Identification of DOT1L inhibitor in a screen for factors that promote dopaminergic neuron survival.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra region of the midbrain. Diagnostic criteria for PD require that at least two of three motor signs are observed: tremor, rigidity, and/or bradykinesia. The most common and effective treatment for PD is Levodopa (L-DOPA) which is readily converted to DA and has been the primary treatment since the 1960's. Dopamine agonists have also been developed but are less effective than L-DOPA. Although the lack of a model system to study PD has hampered efforts to identify treatments, diverse screening strategies have been proposed for identification of new pharmaceutical candidates. Here, we describe a pilot screen to identify candidate molecules from a bioactive compound library, that might increase formation, maintenance and/or survival of DA neurons in vitro. The screen used a previously characterized reporter construct consisting of the luciferase gene inserted downstream of the endogenous tyrosine hydroxylase (TH) gene and neurons differentiated from human pluripotent stem cells for 18 days. The reporter mimics expression of TH and includes a secreted luciferase whose activity can be measured non-invasively over multiple timepoints. Screening of the bioactive compound library resulted in the identification of a single molecule, SGC0946, that is an inhibitor of DOT1L (Disruptor Of Telomeric silencing 1-Like) which encodes a widely-conserved histone H3K79 methyltransferase that is able to both activate and repress gene transcription. Our results indicate that SGC0946 increased reporter luciferase activity with a single treatment for 48-h post-plating being equivalent to continuous treatment. Moreover, data suggested that the total number of neurons differentiated in the assays was comparable from experiment to experiment under different SGC0946 treatments over time. In contrast, data suggested that the survival and/or maintenance of DA neurons might be specifically enhanced by SGC0946 treatment. These results document the feasibility of a set of tools for further exploration of small molecules that may impact DA neuron differentiation, maintenance and/or survival. Results provide evidence in support of other reports that indicate inhibition of DOT1L may play an important role in maintenance and survival of neural progenitor cells (NPCs) and their lineage-specific differentiation.

Parkinson's Disease: Overview of Transcription Factor Regulation, Genetics, and Cellular and Animal Models.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, affecting nearly 7-10 million people worldwide. Over the last decade, there has been considerable progress in our understanding of the genetic basis of PD, in the development of stem cell-based and animal models of PD, and in management of some clinical features. However, there remains little ability to change the trajectory of PD and limited knowledge of the underlying etiology of PD. The role of genetics versus environment and the underlying physiology that determines the trajectory of the disease are still debated. Moreover, even though protein aggregates such as Lewy bodies and Lewy neurites may provide diagnostic value, their physiological role remains to be fully elucidated. Finally, limitations to the model systems for probing the genetics, etiology and biology of Parkinson's disease have historically been a challenge. Here, we review highlights of the genetics of PD, advances in understanding molecular pathways and physiology, especially transcriptional factor (TF) regulators, and the development of model systems to probe etiology and potential therapeutic applications.

Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration.

Spermatogonial stem cells (SSCs) are a group of adult stem cells in the testis that serve as the foundation of continuous spermatogenesis and male fertility. SSCs are capable of self-renewal to maintain the stability of the stem cell pool and differentiation to produce mature spermatozoa. Dysfunction of SSCs leads to male infertility. Therefore, dissection of the regulatory network of SSCs is of great significance in understanding the fundamental molecular mechanisms of spermatogonial stem cell function in spermatogenesis and the pathogenesis of male infertility. Furthermore, a better understanding of SSC biology will allow us to culture and differentiate SSCs in vitro, which may provide novel stem cell-based therapy for assisted reproduction. This review summarizes the latest research progress on the regulation of SSCs, and the potential application of SSCs for fertility restoration through in vivo and in vitro spermatogenesis. We anticipate that the knowledge gained will advance the application of SSCs to improve male fertility. Furthermore, in vitro spermatogenesis from SSCs sets the stage for the production of SSCs from induced pluripotent stem cells (iPSCs) and subsequent spermatogenesis.

Transcriptional control of human gametogenesis.

The pathways of gametogenesis encompass elaborate cellular specialization accompanied by precise partitioning of the genome content in order to produce fully matured spermatozoa and oocytes. Transcription factors are an important class of molecules that function in gametogenesis to regulate intrinsic gene expression programs, play essential roles in specifying (or determining) germ cell fate and assist in guiding full maturation of germ cells and maintenance of their populations. Moreover, in order to reinforce or redirect cell fate in vitro, it is transcription factors that are most frequently induced, over-expressed or activated. Many reviews have focused on the molecular development and genetics of gametogenesis, in vivo and in vitro, in model organisms and in humans, including several recent comprehensive reviews: here, we focus specifically on the role of transcription factors. Recent advances in stem cell biology and multi-omic studies have enabled deeper investigation into the unique transcriptional mechanisms of human reproductive development. Moreover, as methods continually improve, in vitro differentiation of germ cells can provide the platform for robust gain- and loss-of-function genetic analyses. These analyses are delineating unique and shared human germ cell transcriptional network components that, together with somatic lineage specifiers and pluripotency transcription factors, function in transitions from pluripotent stem cells to gametes. This grand theme review offers additional insight into human infertility and reproductive disorders that are linked predominantly to defects in the transcription factor networks and thus may potentially contribute to the development of novel treatments for infertility.

Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation.

Mammalian development begins in transcriptional silence followed by a period of widespread activation of thousands of genes. DNA methylation reprogramming is integral to embryogenesis and linked to Tet enzymes, but their function in early development is not well understood. Here, we generate combined deficiencies of all three Tet enzymes in mouse oocytes using a morpholino-guided knockdown approach and study the impact of acute Tet enzyme deficiencies on preimplantation development. Tet1-3 deficient embryos arrest at the 2-cell stage with the most severe phenotype linked to Tet2. Individual Tet enzymes display non-redundant roles in the consecutive oxidation of 5-methylcytosine to 5-carboxylcytosine. Gene expression analysis uncovers that Tet enzymes are required for completion of embryonic genome activation (EGA) and fine-tuned expression of transposable elements and chimeric transcripts. Whole-genome bisulfite sequencing reveals minor changes of global DNA methylation in Tet-deficient 2-cell embryos, suggesting an important role of non-catalytic functions of Tet enzymes in early embryogenesis. Our results demonstrate that Tet enzymes are key components of the clock that regulates the timing and extent of EGA in mammalian embryos.

Reprogramming of DNA methylation is linked to successful human preimplantation development.

Human preimplantation development is characterized by low developmental rates that are poorly understood. Early mammalian embryogenesis is characterized by a major phase of epigenetic reprogramming, which involves global DNA methylation changes and activity of TET enzymes; the importance of DNA methylation reprogramming for successful human preimplantation development has not been investigated. Here, we analyzed early human embryos for dynamic changes in 5-methylcytosine and its oxidized derivatives generated by TET enzymes. We observed that 5-methylcytosine and 5-hydroxymethylcytosine show similar, albeit less pronounced, asymmetry between the parental pronuclei of human zygotes relative to mouse zygotes. Notably, we detected low levels of 5-formylcytosine and 5-carboxylcytosine, with no apparent difference in maternal or paternal pronuclei of human zygotes. Analysis of later human preimplantation stages revealed a mosaic pattern of DNA 5C modifications similar to those of the mouse and other mammals. Strikingly, using noninvasive time-lapse imaging and well-defined cell cycle parameters, we analyzed normally and abnormally developing human four-cell embryos for global reprogramming of DNA methylation and detected lower 5-methylcytosine and 5-hydroxymethylcytosine levels in normal embryos compared to abnormal embryos. In conclusion, our results suggest that DNA methylation reprogramming is conserved in humans, with human-specific dynamics and extent. Furthermore, abnormalities in the four-cell-specific DNA methylome in early human embryogenesis are associated with abnormal development, highlighting an essential role of epigenetic reprogramming for successful human embryogenesis. Further research should identify the underlying genomic regions and cause of abnormal DNA methylation reprogramming in early human embryos.

Links between age at menarche, antral follicle count, and body mass index in African American and European American women.

OBJECTIVE: To examine the relationships between age at menarche, antral follicle count (AFC), and body mass index (BMI) in a multi-ethnic population of women. DESIGN: Community-based, cross-sectional study. SETTING: Academic setting. PATIENT(S): A total of 245 African American women and 273 European American women, aged 25-45 years, with regular menstrual cycles and no reproductive disorders. The ethnicity of these women was self-reported and genetically validated. INTERVENTION(S): The AFCs were measured by transvaginal ultrasound during the early follicular phase. Anthropometric measurements were taken, and age at menarche was gathered by questionnaire. MAIN OUTCOME MEASURE(S): Determination of the associations between age of menarche and adult AFC and BMI. RESULT(S): Earlier age of menarche was associated with both higher BMIs and higher AFCs in adulthood, with control for female age. The antral follicle difference between early (<12 years) vs. late (≥15 years) initiation of menarche in both white and black women was +3.81 and +3.34 follicles, respectively, which is equivalent to an approximately 20% difference in AFC. CONCLUSION(S): This study provides the first evidence that timing of menarche may influence AFC. Because of limited studies on African American women, this work provides additional needed data and may enhance our ability to prospectively screen and better treat various diseases associated with the female reproductive lifespan.

A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells.

Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. Here, we use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regulators including a zinc finger protein, ZNF207, that plays diverse roles during development. In hESCs, ZNF207 partners with master pluripotency TFs to govern self-renewal and pluripotency while simultaneously controlling commitment of cells towards ectoderm through direct regulation of neuronal TFs, including OTX2. The distinct roles of ZNF207 during differentiation occur via isoform switching. Thus, a distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm.

A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

Derivation of human induced pluripotent stem cell line (HUSTi001-A) from a 40-year-old patient with idiopathic infertility.

Human fibroblasts were isolated from foreskin of a clinically diagnosed 40-year old patient with idiopathic infertility. The fibroblasts were reprogrammed with the Yamanaka KOSM transcriptional factors using the retroviral vectors. The obtained induced pluripotent stem cell (iPSC) line showed pluripotency verified by the expression of pluripotency markers, NANOG, SOX2, OCT4, TRA-1-60, and SSEA-4. And the iPSC line was demonstrated to have the three germ layers differentiation capacity in vivo by teratoma assay. The iPSC line also showed normal karyotype. This patient-specific iPSC line can be used to explore the mechanism for idiopathic male infertility.

Isolation of human testicular cells and co-culture with embryonic stem cells.

Our overall goal is to create a three-dimensional human cell-based testicular model for toxicological and spermatogenesis studies. Methods to purify the major somatic testicular cells, namely Leydig cells (LCs), peritubular myoid cells (PCs) and Sertoli cells (SCs), from rats, mice and guinea pigs have been reported. In humans, the isolation of populations enriched for primary LCs, PCs or SCs also have described. One objective of this study was to determine if populations of cells enriched for all three of these cell types can be isolated from testes of single human donors, and we were successful in doing so from testes of three donors. Testes tissues were enzymatically digested, gravity sedimented and Percoll filtered to isolate populations enriched for LCs, PCs and SCs. LCs and PCs were identified by colorimetric detection of the expression of prototypical enzymes. Division of PCs and SCs in culture has been reported. We observed that primary human LCs could divide in culture by incorporation of 5-ethynyl-2'-deoxyuridine. SCs were identified and their functionality was demonstrated by the formation of tight junctions as shown by the expression of tight junction proteins, increased transepithelial electrical resistance, polarized secretion of biomolecules and inhibition of lucifer yellow penetration. Furthermore, we found that human SC feeder layers could facilitate germ cell progression of human embryonic stem cells (hESCs) by microarray analysis of gene expression.

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.

Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter.

Human pluripotent stem cells provide a powerful human-genome based system for modeling human diseases in vitro and for potentially identifying novel treatments. Directed differentiation of pluripotent stem cells produces many specific cell types including dopaminergic neurons. Here, we generated a genetic reporter assay in pluripotent stem cells using newly-developed genome editing technologies in order to monitor differentiation efficiency and compare dopaminergic neuron survival under different conditions. We show that insertion of a luciferase reporter gene into the endogenous tyrosine hydroxylase (TH) locus enables rapid and easy quantification of dopaminergic neurons in cell culture throughout the entire differentiation process. Moreover, we demonstrate that the cellular assay is effective in assessing neuron response to different cytotoxic chemicals and is able to be scaled for high throughput applications. These results suggest that stem cell-derived terminal cell types can provide an alternative to traditional immortal cell lines or primary cells as a quantitative cellular model for toxin evaluation and drug discovery.

Transcriptional comparison of human induced and primary midbrain dopaminergic neurons.

Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson's disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson's Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression, especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies, including in vitro modeling of PD. However, further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs.

The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming.

Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

Dynamic and social behaviors of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored.

Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis.

Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT-qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n=28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature.

Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

X chromosome inactivation: recent advances and a look forward.

X chromosome inactivation, the transcriptional inactivation of one X chromosome in somatic cells of female mammals, has revealed important advances in our understanding of development, epigenetic control, and RNA biology. Most of this knowledge comes from extensive studies in the mouse; however, there are some significant differences when compared to human biology. This is especially true in pluripotent cell types and, over the past few years, a significant amount of work has been dedicated to understanding these differences. This review focuses specifically on recent advances in the mechanism of Xist spreading, the role of Xist in cancer, the effects of reprogramming on X chromosome inactivation in human induced pluripotent stem cells, and new tools for studying X chromosome inactivation.

Directed dopaminergic neuron differentiation from human pluripotent stem cells.

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson's disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.