Chemicals that modulate stem cell differentiation

Abstract

Important cellular processes such as cell fate are likely to be controlled by an elaborate orchestration of multiple signaling pathways, many of which are still not well understood or known. Because protein kinases, the members of a large family of proteins involved in modulating many known signaling pathways, are likely to play important roles in balancing multiple signals to modulate cell fate, we focused our initial search for chemical reagents that regulate stem cell fate among known inhibitors of protein kinases. We have screened 41 characterized inhibitors of six major protein kinase subfamilies to alter the orchestration of multiple signaling pathways involved in differentiation of stem cells. We found that some of them cause recognizable changes in the differentiation rates of two types of stem cells, rat mesenchymal stem cells (MSCs) and mouse embryonic stem cells (ESCs). Among many, we describe the two most effective derivatives of the same scaffold compound, isoquinolinesulfonamide, on the stem cell differentiation: rat MSCs to chondrocytes and mouse ESCs to dopaminergic neurons.

Fig. 1.

Use of protein kinase inhibitors for the differentiation of rat MSCs. (A) The relative expression level of seven target cell markers (C1–C7) in the presence of various kinase inhibitors are detected by sandwich ELISA, and normalized in the range of 0 to 1 for standardization. Kinase inhibitors used (at 1 μM) are from Calbiochem and are distinguished by the numerals after K. The kinases they preferentially inhibit are in parentheses: K0, no inhibitor; K1 (AKT 1,2), 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one; K2 (AKT), 1L6-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, 2HCl); K8 (CaseinK I), D4476(4-(4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-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)]-1H-pyrazole; K17 (PKA), H-89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl]; K21 (PKC), Gö 6983 (2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)) maleimide; K22 (PKG-1α,β), guanosine 3′,5′-cyclic monophosphorothioate, β-phenyl-1, 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)1H-imidazole]. The target cells tested for each cell type were: C1, MSC positive; C2, MSC negative; C3, osteocyte; C4, chondrocyte; C5, cardiomycyte; C6, hepatocyte; C7, endothelium. The target cell markers assayed for each cell type were: C1, CD105; C2, CD34; C3, osteopontin (OPN); C4, aggrecan (AGG); C5, cardiac troponin T (CTnT); C6, CK-18, C7: CD31. (B) 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 A. We obtained the coordinates of inhibitors and target cell types by 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 PC1, PC2, and PC3. The inhibitors are indicated by red balls and the cell types are shown as green balls. The balls attached to the longer vectors are more efficient differentiation inducers for the cell types nearest to the balls. (C) 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 indicate the quantitation of chondrogenesis. (D) 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. Cotreatment with 1 μM H-89 and 10 μM U0126 did not lead to a change of expression level. During chondrogenesis of MSC, the change of expression level in aggrecan was examined by sandwich ELISA. (E) Activation of ERKs was increased in MSCs treated with 1 μM H-89 but cotreatment with 1 μM H-89 and 10 μM U0126 did not lead to a change in ERK activation for 11 days. (F) 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 periods in the presence of 1 μM H-89. **, P < 0.01 for data from a typical experiment conducted more than three times.

Fig. 2.

Effect of treatment of H-1152, a kinase inhibitor, on differentiation of ESCs into TH-positive neurons. (A) 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. Tuj1-positive cells (neurons) were stained with a green color, and TH-positive cells are shown in red. (Scale bar: 10 μm.) (B) Quantification of the ratios between the numbers of TH-positive and Tuj1-positive cells. TH-positive and Tuj1-positive cells were counted from 10 random fields per sample. Each group represents an average of three samples from independent experiment (*, P < 0.05). Inhibitor concentration was 1 μM. (C) 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. (D) Analyses of midbrain dopamine neuronal markers by semiquantitative RT-PCR. Treatment of ESCs with H-1152 resulted in an increase of dopamine neuronal markers (TH, Nurr1, and Pitx3) compared with the untreated control. The expression level of each gene was normalized to that of GAPDH. (E) Quantification of the number of total viable cells at days 10 and 14 (Left) and the ratios of Tuj1-positive cells among total cells at day 14 (Right). 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 (Tuj1-positive cells per total cells) was determined after staining with Tuj1 antibody and DAPI. Each bar in Fig. 1E was from the counting from 10 random fields (n = 3). Inhibitor concentration was 1 μM. (F) Immunocytochemical analyses of cells obtained after differentiation of H-1152-treated ESCs. The majority of TH-positive neurons coexpressed a midbrain dopamine neuronal marker, En1, but not DBH, PNMT, and GABA. (a, d, g, and j) Cells were stained with anti-TH. (b, e, j, and k) Cells were stained with anti-En1 (midbrain DA neuronal marker, b), anti-DBH (noradrenergic and adrenergic neuronal marker, e), anti-PNMT (adrenergic neuronal marker, h), and anti-GABA (k) antibodies. (c, f, i, and l) Merged images. (Scale bar: 10 μm.) (G) Expression of synaptophysin in TH-positive cells. (a and b) Differentiated cells were stained with antibodies against TH (a) and synaptophysin (b). (c) The coexpression of synaptophysin is an indication of synapse formation in the TH-positive cells. (Scale bar: 10 μm.)