Decellularization techniques support the creation of biocompatible extracellular matrix hydrogels, providing tissue-specific environments for both in vitro cell culture and in vivo tissue regeneration. We obtained endometrium derived from porcine decellularized uteri to create endometrial extracellular matrix (EndoECM) hydrogels. After decellularization and detergent removal, we investigated the physicochemical features of the EndoECM, including gelation kinetics, ultrastructure, and proteomic profile. The matrisome showed conservation of structural and tissue-specific components with low amounts of immunoreactive molecules. EndoECM supported in vitro culture of human endometrial cells in two- and three-dimensional conditions and improved proliferation of endometrial stem cells with respect to collagen and Matrigel. Further, we developed a three-dimensional endometrium-like co-culture system of epithelial and stromal cells from different origins. Endometrial co-cultures remained viable and showed significant remodeling. Finally, EndoECM was injected subcutaneously in immunocompetent mice in a preliminary study to test a possible hypoimmunogenic reaction. Biomimetic endometrial milieus offer new strategies in reproductive techniques and endometrial repair and our findings demonstrate that EndoECM has potential for in vitro endometrial culture and as treatment for endometrial pathologies.
Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids.
Objective
Determining genetic and paracrine mechanisms behind endometrial regeneration in Asherman's syndrome and endometrial atrophy (AS/EA) patients after autologous CD133+ bone marrow‐derived stem cell (CD133+BMDSC) transplantation.
Design
Retrospective study using human endometrial biopsies and mouse models.
Setting
Fundación‐IVI, IIS‐La Fe, Valencia, Spain.
Samples
Endometrial biopsies collected before and after CD133+BMDSC therapy, from eight women with AS/EA (NCT02144987) from the uterus of five mice with only left horns receiving CD133+BMDSC therapy.
Methods
In human samples, haematoxylin and eosin (H&E) staining, RNA arrays, PCR validation, and neutrophil elastase (NE) immunohistochemistry (IHQ). In mouse samples, PCR validation and protein immunoarrays.
Main outcome measures
H&E microscopic evaluation, RNA expression levels, PCR, and growth/angiogenic factors quantification, NE IHQ signal.
Results
Treatment improved endometrial morphology and thickness for all patients. In human samples, Jun, Serpine1, and Il4 were up‐regulated whereas Ccnd1 and Cxcl8 were down‐regulated after treatment. The significant decrease of NE signal corroborated Cxcl8 expression. Animal model analysis confirmed human results and revealed a higher expression of pro‐angiogenic cytokines (IL18, HGF, MCP‐1, MIP2) in treated uterine horns.
Conclusions
CD133+BMDSC seems to activate several factors through a paracrine mechanism to help tissue regeneration, modifying endometrial behaviour through an immunomodulatory milieu that precedes proliferation and angiogenic processes. Insight into these processes could bring us one step closer to a non‐invasive treatment for AS/EA patients.
Tweetable abstract
CD133+BMDSC therapy regenerates endometrium, modifying the immunological milieu that precedes proliferation and angiogenesis.
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