scholarly journals Single-cell roadmap of human gonadal development

2021 ◽  
Author(s):  
Roser Vento-Tormo ◽  
Luz Garcia-Alonso ◽  
Valentina Lorenzi ◽  
Cecilia Mazzeo ◽  
Carmen Sancho-Serra ◽  
...  
Keyword(s):  
Single Cell ◽  
Granulosa Cells ◽  
Mammalian Species ◽  
Supporting Cell ◽  
Gonadal Development ◽  
Chromatin Accessibility ◽  
In Vitro Models ◽  
Second Trimester ◽  
Germ Cell Differentiation

Abstract Gonadal development is a complex process that involves sex determination followed by divergent maturation into ovaries or testes. Historically, limited tissue accessibility and lack of reliable in vitro models have impeded our understanding of human gonadogenesis, despite its importance in gonadal pathologies and infertility. Here, we generated a comprehensive map of first- and second-trimester gonadal development using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and imaging. Using this approach, we identified novel transcription factors and cell states in human germ and supporting cell lineages. We compared them with other mammalian species and found primate-specific regulatory programmes. Our data identified cell context–specific interactions shaping sex specification and development of human germ cells. We defined a novel bipotent progenitor cell (LGR5+, TSPAN8+) in late embryos that can differentiate into early Sertoli in males or pre-granulosa cells in females. In fetal ovaries, we defined two subsets of pre-granulosa cells supporting germ-cell differentiation and distributed across the cortico-medullary axis. We also found a subset of developing granulosa cells appearing during the second trimester of pregnancy that is involved in follicular assembly. In fetal testes, we defined a novel supporting population (sPAX8 cells) located at the poles of the developing testis cords. We also found two tissue-resident myeloid populations that we named microglia-like and SIGLEC15+ fetal testicular macrophages. This study provides an unprecedented spatiotemporal map of human gonadal differentiation that can be utilised as a blueprint for in vitro gametogenesis.

scholarly journals Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karl Olofsson ◽  
Valentina Carannante ◽  
Madoka Takai ◽  
Björn Önfelt ◽  
Martin Wiklund
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Drug Distribution ◽  
In Vitro Models ◽  
Biological Information ◽  
Dimensional Structure ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Nuclear Segmentation

AbstractMulticellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

scholarly journals Mouse Gonad Development in the Absence of the Pro-Ovary Factor WNT4 and the Pro-Testis Factor SOX9

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1103
Author(s):  
Furong Tang ◽  
Nainoa Richardson ◽  
Audrey Albina ◽  
Marie-Christine Chaboissier ◽  
Aitana Perea-Gomez
Keyword(s):  
Granulosa Cells ◽  
Gonad Development ◽  
Supporting Cell ◽  
Gonadal Development ◽  
Ovary Development ◽  
Double Knockout ◽  
Cell Identity ◽  
Knockout Mouse Model ◽  
Steroidogenic Cell ◽  
Male Specific

The transcription factors SRY and SOX9 and RSPO1/WNT4/β-Catenin signaling act as antagonistic pathways to drive testis and ovary development respectively, from a common gonadal primordium in mouse embryos. In this work, we took advantage of a double knockout mouse model to study gonadal development when Sox9 and Wnt4 are both mutated. We show that the XX gonad mutant for Wnt4 or for both Wnt4 and Sox9 develop as ovotestes, demonstrating that ectopic SOX9 function is not required for the partial female-to-male sex reversal caused by a Wnt4 mutation. Sox9 deletion in XY gonads leads to ovarian development accompanied by ectopic WNT/β-catenin signaling. In XY Sox9 mutant gonads, SRY-positive supporting precursors adopt a female-like identity and develop as pre-granulosa-like cells. This phenotype cannot be fully prevented by the deletion of Wnt4 or Rspo1, indicating that SOX9 is required for the early determination of the male supporting cell identity independently of repressing RSPO1/WNT4/β-Catenin signaling. However, in XY Sox9 Wnt4 double mutant gonads, pre-granulosa cells are not maintained, as they prematurely differentiate as mature granulosa cells and then trans-differentiate into Sertoli-like cells. Together, our results reveal the dynamics of the specific and independent actions of SOX9 and WNT4 during gonadal differentiation: SOX9 is essential in the testis for early specification of male-supporting cells whereas WNT4 functions in the ovary to maintain female-supporting cell identity and inhibit male-specific vascular and steroidogenic cell differentiation.

scholarly journals Improved single-cell ATAC-seq reveals chromatin dynamics of in vitro corticogenesis

2019 ◽  
Author(s):  
Ryan M. Mulqueen ◽  
Brooke A. DeRosa ◽  
Casey A. Thornton ◽  
Zeynep Sayar ◽  
Kristof A. Torkenczy ◽  
...  
Keyword(s):  
Single Cell ◽  
Gene Networks ◽  
Regulatory Gene ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Chromatin Dynamics ◽  
Cell Library ◽  
Fate Decision

AbstractDevelopment is a complex process that requires the precise modulation of regulatory gene networks controlled through dynamic changes in the epigenome. Single-cell-omic technologies provide an avenue for understanding the mechanisms of these processes by capturing the progression of epigenetic cell states during the course of cellular differentiation using in vitro or in vivo models1. However, current single-cell epigenomic methods are limited in the information garnered per individual cell, which in turn limits their ability to measure chromatin dynamics and state shifts. Single-cell combinatorial indexing (sci-) has been applied as a strategy for identifying single-cell-omic originating libraries and removes the necessity of single-cell, single-compartment chemistry2. Here, we report an improved sci-assay for transposase accessible chromatin by sequencing (ATAC-seq), which utilizes the small molecule inhibitor Pitstop 2™ (scip-ATAC-seq)3. We demonstrate that these improvements, which theoretically could be applied to any in situ transposition method for single-cell library preparation, significantly increase the ability of transposase to enter the nucleus and generate highly complex single-cell libraries, without altering biological signal. We applied sci-ATAC-seq and scip-ATAC-seq to characterize the chromatin dynamics of developing forebrain-like organoids, an in vitro model of human corticogenesis4. Using these data, we characterized novel putative regulatory elements, compared the epigenome of the organoid model to human cortex data, generated a high-resolution pseudotemporal map of chromatin accessibility through differentiation, and measured epigenomic changes coinciding with a neurogenic fate decision point. Finally, we combined transcription factor motif accessibility with gene activity (GA) scores to directly observe the dynamics of complex regulatory programs that regulate neurogenesis through developmental pseudotime. Overall, scip-ATAC-seq increases information content per cell and bolsters the potential for future single-cell studies into complex developmental processes.

scholarly journals Allele-resolved single-cell multi-omics uncovers the dynamics and transcriptional kinetics of X-chromosome upregulation

2021 ◽  
Author(s):  
Antonio Lentini ◽  
Huaitao Cheng ◽  
Joyce Carol Noble ◽  
Natali Papanicolaou ◽  
Christos Coucoravas ◽  
...  
Keyword(s):  
Single Cell ◽  
X Chromosome ◽  
Dosage Compensation ◽  
Chromatin Accessibility ◽  
Embryonic Cells ◽  
Mouse Development ◽  
Burst Frequency ◽  
Allelic Regulation

X-chromosome inactivation (XCI) and upregulation (XCU) are the major opposing chromosome-wide modes of gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling to finely dissect the separate effects of XCI and XCU on RNA levels during mouse development. We uncover that balanced X dosage is flexibly attained through expression tuning by XCU in a sex- and lineage-specific manner along varying degrees of XCI and across developmental and cellular states. Male blastomeres achieve XCU upon zygotic genome activation while females experience two distinct waves of XCU, upon imprinted- and random XCI, and ablation of Xist impedes female XCU. Contrary to widely established models of mammalian dosage compensation, naïve female embryonic cells carrying two active X chromosomes do not exhibit upregulation but express both alleles at basal level, yet collectively exceeding the RNA output of a single hyperactive allele. We show, in vivo and in vitro, that XCU is kinetically driven by X-specific modulation of transcriptional burst frequency, coinciding with increased compartmentalization of the hyperactive allele. Altogether, our data provide unprecedented insights into the dynamics of mammalian XCU, prompting a revised model of the chain in events of allelic regulation by XCU and XCI in unitedly achieving stable cellular levels of X-chromosome transcripts.

scholarly journals Adult porcine (Sus scrofa) derived inner ear cells possessing multipotent stem/progenitor cell characteristics in in vitro cultures

2021 ◽  
Author(s):  
Printha Wijesinghe ◽  
Anand Sastry ◽  
Elizabeth Hui ◽  
Tristan A. Cogan ◽  
Boyuan Zheng ◽  
...  
Keyword(s):  
Inner Ear ◽  
Sus Scrofa ◽  
Progenitor Cell ◽  
Mammalian Species ◽  
Supporting Cell ◽  
Reproducible Method ◽  
Polymerase Chain ◽  
Regenerative Therapies ◽  
Human Inner Ear

AbstractThe human inner ear compared with that of other mammalian species is very complex. Although the mouse’s cochlea is frequently studied the mouse’s inner ear continues to develop postnatally whilst the human inner ear is fully developed by the third month of gestation which leads one to question the applicability of findings based on research on mice to human regenerative therapies. Here, we report a novel in vitro culture of adult porcine (Sus scrofa) inner ear cells developed from post-mortem labyrinth specimens. Anatomical findings based on maximal transverse and vertical axial diameters and the length of the cochlear duct suggest that the pig’s cochlea is similar to the human cochlea. In vitro cultures of porcine cochlear and vestibular cells showed the persistence of both inner ear hair cell (HC), supporting cell (SC) and stem/progenitor cell characteristics across passages up to 6 based on scanning electron microscopy, fluorescence immunocytochemistry and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings showed that porcine cochlear and vestibular epithelia maintained multipotent stem/progenitor cell populations into adulthood although their regenerative capacities differed across the passages. The development of a viable and reproducible method to culture porcine inner ear cells provides an important investigative tool that can be utilized to study and evaluate the pathophysiological causes and cellular consequences of human inner ear disorders.

Analysis of granulosa cells by single cell PCR—establishment of an in vitro model system

2011 ◽  
Vol 90 (2) ◽  
pp. 176
Author(s):  
J. Pastuschek ◽  
S. Hoelters ◽  
S. Neubeck ◽  
J.S. Fitzgerald ◽  
E. Schleussner ◽  
...  
Keyword(s):  
Single Cell ◽  
Granulosa Cells ◽  
In Vitro Model ◽  
Model System ◽  
Single Cell Pcr ◽  
In Vitro Model System ◽  
Vitro Model

scholarly journals Sex-Specific Isolation and Propagation of Human Premeiotic Fetal Germ Cells and Germ Cell-Like Cells

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1214
Author(s):  
Swati Mishra ◽  
Jasin Taelman ◽  
Yolanda W. Chang ◽  
Annekatrien Boel ◽  
Petra De Sutter ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Cell Sorting ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Gonadal Development ◽  
Surface Markers ◽  
Second Trimester ◽  
Differentiation Efficiency

The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating ‘mitotic’ from ‘retinoic-acid responsive’ female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans.

scholarly journals PAX2+ Mesenchymal Origin of Gonadal Supporting Cells Is Conserved in Birds

2021 ◽  
Vol 9 ◽  
Author(s):  
Martin A. Estermann ◽  
Mylene M. Mariette ◽  
Julie L. M. Moreau ◽  
Alexander N. Combes ◽  
Craig A. Smith
Keyword(s):  
Cell Differentiation ◽  
Sex Differentiation ◽  
Cell Lineage ◽  
Mesenchymal Cell ◽  
Supporting Cell ◽  
Gonadal Development ◽  
Supporting Cells ◽  
Mesenchymal Origin ◽  
Germ Cell Differentiation ◽  
Pax2 Expression

During embryonic gonadal development, the supporting cell lineage is the first cell type to differentiate, giving rise to Sertoli cells in the testis and pre-granulosa cells in the ovary. These cells are thought to direct other gonadal cell lineages down the testis or ovarian pathways, including the germline. Recent research has shown that, in contrast to mouse, chicken gonadal supporting cells derive from a PAX2/OSR1/DMRT1/WNT4 positive mesenchymal cell population. These cells colonize the undifferentiated genital ridge during early gonadogenesis, around the time that germ cells migrate into the gonad. During the process of somatic gonadal sex differentiation, PAX2 expression is down-regulated in embryonic chicken gonads just prior to up-regulation of testis- and ovary-specific markers and prior to germ cell differentiation. Most research on avian gonadal development has focused on the chicken model, and related species from the Galloanserae clade. There is a lack of knowledge on gonadal sex differentiation in other avian lineages. Comparative analysis in birds is required to fully understand the mechanisms of avian sex determination and gonadal differentiation. Here we report the first comparative molecular characterization of gonadal supporting cell differentiation in birds from each of the three main clades, Galloanserae (chicken and quail), Neoaves (zebra finch) and Palaeognathe (emu). Our analysis reveals conservation of PAX2+ expression and a mesenchymal origin of supporting cells in each clade. Moreover, down-regulation of PAX2 expression precisely defines the onset of gonadal sex differentiation in each species. Altogether, these results indicate that gonadal morphogenesis is conserved among the major bird clades.

scholarly journals Single-cell chromatin accessibility profiling of glioblastoma identifies an Invasive cancer stem cell population associated with lower survival

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Paul Guilhamon ◽  
Charles Chesnelong ◽  
Michelle M Kushida ◽  
Ana Nikolic ◽  
Divya Singhal ◽  
...  
Keyword(s):  
Single Cell ◽  
Cancer Progression ◽  
Patient Survival ◽  
Chromatin Accessibility ◽  
Stem Cell Population ◽  
Primary Tumors ◽  
Orthotopic Xenografts

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as Glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

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