentration is increased, or the hybridization temperature is increased. In addition, washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature, and the stringency of the wash increases with decreasing salt concentration and increasing temperature.

Therefore, “stringent conditions” means that the degree of homology between each base sequence is, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more on the average on the whole. It means that the hybrid is specifically formed only between base sequences having high homology. Specifically, for example, the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and a pH of 6 to 8 at a temperature of 60 to 68 ° C. As a specific example of stringent conditions, hybridization is performed under the conditions of 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 1% SDS, 5 x Denhardt's solution 50% formaldehyde, and 42 ° C. Washing is performed under conditions of 0.1 × SSC (15 mM NaCl, 1.5 mM trisodium citrate), 0.1% SDS, and 55 ° C.

  Hybridization may be performed by a method known in the art, such as, for example, the method described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). It can carry out according to the method according to it. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

The gene of the present invention can be prepared by any method known to those skilled in the art using the above-mentioned literature or a database of a public institution known to those skilled in the art or a primer or probe prepared based on the base sequence described herein. I can do it. For example, various PCR and other DNA amplification techniques known to those skilled in the art such as NASBA (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method and SDA (Strand Displacement Amplification) method are used. Thus, it can be easily obtained as cDNA of the gene.

Alternatively, the gene can be isolated by screening the cDNA libraries described herein by methods well known to those skilled in the art. Furthermore, the cDNA of the gene can be prepared by introducing a base mutation using a commercially available mutation system or the like based on site-directed mutagenesis known to those skilled in the art.

In addition, the above gene can be obtained by a known method (for example, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411-418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) ) Nucleic Acid Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90 Synthesized in vitro by well-known chemical synthesis techniques such as described in Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; US Pat. No. 4,458,066) You can also. It can also be produced by a method such as cleaving the polynucleotide of the present invention with an appropriate restriction enzyme.

The gene of the present invention is introduced into an undifferentiated cell by any method known to those skilled in the art, the cell is transformed, and a gene in which each gene is forcibly expressed under certain conditions (for example, tetracycline control). A converter can be obtained. For example, calcium phosphate method, lipofection method, method using transferrin receptor, method using membrane-permeable peptide such as penetratin, physical method such as microinjection, electroporation and particle gun, and retrovirus and adeno A method using an appropriate virus such as a virus can be mentioned.

Depending on the various transformation methods described above, each gene can be a single form (for example, mRNA or cDNA molecule) as it is or a recombinant vector for expression produced by incorporating it into an appropriate vector (same vector or another vector). Introduced in form. Examples of such vectors include various viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, non-viral vectors, and mixed vectors. For such vectors, expression control sequences include appropriate promoters, enhancers, transcription terminators, start codons (ie, ATG) in genes encoding proteins, splicing signals for introns, polyadenylation sites, stop codons, etc. Various elements such as various gene expression regulatory sequences, cloning sites, drug resistance genes and the like are appropriately included, and can be prepared by any method known to those skilled in the art.

Furthermore, as a third aspect of the present invention, the present invention relates to a cilia cell that has been induced to differentiate by the abnormal method of the present invention. The ciliated cells thus obtained can be advantageously used in, for example, a screening method for drugs that regenerate or inhibit cilia.

The screening of the present invention can be performed by any method known to those skilled in the art. By the screening method of the present invention, a drug (compound) that regenerates or inhibits cilia can be identified. The compound may be a substance originally contained in a living body such as a human or a substance synthesized artificially.

The screening method of the present invention can be carried out, for example, by the following steps.
(a) contacting the test compound with ciliated cells;
(b) observing or measuring the state of cilia in the ciliated cells, and
(c) selecting a compound that regenerates or inhibits the cilia.

  In the screening method, the contact with the ciliated cells in the step (a) is performed by bringing them into contact with each other by any means known to those skilled in the art, such as adding a test compound to the cell culture system. Can be implemented. In addition, as shown in the Examples, the ciliary state is the observation of ciliary movement or tubulin (β-tubulin IV), which is a protein constituting cilia, or a protein unique to cilia (marker protein). This can be done by measuring the expression of Foxj1, which is a gene.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is limited and is not interpreted at all by description of a following example. Also, unless otherwise noted, the following examples include routine methods in the art, including immunostaining and Real-time PCR methods, and standard methods known to those skilled in the art, such as Sambrook and Maniatis, in Genetic engineering described in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. And performed according to molecular biological techniques. Moreover, the description content of the literature cited as a reference etc. in this specification constitutes a part of the disclosure content of this specification.

  Hereinafter, the present invention will be described in more detail with reference to examples. The technical scope of the present invention is not limited by these descriptions. In addition, the content of the technical literature quoted in this specification is considered as a part of the content of an indication of this specification.

1. Preparation of ES cells expressing Smad6, Smad7, Foxj1 and Foxa2 by tetracycline regulation <br/> Establishment of transgene-expressing ES cells by tetracycline regulation was in accordance with the paper by Masui et al. Y., Yagi, R., Takahashi, K. and Niwa, H. (2005). An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit. Nucleic Acids Res 33, e43.). In this expression system, the transgene is not expressed when tetracycline is present, and is expressed only when it is not present. In the present invention, Smad6, Smad7, Foxj1, and Foxa2-expressing ES cells were established under the control of tetracycline.

2. Maintenance of Smad6, Smad7, Foxj1, Foxa2-ES cells
ES cells expressing Smad6, Smad7, Foxj1, and Foxa2 were maintained on drug-resistant mouse embryonic fibroblasts [MEF (DR4; ATCC No. SCRC-1045)]. After stopping the cell division and 2 hours at the MEF 10 [mu] g / ml mitomycin C (Sigma), were seeded at a concentration of 2 × 10 6/10 cm dish in culture dishes 0.1% gelatin (Sigma) coated. Maintenance medium includes 15% fetal bovine serum (FBS; ES qualified; Invitrogen), 0.1 mM non-essential amino acid (Sigma), 0.1 mM b-mercaptoethanol (sigma), 100 U / ml penicillin (Sigma), High glucose DMEM (Invitrogen) supplemented with 100 U / ml streptomycin (Sigma), 1,000 U / ml leukemia inhibitory factor (LIF; Chemicon), 1.5 μg / ml puromycin (Sigma), 1 μg / ml tetracycline (Sigma) ) Was used. The medium was changed every day and subcultured every 3 days.

3. Embryoid body formation and ciliary cell differentiation induction Embryoid bodies were formed from ES cells to differentiate into ciliary cells. Embryoid bodies are those differentiated into three germ layers by forming cell masses of ES cells in vitro. In order to form embryoid bodies, undifferentiated and maintained ES cell colonies were treated with a phosphate buffer containing 0.1% trypsin and 0.5 mg / ml EDTA to be dissociated. The dissociated cells were seeded on a culture dish and cultured for 30 minutes. By this operation, the MEF adheres to the culture dish, but the ES cells remain floating. By collecting only the culture supernatant, ES cells and MEF (mouse embryonic fibroblasts) can be separated. The obtained ES cells were seeded in a round-bottomed low-adhesion 96-well plate at 2000 cells per well and cultured for 3 days to form embryoid bodies. Low-glucose DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 100 U / ml penicillin, 100 U / ml streptomycin, 1.5 μg / ml puromycin, 1 μg / ml tetracycline It was. At this time, Smad6, Smad7, Foxj1, and Foxa2 are not expressed. The formed embryoid body was transferred to a gelatin-coated culture dish and adhesion culture was performed. The medium used was low glucose DMEM (Invitrogen) supplemented with 10% FBS, 100 U / ml penicillin, 100 U / ml streptomycin, 1.5 μg / ml puromycin. From this adhesion culture, Smad6, Smad7, Foxj1, and Foxa2 were expressed.

4). Ciliary cell differentiation (inhibition of BMP signaling)
When the BMP signal was suppressed with Smad6 or Smad7, differentiation of ciliated cells could be induced even in a medium containing FBS, which had not differentiated from ciliary cells until now (FIG. 1).
Cilia movement was observed in the differentiated cilia cells. It was confirmed that ciliated cells induced to differentiate by immunostaining express beta-tubulin IV, which is a constituent protein of cilia (left of FIGS. 1 and 2). In addition, differentiation-induced ciliary cells expressed Musashi1, which is known to be expressed in ciliary cells of the ventricle, indicating that they are ciliated cells of the ventricle (right side of FIG. 2). Furthermore, when the BMP signal was suppressed with Smad7, the expression of Foxj1, beta-tubulin IV, and Centrin4, which are genes specific to ciliated cells, was recognized by Real-time PCR (FIG. 3).

5. Expression of ciliary cells (Foxj1, Foxa2) When Foxj1, which is an essential gene for cilia formation, or Foxa2, which plays an important role in the differentiation of respiratory epithelium, was expressed, FBS had not been differentiated until now. Cilia cells could be induced to differentiate even in the medium containing (FIGS. 4 and 5). Furthermore, the expression increase of Foxj1, beta-tubulin IV, and Centrin4 was recognized by Real-time PCR method (FIGS. 6 and 7).

In the culture system using FBS according to the present invention, it is possible to induce differentiation of ventricle-specific cilia cells by suppressing the BMP signal. Ciliary cells in the ventricle have a function of circulating cerebrospinal fluid, and when the function is inhibited, hydrocephalus develops. Transplantation of ventricular ciliated cells induced by inhibition of BMP signaling may be able to treat hydrocephalus.

Furthermore, Foxa2 is a gene expressed in the respiratory epithelium, and cilia cells induced by Foxa2 may be the respiratory system. Ciliary cells of the respiratory system play a role of discharging foreign substances, and when the function is inhibited, serious diseases such as bronchiectasis are caused. Although a fundamental treatment method for bronchiectasis has not been established, there is a possibility that a fundamental treatment can be performed by transplanting ciliated cells induced by the present invention.

Claims (14)

  1. A method of inducing differentiation from an undifferentiated cell to a ciliated cell by suppressing a BMP signal in the undifferentiated cell in a step of culturing an embryoid body formed from an undifferentiated cell in a medium containing serum.
  2. The method according to claim 1, wherein the BMP signal is suppressed by introducing a Smad6 and / or Smad7 gene into an undifferentiated cell and forcibly expressing the gene.
  3. The method according to claim 1, wherein the BMP signal is suppressed by enhancing the expression of Smad6 and / or Smad7 genes in undifferentiated cells.
  4. The method according to claim 3, wherein the Smad6 and / or Smad7 gene is derived from a mammal.
  5. A method of inducing differentiation from an undifferentiated cell to a ciliated cell by enhancing expression of a gene involved in differentiation of the ciliated cell in a step of culturing an embryoid body formed from an undifferentiated cell in a medium containing serum.
  6. The method according to claim 5, wherein the gene involved in ciliary cell differentiation is Foxj1 and / or Foxa2 gene.
  7. The method according to claim 6, comprising introducing Foxj1 and / or Foxa2 genes into undifferentiated cells and forcibly expressing the genes.
  8. The method according to claim 7, wherein the Foxj1 and / or Foxa2 gene is derived from a mammal.
  9. The method according to any one of claims 1 to 8, wherein the undifferentiated cells are embryonic stem cells.
  10. The method according to any one of claims 1 to 9, wherein the undifferentiated cells are derived from a human.
  11. The method according to any one of claims 1 to 10, wherein the serum is fetal bovine serum.
  12. Cilia cells induced to differentiate by the method according to any one of claims 1 to 11.
  13. The ciliated cell according to claim 12, which is a ciliated cell of the ventricle.
  14. A method for screening a drug that regenerates or inhibits cilia, using the ciliated cells according to claim 12 or 13.
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