Reproduction Network

Reproduction Network - Overview

A comprehensive cellular anatomy of normal human prostate is essential for solving the cellular origins of benign prostatic hyperplasia and prostate cancer. The tools used to analyze the contribution of individual cell types are not robust. We provide a cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single-cell RNA sequencing (scRNA-seq) and flow cytometry on ∼98,000 cells taken from different anatomical regions. Immunohistochemistry with newly derived cell type-specific markers revealed the distribution of each epithelial and stromal cell type on whole mounts, revising our understanding of zonal anatomy. Based on discovered cell surface markers, flow cytometry antibody panels were designed to improve the purification of each cell type, with each gate confirmed by scRNA-seq. The molecular classification, anatomical distribution, and purification tools for each cell type in the human prostate create a powerful resource for experimental design in human prostate disease.

A Cellular Anatomy of the Normal Adult Human Prostate and Prostatic Urethra

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

A human cell atlas of fetal gene expression.

Deconvolution of regulatory mechanisms that drive transcriptional programs in cancer cells is key to understanding tumor biology. Herein, we present matched transcriptome (scRNA-seq) and chromatin accessibility (scATAC-seq) profiles at single-cell resolution from human ovarian and endometrial tumors processed immediately following surgical resection. This dataset reveals the complex cellular heterogeneity of these tumors and enabled us to quantitatively link variation in chromatin accessibility to gene expression. We show that malignant cells acquire previously unannotated regulatory elements to drive hallmark cancer pathways. Moreover, malignant cells from within the same patients show substantial variation in chromatin accessibility linked to transcriptional output, highlighting the importance of intratumoral heterogeneity. Finally, we infer the malignant cell type-specific activity of transcription factors. By defining the regulatory logic of cancer cells, this work reveals an important reliance on oncogenic regulatory elements and highlights the ability of matched scRNA-seq/scATAC-seq to uncover clinically relevant mechanisms of tumorigenesis in gynecologic cancers.

A multi-omic single-cell landscape of human gynecologic malignancies

Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here, single cell RNA-sequencing was used to construct an atlas of the human proximal epididymis.

A single cell atlas of the human proximal epididymis reveals cell-type specific functions and distinct roles for CFTR

The authors developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq.

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors.

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for approximately 1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems. Overall design: Applying single cell chromatin accessibility assays to diverse human tissue types from four donors.

A single-cell atlas of chromatin accessibility in the human genome

The placenta and decidua interact dynamically to enable embryonic and fetal development. Here, we report single-cell RNA sequencing of 14,341 and 6754 cells from first-trimester human placental villous and decidual tissues, respectively. Bioinformatic analysis identified major cell types, many known and some subtypes previously unknown in placental villi and decidual context. Further detailed analysis revealed proliferating subpopulations, enrichment of cell type–specific transcription factors, and putative intercellular communication in the fetomaternal microenvironment. This study provides a blueprint to further the understanding of the roles of these cells in the placenta and decidua for maintenance of early gestation as well as pathogenesis in pregnancy-related disorders.

A single-cell survey of the human first-trimester placenta and decidua

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing

Fallopian tube (FT) homeostasis requires dynamic regulation of heterogeneous cell populations and is disrupted in infertility and ovarian cancer. Here we applied single-cell RNAseq to profile 59,738 FT cells from 4 healthy pre-menopausal subjects. The resulting cell atlas contains 12 major cell types representing epithelial, stromal and immune compartments.  Re-clustering of epithelial cells identified 4 ciliated and 6 non-ciliated secretory epithelial subtypes, two of which represent potential progenitor pools: one leading to mature secretory cells, while the other contributing to either ciliated cells or one of the stromal cell types. To understand how FT cell numbers and states change in a disease state, we analyzed 17,798 cells from two hydrosalpinx samples and observed shifts in epithelial and stromal populations, and cell type-specific changes in extracellular matrix and TGF- signaling, underscoring fibrosis pathophysiology. This resource is expected to facilitate future studies to understand fallopian tube homeostasis in normal development and disease.

Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified by scRNA-seq

4 independent pools of iPS cells were differentiated and used to generate kidney organoids. Each pool was used to generate each of the 3 types of conformations: Manual organoids, bioprinted organoids composed of a single deposition of cells (R0) and bioprinted organoids composed a thin line of cells (R40). Manual and two types of bioprinted kidney organoids were analysed by single cell RNA-seq.

Comparison of manual and two types of bioprinted kidney organoids by single cell RNA-seq.

Single cell transcriptome analysis of a cancer tissue can provide objective assessment of subtype population or the activation of each of various microenvironment component cells. In this study, we applied our newly developed technique of single cell analysis to the myometrial infiltration side (M-side) and the endometrial side (E-side) of a human endometrioid adenocarcinoma with squamous differentiation tissues. We also analyzed spherogenic cultures derived from the same tissue to identify putative regulators of stemness in vivo. Cancer cells in the E-side were highly malignant compared with those in the M-side. Many cells on the E-side were positive for spheroid-specific tumorigenesis-related markers including SOX2. In addition, there were higher numbers of epithelial-to-mesenchymal transition (EMT) cells in the E-side compared with the M-side. This study identified a site containing cells with high malignant potential such as EMT and cancer stem-like cells in cancer tissues. Finally, we demonstrate that established endometrioid adenocarcinoma subtype classifiers were variably expressed across individual cells within a tumor. Thus, such intratumoral heterogeneity may be related to prognostic implications.

Comprehensive single-cell transcriptome analysis reveals heterogeneity in endometrioid adenocarcinoma tissues

Single-cell RNA sequencing was used to determine the cell type composition of all major human organs and construct a basic scheme for the human cell landscape (HCL).

Construction of A Human Cell Landscape by Single-cell mRNA-seq

X chromosome dosage compensation is one of the most important epigenetic events in female mammalian development. Although the mouse has served as the primary model to study how genetic equality is established between male and female mammals, it is now appreciated that these mechanisms are not fully conserved in humans. Using human primordial germ cells (hPGCs) as a model together with single cell RNA-sequencing, in vitro differentiation of hPGC like cells and human embryo attachment culture, we show that X-linked gene expression in female hPGCs is compensated to levels found in female somatic cells though a mechanism related to X chromosome dampening rather than X chromosome inactivation. Loss of dampening and decompensation of both X chromosomes occurs when female hPGCs enter meiosis and is coincident with loss of the long non-coding RNAs XIST and XACT. In contrast, differentiation of male hPGCs results in no change in compensation despite loss of XACT. Taken together, future studies of X-linked gene regulation and function in human germ cell development and fertility must take into account the unique and sexually dimorphic dosage compensation occurring during in the prenatal human germline prior to birth. Overall design: In our work, we performed single cell RNA-sequencing (scRNA-seq) with 10x Genomics on 5 different female human prenatal ovaries from 5 different developmental timepoints: Week 7, 9, 10, 13 and 16 post fertilization. In total, we sequenced ~25,000 gonadal somatic and germ cells to uncover X chromosome dynamics during female germline development.

Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation [Female]

X chromosome dosage compensation is one of the most important epigenetic events in female mammalian development. Although the mouse has served as the primary model to study how genetic equality is established between male and female mammals, it is now appreciated that these mechanisms are not fully conserved in humans. Using human primordial germ cells (hPGCs) as a model together with single cell RNA-sequencing, in vitro differentiation of hPGC like cells and human embryo attachment culture, we show that X-linked gene expression in female hPGCs is compensated to levels found in female somatic cells though a mechanism related to X chromosome dampening rather than X chromosome inactivation. Loss of dampening and decompensation of both X chromosomes occurs when female hPGCs enter meiosis and is coincident with loss of the long non-coding RNAs XIST and XACT. In contrast, differentiation of male hPGCs results in no change in compensation despite loss of XACT. Taken together, future studies of X-linked gene regulation and function in human germ cell development and fertility must take into account the unique and sexually dimorphic dosage compensation occurring during in the prenatal human germline prior to birth. Overall design: In our work, we performed single cell RNA-sequencing (scRNA-seq) with 10x Genomics on 6 different male human prenatal testes from 6 different developmental timepoints: Week 6, 7, 8, 12, 15 and 16 post fertilization. In total, we sequenced ~30,000 gonadal somatic and germ cells to uncover X chromosome dynamics during female and male germline development.

Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation [Male]

Human adult spermatogonial stem cells (hSSCs) must balance self-renewal and differentiation. To understand how this is achieved, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4+ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4+ hSSCs and differentiating c-KIT+ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic “poising” in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation.

Genomic profiling of human spermatogonial stem cells

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and 3D endometrial organoid cultures. We dissect the signalling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro . We show in vitro that downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilise our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for a range of common conditions including endometriosis and endometrial carcinoma.

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids.

Here we demonstrate single-cell RNA-5′end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay at no extra cost. We showed enhancer RNAs can be detected using sc-end5-seq methods with either random or oligo(dT) priming.

Profiling of transcribed cis-regulatory elements in single cells.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast–decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand–receptor complexes and a statistical tool to predict the cell-type specificity of cell–cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal–fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics

We measured 720 HeLa-CCL2 cells (288 from the 9th passage, 288 from the 14th passage and 144 from the 20th passage) in this work on an Illumina HiSeq 4000 platform (sequenced by Novogene, China) for 150 bp paired-end sequencing. The protocol was based on Tang’s modification on SMART-Seq2. Single cells were captured by mouth pipette under the stereomicroscope（Nikon SZM745）and followed by the protocol. Every cell was assigned with 250 M raw data on average. 

Single cell RNA-seq of HeLa CCL2

Endometriosis is characterized by growth of endometrial-like tissue outside of the uterus affecting many women during their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, limiting early diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared to matched eutopic endometrium, control endometrium, and organoids derived from these tissues, generating data on over 122,000 cells across 14 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell unique to the peritoneal lesions with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesions microenvironments, and an unreported progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment representing a comprehensive cell atlas of the disease in hormonally treated patients, essential information for advancing therapeutics and diagnostics. Overall design: Biopsies were collected from a total of 27 patients, 19 with confirmed endometriosis and 8 patients control. Biopsies were subjected to single cell experiments, bulk sequencing and cell hashing on patient-derived organoid. Single cells experiement were conducted on 31 biopsies consisting of 3 control and 9 eutopic endometrium, 8 ectopic peritoneal, 6 ectopic peritoneal adjacent, 4 ectopic ovary of endometriosis patient. A total of 24 biopsies were subjected to bulk sequencing, consist of 5 control and 7 eutopic endometrium, 6 ectopic peritoneal, 6 ectopic ovary of endometriosis patient. Single-cell cell hashing experiment were conducted on patient-derived organoid from 2 control and 4 eutopic endometrium, 4 ectopic peritoneum, 1 ectopic peritoneal adjacent of endometriosis patient. All single cell experiments were performed on 10x Genomics platform.

Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues

Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here, we performed single-cell RNA-seq analysis of over 2000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that the female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis and cell cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of BMP and Notch signaling pathways. Our work provides key insights into crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo.

Single-Cell RNA-Seq Analysis Maps Development of Human Germline Cells and Gonadal- Niche Interactions

Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm of reproductive age women which causes cystic lung remodeling and progressive respiratory failure. While LAM lesions are known to contain abnormal smooth muscle-like cells which harbor mTOR activating mutations in TSC1 or TSC2, the tissue origins of the mutant “LAM cells” that invade the lung remain unclear. By employing single cell and single nuclear RNA sequencing on explanted LAM lungs, we identified a unique population of cells and associated signature genes and gene networks which were readily distinguished from those of endogenous lung cells. These unique LAMCORE cells shared closest transcriptomic similarity to normal uterus and share transcriptomic features with neural crest, as identified in uterine LAM lesions by single nuclear RNA-seq. Immunofluorescence microscopy demonstrated the expression of LAMCORE cell signature genes within LAM lesions in both lung and uterus. Serum aptamer proteomics and ELISA identified biomarkers consistent with the signature genes expressed and predicted to be secreted by LAMCORE cells. Single cell transcriptomics strongly supports a uterine neural crest origin of LAMCORE cells; providing insights into disease pathogenesis and informing future treatment strategies for LAM. Overall design: scRNA-seq and snRNA-seq was performed using the 10x Chromium platform on four lung explants from LAM patients undergoing lung transplantation. We also performed scRNA-seq on a renal AML resected from a patient with a sporadic AML (S-AML), and snRNA-seq on uterine tissue obtained at hysterectomy from an S-LAM patient. As controls, we conducted scRNA-seq on a 31 years old female explanted lung, brain dead, beating-heart, organ donor and snRNA-seq on one normal uterine tissue at hysterectomy from a 29 years old female patient with cervical cancer, and obtained scRNA-seq data of six additional female donor lungs from Gene Expression Omnibus (GSE122960) and scRNA-seq data from normal mouse uterus (GSE118180). qRT-PCR, immunofluorescence and immunochemical stains to validate the spatial relationships of LAM cells and LAMCORE signature genes.Serum aptamer proteomics and ELISA assays screening were used for validation of secretome predictions.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell

Maternal fetal immune tolerance is a biological event that plays a key role in pregnancy. In particular, the mechanism by which the maternal immune system does not exclude the semi-identical antigen of the fetus has not been fully answered so far. The most serious result of the maternal fetal immune tolerance balance is the recurrent spontaneous abortion (RSA). The fetus is rejected by the mother in the early pregnancy and cannot continue to develop in the uterus. Based on these scientific questions, we used single-cell sequencing technology to analyze nearly 30,000 leukocytes in the decidua of normal and RSA. A total of 13 immune cell subsets were found, of which dNK cells were more prominent, The lineage differentiation pathway of dNK cells was excavated, and the pathological role of dNK differentiation abnormality in the occurrence of RSA was analyzed. Our results deepen our understanding of the differentiation and function of human decidual leukocytes, especially dNK cells. We clearly recognize that the immune system has dynamic changes compared with normal pregnancy under pathological conditions, which is likely to occur in RSA. The important factors, we expect our results to provide a new basis for RSA's immune diagnosis and treatment strategy. Overall design: Single cell sequencing on decidual immune cellss was performed from normmal pregnancy women and recurrent spontaneous abortion (RSA) patients. CD45+ leukocytes were sorted from decidua at early pregnancy period through MoFLo XDP.

Single-cell RNA-seq to decipher the subpopulations of human decidual leukocytes in normal and RSA pregnancies

Cervical cancer (CC) is the forth leading cause of deaths in gynecological tumor. Although the etiology of CC has been extensively investigated, the exact pathogenesis of CC remains incomplete. Therefore, we applied single-cell RNA sequencing on cancer tissue and normal adjacent tissue from a patient with CC. Together, we identified four tumor cell subpopulations in tumor cells, which had specific signature genes with different biological functions and presented different prognosis. Among them, we identified a subset of cancer stem cell (CSCs), which was related to the developmental hierarchy of tumor progression. These data facilitate the understanding of the CC landscape and provides deeper insights into the development of prognostic biomarkers and therapeutic targets in the CC.

Single-cell RNA-sequencing reveals the landscape of cervical cancer heterogeneity

Unhealthy aging of testis seriously affects fertility and life quality of older men, while its interventions depend on in-depth knowledge of the molecular and functional changes of various testicular cell types. Here, we profile human testicular single-cell transcriptomes from young adult, healthy old men and late-onset hypogonadism (LOH) patients, and identified the somatic cells underwent a greater change than germ cells.

Single-cell Transcriptome Atlas of Testis Aging

Three normal tissue samples were obtained from the tumour margin of penile carcinoma resection, all patients reported good stimulated erections and early morning erections. The five ED CC tissues were obtained from biopsy samples in artificial cavernous body implantation, all the ED patients were diagnosed with organic ED rather than psychological ED by the nocturnal penile tumescence (NPT) and intracavernosal injection (ICI) tests. The DMED patients had a at least 10-year history of type 1 diabetes and had good blood glucose control before surgery.

Single-cell Transcriptome Atlas of the Human Corpus Cavernosum

The ovary is perhaps the most dynamic organ in the human body, only rivaled by the uterus. The molecular mechanisms that regulate follicular growth and regression, ensuring ovarian tissue homeostasis, remain elusive. We have performed single-cell RNA-sequencing using human adult ovaries to provide a map of the molecular signature of growing and regressing follicular populations. We have identified different types of granulosa and theca cells and detected local production of components of the complement system by (atretic) theca cells and stromal cells. We also have detected a mixture of adaptive and innate immune cells, as well as several types of endothelial and smooth muscle cells to aid the remodeling process. Our results highlight the relevance of mapping whole adult organs at the single-cell level and reflect ongoing efforts to map the human body. The association between complement system and follicular remodeling may provide key insights in reproductive biology and (in)fertility.

Single-cell reconstruction of follicular remodeling in the human adult ovary

Study questionWhat are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing?Summary answerWe discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues.What is known alreadyPrevious studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues.Study design, size, durationWe collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas.Participants/materials, setting, methodsWe collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas.Main results and role of chanceOur data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas.Large scale dataThe datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122.Limitations, reasons for cautionOur study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles.Wider implications for the findingsOur study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas.Study funding/competing interest(s)This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Single-cell sequencing reveals novel cellular heterogeneity in uterine leiomyomas.

Anterior vaginal prolapse, as the most common form of POP, severely affect women’s physical and mental health. However, the cellular profiles of vaginal wall and molecular mechanism in pathological process of POP remain unclear. Here, we built the first single-cell transcriptomic atlas of prolapsed vaginal wall. We further revealed prolapse induced aberrant gene expression and transcriptional regulatory networks in cell-type specific manner in POP. Besides extracellular matrix dysregulation, dysfunction of immune cells and immune response were involved with prolapse occur. Computational prediction demonstrated that the abnormal cell-cell communication patterns present among fibroblasts, smooth muscle cells and macrophages in POP, with interplay in extracellular matrix remodeling and regulation of immune reaction. Taken together, our work provides the ﬁrst comprehensive single-cell transcriptomic atlas for deciphering gene expression landscapes of heterogeneous cell types in prolapsed anterior vaginal wall, broadens our understanding of cell identities and cell-type-speciﬁc gene changes in POP and demonstrated the vital role of enhanced interplay between non-immune cells and immune cells in prolapsed vaginal wall. Overall design: scRNA-seq analysis of vaginal wall tissue in pelvic organ prolapse and control sample

Single-cell transcriptome profiling of the vaginal wall in women with severe anterior vaginal prolapse

To unbiasedly and systematically characterize endometrial transformation across the human menstrual cycle in preparation for embryo implantation, we analyzed the transcriptomic transformation of human endometrium at single cell resolution, dissecting multidimensional cellular heterogeneity of the tissue across the entire natural menstrual cycle.  We collected single cell transcriptome profile of endometrium biopsies sampled from 19 healthy and fertile females, at 4-27 days of their menstrual cycle.  10x dataset - collected to validate main findings drawn from the original C1 dataset of this study. In this dataset, we collected additional data from 10 endometrial biopsies using the 10x Chromium system. Importantly, we collected both C1 and 10x data for two biopsies, one from mid-secretory phase and the other early-secretory. They served as anchors for a direct comparison between the two datasets, and were referred as 'anchor biopsies' in the manuscript of this study. Biopsies in the 10x dataset were obtained following the same protocol used for the C1 dataset, as described in the manuscript.

Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function.

The relationship between the human placenta—the extraembryonic organ made by the fetus, and the decidua—the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal–fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids and trophoblast stem cells. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell–cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.

Spatial multiomics map of trophoblast development in early pregnancy

In recent years there has been tremendous progress towards deep molecular characterization of cell types using single cell transcriptome sequencing. Here we report a single cell transcriptomic atlas comprising nearly 500,000 cells from 24 different human tissues and organs. In several instances multiple organs were analyzed from the same donor. Analyzing organs from the same individual controls for genetic background, age, environment, and epigenetic effects, and enables a detailed comparison of cell types that are shared between tissues. This resource provides a rich molecular characterization of more than 400 cell types, their distribution across tissues, and detailed information about tissue specific variation in gene expression. We have used the fact that multiple tissues came from the same donor to study the clonal distribution of T cells between tissues, to understand the tissue specific mutation rate in B cells, and to analyze the cell cycle state and proliferative potential of shared cell types across tissues. Finally, we have also used this data to characterize cell type specific RNA splicing and how such splicing varies across tissues within an individual.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans

Rhabdomyosarcoma (RMS) is the most soft tissue sarcoma of childhood and histologically mimics developing skeletal muscle. To better understand RMS, we performed single-cell RNA-seq of one embryonal subtype RMS (ERMS) and one alveolar subtype RMS (ARMS). We expanded our observations by performing single-nucleus RNA-seq of 18 patient tumors (12 ERMS, 6 ARMS). All 18 of these tumors were used to generate orthotopic patient-derived xenografts, and we performed single-cell RNA-seq of all 18 O-PDXs to compare heterogeneity between the original tumor and the patient-derived model. 15 of the O-PDXs underwent lentiviral barcoding, and lineage could be tracked using a separate dial-out PCR step during the single-cell RNA-seq workflow. We performed single-cell RNA-seq of an ex vivo organoid, showing that we can preserve heterogeneity in this model. We expanded our study to the epigenetic level by using single-cell ATAC-sequencing of 7 O-PDXs. Finally, we longitudinally tracked shifts in heterogeneity in an O-PDX model treated with chemotherapy. Overall design: We performed single-cell RNA-seq from 2 patient RMS tumors, 18 O-PDXs, and 1 organoid. Additionally, we performed single-nucleus RNA-seq from 18 patient RMS tumors and single-cell ATAC-seq from 7 O-PDXs. We also provide dialout PCR data from 15 RMS O-PDX that were used for lentiviral barcode experiments to track clonal lineages within O-PDXs.

The myogenesis program drives clonal selection and drug resistance in rhabdomyosarcoma

The ovary is the first organ to age in the human body, affecting both fertility and overall health in women. However, the biological mechanisms underlying human ovarian ageing remain poorly understood. Here we performed single-cell multi-omics analysis of young and reproductively aged human ovaries to understand the molecular and cellular basis of ovarian ageing in humans. Our analysis reveals coordinated changes in transcriptomic output and chromatin accessibility across cell types during ageing, including elevated mTOR and MAPK signaling, decreased activity of the oxidative phosphorylation and DNA damage repair pathways, and an increased signature of cellular senescence. By constructing cell type-specific regulatory networks, we uncover enhanced activity of the transcription factor CEBPD across cell types in the aged ovary, with a corresponding significant loss of activity of most cell identity-associated transcription factors. Moreover, by performing integrative analyses of our single-nuclei multi-omics data with common genetic variants associated with age at natural menopause (ANM) from genome-wide association studies, we demonstrate a global impact of functional variants on changes in gene regulatory networks across ovarian cell types. Finally, we nominate about a dozen of functional non-coding variants, their target genes and cell types and regulatory mechanisms that underlie genetic association with ANM. This work provides a comprehensive multimodal landscape of human ovarian ageing and mechanistic insights into inherited variation of ANM.

The regulatory landscapes of human ovarian ageing

Primary human endometrial stromal cells (HESCs) from three patients and the Telomerase-immortalized endometrial stromal cell line (THESC) were cultured in the cluture medium and collected for scRNA-Seq. 13 distinct cell clusters were identified among all the cells, which not only reflect the differences between the four samples, but also the heterogeneity within each sample divided into mature, proliferative and active fibroblast. The conserved transcriptomic changes from the three primary HESC were observed in comparison to the THESCs. Cell clustering with batch correction further indicate that the intrinsic cellular distribution were similar among all the primary HESCs and THESCs. These intrinsic cell clustering showed varaiable stages of cell cycle and a cell development lineage between proliferative and mature or active fibroblast. Overall design: Primary human endometrial stromal cells (HESCs) from three patients and the Telomerase-immortalized endometrial stromal cell line (THESC) were cultured in the culture medium and collected for scRNA-Seq.

The single cell atlas of human endometrial stromal cells

The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remains unknown. Using RNA-Seq analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in-vivo and restoring fully competent oocytes in-vitro.

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