A gastruloid model of the interaction between embryonic and extra-embryonic cell types.

Stem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo development. Full control over their initial composition, scalability, and easily measurable dynamics make those systems useful for studying specific developmental processes in isolation. Here we report the formation of gastruloids consisting of mouse embryonic stem cells (mESCs) and extraembryonic endoderm (XEN) cells. These XEN-enhanced gastruloids (XEGs) exhibit the formation of neural epithelia, which are absent in gastruloids derived from mESCs only. By single-cell RNA-seq, imaging, and differentiation experiments, we demonstrate the neural characteristics of the epithelial tissue. We further show that the mESCs induce the differentiation of the XEN cells to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish XEGs to explore heterotypic cellular interactions and their developmental consequences in vitro.

Phiclust: a clusterability measure for single-cell transcriptomics reveals phenotypic subpopulations.

The ability to discover new cell phenotypes by unsupervised clustering of single-cell transcriptomes has revolutionized biology. Currently, there is no principled way to decide whether a cluster of cells contains meaningful subpopulations that should be further resolved. Here, we present phiclust (ϕclust), a clusterability measure derived from random matrix theory that can be used to identify cell clusters with non-random substructure, testably leading to the discovery of previously overlooked phenotypes.

Deciphering Metabolic Heterogeneity by Single-Cell Analysis.

Single-cell analysis provides insights into cellular heterogeneity and dynamics of individual cells. This Feature highlights recent developments in key analytical techniques suited for single-cell metabolic analysis with a special focus on mass spectrometry-based analytical platforms and RNA-seq as well as imaging techniques that reveal stochasticity in metabolism.

Low-Dose Pesticide Mixture Induces Accelerated Mesenchymal Stem Cell Aging In Vitro.

The general population is chronically exposed to multiple environmental contaminants such as pesticides. We have previously demonstrated that human mesenchymal stem cells (MSCs) exposed in vitro to low doses of a mixture of seven common pesticides showed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation toward adipogenesis. Thus, we hypothesized that common combination of pesticides may induce a premature cellular aging of adult MSCs. Our goal was to evaluate if the prolonged exposure to pesticide mixture could accelerate aging-related markers and in particular deteriorate the immunosuppressive properties of MSCs. MSCs exposed to pesticide mixture, under long-term culture and obtained from aging donor, were compared by bulk RNA sequencing analysis. Aging, senescence, and immunomodulatory markers were compared. The protein expression of cellular aging-associated metabolic markers and immune function of MSCs were analyzed. Functional analysis of the secretome impacts on immunomodulatory properties of MSCs was realized after 21 days' exposure to pesticide mixture. The RNA sequencing analysis of MSCs exposed to pesticide showed some similarities with cells from prolonged culture, but also with the MSCs of an aged donor. Changes in the metabolic markers MDH1, GOT and SIRT3, as well as an alteration in the modulation of active T cells and modifications in cytokine production are all associated with cellular aging. A modified functional profile was found with similarities to aging process. Stem Cells 2019;37:1083-1094.

Single-cell transcriptomics reveals gene expression dynamics of human fetal kidney development.

The current understanding of mammalian kidney development is largely based on mouse models. Recent landmark studies revealed pervasive differences in renal embryogenesis between mouse and human. The scarcity of detailed gene expression data in humans therefore hampers a thorough understanding of human kidney development and the possible developmental origin of kidney diseases. In this paper, we present a single-cell transcriptomics study of the human fetal kidney. We identified 22 cell types and a host of marker genes. Comparison of samples from different developmental ages revealed continuous gene expression changes in podocytes. To demonstrate the usefulness of our data set, we explored the heterogeneity of the nephrogenic niche, localized podocyte precursors, and confirmed disease-associated marker genes. With close to 18,000 renal cells from five different developmental ages, this study provides a rich resource for the elucidation of human kidney development, easily accessible through an interactive web application.

Parathyroid Hormone-Related Protein Contributes to Early Healing of Habu Snake Venom-Induced Glomerulonephritis in Mice.

Proliferative glomerulonephritis is characterized by local inflammation and mesangial cell deterioration, followed by mesangial proliferation and glomerular healing. Parathyroid hormone-related peptide (PTHrP) is a mesangial cytokine-like growth factor implicated in mesangial proliferation and survival. No data are available about its role in glomerulonephritis. Herein, we analyzed the expression and role of PTHrP in glomerular inflammation and healing in an experimental model of glomerulonephritis induced by i.v. injection of Habu snake venom in mice. The temporal analysis showed marked renal damage in the first days after venom injection and the beginning of recovery within 7 days. Glomerular expression of PTHrP (transcript and protein) was observed in the early phase after venom injection (from day 1 to day 3), along with an inflammatory environment. The inactivation of secreted PTHrP with PTHrP-neutralizing antibody (PTH2E11; 120 µg i.p. daily) reduced the markers of local inflammation (expression of macrophage chemotactic protein-1; regulated upon activation, normal T cell expressed and secreted; cyclooxygenase 2; IL-6; and macrophage infiltration) and abolished the expression of PTHrP itself. Moreover, the glomerular cell proliferation was hampered, and the healing process was prevented on day 7 after venom injection. These results show that PTHrP has antinomic actions in glomerulonephritis, participating in both the proinflammatory condition and the healing process. Our work reveals the essential role of PTHrP in early glomerular repair in an experimental model of glomerulonephritis.

Parathyroid hormone-related protein modulates inflammation in mouse mesangial cells and blunts apoptosis by enhancing COX-2 expression.

Injury of mesangial cells (MC) is a prominent feature of glomerulonephritis. Activated MC secrete inflammatory mediators that induce cell apoptosis. Parathyroid hormone-related peptide (PTHrP) is a locally active cytokine that enhances cell survival and is upregulated by proinflammatory factors in many cell types. The aim of this study was to analyze the regulation of PTHrP expression by inflammatory cytokines and to evaluate whether PTHrP itself acts as a proinflammatory and/or survival factor on male murine MC in primary culture. Our results showed that IL-1ß (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1ß were both transcriptional and posttranscriptional, with stabilization of the PTHrP mRNA by human antigen R (HuR). Proteome profiler arrays showed that PTHrP itself enhanced cytokines within 2 h in cell lysates, mainly IL-17, IL-16, IL-1α, and IL-6. PTHrP also stimulated sustained expression (2-4 h) of chemokines, mainly regulated upon activation normal T cell expressed and secreted (RANTES)/C-C motif chemokine 5 (CCL5) and macrophage inflammatory protein-2 (MIP-2)/C-X-C motif chemokine 2 (CXCL2), thymus and activation-regulated chemokine (TARC)/CCL17, and interferon-inducible T cell α-chemoattractant (I-TAC)/CXCL11. Moreover, PTHrP markedly enhanced cyclooxygenase-2 (COX-2) expression and elicited its autoinduction through the activation of the NF-κB pathway. PTHrP induced MC survival via the COX-2 products, and PTHrP overexpression in MC blunted the apoptotic effects of IL-1ß and TNF-α. Altogether, these findings suggest that PTHrP functions as a booster of glomerular inflammatory processes and may be a negative feedback loop preserving MC survival.

Low-Dose Pesticide Mixture Induces Senescence in Normal Mesenchymal Stem Cells (MSC) and Promotes Tumorigenic Phenotype in Premalignant MSC.

Humans are chronically exposed to multiple environmental pollutants such as pesticides with no significant evidence about the safety of such poly-exposures. We exposed mesenchymal stem cells (MSC) to very low doses of mixture of seven pesticides frequently detected in food samples for 21 days in vitro. We observed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation towards adipogenesis but did not initiate a tumorigenic transformation. In modified MSC in which a premalignant phenotype was induced, the exposure to pesticide mixture promoted tumorigenic phenotype both in vitro and in vivo after cell implantation, in all nude mice. Our results suggest that a common combination of pesticides can induce a premature ageing of adult MSC, and as such could accelerate age-related diseases. Exposure to pesticide mixture may also promote the tumorigenic transformation in a predisposed stromal environment. Abstract Video Link: https://youtu.be/mfSVPTol-Gk Stem Cells 2017;35:800-811.

Targeting FAK scaffold functions inhibits human renal cell carcinoma growth.

Human conventional renal cell carcinoma (CCC) remains resistant to current therapies. Focal Adhesion Kinase (FAK) is upregulated in many epithelial tumors and clearly implicated in nearly all facets of cancer. However, only few reports have assessed whether FAK may be associated with renal tumorigenesis. In this study, we investigated the potential role of FAK in the growth of human CCC using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as normal/tumoral renal tissue pairs. FAK was found constitutively expressed in human CCC both in culture cells and freshly harvested tumors obtained from patients. We showed that CCC cell growth was dramatically reduced in FAK-depleted cells or after FAK inhibition with various inhibitors and this effect was obtained through inhibition of cell proliferation and induction of cell apoptosis. Additionally, our results indicated that FAK knockdown decreased CCC cell migration and invasion. More importantly, depletion or pharmacological inhibition of FAK substantially inhibited tumor growth in vivo. Interestingly, investigations of the molecular mechanism revealed loss of FAK phosphorylation during renal tumorigenesis impacting multiple signaling pathways. Taken together, our findings reveal a previously uncharacterized role of FAK in CCC whereby FAK exerts oncogenic properties through a non canonical signaling pathway involving its scaffolding kinase-independent properties. Therefore, targeting the FAK scaffold may represent a promising approach for developing innovative and highly specific therapies in human CCC.

Knockdown of parathyroid hormone related protein in smooth muscle cells alters renal hemodynamics but not blood pressure.

Parathyroid hormone-related protein (PTHrP) belongs to vasoactive factors that regulate blood pressure and renal hemodynamics both by reducing vascular tone and raising renin release. PTHrP is expressed in systemic and renal vasculature. Here, we wanted to assess the contribution of vascular smooth muscle cell endogenous PTHrP to the regulation of cardiovascular and renal functions. We generated a mouse strain (SMA-CreERT2/PTHrPL2/L2 or premutant PTHrPSM-/-), which allows temporally controlled, smooth muscle-targeted PTHrP knockdown in adult mice. Tamoxifen treatment induced efficient recombination of PTHrP-floxed alleles and decreased PTHrP expression in vascular and visceral smooth muscle cells of PTHrPSM-/- mice. Blood pressure remained unchanged in PTHrPSM-/- mice, but plasma renin concentration and creatinine clearance were reduced. Renal hemodynamics were further analyzed during clearance measurements in anesthetized mice. Conditional knockdown of PTHrP decreased renal plasma flow and glomerular filtration rate with concomitant reduction in filtration fraction. Similar measurements were repeated during acute saline volume expansion. Saline volume expansion induced a rise in renal plasma flow and reduced filtration fraction; both were blunted in PTHrPSM-/- mice leading to impaired diuresis. These findings show that endogenous vascular smooth muscle PTHrP controls renal hemodynamics under basal conditions, and it is an essential factor in renal vasodilation elicited by saline volume expansion.

Parathyroid hormone-related protein is a mitogenic and a survival factor of mesangial cells from male mice: role of intracrine and paracrine pathways.

Glomerulonephritis is characterized by the proliferation and apoptosis of mesangial cells (MC). The parathyroid-hormone related protein (PTHrP) is a locally active cytokine that affects these phenomena in many cell types, through either paracrine or intracrine pathways. The aim of this study was to evaluate the effect of both PTHrP pathways on MC proliferation and apoptosis. In vitro studies were based on MC from male transgenic mice allowing PTHrP-gene excision by a CreLoxP system. MC were also transfected with different PTHrP constructs: wild type PTHrP, PTHrP devoid of its signal peptide, or of its nuclear localization sequence. The results showed that PTHrP deletion in MC reduced their proliferation even in the presence of serum and increased their apoptosis when serum-deprived. PTH1R activation by PTHrP(1-36) or PTH(1-34) had no effect on proliferation but improved MC survival. Transfection of MC with PTHrP devoid of its signal peptide significantly increased their proliferation and minimally reduced their apoptosis. Overexpression of PTHrP devoid of its nuclear localization sequence protected cells from apoptosis without changing their proliferation. Wild type PTHrP transfection conferred both mitogenic and survival effects, which seem independent of midregion and C-terminal PTHrP fragments. PTHrP-induced MC proliferation was associated with p27(Kip1) down-regulation and c-Myc/E2F1 up-regulation. PTHrP increased MC survival through the activation of cAMP/protein kinase A and PI3-K/Akt pathways. These results reveal that PTHrP is a cytokine of multiple roles in MC, acting as a mitogenic factor only through an intracrine pathway, and reducing apoptosis mainly through the paracrine pathway. Thus, PTHrP appears as a probable actor in MC injuries.