IWP-4

Wnt (IC50 = 25 nM)

IWP-4 是一种小分子 Wnt 抑制剂，IC50 为 25 nM。心脏指标，如心肌肌钙蛋白 I (CTNI) 和心肌肌球蛋白重链白细胞 (MYHhi+)，由 IWP-4 诱导表达。此外，响应 IWP-4 出现脉动病灶（每秒 0.44 ± 0.10 SEM 节拍）；这些病灶不存在于任何未接受 IWP-4 的培养物中。此外，第16天的流式细胞术分析显示，IWP-4处理和未处理的培养物之间的MYHlo + 细胞数量存在显着差异（P<0.0002），分别为17.0±1.3SD%和5.4±1.4SD%。根据 NKX2-5 蛋白表达定量，63% (481/817) 的 IWP-4 处理细胞具有核 NKX2-5 表达 [1]。当在第 7 天将用 IWP-4 处理的间充质前体细胞 (MPC) 与单独的成骨培养基进行比较时，AXIN2、CTNNB1 和 GSK3B 的表达没有显着变化。然而，在第 21 天，用 IWP-4 处理的 MPC 中 DKK1 和 GSK3β 的表达增加。 IWP-4 同样导致 COL1A1 和 SPARC 显着下调 [2]。

基因表达调控[1]
使用这些静态培养，我们随后利用RT-qPCR测量Wnt信号通路的一些关键成员表达的任何变化，并确定它们如何受到CHIR、IWR-1和<强>IWP-4处理的影响。正如预期的那样，由于其作为典型Wnt激动剂的作用，CHIR治疗MPCs导致AXIN2（被视为典型Wnt通路激活的标记物），CTNNB1 （β-catenin）和GSK3B上调，而Wnt抑制剂DKK1在7天和21天下调（图4）。在第7天，与单独成骨培养基相比，IWP-4和IWR-1处理的MPCs中AXIN2、CTNNB1和GSK3B的表达没有显著变化，但在第21天，IWP-4处理的MPCs中DKK1和GSK3B的表达水平升高。在CHIR处理的MPCs中，在第7天和第21天AXIN2的显著上调（高达350倍）提供了一个强有力的迹象，表明CHIR以预期的方式起作用（激活典型的Wnt信号），因此我们接下来分析了成骨不同阶段标志物的表达，以阐明为什么CHIR可能起抑制分化的作用，以及激动剂CHIR与拮抗剂IWR-1和IWP-4之间可能观察到什么差异。[1]

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与MBA筛选结果一致，IWP-4和IWR-1对基因表达水平的影响弱于CHIR。然而，通过CHIR观察，IWR-1和IWP-4均降低了ALP的表达水平，而RUNX2、MSX2和DLX5的表达水平未同时升高（图3C）。21天后，IWR-1处理下的ALP表达与未处理的对照组相似，但在iwr -4处理下仍降低。在这个较晚的时间点，IWP-4也引起了SPARC和COL1A1的显著下调，而使用IWR-4只观察到COL1A1的显著降低（图3D）

CHIR IWP-4和IWR-1处理7天后MPCs的ELF97（绿色）和PI（红色）染色。比例尺，100µm。B CHIR、IWP-4和IWR-1联合处理21天的MPCs茜素红染色。[1]

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晚期成骨标志物[1]
的影响 研究人员进一步研究了每种分子对晚期成骨的影响，使用茜素红染色来确定21天后矿物质沉积的程度。这些结果反映了ELF97染色的结果，在大部分培养表面，成骨补充剂诱导茜素红阳性沉积物的形成。在CHIR的存在下，这几乎完全被消除，并且在测试浓度下，IWP-4或IWR-1在较小程度上被抑制（图3B）。这证实了在MBA和静态板中检测到的效应，使用7天ELF97染色作为早期读数，转化为对MPCs最终成熟为矿化成骨细胞的等效影响。总之，这些数据提供了信心，我们可以使用传统文化来进一步研究MBA屏幕上看到的变化。

[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

164.95

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。