Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5–10%), re-differentiated monolayers with 11–27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.

Efficient hepatic differentiation and regeneration potential under xeno-free conditions using mass-producible amnion-derived mesenchymal stem cells

Background Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. Methods We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. Results We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. Conclusion This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.

Novel Evidence That By Employing a Two-Step Differentiation Protocol in the Presence of UM171 and Nicotinamide Acid Human Very Small Embryonic like Stem Cells (VSELs) Differentiate into Functional Endothelial Cells (ECs)

Background . Human Very small embryonic-like stem cells (VSELs) are CD133 + CD34 + small dormant stem cells with properties of self-renewal and multipotential ability to differentiate in the three-germ layers (Circulation Research 2019; 124: 208-210) and currently, more than 40 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010 , 9.29.1-9.29.15). We have previously reported that human bone marrow (BM)-derived VSELs are able to give rise to vessel formation and endothelial differentiation (Thrombosis and Haemostasis 2015 ; 113 : 1084-1094) and several independent groups confirmed our data with human, mouse, or rat VSELs. Thus, VSELs are a promising source in regenerative medicine for the treatment of vascular diseases. Aim of the study. We aimed to develop an in vitro expansion and differentiation protocol of VSELs into endothelial cells (EC-VSELs) that will provide a clinically relevant cell therapy product without ethical problems and undesirable side effects. Materials and Methods. Highly purified by FACS from umbilical cord blood (UCB), VSELs were sorted as very small lineage-negative, CD45 -, CD34 + cells and then cultured and expanded into EC-VSELs in pro-angiogenic medium supplemented with mesodermal differentiation factors followed by stimulation by endothelial differentiation factors in the presence of UM171 and nicotinamide acid. In parallel, for comparison we expanded ECFCs from MNCs isolated from the same UCB units. The endothelial nature of the expanded VSEL-derived ECs (EC-VSELs) was tested by the expression of typical EC markers as well as by in vitro and in vivo functional angiogenic assays. Results. We report here for the first time a multistep differentiation strategy of highly purified UCB-derived VSELsThese cells after isolation by FACS were small and round, then in the presence of GSK3b inhibitor and BMP4 inducing mesodermal differentiation and high VEGF concentration to induce endothelial differentiation, VSELs enlarged and displayed extended morphology and acquired a characteristic cobblestone morphology. Finally, we have obtained a high number of cells with typical morphology of endothelial cells (EC-VSELs). By inhibiting potential mesodermal transition using TGFb inhibitor, EC-VSELs had a comparable morphology to primary human ECFCs and were characterized by tight junctions, caveolae, and Weibel-Palade Bodies, as demonstrated by transmission electron microscopy analysis of cell cultures conducted on fibrin network on the top of pericardial membranes. ECFCs differentiation was confirmed by analyzing the expression of endothelial markers by flow cytometry, and EC-VSELs were positive for PECAM1, VE-cadherin, VEGFR2, and endoglin. EC-VSELs as compared to ECFCs presented the same levels of expression of all these endothelial markers. What is important at the same time, EC-VSELs, as well as ECFCs, were negative for mesodermal marker Thy-1, confirming their endothelial phenotype. Migration properties of EC-VSELs were studied in basal conditions or in pro-angiogenic conditions using two in vitro models: wound healing assay and Trans well migration assay, and in both models EC-VSELs migration properties were similar to those of ECFCs. Next, we compared paracrine activity by evaluating growth factor and cytokine secretion profile of EC-VSELs, and noticed that the cytokine secretion by expanded VSELs was comparable to that of ECFCs. Moreover, the formation of pseudo-tubes was similar with both conditioned media. Finally, we have assessed the angiogenic capacity of EC-VSELs with a 3D in vitro sprouting assay and in vivo Matrigel plug assay. EC-VSELs display angiogenic properties but with lower potential in comparison with ECFCs which could be explained by their more primitive potential and most likely they need more time to be fully specified into the endothelial lineage. Conclusions. Based on our novel intriguing data, showing that highly purified VSELs expand efficient by employing a two-step differentiation protocol in the presence of UM171 and nicotinamide to EC-VSELs and acquire the same endothelial morphology, phenotype, and secretory potential as ECFCs as well as form functional vessels in in vitro and in vivo angiogenic assays, they could become an alternative source of ECFCs to treat vascular diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Expression of Endogenous Putative TSH Binding Protein in Orbit

Thyroid stimulating antibodies (TSAB) cause Graves’ disease and contribute to Graves’ Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2β5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = −0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. β5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR.

A Short S-Equol Exposure Has a Long-Term Inhibitory Effect on Adipogenesis in Mouse 3T3-L1 Cells

In the search for new drugs against obesity, the chronic disease that threatens human health worldwide, several works have focused on the study of estrogen homologs because of the role of estrogen receptors (ERs) in adipocyte growth. The isoflavone equol, an ERβ agonist, has shown beneficial metabolic effects in in vivo and in vitro assays; however, additional studies are required to better characterize its potential for body weight control. Here, we showed that the treatment of 3T3-L1 cells with 10 μM of S-equol for the first three days of the adipocyte differentiation protocol was able to prevent cells becoming semi-rounded and having a lipid droplet formation until the seventh day of culture; moreover, lipid accumulation was reduced by about 50%. Congruently, S-equol induced a reduction in mRNA expression of the adipogenic markers C/EBPα and PPARγ, and adipokines secretion, mainly Adiponectin, Leptin, Resistin, and MCP-1, while the release of PAI-1 was augmented. Moreover, it also reduced the expression of ERα and attenuated the subexpression of ERβ associated with adipogenesis. Altogether, our data suggested that S-equol binding to ERβ affects the transcriptional program that regulates adipogenesis and alters adipocyte functions. Future efforts will focus on studying the impact of S-equol on ER signaling pathways.

iPS Cell-Based Model for MAPT Haplotype as a Risk Factor for Human Tauopathies Identifies No Major Differences in TAU Expression

The H1 haplotype of the microtubule-associated protein tau (MAPT) gene is a common genetic risk factor for some neurodegenerative diseases such as progressive supranuclear palsy, corticobasal degeneration, and Parkinson’s disease. The molecular mechanism causing the increased risk for the named diseases, however, remains unclear. In this paper, we present a valuable tool of eight small molecule neural precursor cell lines (smNPC) homozygous for the MAPT haplotypes (four H1/H1 and four H2/H2 cell lines), which can be used to identify MAPT-dependent phenotypes. The employed differentiation protocol is fast due to overexpression of NEUROGENIN-2 and therefore suitable for high-throughput approaches. A basic characterization of all human cell lines was performed, and their TAU and α-SYNUCLEIN profiles were compared during a differentiation time of 30 days. We could identify higher levels of conformationally altered TAU in cell lines carrying the H2 haplotype. Additionally, we found increased expression levels of α-SYNUCLEIN in H1/H1 cells. With this resource, we aim to fill a gap in neurodegenerative disease modeling with induced pluripotent stem cells (iPSC) for sporadic tauopathies.

Differentiation protocol for generating functional pancreatic β cells from human pluripotent stem cells

The efficient generation of pancreatic β cells from human pluripotent stem cells may allow us to study their biological characteristics and use them for the treatment of type I diabetes. The protocol we present in the study provides an efficient method for producing β cells using either human embryonic stem cells or human induced pluripotent stem cells as the starting material.

Differentiation of Peritubular Myoid-Like Cells from Human Induced Pluripotent Stem Cells

Spermatogenesis is a complex process requiring intricate cellular interactions between multiple cell types to produce viable sperm. Peritubular myoid cells (PTMs) are smooth muscle cells that line the seminiferous tubules and play a critical role in sperm production by providing mechanical support and molecular signaling factors. In vitro investigation of their contribution to spermatogenesis and their dysfunction in infertility is currently limited by the rare accessibility of human testicular tissue for research. Therefore, this study set forth to generate an alternative source of PTMs using human induced pluripotent stem cells (hiPSCs) - adult cells that have been reprogrammed into a pluripotent state, making them capable of indefinite expansion and the regeneration of any cell type in the body. PTMs and Leydig cells arise from a common progenitor, so we hypothesized that PTMs could be derived by modifying an existing differentiation protocol for Leydig cell differentiation from hiPSCs. These hiPSC-derived cells, or hPTMs, were characterized and compared to hiPSC-derived Leydig cells (hLCs) and human primary Sertoli cells as a negative control. Our findings show that the substitution of the molecular patterning factor Platelet-Derived Growth Factor Subunit B (PDGF-BB) for Platelet-Derived Growth Factor Subunit A (PDGF-AA) in a molecule-based differentiation protocol for deriving Leydig-like cells, is sufficient to derive peritubular myoid-like cells. This study describes a method for generating PTM-like cells from hiPSCs. These cells will allow for ongoing understanding of the cellular interactions required for normal spermatogenesis in an in vitro setting.

Single cell trajectory analysis of human pluripotent stem cells differentiating towards lung and hepatocyte progenitors

ABSTRACTUnderstanding the development of human respiratory tissues is crucial for modeling and treating lung disorders. The molecular details for the specification of lung progenitors from human pluripotent stem cells (hPSCs) are unclear. Here, we use single cell RNA-sequencing with high temporal resolution along an optimized differentiation protocol to determine the transcriptional hierarchy of lung specification from human hPSCs and map out the underlying single cell trajectories. We show that Sonic hedgehog, TGF-β and Notch activation are required in an ISL1/NKX2-1 trajectory that gives rise to lung progenitors during the foregut endoderm stage. Induction of HHEX marks an alternative trajectory at the early definitive endoderm stage, which precedes the lung trajectory and generates a major hepatoblast population. Moreover, neither KDR+ nor mesendoderm progenitors are apparent intermediate states of lung and hepatic lineages. Our hierarchical multistep model predicts mechanisms leading to lung organogenesis, and creates a basis for studying early human lung development, as well as hPSC based disease and drug research.Abstract Figure