Bone Marrow Mesenchymal Stem Cells (BMSCs) Enhance Endometrial Stromal Cell Migration and Epithelial-Mesenchymal Transition in Adenomyosis Through Upregulation of Neuropilin 1

Hormone support (estrogen and progesterone) is a key factor in decidualization and embryo implantation. Elevated levels of estrogen lead to luteal phase defects through Neuropilin 1, a membranecytoskeleton junction protein. This study aimed to explore the effect of BMSCs on endometrial stromal cells (ESCs) in adenomyosis. ESCs obtained from patients with adenomyosis were cocultured with BMSCs in the absence of presence of Neuropilin 1 inhibitor followed by analysis of expression of decidualization-related genes by RT-qPCR and western blot, cell viability by MTT assay, cell invasion and migration by Transwell assay, oxidative stress factors by ROS kit. Treatment with Neuropilin 1 inhibitor significantly decreased ESC proliferation and invasion, blocked epithelialmesenchymal transition (EMT) process, and restrained decidualization with a downregulation of decidualization-related genes. Furthermore, inhibition of Neuropilin 1 exerted effects through estrogen regulation. However, co-culture with BMSCs restored ESC activity by promoting Neuropilin expression and enhanced intrauterine ESC decidualization. In conclusion, Neuropilin 1 inhibitor restrains decidualization through estrogen regulation which can be abrogated by estrogen receptor antagonists. BMSCs restore the damaged ESC decidualization through increasing Neuropilin 1 expression, which provides new insights into the adverse effect of Neuropilin 1 on human ESCs, suggesting that BMSC is a potential therapeutic drug candidate for adenomyosis.

Rostral Hypothalamic Differentiation With Minimal Exogenous Signals in Human Naive Induced Pluripotent Stem Cells

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs) differentiate into AVP neurons, whereas human ESCs/induced pluripotent stem cells (iPSCs) die. Human ES/iPSCs are generally more similar to mouse epiblast stem cells compared to mouse ESCs, which are termed as primed and naive, respectively. In this study, we converted human FNDI-specific iPSCs from primed to naive cells, and found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

Capturing Pluripotency and Beyond

During the development of a multicellular organism, the specification of different cell lineages originates in a small group of pluripotent cells, the epiblasts, formed in the preimplantation embryo. The pluripotent epiblast is protected from premature differentiation until exposure to inductive cues in strictly controlled spatially and temporally organized patterns guiding fetus formation. Epiblasts cultured in vitro are embryonic stem cells (ESCs), which recapitulate the self-renewal and lineage specification properties of their endogenous counterparts. The characteristics of totipotency, although less understood than pluripotency, are becoming clearer. Recent studies have shown that a minor ESC subpopulation exhibits expanded developmental potential beyond pluripotency, displaying a characteristic reminiscent of two-cell embryo blastomeres (2CLCs). In addition, reprogramming both mouse and human ESCs in defined media can produce expanded/extended pluripotent stem cells (EPSCs) similar to but different from 2CLCs. Further, the molecular roadmaps driving the transition of various potency states have been clarified. These recent key findings will allow us to understand eutherian mammalian development by comparing the underlying differences between potency network components during development. Using the mouse as a paradigm and recent progress in human PSCs, we review the epiblast’s identity acquisition during embryogenesis and their ESC counterparts regarding their pluripotent fates and beyond.

Follistatin-like I promotes endometriosis by increasing proinflammatory factors and promoting angiogenesis

Endometriosis (EMS) is a chronic benign inflammatory disease characterized by the growth of endometrial-like tissue in aberrant locations outside of the uterine cavity. Angiogenesis and abnormal immune responses are the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. Follistatin-like I (FSTL1) is a secreted glycoprotein that exhibits varied expression levels in cardiovascular disease, cancer and arthritis. However, the role of FSTL1 in the development of EMS remains to be fully elucidated. Results of the present study demonstrated that the expression of FSTL1 was significantly increased in ectopic endometrial stromal cells (ESCs) and peritoneal fluid from patients with EMS, compared the control group. Both conditions of hypoxia and estrogen treatment induced human ESCs to produce increased levels of FSTL1 and disco-interacting protein 2 homolog A (DIP2A). Furthermore, the expression levels of DIP2A, IL-8 and IL-1β were increased in FSTL1 overexpressed HESCs. Additionally, FSTL1 treatment increased the proliferation of HUVECs in a dose-dependent manner in vitro, and markedly increased the tube formation of HUVECs. Moreover, treatment with FSTL1 facilitated M1 polarization of macrophages, increased the secretion of proinflammatory factors and inhibited the expression of scavenger receptor CD36. Results of the present study suggested that the elevated expression of FSTL1 may play a key role in accelerating the development of EMS via enhancing the secretion of proinflammatory factors and promoting angiogenesis.

The inhibitory effect of AMP-activated protein kinase (AMPK) on chemokine and prostaglandin production in human endometrial stromal cells

Background To investigate the role of adenosine monophosphate (AMP)-activated protein kinase (AMPK) on the production of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, prostaglandin E2 and F2α induced by IL-1β in endometrial stromal cells (ESCs) following treatment with 5-aminoimidazole-4- carboxamide ribonucleoside (AICAR). Methods Endometrial specimens were obtained and cultured. We examined the effects of IL-1β, IL-1 ra and AICAR on the production of IL-8, MCP-1, PGE2 and PGF2α in human ESCs. The phosphorylations of AMPK, IκB, 4EBP-1, p70S6K and S6 ribosomal protein were analyzed by Western immunoblotting. Results Following stimulation by IL-1β, the production of IL-8, MCP-1, PGE2 and PGF2α showed significant increases, and these increases were suppressed by AICAR. The expression of cyclooxygenase-2 (COX-2) induced by IL-1β and suppressed by AICAR. The phosphorylation of IκB, 4EBP-1, p70S6K and S6 ribosomal protein were inhibited via an AMPK-dependent signal transduction. Conclusions The production of IL-8, MCP-1, PGE2 and PGF2α induced by IL-1β in ESCs were involved in the negative regulatory mechanisms of AMPK. The substances that activate AMPK may be promising agents for the treatment of pathological problems such as dysmenorrhea.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Harnessing the Potential of Human Pluripotent Stem Cell-Derived Motor Neurons for Drug Discovery in Amyotrophic Lateral Sclerosis: From the Clinic to the Laboratory and Back to the Patient

Amyotrophic Lateral Sclerosis (ALS) is a motor neurodegenerative disorder whose cellular hallmarks are the progressive death of motor neurons (MNs) located in the anterior horn of the spinal cord, brainstem and motor cortex, and the formation of intracellular protein aggregates. Over the course of the disease, progressive paralysis takes place, leading to patient death within 3–5 years after the diagnosis. Despite decades of intensive research, only a few therapeutic options exist, with a limited benefit on the disease progression. Preclinical animal models have been very useful to decipher some aspects of the mechanisms underlying ALS. However, discoveries made using transgenic animal models have failed to translate into clinically meaningful therapeutic strategies. Thus, there is an urgent need to find solutions to discover drugs that could impact on the course of the disease, with the ultimate goal to extend the life of patients and improve their quality of life. Induced pluripotent stem cells (iPSCs), similarly to embryonic stem cells (ESCs), have the capacity to differentiate into all three embryonic germ layers, which offers the unprecedented opportunity to access patient-specific central nervous system cells in an inexhaustible manner. Human MNs generated from ALS patient iPSCs are an exciting tool for disease modelling and drug discovery projects, since they display ALS-specific phenotypes. Here, we attempted to review almost 2 decades of research in the field, first highlighting the steps required to efficiently generate MNs from human ESCs and iPSCs. Then, we address relevant ALS studies which employed human ESCs and iPSC-derived MNs that led to the identification of compounds currently being tested in clinical trials for ALS. Finally, we discuss the potential and caveats of using patient iPSC-derived MNs as a platform for drug screening, and anticipate ongoing and future challenges in ALS drug discovery.

CHD8 safeguards early neuroectoderm differentiation in human ESCs and protects from apoptosis during neurogenesis

The chromatin remodeler CHD8, which belongs to the ATP-dependent chromatin remodelers CHD family, is one of the most high-risk mutated genes in autism spectrum disorders. However, the role of CHD8 in neural differentiation and the mechanism of CHD8 in autism remains unclear, despite there are a few studies based on the CHD8 haploinsufficient models. Here, we generate the CHD8 knockout human ESCs by CRISPR/Cas9 technology and characterize the effect of loss-of-function of CHD8 on pluripotency maintenance and lineage determination by utilizing efficient directed differentiation protocols. The results show loss-of-function of CHD8 does not affect human ESC maintenance although having slight effect on proliferation and cell cycle. Interestingly, CHD8 depletion results in defective neuroectoderm differentiation, along with severe cell death in neural progenitor stage. Transcriptome analysis also indicates CHD8 does not alter the expression of pluripotent genes in ESC stage, but in neural progenitor cells depletion of CHD8 induces the abnormal expression of the apoptosis genes and suppresses neuroectoderm-related genes. These results provide the evidence that CHD8 plays an essential role in the pluripotency exit and neuroectoderm differentiation as well as the regulation of apoptosis during neurogenesis.

Dermatan-4-O-Sulfotransferase-1 Contributes to the Undifferentiated State of Mouse Embryonic Stem Cells

Mouse embryonic stem cells (mESCs) have the properties of self-renewal and pluripotency. Various signals and growth factors maintain their undifferentiated state and also regulate their differentiation. Glycosaminoglycans are present on the cell surface and in the cell matrix as proteoglycans. Previously, we and other groups reported that the glycosaminoglycan heparan sulfate contributes to both maintenance of undifferentiated state and regulation of mESC differentiation. It has been shown that chondroitin sulfate is needed for pluripotency and differentiation of mESCs, while keratan sulfate is a known marker of human ESCs or induced pluripotent stem cells. We also found that DS promotes neuronal differentiation from mESCs and human neural stem cells; however, the function of DS in the maintenance of mESCs has not yet been revealed. Here, we investigated the role of DS in mESCs by knockdown (KD) or overexpression (O/E) of the dermatan-4-O-sulfotransferase-1 (D4ST1) gene. We found that the activity of the ESC self-renewal marker alkaline phosphatase was reduced in D4ST1 KD mESCs, but, in contrast, increased in D4ST1 O/E mESCs. D4ST1 KD promoted endodermal differentiation, as indicated by an increase in Cdx2 expression. Conversely, Cdx2 expression was decreased by D4ST1 O/E. Wnt signaling, which is also involved in endodermal differentiation, was activated by D4ST1 KD and suppressed by D4ST1 O/E. Collectively, these results demonstrate that D4ST1 contributes to the undifferentiated state of mESCs. Our findings provide new insights into the function of DS in mESCs.

Stemness of Human Pluripotent Cells: Hypoxia-like Response Induced by Low Nitric Oxide

The optimization of conditions to promote the stemness of pluripotent cells in vitro is instrumental for their use in advanced therapies. We show here that exposure of human iPSCs and human ESCs to low concentrations of the chemical NO donor DETA/NO leads to stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) under normoxia, with this effect being dependent on diminished Pro 402 hydroxylation and decreased degradation by the proteasome. Moreover, the master genes of pluripotency, NANOG and OCT-4, were upregulated. NO also induces a shift in the metabolic profile of PSCs, with an increased expression of hypoxia response genes in glycolysis. Furthermore, a reduction in the mitochondrial membrane potential with lower oxygen consumption and increased expression of mitochondrial fusion regulators, such as DRP1, was observed. The results reported here indicate that NO mimics hypoxia response in human PSCs and enhances their stemness properties when cultured under normoxic conditions.