A novel xeno-organoid approach: exploring the crosstalk between human iPSC-derived PGC-like and rat testicular cells.

Specification of germ cell-like cells from induced pluripotent stem cells has become a clinically relevant tool for research. Research on initial embryonic processes is often limited by the access to foetal tissue, and in humans, the molecular events resulting in primordial germ cell (PGC) specification and sex determination remain to be elucidated. A deeper understanding of the underlying processes is crucial to describe pathomechanisms leading to impaired reproductive function. Several protocols have been established for the specification of human pluripotent stem cell towards early PGC-like cells (PGCLC), currently representing the best model to mimic early human germline developmental processes in vitro. Further sex determination towards the male lineage depends on somatic gonadal cells providing the necessary molecular cues. By establishing a culture system characterized by the re-organization of somatic cells from postnatal rat testes into cord-like structures and optimizing efficient PGCLC specification protocols, we facilitated the co-culture of human germ cell-like cells within a surrogate testicular microenvironment. Specified conditions allowed the survival of rat somatic testicular and human PGCLCs for 14 days. Human cells maintained the characteristic expression of octamer-binding transcription factor 4, SRY-box transcription factor 17, and transcription factor AP-2 gamma and were recovered from the xeno-organoids by cell sorting. This novel xeno-organoid approach will allow the in vitro exploration of early sex determination of human PGCLCs.

Single-cell gene expression analysis reveals diversity among human spermatogonia.

STUDY QUESTION: Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? SUMMARY ANSWER: Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. WHAT IS KNOWN ALREADY: Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. STUDY DESIGN, SIZE, DURATION: Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of human spermatogonia at the RNA and protein levels is a snapshot. To further assess the functional meaning of this heterogeneity and the dynamics of stem cell populations, approaches need to be developed to facilitate the repeated analysis of individual cells. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the model of a heterogeneous stem cell population. Future studies will assess the dynamics of spermatogonial populations in fertile and infertile patients. LARGE SCALE DATA: RNA-seq data is published in the GEO database: GSE91063. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft DFG-Research Unit FOR 1041 Germ Cell Potential (grant numbers SCHO 340/7-1, SCHL394/11-2). The authors declare that there is no conflict of interest.

How salt lakes affect atmospheric new particle formation: A case study in Western Australia.

New particle formation was studied above salt lakes in-situ using a mobile aerosol chamber set up above the salt crust and organic-enriched layers of seven different salt lakes in Western Australia. This unique setup made it possible to explore the influence of salt lake emissions on atmospheric new particle formation, and to identify interactions of aqueous-phase and gas-phase chemistry. New particle formation was typically observed at enhanced air temperatures and enhanced solar irradiance. Volatile organic compounds were released from the salt lake surfaces, probably from a soil layer enriched in organic compounds from decomposed leaf litter, and accumulated in the chamber air. After oxidation of these organic precursor gases, the reaction products contributed to new particle formation with observed growth rates from 2.7 to 25.4nmh-1. The presence of ferrous and ferric iron and a drop of pH values in the salt lake water just before new particle formation events indicated that organic compounds were also oxidized in the aqueous phase, affecting the new particle formation process in the atmosphere. The contribution of aqueous-phase chemistry to new particle formation is assumed, as a mixture of hundreds of oxidized organic compounds was characterized with several analytical techniques. This chemically diverse composition of the organic aerosol fraction contained sulfur- and nitrogen-containing organic compounds, and halogenated organic compounds. Coarse mode particles were analyzed using electron microscopy, energy dispersive X-ray spectroscopy and Raman spectroscopy. Ultra-high resolution mass spectrometry was applied to analyze filter samples. A targeted mass spectral analysis revealed the formation of organosulfates from monoterpene precursors and two known tracers for secondary organic aerosol formation from atmospheric oxidation of 1,8-cineole, which indicates that a complex interplay of aqueous-phase and gas-phase oxidation of monoterpenes contributes to new particle formation in the investigated salt lake environment.

A combined approach facilitates the reliable detection of human spermatogonia in vitro.

STUDY QUESTION: Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? SUMMARY ANSWER: Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures. WHAT IS KNOWN ALREADY: The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies. STUDY DESIGN, SIZE, DURATION: Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels. MAIN RESULTS AND THE ROLE OF CHANCE: Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures can be maintained for the period of 10 days. However, marker profiling has to be performed at regular time points as the composition of testicular cell types may continuously change under longer term culture conditions. LIMITATIONS, REASONS FOR CAUTION: There are significant differences regarding the spermatogonial stem cell (SSC) system and spermatogenesis between rodents and primates. It is therefore possible that marker genes that do not reflect the presence of spermatogonia in the human are specific for spermatogonia in other animal models. WIDER IMPLICATIONS OF THE FINDINGS: While some studies have reported that human SSCs can be maintained in vitro and show characteristics of pluripotency, the germ cell origin and the differentiation potential of these cells were subsequently called into question. This study provides critical insights into possible sources for the misinterpretation of results regarding the presence of germ cells in human testicular cell cultures and our findings can therefore help to avoid conflicting reports in the future. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by the Stem Cell Network North Rhine-Westphalia and the Innovative Medical Research of the University of Münster Medical School (Grant KO111014). In addition, it was funded by the DFG-Research Unit FOR 1041 Germ Cell Potential (GR 1547/11-1 and SCHL 394/11-2), the BMBF (01GN0809/10) and the IZKF (CRA 03/09). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Remote-sensing-based analysis of landscape change in the desiccated seabed of the Aral Sea--a potential tool for assessing the hazard degree of dust and salt storms.

With the recession of the Aral Sea in Central Asia, once the world's fourth largest lake, a huge new saline desert emerged which is nowadays called the Aralkum. Saline soils in the Aralkum are a major source for dust and salt storms in the region. The aim of this study was to analyze the spatio-temporal land cover change dynamics in the Aralkum and discuss potential implications for the recent and future dust and salt storm activity in the region. MODIS satellite time series were classified from 2000-2008 and change of land cover was quantified. The Aral Sea desiccation accelerated between 2004 and 2008. The area of sandy surfaces and salt soils, which bear the greatest dust and salt storm generation potential increased by more than 36 %. In parts of the Aralkum desalinization of soils was found to take place within 4-8 years. The implication of the ongoing regression of the Aral Sea is that the expansion of saline surfaces will continue. Knowing the spatio-temporal dynamics of both the location and the surface characteristics of the source areas for dust and salt storms allows drawing conclusions about the potential hazard degree of the dust load. The remote-sensing-based land cover assessment presented in this study could be coupled with existing knowledge on the location of source areas for an early estimation of trends in shifting dust composition. Opportunities, limits, and requirements of satellite-based land cover classification and change detection in the Aralkum are discussed.

[Normalisation of screening HASE for 4- to 4½-year-old children]. / Normierung des Screening HASE für 4- bis 4 ½-jährige Kinder.

For reasons of prevention medical examinations of school beginners are brought forward up to 2 years before school enrollment. In Baden-Württemberg HASE is used as screening for risks in the acquisition of language and in learning to read and write. Up to now norms were insufficient for the age-group of 4;0-4;5 years. Based on the results of 3 354 children the norms for this age-group could be recalculated and are now available as percentile ranks, T-scores, and C-scores.

[Does the SETK 3-5 detect clinically diagnosed language impairment?]. / Werden mit dem SETK 3-5 klinisch diagnostizierte Sprachentwicklungsstörungen erfasst?

BACKGROUND: The German language development test for 3- to 5-year-olds (SETK 3-5) as well as its short form, the language screening for pre-school children (SSV), are widely used tests for language impairment in German mother-tongue children. However, data published on validation are sparse. We investigated to what extent those children who demonstrated language impairment in clinical assessment were also detected by the SETK 3-5 and SSV tests. MATERIAL AND METHODS: A group of 201 children aged 4-5 years was tested using the SETK 3-5, in addition to which assessments in phonology, active vocabulary, grammar, receptive language abilities, and the recall of nonsense syllables and sentences were conducted. Correlation analyses were performed and the clinical assessment based on the aforementioned tests was compared to the results of the SETK 3-5 and the SSV. RESULTS: Raw values of the test results were significantly correlated on different levels of correlation. Those of comparable subtests were high. The SETK 3-5 had good specificity but sensitivity of only 71.9%. CONCLUSION: Children with language impairment are not always detected using the SETK 3-5 test. Standardization with a sufficient number of children should be carried out.

Bcar3 is expressed in sertoli cells and germ cells of the developing testis in mice.

We identified Bcar3 in the course of a screen for developmentally regulated genes at early developmental stages in mouse embryos. In this study, we explored the spatio-temporal expression pattern of Bcar3 during the critical time period of sex determination using in situ hybridization, real-time RT-PCR, and immunohistochemistry. We found that Bcar3 is expressed in XY gonads during early stages of gonad development and that BCAR3 localizes to Sertoli cells and germs cells. In addition, we identified a new alternative Bcar3 transcript in which exons 4-7 are deleted. This deletion could result in the generation of a truncated BCAR3 protein lacking functional domains including the SH2 domain. The data presented here suggest that Bcar3 could play a role in gonad development.

Laser secondary neutral mass spectrometry for copper detection in micro-scale biopsies.

Disease progression and clinical diagnostics of a number of hereditable metabolic diseases are determined by organ involvement in disturbed deposition of certain molecules. Current clinical imaging is unable to visualize this maldistribution with sufficient specificity and sensitivity, such as in Wilson's disease. The quest for understanding cellular Cu distribution in these patients requires element- and molecule-specific images with nanometer-scale spatial resolution. We have used a new cryo-mass spectrometric instrument with an integrated cryosectioning chamber for preparation and analysis of frozen hydrated samples of Wilson's disease tissue. With laser post-ionization secondary neutral mass spectrometry (laser-SNMS), we were able to image Cu and other intrinsic elements and molecules in less than 1 mg of frozen hydrated liver tissue from a murine model of Wilson's disease. A 40-50 times higher Cu concentration was measured in the disease tissue as compared to the control mouse. Furthermore, major histomorphological changes were observed using this advanced nano-science tool. The results showed that the combination of in-vacuum cryosectioning and cryo-laser-SNMS technologies is particularly well suited for identifying specific cell structures and imaging trace element concentrations with subcellular resolution and upper-parts-per-billion sensitivity in biological samples. This technology can provide a novel diagnostic tool for clinical applications in various diseases involving trace elements.

Distinct populations of tumor-initiating cells derived from a tumor generated by rat mammary cancer stem cells.

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo. We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/(WAF1) and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased alpha-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63, was identified as a key gene regulating the transition between CSCs and early progenitor cells.

[A breakthrough in stem cell research? Reprogramming somatic cells into pluripotent stem cells]. / Durchbruch in der Stammzellforschung? Die Reprogrammierung von Körperzellen zu pluripotenten Stammzellen. Ubersicht und Ausblick.

Embryonic stem (ES) cells are capable of generating all cell types and tissues of the body. As such they represent an attractive source for therapeutic approaches. However, transplanted cells may be rejected by the immune system. One way to address this problem is to generate patient-specific ES cells. This, however, requires the transformation of the genetic program of somatic cells back to that of an early embryonic state. The field of stem cell research and reprogramming is rapidly evolving. This article aims at providing background information to understand some of the most exciting recent developments. Subsequently, the different existing strategies of converting somatic cells into ES-like cells are reviewed and evaluated.

[Validated diagnostics for dysgrammatism]. / Validierte Diagnostik des Dysgrammatismus.

BACKGROUND: Non-standardized procedures are used to evaluate, in particular, grammatical performance in most German institutions performing diagnostic procedures on children with impaired speech and language development. This makes a comparison of results difficult. METHODS: We studied 181 boys and 72 girls aged between 5 and 6 years using four subtests of IDIS additionally to the routine procedure. Results were compared to the "degree of dysgrammatism" determined from the traditional evaluation based on expert rating. RESULTS: The new procedure is able to divide the children into groups with normal speech and language ability, with deficits accessible to a traditional logopedic treatment, and with severe speech and language impairment that necessitates intensive treatment. DISCUSSION: The proposed tests allow an accurate evaluation of grammatical performance instead of subjective estimates.

Cell-cell fusion as a means to establish pluripotency.

Embryonic stem cells (ESCs), embryonic germ cells (EGCs), and embryonic carcinoma cells (ECCs) are three types of pluripotent cells derived from mammalian embryos. The three cell types are capable not only of self-renewal, but also of having the potential to give rise to cells of all tissue types in the fetal and adult body. In several reports, ESCs, ECCs, and EGCs have been described to reprogram somatic cells in vitro. After reprogramming caused by fusion, somatic cells exhibit various features of pluripotent cells: expression of pluripotency markers (e.g., Oct4, nanog, and Rex-1), absence of tissue-specific gene expression, reactivation of inactive X chromosome of female somatic cells, demethylation, as well as histone modification. An activity in pluripotent stem cells appears to be capable of inducing the global changes inherent in the reprogramming of somatic cells. Investigations involving pluripotent stem cells will yield substantial insight into various fundamental biological processes, such as cellular differentiation and de-differentiation. Most importantly for the public, however, is that such studies might lead into cell-based therapies and as such have the potential to change regenerative medicine.

Derivation of germ cells from embryonic stem cells.

Embryonic stem cells (ESCs), derivatives of cells of early mammalian embryos, have proven to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germline. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. Amazingly, recently it has been shown that mouse ESCs can also give rise to primordial germ cells (PGCs) in culture that are capable of undergoing meiosis and forming both male and female gametes. While the full potential of these ES-derived germ cells and gametes remains to be demonstrated, these discoveries provide a new approach for studying reproductive biology and medicine.

[Are the language skills of our children declining? Results based on the examinations at school enrollment of the years 1999 to 2004 in Münster]. / Werden die Sprachleistungen unserer Kinder immer schwächer? Beobachtungen an sechs Einschulungsjahrgängen in Münster.

The physical examinations at school enrollment of the years 1999 to 2004 were subjected to comparative analysis to help clarify whether children's language performance at enrollment has significantly decreased in recent years or not. This question has indeed been a matter of public debate for some time. Furthermore, potential influencing factors, in particular social environment, were to be examined as regards their effects on language performance. The results indicate that the children's performance level has not significantly decreased over the six-year period of observation. Instead, rather an increase can be observed over this period of time. If, nevertheless, more and more and, in particular, more significant language deficits continue to be observed, one explanation for this may be seen in a changing assessment of children's needs for therapy or remedial education. The performance level of children who were recommended therapy or means of remedial education in 2004 is significantly higher than that of children for whom such recommendations were given in 1999 or 2000. Perhaps an increased sensitivity for those performance areas where performance levels are found to be deficient should be postulated, and, consequently, a more differentiated assessment of diagnostic findings should be assumed. The results of the examinations once again demonstrate the enormous influence that social factors have on language performance. Children growing up in a less advantaged social environment tend to achieve significantly lower performance marks in language tests than children from more privileged social environments. Also the duration of kindergarten education appears to be relevant to children's language performance: children who have visited institutions of preschool-education for three or more years achieve higher performance marks than children who have spent no ore only little time in these institutions. When seen against the background of circa one in four children with a migration background (around 8% of persons of the same age in Münster's population) having unsatisfactory knowledge of German at school enrollment, there appear to be efficient prevention measures available here.

Recombinant human albumin supports development of somatic cell nuclear transfer embryos in mice: toward the establishment of a chemically defined cloning protocol.

Culturing embryos in different media is a useful approach to characterize their nature in regard to "memory" of the donor nucleus and its "reprogramming" after somatic cell nuclear transfer (SCNT). However, efforts to elucidate the mechanisms of reprogramming are seriously undermined when embryo culture conditions are not completely defined. Using recombinant human albumin (rHA) is a step toward establishing defined culture conditions for mouse cloning. Recombinant HA supports blastocyst formation of cumulus cell-derived clones at a rate comparable with two types of bovine serum albumin (BSA); following transfer of blastocysts to the genital tract, rates of development to midgestation (10.5 dpc) were indistinguishable. rHA also supports the derivation of germline competent embryonic stem (ES) cells from SCNT blastocysts at a substantial rate compared with BSA counterparts and with zygotic blastocysts. Unlike the developmental parameters, the gene expression patterns of clones cultured in rHA or BSA were not superimposed; identical patterns were observed for zygotic blastocysts in the two albumins. In summary, the present study demonstrates that (1) rHA can replace BSA, proving a defined protein source for SCNT in mice; (2) although using rHA is similar to BSA, it is not equal (rHA leaves a mark on gene expression of clones but not zygotes). Future studies that investigate reprogramming after SCNT will need to consider not only the implications of culture media for cloning but also the supplement choice.

[Diagnosis and differentiation of children with language development disorders. What role can be attributed to intelligence?]. / Diagnostik und Differenzierung sprachentwicklungsgestörter Kinder? Welche Rolle spielt das Merkmal Intelligenz?

BACKGROUND: Specific language impairment (SLI) is defined as a developmental disorder in which language comprehension and the child's ability to use expressive spoken language is markedly below the appropriate level for his or her mental age. The intelligence of SLI children is in the normal range, while their language abilities are impaired. "Normal intelligence", the defining feature of SLI is questioned in this study. PATIENTS AND METHODS: Using IDIS (an inventory of diagnostic information in language impairment), we examined 138 children aged 5 and 6 years with severe language impairment; 108 SLI and 30 LI children. Various indicators of speech and language such as articulation, the ability to discriminate sounds, lexicon, grammar and pragmatic abilities but also auditory and visual perception, auditory and visual memory, fine and gross motor function were assessed. RESULTS: The performance of the SLI children was significantly higher in most of the tests than that of the LI children. Factor analysis showed that the two groups also differed in the structure of performance. Auditory short-term memory was reduced in most children irrespective of intelligence. CONCLUSIONS: We propose the retention of the differentiation of subgroups of developmental speech and language disorders depending on the level of intelligence.

Assessment of interfacial mass transfer in water-unsaturated soils during vapor extraction.

This paper presents results of a numerical investigation of soil vapor extraction (SVE) systems at the laboratory scale. The SVE technique is used to remove volatile chlorinated hydrocarbons (VCHC) from the water-unsaturated soil zone. The developed numerical model solves equations of flow, transport and interfacial mass transfer regarding an isothermal n-component and three-phase system. The mathematical model is based on a simple pore network and phase distribution model and designed to be scaled by a characteristic length. All mathematical expressions are structured into VCHC specific and VCHC non-specific parameters. Furthermore, indicators are introduced that help to separate thermodynamic equilibrium from thermodynamic non-equilibrium domains and to determine the controlling physical parameters. For numerical solution, the system of partial differential equations is discretized by a finite volume method and an implicit Euler time stepping scheme. Computational effort is reduced notably through techniques that enable spatial and temporal adaptivity, through a standard multigrid method as well as through a problem-oriented sparse-matrix storage concept. Computations are carried out in two dimensions regarding the laboratory experiment of Fischer et al. [Water Resour. Res. 32 (12) 1996 3413]. By varying the characteristic length scale of the pore network and phase distribution model, it is shown that the experimental gas phase concentrations cannot be explained only by the volatility and diffusivity of the VCHC. The computational results suggest a sorption process whose significance grows with the aqueous activity of the less or non-polar organic compounds.