COMPOSITION AND KIT FOR DIFFERENTIATION OF STEM CELLS INTO NEURAL PROGENITOR CELLS, AND METHOD USING SAME

Abstract

Provided are a composition and a kit for differentiation of stem cells into neural progenitor cells (NPCs), each including a protein kinase C (PKC) inhibitor and a bone morphogenic protein (BMP) inhibitor, and a method using the same. Accordingly, stem cells may be efficiently differentiated into neural progenitor cells using a simple, low-cost composition.

Claims

1. A composition for differentiation of stem cells into neural progenitor cells (NPCs), the composition comprising a protein kinase C (PKC) inhibitor and a bone morphogenic protein (BMP) inhibitor.

2. The composition of claim 1, wherein the PKC inhibitor is a PKC-3 inhibitor.

3. The composition of claim 1, wherein the PKC-3 inhibitor is selected from the group consisting of: 2-[1-(3-dimethylaminopropyl)-5-methoxy indol-3-yl]-3-(1H-indol-3-yl) maleimide; 3-(1-(3-imidazol-1-yl propyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione; (3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione; (3-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide methane sulfonate; 13-hydroxyoctadecadienoic acid; bisindolylmaleimide; 2,6-diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide; 4′-demethylamino-4′-hydroxystaurosporine; and 3-(13-methyl-5-oxo-6,7-dihydro-5H-indolo[2,3-a] pyrrolo [3,4-c] carbazol-12(13H)-yl) propanenitrile.

4. The composition of claim 1, wherein the BMP inhibitor is selected from the group consisting of: dorsomorphin (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine); dorsomorphin homolog 1 (DMH1, 4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline); K 02288 (3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol); LDN 212854 (5-(6-(4-(1-piperazinyl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone); and Noggin polypeptide.

5. The composition of claim 1, further comprising a cell culture medium selected from the group consisting of a Dulbecco Modified Eagle's Medium (DMEM), a DMEM/F12, a F-10 nutrient medium (Nutrient M), a minimum essential medium (MEM), an RPMI medium 1640, an Opti-MEM I reduced serum medium, an Iscove's Modified Dulbecco's Medium (IMDM), an alpha-MEM, and a neurobasal medium.

6. The composition of claim 5, further comprising one selected from the group consisting of a Ham's F12 nutrient mixture, a B27 supplement, an F-10 nutrient mixture, an F-12 nutrient mixture, an N2 supplement, an HT supplement, a G-5 supplement, a lipid supplement, a serum replacement, and an insulin-transferrin-selenium (ITS) supplement.

7. The composition of claim 1, wherein a concentration of the PKC inhibitor in the composition is 0.1 μM to 20 μM.

8. The composition of claim 1, wherein a concentration of the BMP inhibitor in the composition is 0.01 μM to 20 μM.

9. The composition of claim 1, comprising a cell culture medium, an insulin-transferrin-selenium (ITS) supplement, the PKC inhibitor, and 0.25 μM to 5 μM of the BMP inhibitor.

10. The composition of claim 1, comprising a cell culture medium, a serum replacement, the PKC inhibitor, and 5 μM to 10 μM of the BMP inhibitor.

11. The composition of claim 1, wherein the stem cells are embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), or somatic cell nuclear transfer embryonic stem cells.

12. A kit for differentiation of stem cells into neural progenitor cells, the kit comprising the composition of claim 1 and a cell culture dish.

13. A method of differentiating stem cells into neural progenitor cells, the method comprising: suspension-culturing the stem cells in a cell culture medium comprising a PKC inhibitor and a BMP inhibitor to obtain embryoid bodies (EBs); and adherent-culturing cells obtained by chopping the EBs to induce differentiation into neural progenitor cells.

14. The method of claim 13, wherein the suspension culture is performed for 1 day to 10 days.

15. The method of claim 13, wherein the adherent culture is performed for 1 day to 15 days.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0043] FIG. 1A shows an illustration of a culture process of inducing differentiation of human PSCs into NPCs according to an aspect, FIG. 1B shows images of embryoid bodies (EBs) obtained by suspension culture for about 4 days in a defined medium 1 including a PKC-beta inhibitor (PKCI) and DMH1, and cells (rossette formed) obtained by adherent culture of the embryoid bodies for about 5 days (left: embryoid bodies, right: cells forming rosettes), and FIG. 1C shows plots showing results of flow cytometry for detecting SOX1 (NPC marker) and P75 (NCSC marker) in cells obtained by culturing for a total of 9 days;

[0044] FIGS. 2A, 2B, and 2C show images (top) of cells cultured for a total of 9 days, and plots showing results of flow cytometry for detecting SOX1 (middle) and P75 (bottom), when embryonic stem cell lines of CHA15-hESC and CHA6-hESC and an induced pluripotent stem cell line (iPS cell #3) were used, respectively;

[0045] FIG. 3A shows images of cells obtained by suspension culture for about 4 days and adherent culture for about 5 days in a defined medium 2 including a PKC-beta inhibitor (PKCI) and DMH1, and FIG. 3B shows plots showing results of flow cytometry for detecting SOX1 and P75 (left: flow cytometry plot of SOX1, right: flow cytometry plot of P75);

[0046] FIG. 4A shows images of cells obtained by suspension culture for about 4 days and adherent culture for about 5 days in a defined medium 3 including a PKC-beta inhibitor (PKCI) and DMH1, and FIG. 4B shows plots showing results of flow cytometry for detecting SOX1 and P75 (left: flow cytometry plot of SOX1, right: flow cytometry plot of P75);

[0047] FIG. 5A shows images of cells obtained by suspension culture for about 4 days and adherent culture for about 5 days in defined media 1 including a PKC-beta inhibitor (5 μM) and different concentrations of DMH1, and FIGS. 5B, 5C, and 5D show plots showing results of flow cytometry for detecting SOX1 and P75 (upper: flow cytometry plot of SOX1, lower: flow cytometry plot of P75); and

[0048] FIG. 6A shows images of cells obtained by suspension culture for about 4 days and adherent culture for about 5 days in defined media 2 including a PKC-beta inhibitor (5 μM) and different concentrations of DMH1, and FIGS. 6B, 6C, and 6D show plots showing results of flow cytometry for detecting SOX1 and P75 (upper: flow cytometry plot of SOX1, lower: flow cytometry plot of P75).

MODE OF DISCLOSURE

[0049] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1. Differentiation of Human Pluripotent Stem Cells into Neural Progenitor Cells

[0050] 1. Induction of Differentiation into Neural Progenitor Cells in Presence of PKC Inhibitor and BMP Inhibitor

[0051] (1) Use of Defined Medium 1 Including PKC Inhibitor and BMP Inhibitor

[0052] Colonies obtained by culturing H9 human embryonic stem cells (hESCs) (WiCell Research Institute, Inc. Madison, Wis., U.S.A.) in a culture medium were dissociated into single cells using accutase (Life Technologies).

[0053] The cells dissociated into single cells were seeded in a culture plate coated with a protein such as vitronectin (VTN) (vitronectin XF, STEMCELL Technologies), laminine (rhLaminin-521, Thermo Fisher Scientific Inc., Waltham, Mass., USA), fibronectin (Thermo Fisher Scientific Inc.), or Matrigel™ (StemCell Technologies, Inc.). An embryonic stem cell (ESC) culture medium was added to the seeded cells to perform undifferentiation culture. As the ESC culture medium, Essential 8 (E8) medium (StemCell Technologies Inc.), TeSR2 medium (StemCell Technologies Inc), StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany), etc. was used.

[0054] The obtained colonies were finely chopped and suspension-cultured under conditions of 37° C. and 5% CO.sub.2 in a defined medium 1 containing 5 μg/ml of a PKC inhibitor and 0.5 μM of a BMP inhibitor for about 4 days to prepare embryoid bodies (EBs). The defined medium 1 included DMEM/F12 (Life Technologies), 10 μg/ml of insulin (Sigma-Aldrich), 9 μg/ml of transferrin (Sigma-Aldrich), and 14 ng/ml of sodium selenite (Sigma-Aldrich). 3-(1-(3-Imidazol-1-yl propyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione) (PKC-beta inhibitor) (Millipore) was used as the PKC inhibitor. DMH1 (dorsomorphin homolog 1) (Tocris Bioscience, USA) which is a compound inhibiting BMP 1 type receptor was used as the BMP inhibitor. As a negative control, dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used instead of the drug.

[0055] EBs, which are round cell masses produced by suspension culture (defined medium 1) for about 4 days, were finely chopped, and then adhered to the bottom of the culture plate, followed by adherent culture for about 5 days (DMEM-F12, 1×N2 supplement, 25 μg/ml of insulin, 20 ng/ml of bFGF, and a matrigel-coated culture plate were used). Microscopic images of EBs obtained by suspension culture for about 4 days, and cells forming rossettes obtained by adherent culture for about 5 days are shown in FIG. 1B (left: embryoid bodies, right: cells forming rosettes).

[0056] 2% (v/v) paraformaldehyde (Sigma-Aldrich) was added to cells cultured for a total of 9 days (suspension culture for 4 days+adherent culture for 5 days), and allowed to react at room temperature for about 10 minutes to fix the cells. 2% (v/v) normal serum/1×PBS (Vector Laboratories, Inc., Burlingame, Calif.) containing 0.1% (v/v) TRITON™ X-100 (Sigma-Aldrich) was added to the cells, and allowed to react at room temperature for 30 minutes to block the fixation. To analyze a proportion of cells expressing SOX1 which is a neural progenitor cell (NPC) marker or P75 protein which is a neural crest stem cell (NCSC) marker, immunostaining was performed using phycoerythrin (PE)-labeled anti-P75 monoclonal antibody (1:50 dilution) (Miltenyi Biotec), and phycoerythrin (PE)-labeled anti-Sox1 monoclonal antibody (1:100 dilution) (BD Biosciences). The immunostained cells were subjected to flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, Sparks, Md., USA). The results of flow cytometry are shown in FIG. 1C.

[0057] As shown in FIG. 1C, it was confirmed that when hESCs were cultured in the defined medium 1 including the PKC inhibitor and DMH1 by the method of differentiating via embryoid bodies, most of the cultured cells expressed SOX1 which is a NPC marker, and the number of cells expressing P75 which is a neural crest stem cell marker was small. Therefore, it was confirmed that when hESCs are cultured in the defined medium 1 including the PKC inhibitor and DMH1, differentiation into NPC is induced.

[0058] Further, two different kinds of embryonic stem cell lines (CHA15-hESC and CHA6-hESC) (CHA stem cell Institute) and one kind of induced pluripotent stem cell line (iPS cell #3) (prepared by a known method) were used, instead of H9 hESCs, and images (upper) of the cells cultured for a total of 9 days, results of SOX1 flow cytometry (middle), and results of P75 flow cytometry (lower) are shown in FIGS. 2A, 2B, and 2C. As shown in FIGS. 2A, 2B, and 2C, it was confirmed that when stem cells are cultured in the defined medium 1 including the PKC inhibitor and the BMP inhibitor, differentiation into NPCs is induced.

[0059] (2) Use of Defined Medium 2 Including PKC Inhibitor and BMP Inhibitor

[0060] H9-hESCs were cultured by the method of differentiating via embryoid bodies as in Example 1.1(1). However, a defined medium 2 described below was used instead of the defined medium 1 used in Example 1.1(1).

[0061] The defined medium 2 was prepared by mixing knockout DMEM (Invitrogen), 20% (v/v) knockout serum replacement (KSR, Invitrogen), 2 mM L-glutamine (invitrogen), 1% (w/v) penicillin/streptomycin (Invitrogen), and 0.1 mM β-mercaptoethanol (Invitrogen).

[0062] Cells obtained by suspension culture for about 4 days and adherent culture for about 5 days (DMEM-F12, 1×N2 supplement, 25 μg/ml of insulin, 20 ng/ml of bFGF, and a matrigel-coated culture plate were used) were examined using a microscope, and images thereof are shown FIG. 3A.

[0063] In the obtained cells, the number of SOX1-expressing cells (i.e., NPCs) and the number of P75-expressing cells (i.e., neural crest stem cells) were determined by flow cytometry, and the results are shown in FIG. 3B.

[0064] As shown in FIG. 3B, it was confirmed that when hESCs were cultured in the defined medium 2 including the PKC inhibitor, most of the cultured cells expressed SOX1 which is a NPC marker, and the number of cells expressing P75 which is a neural crest stem cell marker was relatively very small. Therefore, it was confirmed that when hESCs are cultured in the defined medium 2 including the PKC inhibitor and BMP inhibitor, differentiation into NPCs is induced.

[0065] (3) Use of Defined Medium 3 Including PKC Inhibitor and BMP Inhibitor

[0066] H9-hESCs were cultured by the method of differentiating via embryoid bodies as in Example 1(1). However, a defined medium 3 was used instead of the defined medium 1 used in Example 1(1). The defined medium 3 was prepared by mixing DMEM/F12 (Invitrogen) and N2 supplement (Invitrogen).

[0067] Cells obtained by suspension culture for about 4 days and adherent culture for about 5 days (DMEM-F12, 1×N2 supplement, 25 μg/ml of insulin, 20 ng/ml of bFGF, and a matrigel-coated culture plate were used) were examined using a microscope, and images thereof are shown FIG. 4A.

[0068] In the obtained cells, the number of SOX1-expressing cells (i.e., NPCs) and the number of P75-expressing cells (i.e., neural crest stem cells) were determined by flow cytometry, and the results are shown in FIG. 4B.

[0069] As shown in FIG. 4B, it was confirmed that when hESCs were cultured in the defined medium 3 including the PKC inhibitor, most of the cultured cells expressed SOX1 which is a NPC marker, and the number of cells expressing P75 which is a neural crest stem cell marker was relatively very small. Therefore, it was confirmed that when hESCs are cultured in the defined medium 3 including the PKC inhibitor and BMP inhibitor, differentiation into NPCs is induced.

[0070] 2. Induction of Differentiation into Neural Progenitor Cells in Presence of PKC Inhibitor and BMP Inhibitor

[0071] (1) Use of Defined Medium 1 Including PKC Inhibitor and BMP Inhibitor

[0072] H9-hESCs were cultured by the method of differentiating via embryoid bodies as in Example 1.1(1). However, 5 μM of the PKC inhibitor and 0 μM to 5 μM of the BMP inhibitor (DMH1) were added to the defined medium 1 in Example 1.1(1).

[0073] Cells obtained by suspension culture for about 4 days and adherent culture for about 5 days were examined using a microscope, and images thereof are shown FIG. 5A. In the obtained cells, the number of SOX1-expressing cells (i.e., NPCs) and the number of P75-expressing cells (i.e., neural crest stem cells) were determined by flow cytometry, and the results are shown in FIGS. 5B, 5C, and 5D.

[0074] As shown in FIGS. 5A to 5D, it was confirmed that when H9-hESCs were cultured in the defined medium 1 including 5 μM of the PKC inhibitor and 0.25 μM to 5 μM of DMH1, differentiation into SOX1-expressing NPCs (about 96% or more) was observed. In contrast, it was confirmed that when H9-hESCs were cultured in the defined medium 1 including only 5 μM of the PKC inhibitor without DMH1, differentiation into P75-expressing neural crest stem cells (about 87% or more) was observed.

[0075] (2) Use of Defined Medium 2 Including PKC Inhibitor and BMP Inhibitor

[0076] H9-hESCs were cultured as described in Example 1.2(1). However, the defined medium 2 was used instead of the defined medium 1.

[0077] Cells obtained by suspension culture for about 4 days and adherent culture for about 5 days were examined using a microscope, and images thereof are shown FIG. 6A. In the obtained cells, the number of SOX1-expressing cells (i.e., NPCs) and the number of P75-expressing cells (i.e., neural crest stem cells) were determined by flow cytometry, and the results are shown in FIGS. 6B, 6C, and 6D.

[0078] As shown in FIGS. 6A to 6D, it was confirmed that when H9-hESCs were cultured in the defined medium 2 including 5 μM of the PKC inhibitor and 5 μM of DMH1, differentiation into SOX1-expressing NPCs (about 96% or more) was observed. In contrast, it was confirmed that when H9-hESCs were cultured in the defined medium 2 including 5 μM of the PKC inhibitor and 0 μM to 0.5 μM of DMH1, differentiation into P75-expressing neural crest stem cells (about 84% or more) was observed.