Human uterine fibroblasts (HuF) isolated from the maternal part (decidua parietalis) of a term placenta provide a useful model of in vitro cell differentiation into decidual cells (decidualization), critical for successful pregnancy. After isolation, the cells adhere to plastic and have either a small round or spindle-shaped morphology which later changes into a flattened pattern in culture. HuF robustly proliferate in culture until passage 20 and form colonies when plated at low densities. The cells express the mesenchymal cell markers fibronectin, integrin-β1, ICAM-1 (CD54), and collagen I. Flow cytometry of HuF detected the presence of CD34, a marker of hematopoietic stem cell lineage, and an absence of CD10, CD11b/Mac, CD14, CD45, and HLA type II. Furthermore, they also express the pluripotency markers SSEA-1, SSEA-4, Oct-4, Stro-1, and TRA-1−81 as detected by confocal microscopy. Treatment for 14−21 days with differentiation-inducing media led to the differentiation of HuF into osteoblasts, adipocytes, and chondrocytes. The presence of α-smooth muscle actin, calponin, and myosin light chain kinase in cultured HuF implies their similarity to myofibroblasts. Treatment of the HuF with DMSO causes reversion back into the spindle-shaped morphology and a loss of myofibroblast characteristics, suggesting a switch into a less differentiated phenotype. The unique abilities of HuF to exhibit multipotency, even with myofibroblast characteristics, and their ready availability and low maintenance requirements make them an interesting cell model for further exploration as a possible tool for regenerative medicine.

The differentiation of uterine stromal fibroblasts into decidual cells is critical for establishing pregnancy. This process, called decidualization, requires the reorganization of the actin cytoskeleton, which mainly depends on actin dynamics and the phosphorylation status of the myosin light chain. We manipulated actin dynamics with jasplakinolide (100 nM) and latrunculin B (1 microM), both of which significantly inhibited the synthesis of decidualization markers induced by 6 days of treatment with embryo-mimicking stimulus interleukin 1beta (IL1B) and steroid hormones (SHs; 17beta-estradiol and medroxyprogesterone acetate) in the human uterine fibroblast (HuF) in vitro model. However, only jasplakinolide had long-lasting effects on the G-actin:F-actin ratio and prevented decidualization induced by the artificial stimulus cAMP (and SHs). Actin-binding protein cofilin mainly colocalized with G-actin in the nucleus as well as the cytoplasm. Only some spots of colocalization between cofilin and F-actin were detected in the cytoplasm. Brief extraction of cytosolic proteins from living cells revealed that in cells treated with IL1B or cAMP (and SHs) for 6 days, cofilin was mainly detected in the nucleus. The translocation of cofilin from cytosol to nucleus was also detected in HuFs treated for 12 days with SHs, IL1B and SHs, and cAMP and SHs. The same significant translocation was confirmed in primary baboon stromal uterine fibroblasts. We conclude that changes in actin dynamics, particularly the stabilization of F-actin, have a significant negative impact on decidualization, and the translocation of cofilin to the nucleus is a key feature of this process in the primate.

Human uterine fibroblasts (HuF) isolated from decidua parietalis of term placenta provide a useful model of in vitro cell differentiation into decidual cells, critical for successful pregnancy. After isolation, cells adhere to plastic and have a spindle‐shaped morphology which later changes into a flattened pattern. HuF robustly proliferate and express fibronectin, integrin‐β1, ICAM‐1 and collagen I. To further explore their potential to transform into other cell types, HuF were treated for 14‐21 days with differentiation‐inducing media. Differentiation of HuF into osteocytes, adipocytes and chondrocytes was identified. Flow cytometry of HuF revealed absence of monocyte surface receptor, but exhibited a marker of heamatopoietic lineage, CD45. CD45 positivity makes HuF cells close to fibrocytes, cells involved in tissue repair. Alpha‐smooth muscle actin, calponin and myosin light chain kinase presence confirms their similarity with myofibroblasts. Treatment of the HuF with 2.5% DMSO caused reversion back into spindle‐shaped morphology and loss of myofibroblast characteristics, suggesting a switch into less differentiated proto‐myofibroblasts. The unique abilities of HuF to exhibit multipotency as well as reverting back into less differentiated status and their easy availability makes them an interesting cell model to further explore as a possible tool for regenerative medicine (NIH HD 044713).

Decidualization (D), the differentiation of stromal fibroblasts (SF) into decidual cells, is critical for pregnancy establishment. D requires the reorganization of the actin cytoskeleton that is governed by phosphorylation (P) of myosin light chain and actin dynamics. Manipulation of actin dynamics by jasplakinolide prevents D of human SF. Actin dynamics are regulated by actin‐binding proteins, one of which is cofilin. The activity of cofilin depends on its P, which is regulated by LIMK1 and LIMK2. D of human SF is associated with changes in the distribution and activity of the proteins of the LIMK‐cofilin pathway. D induced by a stimulus of embryonic origin, interleukin‐1β (IL‐1β) and steroid hormones (H), is accompanied by an upregulation of LIMK1, a downregulation of LIMK2 and the translocation of inactivated (phosphorylated) cofilin from the nucleus into the cytosol. On the other hand, D induced by an artificial stimulus cAMP (with H) is characterized by a significant decrease in phosphorylated cofilin and unchanged amounts of LIMK1 and LIMK2. Despite the differences in molecular mechanisms underlying the D induced by H alone, IL‐1β+H or cAMP+H, the most striking observation during D is the translocation of cofilin from the cytosol to the nucleus. Taken together, the stabilization of F‐actin significantly inhibits D; and the translocation of cofilin to the nucleus is a key feature of D. Supported by HD044713