three-dimensional-organoids-culturing-processing-techniques-for

Three-Dimensional Organoids Culturing and Processing Techniques for Myometrial and Uterine Fibroids Generation

Uterine fibroids are the most common benign, monoclonal, gynecological tumors in a woman's uterus. To overcome the common obstacles related to the methods ...

University of Chicago

Cairo University

Uterine fibroids (UFs; leiomyoma) are the most common benign neoplastic threat to women worldwide, exacting an immense personal burden on female health and a monetary expense to the healthcare system estimated in the hundreds of billions of dollars every year globally. With no long-term non-invasive treatment option currently available to treat UFs, deeper insights regarding tumor etiology are the key for developing newer therapies. Accordingly, in this review, we discuss new mechanistic paradigm to explain UF tumor development through an exquisite model involving developmental reprogramming of myometrial stem cells due to early life endocrine disruptors exposure, inflammation, fibrosis, DNA damage, and eventually tissue stiffness. Further, we propose to utilize shear wave elastography as a potential screening tool for the early identification of women at risk for developing UFs who can benefit from several simple preventive strategies, including the consumption of natural compounds such as vitamin D and green tea as a safe fertility friendly non-hormonal modality to delay or even arrest or reverse UF progression.

Prevention of Uterine Fibroids: molecular mechanisms and potential clinical application.

This is a phase II clinical trial to investigate the immunotherapeutic effect of Curcumin, Piperine, and Taurine (CPT) combination in hepatocellular carcinoma (HCC).

The potential immuno-stimulating effect of curcumin, piperine, and taurine combination in hepatocellular carcinoma; a pilot study.

Uterine fibroids (UFs) are common tumors in women of reproductive age. It is imperative to comprehend UFs' associated risk factors to facilitate early detection and prevention. Simple relying on surgical/pharmacological treatment of advanced disease is not only highly expensive, but it also deprives patients of good quality of life (QOL). Unfortunately, even if the disease is discovered early, no medical intervention is traditionally initiated until the disease burden becomes high, and only then is surgical intervention performed. Furthermore, after myomectomy, the recurrence rate of UFs is extremely high with the need for additional surgeries and other interventions. This confused approach is invasive and extremely costly with an overall negative impact on women's health. Secondary prevention is the management of early disease to slow down its progression or even halt it completely. The current approach of watchful observation for early disease is considered a major missed opportunity in the literature. The aim of this article is to present an approach named the ESCAPE (Evidence-Based Approach for Secondary Prevention) of UF management. It comprises simple, inexpensive, and safe steps that can arrest the development of UFs, promote overall reproductive health, decrease the number of unnecessary surgeries, and save billions of health care systems' dollars worldwide.

Evidence-Based Approach for Secondary Prevention of Uterine Fibroids (The ESCAPE Approach).

Bromodomain-containing proteins (BRDs) are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD dysfunction has been linked to many diseases, including tumorigenesis. However, the role of BRDs in the pathogenesis of uterine fibroids (UFs) is entirely unknown. The present study aimed to determine the expression pattern of BRD9 in UFs and matched myometrium and further assess the impact of a BRD9 inhibitor on UF phenotype and epigenetic/epitranscriptomic changes. Our studies demonstrated that the levels of BRD9 were significantly upregulated in UFs compared to matched myometrium, suggesting that the aberrant BRD expression may contribute to the pathogenesis of UFs. We then evaluated the potential roles of BRD9 using its specific inhibitor, I-BRD9. Targeted inhibition of BRD9 suppressed UF tumorigenesis with increased apoptosis and cell cycle arrest, decreased cell proliferation, and extracellular matrix deposition in UF cells. The latter is the key hallmark of UFs. Unbiased transcriptomic profiling coupled with downstream bioinformatics analysis further and extensively demonstrated that targeted inhibition of BRD9 impacted the cell cycle- and ECM-related biological pathways and reprogrammed the UF cell epigenome and epitranscriptome in UFs. Taken together, our studies support the critical role of BRD9 in UF cells and the strong interconnection between BRD9 and other pathways controlling the UF progression. Targeted inhibition of BRDs might provide a non-hormonal treatment option for this most common benign tumor in women of reproductive age.

Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

Perimenopause significantly impacts women's health globally, often managed with hormone replacement therapy (HRT) despite the associated risks. This study explores a novel alternative exosome therapy, aimed at stimulating estrogen production in ovarian tissues, thus offering a potential non-hormonal treatment for perimenopausal symptoms. Employing ex vivo methodologies, ovarian cortex specimens from perimenopausal women were treated with exosomes derived from human umbilical cord mesenchymal stem cells and cultured under specific conditions (patent number: PCT/US2022/073467). The exosomes were produced under cyclic guanosine monophosphate (cGMP) conditions, ensuring high safety standards. Estrogen levels were quantified using enzyme-linked immunosorbent assay (ELISA), and gene expression changes in estrogen and follicle-stimulating hormone (FSH) receptors were assessed via quantitative polymerase chain reaction (PCR). Immunohistochemistry (IHC) was utilized to evaluate cellular proliferation and apoptotic markers. The results indicated a significant increase in estrogen levels and estrogen receptor-alpha (Erα) expression in treated tissues compared to controls. Additionally, a decrease in apoptotic markers and an increase in cellular proliferation markers were observed. These findings suggest that exosome therapy can effectively enhance estrogen production and modulate receptor sensitivity in perimenopausal ovarian tissues. This approach could serve as a safer alternative to HRT, aligning with the body's natural regulatory mechanisms and potentially offering a more effective treatment option for managing perimenopausal symptoms.

Exosome Therapy: A Novel Approach for Enhancing Estrogen Levels in Perimenopause.

Uterine fibroids (UFs) are the most common benign gynecologic tumours affecting women of reproductive age. This study aims to deepen the understanding of UFs complex aetiology through harnessing the power of 3D organoid models derived from human myometrial stem cells to emulate the in vivo behaviour of these tumours. Isolated SCs were cultured over 7 days under a defined culture system. Immunohistochemistry, Immunofluorescence, organoid stiffness, RNA Sequencing was conducted, and differential gene expression was assessed using RT-PCR. The derived organoids exhibited diverse populations of cells, including stem cells, smooth muscle, and fibroblasts. Excessive ECM deposition was shown via Collagen and Fibronectin expression. We confirmed that our organoids expressed oestrogen receptor in a pattern similar to that in their corresponding tissue, as well as responded to steroid hormone. Interestingly, we revealed significant racial disparities in ECM accumulation within organoids derived from different racial groups. This augmented ECM deposition is theorised to enhance tissue stiffness, as assessed using Young's modulus. Additionally, our research demonstrated significant decreases in fibrotic markers upon treatment with Vitamin D3 and Doxercalciferol. Furthermore, the pro-fibroid effects of environmental phthalates further elucidate the potential factors contributing to UF pathology. The 3D organoid model can serve as a robust platform to study the underlying molecular mechanisms of UFs, besides offering invaluable insights for potential therapeutic interventions.

Utilising Human Myometrial and Uterine Fibroid Stem Cell-Derived Three Dimentional Organoids as a Robust Model System for Understanding the Pathophysiology of Uterine Fibroids.

Cancer cells possess high proliferative ability and usually override apoptosis and metastasize to distant lesions. Autophagy in cancer cells is a double-edged weapon where a cross-regulation postulation between apoptosis and autophagy exists. The aim of the present study was to investigate the effect of adding Thymoquinone (TQ) to Imatinib (IM) in HCT human colorectal cancer cell line model on various apoptotic and autophagy markers. The combination doses of IM and TQ were selected according to our previous study concerned with cytotoxicity and uptake/efflux genes modulation. In the current study, the combination induced autophagy in HCT cell line which in turn enhanced apoptosis. Moreover, early apoptosis was evidenced. The induction of both autophagy and apoptosis resulted in programmed cell death. The assessment of AMPK, Par-4, apoptosis markers, colony formation assays, flow cytometry and autophagy detection by acridine orange proved this rapport.

Promotion of Autophagy and Apoptosis in Colorectal Cancer Exposed to Imatinib and Thymoquinone.

Uterine sarcoma is strongly associated with poor prognosis. However, its treatment options remain limited. Tazemetostat is a potent and selective EZH2 inhibitor with limited clinical application. Entinostat is one of the strong inhibitors for HDAC1 and HDAC3. This study aimed to assess the effect of dual targeting of EZH2 and HDACs on the phenotype of uterine sarcoma cells in both 2D and 3D culture systems. The uterine sarcoma cell line (MES-SA) was treated with varying concentrations of tazemetostat and/or entinostat for 24, 48 and 72 h. For 3D culture conditions, the cells were combined with Matrigel and seeded in V-bottom plates and incubated for 5 days. Cell proliferation, cell cycle progression and apoptosis were evaluated. Additionally, the RNA expression, IHC staining, wound healing assay, DNMT and HDAC activity measurements were conducted. Our data showed that single-inhibitor treatment with entinostat or tazemetostat significantly increased the cytotoxicity and significantly enhanced apoptosis concomitantly. Furthermore, both inhibitors induced cell cycle arrest in 2D and 3D culture conditions. We also demonstrated that entinostat, but not tazemetostat, suppressed the wound healing in the 2D culture. The combination treatment showed a significantly superior effect compared to single-agent treatment. Our studies demonstrate that treatment with either entinostat or tazemetostat alone showed a potent anti-uterine sarcoma effect in 2D and 3D culture conditions. Importantly, the combination of entinostat and tazemetostat produced superior therapeutic effects, suggesting that dual targeting EZH2 and HDACs may provide a promising treatment option for this aggressive cancer.

Dual Targeting EZH2 and Histone Deacetylases in Human Uterine Sarcoma Cells Under Both 2D and 3D Culture Conditions.

Uterine Fibroids (UFs) are the most common benign tumors in women of reproductive age, affecting ~77% of women overall and are clinically manifest in ~25% by age 50. Bromodomain and extra-terminal domain (BET) proteins play key roles in epigenetic transcriptional regulation, influencing many biological processes, such as proliferation, differentiation, and DNA damage response. Although BET dysregulation contributes to various diseases, their specific role in the pathogenesis of UFs remains largely unexplored. The present study aimed to determine the expression pattern of BET proteins in UFs and matched myometrium and further assess the impact of BET inhibitors on UF phenotype and epigenetic changes. Our studies demonstrated that the levels of Bromodomain-containing protein (BRD)2 and detection rate of BRD4 were significantly altered in UFs compared to matched myometrium, suggesting that aberrant BET protein expression may contribute to the pathogenesis of UFs. To investigate the biological effects of BET proteins, two small-molecule inhibitors, JQ1 and I-BET762, were used to assess their impact on UF cell behavior and transcriptomic profiles. Targeted inhibition of BET proteins markedly reduced UF cell viability compared with myometrial cells and induced cell cycle arrest. Unbiased transcriptomic profiling coupled with bioinformatic analysis revealed that BET inhibition altered multiple biological pathways, including G2M checkpoint, E2F targets, mitotic spindle, mTORC1 signaling, TNF-α signaling via NF-κB, and inflammatory response, as well as reprogrammed the UF cell epigenome. Notably, BET inhibition decreased the expression of several genes encoding extracellular matrix (ECM) proteins, a hallmark of UFs. Collectively, these results support that BET proteins play a pivotal role in regulating key signaling pathways and cellular processes in UFs. Targeting BET proteins may therefore represent a promising non-hormonal therapeutic strategy for UF treatment.

Decoding Bromodomain and Extra-Terminal Domain Protein-Mediated Epigenetic Mechanisms in Human Uterine Fibroids.

This study investigates how phthalate exposure contributes to uterine fibroid (UF) development by studying the effects of the Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), a metabolite of Di(2-ethylhexyl) phthalate, on myometrial stem cells (MMSCs). MMSCs from normal (MYON) and at-risk (MYOF) uterine tissues were cultured in 3D organoids and treated with 1.6 μM MEHHP for 48 h. Functional assays investigated cell viability, apoptosis, and mitochondrial activity, whereas RT-PCR, immunohistochemistry (IHC), and RNA sequencing evaluated markers of proliferation, apoptosis, extracellular matrix (ECM), and oxidative stress (OS). Cytokines and growth factors secretion were analyzed using a multiplex ELISA. Results showed that MEHHP exposure significantly increased cell viability and inhibited apoptosis in MYOF compared to MYON organoids. Proliferation markers (PCNA, Ki67), anti-apoptotic markers (BCL2/BAX ratio), and ECM markers (fibronectin and COL1A1) were significantly upregulated, whereas pro-apoptotic markers (Caspase-3) were downregulated in MYOF organoids. MEHHP-treated MYOF organoids exhibited elevated secretion of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-8) and growth factors (e.g., PDGF, VEGF, TGFβ1), indicative of impaired tissue repair and fibrosis. RNA sequencing identified increased OS in MYOF organoids, validated by differential expression of genes such as CA9 and GPX3. Mitochondrial analysis revealed enhanced oxidative phosphorylation (OXPHOS) and elevated oxygen consumption rates, implicating mitochondrial dysfunction as a driver of cytokine release and UF pathogenesis. In conclusion, MEHHP was shown to promote the transformation of MYOF organoids into a UF phenotype by driving proliferation, inhibiting apoptosis, and inducing cytokine-mediated inflammation via mitochondrial dysfunction. These findings related to MYOF-specific effects, as compared to MYON, emphasize that these differences are statistically significant and relevant to UF risk. It can shed insight on how phthalates exposures may impact UF pathogenesis and provide a basis for exploring targeted therapeutic strategies.

Phthalate Metabolite, Mono(2-Ethyl-5-Hydroxyhexyl) Phthalate (MEHHP), Promotes Uterine-Fibroid-Associated Phenotypes in Myometrial Stem Cell-Derived 3D Organoids.

Fertility preservation remains a significant concern for individuals undergoing gonadotoxic treatments. While traditional fertility preservation techniques are well-established, these methods can be time-consuming and limited by various medical or logistical barriers. In recent years, the potential of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) has emerged as a promising, paradigm-shifting approach in fertility preservation. Preclinical studies have demonstrated the protective and regenerative properties of EVs in chemotherapy-induced ovarian and testicular damage in animal models. EVs provide a cell-free therapy that can potentially preserve ovarian function in females and spermatogenesis in males without the need for surgery or delay in cancer treatment. Additionally, using MSC-derived EVs offers advantages over traditional stem cell therapies, such as a reduced risk of immune rejection, targeted treatment, and avoidance of safety concerns associated with stem cell-based therapies. Future directions include enhancing the therapeutic potential of MSC-derived EVs through genetic engineering or cell priming techniques to target specific tissues and further optimize their utilization in fertility preservation. Given the potential of MSC-derived EVs to protect fertility in both females and males, this approach could revolutionize treatment in oncofertility. Further research, including clinical trials, is necessary to confirm the safety and efficacy of MSC-derived EVs, focusing on premature ovarian insufficiency. Looking ahead, MSC-derived EVs could revolutionize fertility preservation, offering hope for cancer patients and individuals exposed to various environmental risks affecting reproduction, including in space exploration, where protection from cosmic radiation is essential.

Role of extracellular vesicles in fertility preservation before gonadotoxic exposures to the ovaries and testes.

The medical use of metal nanoparticles is recommended to reduce adverse effects and improve curative action when compared to their pure ions. Current research discussed the healing effect of topical formulations of silver, gold, and their mixtures of nanoparticles on excision wounds in rat models. The physicochemical factors of the prepared nanoparticles were measured by dynamic light scattering (DLS), Zeta potential, atomic force microscopy (AFM), and transmission electron microscopy (TEM). Thirty-two albino rats were divided into six groups: wound untreated, silver nanoparticles (AgNps) treated, gold nanoparticles (AuNps), silver-gold nanoparticles (Ag/AuNps 1:1) mixture treated, and fusidic acid (reference drug) treated groups, respectively. Management was performed daily for 21 days. In all groups, the percentages of contraction area and histological investigation of the wound area were investigated. Myeloperoxidase (MPO), nitric oxide (NO), vascular endothelial growth factor (VEGF), hydroxyproline, hexosamine, and collagen deposition using Masson's trichrome stain (MT) as a biomarker of wound healing activity were measured. An in-silico study investigated the molecular interaction of the metal nanoformulations with various protease enzymes involved in inflammation and the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the examined metals. The AgNPs and AuNPs have efficient wound-repairing activity, as evident from the wound shrinkage and enhanced wound healing. Metal nanoformulations have effective wound healing activity by enhancing angiogenesis and collagen formation.

Comparative evaluation of the healing potential of excision wound using Nano- formulations of silver, gold, or their mixture: In-silico and in vivo approaches.

Article	Year
Prevention of Uterine Fibroids: molecular mechanisms and potential clinical application. Journal of endometriosis and uterine disorders\| PubMed ID: 37637856	2023
The potential immuno-stimulating effect of curcumin, piperine, and taurine combination in hepatocellular carcinoma; a pilot study. Discover oncology\| PubMed ID: 37704828	2023
Evidence-Based Approach for Secondary Prevention of Uterine Fibroids (The ESCAPE Approach). International journal of molecular sciences\| PubMed ID: 37958957	2023
Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells. International journal of molecular sciences\| PubMed ID: 38255982	2024
Exosome Therapy: A Novel Approach for Enhancing Estrogen Levels in Perimenopause. International journal of molecular sciences\| PubMed ID: 39000181	2024
Utilising Human Myometrial and Uterine Fibroid Stem Cell-Derived Three Dimentional Organoids as a Robust Model System for Understanding the Pathophysiology of Uterine Fibroids. Cell proliferation\| PubMed ID: 40108998	2025
Promotion of Autophagy and Apoptosis in Colorectal Cancer Exposed to Imatinib and Thymoquinone. Journal of biochemical and molecular toxicology\| PubMed ID: 40143604	2025
Dual Targeting EZH2 and Histone Deacetylases in Human Uterine Sarcoma Cells Under Both 2D and 3D Culture Conditions. Journal of cellular and molecular medicine\| PubMed ID: 40464712	2025
Decoding Bromodomain and Extra-Terminal Domain Protein-Mediated Epigenetic Mechanisms in Human Uterine Fibroids. International journal of molecular sciences\| PubMed ID: 41465569	2025
Phthalate Metabolite, Mono(2-Ethyl-5-Hydroxyhexyl) Phthalate (MEHHP), Promotes Uterine-Fibroid-Associated Phenotypes in Myometrial Stem Cell-Derived 3D Organoids. Environmental toxicology\| PubMed ID: 41621087	2026
Role of extracellular vesicles in fertility preservation before gonadotoxic exposures to the ovaries and testes. Human reproduction (Oxford, England)\| PubMed ID: 41632688	2026
Comparative evaluation of the healing potential of excision wound using Nano- formulations of silver, gold, or their mixture: In-silico and in vivo approaches. PloS one\| PubMed ID: 42060644	2026

Mervat M. Omran

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