WO2009116840A2

WO2009116840A2 - Chemical reagents regulating stem cell fate

Info

Publication number: WO2009116840A2
Application number: PCT/KR2009/001443
Authority: WO
Inventors: Dong Wook Kim; Ki-Chul Hwang; Sung Hou Kim; Ji Young Kim; Woo Chul Chang
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2008-03-21
Filing date: 2009-03-20
Publication date: 2009-09-24
Also published as: WO2009116840A3

Abstract

The present invention relates to a method for inducing differentiation of a stem cell, which comprises culturing the stem cell in the presence of an inhibitor to protein kinase A or Rho kinase. The present invention provides novel small molecules having isoquinolinesulfonamide scaffold to effectively induce differentiation of stem cells. The present invention allows for directed differentiation of stem cells to a specific cell type.

Description

CHEMICAL REAGENTS REGULATING STEM CELL FATE

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a method for inducing differentiation of a stem cell and a composition for inducing differentiation of a stem cell.

DESCRIPTION OF THE RELATED ART For practical use of stem cells for regeneration therapy there are at least three prerequisites: (1) the directed differentiation of stem cell to specific cell types, (2) achieving high survival of the cells after transplantation, and (3) prevention of undifferentiated stem cells which are prone to form teratoma or cancer. Since such important cellular processes are likely to be controlled by a complicated orchestration of many signaling pathways, including many as yet undiscovered pathways, common signal modulators, such as protein kinases, are likely to play important role in balancing multiple signals to affect several aspects of these prerequisites. Protein kinases belong to one of the largest protein families in human genome, and they play critical roles in signaling pathways implicated in development, differentiation, proliferation, and death of cells. Several signaling pathways have been known to induce or suppress differentiation of stem cells, such as the pathways involving mitogen-activated kinases, glycogen synthase kinase-3, PI3 kinase, and others (1-5), and a GSK-3 inhibitor synthesized by combinatorial chemistry has been shown to induce neurogenesis (5).

Throughout this application, various patents and publications are referenced, and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION For developing novel chemical compounds capable of inducing differentiation of stem cells, we have broadly tested whether kinase inhibitors may induce differentiation of stem cells to a specific cell type and assembled a small library (41 compounds) of characterized and commercially available inhibitors of 6 major subfamilies (6) of protein kinases that are known to inhibit various cellular processes; TK (tyrosine kinase families), TKL (tyrosine kinase-like families ), CMGC (CDK, MAPK, GSK3, CLK families), CAMK (Ca/calmodulin-dependent protein kinase), AGC (PKA, PKG, and PKC families), and CKI (Casein kinase family). As results, several compounds have been identified for their ability to alter differentiation rates of mesenchymal stem cells (MSCs) to chondrocytes and embryonic stem cells (ESCs) to dopaminergic neurons. Accordingly, it is an object of this invention to provide a method for inducing differentiation of a stem cell.

It is another object of this invention to provide a composition for inducing differentiation of a stem cell.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. Ia-If represent use of protein kinase inhibitors for the differentiation of rat MSCs. (Hg. Ia) The relative expression level of 7 target cell markers (Cl -C7) in the presence of various kinase inhibitors are detected by Sandwitch ELISA, and normalized in the range of 0 to 1 for standardization. Kinase inhibitors used are from Calbiochem (EMD Chemicals, Inc., San Diego, CA) and distinguished by the numerals following K and the kinase they preferentially inhibit are in parentheses: KO: no inhibitor;

Kl(AKT 1,2): l,3-Dihydro-l-(l-((4-(6-phenyl-lH-imidazo[4,5-g]quinoxalin-7- yl)phenyl)methyl)- 4-piperidinyl)-2H-benzimidazol-2-one;

K2(AKT): lL6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn- glycerocarbonate;

K4(CaMK II): Lavendustin (5-(N-2',5'-Dihydroxybenzyl) aminosalicylic Acid); K6(Calcium channel): HA 1077 (Fasudil, 5-Isoquinolinesulfonyl)homopiperazine, 2HCI); K8(CaseinK I): D4476(4-(4-(2,3-Dihydrobenzo[l,4]dioxin-6-yl)-5-pyridin-2-yl-lH- imidazol-2-yl)benzamide;

K12(CDK 1,2): NU6102 (6-Cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine); K13(TGF-βR I Kinase): [3-(Pyridin-2-yl)-4-(4-quinonyl)]-lH-pyrazole; K17(PKA): H-89 (N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCI);

K21(PKC): Go 6983 (2-[l-(3-Dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(lH-indol-

3-yl)) maleimide; K22(PKG-1 ): Guanosine 3',5'-CyCUc Monophosphorothioate, b-Phenyl-l,N2- etheno-8-bromo-, Rp-Isomer, Sodium Salt; K23(EGFR PTK): Compound 56 (4-[(3-Bromophenyl)amino]~6,7-diethoxyquinazoline); K27(ROCK): N-(4-Pyridyl)-N'-(2,4,6-trichlorophenyl) urea; K30(DNA-PK): 4,5-Dimethoxy-2-nitrobenzaldehyde; K35(p38 MAPK): SB 202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4- pyridyl)lH-imidazole). The target cells tested and cell markers assayed for each cell type are: Target cell types:

Cl: MSC positive, C2: MSC negative, C3: Osteocyte, C4: Chondrocyte, C5: Cardiomycyte, C6: Hepatocyte, C7: Endothelium Target cell marker:

Cl: CD105; C2: CD34; C3: Osteopontin (OPN); C4: Aggrecan (AGG); C5: Cardiac troponin T (CTnT); C6: CK-18; C7: CD31

(Fig. Ib) The effectiveness of inhibitors relative to the directed target cell development was analyzed by the principal component analysis (PCA) method using numerical values used to generate Fig. Ia. We obtained the coordinates of inhibitors and target cell types using the first three principal components (for visualization) from PCA and scaled the two sets of the coordinates to plot them together in the map. The three largest principal components of PCA analysis are represented as PCl, PC2, and PC3. The inhibitors are indicated by red balls and the cell types by green balls. The balls attached to the longer vectors are more efficient differentiation inducers for the cell types nearest to the balls. (Fig. Ic) Chondrogenesis of MSCs in vitro was significantly improved in cells treated with 1 μM H-89 for 11 days. The data of Alcian blue staining for sulfated proteoglycan indicates the quantitation of chondrogenesis. (Fig. Id) Aggrecan, one of the extracellular matrix genes in chondrocytes, was induced in MSCs treated with various concentrations of H-89 for 11 days (0.1-1 μM). The effect of U0126, a selective inhibitor of MEK, was examined on the expression of aggrecan. Co- treatment with 1 μM H-89 and 10 μM U0126 did not lead to the change of expression level. During chondrogenesis of MSC, the change of expression level in aggrecan was examined by sandwich ELISA. (Fig. Ie) Activation of ERKs was increased in MSCs treated with 1 μM H-89 but co-treatment with 1 μM H-89 and 10 μM U0126 did not lead to a change in ERKs activation for 11 days. The effect of U0126, a selective inhibitor of MEK, was examined on the expression of aggrecan. After co-treatment with 1 μM H-89 and 10 μM U0126, the change of expression level in aggrecan was examined by sandwich ELISA. (Fig. If) Semiquantitative RT-PCR showed a significantly changed level of cell adhesion molecule during chondrogenesis, using primers to the RNAs indicated. MSCs were cultured for the indicated time period in the presence of 1 μM H-89. **The data from a typical experiment conducted more than three times, /xθ.01.

Fig. 2a-2g represent effect of the treatment of H-1152, a kinase inhibitor, on differentiation of ESCs into TH-positive neurons. (Fig. 2a) Immunocytochemical analyses of TH-positive neuronal generation from the H-1152-treated ESCs. ESCs were treated with H-1152 during days 5-9, which correspond to the neural precursor stage. Tujl-positive cells (neurons) were stained with a green color and TH-positive cells are shown in red. Scale bar, lOμM. (Fig. 2b) Quantification of the ratios between the numbers of TH-positive and Tujl-positive cells. TH-positive and Tujl-positive cells were counted from 10 random fields per sample. Each group represents an average of three samples from independent experiment (*: p<0.05). (Fig. 2c) Dose-dependent effect of H-1152 measured by semiquantitative RT-PCR. TH gene expression went up as the concentration of H-1152 increased. The expression level of each gene was normalized to that of GAPDH. (Fig. 2d) Analyses of midbrain DA neuronal markers by semiquantitative RT-PCR. Treatment of ESCs with H-1152 resulted in an increase of DA neuronal markers (TH, Nurrl and Pitx3) compared to the untreated control. The expression level of each gene was normalized to that of GAPDH. (Fig. 2e) Quantification of the number of total viable cells at days 10 and 14 {left graph) and the ratios of Tujl-positive cells among total cells at day 14 {right graph). The number of viable cells was counted after staining the cells with trypan blue (n=3-4), and the proportion of neurons among total cells (Tujl-positive cells/total cells) was determined after staining with Tujl antibody and DAPI. Each bar in Figure IE was from the counting from 10 random fields (n=3). (Fig. 2f) Immunocytochemical analyses of cells obtained after differentiation of H-1152-treated ESCs. The majority of TH- positive neurons co-expressed a midbrain DA neuronal marker, EnI, but not DBH, PNMT, and GABA. Cells in the left panels were stained with anti-TH (a, d, g, and j), whereas cells in the middle panels were stained with anti-En 1 (midbrain DA neuronal marker, b), anti-DBH (noradrenergic and adrenergic neuronal marker, e), anti-PNMT (adrenergic neuronal marker, h), and anti-GABA (k) antibodies. The right panels are merged images. Scale bar, lOμM. (Fig. 2g) Expression of synaptophysin in TH- positive cells. Differentiated cells were stained with antibodies against TH (a) and synaptophysin (b). The co-expression of synaptophysin is an indication of synapse formation in the TH-positive cells. Scale bar, lOμM.

DETAILED DESCRIPTION OF THIS INVETNION

In one aspect of this invention, there is provided a method for inducing differentiation of a stem cell, which comprises culturing the stem cell in the presence of an inhibitor to protein kinase A or Rho kinase for a sufficient time period for inducing differentiation of the stem cell.

In another aspect of this invention, there is provided a composition for inducing differentiation of a stem cell, which comprises an inhibitor to protein kinase A or Rho kinase as active ingredients.

For developing novel chemical compounds capable of inducing differentiation of stem cells, we have broadly tested whether kinase inhibitors may induce differentiation of stem cells to a specific cell type and assembled a small library (41 compounds) of characterized and commercially available inhibitors of 6 major subfamilies (6) of protein kinases that are known to inhibit various cellular processes; TK (tyrosine kinase families), TKL (tyrosine kinase-like families ), CMGC (CDK, MAPK, GSK3, CLK families), CAMK (Ca/calmodulin-dependent protein kinase), AGC (PKA, PKG, and PKC families), and CKI (Casein kinase family). As results, several compounds have been identified for their ability to alter differentiation rates of mesenchymal stem cells (MSCs) to chondrocytes and embryonic stem cells (ESCs) to dopaminergic neurons. The most prominent feature of the invention is to utilize inhibitors to protein kinase A or Rho kinase for inducing the differentiation of stem cells.

According to a preferred embodiment, the inhibitors of the present invention are isoquinolinesulfonamide derivatives {i.e., a compound containing an isoquinolinesulfonamide scaffold).

More specifically, the compound containing an isoquinolinesulfonamide scaffold useful in the present invention is represented by the following Formula I:

i O= S =O

wherein Ri and R₂ independently represent H or alkyl or R₁ and R₂ may connect to form piperazine or homopiperazine, and R₃ represents H or alkyl.

The term "alkyl" is defined herein to be straight chain or branched chain saturated hydrocarbon group. The alkyl group in Ri or R₂ is preferably substituted. Preferably, the substituent may be alkylamino, arylalkylamino or arylallylamino. Where Ri or R₂ is alkyl, it is more preferably cinnamylaminoethyl. The phenyl group of cinnamylaminoethyl may be preferably substituted with halo, hydroxy, nitro or cyano group, more preferably halo or hydroxyl group, most preferably halo group. The term used herein "halo" means halogen atoms, for instance including fluoro, chloro, bromo, and iodo, preferably fluoro, chloro or bromo, more preferably bromo.

The alkyl group in R₃ is preferably lower alkyl. The term "lower alkyl" is defined herein as Cl to C4 unless otherwise preceded by some other chain length designator. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl and the like. Preferably, the alkyl group in R₃ is methyl, ethyl or propyl, more preferably methyl or ethyl, most preferably methyl.

Ri and R₂ may connect to form piperazine or homopiperazine, preferably homopiperazine. Where Ri and R₂ are connected to form homopiperazine, at least one of carbon atoms of homopiperazine is preferably substituted with lower alkyl. Preferably, the lower alkyl is methyl, ethyl or propyl, more preferably methyl or ethyl, most preferably methyl.

The stem cells differentiated by the present invention may be derived from human, bovine, sheep, ovine, pig, horse, rabbit, goat, mouse, hamster or rat, preferably, human, mouse or rat.

According to a preferred embodiment, the inhibitor to protein kinase A is a compound containing an isoquinolinesulfonamide scaffold.

As inhibitors to protein kinase A for inducing differentiation of stem cells, the compound containing an isoquinolinesulfonamide scaffold is preferably represented by the following Formula II:

wherein Ri represents alkyl and R₂ represents H or alkyl. The term "alkyl" is defined in Formula II to be straight chain or branched chain saturated hydrocarbon group. The alkyl group in Ri is preferably substituted. Preferably, the substituent may be alkylamino, arylalkylamino or arylallylamino, more preferably arylallylamino. Most preferably, Ri is cinnamylaminoethyl. The phenyl group of cinnamylaminoethyl may be preferably substituted with halo, hydroxy, nitro or cyano group, more preferably halo or hydroxyl group, most preferably halo group. The term used herein "halo" means halogen atoms, for instance including fluoro, chloro, bromo, and iodo, preferably fluoro, chloro or bromo, more preferably bromo. The alkyl group in R₂ is preferably lower alkyl. The term "lower alkyl" is defined herein as Cl to C4 unless otherwise preceded by some other chain length designator. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl and the like. Preferably, the alkyl group in R₂ is methyl, ethyl or propyl, more preferably methyl or ethyl, most preferably methyl.

According to a more preferred embodiment, the inhibitor to protein kinase A for inducing differentiation of stem cells is preferably represented by the following Formula III:

wherein Ri represents halo, R₂ represents H or lower alkyl and n represents an integer of 1-10.

Preferably, Ri is fluoro, chloro, bromo, and iodo, more preferably fluoro, chloro or bromo, most preferably bromo. R₂ is preferably H. Where R₂ is lower alkyl, it is preferably methyl, ethyl or propyl, more preferably methyl or ethyl, most preferably methyl.

The term "n" in Formula III is preferably an integer of 1-5, more preferably an integer of 1-3, most preferably 2.

Most preferably, the inhibitor to protein kinase A for inducing differentiation of stem cells is N-[2-((yθ-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide represented by the following Formula IV:

N-[2-((^bromodnnamyl)amino)ethyl]-5-isoquinolinesulfonamide called as H89 has been reported to be a highly specific PKA (protein kinase A) inhibitor with little effect on the activity of protein kinase C.

The isoquinolinesulfonamide derivatives used as inhibitors to protein kinase A for inducing differentiation of stem cells are preferably in a salt form. The salt means those salts of the isoquinolinesulfonamide derivatives which retain activities of interest, i.e., activities to inhibit protein kinase A for inducing differentiation of stem cells.

These salts may be formed using inorganic acids such as hydrochloride, hydrobromide and hydroiodide, or organic acids such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate, butyrate, citrate, camphorate, camphosulfate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethanesulfate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate. Preferably, the salt may be formed using inorganic acids, more preferably hydrochloride, hydrobromide and hydroiodide, most preferably hydrochloride.

According to a preferred embodiment, the compound containing an isoquinolinesulfonamide scaffold as inhibitors to protein kinase A for inducing differentiation of stem cells is very effective in differentiation of multipotent stem cells rather than pluripotent stem cells.

The term used herein "multipotent stem cells" means stem cells having the potential to give rise to cells from multiple, but a limited number of lineages. An example of a multipotent stem cell includes a hematopoietic cell and mesenchymal stem cell. More preferably, the inhibitor to protein kinase A is utilized to differentiation of multipotent stem cells into chondrocyte.

Most preferably, the compound containing an isoquinolinesulfonamide scaffold as inhibitors to protein kinase A is implicated in differentiation of mesenchymal stem cells (MSCs) (preferably, bone marrow-derived MSCs) into chondrocyte.

According to a preferred embodiment, the inhibitor to Rho kinase is a compound containing an isoquinolinesulfonamide scaffold.

As inhibitors to Rho kinase for inducing differentiation of stem cells, the compound containing an isoquinolinesulfonamide scaffold is preferably represented by the following Formula V or VI:

wherein R₁ represents H or alkyl and R₂ represents H or alkyl. The inhibitors to Rho kinase represented by Formula V and Formula VI are isoquinolinesulfonamide derivatives linked to piperazine and homopiperazine, respectively.

The term "alkyl" is defined in Formula V or VI to be straight chain or branched chain saturated hydrocarbon group. Where Ri or R₂ is alkyl, it is preferably lower alkyl. The term "lower alkyl" is defined herein as Cl to C4 unless otherwise preceded by some other chain length designator. Exemplary alkyl groups include methyl, ethyl, n- propyl, isopropyl, isobutyl, n-butyl and the like. Preferably, the alkyl group in R₃ is methyl, ethyl or propyl, more preferably methyl or ethyl, most preferably methyl.

Most preferably, the inhibitor to Rho kinase for inducing differentiation of stem cells is 2-methyl-l-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine represented by the following Formula VII:

The isoquinolinesulfonamide derivatives used as inhibitors to Rho kinase for inducing differentiation of stem cells are preferably in a salt form. The salt means those salts of the isoquinolinesulfonamide derivatives which retain activities of interest, i.e., activities to inhibit Rho kinase for inducing differentiation of stem cells. These salts may be formed using inorganic acids such as hydrochloride, hydrobromide and hydroiodide, or organic acids such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate, butyrate, citrate, camphorate, camphosulfate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethanesulfate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate. Preferably, the salt may be formed using inorganic acids, more preferably hydrochloride, hydrobromide and hydroiodide, most preferably hydrochloride.

According to a preferred embodiment, the compound containing an isoquinolinesulfonamide scaffold as inhibitors to Rho kinase (ROCK) for inducing differentiation of stem cells is very effective in differentiation of pluripotent stem cells rather than multipotent stem cells. The term used herein "pluripotent stem cells" means that cells have the ability to develop into any cell derived from the three main germ cell layers. When transferred into SCID mice, a successful somatic cell-derived pluripotent stem cell will differentiate into cells derived from all three embryonic germ layers. An example of a pluripotent stem cell includes embryonic stem cells, embryonic germ cells and induced pluripotent cells.

More preferably, the inhibitor to Rho kinase is utilized to differentiation of pluripotent stem cells into neuron (preferably dopaminergic neuron).

Most preferably, the compound containing an isoquinolinesulfonamide scaffold as inhibitors to Rho kinase is very effective in differentiation of embryonic stem cells (ESCs) into neuron.

The culture of stem cells in the presence of an inhibitor to protein kinase A or Rho kinase may be carried out in accordance with conventional methods using conventional media. A medium useful includes any conventional medium known in the art. For example, the medium includes Dulbecco's modified Eagle's medium (DMEM), knock DMEM, DMEM containing fetal bovine serum (FBS), DMEM containing serum replacement, Chatot, Ziomek and Bavister (CZB) medium, Ham's F-IO containing fetal calf serum (FCS), Tyrodes-albumin-lactate-pyruvate (TALP), Dulbecco's phosphate buffered saline (PBS), and Eagle's and Whitten's media. The detailed description of media is found in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic

Technique, Alan R. Liss, Inc., New York, WO 97/47734 and WO 98/30679, the teachings of which are incorporated herein by reference in their entities. The period of time for generating differentiated cells from stem cells is not particularly restricted, preferably in the range of 4-20 days.

In still another aspect of this invention, there is provided a compound to induce differentiation of stem cells (preferably mesenchymal stem cells) as follows: 1,3- Dihydro-l-(l-((4-(6-phenyl-lH-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4- piperidinyl)-2H-benzimidazol-2-one; lL6-Hydroxymethyl-chiro-inositol-2-(R)-2-O- methyl-3-O-octadecyl-sn-glycerocarbonate; Lavendustin (5-(N-2',5'-Dihydroxybenzyl) aminosalicylic Acid); Fasudil, (5-Isoquinolinesulfonyl)homopiperazine, 2HCI; (4-(4-(2,3- Dihydrobenzo[l_/4]dioxin-6-yl)-5-pyridin-2-yl-lH-imidazol-2-yl)benzamide; (6-

Cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine); [3-(Pyridin-2-yl)-4-(4-quinonyl)]- lH-pyrazole; (N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCI); (2-[l-(3-Dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(lH-indol-3-yl)) maleimide; Guanosine 3',5'-cyclic Monophosphorothioate, b-Phenyl-l,N2-etheno-8-bromo-,Rp- Isomer, Sodium Salt; (4-[(3-Bromophenyl)amino]-6,7-diethoxyquinazoline); N-(4- Pyridyl)-N'-(2,4_/6-trichlorophenyl) urea; 4_/5-Dimethoxy-2-nitrobenzaldehyde; (4-(4- Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4- pyridyl)lH-imidazole).

The features and advantages of this invention are will be summarized as follows:

(a) The present invention provides novel small molecules having isoquinolinesulfonamide scaffold to effectively induce differentiation of stem cells.

(b) The present invention allows for directed differentiation of stem cells to a specific cell type.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

Materials and Methods Isolation and Culture of MSCs Isolation and primary culture of MSCs from the femoral and tibial bones of donor rats were performed. Bone marrow-derived mesenchymal stem cells were collected from the aspirates of the femurs and tibias of 4-week-old Sprague-Dawley male rats (about 100 g) with 10 ml of MSC medium consisting of Dulbecco's modified Eagle's medium (DMEM)-low glucose supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD) and 1% antibiotic-penicillin and streptomycin solution (Gibco). Mononuclear cells recovered from the interface of Percoll-separated bone marrow were washed twice and resuspended in 10% FBS-DMEM, and plated at 1 x 10⁶cells/100 cm² in flasks. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. After 48 or 72 hrs, nonadherent cells were discarded, and the adherent cells were thoroughly washed twice with phosphate-buffered saline (PBS). Fresh complete medium was added and replaced every 3 or 4 days for about 10 days. To further purify the MSCs, Isolex Magnetic Cell Selection System (Baxter Healthcare Corporation, Irvine, CA) was used. Briefly, cells were incubated with Dynabeads M-450 coated with anti-CD34 monoclonal antibody. A magnetic field was applied to the chamber and the CD34+ cell-bead complexes were separated magnetically from the remaining cell suspension with the CD34- fraction being further cultured. The cells were harvested after incubation with 0.25% trypsin and 1 mM EDTA (Gibco) for 5 minutes at 37°C, replated in 1 x 10⁵/100-cm² plates, and again grown for approximately 10 days.

ESCs culture and in vitro differentiation

Undifferentiated mouse ESCs (Jl) were maintained on gelatin-coated dish in DMEM (Gibco) supplemented with 2 mM glutamine (Gibco), 0.001% β- mercaptoethanol (Sigma, St. Louis), Ix nonessential amino acids (Gibco), 2000 U/ml human recombinant leukemia inhibitory factor (LIF) (Chemicon International, Inc.

Temecula, CA), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco), and 10% FBS

(Gibco). PA6 cells were purchased from Riken (Tsukuba, Japan) and were maintained in α-minimum essential medium (α-MEM) (Gibco) supplemented with penicillin and streptomysin (Gibco), and 10% FBS (Gibco). To differentiate ESCs in vitro, PA6 cells were plated on gelatin-coated culture dishes to make a uniform feeder monolayer 1 day before the addition of Jl, and then ESCs were added at a density of Ix 10³ per well of a 24 well plate and 4 well plate, ES differentiation medium I [Glasgow minimum essential medium (G-MEM) (Gibco) supplemented with 10% knockout serum replacement (Gibco), 0.1 mM nonessential amino acid (Gibco), 1 mM sodium pyruvate (Sigma), 0.1 mM β-mercaptoethanol (Sigma), and PEST (Gibco)] was used for 8 days and then replaced with embryonic stem differentiation medium II [Glasgow minimum essential medium (G-MEM) (Gibco) supplemented with Ix N2 supplement (Gibco), 0.1 mM nonessential amino acid (Gibco), 1 mM sodium pyruvate (Sigma), 0.1 mM β- mercaptoethanol (Sigma), and PEST (Gibco)] for an additional 6 days. The culture medium was changed on day 4 and every other day thereafter.

Screening of protein kinase inhibitors for differentiation of MSCs and ESCs

We assembled a small library (41 compounds) of commercially available protein kinase inhibitors that are known to inhibit various members of protein kinase subfamilies (16). Of these, a subset of compounds was selected based on their low cytotoxicity. These compounds were screened for their activity to differentiate rat MSCs to chondrocytes and mouse ESCs to DA neurons.

Aldan Blue staining

Cells were first rinsed with PBS (Gibco) three times then fixed with 100% methanol (Sigma) for 10 min at -2O⁰C. Staining was accomplished by applying a solution of 1% Alcian blue 8GX (Bio Basic, Ontario, Canada) in 0.1 M HCI (pH 1.0)

(Sigma) to the cells for 2 hrs at room temperature. To quantify the intensity of the staining, the stained culture plates were rinsed with PBS three times and each well was extracted with 1 ml of 6 M guanidine-HCI (Sigma) overnight at room temperature. The optical density of extracted dye was measured at 650 nm.

Sandwich ELISA

The capture antibody was bound to the bottom of each well and then the plate was incubated overnight at 4°C. The plate was washed twice with PBS (Gibco) and treated with 100 μl of 3% BSA (Sigma) /PBS for 2~3 hrs at 37⁰C at room temperature. After washing the plate twice with PBS, cell lysate was added to each well and the plate was incubated for at least 2 hrs at room temperature in a humid atmosphere. The plate was washed four times with PBS containing 0.02% tween-20 (Sigma). Following adding the detector antibody, the plate was incubated for 2 hrs at room temperature in a humid atmosphere. The plate was incubated again with addition of peroxidase conjugated secondary Ab for 1 hr at 37⁰C. Finally, the plate was treated with 100 μl of TMB (tetramethylbenzidine, Sigma) as substrate and 25 μl of 0.1 M H₂SO₄ as stop buffer, then detected immediately at 450 nm on an ELISA plate reader.

Immunostaining

After 14 days of differentiation on PA6 feeder layer, ESCs were fixed with 4% formaldehyde for 30 min, washed with PBS, and perforated with PBS containing 0.1% Triton X-100 for 10 min. Then coverslips were incubated with blocking buffer [PBS containing 5% normal donkey serum (NDS)] for 1 hr. Cells were incubated at room temperature with primary antibodies diluted in PBS containing 5% NDS for 1 hr. The following primary antibodies were used: mouse anti-neuronal class III β-tubulin (1:1000, Covance, Richmond, CA), rabbit anti-TH (1:300, Pel-Freeze, Rogers, AK), mouse anti-TH (1:100, Chemicon), mouse anti-Enl (1:50, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), sheep anti-dopamine β hydroxylase (DBH) (1:250, Chemicon), rabbit anti-phenylethanloamine N-methyltransferase (PNMT) (1:200, Chemicon), and rabbit anti-mammalian γ-aminobutyric acid (GABA) antibodies (1:2500, Sigma). The cells washed with PBS and then incubated with fluorescent- labeled secondary antibodies [Alexa Flour 488 (green) or Alexa Fluor 594 (red)-labeled mouse/rabbit IgG (1:1000, Molecular Probes, Invitrogen, Carlsbad, CA)] in PBS with 5% NDS for 30 min at room temperature. The coverslips were rinsed 3 times for 5 minutes in PBS and mounted onto slides using VECTASHIELD Hardset mounting medium with DAPI (Vector laboratories, Buringame, CA). Images were obtained under a fluorescence microscope Olympus 1X71 and confocal microscope.

Immunoblot analysis Cells were washed once in PBS and lysed in a lysis buffer (Cell signaling,

Beverly, MA, USA) containing 20 mM Tris (pH 7.5), 150 mM NaCI, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mg/ml leupeptin, and 1 mM PMSF. Protein concentrations were determined using the Bradford protein assay kit (BioRad, Hercules, CA, USA). Proteins were separated in a 12% SDS-polyacrylamide gel and transferred to PVDF membrane (Millipore Co, Bedford, MA, USA). After blocking the membrane with Tris- buffered saline-tween 20 (TBS-T, 0.1% tween 20) containing 5% non-fat dried milk for 1 hr at room temperature, membrane was washed twice with TBS-T and incubated with primary antibodies for 1 hr at room temperature or for overnight at 4⁰C. The membrane was washed three times with TBS-T for 10 min, and then incubated for 1 hr at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. After extensive washing, the bands were detected by enhanced chemiluminescence (ECL) reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The band intensities were quantified using Photo-Image System (Molecular Dynamics, Uppsala, Sweden).

RT-PCR analysis

The expression levels of various genes were analyzed by the reverse transcription polymerase chain reaction (RT-PCR) technique. Total RNA was prepared by Ultraspect™-II RNA system (Biotecx Laboratories, Inc., USA) and easy-BLUE™ (Intron Biotechnology, Seoul, South Korea). Single-stranded cDNA was then synthesized from isolated total RNA by Avian Myeloblastosis virus (AMV) reverse transcriptase (Power cDNA Synthesis Kit, Intron Biotechnology). A 20 μl reverse transcription reaction mixture containing 1 μg of total RNA, IX reverse transcription buffer (10 mM Tris-HCI, pH 9.0, 50 mM KCI, 0.1% Triton X-100), 1 mM deoxynucleoside triphosphates (dNTPs) 0.5 unit of RNase inhibitor, 0.5 μg of oligo(dT)i₅, and 15 units of AMV reverse transcriptase was incubated at 42°C for 15 min, heated to 99°C for 5 min, and then incubated at 0-5⁰C for 5 min. PCR was performed using a standard procedure with Taq Polymerase (i-Max™ DNA Polymerase, Intron Biotechnology). The number of cycles varied from 25 to 40 cycles with denaturation at 94°C for 30 seconds, annealing at 58°C to 65°C for 30 seconds, and elongation at 72⁰C for 30 seconds. Primers were as follows: Fibronectin: 5'-CCTT AAGCCTTCTGCTCTGG-3', 5'-CGGCAAAAGAAAGCAGAACT-S' (300 bp); βl integrin: 5 - GCCAGTGTCACCTGGAAAAT-3', 5'-TCGTCCATTTTCTCCTGTCC-3' (344 bp); α5 integrin: 5'-CTTCGGTTCACTGTTCCTC-S', 5'-TGGCTTCAGG GCATTT-3' (283 bp); N-cadherin 5'- GCCACCATATGACTCCCTTTTAGT-3', 5'-CAGAAAACTAATTCCAATCTGAAA-S' (454 bp); TH: 5'-GAGAGGACAGCAT TCCACAG-3', 5'-TCACGGGCAGACAGTAGACC-S' (100 bp); Nurrl: 5'-GCTAA ACAAAACTTGCATGC-3', 5'-CTCATATCATGTGCCATACTAG-S' (208 bp); Pitx3: 5'-CTAGACCTCCCTCCATGGAG-S', 5'-TCTTGAACCACACCCGCACG-S' (348 bp). The GAPDH primers [S'-CTCCCAACGTGTCTGTTGTG-S', 5'-TGAGCTTGA CAAAGTGGTCG- 3' (450 bp) and 5'-ACCACAGTCCATGCCATCAC-S', 5'-TCCACCACCCTGTTGCT GTA-3' (450 bp)] were used as the internal standard. The signal intensity of the amplification product was normalized to its respective actin signal intensity.

Viable cell counting

Cultured cells were washed with PBS, detached from the dish with Ix trypsin/EDTA, and then collected in the medium. After spinning down, the cell pellet was resuspended in trypan blue solution for counting under microscope.

Statistical analysis

Results are expressed as mean ± SEM. Statistical analysis as performed by student's t-test. Relationships were considered statistically significant when p value was less than 0.05.

Results Under proper stimulation, MSCs can be induced to differentiate into chondrocytes, myocytes, adipocytes, osteoblasts, tenocytes and hematopoietic- supporting stroma (7). To examine the effect of these kinase inhibitors on the differentiation of rat MSCs into various cell types, the MSCs were seeded and treated every 3 days with the kinase inhibitors for up to 11 days. Among the 41 inhibitors 14 inhibitors show recognizable indication of inducing various cell types at different degrees. In order to assess the effectiveness of these 14 inhibitors for the directed target cell development, we have constructed a profile matrix, where the rows and columns represent target cell markers and the kinase inhibitors, respectively (Fig. IA). Using the principal component analysis (PCA) method on the profile matrix, we examined the relative strength of the induction and the cross-relationship among them. The size of the profile matrix used in PCA was 7 x 15 in which we obtained the coordinates of inhibitors and target cell types using the first three principal components from PCA and scaled the coordinates to plot them together in the map (Fig. IB) for visualization. From the examination of Fig. IA and IB, we found several good candidates that drive rat MSCs into specific cell types. Among them, H-89, an inhibitor of protein kinase A, was found to be potentially implicated in chondrogenesis of the MSCs. H-89 is a derivative of isoquinolinesulfonamide, N-[2-((/>Bromocinnamyl) amino)ethyl]-5- isoquinolinesulfonamide, 2HCI (Calbiochem, EMD Biosciences, Inc. La JoIIa, CA, USA). The compound is cell-permeable and known to be selective and potent inhibitor of protein kinase A (K, = 48 nM). Among the kinases tested, it inhibits other kinases only at much higher concentrations: CaM kinase II (Ki = 29.7 μM), casein kinase I (K₁ = 38.3 μM), myosin light chain kinase (K₁ = 28.3 μM), protein kinase C (K, = 31.7 μM), and ROCK-II (IC₅₀ = 270 nM) (8-11).

To examine the capability of H-89 in inducing chondrogenesis and mechanisms underlying the process, we carried out several experiments using cell culture assays; (1) Quantification of chondrogenesis by measuring an absorbance of Alcian blue extract indicated that H-89 enhanced chondrogenesis more than 2-fold over the control (Rg. 1C). (2) Rat MSCs treated with various concentrations of H-89 (0.1-1 μM) were differentiated into chondrocytes in a dose-dependent manner as judged by the upregulated expression of aggrecan, a chondrocyte marker (Rg. ID). We used 1 μM concentration of H-89 for the rest of our studies. (3) The induction of chondrogenesis by H-89 is thought to be mediated by (ERK) MAP kinase signaling pathway. H-89 treatment increased the phosphorylation of ERK through unknown pathways (Rg. IE). (4) Furthermore, the treatment with U0126, a selective inhibitor of MEK, was shown to block the phosphorylation of ERK even in the presence of H-89 and nullified the chondrogenic inducibility of H-89 (Rg. IE). (5) Expression level of IM- cadherin, which mediates cell-cell interaction, was highest on the first day of H-89 treatment, but was decreased as the differentiation of MSCs to chondrocytes proceeded (Fig. IF). (6) Since the interaction of cell with extracellular matrix (ECM) in addition to cell-cell interaction is involved in the regulation of chondrogensis, potential involvement of H-89 in the regulation of integrin α5βl and its ligand fibronectin was also examined. During the chondrogenic differentiation of MSCs treated with H-89, the fibronectin-receptor (integrin α5βl) and fibronectin were down- regulated (Rg. IF). (7) During chondrogenesis, H-89 treatment reduced the proliferation of MSCs compared with non-treated cells, indicating that H-89 might modulate the balance between differentiation and proliferation (data not shown). We have also examined the effect of the 41 kinase inhibitors on the differentiation of mouse ESCs into dopamine (DA) neurons. We differentiated Jl, a mouse ESC line, into midbrain DA neurons on PA6 feeder layer for 14 days (12,13). During this procedure, the ESCs were treated with 41 different kinase inhibitors at two different concentrations, 0.1 μM and 1 μM, during days 5-9, which correspond to neural precursor stage. Jl ESCs without the treatment of kinase inhibitors were used as a control. After 14 days, expression of tyrosine hydroxylase (TH), a catecholaminergic neuronal marker, was measured by sandwich ELISA from differentiated cells. Some inhibitors decreased the expression level of TH while others increased the expression level of TH compared to control (data not shown). Among the 41 compounds tested H-1152 (Calbiochem, EMD Biosciences, Inc. La JoIIa, CA, USA), a Rho kinase inhibitor, was the best inducer of the differentiation of ESCs into TH-positive neurons. H-1152 is another cell-permeable isoquinolinesulfonamide derivative that acts as a highly specific, potent, and ATP-competitive inhibitor of G- protein Rho-associated kinase (ROCK; Kj = 1.6 nM). Among the kinases tested, the inhibition is much weaker for other tested serine/threonine kinases (e.g., K₁ = 630 nM for PKA, 9.27 M for PKC, and 10.1 M for MLCK) (14,15).

The number of TH-positive cells among total neurons went up to 55% by H- 1152 treatment which is a significant increase compared to that from the untreated control (25~30%) (Rg. 2A and B). At the concentration between 0.1 M and 2 M, the increase of TH expression by H-1152 occurred in a dose-dependent manner without any indication of cytotoxicity (Fig. 2C). However, we noticed some cytotoxic effect of the H-1152 at high concentration (> 5 μM) (data not shown). Semiquantitative RT-PCR analysis also supported the positive role of H-1152 in the differentiation of ESCs into midbrain DA neurons; the expression levels of three representative midbrain DA neuronal markers, TH, Nurrl and Pitx3, were increased about 1.5- to 2.2-fold in H-1152-treated cells (Fig. 2D). There was no significant difference in the total number of cells as well as neurons between H-1152-treated and untreated cells on both day 10 and day 14. For example, the total number of viable cells and the percentage of neurons among total cells (Tujl⁺cells/total cell) between H-1152-treated and untreated cells were 3.1±1.0 x 10⁶ vs. 3.8±1.1 x 10⁶ and 74.3±2.43% vs. 81.2±4.8%, respectively, on day 14 which was the end point of differentiation process (Fig. 2E). These results indicate that H-1152 may regulate dopaminergic specification and differentiation directly, without affecting cell cycle or cell proliferation.

To confirm that the majority of TH-positive neurons generated in the presence of H-1152 retain characteristics of midbrain DA neurons, we analyzed the TH-positive cell population closely (Rg. 2F). First, we tried to determine whether the TH-positive cell population contains other types of catecholamine neurons such as noradrenergic and adrenergic neurons. To this end, a midbrain DA neuron- specific marker, EnI, and other catecholaminergic neuronal markers, such as dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), were co-immunostained with TH. Our result showed that most TH-positive cells expressed EnI, indicating that DA neurons are the major cell type in the population (Fig. 2F, a-c). This result is in line with the observation that only a few TH-positive cells expressed DBH, an enzyme that converts dopamine into norepinephrine (Fig. 2F, d-f) or PNMT which catalyzes the formation of epinephrine from norepinephrine (Fig. 2F, g-i). These two enzymes are known to be expressed in catecholaminergic neurons except DA neurons. GABA expression was also not detected among the TH-positive cells, indicating that the TH- positive neurons are not the olfactory DA neurons (Fig. 2F, j-l). The TH-positive neurons generated in the presence of H-1152 also expressed synaptophysin, suggesting that the cells have the capability to form s ynapses (Fig. 2G). Taken together, our results suggest that the majority of TH-positive cells generated in the presence of H-1152 are midbrain DA neurons that can form synapses.

Conclusions and Discussions Both observations described above, the enhanced differentiation of rat MSCs to chondrocytes and of mouse ESCs to DA neurons, support the notion that, although cell differentiation may be the result of a complex orchestration of many signals from multiple signaling pathways, even single chemical reagent, a kinase inhibitor in this case, can alter the relative balance of many signals enough to enhance differentiation of stem cells to particular cell types. They also suggest that the process may be optimized by a "cocktail" of various multiple kinase inhibitors. Interestingly, both inhibitors, H-89 and H-1152, are two different derivatives of the same scaffold, isoquinolinesulfonamide.

Since these compounds may interact with "off-target" kinases as well as other unknown proteins our observation should be considered as a practical approach for finding chemical reagents for inducing stem cell differentiation to specific cell type even when most of the signaling pathways are unknown. Thus, the approach described or some variation of it may be applied to other stem cells, including human stem cells, to find chemical molecules that can trigger initiation, inhibition, or even reversion of differentiation process of stem cells or progenitor cells.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

References 1. Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF (2000) J Bio Chem 275:9645-9652.

2. Castelo-Branco G, Rawal N, Arenas E (2004) J CeII Sci 117:5731-5737.

3. Hu X, Jin L, Feng L (2004) J Neurochem 90:1339-1347.

4. Masamichi Koyanagi, Judith Haendeler, Cornel Badorff, RaIf P. Brandes, Jorg Hoffmann, Petra Pandur, Andreas M. Zeiher, Michael Kϋhl, Stefanie Dimmeler

Masamichi K (2005) J Bio Chem 280:16838-9652.

5. Ding S, Tom YH Wu, Brinker A, Peters EC, Hur W, Gray NS, Schultz PG (2003) Proc. Natl. Acad. Sd. USA 100:7632-7637. 6. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. Manning G (2002) Science 298:1912-1934.

7. Pittenger MF₇ Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR.Pittenger (1999) Science 284:143-147. 8. Leemhuis J, Boutillier S, Schmidt G, Meyer DK (2002) J Pharmacol Exp Ther 300 : 1000-1007.

9. Davies SP, Reddy H, Caivano M, Cohen P (2000) Biochem 7351:95-105.

10. Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM (1998) Science 282:2275-2279. 11. Combest WL, Bloom TJ, Gilbert LI (1998) J Neumchem 51 :1581-1591.

12. Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y (2000) Neuron 28:31-40.

13. Kim DW, Chung S, Hwang M, Ferree A, Tsai HC, Park JJ, Chung S, Nam TS, Kang UJ, Isacson O, Kim KS (2006) Stem Cells 24:557-567. 14. Ikenoya M, Hidaka H, Hosoya T, Suzuki M, Yamamoto N, Sasaki Y (2002) J Neumchem 81 :9-16.

15. Sasaki Y, Suzuki M, Hidaka H (2002) Pharmaco. Ther '93:225-232.

16. Manning G, Whyte D, Martinez R, Hunter T, Sudarsanam S (2002) Science 6;298(5600): 1912-34

Claims

What is claimed is:

1. A method for inducing differentiation of a stem cell, which comprises culturing the stem cell in the presence of an inhibitor to protein kinase A or Rho kinase.

2. The method according to claim 1, wherein the inhibitor to protein kinase A or Rho kinase is a compound containing an isoquinolinesulfonamide scaffold.

3. The method according to claim 2, wherein the inhibitor to protein kinase A is a compound containing an isoquinolinesulfonamide scaffold.

4. The method according to claim 3, wherein the compound containing an isoquinolinesulfonamide scaffold is represented by the following formula III:

NH

(CH₂}_n NH

5. The method according to claim 4, wherein the compound containing an isoquinolinesulfonamide scaffold is N-[2-((/>bromocinnamyl)amino)ethyl]-5- isoquinolinesulfonamide.

6. The method according to claim 3, wherein the stem cell is a multipotent stem cell and is differentiated into chondrocyte by the inhibitor to protein kinase A.

7. The method according to claim 6, wherein the multipotent stem cell is mesenchymal stem cell.

8. The method according to claim 1, wherein the inhibitor to Rho kinase is a compound containing an isoquinolinesulfonamide scaffold.

9. The method according to claim 8, wherein the compound containing an isoquinolinesulfonamide scaffold is represented by the following formula V or VI:

wherein Ri represents H or alkyl and R₂ represents H or alkyl.

10. The method according to claim 9, wherein the compound containing an isoquinolinesulfonamide scaffold is 2-methyl-l-[(4-methyl-5- isoquinolinyl)sulfonyl]homopiperazine.

11. The method according to claim 8, wherein the stem cell is a pluripotent stem cell and is differentiated into neuron by the inhibitor to Rho kinase.

12. The method according to claim 11, wherein the neuron is dopaminergic.

13. The method according to claim 11, wherein the pluripotent stem cell is an embryonic stem cell.

14. A composition for inducing differentiation of a stem cell, which comprises an inhibitor to protein kinase A or Rho kinase as active ingredients.

15. The composition according to claim 12, wherein the inhibitor to protein kinase A or Rho kinase is a compound containing an isoquinolinesulfonamide scaffold.

16. The composition according to claim 15, wherein the inhibitor to protein kinase A is a compound containing an isoquinolinesulfonamide scaffold.

17. The composition according to claim 16, wherein the compound containing an isoquinolinesulfonamide scaffold is represented by the following formula III:

NH I

wherein R₁ represents halo, R₂ represents H or lower alkyl and n represents an integer of 1-10.

18. The composition according to claim 17, wherein the compound containing an isoquinolinesulfonamide structure is N-[2-((/τ-bromocinnamyl)amino)ethyl]-5- isoquinolinesulfonamide.

19. The composition according to claim 16, wherein the stem cell is a multipotent stem cell and is differentiated into chondrocyte by the inhibition of protein kinase A.

20. The composition according to claim 19, wherein the multipotent stem cell is mesenchymal stem cell.

21. The composition according to claim 15, wherein the inhibitor to Rho kinase is a compound containing an isoquinolinesulfonamide scaffold.

22. The composition according to claim 21, wherein the compound containing an isoquinolinesulfonamide scaffold is represented by the following formula V or VI:

wherein R₁ represents H or alkyl and R₂ represents H or alky!.

23. The composition according to claim 22, wherein the compound containing an isoquinolinesulfonamide structure is 2-methyl-l-[(4-methyl-5- isoquinolinyl)sulfonyl]homopiperazine.

24. The composition according to claim 21, wherein the stem cell is a pluripotent stem cell and is differentiated into neuron by the inhibition of Rho kinase.

25. The composition according to claim 24, wherein the neuron is dopaminergic.

26. The composition according to claim 24, wherein the pluripotent stem cell is an embryonic stem cell.