Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells.

STUDY QUESTION: Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype? SUMMARY ANSWER: Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features. WHAT IS KNOWN ALREADY: So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture. STUDY DESIGN, SIZE, DURATION: The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives. MAIN RESULTS AND THE ROLE OF CHANCE: Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS. LIMITATIONS, REASONS FOR CAUTION: Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments. WIDER IMPLICATIONS OF THE FINDINGS: As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI 'escapee' genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2-388/2016-40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.

Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States.

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.

Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells.

The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo.

Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.

Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.

Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells.

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.

Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Comparison of biomaterials and extracellular matrices as a culture platform for multiple, independently derived human embryonic stem cell lines.

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins, human and animal sera matrices, and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media, human foreskin fibroblast-conditioned culture medium, chemically defined medium, TeSR1, and modified TeSR1 media. The results showed the undefined, xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins, serum matrices, and the biomaterials tested. A long-term, feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium, but a xeno-free, fully defined, and reproducible feeder cell-free hESC culture method still remains to be developed.

Preparation of human foreskin fibroblasts for human embryonic stem cell culture.

INTRODUCTIONIn order to reduce the risk of immune rejection and zoonosis in human embryonic stem cell (hESC) transplantation recipients, animal-derived components, nonhuman sera, and animal feeder cells need to be removed from hESC culture. To limit the use of animal products, several different human feeder cell types have been tested for hESC culture. These include cells from fetal muscle and skin, as well as adult fallopian tube epithelial cells and adult muscle cells. Human foreskin fibroblasts (hFFs) have been used successfully as feeder cells to support the derivation and undifferentiated growth of hESCs and are commercially available through the American Type Culture Collection (ATCC). This protocol describes a method for preparing hFFs for hESC culture.

Testing of nine different xeno-free culture media for human embryonic stem cell cultures.

BACKGROUND: Human embryonic stem cells (hESC) are excellent candidates for cell replacement therapies. However, currently used culture conditions contain animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hESC. METHODS: Nine xeno-free culture media were compared with the conventional serum replacement (ko-SR) containing media in the culture of hESC on human feeder cells. Cultured hESC were characterized immunocytochemically and by fluorescence-activated cell sorter analysis. The differentiation potential of hESC cultured with xeno-free media was determined with the RT-PCR analysis. RESULTS: The hESC cultured in xeno-free media differentiated or the proliferation decreased substantially. Under some test conditions, the morphology of the feeder cells was altered considerably. The hESC cultured with human serum underwent excessive differentiation in the beginning of culture, but a fraction of hESC was able to adapt to culture conditions containing 20% of human serum. CONCLUSIONS: None of the studied xeno-free media was able to maintain the undifferentiated growth of hESC. The medium containing 20% human serum was found to sustain undifferentiated hESC proliferation to some extent, yet was inferior to the conventional ko-SR-containing medium.