TWI459959B - Selective pharmaceutical composition for the selective removal of undifferentiated universal stem cells and methods thereof - Google Patents

Description

Medicine composition for selectively removing undifferentiated univalent stem cells and method thereof

The present invention relates to the screening of undifferentiated univalent stem cells, and in particular to a pharmaceutical composition for selectively removing undifferentiated univalent stem cells and a method thereof.

By stem cell line is a primitive cell that has not fully differentiated but has the ability to self-renew and differentiate into two or more mature cells. The stem cells are classified into differentiation cells, which can be divided into totipotent stem cells, pluripotent stem cells, multipotent stem cells, and bipotent stem cells: they are differentiated according to stem cell sources. It can be divided into embryonic stem cells, somatic stem cells and induced pluripotent stem cells (iPSC). Among them, the human embryonic stem cell line belongs to the omnipotent stem cell, which is derived from the cell mass in the blastocyst stage before implantation, and has omnipotence and can be differentiated into various adult cells; in addition, the induced univalent stem cell is forced to perform ( Enforced expression) The method of manipulating a specific gene or protein into a differentiated somatic cell, such that the somatic cell is reprogrammed to become an embryonic stem cell.

Since stem cells have the ability to divide, regenerate and regenerate, they can also be induced to differentiate into specific tissues. Therefore, many researchers are currently working on stem cell differentiation, and hope that after stem cells differentiate into specific cells or tissues, they can be used for humans. For the treatment of diseases or regenerative medicine, for example, differentiated cultured dopamine neurons can be used to treat Parkin Sjogren's disease, the organ after differentiation is used for patients with organ damage. In particular, it is necessary for biomedical research such as neurodevelopment, nerve damage, and neurodegenerative diseases, or related drug screening, to be carried out by nerve cells differentiated by stem cells. In addition, many studies have targeted embryonic stem cells to differentiate them into hematopoietic stem cells or islet cells of the pancreas, hoping to solve the problems of blood diseases and diabetes diseases.

However, in the process of stem cell culture differentiation into a predetermined cell, if the cell produced is interspersed with other undifferentiated omnipotent stem cells, and the undifferentiated omnipotent stem cells are moved into an organism, a teratoma is formed. Increases the risk of developing cancer in living organisms. In order to avoid the use of undifferentiated pluripotent stem cells in the cells used, screening for undifferentiated stem cells by fluorescence-activated cell sorting (FACS) is a screening technique, which is a fluorescent marker. An antibody, such as SSEA-5, is added to the univalent stem cells that are being differentiated, the fluorescently labeled antibody is bound to an undifferentiated omnipotent stem cell, and the undifferentiated univalent stem cells are screened by flow cytometry. This method can effectively screen out undifferentiated stem cells, but it takes more time and more manpower to effectively screen out undifferentiated omnipotent stem cells.

Therefore, in order to improve the reliability and safety of stem cells in clinical applications, it is an important subject of current research to find a method for effectively and easily removing undifferentiated univalent stem cells.

The main object of the present invention is to provide a pharmaceutical composition for selectively removing undifferentiated omnipotent stem cells, thereby increasing the concentration of potassium ions in undifferentiated omnipotent stem cells to produce cytotoxicity, thereby causing undifferentiated omnipotent stem cells to die, thereby Cultured by omnipotent stem cells The specific cells obtained are not mixed with undifferentiated omnipotent stem cells.

Wherein, the pharmaceutical composition is a potassium ion carrier or a potassium ion channel promoter.

Among them, the potassium ion carrier is valinomycin or Nigericin.

The undifferentiated univalent stem cell line can be an embryonic stem cell line, an induced omnipotent stem cell or a versatile cell having differentiation into somatic characteristics.

Another object of the present invention is to provide a method for selectively removing undifferentiated univalent stem cells by treating the univalent stem cells with the pharmaceutical composition for selectively removing undifferentiated univalent stem cells.

Wherein, the omnipotent stem cell line is an embryonic stem cell line, an induced omnipotent stem cell or a versatile cell having differentiation into somatic characteristics, and the state of the omnipotent stem cell is differentiated or differentiated.

Wherein the concentration of the pharmaceutical composition is greater than 0.1 nM, and the concentration of the pharmaceutical composition is 5 nM to 500 nM to remove undifferentiated inducible omnipotent stem cells, which may have better efficacy or concentration of the pharmaceutical composition. Screening undifferentiated human embryonic stem cells from 0.1 nM to 500 nM may also have better efficacy.

The first panel A is the observation result under the electron microscope after the culture in the 1-1st group for 3 days.

The first panel B is the observation result under the electron microscope after the culture in the first group for 3 days.

The second panel A is the observation result under the electron microscope after the culture in the 2-1 group for 3 days.

The second panel B is the observation result under the electron microscope after the culture in the 2nd-2 group for 3 days.

The third panel A is the observation result under the electron microscope after the culture in the 3-1rd group for 3 days.

The third panel B shows the results of observation under electron microscope after 3 days of culture in Groups 3-2.

The fourth panel A shows the results of cell growth assay analysis of fixation and staining of TW1 human embryonic stem cells cultured under different treatment conditions.

The fourth panel B is the OD value measured by cell viability analysis of TW1 human embryonic stem cells cultured under different treatment conditions.

Figure 5A shows the results of cell culture survival analysis and fixation by H1 human embryonic stem cells cultured under different treatment conditions.

Figure 5B is the OD value measured by cell viability analysis of TW1 human embryonic stem cells cultured under different treatment conditions.

Fig. 6 is a result of fixation and staining of the induced omnipotent stem cell line IMR90 by cell viability analysis under different treatment conditions.

Figure 6B shows the OD value measured by cell viability analysis of the induced omnipotent stem cell line IMR90 cultured under different treatment conditions.

The seventh panel is the oct4 performance of TW1 human embryonic stem cells cultured on day 8 of differentiation under different conditions.

Figure 8A shows the nerve cells treated with dimethyl hydrazine.

Figure 8B shows the nerve cells treated with valinomycin.

The pharmaceutical composition for selectively removing undifferentiated univalent stem cells disclosed in the present invention is capable of increasing the concentration of potassium ions in undifferentiated omnipotent stem cells to produce cytotoxicity, causing the undifferentiated omnipotent stem cells to die and cannot be subcultured. And the pharmaceutical composition is a potassium ionophore or a potassium ion channel promoter such as valinomycin or Nigericin. Further, by treating the differentiated omnipotent stem cells or the differentiated omnipotent stem cells by the pharmaceutical composition, the undifferentiated omnipotent stem cells are screened out.

Furthermore, the pharmaceutical composition for selectively removing undifferentiated omnipotent stem cells disclosed in the present invention is killed by increasing the concentration of potassium ions in undifferentiated cells, and therefore, the pharmaceutical combination for selectively removing undifferentiated pluripotent stem cells The lines are only cytotoxic to undifferentiated cells and not cytotoxic to differentiated cell lines.

With regard to the nouns used in the present invention, the core of the text is as follows, but the scope of the text is not limited to it: omnipotent stem cells, which are embryonic stem cells containing mammals, and are expressed by genes or proteins. Induced omnipotent stem cells or other versatile cells that differentiate into all somatic characteristics.

Hereinafter, the present invention will be further illustrated by a number of examples and drawings.

It must be explained first that since the pharmaceutical composition for selectively removing undifferentiated univalent stem cells disclosed in the present invention contains a potassium ion carrier or a potassium ion channel promoter, and the types of drugs belonging to the two types are numerous, In the following examples of the invention, only valinomycin is taken as an example, but it is not intended to limit the scope of the invention.

Example 1: Human embryonic stem cells co-cultured with fibroblasts

A human embryonic stem cell TW1 was cultured in a DMEM-F12 medium containing 20% serum substitute (KSR; Invitrogen, USA) at 37 ° C under 5% carbon dioxide to further the human The embryonic stem cells TW1 were treated with different drugs, respectively, wherein no drugs were added in groups 1-1, 2-1, and 3-1; groups 1-2 were added with 5 nM valinomycin; Group 2-2 was a 50 nM calcium ionophore A23187 (Sigma, USA); Group 3-2 was a 50 nM hydrogen ionophore FCCP (Sigma, USA).

After each group was cultured for three days, the results are shown in the first to third figures, wherein the first figure A is the result of the group 1-1; the first figure B is the group 1-2 Results; second panel A is the result of group 2-1; second panel B is the result of group 2-2; third panel A is the result of group 3-1; third panel B is Results of Group 3-2.

From the results of the first to the third figures, it can be seen that the human embryonic stem cell TW1 in the 1-1st group, the 2-1st group, and the 3-1th group is grown into a complete mass with the support of the fibroblast; The human embryonic stem cell TW1 in group 1-2 died, while the co-cultured fibroblasts remained intact and there was no cell death; although groups 2-2 and 3-2 had different ion carriers added, However, the cell type of the human embryonic stem cell TW1 of each group remained intact.

From the above results, it was revealed that the addition of 5 nM of valinomycin caused death of undifferentiated human embryonic stem cells, and it was thus inferred that the addition of a low concentration of potassium ionophore resulted in cytotoxicity of undifferentiated stem cells.

Example 2: Human embryonic stem cells without co-culture (1)

One TW1 human embryonic stem cell was cultured in mTeSR1 (Invitrogen, In the culture medium of USA), the co-culture of fibroblasts is exempted and treated under different conditions, wherein the first group is untreated; the second group is 50 nM calcium ion carrier A23187 ( Sigma, USA); the third group is the addition of 50 nM hydrogen ionophore FCCP (Sigma, USA); the fourth group is the addition of 1 nM valinomycin; the fifth group is the addition of 5 nM valinomycin .

Each group was treated under different conditions for 3 days, and each group of cells was detected by cell viability assay (MTT assay). The results are shown in the fourth panel, wherein the fourth panel A is the cell viability analysis of each group. The results of fixation and staining; the fourth panel B is the result of the OD value measured by cell viability analysis of each group of cells in different culture days.

From the results of the fourth panel A and B, it can be seen that the remaining cell lines of the fourth group and the fifth group are less than those of the first group to the third group, and the cell survival rate of the fifth group is higher than that of the other four groups. The group was significantly reduced. From the above results, it was revealed that the addition of a concentration of at least 1 nM of valinomycin was allowed to cause the death of undifferentiated TW1 human embryonic stem cells.

Implementation 3: Human embryonic stem cells without co-culture (2)

As described in Example 2, the difference is that H1 human embryonic stem cells are used in this example, and according to different treatment conditions, they are divided into six groups, wherein the first group is untreated; the second group is 50 nM calcium ionophore A23187 (Sigma, USA) was added; the third group was a 50 nM hydrogen ionophore FCCP (Sigma, USA); the fourth group was a 0.1 nM valinomycin; the fifth group To add 1 nM of valinomycin; the sixth group was to add 5 nM of valinomycin. The results of detecting the cells of each group by cell viability assay (MTT assay) are shown in the fifth figure, wherein the fifth panel A is fixed and stained by cell survival analysis of each group. The result of coloring; the fifth figure B is the result of the OD value measured by cell viability analysis of each group of cells in different culture days.

From the results of the fifth panel A and B, it can be seen that the remaining cell lines of the fourth group to the sixth group are less than the cells remaining in the first group to the third group, and the cell survival rate of the sixth group is higher than that of the other groups. The five groups were significantly reduced. From the above results, it was revealed that the added concentration of at least 0.1 nM of valinomycin was used to cause the undifferentiated H1 human embryonic stem cells to die.

Example 4: Induced univalent stem cells without co-culture

As described in Example 2, the difference is that in this example, the induced pluripotent stem cells (iPS cells) cell line IMR90 is used, and according to different treatment conditions, it is divided into four groups, wherein the first group The system was untreated; the second group was a 50 nM calcium ionophore A23187 (Sigma, USA): the third group was a 50 nM hydrogen ionophore FCCP (Sigma, USA); the fourth group was added. 5 nM valinomycin. The results of detecting the cells of each group by cell viability assay (MTT assay) are shown in the sixth graph, wherein the sixth panel A is the result of fixation and staining of the cell viability analysis of each group; The result is the OD value measured by cell viability analysis of each group of cells in different culture days.

From the results of the sixth panel A and B, it can be seen that the remaining cell lines in the fourth group are significantly less than the remaining cells in the first group to the third group, indicating that the added concentration of at least 5 nM of valinomycin is undifferentiated. Induced omnipotent stem cell death.

Example 5: TW1 human embryonic stem cells in differentiation

Suspension culture of TW1 human embryonic stem cells in neural cell culture medium (neurobasal medium), after adding 10 mg/ml fibroblast growth factor 2 (FGF2), the TW1 human embryonic stem cells were differentiated, and the TW1 human embryonic stem cells differentiated on the 8th day were treated under different conditions, respectively. Then, oct4 immunofluorescence staining was performed, and the oct4 expression amount was quantified. The results are shown in the seventh figure. Among them, the left straight line in the seventh figure is the control group without added valinomycin, and the right straight line is A control group of valinomycin at a concentration of 50 nM was added.

Since the embryonic stem cell line is expressed by a marker factor such as oct4, undifferentiated embryonic stem cells can be measured by analyzing the performance of oct4. Therefore, the results of the seventh panel A to the seventh panel B show that the performance of oct4 in the case of treatment without valinomycin is significantly greater than that of the person treated with valinomycin, and the image of quinavimycin via the seventh panel C The number of oct4 communities exhibited by the treated TW1 human embryonic stem cells was significantly reduced by about 60% compared with those without valinemycin treatment. From the above results, it was revealed that treatment of the differentiated human embryonic stem cell line with valinomycin causes undifferentiated human embryonic stem cells to die.

Example 6: Cells after differentiation of human embryonic stem cells

Since human embryonic stem cells differentiate into nerve cells, the system expresses β-III tubulin (TuJ1). Therefore, this example is a neuronal cell after differentiation of human embryonic stem cells, respectively, with dimethyl sulfoxide. After treatment with (DMSO) and 50 nM valinomycin, it was found that the neuronal cell line with valinomycin had no obvious death, and the formation of neurites of nerve cells was not significantly inhibited. In the eighth diagram, the eighth panel A is a nerve cell treated with dimethyl hydrazine; the eighth panel B is a nerve cell treated with valinomycin.

It can be seen from the above examples that the pharmaceutical composition for selectively removing undifferentiated univalent stem cells of the present invention does have the function of killing undifferentiated pluripotent stem cells, and at low concentrations, also has the effect of screening undifferentiated pluripotent stem cells. The pharmaceutical composition can be applied to screen out omnipotent stem cells that have indeed completed differentiation to ensure clinical application safety and reduce potential risks in use.

Claims (9)

A pharmaceutical composition for selectively removing undifferentiated univalent stem cells, which is valinomycin, is used as a potassium ion carrier to increase the concentration of potassium ions in undifferentiated univalent stem cells, and to cause undifferentiated omnipotent stem cells to die. The pharmaceutical composition according to claim 1, wherein the undifferentiated pluripotent stem cell line is selected from the group consisting of an embryonic stem cell strain, an induced omnipotent stem cell, and a versatile cell having differentiation into somatic characteristics. A method for selectively removing undifferentiated omnipotent stem cells, which comprises adding a pharmaceutical composition according to claim 1 of the patent application to a culture environment for performing a differentiated omnipotent stem cell, thereby causing a univalent stem cell which is not differentiated into a predetermined cell death. The method according to claim 3, wherein the omnipotent stem cell line is selected from the group consisting of an embryonic stem cell strain, an induced omnipotent stem cell, and a versatile cell having differentiation into somatic characteristics. The method of claim 4, wherein the pharmaceutical composition is added when the pluripotent stem cell is differentiated. The method of claim 4, wherein the pharmaceutical composition is added to the pluripotent stem cell during differentiation. The method of claim 3, 4, 5 or 6, wherein the concentration of the pharmaceutical composition is greater than 0.1 nM. The method of claim 7, wherein the pharmaceutical composition has a concentration of from 5 nM to 500 nM and is used to treat inducible versatile stem cells. According to the method of claim 7, wherein the concentration of the pharmaceutical composition The system is between 0.1 nM and 500 nM and is used to treat human embryonic stem cells.