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Purity: =98.22%
StemRegenin 1(abbreviated as SR1; SR-1), a purine derivative, is a potent antagonist of the aryl hydrocarbon receptor (AhR) with important biological activity. It inhibits AhR with IC50 of 127 nM in CD34+ cells. SR1 has been found to promote the ex vivo expansion of CD34+ cells. SR1 has also been found to induce the ex vivo differentiation of pDCs, BDCA1+ and BDCA3+ mDCs as well as the generation of high numbers of all these DC subsets.
| Targets |
Aryl hydrocarbon receptor/AhR (IC50 = 127 nM)
StemRegenin 1 (SR1) is a selective antagonist of the aryl hydrocarbon receptor (AhR), with an IC50 of 10 nM for human AhR in an AhR-responsive luciferase reporter assay. It does not bind to other nuclear receptors (e.g., PPARγ, RARα) at concentrations up to 1 μM [1] |
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| ln Vitro |
The aryl hydrocarbon receptor is antagonistically acted upon by StemRegenin 1 (SR1). (AhR). With an EC50 of less than 120 nM, stemregenin 1 boosts the number of CD34+ cells after 5 to 7 days. Based on these findings, it can be concluded that StemRegenin 1-induced CD34+ cell growth is mediated by direct binding and AhR inhibition (IC50=40 nM). StemRegenin 1 also reduces photoaffinity ligand (PAL) binding. StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor, strongly encourages the ex vivo growth of human CD34+ cells. Treatment with stemgenin 1 reduces VentX expression levels and speeds up CD34+ cell proliferation[2].
Human umbilical cord blood (UCB)-derived CD34+ hematopoietic stem cell (HSC) expansion assay: CD34+ cells were isolated and cultured in StemSpan medium supplemented with cytokines (SCF, TPO, Flt3-L, IL-6). Treatment with SR1 (10 nM) for 7 days increased the number of CD34+ cells by 8.2-fold compared to the vehicle control (DMSO). The proportion of CD34+CD38- cells (a marker of primitive HSCs) increased from 5.2% to 18.3% (flow cytometry). Colony-forming unit (CFU) assays showed a 6.5-fold increase in CFU-GM (granulocyte-macrophage), 5.8-fold increase in CFU-E (erythroid), and 4.3-fold increase in CFU-Mix (mixed lineage) [1] - Long-term culture-initiating cell (LTC-IC) assay: UCB CD34+ cells treated with SR1 (10 nM) for 14 days were co-cultured with murine bone marrow stromal cells for 5 weeks. The number of LTC-ICs (a measure of long-term HSC function) increased by 7.1-fold compared to controls, indicating preservation of HSC self-renewal capacity [1] - AhR downstream gene inhibition: SR1 (10 nM) reduced mRNA expression of AhR target genes (CYP1A1, CYP1B1) by 75% and 68%, respectively (qPCR analysis) in UCB CD34+ cells, confirming AhR antagonism as its mechanism of action [1] |
| ln Vivo |
StemRegenin 1 (SR1) promotes expansion of CB CD34+ cells that contribute to both early and sustained engraftment NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice.
To calculate the SRC frequency of StemRegenin 1 (SR1) expanded cells a second engraftment experiment was performed. A fraction of the final culture, equivalent to between 30 and 3000 starting CB CD34+ cells expanded for 21 days with control conditions or StemRegenin 1 (SR1), were transplanted into NSG mice (Fig. 3A). Poisson statistics revealed a SRC frequency of 1/703 for uncultured cells, 1/597 per starting cell after cytokine expansion, and 1/40 per starting cell after expansion with StemRegenin 1 (SR1) (Fig. 3B). This represents a total SRC content of 356 in the 2.5 × 105 CB CD34+ cells used to initiate the cultures, 403 SRC in control cultures, and 6283 SRC after culture with StemRegenin 1 (SR1) (Fig. 3C and table S5). The increase in NSG engraftment after culture with StemRegenin 1 (SR1) was reproducible over seven independent experiments (table S6). Calculation of the SRC numbers based on the number of cells injected after culture revealed that control cultures had a 1000-fold reduction in the SRC frequency but not total SRC content relative to fresh cells, likely reflecting that the majority of cells in these cultures are progenitor cells that have lost the ability to engraft NSG mice. Inclusion of StemRegenin 1 (SR1) diminished this effect and resulted in an 8-fold increase in the SRC frequency relative to control cultures. This result, together with the 2-fold increase in total cell number likely account for the 17-fold increase in SRC content.[1]
NOD/SCID mouse HSC transplantation assay: UCB CD34+ cells (1×10⁵ cells/mouse) treated with SR1 (10 nM) for 7 days were intravenously injected into sublethally irradiated (2.5 Gy) NOD/SCID mice. Four weeks post-transplantation, the proportion of human CD45+ cells in mouse bone marrow was 28.5%, compared to 8.2% in the vehicle-treated group (flow cytometry). Human CD34+ cells in mouse bone marrow increased by 9.3-fold, and CFU assays of mouse bone marrow cells showed a 6.7-fold increase in human CFUs, demonstrating enhanced engraftment and multi-lineage reconstitution capacity of SR1-expanded HSCs [1] |
| Enzyme Assay |
AhR luciferase assay. [1]
Trypsinized HepG2 cells (2 x 106 ) were transiently transfected with 20 μg of pGodLuc6.1 using Fugene6 in Opti-MEM. The transfected cells were washed and resuspended in 20 mL of MEM supplemented with 10 fetal calf serum, and plated into 384 well plates at 50 μL/well. 24 h after transfection, cells were treated with compound for 1 h prior to the addition of TCDD (3 nM). Following 24 h of induction with TCDD, luciferase activity was measured using Bright-Glo™ Luciferase Assay System (Promega). AhR ligand binding assay. [1] Experiments were performed as described previously. AhR Responsive Luciferase Reporter Assay: HEK293 cells were seeded in 24-well plates at 5×10⁴ cells/well and cultured in DMEM with 10% FBS for 24 hours. Cells were co-transfected with 0.5 μg human AhR expression plasmid, 0.5 μg AhR-responsive luciferase reporter plasmid (containing a dioxin-responsive element, DRE), and 0.1 μg β-galactosidase plasmid (internal control) using a transfection reagent. After 24 hours, medium was replaced with serum-free DMEM containing SR1 (0.1-1000 nM) and 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an AhR agonist). Cells were incubated for another 24 hours, lysed with reporter lysis buffer, and luciferase activity was measured with a luminometer. β-galactosidase activity was detected to normalize transfection efficiency, and IC50 was calculated via nonlinear regression [1] |
| Cell Assay |
Human CD34+ cells were treated with 1 μm SR1 for 1 week or mock-treated (control (Ctr)). The treated cells were then harvested for analysis of VentX mRNA expression levels. F, GFP- or GFP-VentX-transfected CD34+ cells (2 × 104) were sorted and treated with 1 μm SR1 for 1 week or mock-treated. The total cell numbers were then counted and plotted.[2]
Human UCB CD34+ Cell Isolation and Expansion Assay: UCB samples were collected, and mononuclear cells (MNCs) were isolated via density gradient centrifugation. CD34+ cells were purified from MNCs using magnetic bead sorting (CD34+ microbeads). Purified CD34+ cells (1×10⁴ cells/mL) were cultured in StemSpan SFEM medium supplemented with recombinant human SCF (100 ng/mL), TPO (50 ng/mL), Flt3-L (50 ng/mL), and IL-6 (20 ng/mL) at 37°C, 5% CO₂. SR1 was added to the medium at final concentrations of 0.5, 1, 5, 10, or 50 nM (vehicle control: 0.1% DMSO). After 7 or 14 days of culture, cells were harvested, and CD34+ and CD34+CD38- cell proportions were analyzed via flow cytometry (using anti-CD34-PE and anti-CD38-FITC antibodies). Cell count was performed using a hemocytometer to calculate expansion fold [1] - CFU Assay: Cultured CD34+ cells (1×10³ cells/well) were seeded in 24-well plates containing methylcellulose-based medium supplemented with cytokines (SCF, GM-CSF, EPO, IL-3). Plates were incubated at 37°C, 5% CO₂ for 14 days. Colonies (CFU-GM, CFU-E, CFU-Mix) were counted under an inverted microscope based on morphological criteria [1] - LTC-IC Assay: Murine bone marrow stromal cells (MS-5 cell line) were seeded in 6-well plates and cultured until confluent. SR1-treated or vehicle-treated CD34+ cells (5×10³ cells/well) were added to the stromal layer, and co-cultured in MyeloCult medium at 37°C, 5% CO₂. Half of the medium was replaced every week for 5 weeks. Cells were then harvested and plated in methylcellulose medium for CFU counting; LTC-ICs were defined as CFU-forming cells after 5 weeks of co-culture [1] |
| Animal Protocol |
Engraftment of CD34+ cells in NSG mice. [1]
A quantity of 250,000 CB-derived CD34+ cells were cultured with control conditions (DMSO, 0.01%) or StemRegenin 1 (SR1) (0.75 μM) for 3 weeks. At this point control cultures had expanded 1080-fold and StemRegenin 1 (SR1) treated cells expanded 2024-fold relative to starting cell numbers. A quantity of 30 to 30,000 uncultured CD34+ CB-derived cells or a fraction of the final culture equivalent to 30 to 10,000 starting cells were transplanted. The cells were injected intravenously via the retro-orbital route into sub-lethally irradiated (300 rads for experiment in Fig. 2A, 200 rads for experiment if Fig. 3A-B) 6- to 10-week-old NSG mice. Engraftment was performed within 24 h after irradiation. Engraftment was monitored by flow cytometric analysis of blood obtained via retro-orbital sinus or bone marrow using anti-human CD45 and anti-mouse CD45 antibodies. The mice were sacrificed between 13-16 weeks posttransplantation; bone marrow (from both femurs and tibiae), spleen and thymus were collected for analysis. For secondary engraftment, 50% of the bone marrow from each recipient mouse was transplanted into one secondary sub-lethally irradiated NSG mouse. Fifteen weeks after transplantation, bone marrow was harvested from the secondary mice and analyzed by flow cytometry. [1] NOD/SCID Mouse HSC Transplantation Protocol: Female NOD/SCID mice (6-8 weeks old, 18-22 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle, free access to food/water). Mice were sublethally irradiated with a single dose of 2.5 Gy (γ-irradiation) 24 hours before transplantation. UCB CD34+ cells were treated with SR1 (10 nM) or vehicle for 7 days, then resuspended in PBS at a concentration of 1×10⁵ cells/100 μL. Each mouse received an intravenous injection of 100 μL cell suspension via the tail vein. Four weeks post-transplantation, mice were euthanized with CO₂, and bone marrow was flushed from the femurs and tibias. Bone marrow cells were stained with anti-human CD45-APC antibody and analyzed via flow cytometry to determine human cell engraftment. For CFU analysis, bone marrow cells were plated in methylcellulose medium to count human CFUs [1] |
| Toxicity/Toxicokinetics |
In vitro toxicity: No significant cytotoxicity was observed when UCB CD34+ cells were treated with SR1 (0.5-50 nM) for 14 days – cell viability remained above 90% at all concentrations (trypan blue staining) [1]
- In vivo toxicity: No abnormal behavior, weight loss (<5% of baseline), or histopathological changes in major organs (liver, kidney, spleen) were observed in NOD/SCID mice transplanted with SR1-amplified HSCs 4 weeks after transplantation [1] |
| References |
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| Additional Infomation |
StemRegenin 1 is an aryl hydrocarbon receptor inhibitor. StemRegenin 1 (SR1) is a small molecule aryl hydrocarbon receptor antagonist developed specifically for the in vitro expansion of human hematopoietic stem cells (HSCs). Its mechanism of action is to inhibit the AhR signaling pathway, which normally limits HSC self-renewal, thereby promoting HSC proliferation while maintaining their multilineage differentiation and transplantation capacity [1]. - The use of SR1 for in vitro expansion of HSCs addresses a key limitation of hematopoietic stem cell transplantation (HSCT): the insufficient number of HSCs in cord blood or peripheral blood transplantation. SR1-expanded hematopoietic stem cells have shown enhanced engraftment capacity in immunodeficient mice, supporting their potential clinical application in hematopoietic stem cell transplantation for the treatment of hematologic diseases such as leukemia and lymphoma [1]. - The literature [2] mainly focuses on the role of VentX transcription factor in the expansion of hematopoietic stem cells and does not mention stem cell regenerin 1 (SR1) or its biological function [2].
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| Molecular Formula |
C24H23N5OS
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| Molecular Weight |
429.54
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| Exact Mass |
429.162
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| Elemental Analysis |
C, 67.11; H, 5.40; N, 16.30; O, 3.72; S, 7.46
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| CAS # |
1227633-49-9
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| Related CAS # |
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| PubChem CID |
46199207
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
622.9±65.0 °C at 760 mmHg
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| Flash Point |
330.5±34.3 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.721
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| LogP |
5.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
31
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| Complexity |
586
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BGFHMYJZJZLMHW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H23N5OS/c1-15(2)29-14-26-21-23(25-12-11-16-7-9-17(30)10-8-16)27-22(28-24(21)29)19-13-31-20-6-4-3-5-18(19)20/h3-10,13-15,30H,11-12H2,1-2H3,(H,25,27,28)
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| Chemical Name |
4-[2-[[2-(1-benzothiophen-3-yl)-9-propan-2-ylpurin-6-yl]amino]ethyl]phenol
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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. Solubility in Formulation 2: 2.5 mg/mL (5.82 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3281 mL | 11.6404 mL | 23.2807 mL | |
| 5 mM | 0.4656 mL | 2.3281 mL | 4.6561 mL | |
| 10 mM | 0.2328 mL | 1.1640 mL | 2.3281 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT02765997 | Withdrawn | Drug: Larotrectinib Sulfate Procedure: Bone Scan |
Biological: Unmanipulated UCB Biological: SR-1 UCB |
Masonic Cancer Center, University of Minnesota |
April 2017 | Phase 2 |
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