JP6265385B2 - Cell amplification using amino acid preparations - Google Patents

Description

  The present invention relates to a cell amplification method using an amino acid preparation, and more particularly to a method for amplifying a cell by culturing the cell using the amino acid preparation.

  In recent years, attention has been focused on methods for creating human functional cells useful for drug discovery screening and regenerative medicine by inducing differentiation of pluripotent stem cells such as iPS cells having the ability to differentiate into various functional cells.

  In general, by adding a protein preparation such as a cytokine to a culture system of pluripotent stem cells, maintenance and amplification of stem cells and induction of differentiation into various functional cells have been attempted (Patent Documents 1 to 3). 3. Non-patent document 1).

However, in the conventional culture methods as described above, it is a serious problem that the protein preparation required for inducing differentiation of the target organ cells is extremely expensive. In particular, in the case of organs that require an extremely large number of cells (10 ¹¹ cells) for regenerative medicine and industrial applications, such as the liver, the production of cells requires enormous costs, and applications using pluripotent stem cells are required. It is considered the greatest barrier to block.

  Therefore, in order to realize regenerative medicine / industrial application using pluripotent stem cells such as ES cells or iPS cells, how efficiently and inexpensively purify functional cells in large quantities from undifferentiated organ cells This is considered a serious issue.

JP2011-206064 JP2012-228263A JP 2010-75199 A

Hannan N.R., et al., Nat. Protoc., 8 (2), 430-437 (2013)

  An object of the present invention is to provide a method for efficiently and inexpensively creating functional cells from undifferentiated organ cells.

  The present inventors paid attention to the difference in metabolic characteristics between undifferentiated cells existing in an intermediate differentiation stage and cells existing in other differentiation stages, which are the source of organ cells that attempt mass creation.

  First, metabolome analysis and transcriptome analysis were performed on the liver from the early embryonic stage to adulthood in order to detect the difference in metabolic characteristics in cells at different stages of differentiation. As a result, in the early embryonic liver (E9.5 to 11.5) in which undifferentiated cells are frequently present, the branched-chain aminotransferase (BCAT1), which specifically degrades branched-chain amino acids, is high. It was expressed, and it was found that the degradation of branched chain amino acids was enhanced.

  From these results, it was hypothesized that the metabolic demand of branched chain amino acids was remarkably high in undifferentiated cells with active cell proliferation, and the effects of adding branched chain amino acids to undifferentiated cells were evaluated. Regarding the effect of branched-chain amino acids (L-valine, L-leucine, L-isoleucine) on which BCAT1 functions, and commercially available amino acid cocktails that are frequently used in comparison and conventional culture systems, on the fetus Examination was performed using liver cells.

  As a result, when using undifferentiated liver-derived cells in the early embryonic period (E11.5), the branched chain compared to the amino acid non-addition group, the commercially available essential amino acid preparation added group, and the commercially available non-essential amino acid preparation added group It was found that colonies composed of cells exhibiting high proliferation in the group to which amino acids were added and composed of cells that strongly express albumin, a hepatocyte-specific marker, frequently appear. Furthermore, it was confirmed from the gene expression analysis that albumin expression was increased in the branched chain amino acid addition group. The effect of such branched chain amino acids was not confirmed by analysis using liver cells in the mid-embryonic stage (E15.5). It was discovered that the effect of addition appears.

  From the above results, it was found that by adding a branched chain amino acid to undifferentiated liver cells, the proliferation was enhanced and hepatocytes expressing albumin appeared frequently. Therefore, it is considered that the branched chain amino acid has an action of inducing differentiation of undifferentiated organ cells into functional cells through induction of transient amplification (transit amplification).

The gist of the present invention is as follows.
(1) A method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid having a concentration of 1 mM or more.
(2) The culture of undifferentiated hepatocytes in the presence of a branched-chain amino acid at a concentration of 1 mM or more amplifies undifferentiated hepatocytes and induces differentiation into hepatocytes (1) Method.
(3) The method according to (1) or (2), wherein the branched chain amino acid is selected from the group consisting of valine, leucine, isoleucine and combinations thereof.
(4) A method for inducing differentiation into hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.
(5) Hepatocytes comprising amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. Preparation method.
(6) A method for selecting cells capable of differentiating into hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched-chain amino acid at a concentration of 1 mM or more.
(7) A medium containing a branched chain amino acid at a concentration of 1 mM or more.
(8) The medium according to (7), which is used for amplifying undifferentiated hepatocytes.
(9) The medium according to (7), which is used for inducing differentiation of undifferentiated hepatocytes into hepatocytes.
(10) An undifferentiated hepatocyte amplification promoter containing a branched chain amino acid.
(11) An agent for inducing differentiation into hepatocytes, comprising a branched chain amino acid.

  By the culture method and medium provided by the present invention, it becomes possible to efficiently and efficiently create functional cells from undifferentiated organ cells such as tissue stem cells derived from ES / iPS cells. This is expected to become a cell manipulation technique useful for creating a large amount of human hepatocytes necessary for drug discovery screening.

  Furthermore, in order to clinically apply pluripotent stem cells, in addition to obtaining a large amount of cells, pluripotent stem cells before tissue stem cells that cause tumor formation and ectopic tissue formation and cells that have differentiated into other organs It is essential to remove it. By constructing a metabolic environment in which more undifferentiated cells and unintended organ cells cannot survive according to the present invention, only target undifferentiated organ cells can be purified in large quantities at low cost. It is expected. Since there is no need to use complicated techniques such as genetic modification and FACS as in the prior art, it is an inexpensive and simple method with extremely high advantages.

  INDUSTRIAL APPLICABILITY The present invention provides a culture method that can efficiently amplify and sort cells using an amino acid preparation that is less expensive than conventionally used protein preparations.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2013-106289, which is the basis of the priority of the present application.

Extraction of metabolic pathways that exhibit characteristic activities in the early stages of liver development. (A) Comparison of expression levels of genes related to amino acid metabolism in livers at different stages of differentiation by transcriptome analysis. Each amino acid metabolism pathway in the liver cells of embryonic day 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, 19.5 day mouse fetus, birth day 0, day 3 infant and 8 week old adult mouse calculated from transcriptome analysis The expression level of the gene group involved in is shown in the heat map. The color of the heat map indicates that the average value is black, red: an increase of 2 times or more, and green: a decrease of 2 times or more. It shows that Bcat1 is remarkably expressed in the early stage of liver development among several markers. (B) Confirmation of expression of mouse branching difference amino acid metabolizing enzyme (Bcat1) using quantitative RT-PCR. Similar to the results of (A), it is confirmed that the expression of Bcat1 is increased only in the early stage of liver development. (C) Confirmation of Bcat1 expression in early embryonic mouse fetal liver (embryonic day 11.5) by immunohistochemical staining. Bcat1 expression is confirmed in fetal liver stained with Ck8 / 18. Red: Bcat1, Green: Ck8 / 18 (epithelial cell marker), Blue: DAPI. He: Heart, Li: Liver. Comparison of amino acid content in livers at different stages of differentiation by metabolomic analysis. (A) Branching difference amino acid metabolic pathway diagram. Bcat is responsible for the initial stage of metabolism of branched-chain amino acids (BCAA), and branched-chain amino acid metabolites are finally taken into the citric acid cycle and used for energy production. (B) Comparison of amino acid concentrations in liver tissues of embryonic day 11.5 and 19.5 day mouse fetuses and 8-week-old adult mice, calculated from metabolomic analysis. Leu: leucine, Val: valine, Ile: isoleucine, Arg: arginine, Asn: asparagine, Asp: aspartic acid, Cys: cysteine, Glu: glutamic acid, Phe: phenylalanine, Pro: proline, Ser: serine, Thr: threonine. The unit of the vertical axis is nmol / g. It is confirmed that the content of divergent amino acids (Leu, Val, Ile) is low in the liver on embryonic day 11.5. Evaluation of the effect on fetal liver formation when a diet containing no branched-chain amino acids is given to pregnant mice. (A) Embryonic day 13.5 mice and liver images fed with a diet containing no branching difference amino acids from the 8.5th day of pregnancy. (B) Mouse fetal weight, (C) Liver weight, (D) Liver weight ratio to fetal weight. Liver formation is inhibited in mice fed a diet containing no branching difference amino acids. Evaluation of the effect on fetal liver progenitor cells when a valine-free diet was given to pregnant mice. (A) Flow cytometric analysis of embryonic day 13.5 mouse liver cells fed a valine-free diet from day 8.5 of gestation. CD45 / TER119 was used as a blood cell marker, and Dlk1 was used as a hepatic progenitor cell marker. (B) Percentage of Dlk1-positive cells based on (A). Mouse fetuses fed a valine-free diet also have a reduced proportion of hepatic progenitor cells in the liver. Proliferation of mouse liver cells in the early stages of development is enhanced under culture conditions rich in L-valine among branched chain amino acids. (A) A branched chain amino acid (L-valine, L-isoleucine, L-leucine) is added to a medium having a basic composition at a concentration ranging from 0.4 mM to 4 mM, and embryonic day 11 mouse fetal liver-derived cells are added for 6 days. Change in the number of colonies composed of 90 or more after culturing. Cont indicates a group not added with amino acid, and the rate of increase relative to the value of Cont is indicated in%. (B) Branched-chain amino acids (L-valine, L-isoleucine, L-leucine) and non-essential amino acids were added to the basic composition medium at a concentration of 4 mM, and embryonic day 11.5, 13.5, 15.5 mouse embryonic liver-derived cells Change in the number of colonies composed of 90 or more after 6 days of culture. The left is a photograph of a colony on the 6th day of culture when the control group and L-valine were added. The scale bar is 200 μm. On the right is the change in the number of colonies composed of 90 or more after adding each amino acid and culturing mouse fetal liver-derived cells for 6 days. BCAA represents L-valine, L-isoleucine and L-leucine added at 4 mM each, NEAA represents a non-essential amino acid addition group, and the percentage increase relative to the value of Cont is indicated in%. From the top row, data for embryonic day 11.5, embryonic day 13.5, and embryonic day 15.5. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 3-12. Under culture conditions rich in L-valine, differentiation of mouse hepatic stem / progenitor cells into hepatocytes is promoted. (A) An immunostained image of a colony after adding 6 mM of a branched chain amino acid and culturing for 6 days. Red: Albumin (ALB), Green: Cytokeratin (Ck) 7, Blue: DAPI. From the top, no addition group, L-valine, L-isoleucine, L-leucine addition group. (B) Comparison of the abundance of each lineage cell in the colony when branched-chain amino acids were added, calculated from immunostained images. The black part in the pie chart is the ratio of ALB positive and Ck7 negative cells, the gray part is ALB negative and Ck7 positive cells, and the white part is the ratio of ALB / Ck7 co-positive cells. From the top, the results for the non-added group, L-valine, L-isoleucine and L-leucine are shown. (C) Comparison of gene expression levels of ALB and Ck7 in cells after 6 days in culture after adding amino acids to the basal medium. The value is shown as a relative value when the value of the non-addition group is 1. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 4-8. Under culture conditions rich in branched chain amino acids, proliferation of undifferentiated human liver cells and differentiation into hepatocytes are promoted. (A) Schematic diagram of the differentiation induction protocol used in this experiment. As cells at each differentiation stage, Stages 1, 2, 3, and 4 were defined for each time point at which the factors added to the culture system were changed. (B) Comparison of human BCAT1 gene expression levels in human iPS-derived liver cells and human liver cells in each differentiation stage. In the differentiation process of iPS cells, it was confirmed that the expression of BCAT1 in Stage2 was the highest. (C) Proliferative change of human iPS-derived hepatic endoderm cells upon addition of branched chain amino acids. (D) Change in differentiation of human iPS-derived hepatic endoderm cells upon addition of branched chain amino acids. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 4-8. Verification of the effect of addition of branching amino acids in the process of creating human iPS cell-derived liver buds. (A) Outline of experimental protocol. Liver buds were created by co-culturing human iPS cell-derived liver cells (Stage2), human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC). Thereafter, the liver buds were cultured in a medium containing excess branched-chain amino acids. (B) Hepatic bud morphology after 2 weeks of culture. (C) Average radius of liver buds and percentage of hepatic buds with high proliferation. Examination of the effect of increasing cell proliferation of mouse liver cells when the concentration of branched chain amino acids is changed. (A) A branched chain amino acid (L-valine, L-isoleucine, L-leucine) is added to a medium having a basic composition at a concentration ranging from 0.4 mM to 20 mM, and embryonic day 11 mouse fetal liver-derived cells are added for 6 days. Change in the number of colonies composed of 90 or more after culturing. The horizontal axis shows the concentration of each branched chain amino acid added (mM), and the percentage increase relative to the value of the non-added group (0 mM) is shown in%. Error bars are standard errors. N is 3-12.

  Hereinafter, the present invention will be described in detail.

  The present invention provides a method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.

  In the method of the present invention, the concentration of the branched chain amino acid is 1 mM or more, preferably 1 to 20 mM, and more preferably 2 to 10 mM.

  Examples of branched chain amino acids include amino acids (both D and / or L-amino acids) such as valine, leucine, isoleucine and derivatives thereof, and combinations thereof, and valine is preferred.

“Undifferentiated” of undifferentiated hepatocytes refers to a state in which differentiation is not completely completed, and undifferentiated hepatocytes is a concept including all cells that can differentiate into hepatocytes. Examples of undifferentiated hepatocytes include pluripotent stem cells such as iPS cells and ES cells, and undifferentiated organ (eg, liver) cells derived from living tissue. The undifferentiated hepatocytes are preferably in a stage where the expression of BCAT1 is enhanced. Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of the coating agent for the cell culture container include matrigel, laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO ₂ concentration is preferably 2% to 10%, and most preferably 5%.

By culturing undifferentiated hepatocytes in the presence of a branched chain amino acid having a concentration of 1 mM or more, undifferentiated hepatocytes are amplified and differentiation into hepatocytes is induced. Therefore, the present invention also provides a method for inducing differentiation into hepatocytes, including culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of the coating agent for the cell culture container include matrigel, laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO ₂ concentration is preferably 2% to 10%, and most preferably 5%.

  Amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by the method of the present invention can be used for the preparation of hepatocytes. Therefore, the present invention includes amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. A method for preparing hepatocytes is also provided.

  As shown in the Examples below, in the case of mice, when branched chain amino acids are added to liver-derived undifferentiated cells in the early embryonic period (E11.5), they are composed of cells that strongly express albumin, a hepatocyte-specific marker. Appeared frequently. Therefore, in the method of the present invention, undifferentiated hepatocytes cultured in the presence of a branched chain amino acid at a concentration of 1 mM or more are cells at a differentiation stage level corresponding to the period of E11.5 in mice. It is preferable. This differentiation stage is a stage in which a large amount of liver stem / progenitor cells are present. The presence of cells in such a period means that liver differentiation markers (eg, albumin (ALB), α-fetoprotein (AFP), transthyretin (TTR), forkhead box protein A2 (FOXA2), Hepatocyte nuclear factor 4 alpha (HNF4A), retinol binding protein 4 (RBP4), cytochrome P450 (CYP) 3A4, CYP3A7, glucose-6-phosphatase (G6PC), GATA binding protein 4/6 (GATA4 / 6), asialoglycoprotein receptor 1 (ASGR1) , Angiotensinogen (AGT), transferrin (TRF), apolipoprotein A-1 (APOA1), fibrinogen alpha chain (FGA), homogentisic acid (HGD), Carbamoyl-Phosphate Synthase 1 (CPS1) Can be guessed. Specifically, if the expression level of markers such as GATA4 / 6, HNF4A, HHEX1, and AFP is increased compared to ES cells and iPS cells, the level of differentiation stage corresponding to the time of mouse E11.5 It can be assumed that it is a cell. Also, if the expression level of markers such as ALB, RBP4, ASGR1, AGT, TRF, FGA, APOA1, HGD, CPS1 is reduced compared to cells in other differentiation stages, it corresponds to the time of mouse E11.5 It can be assumed that the cells are at the level of differentiation stage. On the other hand, it is also possible to estimate using the expression of BCAT1 as an index. Specifically, if the expression level of BCAT1 is increased as compared with ES cells or iPS cells, it can be assumed that the cells are at the level of differentiation corresponding to the time of mouse E11.5.

In addition, as shown in Examples below, after culturing undifferentiated liver cells derived from mouse fetuses, immunostaining was performed in order to examine the differentiation state, the branched chain amino acid added group was compared with the non-added group, Albumin-positive hepatocytes increased, and the appearance of cytokeratin 7-positive bile duct epithelial cells decreased. Moreover, gene expression analysis also confirmed that cytokeratin 7 gene expression decreased and albumin gene expression increased when branched chain amino acid (particularly L-valine) was added. Therefore, it is considered that the addition of branched chain amino acids can suppress the appearance of bile duct epithelial cells and efficiently create hepatocytes. Therefore, the present invention also provides a method for selecting cells capable of differentiating into hepatocytes, including culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of coating agents for cell culture containers include laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO ₂ concentration is preferably 2% to 10%, and most preferably 5%.

  Furthermore, the present invention provides a medium containing a branched chain amino acid at a concentration of 1 mM or more.

  The medium of the present invention can be used to amplify undifferentiated hepatocytes. The medium of the present invention can be used for inducing differentiation of undifferentiated hepatocytes into hepatocytes. Furthermore, the culture medium of the present invention can also be used to select cells that can differentiate into hepatocytes.

  The medium of the present invention contains amino acids other than branched chain amino acids (glutamine, alanine, arginine, asparagine, cysteine, glutamic acid, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine, and these Amino acids such as derivatives (both D and / or L-amino acids)), phenol red, pyruvate, HEPES, trace metals, phosphates, acetates, vitamins, ascorbic acid, nicotinamide, 2-mercaptoethanol, Dexamethasone, insulin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), activin A, basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP) 4, oncostatin M, hydrocortisone, heparin, blood vessel Endothelial cell growth factor (VEGF), insulin-like growth factor (R3- IGF) -1, bovine brain extract (BBE), fetal bovine serum (FBS), transferrin, bovine serum albumin (BSA), serum replacement (N2, B27 supplement etc.), buffer, antibiotics (gentamicin, penicillin, Other components such as streptomycin, amphotericin-B and the like may be added.

  As the medium of the medium of the present invention, water, serum, pH buffer solution or the like can be used. Alternatively, commercially available media (eg Dulbecco's Modified Eagle Medium (DMEM), E-MEM, IMDM, lactose-containing glucose-free DMEM, Ham F12, RPMI-1640, Williams E, etc., and mixtures thereof A branched chain amino acid may be added to) so as to contain a branched chain amino acid having a concentration of 1 mM or more.

  The undifferentiated hepatocytes have been described above.

  In the present invention, branched chain amino acids can promote amplification of undifferentiated hepatocytes. Therefore, the present invention provides an undifferentiated hepatocyte amplification promoter comprising a branched chain amino acid.

  In addition, branched chain amino acids can induce differentiation from undifferentiated hepatocytes to hepatocytes. Therefore, the present invention provides an agent for inducing differentiation into hepatocytes containing a branched chain amino acid.

  The present invention provides a basic culture technique capable of drastically reducing costs for industrial production of human organ cells by using an inexpensive and simple method of adding an amino acid preparation (branched chain amino acid). By cooperating with a technique developed in the past by the present inventors (“Tissue / Organ Production Method” WO2013 / 047639), it becomes a cell manipulation technique that is extremely useful for regenerative medicine and industrial applications. For example, by efficiently amplifying human iPS cell-derived human hepatocytes according to the present invention, it becomes possible to produce a large amount of mature human hepatocytes necessary for drug development at low cost. In addition, by using a plurality of iPS cell lines established for each individual, it becomes possible to stably supply a large amount of mature human hepatocytes having clear specifications such as race, sex, and individual differences. This is expected to provide an innovative screening technique for detecting differences in individual responsiveness, which was a challenge in drug development.

Hereinafter, the present invention will be described in more detail with reference to examples. In this example, unless otherwise specified, the amino acid is an L-amino acid.
[Example 1]
result
Comparison of metabolic characteristics in livers of different differentiation stages by metabolomic and transcriptome analysis To detect differences in the expression of metabolism-related genes in cells of different differentiation stages, we used mouse liver cells from early embryonic stages to adulthood. Transcriptome analysis was performed. As a result of comparing the expression of representative molecules in each metabolic pathway, the liver in the early developmental stage (embryonic day 9.5 to 11.5) in which undifferentiated hepatic stem / progenitor cells are frequently present is found in the liver in the later developmental stages. In contrast, the expression of many genes involved in major metabolism in the adult liver including lipid metabolism, bilirubin metabolism, and urea cycle was found to be lower (data not shown). On the other hand, some of the genes related to amino acid metabolism showed high expression only in the early stage of liver development, and in particular, it was confirmed that the expression of branched-chain amino acid metabolism gene Branched-chain aminotransferase 1 (Bcat1) was particularly remarkable (Figure 1A). ). Similarly, when the results were analyzed by quantitative PCR, specific expression enhancement in the early stage of liver development was observed (FIG. 1B). Furthermore, when immunohistochemical analysis was performed on the liver in the early stage of development (embryonic day 11.5), high Bcat1 protein expression was confirmed in the liver.

  In order to confirm the functions of amino acid metabolism-related genes, metabolome analysis was used to compare the amount of each metabolite in the liver development process. As shown in FIG. 2A, Bcat1 is an enzyme responsible for the initial metabolic reaction of branched chain amino acids (L-valine, L-leucine, L-isoleucine). As a result of metabolomic analysis, it was found that the concentration of branched chain amino acids (L-valine, L-leucine, L-isoleucine) among amino acids was low in the early stage of liver development (embryonic day 11) (Fig. 2). Based on the above results, we hypothesized that the metabolic activity of branched-chain amino acids is high in undifferentiated liver cells existing in the early stages of liver development, and functionally verified the branched-chain amino acids.

Decreased maternal L-valine intake reduces fetal liver stem / progenitor cell frequency. To verify the effects of branched-chain amino acids on early fetal liver formation, maternal day 8.5 gestation when liver development begins Mice were fed a diet containing no branched-chain amino acids, and the embryos on embryonic day 13.5 were analyzed. As a result, it was confirmed that the liver weight was significantly reduced in the group fed with the diet not containing branched chain amino acids compared to the control group (FIG. 3). This result suggests that branched-chain amino acids play an important role in the early hepatogenesis process. Similarly, inhibition of liver formation was also confirmed in mouse fetuses fed with L-valine-free diet (data not shown).

  Next, in order to evaluate the effects on mouse fetal liver stem / progenitor cells, the L-valine-free diet was given to the 8.5th day of pregnancy, and the liver stem / progenitor cells contained in the embryonic day 13.5 fetal liver Therefore, flow cytometry analysis was performed using Dlk1 as an index. As a result, it was confirmed that the frequency of Dlk1-positive cells was greatly reduced in the group fed with L-valine-free feed compared to the control group (FIG. 4). These results suggest that L-valine deficiency decreases the frequency of hepatic stem / progenitor cells and inhibits the early process of liver development.

Increased proliferation of mouse hepatic stem / progenitor cells in culture conditions rich in L-valine
Regarding the effect of L-leucine, L-isoleucine, L-valine, which are responsible for metabolic reactions of BCAT1, and commercially available amino acid cocktails that are frequently used in conventional culture systems, on the cell growth, mouse fetal liver It examined using the cell. First, the effects of each branched chain amino acid addition were evaluated using undifferentiated liver-derived cells in the early embryonic period (E11.5). When the growth promotion effect when changing the concentration of branched chain amino acids was examined, when the amino acid addition concentration was 0.4 or 0.8 mM, the appearance frequency of highly proliferative colonies composed of 90 or more cells was equivalent to that of the control. On the other hand, when L-valine 4 mM was added, it was found to increase 31% (FIG. 5A). Therefore, the effect of adding 4 mM of each branched chain amino acid to liver-derived undifferentiated cells in the early embryonic period was compared with the amino acid non-added group, the commercially available essential amino acid preparation added group, and the commercially available non-essential amino acid preparation added group. As a result, it was confirmed that the frequency of colonies exhibiting high proliferative activity increased only in the group to which 4 mM L-valine was added compared to the non-added group (FIG. 5B). At this time, there was no difference in the appearance frequency of colonies showing high growth in the group to which 4 mM of other branched chain amino acids (L-isoleucine, L-leucine) and commercially available non-essential and essential amino acid preparations were added. .

  Next, the effect of addition of amino acids on the proliferation of cells was similarly evaluated for E13.5 and E15.5 liver-derived cells, which were more advanced. As a result, unlike the E11.5 liver-derived cells, the appearance frequency of highly proliferative colonies was the same or decreased compared to the control when any branched-chain amino acid solution was added. Based on the above results, the growth-promoting effect of L-valine was not confirmed by analysis using liver cells after the middle embryonic period (E13.5 ~), so only in undifferentiated liver cells existing in the early embryonic period. It was shown that the growth promoting effect by adding L-valine specifically appears.

L-valine-rich culture conditions enhance the differentiation of mouse hepatic stem / progenitor cells into hepatocytes. Next, we examined the effects of L-leucine, L-isoleucine, and L-valine on cell differentiation. . The effect of each branched-chain amino acid addition was compared with the non-amino acid addition group using liver-derived undifferentiated cells in the early embryonic period (E11.5). After culturing for 6 days in a medium supplemented with 4 mM branched-chain amino acid, the differentiation state of hepatic stem / progenitor cells was evaluated by immunostaining. As a result, when L-valine was added, cells that strongly express only albumin, a hepatocyte-specific marker, were found. It was found that the frequency of colonies present in large numbers increased by 23 ± 15% (FIG. 6AB). Furthermore, when gene expression analysis was performed on colonies after culturing, no increase in the expression level of cytokeratin (Ck7), a bile duct epithelium-specific marker, was observed in the L-valine addition group, whereas albumin It was confirmed that the expression level increased by 60 ± 18% (FIG. 6C). On the other hand, when isoleucine was added, albumin expression was reduced by 39%, and when leucine was added, the expression levels of both albumin and Ck7 were increased by 50% and 51%, respectively (FIG. 6C). From the above results, it was clarified that mouse embryonic cells in the early embryonic stage are enhanced to differentiate into hepatocytes when L-valine is abundant in the medium.

Culture conditions rich in L-valine enhance human liver cell proliferation and differentiation into hepatocytes. Next, we observed the effects of L-valine on the proliferation and differentiation promoting effects observed in early mouse liver cells. It was verified whether it was also applicable to human undifferentiated liver cells. First, when comparing the expression of BCAT1 in each differentiation stage using human iPS cell-derived liver cells, the cell corresponding to the differentiation stage in the early stage of liver development (Stage 2 in FIG. 7A) is low in the cells in the stage of more advanced differentiation. It became clear (Fig. 7A). Then, each branched-chain amino acid was added using Stage 2 iPS cell-derived liver cells, which showed the highest expression of BCAT1, and the effect on proliferation was examined. As a result, it was found that the number of cells on the 6th day of culture increased by 11.3 (± 3.1)% when 4 mM L-valine was added compared to the group without branched chain amino acid. At this time, there was no difference in the number of cells after culture in the group to which 4 mM of a commercially available non-essential and essential amino acid preparation was added. From the above results, it has been clarified that cell proliferation is enhanced by adding branched chain amino acids to cells in a differentiation stage where undifferentiated liver cells are frequently present (FIG. 7B). Next, the effect of branched chain amino acids on the differentiation of iPS cell-derived liver cells was examined. Gene expression analysis was performed on cells cultured for 8 days in a medium supplemented with 4 mM branched-chain amino acids. In the L-valine addition group, the expression levels of albumin and RBP4, which are differentiation markers for hepatocytes, were 52 and 52 respectively. Increases of ± 28% and 58 ± 2% were confirmed (FIG. 7C). On the other hand, it was confirmed that the expression of these markers greatly decreased when isoleucine was added. From the above results, it has been clarified that iPS cell-derived hepatocytes enhance proliferation and differentiation into hepatocytes when L-valine is abundant in the medium.

Addition of L-valine enhances cell proliferation in human iPS cell-derived hepatoblasts Differentiated (Stage2) human iPS cell-derived hepatocytes, human umbilical vein endothelial cells, and mesenchymal stem cells By culture, human iPS cell-derived hepatoblasts were created (FIG. 8A). The resulting hepatic bud was further cultured in a medium containing a large amount of a specific branched chain amino acid, and the size was compared. As a result, the hepatic bud cultured in a medium containing a large amount of L-valine was a medium of normal composition. It was confirmed that larger liver buds were created than when cultured in (FIG. 8BC). From the above results, it was confirmed that a medium containing a large amount of L-valine is effective not only in a two-dimensional culture system but also in cells in three-dimensional tissues against human iPS cell-derived hepatic progenitor cells. It was.

  According to the present invention, it is possible to amplify cells at a specific differentiation stage by adding amino acids.

  By subculturing cells at this differentiation stage under valine-added conditions, amplification is possible about 1.5 times as compared with unadded conditions. Undifferentiated liver cells can be repeatedly used for the creation of functional cells by induction of differentiation according to a previous patent (WO2009 / 139419) by repeating subculture while maintaining the properties.

  Therefore, according to the present invention, the fact that 1.5-fold increase in proliferation can be induced in the undifferentiated cells can be expanded thereafter exponentially (power of 1.5), and a large amount of functional cells It becomes a very useful technique to obtain.

Experimental materials and methods
Experimental animals In this experiment, wild type mice (C57BL6 / J) purchased from Japan SLC were used. All animal experiments were conducted with the approval of the committee based on the guidelines for animal experiments at Yokohama City University Fukuura Campus.

Preparation of mouse fetal liver cells After anesthetizing a wild-type pregnant mouse, the fetus was taken out from the abdominal cavity and the fetal liver was collected under a binocular stereomicroscope. The collected fetal liver was immersed in DMEM / F12 (Invitrogen) containing 0.2% trypsin and 5% fetal bovine serum (FBS: ICN), incubated on ice for 30 minutes, and then shaken at 37 ° C. for 15 minutes. Thereafter, the cells were dispersed by gentle pipetting. After centrifugation, the plate was washed twice with DMEM / F12 containing 5% FBS. The obtained fetal cells were passed through a nylon mesh (diameter 40 μm) to collect only single cells and used for the subsequent experiments.

Culture of liver cells Isolated fetal liver cells were seeded on a 6-well plate (BD) coated with Laminin (BD) at 1000 cells / cm ² and cultured. A uniquely prepared medium was used for cell culture. Culture medium is 10% FBS, 1 μg / mL Insulin (Wako), 1 x 10 ^-7 M Dexamethasone (Sigma), 10 mmol / L nicotinamide (Sigma), 2 mmol / L L-glutamine (Invitrogen), 50 mmol / L 2-mercaptoethanol (Invitrogen), 5 mmol / L HEPES (Dojindo) 2.6 x 10 ^-4 mol / L L-Ascorbic acid 2-Phosphate (Sigma), DMEM / F12 medium containing 1 x penicillin / streptomycin (Invitrogen) Is added to the branched chain amino acid solution (isoleucine: Ile, leucine: Leu, valine: Val), or commercially available essential amino acid solution (GIBCO), non-essential amino acid solution (GIBCO). The influence on culture was evaluated. An equal amount of solvent (PBS) was added to the control. After cell seeding, when the cell adhesion is confirmed, the medium is changed, and the medium before replacement is further added with 20 ng / ml Epidermal growth factor (Sigma) and 25 ng / mL Hepatocyte growth factor (Kringle Pharma). The culture was continued. Incubation was performed at 37 ° C. in a 5% CO 2 gas phase.

After removing the immunostaining medium, it was washed with PBS. The cultured cells were fixed by adding 1: 1 acetone / methanol and reacting at −30 ° C. for 10 minutes or in 4% PFA for 10 minutes at room temperature. Fixed cell samples are washed once with PBS containing 0.05% Tweeen20 for 5 minutes and incubated in PBS solution containing 10% immunized animal serum (Goat serum) (Sigma) for 2 hours at room temperature. did. Thereafter, the primary antibody reaction was performed overnight at 4 ° C. After the primary antibody reaction, the plate was washed 3 times for 5 minutes with PBS containing 0.05% Tween 20, and then the secondary antibody was treated at room temperature for 1 hour under light shielding. After the treatment, the plate was washed three times with PBS containing 0.05% Tween 20, sealed with a trend protector (Vector), and observed under an upright fluorescence microscope (Zeiss Axio system). The primary antibodies used were mouse anti-Ck7 antibody (Dako) (1: 100), rabbit anti-Albumin antibody (Biogenesis) (1: 500), and rabbit anti-AFP antibody (MP bio) (1: 200). Secondary antibodies are Alexa488-labeled goat anti-mouse IgG1 antibody (Molecular Probe) (1: 500) and Aelxa555 (Molecular Probe) -labeled goat anti-rabbit IgG antibody (Molecular Probe) (1: 500).

Total RNA non blood cell fraction obtained from fetal, and infant wild-type mice at each developmental stage preparation of (CD45 ^- Ter119 ^-), or the totalRNA recovered from 8-week-old mouse liver which has been subjected to through portal vein manner blood removal operation Then, the expression of the target gene was examined using a quantitative PCR method.

  Non-hemocyte cells were isolated as follows. Biotin-labeled anti-mouse TER119 antibody (PharMingen) and Biotin-labeled anti-mouse CD45 antibody (PharMingen) were added to the liver cells collected from the wild-type mouse liver, and reacted on ice for 20 minutes. A PBS solution containing 3% FBS was added to perform centrifugation and washing. Thereafter, IMag (BD) was reacted on ice for 30 minutes. The cell suspension was suspended in 2 ml of PBS containing EDTA, and CD45 / Ter119 positive cells were removed with a magnet to obtain non-hemocyte cells.

Total RNA was extracted using TRIzol (Invitrogen). When RNA was extracted from the adult liver, TRIzol was added after the tissue pieces were physically damaged in liquid nitrogen. Total RNA extraction using TRIzol was performed according to the WAKO manual. Genomic DNA mixed in the RNA solution was degraded by Deoxyribonuclease I, Amplification Grade (Invitrogen). Thereafter, a reverse transcription reaction was performed in the presence of a random primer using SuperScript ^™ III Reverse Transcriptase (Invitrogen) to obtain a cDNA solution. These operation methods followed the manual attached to Kit.

Quantitative PCR
Quantification was performed using the cDNA solution as a template. The quantitative analysis used the comparative CT method. In the analysis of mouse cells, relative quantification was performed by correcting the amount of cDNA used in the reaction based on the measured value of the GAPDH gene in each sample. In the analysis of human cells, correction was performed based on the expression level of 18S.

Transcriptome analysis
Agilent microarray analysis was performed using the total RNA solution as a template. The array data was normalized using the Grobal normalization method, and the difference in gene expression between specimens was examined. A heat map was created from the obtained data, and the gene expression level at each developmental stage was compared. Extraction of genes related to each metabolic pathway was performed based on Gene Ontology classification.

Metabolome analysis Mouse liver at each developmental stage was collected and metabolome analysis was performed by Human Metabolome Technologies (HMT). A methanol solution containing a mouse liver sample and an internal standard substance of 50 μM was placed in a crushing tube, and crushed using a desktop crusher (bms) under cooling. Chloroform and Milli-Q water were added to this and stirred, followed by centrifugation. After centrifugation, the aqueous layer was transferred to an ultrafiltration tube. This was centrifuged and subjected to ultrafiltration treatment. The filtrate was dried and dissolved in Milli-Q water again for measurement.

  The measurement was carried out in a cation mode and an anion mode of a capillary electrophoresis-time-of-flight mass spectrometer. In this study, analysis was performed on substances registered in the HMT metabolite database.

  The detected peaks were automatically extracted using automatic integration software (MasterHands ver.2.9.0.9), and mass charge cost, migration time and peak area values were obtained as peak information. The obtained peak area value was converted into a relative area value. These data included adduct ions such as Na + and K + and fragment ions such as dehydration and deammonium, so these molecular weight related ions were deleted. For the examined peaks, the peaks between each sample were collated and aligned.

Flow cytometry analysis Add anti-Dlk1 rat antibody (LSBio), PE-labeled anti-CD45 antibody (PharMingen) and PE-labeled anti-TER119 antibody (PharMingen) to liver cells collected from fetal day 13.5 fetuses, and react on ice for 30 minutes I let you. A PBS solution containing 3% FBS was added to perform centrifugation and washing. Thereafter, Alexa647-labeled anti-rat antibody was added as a secondary antibody and allowed to react for 20 minutes. After washing again, the fluorescence intensity of the cells was measured by FACSAria.

Creation of human iPS cell-derived hepatic buds Differentiated human iPS cell-derived liver cells corresponding to hepatic progenitor cells (liver cells differentiated in this laboratory from iPS cells donated by Professor Nakauchi of the University of Tokyo), human Liver buds were created by co-culturing three types of umbilical vein endothelial cells (Lonza) and mesenchymal stem cells (Lonza). Liver buds created by mixing cells at a ratio of 10: 7: 2 and culturing them in a microwell plate are then a medium rich in specific amino acids or a small amount of amino acids (RPMI). And EGM mixed 1: 1) for 2 weeks, and the size was measured. The size of the liver bud was measured using IN Cell Analyzer 2000 based on the fluorescence exhibited by the liver bud.

[Example 2]
In order to clarify the effective concentration of L-valine for promoting cell growth, L-valine was added at various concentrations, and the effect of promoting cell growth on embryonic day 11.5 mouse fetal liver cells was examined. As a result, the cell growth promoting effect was the highest when 4 mM of L-valine was added, and the tendency to promote the growth was observed when the concentration range was 0.8-20 mM (FIG. 9). From these results, it was suggested that the addition of L-valine is effective in enhancing cell proliferation in the range of 0.8-20 mM.

Experimental materials and methods Experiments were performed in the same manner as in Example 1, the collection of mouse fetal liver cells and the culture of liver cells .

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  The present invention can be used as a cell culture technique or a cell manipulation technique for regenerative medicine or drug screening. The present invention leads to the development of a medium preparation for amplifying organ cells, a medium preparation for sorting hepatocytes induced to differentiate from organ cells, and the like.

Claims (8)

A method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of valine at a concentration of 1.0 to 20 mM, wherein the undifferentiated hepatocytes are in the period of E11.5 of a mouse fetus. Said method wherein the cells are at the level of the corresponding differentiation stage. A method for preparing hepatocytes, comprising amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of valine at a concentration of 1.0 to 20 mM. Wherein the undifferentiated hepatocytes are cells at a differentiation stage level corresponding to the E11.5 period of the mouse fetus. In undifferentiated hepatocytes, the expression level of at least one marker selected from the group consisting of GATA binding protein 4/6, Hepatocyte nuclear factor 4 alpha, HHEX1 and α-fetoprotein is compared with ES cells and / or iPS cells. 14. A method according to claim 12 or 13, wherein the method is increasing. In undifferentiated hepatocytes, selected from the group consisting of albumin, retinol binding protein 4, asialoglycoprotein receptor 1, angiotensinogen, transferrin, fibrinogen α chain, apolipoprotein A-1, homogentisic acid and Carbamoyl-Phosphate Synthase 1 14. The method according to claim 12 or 13, wherein the expression level of at least one marker is reduced compared to cells of other differentiation stages. The method according to claim 12 or 13, wherein the expression level of BCAT1 is increased in undifferentiated hepatocytes compared to ES cells and / or iPS cells. By culturing undifferentiated hepatocytes in the presence of valine at a concentration of 1.0 to 20 mM, it is possible to differentiate into hepatocytes, including inhibiting the appearance of bile duct epithelial cells and creating hepatocytes. Cell sorting method. The method according to any one of claims 12 to 17, wherein the concentration of valine is 2 to 10 mM. The method according to claim 18, wherein the concentration of valine is 4 mM.