With an IC50 of 1.8 nM, CP21R7 (Compound 9) is a selective inhibitor of GSK-3β; its IC50 against PKCα is 1900 nM[1]. With maximum activity, CP21R7 (CP21, 3 μM) strongly stimulates canonical Wnt signaling. CP21 considerably raises intracellular β-catenin total levels. BMP4 and CP21 together cause hPSCs to commit to mesoderm[2].

---

1. Activation of canonical Wnt signaling in human pluripotent stem cells (hPSCs): CP21R7 (a GSK3β inhibitor) treatment activated β-catenin promoter activity in hPSCs, as detected by luciferase assay. The assay used a 6-point 3-fold serial dilution of CP21R7 (10, 3, 1, 0.3, 0.1, 0.03 μM), with the last two concentrations not shown. Immunofluorescent staining revealed altered localization of β-catenin in hPSCs after 24-hour treatment with CP21R7. Quantitative PCR confirmed that CP21R7 upregulated the expression of β-catenin target genes in hPSCs [2]
2. Induction of mesodermal fate in hPSCs: Combined treatment with CP21R7 and BMP4 rapidly committed hPSCs to a mesodermal fate. Immunofluorescence staining showed changes in the expression of pluripotency, mesoderm, and endoderm markers in hPSCs during the first 4 days of differentiation [2]
3. Promotion of vascular cell differentiation: After inducing mesodermal fate with CP21R7 and BMP4, subsequent exposure to VEGF-A led to the differentiation of hPSCs into vascular endothelial cells (hPSC-ECs) with an efficiency exceeding 80% within six days. Exposure to PDGF-BB resulted in the differentiation into vascular smooth muscle cells (hPSC-VSMCs) with a similar high efficiency. Flow cytometry sorting via surface markers (CD144 for ECs, CD140b for VSMCs) purified the cells to 99% [2]
4. Functional characterization of differentiated vascular cells:
- hPSC-ECs expressed EC-specific markers (VE-Cadherin, PECAM1, vWF), with 74.52% of cells expressing vWF. They showed in vitro functionality including monolayer impedance response to thrombin, trans-endothelial electrical resistance (TEER) properties, uptake of fluorescently labeled acLDL, upregulation of ICAM1 upon proinflammatory cytokine (TNFα, IL1β) stimulation, and leukocyte (HL60 cell) adhesion which could be blocked by anti-ICAM1 antibody. In vitro tube formation was observed when plated on matrigel, and this tubulogenesis could be inhibited by anti-angiogenic molecules [2]
- hPSC-VSMCs expressed VSMC-specific markers (SMA, Myosin IIB, SM22a), with 62.08% of cells expressing SMA, 92.75% expressing Myosin IIB, and 100% expressing SM22a. They exhibited functional characteristics such as intracellular calcium increase upon stimulation with vasoconstrictive reagents and contractility in response to U46619 [2]

CP21R7 shows efficacy in an in vivo rat osteoporosis models.

---

1. In vivo functionality of hPSC-ECs differentiated with CP21R7: Fibrinogen grafts containing hPSC-ECs (either alone or combined with MSCs or hPSC-VSMCs) were implanted into mice. Histological analysis (HE staining and human-specific CD31/DAB staining) at 7 or 14 days post-implantation showed the formation of vascular structures, confirming the in vivo functionality of the differentiated ECs [2]

1. Luciferase assay for β-catenin promoter activity: hPSCs were transfected with β-catenin promoter-driven luciferase reporter plasmid. After transfection, cells were treated with CP21R7 at a 6-point 3-fold serial dilution (10, 3, 1, 0.3, 0.1, 0.03 μM). Luciferase activity was measured after a specified incubation time to assess β-catenin promoter activation. The experiment was repeated 5 times independently, and results were expressed as mean ± SD [2]
2. Immunofluorescence staining:
- For β-catenin localization: hPSCs were treated with CP21R7 for 24 hours, fixed, permeabilized, and incubated with β-catenin primary antibody followed by fluorescently labeled secondary antibody. Images were captured under a fluorescence microscope, with 3 independent experiments performed [2]
- For cell lineage markers: hPSCs were subjected to differentiation with CP21R7 and BMP4. During the first 4 days, cells were fixed and stained with antibodies against pluripotency, mesoderm, and endoderm markers. Fluorescence images were obtained, with 3 independent experiments conducted [2]
- For vascular cell markers: Differentiated hPSC-ECs and hPSC-VSMCs were fixed and stained with antibodies against EC-specific markers (VE-Cadherin, PECAM1, vWF) and VSMC-specific markers (SMA, Myosin IIB, SM22a), respectively. Images were captured, and positive cell percentages were quantified [2]
- For ICAM1 expression: hPSC-ECs were treated with TNFα, fixed, and stained with ICAM1 primary antibody and fluorescent secondary antibody. Fluorescence images were taken to observe ICAM1 upregulation [2]
3. Quantitative PCR (qPCR): hPSCs were treated with CP21R7, and total RNA was extracted and reverse-transcribed into cDNA. qPCR was performed using primers specific for β-catenin target genes. Each gene had 3 biological and technical replicates, and results were presented as mean ± SEM from 3 independent experiments [2]
4. Flow cytometry:
- For differentiation efficiency analysis: Differentiated hPSC-ECs and hPSC-VSMCs were stained with CD144 (EC marker) and CD140b (VSMC marker) antibodies. Flow cytometry was used to determine the percentage of positive cells, with 10 independent experiments for hPSC-ECs and 16 for hPSC-VSMCs [2]
- For ICAM1 quantification: hPSC-ECs were stimulated with TNFα or IL1β, stained with ICAM1 antibody, and flow cytometry was used to quantify ICAM1 expression levels [2]
5. Leukocyte adhesion assay: hPSC-ECs were stimulated with TNFα, and HL60 cells were added for co-incubation. After washing, adherent HL60 cells were stained and counted under a microscope. For antibody blockage experiments, hPSC-ECs were pretreated with anti-ICAM1 antibody or control antibody before TNFα stimulation. The experiment was repeated 3 times independently, with results expressed as mean ± SD [2]
6. Calcium imaging: hPSC-VSMCs at day 13 of differentiation were loaded with calcium-sensitive fluorescent dye. Vasoconstrictive reagents were added, and changes in intracellular calcium levels (fold change in RFU) were measured at 50s (maximum peak). Data were from 3 independent assays and analyzed by Student’s t-test [2]
7. Contractility assay: UASMCs and hPSC-VSMCs were treated with U46619 for 48 hours. Contractility was assessed, with 1 well for control and 2 wells for test conditions in a single experiment [2]
8. Tube formation assay: hPSC-ECs were plated on matrigel alone or with primary human brain vascular pericytes (hBVPs) and incubated for 24 hours. Tube formation was observed and imaged, with 10 independent experiments for hPSC-ECs alone and 3 for co-culture with hBVPs. For inhibitory experiments, anti-angiogenic molecules were added, and the inhibitory effect on tubulogenesis was quantified from 3 independent experiments [2]

---

1. In vivo implantation of fibrinogen grafts:
- Preparation of grafts: hPSC-ECs were mixed with fibrinogen to prepare grafts; some grafts were combined with MSCs or hPSC-VSMCs. HUVECs + MSCs were used as a control group [2]
- Animal model: SCID mice or NOD SCID mice were selected as experimental animals [2]
- Implantation procedure: Grafts were implanted into mice, with 5 mice per group and 2 implants per mouse (10 implants per group) [2]
- Sample collection and analysis: Implants were harvested at 7 or 14 days post-implantation. Tissues were fixed, embedded in paraffin, sectioned, and subjected to HE staining and human-specific CD31/DAB staining to observe vascular structure formation [2]
2. Rat osteoporosis model experiment: No detailed protocol for the rat osteoporosis model experiment involving CP21R7 was provided; only the efficacy of compound 34 was mentioned [1]

[1]. Discovery of potent and bioavailable GSK-3beta inhibitors. Bioorg Med Chem Lett. 2010 Mar 1;20(5):1693-6.

[2]. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol. 2015 Aug;17(8):994-1003.

1. Background: GSK-3β inhibitors have potential therapeutic applications. A series of highly effective and selective maleimide GSK-3β inhibitors have been developed, among which compound 34 (belonging to the same series as CP21R7) has an IC50 value of 0.6 nM against GSK-3β and a selectivity of more than 100-fold against other kinases [1]. 2. Background: Human pluripotent stem cells (hPSCs) need to differentiate into functional adult cell types for in vitro disease modeling and clinical applications. GSK3 inhibition combined with BMP4 treatment is a key step for the rapid and efficient differentiation of hPSCs into vascular endothelial cells and smooth muscle cells [2]. 3. Mechanism of action: CP21R7, as a GSK3β inhibitor, activates the classical Wnt signaling pathway by regulating β-catenin activity. This activation, combined with BMP4 treatment, can cause hPSCs to differentiate into mesodermal lineages and further differentiate into vascular endothelial cells under VEGF-A stimulation, or into vascular smooth muscle cells under PDGF-BB stimulation [2]. 4. Therapeutic potential: CP21R7-differentiated hPSC-ECs and hPSC-VSMCs are highly similar to their in vivo counterparts in terms of overall transcriptomic and metabolomic characteristics and exhibit related in vitro and in vivo functions. These cells can be used to construct realistic models of human vascular diseases [2].

C19H15N3O2

317.34

317.116

125314-13-8

5327711

Yellow to orange solid powder

1.4±0.1 g/cm3

627.4±55.0 °C at 760 mmHg

333.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.714

2.4

2

3

2

24

585

0

RGTAEYDIDMGJLX-UHFFFAOYSA-N

InChI=1S/C19H15N3O2/c1-22-10-14(13-7-2-3-8-15(13)22)17-16(18(23)21-19(17)24)11-5-4-6-12(20)9-11/h2-10H,20H2,1H3,(H,21,23,24)

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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