WO2014038653A1

WO2014038653A1 - Production method for kidney-derived somatic stem cells

Info

Publication number: WO2014038653A1
Application number: PCT/JP2013/074028
Authority: WO
Inventors: 誠土方; 達哉山口
Original assignee: 国立大学法人京都大学; 東洋紡株式会社
Priority date: 2012-09-07
Filing date: 2013-09-06
Publication date: 2014-03-13
Also published as: JPWO2014038653A1

Abstract

The present invention addresses the problem of providing a method and the like for efficiently growing kidney-derived somatic stem cells. This production method for kidney-derived somatic stem cells comprises the following steps (A) and (B): a step (A) in which a group of cells including cells that constitute the kidneys are made to pass through the G0 phase or the G1 phase, and a gene for a protein that is active in causing the transition to the S phase are expressed; and a step (B) in which the cells obtained at the step (A) are cultivated in the presence of an extracellular growth factor.

Description

Method for producing kidney-derived somatic stem cells

The present invention relates to a method for producing kidney-derived somatic stem cells and applied technology thereof.

In the disease research and new drug development, the use of cells, especially human cells, is now essential, and the demand is expected to increase in the future. Cells are separated and prepared from tissues collected from living organisms and cultured for wide use in test studies. In this case, differentiated cells according to the purpose are often used. However, differentiated cells taken out of the body may be difficult to grow. In addition, there are many problems such as the loss of the original activity in a short time, the very high cost of collection, and the large variation in the characteristics depending on the donor.

Measures may be taken to immortalize differentiated cells in order to clear these problems, particularly those related to proliferation. For example, many attempts have been made to immortalize cells by various methods such as introducing SV (simian virus) 40 large T antigen gene and human telomerase gene into human hepatocytes (Non-patent Document 1). However, most of the cells obtained by immortalization do not have the original differentiation traits, and no immortalized cells that have sufficiently retained their original functions have been known so far. For example, the telomerase gene is hardly expressed in normal cells of human tissue. In addition, the use of so-called oncogenes for the production of immortalized cells is considered to be unfavorable, including the possibility of deviating from normal cells.

On the other hand, as a means for obtaining a large amount of target normal primary cells, there is a method in which somatic stem cells having high proliferative properties are isolated from a living body and cultured and proliferated outside the body.

As a method for obtaining somatic stem cells, for example, a method has been reported in which somatic cells are collected from a living tissue and somatic stem cells are separated using a cell sorter based on the expression pattern of cell surface molecules. However, it is difficult to obtain a tissue containing a desired somatic stem cell and select an appropriate antibody against the somatic stem cell, and it is not easy to obtain a somatic stem cell population with high purity. In addition, since the number of stem cells present in the tissue is extremely small, in order to collect a certain amount of stem cells using a cell sorter, a very large number of cells (for example, 10 8 or more) are required as a starting material. It will be. Furthermore, even if a somatic stem cell population with high purity is obtained, it is difficult to culture and proliferate it (particularly, culture in a state in which the characteristics of the original somatic stem cells are maintained).

As another means, there is a method in which stem cells having pluripotency such as ES cells and iPS cells are proliferated and then induced to differentiate into target differentiated cells. Although it has been confirmed that some cells of ES cells or iPS cells are differentiated, the differentiation efficiency is not sufficient at the present stage, and there are many cases in which various cells are mixed and the differentiated function has high purity. It is still difficult to obtain a cell population.

Thus, a method that can stably supply a high-purity population of somatic stem cells that retain a certain differentiation potential has been truly desired.

Special table 2002-512788 Special table 2003-501081 Special table 2009-520474

This invention makes it a subject to solve the subject of the said prior art. More specifically, an object of the present invention is to provide a method for obtaining kidney-derived stem cells that can proliferate in a state having differentiation ability.

In light of the above problems, the present inventors have conducted extensive research, and as a result, a group of cells containing cells that constitute the kidney with a gene of a protein having an activity of passing through the G0 phase or G1 phase and shifting to the S phase. Introduced into the cell and cultured in the presence of extracellular growth factor, somatic stem cells with clonal proliferative ability can be proliferated predominantly without introducing other genes. It has been found that only sex stem cells can be obtained simply and efficiently. In addition, the present inventors have confirmed that the kidney-derived somatic stem cells thus obtained can be differentiated into cells constituting the target kidney by culturing them in a medium suitable for differentiation. did. The inventors of the present invention have completed the present invention by further studying and improving the above knowledge.

The representative invention of the present application is as follows.
Item 1
A method for producing kidney-derived somatic stem cells, comprising the following steps (A) and (B):
(A) In a cell group including cells constituting the kidney, a step of expressing a gene of a protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase; and (B) obtained in the step (A). Culturing cultured cells in the presence of extracellular growth factor.
Item 2
Item 2. The method according to Item 1, wherein the expression is transient expression.
Item 3
Item 3. The method according to

Item

1 or 2, wherein the step (A) and the step (B) are repeated twice or more.
Item 4
Item 4. The method according to any one of Items 1 to 3, wherein the protein is a cyclin-dependent kinase.
Item 5
Item 5. The method according to any one of Items 1 to 4, wherein the protein is at least one protein selected from the group consisting of cyclin-dependent kinase 4 and cyclin-dependent kinase 6.
Item 6
The extracellular growth factor is epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like Item 6. The method according to any one of Items 1 to 5, which is at least one extracellular growth factor selected from the group consisting of growth factors (IGFs).
Item 7
Item 7. A kidney-derived somatic stem cell obtainable by the method according to any one of Items 1 to 6.
Item 8
Item 8. The cell according to Item 7, wherein the kidney-derived somatic stem cell is Pax-2 positive and / or Wnt-4 positive.
Item 9
Item 9. A cell in which an exogenous gene is further introduced into the cell according to

Item

7 or 8.
Item 10
Item 10. A method for producing a differentiated cell, comprising the step of differentiating the cell according to any one of Items 7 to 9 into a cell constituting a kidney.
Item 11
Item 11. The method according to Item 10, wherein the step of differentiating comprises a step of performing three-dimensional culture.
Item 12
Item 12. A cell constituting the kidney, which can be obtained by the method according to

Item

10 or 11.
Item 13
Item 13. A pharmaceutical composition comprising the cell according to any one of Items 7 to 9 and 12.
Item 14
Isolating kidney-derived somatic stem cells from a group of cells containing cells constituting the kidney, including an expression vector that expresses a protein having an activity to pass through the G0 phase or G1 phase and enter the S phase, and an extracellular growth factor Kit for.

By the method of the present invention, it is necessary to isolate somatic stem cells using a conventional cell sorter or the like by proliferating kidney-derived somatic stem cells predominantly from the diverse cell populations that make up the kidney. Compared with the method, it is possible to produce kidney-derived somatic stem cells efficiently and with high purity. By using the present invention, even if a large number of cells are not required, such as a cell sorter, if there is a relatively small number of cells constituting the kidney (for example, about 10 5 to the 6th power), the physicality can be efficiently generated from the cells. Stem cells can be obtained. Moreover, the kidney-derived somatic stem cells produced by the method of the present invention can be differentiated into differentiated cells constituting the kidney according to the purpose.

In the present invention, the gene introduced to proliferate kidney-derived somatic stem cells can be transiently expressed. Since the kidney-derived somatic stem cells produced in this way do not contain exogenous genes, they have substantially the same structure and properties as kidney stem cells that proliferate in vivo. Therefore, the kidney-derived somatic stem cells of the present invention are suitable for transplantation into a living body from the viewpoint of safety.

Since the kidney-derived somatic stem cells obtained by the present invention also function as a host cell that expresses an exogenous gene, the exogenous gene is introduced into the kidney-derived somatic stem cell and used as a gene therapy drug. Is also possible.

FIG. 1 shows the results of obtaining cells showing clonal growth obtained in Example 1. FIG. 2 shows the results of obtaining cells showing clonal expansion obtained in Example 2. FIG. 3 shows the result of characterization of cells showing clonal growth in Example 3. FIG. 4 shows the results of induction of differentiation of clonal proliferative cells into kidney cells in Example 4. For each marker, the left bar shows the results for day 0 and the right bar shows the results for day 28.

Hereinafter, specific embodiments of the present invention will be described.

A. Method for Producing Kidney-derived Somatic Stem Cells The method for producing kidney-derived somatic stem cells of the present invention includes the following steps (A) and (B):
(A) In a cell group including cells constituting the kidney, a step of expressing a gene of a protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase; and (B) obtained in the step (A). Culturing cultured cells in the presence of extracellular growth factor.

1. Step (A)
Step (A) is a step of expressing a gene of a protein having an activity of passing through the G0 phase or the G1 phase and shifting to the S phase in a cell group including cells constituting the kidney.

(1) Cell group containing cells constituting the kidney The “cell group containing cells constituting the kidney” used as a raw material only needs to contain cells constituting the kidney, and may contain other cells. The ratio of the cells constituting the kidney in the cell group is not particularly limited, but it is preferable to use a cell group consisting only of cells constituting the kidney from the viewpoint of production efficiency. The cells constituting the kidney include, for example, proximal tubular epithelial cells, distal tubular epithelial cells, glomerular endothelial cells, human renal mesangial cells, renal cortical epithelial cells, and the like, and a few kidneys There are stem cells. It is preferable that a renal stem cell is contained in the cell group containing the cell which comprises a kidney. The ratio of the renal stem cells contained in such a cell group is not particularly limited, but may be contained, for example, at a ratio of 1/1000000 to 1/10000.

As the cell group including cells constituting the kidney, commercially available cells may be used, or cells collected from a living body by a surgical technique may be used. The cell group including the cells constituting the kidney is, for example, a primary cultured cell or a subcultured cell obtained by repeating subculture within a range in which the original function of the cell is maintained after being collected from the living body (this book In the specification, it may be referred to as “early subcultured cell”). When using the initial subculture cells, the number of subcultures is not particularly limited as long as kidney-derived somatic stem cells can be produced, but is preferably within 10 times, more preferably within 5 times. Preferably, it is more preferably within 2 times.

The organism from which the cell group including the cells constituting the kidney is derived can be appropriately selected according to the purpose, for example, mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, goat, Examples include mammals such as monkeys and humans. When the obtained kidney-derived somatic stem cells are used for research or treatment of human diseases, they are preferably derived from humans.

The cell group including cells constituting the kidney preferably has high viability. For example, the viability is 70% or more, more preferably 80% or more, and further preferably 90% or more. The viability of the cells can be measured using a commercially available analyzer. Moreover, it is preferable that the cell group containing the cells constituting the kidney has a high adhesion rate (for example, 70% or more) to a plate coated with collagen or the like.

Viability of a cell group including cells constituting the kidney can be measured according to a known method. For example, a cell group including cells constituting the kidney is treated with trypan blue dye and stained blue. It can be determined by measuring the ratio of dead cells using a microscope or the like.

(2) A protein having an activity of passing through the G0 phase or the G1 phase and transferring to the S phase as a gene introduced into the cell “a protein having an activity of passing through the G0 phase or the G1 phase and transferring to the S phase” (this specification) In the text, “cell cycle reactivation protein”) is not particularly limited as long as it has an activity of passing through the G0 phase or G1 phase and shifting to the S phase.

“Transfer to the S phase by passing the G0 phase or the G1 phase” means (1) acting on the cells in the G0 phase and being in a dormant state by deviating (escaping) from the cell cycle. Or (2) acting on a cell in the G1 phase to shift the cell cycle from the G1 phase to the S phase.

The presence / absence of “activity that passes through the G0 period or G1 period and shifts to the S period” can be confirmed by the following method. That is, confirmation of the transition to S phase (DNA synthesis phase) can be determined by examining the activity of 5-bromo-2-deoxyuridine (BrdU), which is an analog of thymidine, into the cells. Specifically, BrdU is put into a cell culture medium, and then the cell surface is immunostained by reacting with a fluorescently labeled anti-BrdU antibody, and analyzed using a flow cytometer or the like.

As the cell cycle reactivation protein, for example, a protein having an action of promoting phosphorylation of Rb protein can be used. Examples include cyclin-dependent kinases and cyclins. Examples of cyclin-dependent kinases include CDK1, CDK2, CDK3, CDK4, CDK6, and CDK7. An example of a cyclin is cyclin D. Among these, CDK4 and CDK6 are preferable, and CDK4 is more preferable. Only one kind of cell cycle reactivation protein gene may be used alone, or a plurality of kinds may be used in combination.

The origin of the cell cycle reactivation protein gene is not particularly limited as long as the effect of the present invention is exhibited. It may be the same species as the animal species from which the cell group including the cells constituting the kidney used in step (A) is derived, or may be a different animal species. From the viewpoint of expression efficiency and the like, the origin of the cell cycle reactivation protein gene is preferably derived from the same species as the animal species from which the cell group including the cells constituting the kidney is derived.

The means for expressing the cell cycle reactivation protein gene is not limited as long as the cell cycle reactivation protein can be expressed. For example, transient expression may be used, or stable expression may be used. Transient expression means that a gene is introduced into a cell by a DNA transfection method or the like and expressed transiently. Transient usually refers to a period of hours to days. In contrast, stable expression means that a gene to be expressed is stably expressed in a chromosome. From the viewpoint that the kidney-derived somatic stem cell and its differentiated cell produced by the method of the present invention have the same or as similar structure and properties as the kidney-derived somatic stem cell or its differentiated cell in vivo, The gene for the cell cycle reactivation protein is preferably expressed transiently.

Transient expression is not particularly limited, and can be performed, for example, by introducing an expression vector having a target gene downstream of an expression promoter into a cell and expressing the gene from this expression vector. In this case, as an expression promoter, for example, CMV promoter, SV40 promoter and the like can be used, but are not limited thereto. Examples of expression vectors include, but are not limited to, plasmid vectors and liposomes as non-viral vectors, and adenovirus vectors and retrovirus vectors as virus vectors. It is preferable to use a non-viral vector from the viewpoint of safety when using the cells to be produced for pharmaceutical purposes and ensuring that the gene to be introduced is transiently expressed, and in particular, the origin of replication in the host cell. Non-viral vectors that do not contain are preferred. In order to carry out transient expression more reliably, it is possible to add a step of confirming that the introduced cell is not incorporated into the chromosome. Examples of plasmid vectors that can be used from such a viewpoint include pcDNA, pSVL, and the like. As a method for introducing an expression vector into a cell, for example, a lipofection method, an electroporation method, a method in which a gene is incorporated into a viral vector and infected, and the like can be used, but not limited thereto.

Although stable expression is not particularly limited, it can be performed, for example, by the following method. An expression vector having a target gene and a dominant selection marker downstream of the expression promoter is introduced into the cell, and a strain in which the target gene is integrated into the chromosome is established. In this established strain, stable expression is performed. In this case, as an expression promoter, for example, CMV promoter, SV40 promoter and the like can be used, but are not limited thereto. As a dominant selection marker, for example, various drug resistance genes can be used, but not limited thereto. When a drug resistance gene is used as a dominant selection marker, only cell lines that stably express the drug resistance gene can be selected by continuing cell culture in the presence of a drug exhibiting resistance. . Usually, in such a cell line, the target gene is considered to be stably expressed as well. Whether or not the target gene is actually stably expressed can be clarified by analyzing the base sequence of the chromosome by a DNA sequence or the like. Moreover, as a method for introducing an expression vector into a cell, for example, a lipofection method, an electroporation method, or the like can be used, but is not limited thereto. When a viral vector is used, a method of incorporating a gene into a viral vector and infecting it can also be used.

In addition, when gene transfer is performed on primary cultured cells, it is preferable to use a transfection reagent that is considered to be relatively weak in cytotoxicity. In order to improve transfection efficiency, for example, it is preferable to perform transfection on cells in good condition. In the case of primary cells, the cell state is relatively good within 2 to 3 days after seeding on a culture plate.

Step (A) is preferably performed while culturing in a medium that supports the growth of cells (preferably kidney stem cells) constituting the kidney. For example, DMEM (Doulbecco's modified Eagle's Medium) medium (manufactured by Gibco) used for normal mammalian cell culture is used as a basal medium, and a medium supplemented with fetal bovine serum, human serum or the like is preferably used. be able to.

Extracellular growth factor may be further added to the medium used when performing step (A). An extracellular growth factor is a substance having an action of externally supporting the growth of kidney-derived somatic stem cells, and any substance having such action may be used without particular limitation. Examples of extracellular growth factors include cell growth factors and hormones that stimulate cell growth. Examples of the cell growth factor include epidermal growth factor (EGF), hepatocyte growth factor (Hepatocyte Growth Factor; HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial cells. Examples include growth factors (Vascular Endothelial Growth Factor; VEGF) and fibroblast growth factors (FGF). Of these, EGF and HGF are preferred. As hormones, for example, insulin can be preferably used. These may be used alone or in combination of two or more. For example, by using at least one cell growth factor selected from the group consisting of EGF and HGF in combination with at least one cell growth factor selected from the group consisting of VEGF and FGF, Proliferation can be improved synergistically.

The concentration of the extracellular growth factor added in the medium is not particularly limited. For example, it is preferably 0.1 to 200 ng / ml, more preferably 1.0 to 100 ng / ml, and 5 to 50 ng / ml. Further preferred.

In addition to the DNA encoding the cell cycle reactivation protein, other genes can be introduced into the cell group including the cells that constitute the kidney unless the proliferation and pluripotency of kidney-derived somatic stem cells are inhibited. Can be expressed. However, from the viewpoint that the kidney-derived somatic stem cells proliferated by the following step (B) have structural and property characteristics that are the same as or infinitely similar to those of kidney somatic stem cells in vivo. Preferably, exogenous DNA other than the DNA of the activating protein (eg, cyclin-dependent kinase) is substantially not contained. From the same point of view, for example, the gene involved in the regulation of the cell cycle introduced into kidney-derived somatic stem cells is only the gene of a cell cycle activation protein (eg, cyclin-dependent kinase, particularly CDK4 or CDK6). Is preferred. In one embodiment, the kidney-derived somatic stem cells grown according to the present invention preferably do not contain a gene encoding human telomerase reverse transcriptase.

2. Process (B)
Step (B) is a step of culturing the cells obtained in step (A) in the presence of an extracellular growth factor.

In the step (B), kidney-derived somatic stem cells proliferate predominantly from the group of cells containing the cells constituting the kidney as follows. In kidney-derived somatic stem cells, first, the cell cycle transitions from the G0 phase or G1 phase to the S phase by the action of the cell cycle reactivating protein introduced from the outside in the step (A), and further to the M phase (divide). Period), and then proceed to the G1 period again. At the same time, the action of the cell cycle reactivation protein is activated by the action of the extracellular growth factor present in the medium. As a result, kidney-derived somatic stem cells continue to proliferate. On the other hand, in cells different from kidney-derived somatic stem cells, proliferation activation as described above due to the action of cell cycle reactivation protein does not occur, or proliferation is temporary and does not continue. As a result, kidney-derived somatic stem cells proliferate predominantly from a group of cells containing cells that constitute the kidney.

In the step (B), the cell in which the gene for the cell cycle reactivation protein is expressed in the step (A) is cultured in the presence of an extracellular growth factor. When the medium used in the step (A) contains an appropriate extracellular growth factor from the beginning, cells expressing the cell cycle reactivation protein gene may be cultured as they are without changing the medium. Good. Further, the extracellular growth factor may be further added to the medium without changing the medium. The medium may be replaced with a medium containing the extracellular growth factor, or the medium may be replaced with a medium not containing the extracellular growth factor, and then the extracellular growth factor may be added to the medium.

As the extracellular growth factor, the extracellular growth factor (for example, EGF, HGF, VEGF, FGF, PDGF, IGF, etc.) described above with respect to the step (A) can be used.
The concentration of the extracellular growth factor added in the medium is not particularly limited. For example, it is preferably 0.1 to 200 ng / ml, more preferably 1.0 to 100 ng / ml, and 5 to 50 ng / ml. Further preferred.

When a new medium is used in the step (B), a DMEM (Doulbecco's modified Eagle's Medium) medium (manufactured by Gibco) or the like used for normal mammalian cell culture is used as a basal medium, and kidney-derived somatic stem cells are used. What added the component which supports proliferation further can be used.

In step (B), the medium may be changed at an appropriate interval. The frequency of replacing the medium is not particularly limited, but the medium may be replaced once every two or three days. The medium may be replaced with a medium containing the same concentration of extracellular growth factor, or may be replaced with a medium containing a different concentration of extracellular growth factor. The medium may be replaced with a medium containing the same extracellular growth factor, or may be replaced with a medium containing a different extracellular growth factor.

Other culture conditions such as culture temperature can be appropriately set according to known methods according to the type of kidney-derived somatic stem cells to be cultured.

Step (A) and Step (B) may be performed once each, or may be repeated a plurality of times with these as one set. When it is performed a plurality of times, it can be performed preferably 2 to 10 times, more preferably 3 to 8 times, and even more preferably 3 to 5 times. Repeating the steps (A) and (B) in this manner is preferable when the gene encoding the cell cycle reactivation protein is transiently expressed. This is because transient expression is the peak of expression about 3 days after transfection, and in order to shift kidney-derived somatic stem cells to a proliferative state, expression of the gene continued for a certain period of time. This is because it is considered preferable.

Process (A) and process (B) can be performed successively in this order, or other processes may be performed after process (A), and then process (B) may be performed. Step (B) or repetition of steps (A) and (B) can be continued until the required amount of kidney-derived somatic stem cells is obtained. For example, as shown in Example 1 described later, it is preferable to continue the steps (A) and (B) for about 20 to 60 days while repeating the steps (A) and (B).

When the step (B) is performed only once, for example, if the step (B) is continued until colony formation by kidney-derived somatic stem cells is sufficiently performed, a group of kidney-derived somatic stem cells can be easily collected. preferable. The end point of the step (B) is preferably a time point when a colony of kidney-derived somatic stem cells can be confirmed with a microscope or the naked eye, or a time point when a colony of kidney-derived somatic stem cells consisting of 10 to 10,000 cell groups is formed. More preferably, it is the time when a colony of kidney-derived somatic stem cells consisting of a group of 100 to 1000 cells is formed. Cells different from kidney stem cells that were initially included in the group of cells containing kidney-derived somatic stem cells will cease to divide due to the cell division lifetime at the latest by the end of step (B), and most of them will be lost. Even if it remains, the form has changed greatly from the beginning. For this reason, it can be easily distinguished from kidney-derived somatic stem cells forming colonies, and a highly pure kidney-derived somatic stem cell group can be obtained.

When the step (A) and the step (B) are repeated, for example, if the step (A) and the step (B) are repeated until colony formation by the kidney-derived somatic stem cells is sufficiently performed, the kidney-derived somatic stem cell group Is preferable because it can be easily collected. The number of times the step (A) and the step (B) are repeated is preferably the time when a colony of kidney-derived somatic stem cells can be confirmed with a microscope or the naked eye, or the number of colonies of kidney-derived somatic stem cells composed of 10 to 10,000 cell groups. Until formation of a colony of kidney-derived somatic stem cells consisting of a group of 100 to 1000 cells is more preferable. A colony is usually formed within a few weeks to a month from the start of the step (B). Cells different from renal stem cells, which were originally included in the cell group including the cells constituting the kidneys, have stopped cell division because of the cell division lifetime by the end of the last step (B) at the latest, Most of them have dropped out or remain, but the shape has changed greatly from the beginning. For this reason, it can be easily distinguished from kidney-derived somatic stem cells forming colonies, and a highly pure kidney-derived somatic stem cell group can be obtained. In this case, each step (B) can be continued as long as the cells continue to grow. However, it can be terminated during the growth.

As described above, due to the step (B), the renal stem cells eventually form colonies in order to maintain good proliferative ability from the cell group including the cells constituting the kidney. The other cells have a cell division lifetime at the end of the step (B), so that the cell division is stopped, and many of them have dropped or remain, but the shape has changed greatly from the beginning. Therefore, it can be easily distinguished from kidney-derived somatic stem cells forming colonies, and by collecting this colony, the kidney-derived somatic stem cell population can be isolated as a clone.

3. Other Kidney-derived somatic stem cells can be obtained by performing a step of collecting colonies after the step (B). Colony recovery can be performed by a conventionally known method. Although not limited, it can be performed by, for example, a limiting dilution method or a method using a micropipette under a microscope.

According to the production method of the present invention, preferably a kidney-derived somatic stem cell group containing 50% or more of kidney-derived somatic stem cells in terms of the number of cells, more preferably a kidney-derived somatic stem cell group containing 80% or more of kidney-derived somatic stem cells. Can be manufactured. By the production method of the present invention, it is possible to produce a group of somatic stem cells, more preferably substantially composed of only kidney-derived somatic stem cells, and more preferably an isolated kidney-derived somatic stem cell group.

In the present invention, determination of kidney-derived somatic stem cells is not particularly limited, but can be performed, for example, by examining whether or not they differentiate into target cells. In the present invention, kidney-derived somatic stem cells can also be determined by confirming the presence or absence of a cell surface marker. For example, the determination of being a kidney-derived somatic stem cell can be performed based on whether any of molecular markers such as Pax-2 and Wnt-4 positive is positive. Preferably, the determination is performed based on two or more of the molecular markers being positive.

Since the kidney-derived somatic stem cells obtained by the method of the present invention have the same properties as in vivo kidney stem cells, they can be used to stably supply a high-purity population of differentiated kidney cells. The kidney-derived somatic stem cells obtained by the method of the present invention are not only cells derived from somatic stem cells originally present in the kidney but also cells other than somatic stem cells in the above steps (A) and (B). Also included are cells that have acquired clonal proliferation and pluripotency. Kidney differentiated cells can finally be utilized in clinical applications such as cell preparations, or in various research and development such as new drug development or disease research.

B. Method for producing differentiated cells constituting kidney The method for producing differentiated cells constituting the kidney includes a step of differentiating the kidney-derived somatic stem cells obtained by the method A described above.

The step of differentiation can be performed in vitro or in vivo according to a known method. As a method for differentiation in vitro, for example, kidney-derived somatic stem cells can be cultured in a medium suitable for differentiation, and differentiation into various differentiated cells constituting the kidney can be induced. A medium and other culture conditions suitable for differentiation can be appropriately selected and set from known conditions according to the type of target differentiated cells. As a technique for differentiation in vivo, for example, as shown in Example 4 described later, stem cell spheroids are formed on a cell culture plate, transplanted under the kidney capsule of an animal, and maintained for a certain period of time. Can be implemented. Confirmation of differentiation can be confirmed by, for example, expression of AQP-1 which is a proximal tubular marker, THP which is a distal tubular marker, WT1 which is a glomerular epithelial cell marker, and the like.

The culture for differentiating the kidney-derived somatic stem cells may be two-dimensional culture or three-dimensional culture, but three-dimensional culture is preferable from the viewpoint of inducing differentiation faster. The three-dimensional culture is literally a three-dimensional culture of cells, and various substrates and kits for three-dimensional culture can be used. The culture time can be appropriately set according to the type of differentiated cells for the purpose.

It is preferable that the kidney-derived somatic stem cells are preliminarily cultured for a certain period (priming culture) in the presence of an activating substance prior to culture in a medium suitable for differentiation. The priming culture is a process in which differentiation is advanced by one step, and this makes it possible to shorten the culture period necessary for subsequent differentiation. The priming culture can be performed, for example, in the presence of BMP or FGF.

Cells that make up differentiated kidneys can be used for various research materials and kidney transplantation. For example, a kidney-derived somatic stem cell is produced from a cell group containing cells constituting the kidney obtained from a patient suffering from renal failure, etc., and differentiated into cells constituting the kidney, or undifferentiated stem cells The transplantation treatment in which rejection is suppressed can be performed by transplanting the patient as it is.

C. Pharmaceutical Composition Another embodiment of the present invention is a pharmaceutical composition (cell preparation) comprising kidney-derived somatic stem cells obtained by the method of the present invention and / or kidney differentiated cells obtained by further differentiation thereof. The pharmaceutical composition of the present invention can be used for the treatment of various renal dysfunctions (eg, renal failure).

The cells contained in the pharmaceutical composition of the present invention may be those into which an exogenous gene has been introduced. Introduction of exogenous genes into kidney-derived somatic stem cells or kidney differentiated cells produced by the method of the present invention can be performed using methods known in the art. The exogenous gene to be introduced can be appropriately selected according to the purpose of use of the cell preparation (for example, gene therapy).

The dosage form of the pharmaceutical composition of the present invention is appropriately set according to the affected area to be applied. Examples include intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, submucosal injection, intraperitoneal injection, and the like. In addition to this, for example, a method of administration by attaching cells formed into a sheet form by culturing to the affected area to be applied can also be mentioned. Alternatively, a tissue-like structure in which cells are three-dimensionally cultured using a so-called scaffold having biocompatibility can be attached or transplanted to the affected tissue.

The dosage form of the pharmaceutical composition of the present invention is appropriately set according to the dosage form and the like. Examples thereof include a liquid agent in which cells are suspended in a liquid, a gel agent in which cells are suspended in a gel, a cell sheet, and a tissue-like cell aggregate.

The dosage and administration frequency of the pharmaceutical composition of the present invention are appropriately set according to the administration form, dosage form, recipient's condition, cell activity level, disease type, and the like. The dose per administration may be a therapeutically effective amount. For example, the pharmaceutical composition of the present invention may be administered at a frequency of once per day or divided into 2 or 3 times, and doses for 2 days to 1 week may be administered at a time. Also good. The proportion of kidney-derived somatic stem cells or kidney differentiated cells contained in the pharmaceutical composition can be appropriately set according to the dosage form, dosage form, dosage, administration frequency, and the like.

The cell preparation of the present invention may further contain other components as necessary in addition to the active ingredients (kidney-derived somatic stem cells or kidney differentiated cells). Examples of such components include excipients necessary for formulation depending on the dosage form, storage-stable components necessary for storage stability, and other medicinal ingredients. Examples of other medicinal ingredients include anti-inflammatory agents, antibacterial agents, immunosuppressive agents, cell growth factors, hormones and the like.

D. Kidney-derived somatic stem cell isolation kit The kit for isolating kidney-derived somatic stem cells of the present invention comprises a cell comprising cells constituting the kidney, comprising an expression vector for expressing a cell cycle reactivation protein and an extracellular growth factor. A kit for isolating kidney-derived somatic stem cells from a group.

The cell group including the cells constituting the kidney, the extracellular growth factor, the cell cycle reactivation protein, and the expression vector are described in A. This is the same as described in. In addition, the kit includes A.I. It may contain a suitable extracellular growth factor described in.

Hereinafter, although an example and a test example are shown and the present invention is explained in detail, the present invention is not limited to these examples.

Example 1. Acquisition of somatic stem cells derived from human kidney cells (1) Culture of primary human kidney cells Resuscitation of human proximal tubular epithelial cells (DS Pharma Biomedical, RPCT-F) and human kidney mesangial cells (Cell Systems, ACBRI127) Thereafter, the human proximal tubular epithelial cells and human kidney mesangial cells were mixed so that the number of cells was approximately 1: 1, suspended in DMEM medium containing 10% FCS, and then coated on a collagen-coated 12-well cell culture plate. The cells were seeded at a cell density of about 1.0 × 10 ⁵ cells / well. The seeded cells were cultured in an incubator under conditions of 37 ° C. and 5% CO ₂ .

(2) Transfection Two days after seeding with human proximal tubular epithelial cells and human renal mesangial cells, transfection was performed under the conditions of the following medium and transgene. For transfection, a commercially available protein expression plasmid pcDNA3 (Invitrogen) was used, and human CDK4 was cloned into the cloning site between EcoRI and XbaI, or DNA encoding CDK6 was cloned into the cloning site between HindIII and BamHI. Inserted. DNA coding for human CDK4 or CDK6 is based on the nucleotide sequence registered in NCBI (National Center for Biotechnology Information) (CDK4 gene accession number: CAG47043, CDK6 gene accession number: NP-001138778). RT using the total RNA designed and purified from HuS-E / 2 cells (human hepatocyte-derived cells and deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology: FERM ABP-10908) Obtained by performing PCR. As a primer for obtaining DNA encoding CDK4, a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 were used. As a primer for obtaining DNA encoding CDK6, a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 were used. The base sequences of SEQ ID NOs: 1 and 3 include a sequence corresponding to Flag Tag.

0.2 μg per well of a plasmid (pcDNA-FLAG-CDK4 or pcDNA-FLAG-CDK6) in which a FLAG tag is introduced on the N-terminal side of the open reading frame of the DNA encoding human CDK4 or CDK6 obtained in this way It was added together with Effectene transfection reagent (Qiagen) so that the gene was introduced into the cells.

Transfection was performed under the following two conditions, and was repeated 3 times at a frequency of once every 3 to 5 days.
Group 1: 20 ng / ml hepatocyte growth factor (HGF) in DMEM-based medium containing 5% fetal bovine serum, 5% human serum (human HGF, Toyobo, Code: HGF-101, CHO cell recombinant) Medium supplemented with The transgene is CDK4 or CDK6 (pcDNA-FLAG-CDK4 or pcDNA-FLAG-CDK6).
Group 2: Same medium as Group 1. The only transgene is a plasmid (pcDNA3).

(3) Acquisition of somatic stem cells The cell preparation of (1) above was performed a total of three times, changing the lot of human cells, and the transfection of (2) was performed on each cell.

On the 48th day of culture, all the transfected cells under the condition of the second group (negative control) were lost. At this point in time, several colonies consisting of cells showing clonal growth were seen from the pcDNA-FLAG-CDK4 transfected cells. Similarly, several colonies were formed from cells transfected with pcDNA-FLAG-CDK6. These colonies were cloned by the limiting dilution method to obtain cells exhibiting clonal growth. The clones obtained by this series of operations were confirmed to have a self-replicating ability because they continued to grow for more than half a year after the establishment. The number of clonal cells obtained as a result of three experiments is shown in FIG.

Example 2 Obtaining human kidney cell-derived somatic stem cells In Example 1, the growth factor added to the medium used for transfection was 10 ng / ml FGF (fibroblast growth factor), and the same experimental procedure was performed. The acquired number of kidney-derived somatic stem cells is shown in FIG.

Example 3 Characterization of human kidney cell-derived somatic stem cells Some of the clones obtained in Examples 1 and 2 were examined with a focus on differentiation markers related to kidney stem cells. That is, for Pax-2 and Wnt-4, RT-PCR was performed using total RNA extracted by collecting cells, and mRNA expression was examined. The result is shown in FIG. In the figure, positive is represented as (+) and negative as (−).

The obtained cells showing clonal growth were positive for either Pax-2, Wnt-4, or both. From this result, it was found that the cells showing clonal proliferation obtained by the present invention present markers that are characteristic of kidney stem cells and kidney progenitor cells.

Example 4 Induction of differentiation of cells exhibiting clonal growth derived from human kidney cells into kidney cells The cells exhibiting clonal proliferation obtained by the present invention were added to a cell culture plate (Sumitomo Bakelite, MS-9096U) having a low adhesion property. Cultured for ~ 2 weeks to form spheroids (spherical cell mass). Next, this spheroid was transplanted under the kidney capsule of NOD SCID mice (male, 3 weeks old, Nippon Charles River). After transplantation, the transplanted tissue piece (a part of the cell mass) was collected 4 weeks later. RNA was extracted from the collected tissue pieces, and gene expression analysis was performed by real time PCR. The expression intensity of each marker was calculated as a relative value with the expression intensity in the spheroid immediately before transplantation as 100%. As a result, it was confirmed that the expression of AQP-1, which is a proximal tubule marker, THP, which is a distal tubule marker, and WT1, which is a glomerular epithelial cell marker, was strongly induced. The results are shown in FIG.

This result shows that the cells showing clonal proliferation obtained by the present invention have the ability to differentiate into a plurality of types of cells constituting the kidney, that is, are kidney-derived somatic stem cells.

Claims

A method for producing kidney-derived somatic stem cells, comprising the following steps (A) and (B):
(A) In a cell group including cells constituting the kidney, a step of expressing a gene of a protein having an activity of passing through the G0 phase or G1 phase and transferring to the S phase; and (B) obtained in the step (A). Culturing cultured cells in the presence of extracellular growth factor.
The method of claim 1, wherein the expression is transient expression.
The method according to claim 1 or 2, wherein the step (A) and the step (B) are repeated twice or more.
The method according to any one of claims 1 to 3, wherein the protein is a cyclin-dependent kinase.
The method according to any one of claims 1 to 4, wherein the protein is at least one protein selected from the group consisting of cyclin-dependent kinase 4 and cyclin-dependent kinase 6.
The extracellular growth factor is epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like The method according to any one of claims 1 to 5, wherein the method is at least one extracellular growth factor selected from the group consisting of growth factors (IGFs).
A kidney-derived somatic stem cell obtainable by the method according to any one of claims 1 to 6.
The cell according to claim 7, wherein the kidney-derived somatic stem cell is Pax-2 positive and / or Wnt-4 positive.
A cell in which an exogenous gene is further introduced into the cell according to claim 7 or 8.
A method for producing a differentiated cell, comprising a step of differentiating the cell according to any one of claims 7 to 9 into a cell constituting a kidney.
The method according to claim 10, wherein the step of differentiating includes a step of performing three-dimensional culture.
A cell constituting the kidney, which can be obtained by the method according to claim 10 or 11.
A pharmaceutical composition comprising the cell according to any one of claims 7 to 9 and 12.
Isolating kidney-derived somatic stem cells from a group of cells containing cells constituting the kidney, including an expression vector that expresses a protein having an activity to pass through the G0 phase or G1 phase and enter the S phase, and an extracellular growth factor Kit for.