StemRegenin 1 Research Articles

Extensive immunophenotypic sub-population analysis of StemRegenin1 expanded haematopoietic stem/progenitor cells

BackgroundEx vivo haematopoietic stem/progenitor cell (HSPCs) expansion constitutes an important area of research, and has the potential to improve access to umbilical cord blood (UCB) as a source of stem cells for haematopoietic stem cell transplantation (HSCT). The ability to improve stem cell dose and thereby reduce delayed engraftment times, which has plagued the use of UCB as a stem cell source since inception, is a recognised advantage. The extent to which cluster of differentiation (CD)34 sub-populations are affected by expansion with StemRegenin1 (SR1), and whether a particular subtype may account for better engraftment than others, is currently unknown. The purpose of this study was to determine the impact of SR1-induced HSPC expansion on CD34+ immunophenotypic subsets and gene expression profiles.MethodsUCB-derived CD34+ HSPCs were characterised before (D0) and after expansion (D7) with SR1 using an extensive immunophenotypic panel. In addition, gene expression was assessed and differentially expressed genes were categorised into biological processes.ResultsA dose-dependent increase in the number of CD34+ HSPCs was observed with SR1 treatment, and unbiased and extensive HSPC immunophenotyping proved to be a powerful tool in identifying unique sub-populations within the HSPC repertoire. In this regard, we found that SR1 promotes the emergence of HSPC subsets which may aid engraftment post expansion. In addition, we observed that SR1 has a minimal effect on the transcriptome of 7-day expanded CD34+ HSPCs when compared to cells expanded without SR1, with only two genes being downregulated in the former.ConclusionThis study revealed that SR1 selects for potentially novel immunophenotypic HSPC subsets post expansion and has a minimal effect on the transcriptome of 7-day expanded HSPCs when compared to vehicle controls. Whether these distinct immunophenotypic sub-populations possess greater engraftment capacity remains to be tested in animal models.

StemRegenin-1 Reverses Drug Resistance of MCF-7/ADR Cells via AhR/ABC Transports and AhR/UGTs Pathways

Objectives: Drug resistance reduces the antitumor efficacy of chemotherapy. Therefore, it is important to know how to reverse drug resistance. In this work, we investigated drug resistance reversal by StemRegenin-1(SR-1) in MCF-7/ADR cells and the mechanism by which it exerts its drug resistance effect. Methods: MTT test and protein blot were employed as the two main in vitro cell tests. The cells were treated with SR-1 and ADM to detect the changes in their proteomics, and then the effects of AhR downstream proteins, glucuronidase, and drug-resistant proteins were verified. The accumulation of ADM in the combined cells and its effect on the cell cycle were detected by flow cytometry. In vivo, a BALB/C mice xenograft test was conducted to observe the anti-tumor effect and side effects of the drug combination. Results: SR-1 combined with ADM inhibited cell proliferation and significantly decreased the expression of CYP1A1, UGT1A6, P-gP (ABCB1), and MRP1 (ABCC1). Furthermore, SR-1 caused apoptosis and cell cycle arrest. In vivo experiments showed that SR-1 significantly enhanced the antitumor effects of ADM and reduced the toxic effects of ADM. Conclusion: SR-1 inhibited AhR activity, decreased its downstream protein CYP1A1 and the expression of UGT1A6, P-gP, and MRP1 in MCF-7/ADR cells, and reversed drug resistance in MCF-7/ADR cells through AhR/ABC transports and AhR/UGTs pathways.

Hsa_circ_0008500 regulates Benzo(a)pyrene-loaded gypsum-induced inflammation and apoptosis in human bronchial epithelial cells via activation of Ahr/C-myc pathways

Polycyclic aromatic hydrocarbons (PAHs) are major organic pollutants attached to fine particulate matter in the atmosphere. They induce lung inflammation, asthma, and other lung diseases. Exploring the toxic mechanism of PAHs on lung epithelial cells may provide a theoretical basis for the prevention and treatment of respiratory diseases induced by PAHs. In our study, 16 human bronchial epithelial (16HBE) cells were exposed to different concentrations of gypsum dust, Benzo(a)pyrene (BaP), and BaP-loaded gypsum dust for 24 hours. Gypsum dust loaded with BaP significantly increased the cytotoxicity of 16HBE cells, enhanced the production of lactate dehydrogenase (LDH), interleukin-6 (IL-6) and interleukin-8 (IL-8), induced cell apoptosis, and upregulate the expression of hsa_circ_0008500 (circ_0008500). The mechanism was studied with a BaP-loaded gypsum dust concentration of 1.25 mg/mL. StemRegenin 1 (SR1) pretreat significantly reduced the release of LDH, IL-6, and IL-8 and decreased the protein levels of Ahr、XAP2, C-myc, and p53. Second-generation sequencing indicated that circ_0008500 was highly expressed after 16HBE induced by BaP-loaded gypsum dust. Functional experiments confirmed that circ_0008500 promoted the inflammation and apoptosis of 16HBE cells induced by BaP-loaded gypsum dust by regulating the Ahr signaling pathway. Our study showed that fine particulate matter adsorption of BaP significantly increased the toxic effect of BaP on cells. By activating the Ahr/C-myc pathway, circ_0008500 promoted inflammation and apoptosis of 16HBE cells induced by BaP-loaded gypsum dust.

3D-bioprinted bone scaffolds incorporating SR1 nanoparticles enhance blood vessel regeneration in rat calvarial defects

The inherent limitations of bone grafting in the treatment of critical-sized bone defects have led to a growing demand for bone repair implants. Three-dimensional (3D) bioprinting has emerged as a promising manufacturing technique for implants, offering flexibility in their structural design and the use of applicable materials. Although numerous 3D-bioprinted bone scaffolds have been developed to enhance osteogenesis, angiogenesis remains a challenge. Angiogenesis is crucial for successful bone healing because the process forms blood vessels to deliver essential nutrients and oxygen. Endothelial progenitor cells (EPCs) play a pivotal role in the early stages of vascularization. These cells, capable of differentiating into endothelial cells (ECs), are recruited from the bone marrow to the injured area during the healing process. CD34+ cells, a subset of EPCs, have gained attention because of their neovascularization potential and ability to contribute to bone regeneration. The incorporation of CD34+ cell-enhancing factors into 3D-printed bone scaffolds may facilitate successful bone healing in critical defects. StemRegenin-1 (SR1), a molecule that promotes CD34+ cell expansion, has shown promising results in increasing CD34+ hematopoietic stem and progenitor cell populations. This study aimed to investigate the sustained release of SR1 from a collagen-based scaffold integrated with mesoporous silica nanoparticles (MSNs) to promote angiogenesis and enhance bone healing. The sustained release of SR1 from the collagen scaffold is hypothesized to promote angiogenesis, thereby facilitating bone repair. In vitro studies have demonstrated the angiogenic potential of SR1; however, further in vivo investigations are required to establish its clinical efficacy. This study contributes to the development of novel therapies targeting CD34+ cells and demonstrates the potential of SR1 as a promising agent for promoting angiogenesis and enhancing bone healing in critical defects.

Qufeng Xuanbi Formula inhibited benzo[a]pyrene-induced aggravated asthma airway mucus secretion by AhR/ROS/ERK pathway

Ethnopharmacological relevanceExcessive secretion of airway mucus may be an important pathological factor of air pollution-induced acute asthma attacks. Treatment of airway mucus hypersecretion improves asthma aggravated by air pollutants. Qufeng Xuanbi Formula (QFXBF) has been used to treat asthma for more than 30 years. However, whether QFXBF inhibits asthmatic mucus secretion exacerbated by air pollutants has not yet been established. Aim of the studyThis study aimed to evaluate the effect of QFXBF on airway mucus secretion and the mechanism of action in an air pollutant benzo[a]pyrene (BaP)-induced mouse model of aggravated asthma. Materials and methodsOvalbumin (OVA) and BaP co-exposure were used to establish the aggravated asthma model. The average enhanced pause (Penh), serum OVA-specific IgE, and changes in lung histopathology were determined. 16HBE cells exposed to BaP, treatment with QFXBF, arylhydrocarbon receptor (AhR) signal antagonist SR1, reactive oxygen species (ROS) antagonist NAC, or extracellular signal-regulated kinase (ERK1/2) signal antagonist U0126 were established to investigate the effect of QFXBF on BaP-induced mucus secretion and its target. The mRNA and protein expression levels of MUC5AC in the lung tissue and 16HBE cells were examined. We also studied the effect of QFXBF on ROS production. Finally, the protein expression of AhR, phospho-extracellular signal-regulated kinases (p-ERK1/2), and ERK1/2 in 16HBE cells and lung tissues was determined by western blotting. ResultsAdministration of QFXBF significantly alleviated the pathological symptoms, including Penh, serum OVA-specific IgE, and changes in lung histopathology in a BaP-induced mouse model of aggravated asthma. QFXBF inhibited MUC5AC expression in asthmatic mice and 16HBE cells exposed to BaP. ROS production, AhR expression, and ERK1/2 phosphorylation were significantly increased in BaP-induced asthmatic mice and 16HBE cells. Signaling pathway inhibitors StemRegenin 1 (SR1), NAC, and U0126 significantly inhibitedBaP-induced MUC5AC expression in 16HBE cells. SR1 reversed Bap-induced ROS production and ERK activation, and NAC inhibited Bap-induced ERK activation. In addition, QFXBF regulated AhR signaling, inhibited ROS production, reversed ERK activation, and downregulated mucus secretion to improve asthma aggravated by air pollutant BaP. ConclusionsQFXBF can ameliorate mucus secretion in BaP-induced aggravated asthmatic mice and 16HBE cells, and the specific mechanism may be related to the inhibition of the AhR/ROS/ERK signaling pathway.

StemRegenin-1 Attenuates Endothelial Progenitor Cell Senescence by Regulating the AhR Pathway-Mediated CYP1A1 and ROS Generation.

Endothelial progenitor cell (EPC)-based stem cell therapy is a promising therapeutic strategy for vascular diseases. However, continuous in vitro expansion for clinical studies induces the loss of EPC functionality due to aging. In this study, we investigated the effects of StemRegenin-1 (SR-1), an antagonist of aryl hydrocarbon receptor (AhR), on replicative senescence in EPCs. We found that SR-1 maintained the expression of EPC surface markers, including stem cell markers, such as CD34, c-Kit, and CXCR4. Moreover, SR-1 long-term-treated EPCs preserved their characteristics. Subsequently, we demonstrated that SR-1 showed that aging phenotypes were reduced through senescence-associated phenotypes, such as β-galactosidase activity, SMP30, p21, p53, and senescence-associated secretory phenotype (SASP). SR-1 treatment also increased the proliferation, migration, and tube-forming capacity of senescent EPCs. SR-1 inhibited the AhR-mediated cytochrome P450 (CYP)1A1 expression, reactive-oxygen species (ROS) production, and DNA damage under oxidative stress conditions in EPCs. Furthermore, as a result of CYP1A1-induced ROS inhibition, it was found that accumulated intracellular ROS were decreased in senescent EPCs. Finally, an in vivo Matrigel plug assay demonstrated drastically enhanced blood vessel formation via SR-1-treated EPCs. In summary, our results suggest that SR-1 contributes to the protection of EPCs against cellular senescence.

StemRegenin 1 Mitigates Radiation-Mediated Hematopoietic Injury by Modulating Radioresponse of Hematopoietic Stem/Progenitor Cells

Hematopoietic injury resulting from the damage of hematopoietic stem/progenitor cells (HSPCs) can be induced by either nuclear accident or radiotherapy. Radiomitigation of HSPCs is critical for the development of medical countermeasure agents. StemRegenin 1 (SR1) modulates the maintenance and function of HSPCs under non-stress conditions. However, the impact of SR1 in radiation-induced hematopoietic injury both in vivo and in vitro remains unknown. In this study, we found that treatment with SR1 after irradiation of C57BL/6 mice significantly mitigates TBI-induced death (80% of SR1-treated mice survival vs. 30% of saline-treated mice survival) with enhanced recovery of peripheral blood cell counts, with the density and cell proliferation of bone marrow components as observed by Hematoxylin and Eosin (H&E) and Ki-67 staining. Interestingly, in vitro analysis of human HSPCs showed that SR1 enhanced the population of human HSPCs (CD34+) under both non-irradiating and irradiating conditions, and reduced radiation-induced DNA damage and apoptosis. Furthermore, SR1 attenuated the radiation-induced expression of a member of the pro-apoptotic BCL-2 family and activity of caspase-3. Overall, these results suggested that SR1 modulates the radioresponse of HSPCs and might provide a potential radiomitigator of hematopoietic injury, which contributes to increase the survival of patients upon irradiation.

The monoculture of cord-blood-derived CD34+ cells by an automated, membrane-based dynamic perfusion system with a novel cytokine cocktail.

Human leukocyte antigen (HLA)-matched cord blood (CB) transplantation is a procedure for the treatment of certain hematological malignancies, hemoglobinopathies, and autoimmune disorders. However, one of the challenges is to provide a sufficient number of Tcell-depleted hematopoietic stem and progenitor cells. Currently, only 4%-5% of the CB units stored in CB banks contain enoughCD34+ cells for engrafting 70-kg patients. To support this clinical need, we have developed an automated expansion protocol for CB-derived CD34+ cells in the Quantum system's dynamic perfusion bioreactor using a novel cytokine cocktail comprised of stemcell factor (SCF), thrombopoietin (TPO), fms-like tyrosine kinase 3 ligand (Flt-3L), interleukin-3 (IL-3), IL-6, glial cell line-derived neurotrophic factor (GDNF), StemRegenin 1 (SR-1), and a fibronectin-stromal-cell-derived factor-1 (SDF-1)-coated membrane. In an 8-day expansion of a 2×106 positively selected CD34+ cell inoculum from 3 donor lineages, the mean cell harvest and cell viability were1.02×108 cells and 95.5%, respectively, and the mean frequency of the CD45+CD34+ immunophenotype was 54.3%. Themean differentiated cell frequencies were 0.5% for lymphocytes, 15.8% for neutrophils, and 15.4% for platelets. These resultsdemonstrate that the automated monoculture protocol can support the expansion of CD34+ cells with minimal lymphocyte residual.

Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here, we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract-induced apoptosis decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

Inhibition of aryl hydrocarbon receptor signaling promotes the terminal differentiation of human erythroblasts.

The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation. RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes, favoring their maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.

AhR Antagonist Promotes Differentiation of Papillary Thyroid Cancer via Regulating circSH2B3/miR-4640-5P/IGF2BP2 Axis.

Abnormally high expression of aryl hydrocarbon receptor (AhR) has been implicated in dedifferentiation of radioiodine-refractory papillary thyroid cancer (RR-PTC). This study aimed to evaluate the differentiation effect of AhR antagonist in PTC, and to explore the potential mechanism of it. Results showed that AhR antagonists promoted differentiation of PTC, as shown as increase in 125I uptake and Na/I symporter (NIS) expression level. CircRNA microarray in K1 cells treated with StemRegenin 1(SR1) revealed that hsa_circ_0006741 (circSH2B3) was down-regulated in SR1 treated K1 cells. Downregulation of circSH2B3 increased 125I uptake and NIS expression levels. CircSH2B3 acted as an endogenous sponge of hsa-miR-4640-5p and modulated IGF2BP2 expression. IGF2BP2 overexpression induced dedifferentiation of PTC, while silencing IGF2BP2 accelerated differentiation of PTC cells. Rescue studies showed that the dedifferentiation activity of AhR was modulated by the circSH2B3/miR-4640-5p/IGF2BP2 axis. Our findings confirmed for the first time that AhR antagonists promote differentiation of PTC via inhibiting the circSH2B3/miR-4640-5p/IGF2BP2 axis, offering a novel therapeutic approach and a potential marker for differentiation of PTC.

AHR:IKAROS Interaction Promotes Platelet Biogenesis in Response to SR1

In vitro, the differentiation of megakaryocytes (MKs) is improved by aryl-hydrocarbon receptor (AHR) antagonists such as StemRegenin 1 (SR1), an effect physiologically recapitulated by the presence of stromal mesenchymal cells (MSC). This inhibition promotes the amplification of a CD34+CD41low population able to mature as MKs with a high capacity for platelet production. In this short report, we showed that the emergence of the thrombocytogenic precursors and the enhancement of platelet production triggered by SR1 involved IKAROS. The downregulation/inhibition of IKAROS (shRNA or lenalidomide) significantly reduced the emergence of SR1-induced thrombocytogenic population, suggesting a crosstalk between AHR and IKAROS. Interestingly, using a proximity ligation assay, we could demonstrate a physical interaction between AHR and IKAROS. This interaction was also observed in the megakaryocytic cells differentiated in the presence of MSCs. In conclusion, our study revealed a previously unknown AHR/ IKAROS -dependent pathway which prompted the expansion of the thrombocytogenic precursors. This AHR- IKAROS dependent checkpoint controlling MK maturation opens new perspectives to platelet production engineering.

Reducing TGF-β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling.

ObjectiveAs an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34− cell numbers. These CD34− cells influenced the ex vivo expansion of CD34+ cells. In this work, the effects of periodically removing CD34− cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated.Materials and methodsCD34− cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents.ResultsAfter 10‐day culture, the expansion of CD34+ cells in the CD34− cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34− cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self‐renewal symmetric division of CD34+ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34− cells with cooperation from SR1 further reduced the expression of AhR‐related genes.ConclusionsPeriodic removal of CD34− cells plus cooperation with SR1 improved the expansion of CD34+ cells, maintained better biological function of expanded CD34+ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

Coinhibition of activated p38 MAPKα and mTORC1 potentiates stemness maintenance of HSCs from SR1-expanded human cord blood CD34+ cells via inhibition of senescence.

The stemness of ex vivo expanded hematopoietic stem cells (HSCs) is usually compromised by current methods. To explore the failure mechanism of stemness maintenance of human HSCs, which were expanded from human umbilical cord blood (hUCB) CD34+ cells, by differentiation inhibitor Stem Regenin 1 (SR1), an antagonist of aryl hydrocarbon receptor, we investigated the activity of p38 mitogen‐activated protein kinase α (p38 MAPKα, p38α) and mammalian target of rapamycin complex 1 (mTORC1), and their effect on SR1‐expanded hUCB CD34+ cells. Our results showed that cellular senescence occurred in the SR1‐expanded hUCB CD34+ cells in which p38α and mTORC1 were successively activated. Furthermore, their coinhibition resulted in a further decrease in hUCB CD34+ cell senescence without an effect on apoptosis, promoted the maintenance of expanded phenotypic HSCs without differentiation inhibition, increased the hematopoietic reconstitution ability of multiple lineages, and potentiated the long‐term self‐renewal capability of HSCs from SR1‐expanded hUCB CD34+ cells in NOD/Shi‐scid/IL‐2Rγnull mice. Our mechanistic study revealed that senescence inhibition by our strategy was mainly attributed to downregulation of the splicesome, proteasome formation, and pyrimidine metabolism signaling pathways. These results suggest that coinhibition of activated p38α and mTORC1 potentiates stemness maintenance of HSCs from SR1‐expanded hUCB CD34+ cells via senescence inhibition. Thus, we established a new strategy to maintain the stemness of ex vivo differentiation inhibitor‐expanded human HSCs via coinhibition of multiple independent senescence initiating signal pathways. This senescence inhibition‐induced stemness maintenance of ex vivo expanded HSCs could also have an important role in other HSC expansion systems.

Forkhead Box M1 promotes the growth and tube formation of human malignant meningioma cells via the aryl hydrocarbon receptor signaling pathway.

Forkhead Box M1 (FOXM1) and aryl hydrocarbon receptor (AHR) signaling pathway participate in meningioma development, but their correlation was inadequately studied. The study is aimed to uncover their functions and correlation in malignant meningioma. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect FOXM1 expression in malignant meningioma and adjacent tissues. The viability, proliferation, apoptosis and tube formation of meningioma IOMM-Lee and CH157-MN cells transfected with overexpressed FOXM1 were examined with MTT assay, clone formation assay, flow cytometry and tube formation assay, respectively. The expressions of AHR and cytochrome P450 family 1 subfamily A member 1 (CYP1A1) in meningioma and adjacent tissues were detected using qRT-PCR, and the correlation of AHR with FOXM1 was analyzed with Pearson's correlation analysis. Western blot was conducted for measuring the expressions of vascular endothelial growth factor A (VEGFA), AHR and CYP1A1. The cell viability, proliferation, apoptosis and tube formation capability were further determined after treatment with StemRegenin 1 (SR1) (an AHR signaling pathway inhibitor), and transfected with or without overexpressed FOXM1. FOXM1, AHR and CYP1A1 expressions were upregulated in malignant meningioma tissues. Overexpressed FOXM1 promoted meningioma cell viability, proliferation, tube formation, upregulated expressions of AHR, CYP1A1 and VEGFA, and inhibited the cell apoptosis. AHR was positively correlated with FOXM1. SR1 suppressed meningioma cell growth and the AHR signaling pathway, and also reversed the active effect of FOXM1 on meningioma cells. FOXM1 may promote malignant meningioma via the AHR signaling pathway, which improved the current understanding of the role of FOXM1 in meningioma.

Generation and function of progenitor T cells from StemRegenin-1–expanded CD34+ human hematopoietic progenitor cells

AbstractBroader clinical application of umbilical cord blood (UCB), as a source of hematopoietic stem/progenitor cells (HSPCs), is limited by low CD34+ and T-cell numbers, contributing to slow lymphohematopoietic recovery, infection, and relapse. Studies have evaluated the safety, feasibility, and expedited neutrophil recovery associated with the transplantation of CD34+ HSPCs from ex vivo expansion cultures using the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1). In a phase 1/2 study of 17 patients who received combined SR1-expanded and unexpanded UCB units, a considerable advantage for enhancing T-cell chimerism was not observed. We previously showed that progenitor T (proT) cells generated in vitro from HSPCs accelerated T-cell reconstitution and restored immunity after hematopoietic stem cell transplantation (HSCT). To expedite immune recovery, we hypothesized that SR1-expanded HSPCs together with proT cells could overcome the known T-cell immune deficiency that occurs post-HSCT. Here, we show that SR1-expanded UCB can induce >250-fold expansion of CD34+ HSPCs, which can generate large numbers of proT cells upon in vitro differentiation. When compared with nonexpanded naive proT cells, SR1 proT cells also showed effective thymus-seeding and peripheral T-cell functional capabilities in vivo despite having an altered phenotype. In a competitive transfer approach, both naive and SR1 proT cells showed comparable thymus-engrafting capacities. Single-cell RNA sequencing of peripheral CD3+ T cells from mice injected with either naive or SR1 proT cells revealed functional subsets of T cells with polyclonal T-cell receptor repertoires. Our findings support the use of SR1-expanded UCB grafts combined with proT-cell generation for decreasing T-cell immunodeficiency post-HSCT.

Abstract 4675: Aryl hydrocarbon receptor antagonist enhances cord blood-derived human hematopoietic stem cell expansion and platelet formation

Abstract Background: Cord blood transplantation is one of the alternatives for hematopoietic stem cell transplantation (HSCT). However, cord blood transplantation has a crucial problem with the small number of hematopoietic stem cells (HSC) in a single cord blood unit which results in graft failure. To overcome this problem with the paucity of HSCs, here we tested the effect of two aryl hydrocarbon receptor (AHR) antagonists on the HSC ex vivo expansion and explored the mechanism of different effect by analyzing the gene expression profiles related with lineage-specific commitment in expanded human HSPCs. Methods & Results: For ex vivo expansion culture, we enriched CD34 positive cell proportion up to 90% from cord blood units. We demonstrated ex vivo expansion culture with two AHR antagonist, StemRegenin 1 (SR1) and CH223191. Lin-/CD34+/CD38-, Lin-/CD34+/CD90+ and Lin-/CD34+/CD45RA+cell proportions are increased by SR1 (x6.5, 4.1, 2.9) and CH223191 (x3.6, 2.5, 2.6) at 14 culture days. With repeated analysis, the absolute number of Lin-/CD34+/CD38-/CD90+/CD45RA-cells were increased more than initial seeding cell numbers. We further performed the colony forming assay (CFA) with expanded HSPCs to functionally validate of expanded HSPCs’ stemness. The number of each colony distinguished by their morphologies were counted; BFU, CFU-G, -M, -GM, -GEMM. SR1 and CH223191 treatment increased the total number of colonies compared to control, and the number of progenitor cells was increased by antagonists. Fourteen days after CFA culture, colonies were harvested and analyzed by FACS with various hematopoietic lineage cell markers (CD45, CD33, CD235a, and CD19). There were differences between SR1- and CH223191-expanded HSPC by FACS analysis and CFA, though two molecules had the similar ability to expand hematopoietic stem/progenitor cell number with the differentiation capacity. To explore the putative cause of the different effect on HSPC expansion capacities of SR1 and CD223191, we performed total-RNA sequencing (Illumina NextSeq) using expanded progenitor cells and examined results in various ways. By differential expressed gene (DEG) analysis, we selected genes significantly changed their expression. Comparing with control, SR1 and CH223191 decreased the expression of genes which are known to be related with the AhR pathway. On the other hand, we found that CH223191 treatment increased gene expression related to platelet activation and formation, coagulation cascade and complement. Conclusions: In this study, we show that the blockade of aryl hydrocarbon receptor results in HSPCs expansion. CH223191 induces the expansion of HSPCs and the gene expression which are related to platelet formation. On this point, further investigation for the therapeutic use of CH223191 for expanded cord blood transplant and the treatment amegakaryocytic thrombocytopenia is needed. Citation Format: Dong Chan Kim, Sung-Soo Yoon, Dong-Yeop Shin. Aryl hydrocarbon receptor antagonist enhances cord blood-derived human hematopoietic stem cell expansion and platelet formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4675.

Abstract 4668: Therapeutic efficacy of aryl hydrocarbon receptor agonist in acute myeloid leukemia

Abstract Background The presence of leukemic stem cells (LSCs) is known to be a major cause of treatment failure and recurrence in acute myeloid leukemia (AML) patients. Therefore, it is important to develop a treatment method of targeting LSCs. Based on several research, aryl hydrocarbon receptor (AhR) antagonist, StemRegenin 1 (SR1), supports LSC activity. Thus, we speculated that the AhR agonist with the opposite of SR1 could trigger LSC differentiation which may result in the anti-cancer effects. We also hypothesize that the combination of the AhR agonist and ASP2215 targeting FMS-like tyrosine kinase 3(FLT3) mutation, which is known to be present at the LSC level shows the augmented effect on cancer therapy. Here we show the biological significance of AhR and its contribution to leukemic cells, and the therapeutic applicability of this approach. Methods and Results The effects of AhR agonists of 6-formylindolo[3,2-b]carbazole(FICZ), and (2′Z)-Indirubin, and AhR antagonist, SR1 were tested on AML cell lines (MV4-11, MOLM14, HL60, NB4, KG-1) using western blot and flow cytometry. AhR modulation was confirmed by changes in AhR and CYP1B1 protein expression levels at 24hours. . When AML cells were exposed to FICZ for 72hours, surface markers for myeloid differentiation, increased significantly. Mononuclear cells isolated from AML patients’ leukapheresis blood samples(n=5) were cultured with stem culture medium containing growth factors with agonists or antagonist of AhR to determine the effect on LSC differentiation and stem-ness. The cell surface markers Lin-CD34+CD38- represent the stem/progenitor cell fraction were analyzed by FACS, FICZ and indirubin reduces this cell proportion by 2.4-fold 1.8-fold respectively compared to the control. In addition, multipotential progenitors-like LSCs(Lin- CD34+CD38-CD90-CD45RA-) and lymphoid-primed multipotential progenitors-like LSCs (Lin- CD34+CD38-CD90-CD45RA+) was reduced by 2.8 and 2.6-fold in the FICZ treatment group and 1.8 and 2.2-fold in the Indirubin treatment group, respectively. Colony formation assay was performed to verify the effect of AhR modulating on LSC function. FICZ and indirubin reduced the stem-ness of cultured leukemic cells compared with control and SR1. We investigated whether the AhR agonist, FICZ, has an anti-leukemia effect through a cell viability assay. As a single agent, FICZ did not affect AML cells. However, FICZ improves the cytotoxicity of ASP2215, a potent FLT3 inhibitor, in FLT3 mutant AML cells. And more, the combination enhances the effect of ASP2215 on cancer cell signaling pathways AKT-mTOR and MEK-ERK. Conclusion We found that FICZ, an AhR agonist not only results in the LSCs differentiation, but also enhances the inhibitory effect of ASP2215 on cancer cell survival through the AKT-mTOR and MEK-ERK signaling. This cross-talk has the multiple meaning on cancer therapy in that it can simultaneously target cancer cells as well as LSCs. Citation Format: Hee Joo Han, Dong-Yeop Shin, Sung-Soo Yoon. Therapeutic efficacy of aryl hydrocarbon receptor agonist in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4668.

Environmental Benzopyrene Attenuates Stemness of Placenta-Derived Mesenchymal Stem Cells via Aryl Hydrocarbon Receptor

The toxic effects of particulate matter have been linked to polycyclic aromatic hydrocarbons (PAHs) such as benzopyrene. PAHs are potent inducers of the aryl hydrocarbon receptor (AhR), which is an expressed nuclear receptor that senses environmental stimuli and modulates gene expression. Even though several studies have shown that the benzopyrene (BP) of chemical pollutants significantly impaired stem cell activity, the exact molecular mechanisms were not clearly elucidated. In the present study, we aimed to investigate the effects of BP on placenta-derived mesenchymal stem cells (PD-MSCs) in vitro. We found that the AhR in PD-MSCs was expressed under the treatment of BP, and its activation markedly disrupted osteogenic differentiation through the alteration of stemness activity of PD-MSCs. Moreover, BP treatment significantly reduced the proliferation activity of PD-MSCs and expression of pluripotent markers through the induction of AhR. Treatment with StemRegenin 1 (SR1), a purine derivative that antagonizes the AhR, effectively prevented BP-induced reduction of the proliferation and differentiation activity of PD-MSCs. In this study, we found that BP treatment in PD-MSCs markedly obstructs PD-MSC stemness through AhR signaling. Noteworthy, SR1-mediated MSC application will contribute to new perspectives on MSC-based therapies for air pollution-related bone diseases.

Effect of Stemregenin1 and SB431542 Small Molecules on Ex Vivo Expansion of Umbilical Cord Blood Hematopoietic Stem Cells on Biocompatible Polyethersulfone Nanofiber Scaffolds

Cord blood Hematopoietic stem cells (HSCs) with several advantages including low chance of viral contamination and low rate of Graft versus host disease (GVHD) are appropriate candidate for vast medical applications such as transplantation. The main obstacle of cord blood HSCs is the low number cells. To improve ex-vivo expansion of umbilical cord HSCs we introduced a new culture system. Isolated HSCs were seeded in Three-dimensional(3D) on Polyethersulfone(PES) scaffolds and Two-dimensional(2D) culture conditions and treated with SB431542 and Stemregenin1(SR1) small molecules. On the fifth and tenth days the expanded cells in different groups were investigated for expression of specific markers by flow cytometry, expression of some stemness genes by qRT-PCR and colony formation by methocult medium. SR1 molecule significantly increased expansion of CD34+ cells while SB431542 induced more CD34+/38+ cells. Also SB431542 treated cells showed higher colony formation capacity. SR1 increased the expression of c-Myc, HOXB4 and SALL4 while SB431542 seemed to inhibit HOXB4 expression and increase SALL4.Together all, this study introduced a new ex vivo culture setting for further medical application of HSCs. Our data showed simultaneous use of these two small molecules can provide appropriate outcome for HSCs transplantation includes both of engraftment and repopulation.