Wnt (IC50 = 25 nM)

IWP-4 is a small molecule Wnt inhibitor that has a 25 nM IC50. Heart indicators, such as cardiac troponin I (CTNI) and cardiac myosin heavy chain leukocytes (MYHhi+), are induced to express by IWP-4. Additionally, pulsatile foci (0.44 ± 0.10 SEM beats per second) appeared in response to IWP-4; these foci were not present in any of the cultures that did not receive IWP-4. Additionally, day 16 flow cytometry analysis revealed a significant difference (P<0.0002) in the number of MYHlo + cells between IWP-4-treated and untreated cultures, with 17.0 ± 1.3 SD% and 5.4±1.4 SD%, respectively. 63% (481/817) of the IWP-4-treated cells had nuclear NKX2-5 expression, according to NKX2-5 protein expression quantification [1]. When mesenchymal precursor cells (MPCs) treated with IWP-4 were compared to osteogenic media alone at day 7, the expression of AXIN2, CTNNB1, and GSK3B did not change significantly. However, at day 21, the expression of DKK1 and GSK3β increased in MPC treated with IWP-4. Significant downregulation of COL1A1 and SPARC was likewise brought on by IWP-4 [2].

Modulation of Gene Expression[1]
Using these static cultures, we then utilised RT-qPCR to measure any changes in the expression of a number of key members of the Wnt signaling pathway and determine how they were influenced by CHIR, IWR-1 and IWP-4 treatments. As would be expected due to its role as a canonical Wnt agonist, CHIR treatment of MPCs caused upregulation of AXIN2 (regarded as a marker of canonical Wnt pathway activation), as well as CTNNB1 (β-catenin) and GSK3B, whilst the Wnt inhibitor DKK1 was downregulated at both 7 and 21 days ( Fig. 4 ). MPCs treated with IWP-4 and IWR-1 showed no significant changes in the expression of AXIN2, CTNNB1 and GSK3B as compared to osteogenic medium alone on day 7, but MPCs treated with IWP-4 expressed elevated levels of DKK1 and GSK3B on day 21. The significant upregulation (up to 350-fold) of AXIN2 in CHIR-treated MPCs at both day 7 and 21 provided a strong indication that CHIR was working in the manner expected (to activate canonical Wnt signaling) and so we next analysed the expression of markers of different stages of osteogenesis to elucidate why CHIR may be acting to inhibit differentiation and what differences may be observed between the agonist CHIR, and antagonists IWR-1 and IWP-4.[1]

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Consistent with the results from the MBA screen, the effects of IWP-4 and IWR-1 upon gene expression levels were weaker than that of CHIR. However, both IWR-1 and IWP-4 decreased expression levels of ALP without the simultaneous increase in RUNX2, MSX2 and DLX5 observed using CHIR ( Fig. 3C ). After 21 days, ALP expression under IWR-1 treatment was similar to untreated controls but was still reduced with IWP-4 treatment. At this later timepoint, IWP-4 also caused a significant downregulation of SPARC and COL1A1, whilst only a significant reduction in COL1A1 was observed using IWR-4 ( Fig. 3D ).[1]

A ELF97 (green) and PI (red) staining of MPCs treated with CHIR, IWP-4 and IWR-1 for 7 days. Scale bar, 100 µm. B Alizarin red staining of MPCs treated with combinations of CHIR, IWP-4 and IWR-1 for 21 days. [1]

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Effects on Late Osteogenesis Markers[1]
Researchers further investigated each molecule’s effects on late osteogenesis, using Alizarin red staining to determine the extent of mineral deposition after 21 days. These results mirrored those of the ELF97 staining, with osteogenic supplements inducing the formation of Alizarin red-positive deposits across the majority of the culture surface. This was almost completely abolished in the presence of CHIR and inhibited to a lesser extent by either IWP-4 or IWR-1 at the concentrations tested ( Fig. 3B ). This confirmed that effects detected in the MBA and static plate, using 7 days ELF97 staining as an early readout, translated through to an equivalent influence on the final maturation of MPCs into mineralizing osteoblasts. Together these data provided confidence that we could use conventional cultures to further investigate the changes seen in the MBA screen.[1]

[1]. Primitive cardiac cells from human embryonic stem cells. Stem Cells Dev. 2012 Jun 10;21(9):1513-23.

[2]. Microbioreactor array screening of Wnt modulators and microenvironmental factors in osteogenic differentiation of mesenchymal progenitor cells. PLoS One. 2013 Dec 23;8(12):e82931.

Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.[1]

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Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.[2]

C23H20N4O3S3

496.618

496.069

C, 55.63; H, 4.06; N, 11.28; O, 9.66; S, 19.37

686772-17-8

686772-17-8

2155264

White to off-white solid powder

1.5±0.1 g/cm3

1.762

5.16

1

8

6

33

851

0

RHUJMHOIQBDFQR-UHFFFAOYSA-N

InChI=1S/C23H20N4O3S3/c1-13-7-8-14-18(11-13)33-22(24-14)26-19(28)12-32-23-25-15-9-10-31-20(15)21(29)27(23)16-5-3-4-6-17(16)30-2/h3-8,11H,9-10,12H2,1-2H3,(H,24,26,28)

N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide

IWP4; IWP 4; 686772-17-8; IWP-4; IWP 4; 2-((3-(2-methoxyphenyl)-4-oxo-3,4,6,7-tetrahydrothieno[3,2-d]pyrimidin-2-yl)thio)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide; wnt inhibitor iwp-4; CHEMBL1257090; N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide; 2-{[3-(2-methoxyphenyl)-4-oxo-3H,4H,6H,7H-thieno[3,2-d]pyrimidin-2-yl]sulfanyl}-N-(6-methyl-1,3-benzothiazol-2-yl)acetamide; IWP-4

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~1.3 mg/mL (~2.62 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.)