Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.

Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-ß/activin/nodal signalling inhibition.

Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 (BMP4) and/or inhibitors of fibroblast growth factor 2 (FGF2) and the transforming growth factor beta (TGF-ß)/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin (HCG) and its hyperglycosylated form (HCG-H) were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes (CGA, CGB and LGALS16), induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-ß/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy.

Copy number variation and selection during reprogramming to pluripotency.

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

Notch signaling regulates the differentiation of neural crest from human pluripotent stem cells.

Neural crest cells are specified at the border between the neural plate and the epiderm. They are capable of differentiating into various somatic cell types, including craniofacial and peripheral nerve tissues. Notch signaling plays important roles during neurogenesis; however, its function during human neural crest development is poorly understood. Here, we generated self-renewing premigratory neural-crest-like cells (pNCCs) from human pluripotent stem cells (hPSCs) and investigated the roles of Notch signaling during neural crest differentiation. pNCCs expressed various neural-crest-specifier genes, including SLUG (also known as SNAI2), SOX10 and TWIST1, and were able to differentiate into most neural crest derivatives. Blocking Notch signaling during the pNCC differentiation suppressed the expression of neural-crest-specifier genes. By contrast, ectopic expression of activated Notch1 intracellular domain (NICD1) augmented the expression of neural-crest-specifier genes, and NICD1 was found to bind to their promoter regions. Notch activity was also required for the maintenance of the premigratory neural crest state, and the suppression of Notch signaling led to the generation of neural-crest-derived neurons. Taken together, we provide a protocol for the generation of pNCCs and show that Notch signaling regulates the formation, migration and differentiation of neural crest from hPSCs.

Activin A and Wnt-dependent specification of human definitive endoderm cells.

Activin/Nodal and Wnt signaling are known to play important roles in the regional specification of endoderm. Here we have investigated the effect of the length of stimulation with Activin A plus Wnt3a on the development of hepatic and pancreatic progenitors from the definitive endoderm (DE) cells derived from human pluripotent stem cells (hPSC). We show that DE-cells derived from hPSC with 3 days high Activin A and Wnt3a treatment were able to differentiate further into both tested endodermal lineages. When prolonging the DE-induction protocol from 3 to 5 or 7 days, almost pure DE-marker positive cell populations were obtained. However, these cells had an impaired pancreatic differentiation capacity, while they still developed into hepatocyte-like cells. Further propagation of the DE-cells in the presence of Wnt3a and Activin A led to the complete loss of differentiation capacity into hepatic or pancreatic lineages. When Wnt3a was removed after 24h from the initiation of the differentiation, the cells were able to differentiate into PDX1+/NKX6.1+ pancreatic progenitors even with longer DE induction time while efficiency of hepatic differentiation was lower. Our results suggest that both the length and the timing of Wnt3a treatment during DE induction are crucial for the final differentiation outcome. Although it is possible to derive apparently pure DE cells with prolonged Activin A/Wnt-stimulation, their progenitor capacity is restricted to a limited time window.

Induced Pluripotent Stem Cells as a Possible Approach for Exploring the Pathophysiology of Polycystic Ovary Syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine condition among women with pleiotropic sequelae possessing reproductive, metabolic, and psychological characteristics. Although the exact origin of PCOS is elusive, it is known to be a complex multigenic disorder with a genetic, epigenetic, and environmental background. However, the pathogenesis of PCOS, and the role of genetic variants in increasing the risk of the condition, are still unknown due to the lack of an appropriate study model. Since the debut of induced pluripotent stem cell (iPSC) technology, the ability of reprogrammed somatic cells to self-renew and their potential for multidirectional differentiation have made them excellent tools to study different disease mechanisms. Recently, researchers have succeeded in establishing human in vitro PCOS disease models utilizing iPSC lines from heterogeneous PCOS patient groups (iPSCPCOS). The current review sets out to summarize, for the first time, our current knowledge of the implications and challenges of iPSC technology in comprehending PCOS pathogenesis and tissue-specific disease mechanisms. Additionally, we suggest that the analysis of polygenic risk prediction based on genome-wide association studies (GWAS) could, theoretically, be utilized when creating iPSC lines as an additional research tool to identify women who are genetically susceptible to PCOS. Taken together, iPSCPCOS may provide a new paradigm for the exploration of PCOS tissue-specific disease mechanisms.

CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate.

The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms' tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Transient DUX4 expression in human embryonic stem cells induces blastomere-like expression program that is marked by SLC34A2.

Embryonic genome activation (EGA) is critical for embryonic development. However, our understanding of the regulatory mechanisms of human EGA is still incomplete. Human embryonic stem cells (hESCs) are an established model for studying developmental processes, but they resemble epiblast and are sub-optimal for modeling EGA. DUX4 regulates human EGA by inducing cleavage-stage-specific genes, while it also induces cell death. We report here that a short-pulsed expression of DUX4 in primed hESCs activates an EGA-like gene expression program in up to 17% of the cells, retaining cell viability. These DUX4-induced cells resembled eight-cell stage blastomeres and were named induced blastomere-like (iBM) cells. The iBM cells showed marked reduction of POU5F1 protein, as previously observed in mouse two-cell-like cells. Finally, the iBM cells were successfully enriched using an antibody against NaPi2b (SLC34A2), which is expressed in human blastomeres. The iBM cells provide an improved model system to study human EGA transcriptome.

DUX4 is a multifunctional factor priming human embryonic genome activation.

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression.

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1-expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.

Complement in Human Pre-implantation Embryos: Attack and Defense.

It is essential for early human life that mucosal immunological responses to developing embryos are tightly regulated. An imbalance of the complement system is a common feature of pregnancy complications. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus, far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation against developing human embryos takes place already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products on healthy developing embryos, which highlights the need for strict complement regulation. We show that embryos express complement membrane inhibitors and bind soluble regulators. These findings show that mucosal complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing revealed cellular expression of complement activators. This shows that the embryonic cells themselves have the capacity to express and activate C3 and C5. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and furthermore may impact cellular homeostasis and differentiation at the earliest stages of life.

Hyperglycosylated hCG activates LH/hCG-receptor with lower activity than hCG.

While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it is controversial whether the hyperglycosylated form of hCG (hCG-h), which is the major hCG isoform during the first 4-5 weeks of pregnancy, is able to activate LH/hCG receptor (LHCGR). To address this, we utilized different extensively characterized hCG and hCGß reference reagents, cell culture- and urine-derived hCG-h preparations, and an in vitro reporter system for LHCGR activation. The WHO hCG reference reagent (99/688) was found to activate LHCGR with an EC50-value of 3.3 ±â€¯0.6 pmol/L (n = 9). All three studied hCG-h preparations were also able to activate LHCGR, but with a lower potency (EC50-values between 7.1 ±â€¯0.5 and 14 ±â€¯3 pmol/L, n = 5-11, for all P < 0.05 as compared to the hCG reference). The activities of commercial urinary hCG (Pregnyl) and recombinant hCG (Ovitrelle) preparations were intermediate between those of the hCG reference and the hCG-h. These results strongly suggest that the hCG-h is functionally similar to hCG, although it has lower potency for LHCGR activation. Whether this explains the reduced proportion of hCG-h to hCG reported in patients developing early onset pre-eclampsia or those having early pregnancy loss remains to be determined.

Maturation of in vitro-generated human islets after transplantation in nude mice.

The long-term function of human pancreatic islet grafts may depend on the neogenesis of beta cells from epithelial precursors within the grafted tissue. We have developed an in vitro model for human islet neogenesis. In this study, we have investigated the morphological signs of maturation in cultivated human islet buds (CHIBs) before and after transplantation. Clusterin is a molecule associated with beta-cell differentiation in rodents. In adult human islets, clusterin expression was located only in alpha- and PP-cells, but in CHIBs and human fetal islets, it was distributed in all four types of endocrine cells. Some immature endocrine cells in the CHIBs co-expressed insulin and glucagon. After transplantation, CHIBs became mature with one type of hormone per endocrine cell, and clusterin expression became restricted in alpha-cells. Cells co-expressing endocrine markers and cytokeratin 19, as a sign of ductal to endocrine cell transition, were frequently detected in both fresh islets and CHIBs after transplantation. We conclude that clusterin may be involved in the development of islets, and the in vitro-derived islets become mature after transplantation into nude mice. Ductal cell differentiation into endocrine cells may be an important factor in sustaining the long-term function of islet transplants.

The Role of Sequential BMP Signaling in Directing Human Embryonic Stem Cells to Bipotential Gonadal Cells.

Context: Human gonads arise as a pair of epithelial ridges on the surface of intermediate mesoderm (IM)-derived mesonephros. Toxic environmental factors and mutations in various genes are known to disturb normal gonadal development, but because of a lack of suitable in vitro models, detailed studies characterizing the molecular basis of the observed defects have not been performed. Objective: To establish an in vitro method for studying differentiation of bipotential gonadal progenitors by using human embryonic stem cells (hESCs) and to investigate the role of bone morphogenetic protein (BMP) in gonadal differentiation. Design: We tested 17 protocols using activin A, CHIR-99021, and varying durations of BMP-7 and the BMP inhibitor dorsomorphin. Activation of activin A, WNT, and BMP pathways was optimized to induce differentiation. Setting: Academic research laboratory. Main Outcomes Measures: Cell differentiation, gene expression, and flow cytometry. Results: The two most efficient protocols consistently upregulated IM markers LHX1, PAX2, and OSR1 at days 2 to 4 and bipotential gonadal markers EMX2, GATA4, WT1, and LHX9 at day 8 of culture. The outcome depended on the combination of the duration, concentration, and type of BMP activation and the length of WNT signaling. Adjusting any of the parameters substantially affected the requirements for other parameters. Conclusions: We have established a reproducible protocol for directed differentiation of hESCs into bipotential gonadal cells. The protocol can be used to model early gonadal development in humans and allows further differentiation to mature gonadal somatic cells.

Distinct differentiation characteristics of individual human embryonic stem cell lines.

BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. CONCLUSION: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

The murine thymic stromal lymphopoietin receptor is partially expressed with an extracellular C-terminus.

The gene encoding the murine thymic stromal lymphopoietin receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica Nuclear Polyhedrosis Virus and expressed with an N-terminal poly-histidine tag and a C-terminal FLAG-tag in the Spodoptera frugiperda insect cell line Sf9 during viral infection. Flow cytometer analysis of cells infected with the produced recombinant virus FastBacHisB-mdelta1-FLAG demonstrated that a majority of the infected cells expressed the mTSLPR with an extracellular C-terminal end. A similar observation was noticed in COS cells transfected with pSVL-mTSLPR-FLAG. Immunoblotting with monoclonal anti-FLAG or anti-his antibodies indicated that the corresponding receptor protein migrated as an approximately 50 kDa protein. mTSLPR produced in presence of tunicamycin migrated with a molecular weight around 40 kDa. The genetically fused poly-histidine tag was also demonstrated to be functional using a Ni-NTA purification system, indicating this protein otherwise to have normal biochemical properties.

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.

We have reproduced a previously described method for the in vitro generation of endocrine cells in adult human pancreatic tissue culture. The aim of this study was to characterize the nature of pancreatic progenitor cells and to identify the factors necessary for their differentiation in this model. During monolayer expansion, two types of cells proliferated sequentially; first cytokeratin 19 (CK19)-positive ductal epithelial cells and then nestin-positive fibroblastoid cells. After the bromodeoxyuridine-labeled cells were traced in differentiated islet buds, some of the proliferating ductal cells had differentiated into endocrine cells, whereas nestin-positive cells could not give rise to endocrine tissue. Serum-free culture was found to be an absolute requirement for the endocrine differentiation to occur. Also, overlay of the cells with Matrigel was essential, whereas nicotinamide had a potentiating effect. The in vitro-generated islet buds released insulin in response to glucose nearly as efficiently as native islets. When transplanted under the kidney capsule of nude mice, only one of five grafts demonstrated further growth with foci of both endocrine and exocrine differentiation. Our results support the previous notion that pancreatic progenitor cells represent a subpopulation of ductal epithelial cells. No evidence was found for the development of endocrine cells from nestin-positive stem cells.

No soluble common cytokine receptor gamma chain (gamma(c)) in activated human lymphocyte cultures-comparison with soluble IL-2Ralpha.

The presence of a soluble form of the common cytokine receptor gamma chain (gamma(c)) in cell free supernatants from unstimulated and 1-6 days PHA stimulated peripheral blood lymphocyte (PBL) cultures was analyzed using a sandwich ELISA. No naturally produced soluble gamma(c) could be detected in these cell free culture supernatants, although a sensitivity in the nanogram range was achieved for recombinant baculovirus expressed human soluble gamma(c) with this assay (detection limit 0.2 ng hIL-2 sRgamma). Analysis of the very same supernatants for soluble IL-2Ralpha demonstrated increased concentrations (up to 20.4 ng/ml) of this other IL-2R member. The membrane-associated form of the common cytokine receptor gamma chain was detected in cell lysates prepared from stimulated PBL at a concentration of 3.5 ng per 0.5 x 10(6) cells. Analysis of a small panel of serum samples from patients with different disorders verified that the soluble form of hIL-2Ralpha, but not hIL-2 sRgamma, can be detected, which thereby strongly suggests that the human soluble gamma(c) seems to be a valuable marker only for a limited number of clinical disorders.