Abstract
The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.
Highlights
The human endometrium is characterized by a high proliferative potential, as it undergoes approximately 450 regenerative cycles during woman’s reproductive lifespan
Some studies have suggested the ability of human endometrial mesenchymal stromal cells (E-MSCs) to repair endometrial damage both in animal models and in patients with Asherman’s Syndrome (AS), a pathological condition characterized by extensive endometrial disruption and intrauterine adhesions leading to hardly reversible infertility [14]
E-MSCs are considered as advanced therapy medicinal products (ATMP), and, as a consequence, must be produced in compliance with Good Manufacturing Practice (GMP) rules [20], defining the highest standards of sterility, quality control, and documentation to ensure that cultured cells are safe
Summary
The human endometrium is characterized by a high proliferative potential, as it undergoes approximately 450 regenerative cycles during woman’s reproductive lifespan. EMSCs were previously isolated by our group, both from the healthy endometrium and from peritoneal, pelvic endometriosis; when cultured, they displayed the ability to form plasticadherent colonies with high proliferative potential, as well as the property of undergoing multi-lineage differentiation into osteoblasts, chondrocytes, adipocytes, or endothelial cells in response to specific culture conditions [9,10] Due to their capacity of multi-lineage differentiation and their immunosuppressive properties, E-MSCs are considered suitable candidates for performing stem cell therapy [11,12]. E-MSCs spheroids transplanted in the uterus of rats with induced AS were able to restore fertility, allowing spontaneous pregnancies in which the litter size was higher than in control AS-affected rats receiving autologous bone marrow cells [18] Taken together, these data suggest that cellular therapy with E-MSCs, might represent a promising option to treat severe endometrial defects causing infertility. A single preparation of 108 human MSCs expanded in standard condition, consisting of the use of FBS as additive in the culture medium, brings
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