Towards a "Testis in a Dish": Generation of Mouse Testicular Organoids that Recapitulate Testis Structure and Expression Profiles.

The testis is responsible for sperm production and androgen synthesis. Abnormalities in testis development and function lead to disorders of sex development and male infertility. Currently, no in vitro system exists for modelling the testis. Here, we generated testis organoids from neonatal mouse primary testicular cells using transwell inserts and show that these organoids generate tubule-like structures and cellular organization resembling that of the in vivo testis. Gene expression analysis of organoids demonstrates a profile that recapitulates that observed in in vivo testis. Embryonic testicular cells, but not adult testicular cells are also capable of forming organoids. These organoids can be maintained in culture for 8-9 weeks and shows signs of entry into meiosis. We further developed defined media compositions that promote the immature versus mature Sertoli cell and Leydig cell states, enabling organoid maturation in vitro. These testis organoids are a promising model system for basic research of testes development and function, with translational applications for elucidation and treatment of developmental sex disorders and infertility.

Generation and characterization of iPSC lines from two nuclear envelopathy patients with a homozygous nonsense mutation in the TOR1AIP1 gene.

LAP1 is an inner nuclear membrane protein encoded by TOR1AIP1. A homozygous c.961C > T loss of function mutation in TOR1AIP1 that affects both isoforms of LAP1 was recently described. This mutation leads to the development of a severe multisystemic nuclear envelopathy syndrome. Here we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines derived from skin fibroblasts of two patients carrying the homozygous c.961C > T mutation. These novel lines can be used as a powerful tool to investigate the molecular mechanism by which LAP1 deficiency leads to the development of this severe hereditary disorder.

Apoptosis induction by the stem cell factor LIN28A.

BACKGROUND INFORMATION: Lin28A and its paralog Lin28B are RNA binding proteins expressed in stem and progenitor cells, regulating the balance between their proliferation and differentiation. In-vivo and in-vitro experiments have shown that overexpression of these genes leads to abnormal cell proliferation, which results in many cases in cell transformation and tumor formation. RESULTS: Here we show, for the first time, that Lin28A overexpression can also lead to the opposite effect, i.e. apoptosis induction. We further demonstrate that this effect is specific to Lin28A but not to Lin28B and that it is mediated via the Let-7 independent pathway in a complex mechanism that involves at least several proteins. CONCLUSIONS AND SIGNIFICANCE: This unexpected observation suggests that cell fate regulation by Lin28 is dependent on a specific cellular/genetic context. Unraveling the cellular and molecular mechanisms underlying this Lin28A overexpression effect may pave the way for novel tumor therapeutic strategies, as Lin28 is commonly expressed in many types of tumors but not in most normal adult cells.

Human Pluripotent Stem Cell Fate Regulation by SMARCB1.

Epigenetic regulation by the SWI/SNF complex is essential for normal self-renewal capacity and pluripotency of human pluripotent stem cells (hPSCs). It has been shown that different subunits of the complex have a distinct role in this regulation. Specifically, the SMARCB1 subunit has been shown to regulate the activity of enhancers in diverse types of cells, including hPSCs. Here, we report the establishment of conditional hPSC lines, enabling control of SMARCB1 expression from complete loss of function to significant overexpression. Using this system, we show that any deviation from normal SMARCB1 expression leads to cell differentiation. We further found that SMARCB1 expression is not required for differentiation of hPSCs into progenitor cells, but rather for later stages of differentiation. Finally, we identify SMARCB1 as a critical player in regulation of cell-cell and cell-ECM interactions in hPSCs and show that this regulation is mediated at least in part by the WNT pathway.

Single-Cell RNA Sequencing Reveals mRNA Splice Isoform Switching during Kidney Development.

BACKGROUND: During mammalian kidney development, nephron progenitors undergo a mesenchymal-to-epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, Drop-seq single-cell RNA sequencing technology for measuring gene expression from thousands of individual cells identified the different cell types in the developing kidney. However, that analysis did not include the additional layer of heterogeneity that alternative mRNA splicing creates. METHODS: Full transcript length single-cell RNA sequencing characterized the transcriptomes of 544 individual cells from mouse embryonic kidneys. RESULTS: Gene expression levels measured with full transcript length single-cell RNA sequencing identified each cell type. Further analysis comprehensively characterized splice isoform switching during the transition between mesenchymal and epithelial cellular states, which is a key transitional process in kidney development. The study also identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. CONCLUSIONS: Discovery of the sets of genes that are alternatively spliced as the fetal kidney mesenchyme differentiates into tubular epithelium will improve our understanding of the molecular mechanisms that drive kidney development.

Heterochronic regulation of lung development via the Lin28-Let-7 pathway.

The heterochronic gene Lin28 regulates diverse developmental processes. It was shown previously that global Lin28A overexpression during mouse embryogenesis results in perinatal lethality. However, the reason for this early lethality has not been elucidated. Here, we showed that Lin28A overexpression prevents normal lung development via the inhibition of the Let-7 micro RNAs, thus causing the perinatal lethality. We further found that Lin28A overexpression in lung mesenchymal cells, but not epithelial cells, is sufficient to recapitulate the lung phenotype. Moreover, we defined the specific time window wherein Lin28A expression exerts its effect. Deep characterization of the transgenic lungs suggests that the Lin28A-Let-7 pathway delays the transition from one developmental stage to another but does not completely abrogate the differentiation capacity of the lung progenitor cells. Finally, we suggested that the effect of Lin28A-Let-7 on embryonic lung development is mediated at least in part through the TGF-ß1-signaling pathway. Altogether, these findings define for the first time the Lin28-Let-7 pathway as a critical heterochronic regulator of lung development.-Komarovsky Gulman, N., Armon, L., Shalit, T., Urbach, A. Heterochronic regulation of lung development via the Lin28-Let-7 pathway.

Peroxisome proliferator-activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target

Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML-xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARγ-pathway activation. Similarly, immunostaining revealed strong PPARγ expression in human AML specimens. Accordingly, we demonstrate that while PPARγ agonism accelerates AML growth, PPARγ antagonism is inhibitory, strongly suppressing AML proliferation and tumor-initiating capacity, via a TGFB-mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARγ-activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARγ as a regulator of AML growth, which could serve as an attractive therapeutic target.

The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

LIN28: A Stem Cell Factor with a Key Role in Pediatric Tumor Formation.

Differentiation and development are normally unidirectional processes in which progenitor/stem cells differentiate into more mature cells. Transformation of adult cells into cancer cells is accompanied in many cases by dedifferentiation of the adult cell, while differentiation failure of progenitor cells can result in the formation of unique type of cancers called pediatric cancer. LIN28A and its paralog LIN28B are pluripotent genes that are expressed mainly in stem/progenitor cells. Since the first identification of LIN28 in mammals, numerous studies demonstrated the general oncogenic features of these genes. In this review, we emphasize the unique role of LIN28 in pediatric tumor formation. We show, based on comprehensive literature screen and analysis of published microarray data, that LIN28 expression in pediatric tumors is even more common than in adult tumors, and discuss the possibility that in the case of pediatric cancers, LIN28 acts by preventing normal development/differentiation rather than by transformation of mature cells into cancer cells. Overall, this review highlights the role of LIN28 as a bridge point between embryonic development, stem cell biology, and cancer.

Human oocyte-derived sperm chemoattractant is a hydrophobic molecule associated with a carrier protein.

OBJECTIVE: To characterize the nature of the human oocyte-derived chemoattractant. DESIGN: Laboratory in vitro study. SETTING: Academic research institute. PATIENT(S): Ten healthy sperm donors. Oocyte-conditioned media from women undergoing IVF treatment because of male factor infertility. INTERVENTION(S): Sperm samples were processed by the migration-sedimentation technique. Oocyte-conditioned media were collected 2-3 hours after oocyte stripping. MAIN OUTCOME MEASURE(S): Sperm chemotaxis was assayed in a µ-slide chamber according to the direction of swimming relative to that of the chemical gradient. RESULT(S): Oocyte-conditioned media treated with proteases did not lose their chemotactic activity; on the contrary, they became more active, with the activity shifted to lower concentrations. When oocyte-conditioned media were subjected to hexane extraction, chemotactic activity was found in both the hydrophobic and aqueous phases. Known mammalian sperm chemoattractants were ruled out as oocyte-derived chemoattractants. CONCLUSION(S): Our results suggest that the oocyte-derived chemoattractant is a hydrophobic nonpeptide molecule that, in an oocyte-conditioned medium, is associated with a carrier protein that enables its presence in a hydrophilic environment.

Testing human sperm chemotaxis: how to detect biased motion in population assays.

Biased motion of motile cells in a concentration gradient of a chemoattractant is frequently studied on the population level. This approach has been particularly employed in human sperm chemotactic assays, where the fraction of responsive cells is low and detection of biased motion depends on subtle differences. In these assays, statistical measures such as population odds ratios of swimming directions can be employed to infer chemotactic performance. Here, we report on an improved method to assess statistical significance of experimentally determined odds ratios and discuss the strong impact of data correlations that arise from the directional persistence of sperm swimming.

Behavioral mechanism during human sperm chemotaxis: involvement of hyperactivation.

When mammalian spermatozoa become capacitated they acquire, among other activities, chemotactic responsiveness and the ability to exhibit occasional events of hyperactivated motility--a vigorous motility type with large amplitudes of head displacement. Although a number of roles have been proposed for this type of motility, its function is still obscure. Here we provide evidence suggesting that hyperactivation is part of the chemotactic response. By analyzing tracks of spermatozoa swimming in a spatial chemoattractant gradient we demonstrate that, in such a gradient, the level of hyperactivation events is significantly lower than in proper controls. This suggests that upon sensing an increase in the chemoattractant concentration capacitated cells repress their hyperactivation events and thus maintain their course of swimming toward the chemoattractant. Furthermore, in response to a temporal concentration jump achieved by photorelease of the chemoattractant progesterone from its caged form, the responsive cells exhibited a delayed turn, often accompanied by hyperactivation events or an even more intense response in the form of flagellar arrest. This study suggests that the function of hyperactivation is to cause a rather sharp turn during the chemotactic response of capacitated cells so as to assist them to reorient according to the chemoattractant gradient. On the basis of these results a model for the behavior of spermatozoa responding to a spatial chemoattractant gradient is proposed.

Behavioral response of human spermatozoa to a concentration jump of chemoattractants or intracellular cyclic nucleotides.

BACKGROUND: A major question in mammalian sperm chemotaxis is whether the cells sense a chemoattractant gradient by comparing the chemoattractant concentration between time points or between spatial points. METHODS: To resolve this question, we exposed human spermatozoa to a temporal chemoattractant gradient under conditions of no spatial gradient by rapidly mixing the cells with progesterone or bourgeonal on a microscope slide and analyzing their swimming with motion analysis software. RESULTS: The cells responded within seconds with an increase in velocity and lateral head displacement, and with a decrease in the linearity of swimming, becoming hyperactivated at the peak of the response. All the responses were transient, lasting for a number of seconds. Essentially similar results were obtained upon intracellular photorelease of cyclic adenosine monophosphate or cyclic guanosine monophosphate, which are thought to be involved in mediating the chemotactic response. CONCLUSION: These results suggest that human spermatozoa sense and respond to a temporal chemoattractant gradient. On the basis of these observations, we propose a potential model for the chemotactic response of spermatozoa in a spatial chemoattractant gradient.

Novel function of ovarian growth factors: combined studies by DNA microarray, biochemical and physiological approaches.

Owing to the development of the DNA microarray technique, modulation of gene function can be studied systematically. Considerable attention has been focused on members of the growth factor family to elucidate the main regulators of oocyte maturation and ovarian follicle rupture. Among these growth factors, it was found both in rodents and in humans that amphiregulin (Ar) and epiregulin (Ep) of the epidermal growth factor (EGF) family were dramatically up-regulated by gonadotrophins in the intact ovary and in primary granulosa cells, respectively. Their role in cumulus expansion and oocyte maturation was established in rodents, and their formation under LH stimulation in granulosa cells was demonstrated in humans. To be activated, Ar and Ep must be cleaved by A Disintegrin And Metalloproteinases (ADAMs) family. However, the precise processing of Ar and Ep by the cumulus cells is still obscure. Future investigations using DNA microarray technique may reveal the repertoire of genes activated in Ar- and Ep-stimulated cumulus cells and may help elucidate the molecular basis of ovulation.

Differential expression of genes coding for EGF-like factors and ADAMTS1 following gonadotropin stimulation in normal and transformed human granulosa cells.

We have demonstrated previously that the synthesis of epiregulin and amphiregulin, of the EGF-like growth factor family, is stimulated by luteinizing hormone in human follicular (granulosa) cells obtained from in vitro fertilization program. In the present work, we demonstrate that H89, a PKA inhibitor, attenuated the expression of these growth factors both in the mRNA and the protein levels, suggesting PKA involvement in this signaling pathway. SV40-transformed human granulosa cells showed higher basal levels of epiregulin and amphiregulin than normal cells, which were still elevated following cAMP stimulation by Forskolin. Cleavage by a disintegrin and metalloproteinases (ADAMs) is essential for activation of these growth factors, allowing their interaction with EGF receptor. Expression of ADAMTS1 and ADAM12 was downregulated by cAMP in normal, but not in SV40-transformed cells, suggesting that in normal cells epiregulin and amphiregulin activity is downregulated by a feedback mechanism that may be lost in SV40-transformed cells and their loss of downregulation may be involved in the development of ovarian tumors.