JP2010200676A - Method for sorting multipotent stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently and simply sorting multipotent stem cells maintaining pluripotent differentiation. <P>SOLUTION: The method includes: sorting cells maintaining pluripotent differentiation from a cell group regarded to have pluripotent differentiation using expression of E-cadherin or E-cadherin and c-Kit as an index; and preparing multipotent stem cells. Further, the method includes collecting candidate cells of multipotent stem cells on the basis of cell size, cell density, cell autofluorescence and/or cell life and death; sorting cells maintaining pluripotent differentiation from candidate cells of the collected multipotent stem cells using expression of E-cadherin and c-Kit on the cell surface and preparing multipotent stem cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for selecting pluripotent stem cells.

A cell having pluripotency is called a pluripotent stem cell, etc., and retains the ability to differentiate into all organs and tissues constituting the living body. It is expected as an extremely effective means. At first, as a cell having differentiation pluripotency, attention has been paid to the pluripotency of ES cells (embryonic stem cells), and vigorous research has been conducted on its utility in the medical field or basic research field. It was. However, since ES cells are obtained from embryos that are the origin of life, ethical problems have been encountered in their use. In addition, tissues prepared from ES cells may cause rejection at the stage of transplantation, and it has been necessary to overcome such immunological problems. Therefore, iPS cells (induced pluripotent stem cells) are recently used as differentiated pluripotent cells that can solve ethical problems and immunological problems that may occur when ES cells are used. Many expectations have been raised.
Mouse iPS cells were first established by Yamanaka et al. By introducing four genes Oct3 / 4, Sox2, Klf4, and c-Myc into mouse fibroblasts using the expression of Nanog gene as an index. Furthermore, regarding human iPS cells, Thomson et al. Established human iPS cells by introducing OCT3 / 4, SOX2, Nanog, LIN28 into human fibroblasts, and Yamanaka et al. SPS2, KLF4, and C-MYC were introduced into human fibroblasts to establish iPS cells.

Although iPS cells are ideal pluripotent cells that have overcome the ethical and immunological problems of ES cells, they are artificially produced, so they are safe (for example, cancer) Further research is needed. The ES cell still has an important position as a research object because it is a cell having inherent pluripotency while including the above problems.
As described above, in the current situation where opportunities for studying pluripotent stem cells such as ES cells and iPS cells are expected to increase more and more, cells having the original properties of pluripotent stem cells can be rapidly It is one of the important issues to obtain a simple method.
ES cells and iPS cells seem to form a uniform cell population at first glance. For example, ES cells are divided into several fractions depending on the expression level of membrane surface antigens and transcription factors. Has been suggested. In addition, it has been found that undifferentiated colonies generated after gene introduction in the process of establishing iPS cells also include colonies generated by incomplete initialization. Therefore, preparing a cell population that retains the same differentiation pluripotency from such a heterogeneous cell population is important in promoting future research and development.

  So far, regarding the use of a marker for identifying the undifferentiation of pluripotent stem cells, for example, c-Kit positive cells are selected from chorionic villi, amniotic fluid, placenta, etc. to prepare pluripotent fetal stem cells Method (Patent Document 1), a method of preparing pluripotent stem cells by selecting p75NTR and c-Kit positive cells from adipose tissue, etc. (Patent Document 2), and testicular cells as glial cell-derived neurotrophic factor (GDNF) It has been reported that pluripotent stem cells can be obtained using the expression of markers such as SSEA-1, CD9, c-Kit as a clue (Patent Document 3). There is a need for markers that enable the preparation of specific pluripotent stem cells.

WO2003 / 042405 JP 2006-230235 A WO2005 / 100548

To date, many methods for preparing ES cells and iPS cells have been reported as pluripotent stem cells that retain differentiation pluripotency, but some of the cell populations prepared by these methods are not yet available. Not a few cells that have already lost their differentiation state are included, which may be an obstacle to efficient differentiation induction.
Accordingly, an object of the present invention is to provide a method for efficiently preparing pluripotent stem cells by selecting cells that maintain pluripotency.

As a result of intensive studies, the present inventors have succeeded in efficiently selecting cells that maintain pluripotency using the expression of E-cadherin and c-Kit as an index, and completed the present invention. I let you.
So far, there has been reported a method for selecting cells that maintain differentiation pluripotency using the expression of c-Kit on the cell surface as an index, but when only c-Kit is used as an index, It was difficult to prevent contamination with c-Kit positive cells (eg, germ cells, hematopoietic cells, etc.) other than pluripotent stem cells. Therefore, the inventors succeeded in removing the contaminated cells by adding the expression of E-cadherin on the cell surface in addition to c-Kit as an index for selecting pluripotent stem cells.
Therefore, the present invention selects cells that maintain pluripotency from the population of cells that are said to have pluripotency, using the expression of E-cadherin and c-Kit on the cell surface as an index. A method for preparing pluripotent stem cells is provided.
Furthermore, the present inventors sort out candidate cells for pluripotent stem cells based on differences in cell size, cell density, cell autofluorescence, and / or cell viability. If cells expressing E-cadherin and c-Kit on the cell surface are selected from candidate pluripotent stem cells, E-cadherin or c-Kit expressing cells other than pluripotent stem cells and / or death It has been found that cells can be effectively removed and pluripotent stem cells can be prepared more efficiently.
Therefore, the present invention sorts pluripotent stem cell candidate cells based on cell size, cell density, cell autofluorescence and / or cell viability, and the sorted pluripotent stem cell candidates. Provided is a method for preparing pluripotent stem cells by selecting cells that maintain differentiation pluripotency from cells, using the expression of E-cadherin and c-Kit on the cell surface as an index.
The present invention also provides a kit and apparatus for carrying out the above-described method of the present invention.

That is, it relates to the following (1) to (15) of the present invention.
(1) A first aspect of the present invention is “a method for preparing pluripotent stem cells by selecting E-cadherin positive and c-Kit positive cells from a cell population”.
(2) According to a second aspect of the present invention, “the cell population is classified as a candidate cell for a pluripotent stem cell, using cell size, cell density, cell autofluorescence and / or cell viability as an index. “The method according to (1) above”.
(3) A third aspect of the present invention is “the method according to (2) above, wherein the sorting of the candidate cells is performed by a flow cytometer”.
(4) A fourth aspect of the present invention is “the method according to (3) above, wherein the sorting is performed by setting gates for forward scattered light and side scattered light”.
(5) A fifth aspect of the present invention is “the method according to (3) or (4) above, wherein the sorting is performed by setting a gate for fluorescence”.
(6) A sixth aspect of the present invention is “the method according to any one of (3) to (5) above, wherein the fractionation is performed by staining cells with propidium iodide (PI)”. .
(7) A seventh aspect of the present invention is “the method according to (5) or (6) above, wherein the fractionation is performed by setting a gate to fluorescence in a wavelength range of 580 nm to 640 nm”.
(8) According to an eighth aspect of the present invention, “the selection of the E-cadherin positive and c-Kit positive cells is performed using an antibody against E-cadherin and an antibody against c-Kit” (1) to (7 ) ”.
(9) A ninth aspect of the present invention is “the method according to (8) above, wherein the antibody is a conjugate of a fluorescent dye or an enzyme”.
(10) A tenth aspect of the present invention is “the method according to any one of (1) to (9) above, wherein the selection is performed by a flow cytometer”.
(11) An eleventh aspect of the present invention is “the method according to any one of (1) to (9) above, wherein the antibody is carried on an immobilizable carrier”.
(12) The twelfth aspect of the present invention is “the method according to any one of (1) to (11) above, wherein the cell population is a cell population containing ES cells”.
(13) A thirteenth aspect of the present invention is “the method according to any one of (1) to (11) above, wherein the cell population is a cell population containing iPS cells”.
(14) A fourteenth aspect of the present invention is “a kit for selecting pluripotent stem cells containing an E-cadherin antibody and a c-Kit antibody”.
(15) According to a fifteenth aspect of the present invention, “the E-cadherin antibody and the c-Kit antibody are immobilized on a carrier, and a holding part that immobilizes the antibody-carrier conjugate is provided. Is a device for use in the method according to any one of the above (1) to (13), which has a structure capable of contacting a liquid.

  According to the present invention, a pluripotency in which cells that maintain differentiation pluripotency are easily and efficiently selected from a cell population including cells that have lost differentiation pluripotency, and complete differentiation pluripotency is maintained. Sexual stem cells can be prepared.

FIG. 2 shows a conceptual diagram of a method for preparing pluripotent stem cells using cell size, cell density, autofluorescence and cell surface antigen as indices. A comparison of the homogeneity of pluripotent stem cells obtained when (A) and without (B) gate setting is shown. The expression analysis result of the membrane surface antigen using E14tg2a ES cell line is shown. The correlation of the cell surface antigen and Nanog using EB3-DsRed-NVtT cell line is shown. Measurement results using Mo-Flo and Flow-jo, the horizontal axis is the expression level of Nanog, and the vertical axis is the expression level of various transcription factors (c-Kit, sca-1, SSEA1, SSEA4, CD9 and E-cadherin) As displayed. c-Kit positive cells (c-Kit ⁺ ), c-Kit negative cells (c-Kit ⁻ ), SSEA positive cells (SSEA ⁺ ), SSEA negative cells (SSEA−), E-cadherin positive cells (E-cadherin ⁻ ) Shows the results of RT-PCR analysis of the expression status of differentiation pluripotency factors (Nanog, Rex1, Oct3 / 4, Sox2, Klf4). MEF is the result of the same analysis for mouse fetal fibroblasts, and DW is the result of the same analysis for water-only control. The result of having fractionated c-Kit positive and c-Kit negative cells with a flow cytometer and evaluating the undifferentiation by alkaline phosphatase staining is shown. The outline of the evaluation method of the chimera formation ability using the blastocyst injection method is shown. The change of the expression of c-Kit and E-cadherin over time in the iPS cell establishment process is shown. Factor 3 shows the results of iPS cells prepared by introducing Klf4, Sox2, and Oct3 / 4. Factor 4 shows the results of iPS cells prepared by introducing Klf4, Sox2, Oct3 / 4, and c-Myc. The change image of the cell shape in the process of iPS cell establishment is observed with a phase contrast microscope. The result of having performed the undifferentiation evaluation by the alkaline phosphatase dyeing | staining in the cultured cell of each fraction is shown. A is a colony photograph detected, and B is the number of colonies stained with alkaline phosphatase and a microscopic image of the stained cells.

The present invention is a method for preparing pluripotent stem cells (or simply referred to as stem cells) that maintain complete differentiation pluripotency using the expression of E-cadherin and c-Kit on the cell surface as an index. . According to the present invention, a stem cell that maintains differentiation pluripotency and a cell that has lost differentiation pluripotency are distinguished from a seemingly uniform cell population. It becomes possible to select pluripotent stem cells to be maintained. That is, E-cadherin positive and c-Kit positive cells can be identified in a certain cell population, and these positive cells can be selected as stem cells that maintain complete pluripotency. If the stem cells selected in this way are cultured in a state that maintains differentiation pluripotency and is capable of dividing proliferation according to a standard method, a population of stem cells that maintain complete differentiation pluripotency can be prepared. it can.
In the present specification, the term “cell population” or “population of cells” has a meaning normally understood in the art, and refers to a collection of a plurality of cells, for example, the pluripotency is lost. A collection of cells, including cells (including cells that have lost some of their pluripotency) and / or cells that have maintained full differentiation pluripotency (ie, pluripotent stem cells or stem cells in the original sense) It is. The collection of such cells is not particularly limited, but is, for example, an ES cell population including cells that have lost pluripotency, in an incompletely initialized state (not in a completely undifferentiated state). An iPS cell population containing cells can be mentioned. Moreover, it is a concept including not only a population of cells in a cultured state in vitro but also a population of cells contained in a living body as part of a tissue or organ.
In general, “ES cells” are obtained by cultivating fertilized eggs at the blastocyst stage together with feeder cells, disaggregating the cells derived from the proliferated inner cell mass, and further repeating the operation of inoculating the cells. Can be established as an “ES cell line”. As described above, ES cells are often obtained from fertilized eggs, but in addition, adipose tissue (refer to Patent Document 2), chorionic villi, amniotic fluid, placenta (refer to Patent Document 1), testis ES cell-like cells obtained from other than fertilized eggs such as cells (see Patent Document 3) and having ES cell-like characteristics and differentiation pluripotency are also known. It is included in the “cell population” or “cell population” according to the invention.

  An “iPS” cell is a cell that has acquired differentiation pluripotency, also called an induced pluripotent stem cell or induced pluripotent stem cell, and is a pluripotent differentiation into a somatic cell (eg, fibroblast). It is a cell that has acquired differentiation pluripotency equivalent to that of an ES cell by introducing several types of transcription factors (hereinafter referred to as “differentiation pluripotency factors”) that impart sex. Many factors have already been reported as “pluripotency factors” and are not limited, but include, for example, the Oct family (eg, Oct3 / 4), the Sox family (eg, Sox2, Sox1, Sox3, Sox15 and Sox17), Klf family (for example, Klf4, Klf2, etc.), Myc family (for example, c-Myc, N-Myc, L-Myc, etc.), Nanog, LIN28, and the like. Since many literatures are published about the iPS cell establishment method, they can be referred (for example, Takahashi et al., Cell 2006, 126: 663-676; Okita et al., Nature 2007, 448: 313). -317; Wernig et al., Nature 2007, 448: 318-324; Maherali et al., Cell Stem Cell 2007, 1: 55-70; Park et al., Nature 2007, 451: 141-146; Nakagawa et al., Nat Biotechnol 2008, 26: 101-106; Wernig et al., Cell Stem Cell 2008, 10: 10-12; Yu et al., Science 2007, 318: 1917-1920; Takahashi et al., C ll 2007,131: 861-872; Stadtfeld et al., Science 2008 322: 945-949 that of reference, etc.).

  The origin of the “cell” described in the present specification is a human or non-human animal (eg, mouse, rat, cow, horse, pig, sheep, monkey, dog, cat, bird, etc.) and is not particularly limited. . Further, the term “pluripotent stem cell” or “stem cell” does not differ from the meaning understood by those skilled in the art. For example, the same differentiation ability is maintained even after cell division, and theoretically in all living organisms. It means a cell that can differentiate into a tissue (cell).

“E-cadherin” is classified into the classic cadherin subfamily, and is a sugar chain-linked membrane protein that is present on the cell surface and plays a role of adhering cells to each other. There are about 20 types of classic cadherins, such as LCAM, N-cadherin, P-cadherin, in addition to E-cadherin. Classic cadherin has five domains (EC domain) outside the cell, and has a structure consisting of one transmembrane segment and an intracellular domain. Catenin binds to the intracellular domain and is linked to the cytoskeleton.
“C-Kit” is also referred to as CD117, and is a cytokine receptor (receptor tyrosine kinase type III) having tyrosine kinase activity expressed on the surface of cells such as hematopoietic stem cells and germ cells. To date, a group of proteins that begin with “CD (Cluster of differentiation)” has been used as an indicator of the differentiation stage of cells, and c-Kit identifies specific types of hematopoietic cell precursors in the bone marrow. Has been used as a cell surface marker. Cytokine stem cell factor (SCF) is known as a ligand for c-Kit. When c-Kit binds to SCF, it forms a dimer and activates intracellular signals via a second messenger.

  The present invention recognizes or identifies cells expressing E-cadherin on the cell surface (E-cadherin positive cells) or cells expressing c-Kit (c-Kit positive cells), and these cells. Including a step of sorting. The means for recognizing E-cadherin and c-Kit on the cell surface is not particularly limited, and molecules that specifically bind to these proteins, such as antibodies, can be used. Commercially available antibodies against E-cadherin and c-Kit can be purchased and used (for example, purchased from Pharmingen, TAKARA, eBioscience, etc.).

In addition, antibodies against E-cadherin and c-Kit can be prepared by themselves in order to obtain antibodies with desired antigen recognizability and affinity.
The antibodies that can be used in the present invention include monoepitope specific antibodies against E-cadherin or C-Kit, polyepitope specific antibodies, single chain antibodies, and fragments thereof (for example, Fv, F (ab ′) ₂ , Fab Etc.). These antibodies include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies and the like.
Polyclonal antibodies can be prepared, for example, by injecting a mixture of immunogen and adjuvant into a mammalian host animal. Usually, the immunogen and / or adjuvant is injected multiple times subcutaneously or intraperitoneally into the host animal. As the immunogen, E-cadherin or c-Kit, or a part of these proteins (for example, a part exposed on the cell surface) can be used. Examples of the adjuvant include complete Freud and monophosphoryl lipid A synthesis-trehalose dicorynomycolate (MPL-TDM).

A monoclonal antibody can be prepared using, for example, a hybridoma method.
This method includes the following four steps: (i) immunizing a host animal or lymphocytes from the host animal, (ii) monoclonal antibody secreting (or potentially secreting) lymphocytes Harvest, (iii) fuse lymphocytes to immortalized cells, (iv) select cells that secrete the desired monoclonal antibody.
A mouse, rat, guinea pig, hamster, or other suitable host animal is selected as the immunized animal and the immunogen is injected. Alternatively, lymphocytes obtained from immunized animals may be immunized in vitro.
After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusing agent such as polyethylene glycol in order to establish hybridoma cells. As fusion cells, rodent, bovine, or human myeloma cells immortalized by transformation are used, or rat or mouse myeloma cell lines are used. After cell fusion, the cells are grown in a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines. Conventional techniques use parent cells that lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine are added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hybridomas. A hybridoma that produces a desired antibody is selected from the hybridomas thus obtained, and the target monoclonal antibody can be obtained from the medium in which the hybridoma grows according to a conventional method.

The selection of pluripotent stem cells that maintain differentiation pluripotency using an antibody is not limited. For example, a fluorescent dye or the like is bound to the antibody, followed by observation by an immunohistochemical method, and labeled with the fluorescent dye. A method for identifying the obtained cells as E-cadherin-positive or c-Kit-positive cells, binding an antibody (eg, alkaline phosphatase, peroxidase, etc.) substrate to the antibody, and developing the color by an enzymatic method, and E-cadherin positive or c -It can be performed using a method for identifying Kit positive cells.
The “antibody” in the present invention includes an antibody bound with a detection molecule such as a fluorescent dye or an enzyme used for recognizing the antibody.

  E-cadherin-positive cells and c-Kit-positive cells are obtained by combining the antibody-carrier conjugate in which E-cadherin antibody and c-Kit antibody are immobilized on a carrier with a cell population containing pluripotent stem cells. Suspend and mix under conditions (eg, physiological ionic strength, pH), and incubate at an appropriate time (eg, 1 hour to overnight) and temperature (eg, about 4 ° C. to 37 ° C.). Thus, E-cadherin positive cells or c-Kit positive cells and an antibody are bound to each other, and the cell-antibody-carrier complex can be recovered and prepared by an appropriate method. Here, the carrier for immobilizing and carrying the antibody may be of any shape and composition as long as the antibody is immobilized. For example, magnetic beads, agarose to which bound Protein A or Protein G is bound. In addition, a resin such as Sepharose, or a plate-like member (plate) in which an antibody is bound to a plane, a membrane, a tube, or the like can be used. For example, when magnetic beads are used, E-cadherin antibody or c-Kit antibody to which magnetic beads are bound (utilizing the binding of avidin or streptavidin and biotin), E-cadherin positive cells or c- It can be prepared by binding to each of Kit positive cells, specifically sorting these positive cells by magnetic force, and further culturing the cells. Alternatively, in addition to the method using magnetic force, E-cadherin antibody or c-Kit antibody bound to E-cadherin positive cells or c-Kit positive cells, respectively, and Protein A or Protein G bound to agarose or Sepharose are used. Thus, it is possible to select these positive cells.

Furthermore, the selection of E-cadherin positive cells or c-Kit positive cells was performed using a method using a fluorescent dye or the like and separating cells using fluorescence as an indicator, such as flow cytometry. It is also possible to use a method well known in the art, such as a cell sorter system (flow cytometer such as FACS). Any flow cytometer can be used. For example, Becton Dickinson (FACS Vantage, FACSAria, FACSCalibur, etc.), Dako Japan (Mo-Flo, etc.), Beckman Coulter (EPICS ALTRA, etc.) ) Can be purchased from. Furthermore, as the software used for the use of the flow cytometer in selecting and sorting the target cells with the flow cytometer, for example, Flow-jo (Tree Star), CellQuest (Becton Dickinson), etc. It can be used according to the device to be used.
In the present specification, the “flow cytometer” means both those having a cell sorter (cell device) function and those having no cell sorter function.

Furthermore, the present invention is based on cell size differences, cell density differences, cell autofluorescence differences and / or cell viability, based on pluripotent stem cell candidate live cells. Candidate cells) are collected, and cells that maintain differentiation pluripotency are expressed from the sorted live pluripotent stem cell candidates using the expression of E-cadherin and c-Kit on the cell surface as an index. A method for sorting and preparing pluripotent stem cells is provided.
Pluripotent stem cells have characteristics different from cells other than pluripotent stem cells with respect to the size, cell density, and autofluorescence (fluorescence characterizing the type and amount of functional groups on the cell surface). Therefore, by combining the selection based on the cell size, cell density, and autofluorescence characteristic of pluripotent stem cells with the selection based on the expression of E-cadherin and c-Kit, the pluripotency can be effectively improved. It becomes possible to select stem cells.
In addition, it is possible to efficiently select live pluripotent stem cells by combining cells with a suitable dye that can distinguish between live and dead cells and further sorting live cells. It becomes.
A method for selecting cells having characteristic cell size, cell density and autofluorescence characteristic of pluripotent stem cells can be carried out by appropriately selecting a method known in the art by those skilled in the art. Can be carried out using a flow cytometer. When using a flow cytometer, but not limited, for example, the selection of candidate cells for pluripotent stem cells by cell size is based on forward scatter (FSC), and the restriction by cell density is It can be based on side scatter (SSC). When using a flow cytometer, set the gate of FSC and SSC for pluripotent stem cells for cell size and cell density (Figure 1, left), and set the gate for the intensity of fluorescence in a certain wavelength region for autofluorescence. By doing so (FIG. 1, central view), pluripotent stem cell candidates can be narrowed down. When the pluripotent stem cell candidates narrowed down in this way are further selected using the expression of c-Kit and E-cadherin as an index (FIG. 1, right figure), uniform cells from which contaminating cells have been removed are obtained. A population of pluripotent stem cells can be prepared. For example, when contamination with MEF (mouse fetal fibroblast) is expected, FSC is in the range of about 10 ¹ to 10 ³ , SSC is in the range of about 10 ¹ to 10 ² , and self Regarding fluorescence, for example, in the fluorescence wavelength range of 580 nm to 640 nm (610 ± 30 nm), the fluorescence intensity from pluripotent stem cells is weaker than the fluorescence intensity from MEF, so the fluorescence intensity is in the range of about 10 ⁰ to 10 ^1. When the gate is set, it is possible to prevent MEF contamination after the selection with c-Kit and E-cadherin (cell population surrounded by an ellipse in FIG. 2A) (FIG. 2A, lower row, comparison of MEF and ES, iPS). (The above settings can be used, for example, when using Mo-Flo and Flow-jo).
In addition, live cells and dead cells are dyes that differ in the degree of staining depending on whether the cells are viable or not. For example, the cells are stained with propidium iodide (PI), and the difference in fluorescence intensity from the cells. It is possible to distinguish between live cells and dead cells.
Note that the “gate” setting is not different from the usual meaning used in flow cytometry. For example, FSC, SSC, fluorescence intensity at a specific fluorescence wavelength is used to sort the target cell population. Is to set a selection range (fractional range) to

Cell size and cell density gates can be easily set using the measured values as indicators by measuring the FSC and SSC intensity ranges of pluripotent stem cells in advance using the flow cytometer. it can.
Similarly, for the gate related to autofluorescence, a flow site that uses the autofluorescence intensity range of pluripotent stem cells in an arbitrary fluorescence wavelength range and the autofluorescence intensity range of a cell (eg, MEF) expected to be mixed. By measuring in advance with a meter, a gate capable of sorting only pluripotent stem cells can be easily set using the measured value as an index.
In addition, as to the distinction between the life and death of cells, any dye can be used as long as the dye has a different degree of staining depending on whether the cells are viable or not, and fluorescence in a wavelength range suitable for the excitation fluorescence wavelength of the dye is detected. It can be easily distinguished by the difference in strength. Furthermore, the distinction based on the difference in autofluorescence and the distinction based on the life or death of the cells can be simultaneously performed by appropriately selecting the wavelength range of the fluorescence to be detected. For example, in the case where contamination with MEF is expected, in order to distinguish between the life and death of cells, fluorescence in a specific wavelength region obtained by excitation with specific excitation light (for example, 488 nm for PI) (for example, for PI) Since the intensity of 580 nm to 640 nm (610 ± 30 nm) increases in the order of live pluripotent stem cells, MEF, and dead cells, gate setting is possible using the obtained intensity as an index.

In addition, the present invention can provide a kit for preparing pluripotent stem cells. Such a kit contains c-Kit antibody and E-cadherin antibody (fluorescent dye may be bound in advance), washing reagent, fluorescent dye, enzyme, chromophore, magnetic beads, protein A or protein G. In addition to the binding carrier, a plate-like, membrane-like, tube-like carrier, antibody binding reagent (for example, a reagent necessary for performing biotin-avidin binding, etc.) and the like can be included. Reagents and the like contained in the kit are supplied into any type of container in which the constituents maintain the activity effectively for a long period of time, are not adsorbed by the material of the container, and do not undergo alteration. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents.
The kit also includes instructions for use. Instructions for using the kit are printed on paper or other material and / or electrically or electromagnetically, such as floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as a readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

Furthermore, the present invention provides an apparatus suitable for performing the method of the present invention. The apparatus includes a carrier on which an E-cadherin antibody and a c-Kit antibody are immobilized, and a holding unit that immobilizes the carrier, and has a configuration that allows the antibody-carrier conjugate to come into contact with a liquid. . Here, the “carrier” may be of any shape and composition as long as the antibody is immobilized. For example, a magnetic bead, a resin such as agarose or sepharose to which bound Protein A or Protein G is bound. Alternatively, a plate-like member (plate), a membrane, a tube or the like in which antibodies are bound to a plane can be used. In addition, the “holding part” is a part having a function of holding a carrier to which an antibody is bound. For example, if the carrier is in the form of beads, it is a column-like part that can be packed. Yes, any material such as metal or glass may be used. In the case where the carrier is a plate-like member (such as a plate), or a film-like or tube-like member, even if it is a clamp that directly holds the plate-like or film-like or tube-like member, It may be a container that accommodates the plate-like, membrane-like, or tube-like member therein. Furthermore, the “configuration in which the liquid can be contacted” means that a contaminant other than the target E-cadherin positive and c-Kit positive cells (for example, free antibodies, cells other than pluripotent stem cells, etc.) Any configuration can be used as long as it can be washed away and can be recovered with a liquid in order to obtain the target cells. For example, when a column or the like is used as the holding portion, it is sufficient that the antibody (or antibody-carrier conjugate) and a liquid such as a buffer can be contacted inside the column, and the plate or membrane or tube is supported on the carrier. When used as a plate, the plate or the membrane or tube is covered with a closed space in the form of a container in which the antibody (or antibody-carrier conjugate) immobilized on the plate or the membrane or tube can come into contact with the liquid. Just do it.
Furthermore, the apparatus of the present invention is appropriately provided with an inlet for filling or injecting a solution such as a carrier, a sample (cells, etc.) and a buffer into the holder, and an outlet for discharging or collecting from the holder. Also good.
The device of the present invention may be a component of the kit of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

1. Analysis of ES cell line 1-1. Culture of ES cell line E14tg2a, EB3-DsRed, and EB3-DsRed-NVtT of mouse embryonic stem cell (mouse embryonic stem cell; mESc) are Glaco's Modified in a gelatin-coated dish. Eagle's Medium (GMEM; Sigma, St. Louis, Mo.), 10% fetal bovine serum (FBS; Nichirei), 0.1 mM 2-mercaptomethanol (Invitrogen), 0.1 mM non-essential amino acid (Invitrogen), 1 mM Pyruvate sodium salt (Invitrogen), 1% L-glutamine-penicillin-streptomycin (Sigma), 1,000 U / ml leukiemia inh Were cultured in; (Millipore LIF) (Feeder-Free medium) medium supplemented with bitory factor.
In EB3-DsRed-NVtT, a sequence connecting yellow fluorescent gene Venus and Neomycin resistance gene via IRES sequence is sandwiched by loxP downstream of Nanog promoter of EB3-DsRed cells, and tetracycline transactivation protein (tTA) is downstream of it. A cell into which a vector to which a gene has been linked has been introduced by electroporation.
E14tg2a cells are 129 / ola-derived cells and are HPRT-negative ES cell lines.
The EB3-DsRed cells were provided by Dr. Hitoshi Niwa (RIKEN Research Center for Development and Regenerative Science), and are cells in which the CAGp-DsRed vector was introduced into the EB3 ES cell line and DsRed was forcibly expressed. . The EB3 cell is a cell obtained by knocking in the drug resistance gene BSD downstream of the Oct-3 / 4 locus of the E14tg2a cell using the IRES sequence.

1-2. Expression analysis of membrane surface antigen of ES cell using flow cytometer E14tg2a cells were seeded in a φ10 cm dish at a concentration of 2.0 × 10 ⁵ and cultured for 2 days. After washing with phosphate buffered saline (PBS), trypsinization was performed to suspend the cells. The antibody (see Table 1) was added to the cell suspension so that the concentration was 0.02 μg / 10 ⁶ cells, and the mixture was allowed to stand on ice for 30 minutes. After washing with PBS (staining medium; SM) containing 3% FBS, resuspended in SM (PI / SM) containing 1 μg / ml propidium iodide (PI), and Mo-Flo (Beckman Coulter) And analysis software, Flow-jo.
As a result of analysis, FSC is about 10 ¹ to 10 ³ , SSC is about 10 ¹ to 10 ² , and PI staining is performed as a cell having a cell size, cell density, and autofluorescence specific to pluripotent stem cells. fluorescence obtained by (excitation wavelength; 488 nm, fluorescence wavelength; 610 ± 30 nm) ^has a 10 0 - 10 ¹ about the cell a candidate cell of pluripotent stem cells (Figure 2).
When a rat-derived purified antibody (without dye labeling) or a biotin-conjugated antibody is used, Alexa 647 conjugated goat anti Rat IgG (Invitrogen) is used as a secondary antibody after washing with SM. 0.2 μg / 10 ⁶ cells, phycoerythrin (PE) conjugated streptavidin was added to the biotin-conjugated antibody so as to be 0.04 μg / 10 ⁶ cells, and left on ice for 30 minutes. After washing twice with SM, it was resuspended in PI / SM and analyzed.

After conducting a primary analysis using this method, the same amount is used for the antibody to be stained, and for an antibody with a poor degree of staining, a better staining condition is determined while examining the amount and reaction time of the antibody. Analysis was performed (Table 2). As a result of the analysis by the antibody, the proportion of cells stained in the E14tg2a ES cell line was classified by the antibody, and the frequency with which the antigen corresponding to each antibody was expressed on the surface of the ES cell line was analyzed (FIG. 3). .

Based on the examination results of the staining conditions and the analysis results of the expression frequency of the antigen for each antibody, the transcription factor Nanog, which is considered to be highly expressed in undifferentiated cells, was selected from these results using EB3-DsRed-NVtT cells. The correlation with c-Kit, sca-1, SSEA1, SSEA4, CD9, and E-cadherin was analyzed using Mo-Flo and Flow-jo. (FIG. 4).
Note that PI is a dye that binds to intracellular DNA, and because it has low cell membrane permeability, live cells cannot be stained, and dead cells with enhanced cell membrane permeability are stained. Using this characteristic, dead cells were identified and removed.

1-3. Cell sorting and RT-PCR
Next, c-Kit positive cells (c-Kit ⁺ ), c-Kit negative cells (c-Kit ⁻ ), SSEA positive cells (SSEA ⁺ ), SSEA negative cells (SSEA ⁻ ), E-cadherin positive cells (E The expression status of pluripotency factors (Nanog, Rex1, Oct3 / 4, Sox2, Klf4) in -cadherin ⁺ ) was examined.
Rat antimouse E-cadherin (TAKARA) was added to the suspension of E14tg2a cells at a ratio of 1 μg / 10 ⁶ cells and allowed to stand on ice for 30 minutes. After washing with SM, Alexa Fluor 647 conjugated goat anti Rat IgG (Invitrogen) was added at a ratio of 2 μg / 10 ⁶ cells and allowed to stand on ice for 30 minutes. After twice washing with SM, in a ratio of PE conjugated anti human / mouse SSEA1 ( R & D systems) to 0.1 [mu] g / 10 ⁶ cells and PE-Cy7 conjugated anti mouse c- Kit (eBioscyence) a 0.06 .mu.g / 10 ⁶ cells In addition, it was left on ice for 30 minutes. After washing with SM, the suspension was resuspended with 1 μg / ml PI / SM, and c-Kit positive / negative, SSEA1 positive / negative cells and E-cadherin positive cells were sorted into TRIZOL (Invitrogen) by Mo-Flo. .
RNA was isolated by phenol / chloroform method, purified by ethanol precipitation, and reverse transcribed using Oligo (dT) ₂₀ primer of Thermo Script RT-PCR system (invitrogen) to prepare cDNA. PCR was performed by Gene Amp PCR system 9700 (Applied Biosystems; AB) using Gapdh as an internal standard and Taq polymerase ^HS (TAKARA) (FIG. 5). As a result, c-Kit positive ES cells show a high expression level of genes important for maintaining undifferentiation, and E-cadherin is also undifferentiated although the expression level is lower than that of c-Kit. Among related genes, particularly high expression of Sox2 and Oct3 / 4 was observed.

Primer sequences used in RT-PCR are as follows.
Nanog (Forward): 5'-CAGGAGTTTGAGGGTAGCTC-3 '(SEQ ID NO: 1)
Nanog (Reverse): 5'-CGGTTCATCATGGTACAGTC-3 '(SEQ ID NO: 2)
Rex1 (Forward): 5'-ACGAGTGGCAGTTTCTTCTTGGGA-3 '(SEQ ID NO: 3)
Rex1 (Reverse): 5'-TATGACTCACTTCCAGGGGGCACT-3 '(SEQ ID NO: 4)
Oct3 / 4 (Forward): 5'-TGCGGGCGGACATGGGGAGATCC-3 '(SEQ ID NO: 5)
Oct3 / 4 (Reverse): 5'-TCTTTCCACCAGGCCCCCGGCTC-3 '(SEQ ID NO: 6)
sox2 (Forward): 5'-TTGCCTTAAACAAGACCACGAAA-3 '(SEQ ID NO: 7)
sox2 (Reverse): 5'-TAGAGCTAGACTCCGGGCGATGA-3 '(SEQ ID NO: 8)
Klf4 (Forward): 5'-TCGCTTCCTCTTCCTCCGACACA-3 '(SEQ ID NO: 9)
Klf4 (Reverse): 5'-GCGAACTCACTCACACAGGCGAGAAACC-3 '(SEQ ID NO: 10)
Gapdh (Forward): 5'-TGCACCACCAACTGCTTA G-3 '(SEQ ID NO: 11)
Gapdh (Reverse): 5'-GGATGCAGGGATGATGTTC-3 '(SEQ ID NO: 12)

1-4. Evaluation of undifferentiation by sorting of c-Kit positive and negative cells and staining with alkaline phosphatase To a suspension of E14tg2a cells, 0.06 μg / 10 ⁶ cells of Allophycocyanin (APC) conjugated anti-mouse c-Kit (eBioscience) was added. It added in the ratio and left still on ice for 30 minutes. After washing with SM, the cells were resuspended with PI / SM, and 4000 c-Kit positive and c-Kit negative cells were sorted into each well of a gelatin-coated 6-well plate using Mo-Flo. The medium was changed the next day, and after 3 days, the expression of c-Kit in each well was analyzed using Mo-Flo and Flow-jo. Some wells were fixed with methanol containing 10% formalin, washed with 0.1 M Tris-HCl (pH 9.5), and then stained with Alkaline Phosphatase Substrate Kit II (VECTOR) for 15 minutes. gave. After washing with 0.1 M Tris-HCl (pH 9.5), it was replaced with PBS and observed under a microscope (FIG. 6). As a result, it was revealed that c-Kit positive ES cells formed a dense undifferentiated colony.

1-5. Evaluation of chimera formation ability using blastocyst injection method c-Kit positive, c-Kit negative, c-Kit positive and SSEA1 positive, c-Kit positive and SSEA1 negative, c-Kit positive and Sca of EB3-DsRed cells -1 positive, c-Kit positive, Sca-1 negative, and E-cadherin positive cells were prepared as ES cells that were sorted into each well of a 96-well plate using Mo-Flo and injected into blastocysts. (FIG. 7).
As the blastocyst used in this experiment, a morula derived from BDF1 × C57BL6 that had been cryopreserved as a host embryo was cultured into a blastocyst (1 day) using KSOM-AA (Millipore). A thing was used.
ES cells sorted using a micromanipulator under a microscope were injected into the cavity at a ratio of 5 to one blastocyst. After waiting for the recovery of the embryo, it was transplanted into the uterus of a pseudopregnant ICR strain mouse 2.5 days after mating, and a cesarean section was performed on the 11th day after the transplantation (13.5 days of gestation), and the fetus was taken out. The obtained fetus was observed under a fluorescence microscope to determine chimera formation (Table 3).

2. 2. Analysis on iPS cell line 2-1. Establishment of iPS cells Mouse fetal fiber cryopreserved 2 days before infection with retrovirus for gene transfer (Klf4, Sox2, Oct3 / 4, c-Myc) (see Yamanaka et al., Cell 2006 126: 663-676) Dulbecco's Modif supplemented with 10% fetal bovine serum (FBS; HANA-NESCO BIO CORP), 1% L-glutamine-penicillin-streptomycin (Sigma) on a gelatin-coated dish of blast cells (Mouse embryonic fibroblast; MEF) Eagle's (DMEM; Sigma, St. Louis, MO) (MEF medium) was used to wake from cryopreservation and culture.
Gene transfer was performed as follows.
One day before virus infection, MEF medium was re-sown in 6-well plates, 1 × 10 ⁵ cells per well.
Day 0 (day of virus infection): Before infection, MEF was replaced with a medium supplemented with protamine sulfate (10 mg / ml) at a rate of 1 μl / ml. Each virus stock (Klf4, Sox2, Oct3 / 4, c-Myc) was melted one by one and 25 μl was added to each well.
Day 1; replaced with fresh MEF medium.
On day 2; 1 well of infected MEF medium infected with the virus was divided into 12 equal portions into 6 well plates and re-spread.
3-21 days; from MEF medium, 15% knockout serum replacement (KSR; invitrogen), 0.1 mM 2-mercaptomethanol (Invitrogen), 0.1 mM non-essential amino acid (Invitrogen), HEPES buffer (Invitrogen), 1 % L-glutamine-penicillin-streptomycin (Sigma), Dulbecco's Modified Eagle's Medium (InK) medium exchanged with 1,000 U / ml leukiemia inhibitory factor (LIF; Millipore). The medium was changed once every two days.

Mouse fetal fibroblasts used in the experiment were prepared from B6 mouse embryos at 13.5 days of gestation. Specifically, one φ10 cm dish was prepared for one fetus, and cells that had been transplanted into one φ15 cm dish at the first and second passages, and three of them were stored frozen were used.
The virus used for infection was used after concentrating the supernatant produced from 293GPG cells. Specifically, using a medium supplemented with 1 μg / ml tetracycline (SIGMA), 2 μg / ml puromycin (SIGMA), and 0.3 mg / ml G418 in MEF medium, a poly L-Lysine (Invtrogen) coated φ10 cm After culturing in a dish and -80% confluent, the seeds were re-sown into 4 dishes, and then the medium was changed to MEF medium when it became -80% confluent again. On the second and third days after the medium change, the supernatant was collected, passed through a 0.45 μm filter, and ultracentrifuged at 6,000 g at 4 ° C. for 24 hours. The supernatant was removed, suspended in 500 μl of KSR ES medium, and stored frozen in 50 μl aliquots.

2-2. Analysis of time-dependent changes in cell membrane surface antigen by flow cytometer and evaluation of undifferentiation by alkaline phosphatase staining The prepared iPS cells were detached from the dish on days 7, 14, and 21, and rat anti mouse E-cadherin (TAKARA). Was added to 1 μg / 10 ⁶ cells and allowed to stand on ice for 30 minutes. After washing with SM, Alexa Fluor 647 conjugated goat anti Rat IgG (Invitrogen) was added to 2 μg / 10 ⁶ cells, and allowed to stand on ice for 30 minutes. After washing twice with SM, PE-Cy7 conjugated anti-mouse c-Kit (eBioscience) was added to 0.06 μg / 10 ⁶ cells and allowed to stand on ice for 30 minutes. Washed with SM, resuspended in PI / SM and analyzed with Mo-Flo (FIG. 8). Moreover, the morphological change of the cell was observed under the microscope (FIG. 9).
C-Kit positive and E-cadherin positive, c-Kit positive and E-cadherin negative, c-Kit negative and E-cadherin positive, c-Kit negative and E-cadherin negative cells, respectively, on day 14 and 21 The cells were sorted one by one with Mo-Flo in a 24-well plate in which feeder cells had been cultured in advance. The cells were cultured for 5 days in KSR ES medium and stained using Alkaline Phosphatase Substrate Kit II (VECTOR) in the same manner as when E14tg2a cells were stained. Under the microscope, count the number of alkaline phosphatase positive colonies, classify the intensity of staining into strong positive (++), weak positive (+), and negative (-), and determine how many undifferentiated-like cells are from the number of colonies in each classification. It was evaluated whether the fractions were sorted more efficiently (FIG. 10).
From this result, it became clear that, on the 21st day, colonies of undifferentiated-like cells were selectively generated from cells positive for both c-Kit and E-cadherin (FIG. 10, day 21).

  The present invention is a method for efficiently selecting pluripotent stem cells that maintain differentiation pluripotency, and is an extremely effective means for efficient preparation of differentiated pluripotent cells used in the medical field and the like. It provides a simple means.

Claims (15)

  A method of preparing pluripotent stem cells by selecting E-cadherin positive and c-Kit positive cells from a cell population.   2. The method according to claim 1, wherein the cell population is sorted as a candidate cell for a pluripotent stem cell using cell size, cell density, cell autofluorescence and / or cell viability as an index. .   The method according to claim 2, wherein the sorting of the candidate cells is performed by a flow cytometer.   The method according to claim 3, wherein the sorting is performed by setting a gate for forward scattered light and side scattered light.   The method according to claim 3 or 4, wherein the sorting is performed by setting a gate for fluorescence.   The method according to any one of claims 3 to 5, wherein the sorting is performed by staining cells with propidium iodide (PI).   The method according to claim 5 or 6, wherein the sorting is performed by setting a gate to fluorescence in a wavelength range of 580 nm to 640 nm.   The method according to any one of claims 1 to 7, wherein the selection of the E-cadherin positive and c-Kit positive cells is performed using an antibody against E-cadherin and an antibody against c-Kit.   The method according to claim 8, wherein the antibody is conjugated with a fluorescent dye or an enzyme.   The method according to claim 1, wherein the sorting is performed by a flow cytometer.   The method according to claim 1, wherein the antibody is carried on an immobilizable carrier.   The method according to any one of claims 1 to 11, wherein the cell population is a cell population containing ES cells.   The method according to any one of claims 1 to 11, wherein the cell population is a cell population containing iPS cells.   A kit for selecting pluripotent stem cells containing an E-cadherin antibody and a c-Kit antibody.   A carrier having an E-cadherin antibody and a c-Kit antibody immobilized thereon and a holding part for immobilizing the antibody-carrier conjugate, wherein the antibody-carrier conjugate is in contact with a liquid. An apparatus for use in the method according to any one of 1 to 13.