SJA710-6 at 5 μM induced rMSCs to exhibit polygonal hepatocyte-like morphology with granular cytoplasm and refractile borders after 14 days of differentiation [1].
Periodic acid-Schiff (PAS) staining showed that SJA710-6-treated rMSCs accumulated glycogen (magenta staining) comparable to the positive control (growth factors), while DMSO control showed no glycogen storage [1].
Albumin secretion measured by ELISA: SJA710-6-treated cells secreted 36.54 ± 7.73 μg/mL albumin, significantly higher than DMSO control (13.77 ± 3.16 μg/mL, p < 0.01) [1].
Urea production after exposure to 6 mM NH₄Cl for 24 h: SJA710-6-differentiated rMSCs produced 12.63 ± 3.17 μg/mL urea, compared to 7.61 ± 1.04 μg/mL for DMSO control (p < 0.01) [1].
LDL uptake: SJA710-6-treated cells showed significantly increased uptake of Dil-Ac-LDL (1641.39 ± 326.62 ng/well) versus DMSO control (352.48 ± 88.26 ng/well, p < 0.01) [1].
Flow cytometry analysis after 24 days of induction: 47.14% of SJA710-6-treated cells exhibited hepatic differentiation, comparable to the growth factor positive control [1].
RT-PCR analysis after 28 days: SJA710-6 treatment significantly increased mRNA expression of hepatocyte-specific genes albumin, AFP, CK18, c-Met, CYP1A1, CYP2B1, and HNF3β, whereas DMSO control expressed none of these genes [1].
Western blot analysis: SJA710-6 treatment significantly enhanced FoxH1 protein expression compared to DMSO control (p < 0.01), similar to the growth factor positive control [1].

Cell isolation: rMSCs were obtained by flushing femurs and tibias of 6-week-old female Sprague-Dawley rats with D-Hanks' solution. Cells were cultured in low-glucose DMEM with 10% FBS, 10 mM HEPES, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C, 5% CO₂ [1].
Hepatic differentiation protocol (4 steps): Step 1 (pre-induction): rMSCs in serum-free DMEM for 24 h. Step 2 (induction): 2% FBS/DMEM with 100 ng/mL activin A and 10 ng/mL FGF4 for 2 days. Step 3 (differentiation): 2% FBS/DMEM with 1× ITS and either DMSO (negative control), growth factors (10 ng/mL FGF4 + 20 ng/mL HGF, positive control), or 5 μM SJA710-6 for 7 days. Step 4 (maturation): 2% FBS/DMEM with 1 μM dexamethasone, 1× ITS and same treatments for 14 days. Media changed every 3 days [1].
PAS staining: Cells fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, oxidized with 1% periodic acid for 1 h at 22 °C, rinsed, treated with Schiff's reagent for 1 h, then examined under light microscope [1].
Albumin ELISA: Culture medium collected after 24 h was analyzed using an ELISA quantitation kit according to manufacturer's instructions; absorbance read on a plate reader [1].
Urea production assay: Cells exposed to 6 mM NH₄Cl for 24 h; culture medium urea concentration measured using an automated chemistry analyzer with a urea assay kit [1].
LDL uptake assay: Differentiated cells incubated with 10 μg/mL Dil-Ac-LDL in high-glucose DMEM for 24 h at 37 °C; supernatant collected and assayed per manufacturer's instructions [1].
Flow cytometry: After 24 days of differentiation, cells treated with brefeldin A (3 μg/mL) for 6 h, fixed in 70% cold ethanol overnight, permeabilized with D-Hanks' solution containing 0.3% Triton X-100, 0.1% NaN₃, 0.1% saponin, 10 mM HEPES, 1% normal serum for 15 min. Cells incubated with primary goat anti-albumin antibody, then with FITC-conjugated rabbit anti-goat secondary antibody, and analyzed by flow cytometry [1].
RT-PCR: Total RNA extracted with TRIzol, reverse transcribed to cDNA. PCR amplification at 94 °C for 40 s, 56–62 °C for 50 s, 72 °C for 60 s for 35 cycles after initial denaturation at 94 °C for 5 min. Primers for albumin, AFP, CK18, c-Met, CYP1A1, CYP2B1, PEPCK, FoxH1, HNF4α, HNF3β, and GAPDH were used [1].
Western blot: Cell lysates prepared with protein extraction reagent plus protease inhibitors. Protein samples heated at 100 °C for 5 min, separated on 20% SDS-PAGE, transferred to PVDF membrane. Membrane blocked for ≥1.5 h, then incubated overnight at 4 °C with primary antibodies against FoxH1 or β-actin. After washing, incubated with HRP-conjugated secondary antibodies for ≥1.5 h at room temperature, developed using chemiluminescence, and images captured on X-ray film [1].

ChemMedChem.2012 Aug;7(8):1447-52.

SJA710-6 was identified through a phenotypic screen using PAS staining as a readout for glycogen accumulation, a hallmark of hepatocyte differentiation. The compound induces rMSCs to differentiate into hepatocyte-like cells with functional characteristics comparable to those induced by growth factors (FGF4 and HGF). The differentiation efficiency with SJA710-6 (47.14%) was similar to that of growth factors. Mechanistically, SJA710-6 enhances FoxH1 expression, suggesting involvement of the Nodal signaling pathway in hepatic fate specification. No in vivo animal efficacy, pharmacokinetic, or toxicity data are reported in this study [1].

C22H20BRFN4

439.323407173157

438.09

C, 60.15; H, 4.59; Br, 18.19; F, 4.32; N, 12.75

1397255-09-2

71466685

Solid powder

5.8

1

4

6

28

479

0

CCCN1C2=C(C=CC(=N2)NCC3=CC=C(C=C3)F)N=C1C4=CC=C(C=C4)Br

RPNQWWSSADJSGS-UHFFFAOYSA-N

InChI=1S/C22H20BrFN4/c1-2-13-28-21(16-5-7-17(23)8-6-16)26-19-11-12-20(27-22(19)28)25-14-15-3-9-18(24)10-4-15/h3-12H,2,13-14H2,1H3,(H,25,27)

2-(4-bromophenyl)-N-[(4-fluorophenyl)methyl]-3-propylimidazo[4,5-b]pyridin-5-amine

SJA710 6; SJA710-6; SJA-710-6; SJA 710-6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~569.06 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)