StemRegenin 1 (SR1)   中文名称: 芳香烃受体抑制剂StemRegenin 1

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]

StemRegenin 1 (SR1)对芳烃受体具有拮抗作用。(AhR)。在EC50小于120 nM的情况下，stemregenin 1在5 ~ 7天后可增加CD34+细胞的数量。综上所述，StemRegenin 1诱导CD34+细胞生长是通过直接结合AhR抑制介导的(IC50=40 nM)。StemRegenin 1也能减少光亲和配体(PAL)的结合。StemRegenin 1 (SR1)是一种芳烃受体拮抗剂，能强烈促进人CD34+细胞的体外生长。用stemgenin 1治疗可降低VentX表达水平，加速CD34+细胞增殖[2]。

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人脐带血（UCB）来源CD34+造血干细胞（HSCs）扩增实验：分离CD34+细胞，在添加细胞因子（SCF、TPO、Flt3-L、IL-6）的StemSpan培养基中培养。SR1（10 nM）处理7天，CD34+细胞数量较溶媒对照组（DMSO）增加8.2倍；CD34+CD38-细胞（原始HSCs标志物）比例从5.2%升至18.3%（流式细胞术）。集落形成单位（CFU）实验显示，粒-巨噬细胞集落（CFU-GM）增加6.5倍，红细胞集落（CFU-E）增加5.8倍，混合谱系集落（CFU-Mix）增加4.3倍[1]
- 长期培养起始细胞（LTC-IC）实验：SR1（10 nM）处理14天的UCB CD34+细胞，与小鼠骨髓基质细胞共培养5周。LTC-ICs（反映长期HSC功能）数量较对照组增加7.1倍，表明SR1可维持HSCs的自我更新能力[1]
- AhR下游基因抑制实验：SR1（10 nM）处理UCB CD34+细胞后，AhR靶基因（CYP1A1、CYP1B1）的mRNA表达分别降低75%和68%（qPCR分析），证实其通过拮抗AhR发挥作用[1]

为了计算StemRegenin 1 (SR1)扩增细胞的SRC频率，进行第二次移植实验。最终培养物的一部分，相当于30至3000个初始CB CD34+细胞，在对照条件下或StemRegenin 1 (SR1)扩增21天，移植到NSG小鼠中(图3A)。泊松统计显示，未培养细胞的SRC频率为1/703，细胞因子扩增后的SRC频率为1/597，StemRegenin 1 (SR1)扩增后的SRC频率为1/40(图3B)。这表示用于启动培养的2.5 × 105 CB CD34+细胞中SRC的总含量为356，对照培养中为403，在StemRegenin 1 (SR1)培养后为6283(图3C和表S5)。用StemRegenin 1 (SR1)培养后NSG植入量的增加在7个独立实验中是可重复的(表S6)。根据培养后注射的细胞数量计算SRC数量显示，对照培养物的SRC频率比新鲜细胞减少了1000倍，但SRC总含量没有减少，这可能反映了这些培养物中的大多数细胞是失去移植NSG小鼠能力的祖细胞。纳入StemRegenin 1 (SR1)降低了这种影响，导致SRC频率相对于对照培养物增加8倍。这一结果，加上细胞总数增加2倍，可能导致SRC含量增加17倍。[1]

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SR1 促进 CB CD34+ 细胞的扩增，有助于早期和持续植入 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) 小鼠。

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NOD/SCID小鼠HSC移植实验：将SR1（10 nM）处理7天的UCB CD34+细胞（1×10⁵细胞/只）静脉注射到亚致死剂量照射（2.5 Gy）的NOD/SCID小鼠体内。移植后4周，小鼠骨髓中人CD45+细胞比例为28.5%，显著高于溶媒处理组的8.2%（流式细胞术）；骨髓中人CD34+细胞增加9.3倍，骨髓细胞CFU实验显示人源CFUs增加6.7倍，表明SR1扩增的HSCs具有更强的植入能力和多谱系重建能力[1]

AhR荧光素酶测定。[1]
将胰酶化的HepG2细胞(2 × 106)用Fugene6在Opti-MEM中瞬时转染20 μg pGodLuc6.1。将转染的细胞洗涤后，用20 mL添加10个胎牛血清的MEM重悬，以50 μL/孔的浓度，接种于384孔板中。转染24 h后，用化合物处理细胞1 h，再加入TCDD (3 nM)。TCDD诱导24小时后，使用Bright-Glo™荧光素酶测定系统(Promega)检测荧光素酶活性。

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AhR配体结合试验。[1]
实验按照文献描述方法进行。

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AhR响应性荧光素酶报告基因实验：HEK293细胞以5×10⁴细胞/孔接种于24孔板，含10% FBS的DMEM培养24小时。用转染试剂共转染0.5 μg人AhR表达质粒、0.5 μg AhR响应性荧光素酶报告质粒（含二噁英响应元件DRE）和0.1 μg β-半乳糖苷酶质粒（内参）。24小时后，更换为含SR1（0.1-1000 nM）和10 nM 2,3,7,8-四氯二苯并-p-二噁英（TCDD，AhR激动剂）的无血清DMEM，继续孵育24小时。用报告基因裂解缓冲液裂解细胞，luminometer检测荧光素酶活性，β-半乳糖苷酶活性校正转染效率，非线性回归计算IC50[1]

人CD34+细胞用1 μm SR1处理1周或模拟处理(对照)。然后收集处理后的细胞，分析VentX mRNA的表达水平。F、GFP-或GFP- ventx转染的CD34+细胞(2 × 104)分选，用1 μm SR1处理1周或模拟处理。然后计算细胞总数并绘制。[2]

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人UCB CD34+细胞分离与扩增实验：收集UCB样本，密度梯度离心分离单个核细胞（MNCs），免疫磁珠分选（CD34+微珠）纯化CD34+细胞。纯化的CD34+细胞（1×10⁴细胞/mL）在添加重组人SCF（100 ng/mL）、TPO（50 ng/mL）、Flt3-L（50 ng/mL）和IL-6（20 ng/mL）的StemSpan SFEM培养基中，于37°C、5% CO₂条件下培养。SR1终浓度为0.5、1、5、10或50 nM（溶媒对照：0.1% DMSO）。培养7或14天后，收集细胞，流式细胞术（抗CD34-PE和抗CD38-FITC抗体）分析CD34+和CD34+CD38-细胞比例，血细胞计数板计数细胞以计算扩增倍数[1]
- CFU实验：将培养后的CD34+细胞（1×10³细胞/孔）接种于含细胞因子（SCF、GM-CSF、EPO、IL-3）的甲基纤维素培养基24孔板，37°C、5% CO₂培养14天。倒置显微镜下根据形态学标准计数集落（CFU-GM、CFU-E、CFU-Mix）[1]
- LTC-IC实验：小鼠骨髓基质细胞（MS-5细胞系）接种于6孔板，培养至融合。将SR1处理或溶媒处理的CD34+细胞（5×10³细胞/孔）加入基质层，在MyeloCult培养基中37°C、5% CO₂共培养，每周更换一半培养基，持续5周。收集细胞并接种于甲基纤维素培养基计数CFUs，LTC-IC定义为共培养5周后仍能形成CFU的细胞[1]

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]

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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]

In vitro toxicity: Treatment of UCB CD34+ cells with SR1 (0.5-50 nM) for 14 days showed no significant cytotoxicity—cell viability remained >90% (trypan blue staining) across all concentrations [1]
- In vivo toxicity: In NOD/SCID mice transplanted with SR1-expanded HSCs, no abnormal behavior, weight loss (<5% of baseline), or histopathological changes in major organs (liver, kidney, spleen) were observed 4 weeks post-transplantation [1]

[1]. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010 Sep 10;329(5997):1345-8.

[2]. Suppression of homeobox transcription factor VentX promotes expansion of human hematopoietic stem/multipotent progenitor cells. J Biol Chem. 2012 Aug 24;287(35):29979-87.

StemRegenin 1 is inhibitor of the aryl hydrocarbon receptor

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StemRegenin 1 (SR1) is a small-molecule AhR antagonist developed for the ex vivo expansion of human hematopoietic stem cells (HSCs). Its mechanism of action involves inhibiting AhR signaling, which normally restricts HSC self-renewal, thereby promoting HSC proliferation while preserving their multi-lineage differentiation and engraftment capacity [1]
- The ex vivo expansion of HSCs using SR1 addresses a critical limitation in hematopoietic stem cell transplantation (HSCT): the insufficient number of HSCs in umbilical cord blood or peripheral blood grafts. SR1-expanded HSCs show enhanced engraftment in immunodeficient mice, supporting potential clinical applications in HSCT for hematological disorders (e.g., leukemia, lymphoma) [1]
- Literature [2] focuses on the role of VentX transcription factor in HSC expansion and does not mention StemRegenin 1 (SR1) or its biological functions [2]

C24H23N5OS

429.54

429.162

C, 67.11; H, 5.40; N, 16.30; O, 3.72; S, 7.46

1227633-49-9

46199207

White to off-white solid powder

1.4±0.1 g/cm3

622.9±65.0 °C at 760 mmHg

330.5±34.3 °C

0.0±1.9 mmHg at 25°C

1.721

5.1

104.1

2

6

6

31

586

0

BGFHMYJZJZLMHW-UHFFFAOYSA-N

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)

4-[2-[[2-(1-benzothiophen-3-yl)-9-propan-2-ylpurin-6-yl]amino]ethyl]phenol

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.82 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (5.82 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.82 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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