Patent Application
20120021448
The present application claims priority under Japanese Patent Application 2008-298819, filed on Nov. 21, 2008, the entire contents of which are hereby incorporated by reference.
The present invention relates to a method for evaluating the state of cells, such as a method for evaluating the level of differentiation of stem cells or precursor cells in which differentiation has been induced. More particularly, the present invention relates to a method for evaluating the level of differentiation of stem cells or precursor cells that have been induced to differentiate into cardiomyocytes or neurons.
With the development in cellular engineering and regenerative therapy has come a need for methods of quantitatively and rapidly evaluating the state of cells. Conventionally, methods of evaluating the state of cells based on the expression levels of specific genes or proteins have been widely employed.
An example will be given below. Research on inducing differentiation in stem cells (known as, for example, iPS cells and ES cells) and precursor cells has been conducted since the 1970s. Through the 1990s, discussion of the level of differentiation of cells revolved primarily around the detection of levels of protein expression by flow cytometry, tissue staining with specific antibodies, and the like. Thus, only semi-quantitative information was obtained. However, since 2000, use of the quantitative RT-PCR method has become widespread. The absolute quantitative detection of the transcription of genes specific to differentiation has become possible (Nonpatent Reference 1). However, there are genes that are transcribed but not translated in the course of cell differentiation, and it has been suggested that the gene transcription level in and of itself does not reflect the level of differentiation (Nonpatent Reference 2).
There are also methods of absolute quantification of the level of differentiation by detecting specific proteins released into the medium after differentiation has been induced (Patent Reference 1). However, the deterioration of detection sensitivity is a concern in the detection of specific proteins. In addition, when no suitable secreted protein is present, the introduction of a reporter gene becomes necessary (Patent Reference 2).
As set forth above, tracking the state of a cell—for example, tracking changes in the genes transcribed in the course of cell differentiation—will generally afford good quantitative properties, but will not necessarily reflect the state of the cell (Nonpatent Reference 2).
Additionally, the detection of proteins, which are the products of translation, is a solid basis of evaluation in detecting the state of cells, such as differentiation. However, when no suitable specific antibody exists, or the detection sensitivity is poor, quantitative analysis becomes difficult, compromising general applicability.
Accordingly, the problems to be solved by the present invention are those of discovering a new indicator of the state of cells instead of genes or proteins, such as the level of differentiation; discovering a means permitting the quantitative determination, to the extent possible, of the new indicator; and providing a means of determining the level of differentiation of cells using the new indicator when differentiation is induced in specific cells.
The present invention takes note of the fact that changes in the state of cells, such as changes in the state of differentiation, produce quantitative changes in the sugar chain profile (Nonpatent Reference 4).
The conventional method of classifying sugar chains distinguishes the three types of high mannose, hybrid, and complex based on the biosynthesis pathway. However, the present inventors discovered that, as shown in
Accordingly, in the present invention, changes in the state of cells are clarified by, for example, exhaustively capturing sugar chains from cells before and after differentiation by the high-speed exhaustive sugar chain enrichment technique (glycoblotting method) (Patent Reference 3, Nonpatent Reference 3) developed by Nishimura et al., and clarifying the quantitative expression profile of sugar chains present in individual cells by mass spectrometry, such as MALDI-TOF/MS. On that basis, sugar chains were discovered that were involved in the level of cell differentiation (cell differentiation stages or types) and that varied. That is, a method of identifying sugar chains functioning as differentiation markers and varying sugar chains, and quantitatively determining the level of differentiation with high sensitivity and specificity was established. It was discovered that by introducing the concept of “sugar type” based on the information thus obtained, it became possible to divide the cells into sub-groups, and on that basis, to quantitatively determine the degree of cell differentiation. The present invention was devised on that basis (
Specifically, it was discovered that cell differentiation could be quantitatively determined by the method of dividing sugar chains into the two categories of mannose-type and other (non-mannose-type), and further dividing the non-mannose-type into categories based on (1) the presence or absence of bisects, (2) the number of fucoses (0, 1, or 2 or more), (3) the presence or absence of sialic acid, and (4) the degree of branching (2 or 3 or more)
Based on the present invention, the state of cells, for example, the level of differentiation of precursor cells, stem cells, and the like—that is, their quality—can be quantitatively determined and managed by means of a method exceeding conventional methods in terms of sensitivity and specificity, and affording mutually complementary superiority. By permitting the quantitative measurement with high sensitivity of the level of differentiation of stem cells and the like, quality and safety management systems are enhanced and contributions to regenerative therapy can be anticipated.
The first aspect of the present invention is a method for categorizing sugar chains employed to evaluate the state of cells. This method comprises the following steps:
(1-1) obtaining a quantitative profile of the N-bond-type sugar chains contained in cells before a change in state;
(1-2) obtaining a quantitative profile of the N-bond-type sugar chains contained in cells after a change in state;
(1-3) extracting at least a part of the group of sugar chains in which the quantities present in the two quantitative profiles have varied;
(1-4) dividing the various sugar chains contained in the group of sugar chains extracted based on the type of sugar chain into high mannose-type and non-high mannose type and further dividing the non-mannose type into categories based on (1) the presence or absence of bisects, (2) the number of fucoses (0, 1, or 2 or more), (3) the presence or absence of sialic acid, and (4) the degree of branching (2 or 3 or more); and
(1-5) determining at least one category of sugar chain from among the five categories as being suited to evaluation of the state of the cells.
In addition to the differentiation of stem cells and precursor cells, examples of the change in cells in the method for categorizing sugar chains of the present invention are cell division, morphologic change, cell death, and carcinogenesis. The present invention is not limited thereto. The state of the cell also includes static and dynamic states of cells (time-course changes in state).
Steps (1-1) to (1-5) above will be described in the method for categorizing sugar chains for the evaluation of differentiation levels, which is described below, by referring to an example of the categorizing of sugar chains for the evaluation of differentiation levels.
The second aspect of the present invention is a method for evaluating the state of cells. This is a method for evaluating the state of cells using at least one of the categories of sugar chains determined in the method for categorizing sugar chains in the first aspect of the present invention set forth above.
The method for evaluating the state of cells of the second aspect of the present invention comprises:
(2-1) obtaining quantitative profiles of N-bond sugar chains contained in cells before and after a change in state; and
(2-2) determining the change in the ratio or the quantity of the sugar chain (group) that is present before and after the change in state for the at least one category of sugar chain determined by the method described in claim 1 from the above quantitative profiles.
The method for evaluating the state of cells, the second aspect of the present invention, will be described in the method for evaluating the differentiation levels of stem cells or precursor cells in which differentiation has been induced, which is described below, by referring to an example of the evaluation of the differentiation levels.
The third aspect of the present invention is a method for categorizing sugar chains employed to evaluate the level of differentiation of stem cells or precursor cells in which differentiation has been induced. This method is comprised of the following steps:
(3-1) obtaining a quantitative profile of the N-bond-type sugar chains contained in stem cells or precursor cells cultivated in the absence of a differentiation-inducing agent;
(3-2) obtaining a quantitative profile of the N-bond-type sugar chains contained in stem cells or precursor cells cultivated in the presence of a differentiation-inducing agent;
(3-3) extracting at least a part of the group of sugar chains in which the quantities present in the two quantitative profiles have varied;
(3-4) categorizing the various sugar chains contained in the group of sugar chains extracted based on type of sugar chain into high mannose-type and non-high mannose type and further dividing the non-mannose type into categories based on (1) the presence or absence of bisects, (2) the number of fucoses (0, 1, or 2 or more), (3) the presence or absence of sialic acid, and (4) the degree of branching (2 or 3 or more); and
(3-5) determining at least one category of sugar chain from among the five categories as being suited to evaluation of the level of differentiation of stem cells or precursor cells.
In step (3-1), a quantitative profile of the N-bond-type sugar chains contained in stem cells or precursor cells cultivated in the absence of a differentiation-inducing agent is obtained. In step (3-2), a quantitative profile of the N-bond-type sugar chains contained in stem cells or precursor cells cultivated in the presence of a differentiation-inducing agent is obtained. Steps (3-1) and (3-2) can be successively conducted, or simultaneously conducted in parallel. When conducted successively, either step can be conducted first.
In the present invention, including additional aspects thereof set forth below, the term “stem cell” means a cell having multipotency or pluripotency. Examples of “stem cells” are embryonic stem cells, tissue stem cells, adult stem cells, and induced pluripotent stem cells (iPS cells). However, any cell that is multipotent or pluripotent is included as a stem cell, without limitation to the above examples.
In the present invention, including additional aspects thereof set forth below, the term “precursor cell” means a cell that is obtained by inducing differentiation in the above stem cell and that is pluripotent. When differentiation is induced in a stem cell, depending on the degree of differentiation, there are cells that have a lower degree of undifferentiation than stem cells while still being pluripotent. In the present invention, such cells are collectively referred to as “precursor cells.” “Precursor cells” are not specifically limited other than that they be pluripotent. The P19CL6 and P19C6 cell strains employed in Embodiments 1 and 2 are both precursor cells.
The above “stem cells” and “precursor cells” can be derived from animals such as mammals and fish. Examples of mammals are: humans, mice, rats, sheep, pigs, and monkeys. Examples of fish are: killifish and zebrafish. However, they are not limited to these examples.
In the third aspect of the present invention, a known method of culturing stem cells or precursor cells can be used as the method of culturing stem cells or precursor cells in the presence or absence of a differentiation-inducing agent. The differentiation-inducing agent is not specifically limited, and can be suitably selected based on the type of stem cell or precursor cell. However, since the first aspect of the present invention is a method for categorizing sugar chains for use in evaluating the level of differentiation of stem cells or precursor cells, the use of a differentiation-inducing agent that is capable of highly efficiently and reliably inducing differentiation is desirable from the perspective facilitating sugar chain categorization. However, there are cases where the differentiation level (the differential stage or type of the cell) varies with the type of differentiation-inducing agent. Thus, the use of a differentiation-inducing agent that is capable of reliably inducing differentiation is not always called for; different differentiation-inducing agents can be used based on the differentiation level being targeted. A single differentiation-inducing agent or a combination of such agents can be employed in culturing. The concentration of the differentiation-inducing agent in the culture (medium) can also be suitably selected.
The cultured stem cells or precursor cells are subjected to processing to separate and purify the N-bond-type sugar chains contained in the cells. Quantitative profiles of the separated and purified N-bond-type sugar chains are suitably obtained. By way of example, the N-bond-type sugar chains are desirably separated and purified by the glycoblotting method described in Patent Reference 3 and Nonpatent Reference 3 from the perspectives of rapid separation and purification without loss of N-bond-type sugar chains. Quantitative profiling is suitably conducted to a certain degree or higher of differentiation of stem cells or precursor cells that have been cultured with a differentiation-inducing agent, that is, to a degree where the progress of differentiation is evident. However, the degree at which the progress of differentiation becomes evident varies with the type of cell, type of differentiation-inducing agent, culturing conditions, and the like. Thus, a suitable determination is made taking these conditions into account.
The quantitative profiles are desirably obtained for separated, purified N-bond-type sugar chains by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) or liquid chromatography-electrospray ionization/sonic spray ionization-time of flight/mass spectrometry (LC-ESI/SSI-TOF/MS). N-bond-type sugar chains can be rapidly quantified with high precision and quantitative profiles can be obtained through the use of MALDI-TOF/MS or LC-ESI/SSI-TOF/MS.
In step (3-3), at least a part of the group of sugar chains in which the quantities present in the two quantitative profiles obtained in steps (3-1) and (3-2) have varied is extracted. In step (3-4), the various sugar chains contained in the group of sugar chains extracted based on type of sugar chain are categorized into high mannose-type and non-mannose type and the non-mannose type is further divided into categories based on (1) the presence or absence of bisects, (2) the number of fucoses (0, 1, or 2 or more), (3) the presence or absence of sialic acid, and (4) the degree of branching (2 or 3 or more).
As a result of experimentation, the present inventors discovered that these five categories of sugar chains could be used to evaluate the differentiation levels of stem cells and precursor cells.
There are cases in which the group of sugar chains in the quantitative profiles differs with the type of cell and the direction and degree of differentiation. However, it is normally comprised of about several tens to several hundred sugar chains. Of these, at least a part of the group of sugar chains in which the quantity present varies based on the differentiation level is extracted, and the group of sugar chains that is extracted is divided into five categories based on the above types of sugar chain.
In step (3-5), at least one category of sugar chain from among the five categories is determined to be suited to evaluation of the state of the cells. For example, the category of sugar chain that is suited to the evaluation of the state of the cells can be one such that time-course variation in the quantity present or the ratio present is not significant in stem cells or precursor cells that have been cultured in the absence of a differentiation-inducing agent, but time-course variation in the quantity present or the ratio present is significant in stem cells or precursor cells that have been cultured in the presence of a differentiation-inducing agent.
The category of sugar chain that is selected and determined here can be used as an evaluation index of the level of differentiation in the method for evaluating the level of differentiation of stem cells or precursor cells in which differentiation has been induced as set forth below.
[The Method for Evaluating the Level of Differentiation of Stem Cells or Precursor Cells in which Differentiation has been Induced]
The fourth aspect of the present invention is a method for evaluating the level of differentiation of stem cells or precursor cells in which differentiation has been induced. This method comprises the following steps:
(4-1) obtaining time-course quantitative profiles of N-bond-type sugar chains contained in stem cells or precursor cells that have been cultured in the presence of a differentiation-inducing agent; and
(4-2) determining from the quantitative profiles the time-course change in the present quantity or present ratio of the sugar chains (groups) contained in at least one of the categories of sugar chain determined by the method of the third aspect of the present invention.
In step (4-1), the time-course quantitative profiles of N-bond-type sugar chains contained in stem cells or precursor cells that have been cultured in the presence of a differentiation-inducing agent can be obtained in the same manner as in step (3-2) above. However, the time-course quantitative profiles is determined in step (4-1). Specifically, the cultured product is sampled over the course of the culturing period, and quantitative profiles are obtained for N-bond-type sugar chains that have been separated and purified from the sampled cultured product. The time-course quantitative profiles that are obtained are obtained at least once, desirably two or more times, and for example, two, three, or four times, following the start of culturing. They can also be obtained more than four times.
In step (4-2), the time-course change in the quantity or ratio of sugar chains (groups) present in the quantitative profiles is determined for at least one of the categories of sugar chain determined by the method of the third aspect of the present invention.
A sugar chain type (sugar chain category) that can be employed as a differentiation marker in the method of evaluating the level of neuron differentiation and in the method of evaluating the level of cardiac muscle differentiation described further below was discovered by means of the sugar chain category-determining method of the third aspect of the present invention. The details are given in the embodiments. However, it was discovered that at least one member selected from the group consisting of the group of sugar chains having one fucose and the group of sugar chains having two fucoses could be employed as a differentiation marker in the method of evaluating the level of differentiation of cardiac muscle. Further, it was discovered that at least one member selected from the group of sugar chains having a bisect structure and the group of sugar chains not having a bisect structure could be employed as a differentiation marker in the method of evaluating the level of differentiation of neurons.
The method for evaluating the level of differentiation using the above differentiation markers will be described below.
The fifth aspect of the present invention is a method for evaluating the level of differentiation of stem cells or precursor cells into cardiac muscle.
This method comprises:
(5-1) obtaining a time-course quantitative profile of N-bond-type sugar chains contained in stem cells or precursor cells cultured in the presence of a cardiac muscle differentiation-inducing agent; and
(5-2) determining a time-course change in at least one member selected from the group consisting of the quantity or the ratio of the group of sugar chains having one fucose, the quantity or the ratio of the group of sugar chains having two fucoses, and the quantity or the ratio of the group of sugar chains of the high mannose type present in the quantitative profile.
Examples of cardiac muscle differentiation-inducing agents include dimethylsulfoxide (DMSO) and oxytocin. The time-course quantitative profile of N-bond-type sugar chains can be obtained in the same manner as in step (3-2) in the third aspect set forth above. However, the differentiation-inducing agent employed is a cardiac muscle differentiation-inducing agent.
The stem cells or precursor cells employed in the method of the present invention are the same as those employed in the first aspect. In particular, P19CL6 cells, ES cells, iPS cells, and the like are preferred.
At least one member selected from the group consisting of the quantity or the ratio of the group of sugar chains having one fucose, the quantity or the ratio of the group of sugar chains having two fucoses, and the quantity or the ratio of the group of sugar chains of the high mannose type present in the quantitative profile is determined. In the present invention, the level of differentiation of cardiac muscle can be evaluated based on the quantity or ratio of any of the group of sugar chains present. However, it is desirably collectively evaluated based on the quantities or ratios of all three of these groups of sugar chains that are present.
An example of a group of sugar chains having one fucose is Chem. 3, Chem. 19, and Chem. 44 in Table 1.
An example of a group of sugar chains having two fucoses is Chem. 20, Chem. 41, and Chem. 49 in Table 1.
An example of a group of sugar chains of the high mannose type is Chem. 5, Chem. 8, and Chem. 14 in Table 1.
As indicated in Embodiment 1, cells presenting a quantitative profile in which the quantity or ratio of the group of sugar chains having one fucose that is present increases with culturing are cells that have undergone an increase in the level of differentiation of cardiac muscle. Similarly, cells presenting a quantitative profile in which the quantity or ratio of the group of sugar chains having two fucoses is decreased by culturing are cells that have undergone an increase in the level of differentiation of cardiac muscle.
The sixth aspect of the present invention is a method for evaluating the level of differentiation of stem cells or precursor cells derived from mice into neurons.
This method comprises:
(6-1) obtaining a time-course quantitative profile of N-bond-type sugar chains contained in stem cells or precursor cells cultured in the presence of an agent inducing differentiation into neurons; and
(6-2) determining a time-course change in at least one member selected from the group consisting of the quantity or the ratio of the group of sugar chains having a bisect structure, the quantity or the ratio of the group of sugar chains not having a bisect structure, present in the quantitative profile.
Examples of agents inducing differentiation into neurons include retinoic acid and DMSO. The time-course quantitative profile of N-bond-type sugar chains can be obtained in the same manner as in step (3-2) of the first aspect. However, the differentiation-inducing agent employed is an agent inducing differentiation into neurons.
The stem cells or precursor cells employed in the method of the present invention are the same as those employed in the first aspect. In particular, P19 cells, P19C6 cells, ES cells, iPS cells, and the like are preferred.
At least one member selected from the group consisting of the quantity or the ratio of the group of sugar chains having a bisect structure, the quantity or the ratio of the group of sugar chains not having a bisect structure, and the quantity or ratio of the group of sugar chains of the high mannose type present in the quantitative profile is determined. In the present invention, the level of differentiation into neurons can be evaluated based on the quantity or ratio of any one group of sugar chains present, but is desirably collectively evaluated based on the quantities or ratios of all three groups of sugar chains present.
The term “bisect structure” refers to the structure of a sugar chain having a bisect GlcNAc. When a bisect GlcNAc is added to the structure of the skeletal sugar chain, the extension of further branch divisions and the severing of sugar chains are known to cease. The reaction that adds a bisect GlcNAc to the sugar chain structure is catalyzed by an enzyme known as GnT-III (Ikeda, Y., et al., Trends in Glycoscience and Glycotechnology 13: 167-176, the entire contents of which are hereby incorporated by reference).
An example of a group of sugar chains having a bisect structure is: Chem. 15, Chem. 45, and Chem. 68 in Table 2.
An example of a group of sugar chains not having a bisect structure is: Chem. 17, Chem. 21, and Chem. 71 in Table 2.
An example of a group of sugar chains of the high mannose type is the same as that given by way of example for the fifth aspect above.
As indicated in Embodiment 2, in mouse precursor cells, cells in which the quantity or ratio of the group of cell chains having a bisect structure that is present increases with culturing are cells in which the level of differentiation into neurons has increased. Cells in which the quantity or ratio of the group of cell chains not having a bisect structure that is present decreases with culturing are cells in which the level of differentiation into neurons has increased. And cells in which the quantity or ratio of the group of cell chains of the high mannose type that is present decreases with culturing are cells in which the level of differentiation into neurons has increased.
As indicated in Embodiment 3, in mouse ES cells, cells in which the quantity or ratio of the group of cell chains having a bisect structure that is present increases with culturing are cells in which the level of differentiation into neurons has increased. Cells in which the quantity or ratio of the group of cell chains not having a bisect structure that is present decreases with culturing are cells in which the level of differentiation into neurons has increased. And cells in which the quantity or ratio of the group of cell chains of the high mannose type that is present decreases with culturing are cells in which the level of differentiation into neurons has increased.
[The Method for Evaluating the Level of Differentiation into Neurons (2)]
The seventh aspect of the present invention is a method for evaluating the level of differentiation of stem cells or precursor cells derived from humans into neurons.
This method comprises:
(7-1) obtaining a time-course quantitative profile of N-bond-type sugar chains contained in stem cells or precursor cells cultured in the presence of an agent inducing differentiation into neurons; and
(7-2) determining a time-course change in at least one member selected from the group consisting of the quantity or the ratio of the group of sugar chains having one fucose, the quantity or the ratio of the group of sugar chains not having sialic acid, and the quantity or ratio of the group of sugar chains of the high mannose type present in the quantitative profile.
(7-1) As in step (6-1), examples of agents inducing differentiation into neurons include retinoic acid and DMSO. A time-course quantitative profile of N-bond-type sugar chains can be obtained in the same manner as in step (3-2) of the first aspect. However, the differentiation-inducing agent employed is an agent inducing differentiation into neurons.
The human-derived stem cells or precursor cells employed in the method of the present invention are the same as those indicated in the first aspect above. In particular, ES cells, iPS cells, and the like are preferred.
Time-course change in at least one member selected from the group consisting of the quantity or the ratio of the group of sugar chains having one fucose, the quantity or the ratio of the group of sugar chains not having sialic acid, and the quantity or ratio of the group of sugar chains of the high mannose type present in the quantitative profile is determined. In the present invention, the level of differentiation into neurons can be evaluated based on the quantity or ratio of any one group of sugar chains present, but is desirably collectively evaluated based on the quantities or ratios of all three groups of sugar chains present.
Examples of sugar chains having one fucose, sugar chains not having sialic acid, and sugar chains of the high mannose type are the sugar chains indicated in Table 4 of Embodiment 4.
As indicated in Embodiment 4, in human ES cells, cells in which the quantity or ratio of the group of sugar chains having one fucose that is present increases with culturing are cells in which the level of differentiation into neurons has increased. Cells in which the quantity or ratio of the group of sugar chains not having sialic acid (Non-SA) that is present increases with culturing are cells in which the level of differentiation into neurons has increased. And cells in which the quantity or ratio of the group of sugar chains of the high mannose type that is present decreases with culturing are cells in which the level of differentiation into neurons has increased.
In the fifth through seventh aspects, attention is paid in the embodiments to the tendency of the quantity or ratio of the various groups of sugar chains present to time-course change. However, by obtaining and gathering information relating to the relation between the stage or type of differentiation and the time-course change of the quantities or ratios of the various groups of sugar chains that are present, it is possible to quantitatively determine the stage and type of differentiation from the quantity or ratio of the various groups of sugar chains that are present. For example, when employing differentiating cells in regenerative therapy, it is desirable in terms of safety not to incorporate cells that are undifferentiated. Accordingly, it is important to correctly determine the stage of differentiation of the cells obtained by culturing. It is also important to confirm that the cells are differentiated into the targeted type, and thus, to correctly determine the type of differentiation. The method of the present invention permits quantitative determination of the stage and type of differentiation from the quantities or ratios of the groups of sugar chains that are present.
The present invention will be described in greater detail below through embodiments.
The cell glycomics employed in the embodiments were implemented according to the following protocols.
In 10 mM EDTA/PBS, a cell scraper is used to recover cells from a dish. The cells are washed with PBS.
On percent (final concentration) of TritonX-100 is added, the cells are incubated for one hour on ice, and the cells are solubilized.
Eighty percent (final concentration) of acetone is added and incubation is conducted over night at −20° C. to cause the protein fraction to precipitate.
Following centrifugation, the precipitate is washed with acetonitrile and the acetone is completely removed.
The precipitate is blown dry, 50 μL of 80 mM ammonium bicarbonate containing 0.2% PHM (1-propanesulfonic acid, 2-hydroxyl-3-myristamido) is added, and the mixture is incubated at 60° C. for 10 minutes and solubilized.
DTT is added to a final concentration of 10 mM to the protein fraction following solubilization, and the mixture is reduced at 60° C. for 30 minutes.
To this is added 20 mM iodoacetamide and the mixture is incubated for 30 minutes at room temperature while being shielded from light to alkylate the amino acid residue reduced in 6.
800 U of trypsin is added and the mixture is incubated over night at 37° C.
The trypsin is activated for 10 minutes at 90° C., 2 U of peptide N-glycanase F is added, and N-bond-type sugar chains were released.
An internal standard in the form of 15 pmol of A2 amide glycan is added to the sugar chains that had been released, after which the mixture is concentrated in a SpeedVac and adjusted to a final volume of 20 μL.
The N-bond-type sugar chains obtained in 10. above were enriched by the glycoblotting method using a BlotGlyco(R) H (Sumitomo Bakelite Co., Ltd.). This method was implemented in accordance with the methods described in the following references. The specific method is described in the references cited below. The following four references are hereby incorporated in their entirety by reference.
N-bond-type sugar chains, which have been released with peptide N-glycanase F, to which 15 pmol of A2 amide glycan has been added as an internal standard, which has been concentrated with a SpeedVac, and the final volume of which has been adjusted to 20 μL, are added to 5 mg of BlotGlyco(R) H beads.
A 180 μL quantity of acetonitrile containing 2% of acetic acid is added to 1.
The mixture is left standing at 80° C. for 45 minutes.
The beads are washed twice with 200 μL each of 16.6 mM ammonium bicarbonate solution containing 2 M guanidine-hydrochloric acid, pure water, and a 1% triethylamine methanol solution.
A 100 μL quantity of a methanol solution containing 10% acetic anhydride is added and the mixture is left standing at room temperature for 30 minutes.
After removing the solution, the beads are washed twice with 200 μL each of 10 mM hydrochloric acid, methanol, and dioxane.
A 100 μL quantity of dioxane solution containing 100 mM 3-methyl 1-p-tolyltriazene is added and the mixture is left standing at 60° C. for an hour to an hour and a half.
The mixture is sequentially washed with 200 μL each of dioxane, pure water, methanol, and pure water.
A 180 μL quantity of acetonitrile containing 2% acetic acid and 20 μL of 20 mM aminooxy-WR is added.
The mixture is left standing at 80° C. for 45 minutes.
The mixture is eluted from 100 μL of pure water.
Differentiation into cardiomyocytes was induced in P19CL6 cells derived from mouse embryonal carcinoma cells, and glycomixes of undifferentiated and differentiated cells were prepared.
Mouse embryonal carcinoma cell-derived P19C16 cells were inoculated onto DMEM medium containing 10% bovine fetal serum to 3.7×105 cells/6 cm dish. To the group in which differentiation was being induced was added 1% dimethylsulfoxide (DMSO) and culturing was conducted. The control was cultured as is, without addition. On day 16 following the start of culturing, pulsation of the entire differentiation-induced group was confirmed and cells were collected from both groups. The above glycoblotting method was used to capture and purify all the N-bond-type sugar chains in the cells. A quantity of total cell protein corresponding to 200 μg was analyzed, and a quantitative profile was obtained by MALDI-TOF/MS. The expression quantities of the various sugar chains shown in
Immunocyte staining was conducted under the following conditions on cells in which differentiation had been induced and on cells that had been cultured without inducing differentiation. The results are given in
Primary antibody: MF20 (mouse monoclonal anti-sarcomeric myosin)
Secondary antibody: HRP-polymer conjugated anti-IgG
Color-developing substrate: Diaminobenzidine (DAB)
Pulsation was confirmed (by visual evaluation) in cells in which differentiation had been induced and in cells that had been cultured without inducing differentiation. As a result, pulsation was not observed in the group in which differentiation had not been induced, but was observed in about 80% of the cells in the group in which differentiation had been induced.
Of the 42 sugar chains in which significant variation was observed, the 11 sugar chains having one Fuc, the six sugar chains having two Fucs, and the sugar chains of the high mannose type are indicated below.
The group of sugar chains having one fucose is: Chem. 2, Chem. 3, Chem. 6, Chem. 10, Chem. 12, Chem. 15, Chem. 16, Chem. 19, Chem. 24, Chem. 25, Chem. 27, Chem. 30, Chem. 31, Chem. 33, Chem. 36, Chem. 37, Chem. 39, Chem. 40, Chem. 41, and Chem. 42 in Table 1.
The group of sugar chains having two fucoses is: Chem. 20, Chem. 22, Chem. 28, Chem. 29, Chem. 34, Chem. 35, and Chem. 38 in Table 1.
The sugar chains of the high mannose type are: Chem. 5, Chem, 8, Chem. 14, and Chem. 17.
Mouse embryonic carcinoma P19C6 cells were induced to differentiate into neurons and glycomixes were prepared for three steps of undifferentiated, intermediate differentiation, and differentiated cells.
P19C6 cells were inoculated and cultured for several days in a 15% FBS/DMEM medium in a cell culture dish. When 80 to 90% confluence was reached, the cells were recovered and employed to prepare a glycomix or used in step 2 for the induction of differentiation.
Cells that had reached 80 to 90% confluence in step 1 were processed with trypsin to prepare a cell suspension and hanging drop cultured in a bacteria grade Petri dish. At the time, 100 spots of 10 μL cell suspension (105 cells/mL) were applied to the cover of a 10 cm dish and float-culturing was conducted to form lumps of cell aggregation. Ten percent FBS/αMEM to which 1 μM retinoic acid had been added to induce differentiation was employed as the medium. The cell aggregates were collected after four days. They were employed to prepare a glycomix and in step 3 to induce differentiation.
The recovered lumps of cell aggregate were processed with trypsin to prepare a cell suspension. The cells were inoculated to 5×104 cells/cm2 on a 6 cm dish coated with polylysine and adhesion cultured. DMEM/F12 to which N-2 supplement and fibronectin had been added was employed as the medium. The cells were recovered after five days and used to prepare a glycomix.
Proteins in the pellets of the recovered cells were solubilized with 1% Triton/PBS. The equivalent of 200 μg of protein was precipitated from acetone, reduced/alkylated, and digested with trypsin. Subsequently, the N-bond-type sugar chains were released with PNGaseF, glycoblotting was used to capture and purify the sugar chains, and a quantitative profile was obtained by MALDI-TOF/MS. The expression levels of the sugar chains were quantitatively analyzed by comparing the areas of the signals derived from the sugar chains expressed by the cells to the area of the signal derived from an oligosaccharide of known concentration that was added as an internal standard in the spectra of the undifferentiated, intermediate differentiation, and differentiated cells (
Immunocyte staining was conducted under the following conditions on cells in which differentiation had been induced and on P19C6 cells in an undifferentiated state. The results, as given in
Primary antibody: mouse monoclonal anti-neurofilament 160
Secondary antibody: HRP-polymer conjugated IgG
Color-developing substrate: Diaminobenzidine (DAB)
The sugar chains having bisect GlcNAc, the sugar chains not having bisect GlcNAc, and the sugar chains of the high mannose type were as follows.
The group of sugar chains having a bisect structure is: Chem. 15, Chem. 22, Chem. 23, Chem. 30, Chem. 36, Chem. 37, Chem. 38, Chem. 44, Chem. 45, Chem. 46, Chem. 48, Chem. 53, Chem. 55, Chem. 56, Chem. 60, Chem. 61, Chem. 64, and Chem. 67 in Table 2.
The group of sugar chains not having a bisect structure is: Chem. 6, Chem. 8 to 10, Chem. 12 to 14, Chem. 17 to 21, Chem. 25 to 29, Chem. 32 to 35, Chem. 40 to 43, Chem. 47, Chem. 50 to 52, Chem. 54, Chem. 57 to 59, Chem. 62, Chem. 63, Chem. 65, and Chem. 66 in Table 2.
The group of sugar chains of the high mannose type is Chem. 5, Chem. 7, Chem. 11, Chem. 16, Chem. 24, Chem. 31, and Chem. 39 in Table 2.
Differentiation of mouse ES cells was induced with retinoic acid, and glycomixes of undifferentiated and differentiated cells were prepared. The induction of differentiation and the quantitative analysis of the levels of sugar chains expressed were conducted in the same manner as in Embodiment 2. The results of the quantitative analysis of the levels of sugar chains expressed are given in
The sugar chains having bisect GlcNAc, the sugar chains not having bisect GlcNAc, and the sugar chains of the high mannose type were as follows.
The group of sugar chains having a bisect structure is: Chem. 29, Chem. 37, Chem. 38, Chem. 58, Chem. 59, Chem. 60, and Chem. 67 in Table 3.
The group of sugar chains not having a bisect structure is: Chem. 4, Chem. 7, Chem. 8, Chem. 11, Chem. 13, Chem. 16, Chem. 18, Chem. 19, Chem. 20, Chem. 26, Chem. 27, Chem. 31, Chem. 32, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem. 40, Chem. 41, Chem. 42, Chem. 43, Chem 44, Chem. 45, Chem. 46, Chem. 47, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 69, Chem. 70, Chem. 71, and Chem. 72 in Table 3.
The group of sugar chains of the high mannose type is: Chem. 12, Chem. 17, Chem. 22, Chem. 30, Chem. 39, Chem. 51, and Chem. 61 in Table 3.
Differentiation was induced in human ES cells by adding retinoic acid and glycomixes were prepared for undifferentiation prior to the addition of retinoic acid and differentiation following addition. The induction of differentiation and the quantitative analysis of the levels of sugar chains expressed were conducted in the same manner as in Embodiment 2. The results of the quantitative analysis of the levels of sugar chains expressed are given in
The sugar chains having one fucose, the sugar chains not having sialic acid, and the sugar chains of the high mannose type were as follows.
The group of sugar chains having one fucose is: Chem. 6, Chem. 11, Chem. 13, Chem. 17, Chem. 19, Chem. 32, Chem. 36, Chem. 37, Chem. 39, Chem. 44, Chem. 47, Chem. 50, Chem. 51, and Chem. 52 in Table 4.
The group of sugar chains not having sialic acid is: Chem. 6, Chem. 7, Chem. 8, Chem. 11, Chem. 13, Chem. 14, Chem. 17, Chem. 19, Chem. 20, Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem. 39, Chem. 42, Chem. 44, Chem. 46, Chem. 47, Chem. 49, and Chem. 52 in Table 4.
The group of sugar chains of the high mannose type is: Chem. 12, Chem. 18, Chem. 22, Chem. 26, Chem. 31, Chem. 40, and Chem. 48 in Table 4.
The present invention is useful in the fields of manufacturing and developing pharmaceuticals and regenerative therapy relating to, for example, the differentiation of stem cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-298819 | Nov 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/006293 | 11/20/2009 | WO | 00 | 8/1/2011 |
