Adipose-derived mesenchymal stem cells (ASCs) are considered to be a useful tool for regenerative medicine, owing to their capabilities in differentiation, self-renewal, and immunomodulation. These cells have become a focus in the clinical setting due to their abundance and easy isolation. However, ASCs from different depots are not well characterized. Here, we analyzed the functional similarities and differences of subcutaneous and visceral ASCs. Subcutaneous ASCs have an extraordinarily directed mode of motility and a highly dynamic focal adhesion turnover, even though they share similar surface markers, whereas visceral ASCs move in an undirected random pattern with more stable focal adhesions. Visceral ASCs have a higher potential to differentiate into adipogenic and osteogenic cells when compared to subcutaneous ASCs. In line with these observations, visceral ASCs demonstrate a more active sonic hedgehog pathway that is linked to a high expression of cilia/differentiation related genes. Moreover, visceral ASCs secrete higher levels of inflammatory cytokines interleukin-6, interleukin-8, and tumor necrosis factor α relative to subcutaneous ASCs. These findings highlight, that both ASC subpopulations share multiple cellular features, but significantly differ in their functions. The functional diversity of ASCs depends on their origin, cellular context and surrounding microenvironment within adipose tissues. The data provide important insight into the biology of ASCs, which might be useful in choosing the adequate ASC subpopulation for regenerative therapies.

The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it’s up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.

Obesity impairs a variety of cell types including adipose-derived mesenchymal stem cells (ASCs). ASCs are indispensable for tissue homeostasis/repair, immunomodulation, and cell renewal. It has been demonstrated that obese ASCs are defective in differentiation, motility, immunomodulation, and replication. We have recently reported that some of these defects are linked to impaired primary cilia, which are unable to properly convey and coordinate a variety of signaling pathways. We hypothesized that the rescue of the primary cilium in obese ASCs would restore their functional properties. Obese ASCs derived from subcutaneous and visceral adipose tissues were treated with a specific inhibitor against Aurora A or with an inhibitor against extracellular signal-regulated kinase 1/2 (Erk1/2). Multiple molecular and cellular assays were performed to analyze the altered functionalities and their involved pathways. The treatment with low doses of these inhibitors extended the length of the primary cilium, restored the invasion and migration potential, and improved the differentiation capacity of obese ASCs. Associated with enhanced differentiation ability, the cells displayed an increased expression of self-renewal/stemness-related genes like SOX2, OCT4, and NANOG, mediated by reduced active glycogen synthase kinase 3 β (GSK3β). This work describes a novel phenomenon whereby the primary cilium of obese ASCs is rescuable by the low-dose inhibition of Aurora A or Erk1/2, restoring functional ASCs with increased stemness. These cells might be able to improve tissue homeostasis in obese patients and thereby ameliorate obesity-associated diseases. Additionally, these functionally restored obese ASCs could be useful for novel autologous mesenchymal stem cell-based therapies.

Tumor progression and pregnancy have several features in common. Tumor cells and placental trophoblasts share many signaling pathways involved in migration and invasion. Preeclampsia, associated with impaired differentiation and migration of trophoblastic cells, is an unpredictable and unpreventable disease leading to maternal and perinatal mortality and morbidity. Like in tumor cells, most pathways, in which p21 is involved, are deregulated in trophoblasts of preeclamptic placentas. The aim of the present study was to enlighten p21’s role in tumorigenic choriocarcinoma and trophoblastic cell lines. We show that knockdown of p21 induces defects in chromosome movement during mitosis, though hardly affecting proliferation and cell cycle distribution. Moreover, suppression of p21 compromises the migration and invasion capability of various trophoblastic and cancer cell lines mediated by, at least partially, a reduction of the extracellular signal-regulated kinase 3, identified using transcriptome-wide profiling, real-time PCR, and Western blot. Further analyses show that downregulation of p21 is associated with reduced matrix metalloproteinase 2 and tissue inhibitor of metalloproteinases 2. This work evinces that p21 is involved in chromosome movement during mitosis as well as in the motility and invasion capacity of trophoblastic and cancer cell lines.

Background: The development of the human placenta is tightly coordinated by a multitude of placental cell types, including human chorionic villi mesenchymal stromal cells (hCV-MSCs). Defective hCV-MSCs have been reported in preeclampsia (PE), a gestational hypertensive disease characterized by maternal endothelial dysfunction and systemic inflammation. Our goal was to determine whether hCV-MSCs are ciliated and whether altered ciliation is responsible for defective hCV-MSCs in preeclamptic placentas, as the primary cilium is a hub for signal transduction, which is important for various cellular activities. Methods: In the present work, we collected placental tissues from different gestational stages and we isolated hCV-MSCs from 1st trimester, term control, and preeclamptic placentas. We studied their ciliation, functionality, and impact on trophoblastic cell lines and organoids formed from human trophoblast stem cells (hTSCs) and from the trophoblastic cell line JEG-3 with various cellular and molecular methods, including immunofluorescence staining, gene analysis, spheroid/organoid formation, motility, and cellular network formation assay. The statistical evaluation was performed using a Student’s t test (two-tailed and paired or homoscedastic) or an unpaired Mann–Whitney U test (two-tailed). Results: The results show that primary cilia appeared abundantly in normal hCV-MSCs, especially in the early development of the placenta. Compared to control hCV-MSCs, the primary cilia were truncated, and there were fewer ciliated hCV-MSCs derived from preeclamptic placentas with impaired hedgehog signaling. Primary cilia are necessary for hCV-MSCs’ proper signal transduction, motility, homing, and differentiation, which are impaired in preeclamptic hCV-MSCs. Moreover, hCV-MSCs derived from preeclamptic placentas are significantly less capable of promoting growth and differentiation of placental organoids, as well as cellular network formation. Conclusions: These data suggest that the primary cilium is required for the functionality of hCV-MSCs and primary cilia are impaired in hCV-MSCs from preeclamptic placentas.

Preeclampsia (PE), a gestational hypertensive disease originating from the placenta, is characterized by an imbalance of various cellular processes. The cell cycle regulator p21^Cip1/CDKN1A (p21) and its family members p27 and p57 regulate signaling pathways fundamental to placental development. The aim of the present study was to enlighten the individual roles of these cell cycle regulators in placental development and their molecular involvement in the pathogenesis of PE. The expression and localization of p21, phospho-p21 (Thr-145), p27, and p57 was immunohistochemically analyzed in placental tissues from patients with early-onset PE, early-onset PE complicated by the HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome as well as late-onset PE compared to their corresponding control tissues from well-matched women undergoing caesarean sections. The gene level was evaluated using real-time quantitative PCR. We demonstrate that the delivery mode strongly influenced placental gene expression, especially for CDKN1A (p21) and CDKN1B (p27), which were significantly upregulated in response to labor. Cell cycle regulators were highly expressed in first trimester placentas and impacted by hypoxic conditions. In support of these observations, p21 protein was abundant in trophoblast organoids and hypoxia reduced its gene expression. Microarray analysis of the trophoblastic BeWo cell line depleted of p21 revealed various interesting candidate genes and signaling pathways for the fusion process. The level of p21 was reduced in fusing cytotrophoblasts in early-onset PE placentas and depletion of p21 led to reduced expression of fusion-related genes such as syncytin-2 and human chorionic gonadotropin (β-hCG), which adversely affected the fusion capability of trophoblastic cells. These data highlight that cell cycle regulators are important for the development of the placenta. Interfering with p21 influences multiple pathways related to the pathogenesis of PE.