Differentiation of mesenchymal stem cells into vascular endothelial cells in 3D culture: a mini review.

Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in morphology, inlining, and protecting blood vessel tissue, as well as maintaining homeostatic conditions. ECs are essential in vascularization and blood vessels formation. The differentiation process, generally carried out in 2D culture systems, were relied on growth factors induction. Therefore, an artificial extracellular matrix with relevant mechanical properties is essential to build 3D culture models. Various 3D fabrication techniques, such as hydrogel-based and fibrous scaffolds, scaffold-free, and co-culture to endothelial cells were reviewed and summarized to gain insights. The obtained MSCs-derived ECs are shown by the expression of endothelial gene markers and tubule-like structure. In order to mimicking relevant vascular tissue, 3D-bioprinting facilitates to form more complex microstructures. In addition, a microfluidic chip with adequate flow rate allows medium perfusion, providing mechanical cues like shear stress to the artificial vascular vessels.

Synergistic Therapeutic Potential of Dual 3D Mesenchymal Stem Cell Therapy in an Ischemic Hind Limb Mouse Model.

Three-dimensional (3D) culture systems have been widely used to promote the viability and metabolic activity of mesenchymal stem cells (MSCs). The aim of this study was to explore the synergistic benefits of using dual 3D MSC culture systems to promote vascular regeneration and enhance therapeutic potential. We used various experimental assays, including dual 3D cultures of human adipose MSCs (hASCs), quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vitro cell migration, Matrigel tube network formation, Matrigel plug assay, therapeutic assays using an ischemic hind limb mouse model, and immunohistochemical analysis. Our qRT-PCR results revealed that fibroblast growth factor 2 (FGF-2), granulocyte chemotactic protein-2 (GCP-2), and vascular endothelial growth factor-A (VEGF-A) were highly upregulated in conventional 3D-cultured hASCs (ASC-3D) than in two-dimensional (2D)-cultured hASCs. Hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and stromal-cell-derived factor-1 (SDF-1) showed higher expression levels in cytokine-cocktail-based, 3D-cultured hASCs (ASC-3Dc). A conditioned medium (CM) mixture of dual 3D ASCs (D-3D; ASC-3D + ASC-3Dc) resulted in higher migration and Matrigel tube formation than the CM of single 3D ASCs (S-3D; ASC-3D). Matrigel plugs containing D-3D contained more red blood cells than those containing S-3D. D-3D transplantation into ischemic mouse hind limbs prevented limb loss and augmented blood perfusion when compared to S-3D transplantation. Transplanted D-3D also revealed a high capillary density and angiogenic cytokine levels and transdifferentiated into endothelial-like cells in the hind limb muscle. These findings highlight the benefits of using the dual 3D culture system to optimize stem-cell-based therapeutic strategies, thereby advancing the therapeutic strategy for ischemic vascular disease and tissue regeneration.

Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts.

PURPOSE: This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. METHODS: A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation. RESULTS: Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2- cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes. CONCLUSION: We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

Physical and biological advances in endothelial cell-based engineered co-culture model systems.

Scientific knowledge in the field of cell biology and mechanobiology heavily leans on cell-based in vitro experiments and models that favor the examination and comprehension of certain biological processes and occurrences across a variety of environments. Cell culture assays are an invaluable instrument for a vast spectrum of biomedical and biophysical investigations. The quality of experimental models in terms of simplicity, reproducibility, and combinability with other methods, and in particular the scale at which they depict cell fate in native tissues, is critical to advancing the knowledge of the comprehension of cell-cell and cell-matrix interactions in tissues and organs. Typically, in vitro models are centered on the experimental tinkering of mammalian cells, most often cultured as monolayers on planar, two-dimensional (2D) materials. Notwithstanding the significant advances and numerous findings that have been accomplished with flat biology models, their usefulness for generating further new biological understanding is constrained because the simple 2D setting does not reproduce the physiological response of cells in natural living tissues. In addition, the co-culture systems in a 2D stetting weakly mirror their natural environment of tissues and organs. Significant advances in 3D cell biology and matrix engineering have resulted in the creation and establishment of a new type of cell culture shapes that more accurately represents the in vivo microenvironment and allows cells and their interactions to be analyzed in a biomimetic approach. Contemporary biomedical and biophysical science has novel advances in technology that permit the design of more challenging and resilient in vitro models for tissue engineering, with a particular focus on scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips, which cover the purposes of co-cultures. Even these complex systems must be kept as simplified as possible in order to grasp a particular section of physiology too very precisely. In particular, it is highly appreciated that they bridge the space between conventional animal research and human (patho)physiology. In this review, the recent progress in 3D biomimetic culturation is presented with a special focus on co-cultures, with an emphasis on the technological building blocks and endothelium-based co-culture models in cancer research that are available for the development of more physiologically relevant in vitro models of human tissues under normal and diseased conditions. Through applications and samples of various physiological and disease models, it is possible to identify the frontiers and future engagement issues that will have to be tackled to integrate synthetic biomimetic culture systems far more successfully into biomedical and biophysical investigations.

Advancements in 3D Cell Culture Systems for Personalizing Anti-Cancer Therapies.

Over 90% of potential anti-cancer drug candidates results in translational failures in clinical trials. The main reason for this failure can be attributed to the non-accurate pre-clinical models that are being currently used for drug development and in personalised therapies. To ensure that the assessment of drug efficacy and their mechanism of action have clinical translatability, the complexity of the tumor microenvironment needs to be properly modelled. 3D culture models are emerging as a powerful research tool that recapitulates in vivo characteristics. Technological advancements in this field show promising application in improving drug discovery, pre-clinical validation, and precision medicine. In this review, we discuss the significance of the tumor microenvironment and its impact on therapy success, the current developments of 3D culture, and the opportunities that advancements that in vitro technologies can provide to improve cancer therapeutics.

Three-Dimensional Culture Systems for Dissecting Notch Signalling in Health and Disease.

Three-dimensional (3D) culture systems opened up new horizons in studying the biology of tissues and organs, modelling various diseases, and screening drugs. Producing accurate in vitro models increases the possibilities for studying molecular control of cell-cell and cell-microenvironment interactions in detail. The Notch signalling is linked to cell fate determination, tissue definition, and maintenance in both physiological and pathological conditions. Hence, 3D cultures provide new accessible platforms for studying activation and modulation of the Notch pathway. In this review, we provide an overview of the recent advances in different 3D culture systems, including spheroids, organoids, and "organ-on-a-chip" models, and their use in analysing the crucial role of Notch signalling in the maintenance of tissue homeostasis, pathology, and regeneration.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro.

Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of hepatocytes often fail to reliably predict DILI: hepatoma cell lines such as HepG2 do not reflect important primary-like hepatic properties and primary human hepatocytes (pHHs) dedifferentiate quickly in vitro and are, therefore, not suitable for long-term toxicity studies. More predictive liver in vitro models are urgently required in drug development and compound safety evaluation. This review discusses available human hepatic cell types for in vitro toxicology analysis and their usage in established and emerging three-dimensional (3D) culture systems. Generally, 3D cultures maintain or improve primary hepatic functions (including expression of drug-metabolizing enzymes) of different liver cells for several weeks of culture, thus allowing long-term and repeated-dose toxicity studies. Spheroid cultures of pHHs have been comprehensively tested, but also other cell types such as HepaRG benefit from 3D culture systems. Emerging 3D culture techniques include usage of induced pluripotent stem-cell-derived hepatocytes and primary-like upcyte cells, as well as advanced culture techniques such as microfluidic liver-on-a-chip models. In-depth characterization of existing and emerging 3D hepatocyte technologies is indispensable for successful implementation of such systems in toxicological analysis.

Inhibition of cell proliferation and promotion of acinus-like structure formation from goat mammary epithelial cells via Wnt/ß-catenin signaling.

Mammary epithelial cells have been suggested to be central to control the expansion and remodeling of mammary gland. Wnt/ß-catenin signaling modulates cell fate in animals throughout their life span, and represents indispensable roles in tissue homeostasis, cell renewal, and regeneration in organs. Here, we utilized the small molecule 6-bromoindirubin-3'-oxime (BIO), an activator of Wnt/ß-catenin signaling, and investigated whether Wnt/ß-catenin signaling regulated the proliferation and acinus-like structure formation of goat mammary epithelial cells (GMECs). We showed that isolated GMECs displayed the typical epithelial cobblestone morphology and expressed specific markers of mammary epithelial cells. BIO inhibited the proliferation of GMECs and decreased the expression of proliferation marker c-myc and cell cycle protein cyclin D1. However, the ability of GMECs to form spheroids was accelerated, and the level of E-cadherin mRNA was upregulated with BIO treatment. E-cadherin showed a bright cytomembrane with DMSO treatment, yet E-cadherin was present in cytomembrane and cytoplasm in GMECs with BIO treatment. Meanwhile, BIO increased the protein level of ß-catenin and enhanced the translocation of ß-catenin into the nucleus in GMECs. Furthermore, the mRNA level of Axin2 was also upregulated. This study suggested that Wnt/ß-catenin signaling may play an important role in the proliferation and the acinus-like formation of GMECs.

From 2D to 3D Cancer Cell Models-The Enigmas of Drug Delivery Research.

Over the past decades, research has made impressive breakthroughs towards drug delivery systems, resulting in a wide range of multifunctional engineered nanoparticles with biomedical applications such as cancer therapy. Despite these significant advances, well-designed nanoparticles rarely reach the clinical stage. Promising results obtained in standard 2D cell culture systems often turn into disappointing outcomes in in vivo models. Although the overall majority of in vitro nanoparticle research is still performed on 2D monolayer cultures, more and more researchers started acknowledging the importance of using 3D cell culture systems, as better models for mimicking the in vivo tumor physiology. In this review, we provide a comprehensive overview of the 3D cancer cell models currently available. We highlight their potential as a platform for drug delivery studies and pinpoint the challenges associated with their use. We discuss in which way each 3D model mimics the in vivo tumor physiology, how they can or have been used in nanomedicine research and to what extent the results obtained so far affect the progress of nanomedicine development. It is of note that the global scientific output associated with 3D models is limited, showing that the use of these systems in nanomedicine investigation is still highly challenging.

MicroRNA-7 promotes neural differentiation of trabecular meshwork mesenchymal stem cell on nanofibrous scaffold.

The purpose of this study was to investigate miR-7 overexpression effects on neural differentiation of mesenchymal stem cells (MSCs) on both two-dimensional (2D) and three-dimensional (3D) culture systems. We upregulated miR-7 through lentiviral vector in trabecular meshwork MSCs (TMMSCs) and polymers of poly l-lactic acid/polycaprolactone fibrous scaffold were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). Neural markers expression was evaluated through quantitative-polymerase chain reaction (q-PCR) and immunostaining. The results showed that the high percentage of cell transduction (84.9%) and miR-7 expression (folds: 677.93 and 556.4) was detected in TMMSCs-miR-7(+). SEM and FTIR established the fabrication of the hybrid scaffold. q-PCR analysis showed that on days 14 and 21 of transduction, the expression level of Nestin and glial fibrillary acidic protein (GFAP) genes were significantly higher in the scaffold (3D) compared with tissue culture polystyrene (2D) culture. The expression of microtubule-associated protein-2 (MAP-2) and GFAP genes in TMMSCs-miR-7(+) cells were significantly higher than those miR-7(-) cells after 21 days of cell culture. Also, MAP-2 and Nestin proteins were detected in TMMSCs-miR-7(+) cells. Our results demonstrate that miR-7 is involved in neural differentiation of TMMSCs and scaffold can improve differentiate into glial and neural progenitor cells. These findings provided some information for future use of microRNAs and scaffold in tissue engineering and cell therapy for neurological diseases.

A Magnesium-Enriched 3D Culture System that Mimics the Bone Development Microenvironment for Vascularized Bone Regeneration.

The redevelopment/regeneration pattern of amputated limbs from a blastema in salamander suggests that enhanced regeneration might be achieved by mimicking the developmental microenvironment. Inspired by the discovery that the expression of magnesium transporter-1 (MagT1), a selective magnesium (Mg) transporter, is significantly upregulated in the endochondral ossification region of mouse embryos, a Mg-enriched 3D culture system is proposed to provide an embryonic-like environment for stem cells. First, the optimum concentration of Mg ions (Mg2+) for creating the osteogenic microenvironment is screened by evaluating MagT1 expression levels, which correspond to the osteogenic differentiation capacity of stem cells. The results reveal that Mg2+ selectively activates the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway to stimulate osteogenic differentiation, and Mg2+ influx via MagT1 is profoundly involved in this process. Then, Mg-enriched microspheres are fabricated at the appropriate size to ensure the viability of the encapsulated cells. A series of experiments show that the Mg-enriched microenvironment not only stimulates the osteogenic differentiation of stem cells but also promotes neovascularization. Obvious vascularized bone regeneration is achieved in vivo using these Mg-enriched cell delivery vehicles. The findings suggest that biomaterials mimicking the developmental microenvironment might be promising tools to enhance tissue regeneration.

Technological advancements for the development of stem cell-based models for hepatotoxicity testing.

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.

Glioblastoma's Next Top Model: Novel Culture Systems for Brain Cancer Radiotherapy Research.

Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, remains one of the least treatable cancers. Current standard of care-combining surgical resection, radiation, and alkylating chemotherapy-results in a median survival of only 15 months. Despite decades of investment and research into the development of new therapies, most candidate anti-glioma compounds fail to translate into effective treatments in clinical trials. One key issue underlying this failure of therapies that work in pre-clinical models to generate meaningful improvement in human patients is the profound mismatch between drug discovery systems-cell cultures and mouse models-and the actual tumors they are supposed to imitate. Indeed, current strategies that evaluate the effects of novel treatments on GBM cells in vitro fail to account for a wide range of factors known to influence tumor growth. These include secreted factors, the brain's unique extracellular matrix, circulatory structures, the presence of non-tumor brain cells, and nutrient sources available for tumor metabolism. While mouse models provide a more realistic testing ground for potential therapies, they still fail to account for the full complexity of tumor-microenvironment interactions, as well as the role of the immune system. Based on the limitations of current models, researchers have begun to develop and implement novel culture systems that better recapitulate the complex reality of brain tumors growing in situ. A rise in the use of patient derived cells, creative combinations of added growth factors and supplements, may provide a more effective proving ground for the development of novel therapies. This review will summarize and analyze these exciting developments in 3D culturing systems. Special attention will be paid to how they enhance the design and identification of compounds that increase the efficacy of radiotherapy, a bedrock of GBM treatment.

Low oxygen tension modulates the osteogenic differentiation of mouse embryonic stem cells.

This study examined the effects of low oxygen tension on the osteogenic differentiation of embryonic stem cells (ESCs) in a three-dimensional culture system. The high expression levels of hypoxia-related proteins hypoxia-inducible factor-1α and vascular endothelial growth factor were first validated in ESCs subjected to hypoxic conditions compared with normoxic controls. The osteogenic differentiation of hypoxic ESCs with either osteogenic or osteogenic factor-free media was subsequently evaluated by measuring alkaline phosphatase activity, intracellular calcium levels, matrix mineralization, and the protein levels of osteogenic markers Runt-related transcription factor 2 and osterix. We confirmed that hypoxia significantly stimulated ESC osteogenic activity; the strongest stimulation of ESC osteogenesis was exerted when cells were grown in osteogenic media. To identify differentially expressed genes associated with hypoxia-induced ESC differentiation, we performed microarray analysis of ESCs cultured in osteogenic media under normoxic and hypoxic conditions. This study demonstrated that differences in oxygen tension induced the differential expression of genes known to play roles in such processes as skeletal system development and signaling pathways for bone morphogenetic protein, Wnt, Notch, mitogen-activated protein kinase, and integrin. These findings reveal the effects of low oxygen tension on osteogenic progression in ESCs and provide insight into the molecular pathways that regulate ESC differentiation following exposure to hypoxia.

Liver 'organ on a chip'.

The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems.

Hepatocellular carcinoma-targeted drug discovery through image-based phenotypic screening in co-cultures of HCC cells with hepatocytes.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in HCC patients. METHODS: Here, we developed a fluorescence image-based phenotypic screening system in vitro to identify HCC-specific drugs in co-cultures of HCC cells with hepatocytes. To this end, we identified two distinctive markers of HCC, CHALV1 and AFP, which are highly expressed in HCC cell lines and liver cancer patient-derived materials. We applied these markers to an HCC-specific drug screening system. RESULTS: Through pilot screening, we identified three anti-folate compounds that had HCC-specific cytotoxicity. Among them, pyrimethamine exhibited the greatest HCC-specific cytotoxicity. Interestingly, pyrimethamine significantly increased the size and number of lysosomes and subsequently induced the release of cathepsin B from the lysosome to the cytosol, which triggered caspase-3-dependent apoptosis in Huh7 (HCC) but not Fa2N-4 cells (immortalized hepatocytes). Importantly, Fa2N-4 cells had strong resistance to pyrimethamine relative to Huh7 cells in 2D and 3D culture systems. CONCLUSION: These results demonstrate that this in vitro image-based phenotypic screening platform has the potential to be widely adopted in drug discovery research, since we promptly estimated anticancer activity and hepatotoxicity and elucidated functional roles of pyrimethamine during the apoptosis process in HCC.

Profiling stem cell states in three-dimensional biomaterial niches using high content image informatics.

A predictive framework for the evolution of stem cell biology in 3-D is currently lacking. In this study we propose deep image informatics of the nuclear biology of stem cells to elucidate how 3-D biomaterials steer stem cell lineage phenotypes. The approach is based on high content imaging informatics to capture minute variations in the 3-D spatial organization of splicing factor SC-35 in the nucleoplasm as a marker to classify emergent cell phenotypes of human mesenchymal stem cells (hMSCs). The cells were cultured in varied 3-D culture systems including hydrogels, electrospun mats and salt leached scaffolds. The approach encompasses high resolution 3-D imaging of SC-35 domains and high content image analysis (HCIA) to compute quantitative 3-D nuclear metrics for SC-35 organization in single cells in concert with machine learning approaches to construct a predictive cell-state classification model. Our findings indicate that hMSCs cultured in collagen hydrogels and induced to differentiate into osteogenic or adipogenic lineages could be classified into the three lineages (stem, adipogenic, osteogenic) with ⩾80% precision and sensitivity, within 72h. Using this framework, the augmentation of osteogenesis by scaffold design exerted by porogen leached scaffolds was also profiled within 72h with ∼80% high sensitivity. Furthermore, by employing 3-D SC-35 organizational metrics, differential osteogenesis induced by novel electrospun fibrous polymer mats incorporating decellularized matrix could also be elucidated and predictably modeled at just 3days with high precision. We demonstrate that 3-D SC-35 organizational metrics can be applied to model the stem cell state in 3-D scaffolds. We propose that this methodology can robustly discern minute changes in stem cell states within complex 3-D architectures and map single cell biological readouts that are critical to assessing population level cell heterogeneity. STATEMENT OF SIGNIFICANCE: The sustained development and validation of bioactive materials relies on technologies that can sensitively discern cell response dynamics to biomaterials, while capturing cell-to-cell heterogeneity and preserving cellular native phenotypes. In this study, we illustrate the application of a novel high content image informatics platform to classify emergent human mesenchymal stem cell (hMSC) phenotypes in a diverse range of 3-D biomaterial scaffolds with high sensitivity and precision, and track cell responses to varied external stimuli. A major in silico innovation is the proposed image profiling technology based on unique three dimensional textural signatures of a mechanoreporter protein within the nuclei of stem cells cultured in 3-D scaffolds. This technology will accelerate the pace of high-fidelity biomaterial screening.

Co-operation between the AKT and ERK signaling pathways may support growth of deep endometriosis in a fibrotic microenvironment in vitro.

STUDY QUESTION: How can deep endometriotic stromal cells proliferate and persist in a fibrotic environment? SUMMARY ANSWER: The serine/threonine kinase AKT and extracellular regulated kinase (ERK) signaling pathways may co-operate to support growth of deep endometriotic lesions by enhancing endometriotic stromal cell proliferation and survival in a fibrotic microenvironment in vitro. WHAT IS KNOWN ALREADY: Endometriosis, particularly deep infiltrating endometriosis, is characterized histologically by dense fibrous tissue that is primarily composed of type I collagen. This tissue may cause pelvic pain and infertility, which are major clinical issues associated with endometriosis. Proliferation of normal fibroblasts is tightly regulated, and fibrillar, polymerized type I collagen inhibits normal fibroblast proliferation. However, no studies to date have investigated how deep endometriotic stromal cells can proliferate and persist in a fibrotic environment. STUDY DESIGN, SIZE, DURATION: Endometrial and/or endometriotic tissues from 104 patients (61 with and 43 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 25 nude mice received a single injection of endometrial fragments from a total of five samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated cell proliferation, caspase 3/7 activity, and the AKT and ERK signaling pathways in endometrial and endometriotic stromal cells on three-dimensional (3D) polymerized collagen matrices in vitro. In addition, to determine whether aberrant activation of the AKT and ERK pathways is involved during progression of fibrosis in endometriosis in vivo, we evaluated the expression of phosphorylated AKT and ERK1/2 in endometriotic implants in a nude mouse model of endometriosis. Finally, we evaluated the effects of MK2206 (an AKT inhibitor) and U0126 (a MEK inhibitor) on cell proliferation, caspase 3/7 activity, and phosphorylation of AKT and ERK1/2 of endometriotic stromal cells on 3D polymerized collagen matrices. MAIN RESULTS AND THE ROLE OF CHANCE: Proliferation of endometriotic stromal cells was significantly less inhibited than that of endometrial stromal cells (P < 0.05) on 3D polymerized collagen. Levels of phosphorylated AKT, phosphorylated p70S6K and phosphorylated ERK1/2 were significantly higher in endometriotic stromal cells than in endometrial stromal cells at 24 h (P < 0.05) and at 72 h (P < 0.05) on 3D polymerized collagen. Phosphorylated AKT expression was significantly increased on Days 21 and 28 compared with those on Days 3 and 7 (all P < 0.05) in endometriotic implants during progression of fibrosis in a nude mouse model of endometriosis. Inhibition of AKT or ERK1/2 with MK2206 or U0126, respectively, did not significantly increase caspase 3/7 activity in endometriotic stromal cells on either two-dimensional or 3D collagen matrices. Western blot analysis showed that MK2206 alone decreased levels of phosphorylated AKT; however, it increased levels of phosphorylated ERK in endometriotic cells compared with vehicle-treated cells (both P < 0.05). In addition, U0126 treatment decreased levels of phosphorylated ERK; however, it resulted in increased levels of phosphorylated AKT in endometriotic stromal cells compared with vehicle-treated cells (both P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Endometriosis involves a number of processes, such as invasion, metastasis, angiogenesis, and apoptosis resistance, and a variety of signaling pathways may be involved in promoting development and progression of the disease. In addition, further animal experiments are required to determine whether the AKT and ERK signaling pathways co-operate to support growth of endometriotic lesions in a fibrotic microenvironment in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Co-targeting the AKT and ERK pathways may be an effective therapeutic strategy for endometriosis treatment. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.