JP2011115161A - Method for inducing differentiation of cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for inducing differentiation of an ES (embryonic stem) cell or an iPS (induced pluripotent stem) cell by which the differentiation of the ES cell or iPS cell into an endoderm system, a mesoderm system and an ectoderm system can be induced without using a supporting cell. <P>SOLUTION: The method for inducing the differentiation from the ES cell or iPS cell into the endoderm system, mesoderm system or ectoderm system includes culturing the ES cell or iPS cell derived from a mammal on a matrix composed of nano-size fibers or particles or a positively charged matrix. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method for inducing differentiation of ES cells and iPS cells. More specifically, the present invention relates to a method for inducing differentiation of ES cells or iPS cells into endoderm or the like using a synthetic nanofiber substrate without using supporting cells.

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of a blastocyst. Regarding liver differentiation, in vitro methods form embryoid bodies and mimic the inducible microenvironment required for liver regeneration, or are treated with specific growth factors and cytokines essential for hepatocyte differentiation. . It has been shown that when ES cells are cultured with embryonic mesenchymal cells, ES cells are directed to the pancreatic and liver lineages. In vitro generation of ES cell-derived hepatocytes
Reported using 4.

The present inventors have reported that differentiation into the liver can be induced with high efficiency by culturing ES cells on M15 cells, which are a feeder cell line (Non-patent Document 1). The present inventors have established a high-efficiency differentiation induction technique from ES cells to embryonic endoderm respiratory and digestive organs by planar culture by a method using feeder cells (Patent Documents 1 and 2). By using M15 cells, ES cells can be induced in vitro into organs derived from mesendoderm, definitive endoderm, and finally region-specific definitive endoderm. The M15 feeder is a useful tool for generating lineage-specific cell types derived from ES cells belonging to three types of germ layers (neural ectoderm, mesoderm and definitive endoderm) (Non-patent Document 3).
However, the above method is a method using living support cells, and it is desirable to develop a method using a cell-free system in consideration of application to humans in the future.

WO 2006/126574 WO2008 / 149807

Shiraki N, Umeda K, Sakashita N, et al. Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells. 2008; 13: 731-746. Shiraki N, Yoshida T, Araki K, et al. Guided differentiation of embryonic stem cells into Pdx1-expressing regional-specific definitive endoderm. Stem Cells. 2008; 26: 874-885. Shiraki N, Higuchi Y, Harada S, et al. Differentiation and characterization of embryonic stem cells into three germ layers. BiochemBiophys Res Commun. 2009; 381: 694-699.

The present invention is a novel induction induction of ES cells or iPS cells that enables differentiation of ES cells or iPS cells into endoderm, mesoderm, and ectoderm without using feeder cells. Providing a method was an issue to be solved.

As a result of intensive studies to solve the above problems, the present inventors have cultivated ES cells or iPS cells using a synthetic nanofiber (sNF) matrix, and without using support cells, endoderm It was found that differentiation can be induced into a mesoderm, mesoderm, and ectoderm. That is, ES cells grown on a synthetic nanofiber matrix are induced in the endoderm,
It can be induced into the pancreas. Since the induction efficiency in the method of the present invention is sufficiently high,
Pdx1 / GFP ES cell line or high-throughput screening of small molecule compounds
By using the Ins1 / GFP ES cell line, a candidate compound that increases the ratio of insulin-expressing cells can be obtained. Furthermore, as a result of examining the usefulness of the synthetic nanofiber (sNF) matrix in the induction of ES cell differentiation into liver or germ layer-specific differentiation, as a result of culture on sNF matrix, differentiation from ES / iPS cells to liver, Differentiation into neuroectodermal and mesoderm could be achieved. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) From ES cells or iPS cells, including endodermal cells, mesoderm, comprising culturing mammalian-derived ES cells or iPS cells on a matrix composed of nano-sized fibers or particles or a positively charged matrix A method for inducing differentiation into a cell line or ectoderm cell line.
(2) The method according to (1), wherein the matrix composed of nano-sized fibers or particles is a synthetic nanofiber matrix.
(3) The synthetic nanofiber matrix has an average diameter of 200-400 nm and a pore size of 5
The method according to (2), which is 00 nm to 1000 nm.
(4) The method according to (2) or (3), wherein the synthetic nanofiber matrix is composed of at least one polyamide polymer.
(5) By culturing a mammal-derived ES cell or iPS cell in the presence of a growth factor on a matrix composed of nano-sized fibers or particles or a positively charged matrix, the endoderm system from the ES cell or iPS cell Induce differentiation into cells, from (1) to (
The method according to any one of 4).
(6) The method according to (5), wherein the endoderm cells are pancreatic or liver cells.
(7) Mammalian-derived ES cells or iPS cells are mouse or human-derived ES cells or iP
The method according to any one of (1) to (6), which is an S cell.
(8) An endoderm cell, mesoderm cell or ectoderm cell obtained by the method according to any one of (1) to (7) and induced to differentiate from an ES cell or iPS cell.

(9) Presence of a test substance when differentiation induction from ES cells or iPS cells into endoderm cells, mesoderm cells or ectoderm cells by the method according to any one of (1) to (7) ES cells or iPS cells under culture, the degree of differentiation induction into endoderm cells, mesoderm cells or ectoderm cells when ES cells or iPS cells are cultured in the absence of the test substance, From comparing ES cells or iPS cells with ES cells or iPS cells when compared to the degree of differentiation induction into endoderm cells, mesodermal cells or ectoderm cells when cultured in the presence of a test substance A method for screening for a substance that promotes or inhibits differentiation into endoderm cells, mesoderm cells, or ectoderm cells.
(10) The screening method according to (9), wherein the test substance is a growth factor or a low molecular compound.
(11) An endoderm cell, mesodermal cell, or ectoderm cell, using as an index the amount or protein amount of a marker transcript expressed in an endoderm cell, mesodermal cell, or ectoderm cell, or both The screening method according to (9) or (10), wherein the degree of differentiation induction is measured.

ES cells and iPS cells are characterized by being capable of proliferating indefinitely and having pluripotency capable of differentiating into any cell tissue. In the present invention, by a method that does not use feeder cells (supporting cells), the present inventors have succeeded in establishing a technique for inducing differentiation of liver cells and pancreatic cells into endoderm system, mesoderm system, and ectoderm system. The method of the present invention is useful for the creation of model cells that can be applied to basic research of drug discovery such as safety evaluation of new drugs, and the creation of transplanted cell sources for regenerative medicine, and in particular, ES cells and iPS cells by the method of the present invention. The method of inducing differentiation into mature hepatocytes and mature pancreatic cells is useful in the fields of drug discovery and regenerative medicine.

FIG. 1 shows the induction of ES cell differentiation into definitive endoderm by sNF matrix. ES cells were assayed for definitive endoderm development or reduction of E-cadherin + / SSEA1 + cells by FCM analysis. A) The numbers in the left panel indicate the percentage of E-cadherin + / SSEA1 + ES cells. The numbers in the right panel represent the percentage of E-cadherin + / Cxcr4 + cells in total cells. B) Percentage of E-cadherin + / SSEA1 + ES cells (white bars) or SSEA1- / E-cadherin + / Cxcr4 + definitive endoderm cells (black bars). FIG. 2 shows the induction of ES cell differentiation into the pancreas by the sNF matrix. (A) Pdx1 / GFP ES cells (SK7 ES cells) grown on the sNF matrix differentiate into Pdx1 / GFP expressing cells (green). (B) To quantify definitive endoderm or pancreatic progenitor cells, FCM analysis was performed using SK7 ES cells and anti-E-cadherin or anti-Cxcr4 antibodies. The numbers in the upper panel represent the percentage of E-cadherin + / Cxcr4 + definitive endoderm cells and the numbers in the lower panel represent the percentage of Pdx1 / GFP + cells within the definitive endoderm population. FIG. 3 shows that ES cell-derived Pdx1 + cells further develop into mature pancreatic endocrine cells, mature pancreatic exocrine cells, and mature pancreatic duct cells. (A, B) Immunocytochemical analysis of insulin positive cells in differentiated SK7 ES cells grown on sNF matrix for 20 days (A), and differentiated Ins1 / GFP ES cells grown on sNF matrix for 20 days ( (ING ES cells) in which insulin or glucagon-expressing cells (B) are shown. (C) The gene expression levels of differentiated Ins1 / GFP ES cells grown on sNF matrix for 20 days were deanalyzed by RT-PCR. For comparison, undifferentiated ES cells were used. FIG. 4 shows the differentiation of ES cells on the sNF matrix into the pancreatic lineage. A) Schematic of the medium used. Mouse ES cells (SK7 ES cells or ING ES cells) were seeded on sNF matrix on day 0 (d0). Medium 1 was used for d1 to d7, and medium 2 was used for d7 to d11. These media were changed every 2 days (d3, 5, 7, and 9). Exchanges were made every 2 days (d11, 13, and 15). The efficiency of cell differentiation was analyzed by insulin / GFP observation or insulin immunocytochemistry. B) Gene expression level of differentiating SK7 ES cells was analyzed by quantitative RT-PCR. C) In SK7 ES cells, Pdx1 / GFP expression was detectable at d9 but not at d11. GFP expression then became detectable again at d13 and increased again at d16. D) GFP expressing cells became detectable in d16 at Ins1 / GFP ES cells. E) Pdx1 / GFP expression and / or Ins1 expression in d16 SK7 ES cells were detected by immunocytochemistry. F) Efficacy as a screening system for drugs that promote the differentiation induction of ES cells into the pancreas by sNF matrix. The result of having analyzed the insulin expression level in the mouse ING ES cell grown and differentiated on the sNF matrix is shown. Various humoral factors and inhibitors shown in the figure were added to mouse ING ES cells differentiated by the method of FIG. 4A during the period of D11-20 to search for factors involved in pancreatic differentiation. FIG. 5 shows the differentiation of human ES cells into pancreatic PDX1-expressing cells on a synthetic sNF matrix. (A) A schematic representation of the stepwise differentiation procedure for human ES cells is shown. This protocol is divided into several steps. Media days and duration, growth factors, chemicals, and other additives are shown. Human ES cells were planarly cultured on sNF matrix and cultured to 80-90% confluence (d0). ES cells differentiated into PDX1-positive pancreatic progenitor cells via SOX17-positive definitive endoderm. Act A; Activin A, RA; Tinoic acid, Cyc; KAAD cyclopamine, ILV; Indolactam V, ITS; DMEM containing insulin, transferrin, selenium and 2000 mg / L glucose. (B, C) Immunostaining showed that d5 differentiated human ES cells were SOX17 positive (red) (B), and d13 was PDX1 positive (red) (C). SOX17 staining was performed using cells adhered on a slide glass with a cytospin cell centrifuge. PDX1 staining was performed by whole mount immunocytochemistry. PDX1 protein production was observed in the monolayer and localized in the nucleus (blue; DAPI). (D) RT-PCR analysis showed that SOX17 and PDX1 transcripts were expressed in d13 (d13) in differentiated ES cells but not in undifferentiated ES cells (ES). FIG. 6 shows liver differentiation of mouse ES cells grown on sNF matrix. (A) Differentiated cells began to produce albumin transcripts at d12, and albumin protein was first detected at d16. Albumin expression and albumin secretion levels reached a plateau around d20. (B) Comparison of albumin transcript levels between differentiated cells grown on sNF matrix and differentiated cells grown on other substrates for 24 days. The inducing action of albumin expression is in the order of sNF matrix> M15> Matrigel> Collagen I. Albumin transcript levels were normalized using β-actin transcript levels. Values are normalized using fetal liver, and the graph represents relative gene expression levels where fetal liver level is taken as 100. (C) Time course of albumin secretion from ES-derived hepatocytes grown on sNF matrix or other substrate. 24 hours after changing to a new medium, the amount of albumin secreted into the medium was measured. Albumin secretion from cells grown on sNF matrix was significantly higher than the others at any measurement point. (D) CYP activity of differentiated ES cells cultured on each substrate and sensitivity to inducers. Differentiated cells on each matrix were treated with 3-methylcholanthrene for 48 hours for induction of CYP1A and for 48 hours with dexamethasone for induction of CYP2C and 3A. To the cells, luciferin-CEE was added to detect CYP1A, luciferin-H was added for CYP2C, and luciferin-PFBE was added to CYP3A. Values were normalized to cell number using ATP activity of living cells grown on sNF matrix. The graph shows the relative induction level of each CYP activity when the level of the cells cultured on the sNF matrix is 1 when no drug is added. (E) PAS staining of differentiated cells on day 24 of culture. Magenta in the cytoplasm reflects glycogen storage, one of the functions of hepatocytes. (F) Indocyanine green (ICG) test as hepatocyte function analysis. At d24, differentiated cells were treated with ICG for 30 minutes at 37 ° C. Compared with cells grown on Matrigel or Collagen I, differentiated cells grown on sNF matrix were more likely to have ICG uptake. FIG. 7 shows the results of quantification of transcripts of liver marker genes by real-time PCR analysis. For differentiation of human ES cells using M15 cells, the cells were cultured in a medium supplemented with activin and bFGF, FBS, and 4500 mg / L glucose for 10 days. From d10 to d20, the additives were switched to Dex and HGF, 10% KSR, and 2000 mg / L glucose. For differentiation using the sNF matrix, human ES cells were cultured in a plane on the sNF matrix and cultured to 80-90% confluence (d0). Next, the cells were cultured at d0 to d5 in a medium supplemented with activin, bFGF, ITS, and 4500 mg / L glucose. From d5 to d20, the medium was switched to Dex and HGF, 10% KSR, and 2000 mg / L glucose. Transcription levels were normalized to cell numbers using GAPDH transcription levels. The values in the graph represent relative gene expression levels when the human fetal liver (FL) level is 100. FIG. 8 shows the differentiation of ES cells into mesoderm and ectoderm cell lineages using sNF matrix plates. A) FCM analysis using SSEA1- / PDGFRα + to quantify paraxial mesoderm. Numbers indicate the fraction of each fraction within the total ES cell culture. ES cells were cultured on FBS-containing differentiation medium (left panel) or ITS-containing medium (right panel). B) Immunocytochemical analysis using βIII tubulin antibody to confirm neural differentiation. ES cells were differentiated for 5 days in a medium supplemented with KSR and retinoic acid. FIG. 9 shows the results of an ES cell differentiation induction experiment using UpCell. FIG. 10 shows the results of an ES cell differentiation induction experiment using RepCell. FIG. 11 shows the results of an ES cell differentiation induction experiment using BD PureCoat Amine plate. FIG. 12 shows the results of an ES cell differentiation induction experiment using various nanofibers.

Hereinafter, embodiments of the present invention will be described in more detail.
In the present invention, a synthetic nanofiber (sNF) matrix was used for the purpose of establishing a technique for differentiating ES cells without using feeder cells. By using sNF matrix, the induction efficiency of insulin positive cells from ES cells or iPS cells was improved. In addition, the sNF matrix can be applied to high content screening for drugs that promote differentiation of ES cells into insulin-expressing cells. In addition, UpCell and RepCell sold by Cell Seed, as well as BD PureCoat Amine
Even when the plate was used, insulin-positive cells could be induced from ES cells.

In addition to pancreas differentiation, ES / iPS cell to liver differentiation could also be induced by culturing ES cells on sNF matrix. Similar to the pancreatic lineage, a high percentage of mouse ES cells differentiate into albumin producing cells. The albumin transcript level is E12.5
Increase to about 8 times fetal liver. ES cell-derived hepatocytes respond to drug inducers in response to Cy
It showed increased metabolic activity of p1A1, Cyp2C9, and Cyp3A.

Furthermore, as a result of investigating whether sNF can be used for differentiation into other germ layers, it was found that not only endoderm cells but also neuroectodermal cells and mesoderm cells can be induced to differentiate. That is, the cell differentiation induction method of the present invention using sNF can be applied to the generation of neuroectodermal lineage, mesoderm lineage, and endoderm lineage from ES / iPS cells.

In the present invention, endoderm cells, mesoderm cells, or ectoderm cells can be induced to differentiate from ES cells or iPS cells by a method using a synthetic nanofiber (sNF) matrix. In the conventional method, differentiation from ES cells to the liver or pancreas has been induced by a method using M15 cells, which are feeder cells. However, according to the present invention, differentiation can be induced without using feeder cells (support cells). It became possible.

The present invention is a method for inducing differentiation from ES cells or iPS cells into endoderm cells, mesoderm cells, or ectoderm cells, which are derived from mammals on a synthetic nanofiber (sNF) matrix. It is a method characterized by culturing iPS cells. ES cells or iPS cells can be cultured preferably in the presence of a growth factor.

In the differentiation-inducing method of the present invention, digestive cells derived from endoderm such as liver or pancreas from ES cells or iPS cells on a synthetic nanofiber (sNF) matrix without using supporting cells, and mesodermal cells Alternatively, differentiation of ectoderm cells can be induced. In the present invention, ES cells or iPS cells are easily induced to differentiate by culturing on a synthetic nanofiber (sNF) matrix. Therefore, when a specific growth growth factor is added, differentiation induction is further promoted. Therefore, the method of the present invention can also be used as a screening for unknown differentiation-inducing factors.

The ES cell used in the present invention is not particularly limited as long as it is a mammal-derived ES cell, and for example, a mouse, monkey or human-derived ES cell can be used. As ES cells, for example, in order to facilitate confirmation of the degree of differentiation, Pdx
Cells in which a reporter gene is introduced in the vicinity of one gene can be used. For example, Pd
129 / Sv-derived ES cell line incorporating the LacZ gene at the x1 locus or under the control of the Pdx1 promoter
ES cell line SK7 having a GFP reporter transgene can be used. Alternatively, an ES cell PH3 strain having an mRFP1 reporter transgene under the control of a Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of a Pdx1 promoter can also be used.

Mammal-derived ES cells can be cultured by conventional methods, for example, 1000 unit / ml leukemia inhibitory factor (LIF) in the presence of mitomycin C-treated mouse embryonic fibroblasts (MEF) as feeder cells if necessary. Chemicon), 15% knockout serum replacement (KSR; Gibco), 1% fetal bovine serum (FBS; Hyclone), 100 μM non-essential amino acids (
NEAA; Invitrogen), 2 mM L-glutamine (L-Gln; Invitrogen), 1 mM sodium pyruvate (Invitrogen), 50 units / ml penicillin and 50 μg / ml streptomycin (PS;
Invitrogen) and Glasgow minimal essential medium (Invitrogen) with 100 μM β-mercaptoethanol (β-ME; Sigma).

An iPS cell (induced pluripotent stem cell) is a pluripotent cell obtained by reprogramming somatic cells. The generation of induced pluripotent stem cells is done by a group of Prof. Shinya Yamanaka from Kyoto University and Rudolf Massachusetts Institute of Technology.
Several groups have been successful, including the group of Rudolf Jaenisch et al., The group of James Thomson et al. At the University of Wisconsin, the group of Konrad Hochedlinger et al. At Harvard University. Artificial pluripotent stem cells are highly expected as ideal pluripotent cells without rejection and ethical problems. For example, International Publication No. WO 2007/069666 discloses somatic cell nuclear reprogramming factors including Oct family genes, Klf family genes, and Myc family gene gene products, as well as Oct family genes, Klf family genes, Sox family genes and A somatic cell nuclear reprogramming factor containing a gene product of a Myc family gene is described, and further, a pluripotent stem cell induced by somatic cell nuclear reprogramming, comprising a step of contacting the nuclear reprogramming factor with the somatic cell. A method of manufacturing is described.

IPS cells used in the present invention can be produced by reprogramming somatic cells. The type of somatic cell used here is not particularly limited, and any somatic cell can be used. That is, the somatic cell referred to in the present invention includes all cells other than the internal germ cells of the cells constituting the living body, and may be a differentiated somatic cell or an undifferentiated stem cell. The origin of the somatic cell may be any of mammals, birds, fishes, reptiles and amphibians, but is not particularly limited, but is preferably a mammal (for example, a rodent such as a mouse or a primate such as a human). A mouse or a human is preferable. When using human somatic cells,
Any neonatal or adult somatic cell may be used.

The iPS cells referred to in the present invention have a self-replicating ability over a long period of time under predetermined culture conditions (for example, conditions under which ES cells are cultured), and are also ectoderm, mesoderm and endoderm under predetermined differentiation-inducing conditions. This refers to stem cells that have pluripotency. The induced pluripotent stem cell in the present invention may be a stem cell capable of forming a teratoma when transplanted to a test animal such as a mouse.

In order to produce iPS cells from somatic cells, first, at least one reprogramming gene is introduced into the somatic cells. The reprogramming gene is a gene encoding a reprogramming factor that has the action of reprogramming somatic cells into iPS cells. Specific examples of the combination of reprogramming genes include the following combinations, but are not limited thereto.
(I) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 large
T gene (iv) Oct gene, Klf gene, Sox gene

  In the present invention, ES cells or iPS cells are cultured on a synthetic nanofiber matrix. The synthetic nanofiber (sNF) matrix used in the present invention is not particularly limited as long as it can induce differentiation of ES cells or iPS cells into endoderm cells, mesoderm cells, or ectoderm cells. The average diameter of the synthetic nanofiber matrix is preferably 200 to 400 nm, and the pore size of the synthetic nanofiber matrix is preferably 500 nm to 1000 nm. The type of polymer constituting the synthetic nanofiber matrix is not particularly limited. For example, polyamide polymer, polylactic acid, poly (lactic acid-glycolic acid) (PLGA), polycaprolactone, polyvinylidene succinate, or protein (polypeptide): gelatin , Collagen, water-soluble polymer: polyvinyl alcohol, polyethylene glycol, poly (N-vinylpyrrolidone), Nafion. General-purpose polymer: Polystyrene, poly (methyl methacrylate) (PMMA) and the like can be prepared by mixing at a specific ratio. Preferably, at least one kind of polyamide polymer can be used.

The method for producing the synthetic nanofiber is not particularly limited, but can be produced, for example, by electrospinning. For example, synthetic nanofiber (sNF) matrices can be made by electrospinning using an industrial electrospinning process with an electric field strength of 30 kV.

A method for culturing mammalian-derived ES cells or iPS cells on a synthetic nanofiber (sNF) matrix is not particularly limited. For example, undifferentiated ES cells or iPS cells are dissociated with trypsin, seeded on a synthetic nanofiber (sNF) matrix, and grown in a differentiation medium. Hereinafter, by culturing for several days, differentiation can be induced into endoderm cells, mesoderm cells, or ectoderm cells.

In the differentiation induction method of the present invention, ES is formed on a synthetic nanofiber (sNF) matrix.
When culturing cells or iPS cells, growth factors can be added and cultured.

According to the method for inducing differentiation of ES cells or iPS cells on a synthetic nanofiber (sNF) matrix according to the present invention, from ES cells or iPS cells, undifferentiated endoderm progenitor cells, immature cells of endoderm-derived organs Or can be induced to differentiate into mature cells of an endoderm-derived organ. Examples of endoderm-derived organs include, but are not limited to, pancreas and liver. The differentiation from ES cells to endoderm cells can be confirmed by measuring the expression level of a marker specific to endoderm.

Furthermore, according to the present invention, by culturing a mammal-derived ES cell or iPS cell on a synthetic nanofiber (sNF) matrix, the endoderm cell, mesodermal cell or ectoderm system is derived from the ES cell or iPS cell. Endodermal cells and mesodermal cells when ES cells or iPS cells are cultured in the presence of a test substance and ES cells or iPS cells are cultured in the absence of the test substance when differentiation induction into cells Or, compare the degree of differentiation induction into ectoderm cells and the degree of differentiation induction into endoderm cells, mesoderm cells, or ectoderm cells when ES cells are cultured in the presence of the test substance. A method for screening a substance that promotes or inhibits differentiation from ES cells or iPS cells into endoderm cells, mesoderm cells, or ectoderm cells is provided. As a test substance, a growth factor or a low molecular weight compound can be used. At this time, differentiation induction into endoderm cells, mesoderm cells or ectoderm cells is performed using the transcript or protein amount of a marker expressed in endoderm cells, mesoderm cells or ectoderm cells as an index. It is possible to measure the degree.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

Example 1:
(Method)
(1) ES cell lines Two ES cell lines were used. One is a cell that expresses GFP under the control of the insulin 1 promoter and is named ING [Hara M, Wang X, Kawamura T, et al. Transgen
ic mice with green fluorescent protein-labeled pancreatic beta -cells. Am J
Physiol Endocrinol Metab. 2003; 284: E177-183]. The other is a cell that expresses green fluorescent protein (GFP) under the control of the Pdx1 promoter and is named SK7 [Shiraki N,
Yoshida T, Araki K, et al. Guided differentiation of embryonic stem cells
into Pdx1-expressing regional-specific definitive endoderm. Stem Cells. 200
8; 26: 874-885]. Both cell lines are 1000 units / ml leukemia inhibitory factor (LIF; Chemicon), 15%
Knockout serum replacement (KSR; Gibco), 1% fetal bovine serum (FBS; Hyclone), 1
00 μM non-essential amino acids (NEAA; Invitrogen), 2 mM L-glutamine (L-Gln; Invitrogen), 1
mM sodium pyruvate (Invitrogen), 50 units / ml penicillin and 50 μg / ml streptomycin (PS; Invitrogen), and 100 μM β-mercaptoethanol (β-ME; S
maintained on mouse embryonic fibroblast (MEF) feeders in Glasgow minimal essential medium (Invitrogen).

(2) Fabrication of synthetic nanofibers by electrospinning Synthetic nanofiber (sNF) matrices were fabricated by electrospinning using an industrial electrospinning process with a 30 kV field strength. The sNF matrix consists of two polyamide polymers 1A (C ₂₈ O ₄ N ₄ H) crosslinked in the presence of an acid catalyst.
₄₇₎ n and _{B (C 28 O 4.4 N 4} H 47) consisting of n. The sNF matrix has a diameter of 200-400 nm and an average diameter of 280 nm. The pore size is about 700 nm, which is similar to the cell basement membrane.

(3) Differentiation of ES cells into pancreatic lineage on sNF matrix For differentiation studies, ES cells were planarly cultured at 5,000 cells per well in a 96-well plate coated with sNF matrix (Ultra-Web Synthetic Polyamine Surface
(# 3873XX1, Corning Coster, Cambridge, MS). Cells contain 4500 mg / L glucose, NEAA, L-Gln, PS, β-ME, 10 μg / ml insulin (Sigma-Aldrich), 5.5 μg / ml transferrin (Sigma-Aldrich), 6.7 pg / ml selenium ( Sigma-Aldrich), and 0.25% Albm
ax (Invitrogen), 10 ng / mL recombinant human activin-A (R & D Systems, Minneapolis, M
N), 5 ng / mL; Dulbecco's modified Eagle medium (DMEM: Invitrogen) containing recombinant human bFGF
, Glasgow, UK) for 7 days (medium 1, FIG. 4A). The medium is then 2000 mg / L glucose (Sigma, St Louis, MO), 1 μM retinoic acid (Sigma-Aldrich), 50 ng / ml human recombinant fibroblast growth factor-10 (Peprotech, Rocky Hill, NJ), B27 SUPPLEMENT (Invitr
ogen), and 0.25 μM 3-keto-N- (aminoethyl-aminocaproyl-dihydrocinnamoyl) cyclopamine Shh signaling antagonist II (Calbiochem, San Diego, C
The RPMI 1640 medium (Invitrogen) containing A) was replaced with d7-d11 (medium 2, FIG. 4A). Finally, the medium contains 1000 mg / L glucose, NEAA, L-Gln, PS, β-ME, 10 μg / ml insulin (Sigma-Aldrich), 5.5 μg / ml transferrin (Sigma-Aldrich), 6.7 pg / ml
Selenium (Sigma-Aldrich), 0.25% Albmax (Invitrogen), and 10 mM nicotinamide (
D11 to d15 were switched to DMEM containing (Sigma-Aldrich) (Medium 3, FIG. 4A). The medium was changed every 2 days with fresh medium and growth factors until d15.

(4) Screening of factors involved in pancreatic differentiation of ES cells using nanofiber differentiation induction system Mouse ES cells (Ins1 / GFP ES cells) were planarized at 5,000 cells per well in a 96-well plate coated with sNF matrix Cultured (Ultra-Web Synthetic Polyamine S
urface (# 3873XX1, Corning Coster, Cambridge, MS). Cells contain 4500 mg / L glucose, NEAA, L-Gln, PS, β-ME, 10 μg / ml insulin (Sigma-Aldrich), 5.5 μg
/ ml transferrin (Sigma-Aldrich), 6.7 pg / ml selenium (Sigma-Aldrich), and 0.
25% Albmax (Invitrogen), 10 ng / mL recombinant human activin-A (R & D Systems, Minneap
olis, MN), 5 ng / mL; Dulbecco's modified Eagle medium (DMEM: Inv) containing recombinant human bFGF
Itrogen, Glasgow, UK) for 7 days (medium 1, FIG. 4A). The medium is then 2000 mg
/ L glucose (Sigma, St Louis, MO), 1 μM retinoic acid (Sigma-Aldrich), 50 ng / ml
Human recombinant fibroblast growth factor-10 (Peprotech, Rocky Hill, NJ), B27 SUPPLEMEN
T (Invitrogen), and 0.25 μM 3-keto-N- (aminoethyl-aminocaproyl-dihydrocinnamoyl) cyclopamine Shh signaling antagonist II (Calbiochem, San
Diego, CA) was replaced with dMI-d11 in RPMI 1640 medium (Invitrogen) (Medium 2, FIG. 4
A). Finally, the medium contains 1000 mg / L glucose, NEAA, L-Gln, PS, β-ME, 1
0 μg / ml insulin (Sigma-Aldrich), 5.5 μg / ml transferrin (Sigma-Aldrich),
D11-d15 was switched to DMEM containing 6.7 pg / ml selenium (Sigma-Aldrich), 0.25% Albmax (Invitrogen), and 10 mM nicotinamide (Sigma-Aldrich) (Medium 3, FIG. 4A). Medium is d
Up to 20 were replaced every 2 days with fresh media and growth factors. In the period of D11-20, various humoral factors and inhibitors shown in FIG. 4F were added to search for factors involved in pancreatic differentiation. Evaluation was performed by real-time PCR on the 20th day. The expression level of insulin 1 in each sample was corrected with the expression level of β-actin. The value shows a relative value when the expression level of insulin 1 in the pancreas on the 12.5th day of embryo is 100.

1. Control
2. Metastin (Sigma) 50ng / ml
3. GDNF (Peprotech) 50ng / ml
4. VEGF (Peprotech) 20ng / ml
5. DAPT (Peptide) 10μg / ml
6. SB203580 (Calbiochem) 10μg / ml
7. Activin A (R & D) 20ng / ml
8.SB431542 (Sigma) 1μg / ml
9. BMP4 (Prime Mune) 25ng / ml
10. Noggin (R & D) 100ng / ml
11. Dorsomophin (Sigma) 0.2μg / ml
12. Retinoic acid (RA) (Sigma) 1μg / ml
13. LE540 (WAKO) 1μg / ml
14. BIO (Calbiochem) 5μg / ml
15. SU5402 (Calbiochem) 10μg / ml
16. HB-EGF (R & D systems) 10ng / ml
17. Exndin4 (Sigma) 1ng / ml
18. Betacellulin (Wako) 1ng / ml
19. IgfII (R & D systems) 25ng / ml
20. Smothened Antagonist (Calbiochem) 0.3μg / ml

(5) Differentiation of human ES cells into pancreatic lineage Human ES cells (KhES-3) were produced by Dr. N. Nakatsuji and Dr. H. Suemori (Kyoto University
, Kyoto, Japan). ES cells are 20% KSR, NEAA, L-Gln, PS, βME, 5ng
Cultured on MEF feeder cells in DMEM / F12 (Sigma) containing / ml bFGF. The cell is 0.2
Subcultured in a ratio of 1: 2 to 1: 3 every 3-4d with 5% trypsin and 0.1 mg / ml collagenase IV.

For differentiation, human ES cells were plated at 20,000-40,000 cells per well in sNF matrix plates coated with Matrigel in a thin phase. Cells were cultured to 80-90% confluence. To generate a pancreatic population, cells were cultured for 1 day in RPMI 1640 medium containing 100 ng / mL activin A, NEAA, L-Gln, PS, βME, then 100
The cells were further cultured for 2 days in RPMI 1640 containing mg / ml activin A, NEAA, L-Gln, PS, βME, and 0.2% FBS. The medium then contains 2000 mg / L glucose, NEAA, L-Gln,
It was replaced with DMEM containing PS, β-ME, 10 μg / ml insulin, 5.5 μg / ml transferrin, 6.7 pg / ml selenium, and 0.25% Albmax, 20 ng / mL activin A, 50 ng / mL bFGF for another 2 days. The medium then contains 2000 mg / L glucose, 1 μM RA, 0.25 μM KAAD cyc
, 50 ng / ml FGF10, NEAA, L-gln, and DMEM containing PS, βME, B27 were replaced for an additional 4 days. Finally, the medium contains 2000mg / L glucose and 300nM indolactam V [Ch
en S, Borowiak M, Fox JL, et al. A small molecule that directs differentiat
ion of human ESCs into the pancreatic lineage. Nat Chem Biol. 2009; 5: 258-26
5], DMEM containing 50 ng / ml Fgf10, NEAA L-gln, PS, NEAA, B27 was replaced for another 4 days. Medium with growth factors was changed daily until d13.

(6) Differentiation of mouse ES cells (or iPS cells) into liver lineage on NF For differentiation studies, ES cells were plated at 5,000 cells per well in a Corning 96 well plate with sNF matrix (Ultra -Web Synthetic Polyamine Surfa
ce # 3873XX1, Corning Coster, Cambridge, MS). The cells then contain 4500 mg / L glucose, NEAA, L-Gln, PS, β-ME, 10 mg / ml insulin (Sigma-Aldrich), 5.5
mg / ml transferrin (Sigma-Aldrich), 6.7 pg / ml selenium (Sigma-Aldrich), 0.25% A
lbmax (Invitrogen), 10 ng / mL recombinant human activin-A (R & D Systems, Minneapolis
, MN), 5 ng / mL; Dulbecco's modified Eagle's medium containing recombinant human bFGF (DMEM: Inv
Itrogen, Glasgow, UK) and cultured for 9 days. In d9, the medium was 2000 mg / L glucose (Sigma, St Louis, MO) 10% KSR, 10 ng / ml recombinant human hepatocyte growth factor (Peprotec
), Changed to DME medium containing 10 μM dexamethasone (Sigma-Aldrich), and cultured to d11. Finally, 10 mM nicotinamide (Sigma-Aldrich) and 1% dimethyl sulfoxide were added to the above medium to remove KSR. The medium was changed every 2 days with fresh medium and growth factors until d24.

(7) Differentiation of mouse ES cells into neuroectodermal and mesodermal lineages on sNF matrix For differentiation into neuronal cells, SK7 ES cells were treated with 10% KSR and 1 μM retinoic acid (Sigma-
The cells were cultured in a differentiation medium containing Aldrich). For mesoderm differentiation, ES cells are 10%
The cells were cultured in a differentiation medium containing FBS.

(8) Hepatocyte function assay in differentiated ESC (periodic acid Schiff staining)
For detection of glycogen stores in differentiated cells, a periodic acid Schiff (PAS) staining kit (Muto Pure Chemicals, Tokyo, Japan) was used. Cells cultured for 24 days were fixed in 3.3% formalin for 10 minutes and stained.

(Albumin secretion assay)
The medium was replaced with fresh medium every 2 days and the supernatant was collected after 24 hours. Mouse (human) albumin secreted into the supernatant was measured with a mouse (human) ELISA quantification kit (Bethyl).

(Indocyanine Green (ICG) test)
Indocyanine green (Daiichi-sankyo pharm. Tokyo, Japan) was diluted with the above medium to a final concentration of 1 mg / dl. The ICG test solution was added to differentiated ES cells cultured for 24 days, and undifferentiated ES cells were used as a control and incubated at 37 ° C. for 30 minutes.
The cells were then washed three times with phosphate buffered saline (PBS) and examined for ICG cell uptake by microscopy.

(9) CYP induction To confirm the ability to induce cytochrome P450 activity in response to inducers, a P450-Glo ™ CYP assay kit (Promega, Madison, WI) was used. Differentiated ES cells were used with 5 μM 3-methylcholanthrene as an inducer of CYP1A1. 50 μM dexamethasone was used as an inducer of CYP2C9 and CYP3A4. Each medium containing the inducer was changed every 24 hours. After 48 hours induction, luminescent CYP substrate (e.g. luciferin-C for CYP1A)
EE and CYP2C9 were changed to medium containing luciferin-H, and CYP3A4 was changed to luciferin-PFBE. The cells were incubated for 3 hours at 37 ° C., then the supernatant was mixed with an equal volume of detection reagent according to the manufacturer's instructions. Luminescence was measured using a GloMax96 microplate luminometer (Promega). CellTiter-Glo Luminescent Cell Viability Assay (
Promega) was used to calculate cell number and P450-Glo assay values were normalized to cell number.

(10) Immunocytochemistry
After 15 days of cell culture, differentiated ES cells were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature and rinsed several times with PBS. The cells are then placed in a humidified chamber in PBST (0.
Primary antibody diluted with Blocking One (Nacalai tesque, Tokyo, Japan) diluted 5-fold with 1% Tween-20) was added and incubated overnight at 4 ° C. After washing the cells in PBST, the cells were incubated with a secondary antibody diluted in 20% Blocking One for 2 hours at room temperature in the dark. After washing out the secondary antibody in PBST, cell nuclei were counterstained with 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Basel, Switzerland). The following antibodies were used as primary antibodies; rabbit anti-GFP (MBL International C
orp, Woburn, MA), mouse anti-insulin (Sigma), mouse anti-glucagon (Sigma), mouse anti-βIII-tubulin (Sigma-Aldrich): secondary antibodies used were Alexa568 binding antibody and Alexa488 binding antibody (Invitrogen) It was.

For human ES cell culture, goat anti-human SOX17 and goat anti-human PDX1 (R & D systems
) Was used as the primary antibody. At d5, the cells are separated into single cells using 0.25% trypsin, then 20000-50000 cells are allowed to adhere on a glass slide using a Cytospin 3 cell centrifuge (Shandon) and then as described above. Fixing, washing and immunostaining were performed.

(11) Flow cytometry The following antibodies were used: biotin-conjugated anti-E-cadherin monoclonal antibody (mAb) ECC
D2, phycoerythrin (PE) -conjugated anti-Cxcr4 mAb (BD), biotin-conjugated anti-PDGFRα mAb (BD),
PE-conjugated anti-SSEA1 (R & D), and streptavidin-allophycocyanin (BD). Stained cells were analyzed using FACS Canto (BD). Data was recorded using the BD FACS Diva software program (BD) and analyzed using the Flowjo program (Tree Star).

(12) RT-PCR analysis
RNA was extracted from ES cells or mouse pancreas and liver using RNeasy mini kit (Qiagen) and then treated with DNase (Qiagen). 3μ for reverse transcription
g RNA was reverse transcribed using ReverTra Ace (Toyobo) and oligo dT primer (Toyobo). 1 μl of 5-fold diluted cDNA (1% of RT product) was used for PCR analysis. The primer sequences of each primer set are shown in Table 1. For real-time PCR analysis, mRNA expression is ABI
Quantification was performed using SyberGreen on a 7500 thermal cycler (Applied Biosystems). P
The CR conditions were as follows: denaturation at 95 ° C. for 15 seconds, and annealing and extension at 60 ° C. for 60 seconds up to 40 cycles. Each measured value is the average β-actin (mouse) Ct
The value and Gapdh (human) Ct value (threshold cycle) are subtracted from the average of each gene Ct value to obtain the Ct
For each sample, β-actin (mouse) and Gapdh (
Normalized to human). Each target mRNA level expressed as arbitrary units was determined by the standard curve method.

(result)
(1) ES cells grown on a synthetic nanofiber matrix were induced to differentiate into the pancreatic definitive endoderm cell lineage.
ES cell differentiation is initiated in ITS-based medium containing activin and bFGF.
This was done by culturing ES cells on plates coated with sNF matrix. Next, flow cytometry (FCM) analysis was performed to examine the time course of E-cadherin + / Cxcr4 + definitive endoderm cell generation or E-cadherin + / SSEA1 + ES cell loss (Figure 1). In FCM analysis, definitive endoderm was first detectable at d5 (2.08%
1B), increased to d7 (11.36%) and E-cadherin + / SSEA1 + population decreased simultaneously (8
1.33% to 30.26%). At d9, E-cadherin + / SSEA1 + ES cells almost disappeared (5.21%), whereas definitive endoderm cells reached a plateau (41.40%). These results indicated that ES cells grown on the sNF matrix were efficiently induced into definitive endoderm cells under this culture condition.

FCM analysis was then performed to examine the effect of sNF matrix on pancreatic differentiation of mouse ES cells (FIG. 2). The expression of Pdx1 / GFP first appeared at d7. Pdx1 / GFP + cells showed a maximum in d10 (23.5% in E-cadherin + / Cxcr4 + cells) and then decreased. This demonstrated that the sNF matrix can support the development of Pdx1-positive pancreatic progenitor cells from ES cells.

Differentiated cells grown on the sNF matrix were tested for differentiation into mature pancreatic cells such as insulin-expressing pancreatic β cells (FIG. 3). Mouse Pd on sNF matrix
By culturing x1 / GFP (SK7) ES cell line or mouse Ins1 / GFP (ING) ES cell line
ES cells can differentiate and become able to express insulin or glucagon at differentiated d20. In the case of the Pdx1 / GFPES cell line, it can be seen that insulin expression overlaps with Pdx1 / GFP expression (FIG. 3A). When Ins1 / GFP ES cells are used, insulin positive staining is detected in GFP positive cells rather than glucagon positive staining (FIG. 3B). RT-PCR analysis shows insulin 1 (In
s1), glucagon (Gcg), pancreatic polypeptide (Ppy), somatostatin (Sst), and cytokeratin 19 (Krt19) transcripts were found to be expressed in differentiated ES cells
(Figure 3C). The above results indicate that ES cells grown on sNF matrix can differentiate into Pdx1 / GFP positive cells and then differentiate into the whole pancreatic lineage, namely endocrine cells, exocrine cells, and duct cells. It was.

(2) High content compound screening for drugs that promote the differentiation of ES cells into insulin-expressing cells.
The culture conditions were modified so that reproducible results were obtained with the high-throughput assay. An outline of the assay procedure is shown in FIG. 4A. SK7 Pdx1 / GFP ES cells or ING Ins1 / GFP ES cells were used. ES cells are seeded on sNF on day 0 (d0). d0 to d7, d7 to d11, d11 to 1
The medium used in 5 was optimized by observation of Pdx1 / GFP positive cells, Ins1 / GFP positive cells, or immunostaining using anti-insulin antibodies. Expression of the SOX17 transcript is detected at a substantial level from d7 to d11 and decreases after d13 (FIG. 4B). Insulin 1 transcript, d13
It was detectable at d15 and increased to a substantial level at d15 (FIG. 4B).

Pdx1 / GFP expression was found to be biphasic. Pdx1 / GFP positive cells are first observed at d9, then downregulated and upregulated again at d16 (Figure 4
C). Ins1 / GFP expression became detectable at d16 (FIG. 4D). D16 detected by anti-GFP
Part of the positive signal in Pdx1 / GFP ES cells overlapped with the positive signal due to anti-insulin antibody. Thus, insulin positive appears to emerge from Pdx1 / GFP positive cells. From these results, it was shown that this assay system can be used for detection of Pdx1 / GFP positive cells and Ins1 / GFP positive cells.

Mouse ES cells (Ins1 / GFP ES cells) are cultured in a 96-well plate coated with sNF matrix in a flat plate at 5,000 cells per well, and various humoral factors and inhibitors shown in FIG. In addition, we searched for factors involved in pancreatic differentiation. Evaluation is 2
On day 0, real-time PCR was performed. The expression level of insulin 1 in each sample is β
Correction was made with the expression level of actin. The value shows a relative value when the expression level of insulin 1 in the pancreas on the 12.5th day of embryo is 100. As a result, the amount of insulin increased when Noggin (# 10) and Dorsomophin (# 11), both of which inhibit BMP signaling, were added, suggesting that this evaluation system can be used for high-content screening. It was done.

(3) Human ES cells grown with sNF are also induced in the pancreatic lineage.
Differentiation from human ES cells to the pancreas was examined. A schematic diagram of the differentiation procedure is shown in FIG. 5A. Kh
ES-3 human ES cells were seeded onto a Matrigel thin phase coated sNF matrix and 80-90
The cells were cultured until confluence of% (d0). Optimal culture conditions are RT-PCR analysis or anti-SOX1
Tested by immunostaining using 7 antibody or anti-PDX1 antibody.
After 13 days of differentiation shown in FIG. 5A, a pancreas derived from definitive endoderm developed (FIG. 5B).
, C, D). After 5 days of culture, SOX17 positive cells were detected (FIG. 5B). Next, PDX1-positive cells appeared in d13 (FIG. 5C). SOX17 and PDX1 transcripts were also detected at d13 by RT-PCR analysis (FIG. 5D).

(4) Mouse ES cells grown on the sNF matrix differentiate into hepatocytes and produce albumin.
Regarding the above-described ES cell differentiation procedure, the induction of differentiation into the liver was examined. Pdx1 / GFP
ES cells were seeded directly on sNF matrix or M15 and differentiated to d24. FIG.
ES cells differentiated on sNF matrix began to express albumin transcripts after 6 days of culture, indicating that albumin protein secretion increased in proportion to transcript expression. Albumin protein secreted into the supernatant became detectable on day 16. (Fig. 6
A).

Albumin transcripts and proteins in differentiated cells cultured on sNF matrix were compared to M15 cells, collagen I, Matrigel. Albumin secretion is d18
Measurements were made every 2 days from ˜d24 (FIG. 6C). At any given time, not only did the kinetics of differentiated cells appear earlier in the sNF matrix than the other substrates, but the amount of albumin protein secreted from these cells was also higher.

(5) Differentiated cells from mouse ES cells have a specific function of hepatocytes.
Differentiated cells were treated with d24-26 for 48 hours using 3-methylcholanthrene, dexamethasone, which is known as an inducer of CYP1A1, 2C9, 3A4. After induction with these agents, a luminescent substrate was added and incubated for 3 hours. Since luciferin, which is a substrate product metabolized by the P450 enzyme, diffuses into the culture medium, an aliquot of the supernatant was measured with a luciferin detection reagent. As can be seen from FIG. 6D, the sNF matrix was more active even when not induced than the differentiated cells cultured on collagen I and Matrigel, and also showed a higher response ability to the inducer. In particular, the Cyp2C9 activity of differentiated cells on collagen I or Matrigel was not induced by dexamethasone, but only differentiated cells could be generated in which only the sNF matrix responded to dexamethasone (FIG. 6D).

Periodic acid Schiff staining for glycogen storage and indocyanine green (ICG) test for cell uptake ability were performed to confirm hepatocyte function. Glycogen storage was observed as the accumulation of magenta staining in the cytoplasm of differentiated cells (FIG. 6E). Cells on collagen or matrigel had a lower population of positive cells than cells on sNF matrix. The tendency was remarkable in the ICG test (FIG. 6F).

(6) Differentiation of human ES cells into liver lineage using sNF matrix.
In order to apply the above culture procedure to human ES cells, KhES-3 was cultured on sNF matrix. KhES-3 became AFP-expressing cells and albumin-expressing cells (FIG. 7). The expression level of AFP was higher than that of fetal liver, but the level of albumin transcript was lower. This result shows that human ES cell-derived hepatocytes were able to differentiate into the liver, although a slightly larger number of culture days were required than in mice.

(7) Mouse ES cells grown on the sNF matrix differentiate into three germ layers.
The sNF matrix was used to test whether ES cells could be differentiated into neuroectodermal and mesoderm. In order to differentiate into nerve cells, the cells were cultured on a medium containing retinoic acid and KSR. In order to identify neuroectodermal cells generated by the above culture procedure, immunocytochemical analysis was performed (FIG. 8B). βIII tubulin-positive neurons appeared on day 5. In order to differentiate into mesoderm, the cells were cultured in a differentiation medium containing FBS. To identify mesoderm cells, flow cytometric analysis was performed to examine the expression of the paraxial mesoderm marker PDGFRα (FIG. 8A). On medium containing FBS
By culturing for 5 days, about 40% of all cells differentiated into PDGFRa + / SSEA1-axial mesoderm cells. From these results, it was shown that ES cells can be differentiated not only into endoderm cells but also into neuroectodermal and mesoderm cells using sNF matrix.

(Summary)
In this example, it was examined whether ES cells could be differentiated using an sNF matrix.
As a result, it was found that by culturing ES cells using an sNF matrix, it was possible to differentiate into definitive endoderm-derived tissue (ie, pancreas and liver), and neuroectodermal tissue and mesoderm tissue. For human ES as well, the sNF matrix was demonstrated to be useful as an alternative substrate for feeder cells, and human ES cells could be differentiated into the pancreas. Further, in this example, it was shown that the sNF matrix is useful as an assay system for screening of low molecular compounds. In addition, differentiation from ES cells to albumin-secreting hepatocytes was promoted by culturing ES cells on sNF matrix.

Example 2:
UpCell and RepCell, which are sold from Cell Seed, are temperature-responsive cell culture dishes for cell sheet collection, and a temperature-responsive polymer (PIPAAm) is immobilized on the surface of the equipment by nano-surface design. The surface of the equipment reversibly changes to hydrophobic (cell adhesion surface) and hydrophilic (cell release surface) at 32 ° C, and the temperature is set to 20 to 25 ° C without using enzymes that damage cells such as trypsin. It is a culture dish that can recover intact cells in sheet form by simply waiting for about 10-30 minutes. At a fine level, it has a structure in which fibers or particles of 20-70 nm are spread (Akiyama Y, Kushida A, Yamato M, Kikuchi A, Okano T.
Surface characterization of poly (N-isopropylacrylamide) grafted tissue cul
ture polystyrene by electron beam irradiation, using atomic force microscop
y, and X-ray photoelectron spectroscopy. SurJ Nanosci Nanotechnol. 2007 Mar
; 7 (3): 796-802.)

The BD PureCoat Amine plate, on the other hand, is an animal-free cell culture surface plate sold by BD. It is a positively charged plate, and has better performance in cell adhesion and cell growth than a normal cell culture treatment surface. Proven to improve.

(1) UpCell
Mouse ES cells (Ins1 / GFP ES cells) 2 × 10 ^ 4ce per well of UpCell 24well plate
ll seeded. After sowing, the cells were cultured in the following culture solution. The culture solution was changed every other day. On days 0-7, serum-free medium (high glucose D) containing Activin (10 ng / ml) and bFGF (5 ng / ml)
MEM + ITS + 2.5mg / ml Albumax II), Retinoic Acid (10 ^ -6M) Activin on culture day 7-9
(10ng / ml), serum-free medium containing bFGF (5ng / ml) (high glucose DMEM + ITS + 2.5mg / ml
ml AlbumaxII), and after 9 days of culture, the cells were cultured in a serum-free medium (low glucose DMEM + ITS + 2.5 mg / ml AlbumaxII) containing GLP1 (10 nM) and Nicotinamide (10 mM). The culture results are shown in FIG.
Shown in Insulin / GFP positive cells were confirmed from the 15th day of culture, and the number increased as the culture progressed.

(2) RepCell
Mouse ES cells (Ins1 / GFP ES cells) were seeded at 5 × 10 5 cells per RepCell 60 mm dish. After sowing, the cells were cultured in the following culture solution. Until the 8th day of culture, the culture solution was changed every other day, and after the 8th day, it was changed daily. Activin (10ng / ml), bFGF (5n
serum-free medium (high glucose DMEM + ITS + 2.5 mg / ml AlbumaxII), culture 7
-8 days, serum-free medium (high glucose DMEM + ITS + 2.5mg / ml AlbumaxII) containing Retinoic Acid (10 ^ -6M) Activin (10ng / ml), bFGF (5ng / ml), culture day 8 After that HGF (10ng
/ ml), GLP1 (10 nM), Nicotinamide (10 mM), serum-free medium (low glucose DMEM + I
The cells were cultured with TS + 2.5 mg / ml AlbumaxII). The culture results are shown in FIG. I on the 16th day of culture
nsulin / GFP positive cells were confirmed, and they existed in clusters near the grid wall.

(3) BD PureCoat Amine plate
Mouse ES cells (Ins1 / GFP ES cells) 5 × 10 per well of BD purecoat amine plate
^ 4 cells were seeded. After sowing, the cells were cultured in the following culture solution. Up to 8 days of culture, the culture solution is 1
It changed every other day, and changed every day after the 8th day. Activin (10ng / ml) on days 0-7 of culture,
Serum-free medium containing bFGF (5ng / ml) (high glucose DMEM + ITS + 2.5mg / ml AlbumaxI
I), serum culture medium (high glucose DMEM + ITS + 2.5mg / ml AlbumaxII) containing Retinoic Acid (10 ^ -6M) Activin (10ng / ml), bFGF (5ng / ml) on days 7-8 , After the 8th day of culture
Serum-free medium (low glucose DMEM + ITS + 2) containing GLP1 (10 nM), Nicotinamide (10 mM)
.5 mg / ml Albumax II). The culture results are shown in FIG. Insulin on the 16th day of culture
/ GFP positive cells were confirmed.

Example 3:
An ES cell differentiation induction experiment was performed using the following nanofibers.
(1) Ultraweb (corning): 96 well plate, polyamide, diameter 200-400n
m, thin (2) polyacetic acid nanofiber (thin, Teijin Limited prototype)
(3) Nanofiber: gelatin, gelatin; polyacetic acid = 1: 1, and three types of polyacetic acid, diameter 100-2000 nm. (Thickness equivalent to cardboard. Prototype of Uyama Laboratory, Osaka University)

Mouse ES cells (Pdx1 / GFP ES cells) are added to (2) and (3) NF (24 well equivalent).
5x10 ^ 4 cells were seeded per well. After sowing, the cells were cultured in the following culture solution. The culture solution was changed every other day. On days 0-7, serum-free medium (high glucose DMEM + ITS + 2.5 mg / ml Albumax II) containing Activin (10 ng / ml) and bFGF (5 ng / ml); on days 7-11, Retinoic Aci
d (10 ^ -6M) Serum-free medium containing Fgf10 and KAAD-cyclopamine (RPMI + B27), and serum-free medium containing GLP1 (10 nM) and Nicotinamide (10 mM) after the 11th day of culture (low glucose DMEM + I
The cells were cultured with TS + 2.5 mg / ml AlbumaxII). The results of RT-PCR analysis are shown in FIG. (1)
Compared with Corning's Ultraweb, it became clear that it has the same or better differentiation induction effect. High insulin 1 transcripts were detected when using thick nanofibers. For thin materials, it is better to keep the diameter low, but when the diameter is large, it was estimated that a certain thickness is required.

Claims (11)

From culturing ES cells or iPS cells derived from mammals on a matrix composed of nano-sized fibers or particles or on a positively charged matrix, from ES cells or iPS cells, endoderm cells, mesodermal cells or A method of inducing differentiation into ectoderm cells. The method of claim 1, wherein the matrix of nano-sized fibers or particles is a synthetic nanofiber matrix. The method of claim 2, wherein the synthetic nanofiber matrix has an average diameter of 200 to 400 nm and a pore size of 500 nm to 1000 nm. The method according to claim 2 or 3, wherein the synthetic nanofiber matrix is composed of at least one polyamide polymer. By culturing ES cells or iPS cells derived from mammals in the presence of growth factors on a matrix composed of nano-sized fibers or particles or a positively charged matrix, ES cells or iPS cells are transformed into endoderm cells. The method according to any one of claims 1 to 4, wherein differentiation is induced. The method according to claim 5, wherein the endoderm cells are pancreatic or liver cells. The method according to any one of claims 1 to 6, wherein the mammal-derived ES cell or iPS cell is a mouse or human-derived ES cell or iPS cell. An endoderm cell, mesodermal cell, or ectoderm cell obtained by the method according to claim 1 and induced to differentiate from an ES cell or iPS cell. When differentiation induction from ES cells or iPS cells into endoderm cells, mesoderm cells, or ectoderm cells by the method according to any one of claims 1 to 7, ES cells or When iPS cells are cultured and ES cells or iPS cells are cultured in the absence of the test substance, the degree of induction of differentiation into endoderm cells, mesoderm cells or ectoderm cells, and the presence of the test substance An ES cell or iPS cell, and an endoderm cell in the case of culturing ES cells or iPS cells, comparing the degree of differentiation induction into mesodermal cells or ectodermal cells, endoderm cells from ES cells or iPS cells, A method for screening for a substance that promotes or inhibits differentiation into mesoderm cells or ectoderm cells. The screening method according to claim 9, wherein the test substance is a growth factor or a low molecular weight compound. Differentiation into endoderm cells, mesodermal cells, or ectoderm cells, using the amount of marker transcripts and / or protein expressed in endoderm cells, mesoderm cells, or ectoderm cells, or both as indicators The screening method according to claim 9 or 10, wherein the degree of induction is measured.