Follicular Cell Differentiation Research Articles

Osteogenic differentiation of follicular stem cells on nano-Saghez scaffold containing BMP2

BackgroundBone tissue is one of the tissues that are capable of self-regeneration. However, bone self-regeneration is defeated in the case of broad lesion of bone structure. Isolated stem cells from wisdom tooth follicles can potentially differentiate into ectodermal and mesodermal cells. Saghez is a natural substance that has been extracted from Pistacia terebinthus with unique features, such as high temperature and mechanical stability, adhesive structure, biocompatibility, and anti-neoplastic properties.MethodsIn this study, Saghez-encapsulated BMP2 was applied as a scaffold for wisdom tooth follicle stem cell differentiation into the osteocyte. A total of three wisdom tooth follicles were obtained for stem cell isolation. For verification of differentiation of the isolated stem cells into osteocyte and adipocyte, Oil Red and Alizarin staining were applied, respectively. Moreover, mesenchymal stem cells were distinguished by profiling their cell surface markers, includingCD73, CD90, CD44, and CD105, by flow cytometry. Saghez scaffold loaded with BMP2 factor was prepared using sol-gel method. Four experimental groups were considered in this study: cells seeded on BMP2 encapsulated in Saghez scaffold, Saghez scaffold, osteogenic medium, and DMEM medium.ResultsMechanical properties of Saghez scaffold, including tensile Young’s modulus, ultimate tensile stress, compression Young’s modulus, and complex shear modulus, were 19 MPa, 32 MPa, 0.42 MPa, and 0.9 MPa, respectively. The porosity of the scaffold was 70–140 μm, and the percentage of porosity was 75–98%. The results of flow cytometry studies indicated that CD44, CD73, CD90, and CD105 were positively expressed on the membrane of the tooth follicles’ stem cell. The results indicated that the rate of differentiation of the follicle stem cells into osteocyte was the highest in the Saghez-BMP2 scaffold containing differentiation medium groups. These findings were verified by morphological studies, osteoblast and osteocalcin gene and protein expression investigations, and alkaline phosphatase activity measurement. The highest osteopontin and osteocalcin genes expression levels (1.7 and 1.9) were seen in positive control, followed by DMEM + differentiation factor (1.5 and 1.6), scaffold + BMP2 (1.2 and 1.4), DMEM + stem cell (1 and 1) and scaffold (0.4 and 0.5), and negative control respectively.ConclusionThis study provides a novel system for differentiation of the stem cell into osteocytes. The results of this study suggest that loaded BMP2 in Saghez scaffold possibly acts as an osteocyte differentiator factor.

Activation of Notch Signaling by Oocytes and Jag1 in Mouse Ovarian Granulosa Cells.

The Notch pathway plays diverse and complex roles in cell signaling during development. In the mammalian ovary, Notch is important for the initial formation and growth of follicles, and for regulating the proliferation and differentiation of follicular granulosa cells during the periovulatory period. This study seeks to determine the contribution of female germ cells toward the initial activation and subsequent maintenance of Notch signaling within somatic granulosa cells of the ovary. To address this issue, transgenic Notch reporter (TNR) mice were crossed with Sohlh1-mCherry (S1CF) transgenic mice to visualize Notch-active cells (EGFP) and germ cells (mCherry) simultaneously in the neonatal ovary. To test the involvement of oocytes in activation of Notch signaling in ovarian somatic cells, we ablated germ cells using busulfan, a chemotherapeutic alkylating agent, or investigated KitWv/Wv (viable dominant white-spotting) mice that lack most germ cells. The data reveal that Notch pathway activation in granulosa cells is significantly suppressed when germ cells are reduced. We further demonstrate that disruption of the gene for the Notch ligand Jag1 in oocytes similarly impacts Notch activation and that recombinant JAG1 enhances Notch target gene expression in granulosa cells. These data are consistent with the hypothesis that germ cells provide a ligand, such as Jag1, that is necessary for activation of Notch signaling in the developing ovary.

Differentiation of follicular epithelium in polytrophic ovaries of Pieris napi (Lepidoptera: Pieridae)\u2014how far to Drosophila model

Lepidoptera together with its sister group Trichoptera belongs to the superorder Amphiesmenoptera, which is closely related to the Antliophora, comprising Diptera, Siphonaptera, and Mecoptera. In the lepidopteran Pieris napi, a representative of the family Pieridae, the ovaries typical of butterflies are polytrophic and consist of structural ovarian units termed ovarioles. Each ovariole is composed of a terminal filament, germarium, vitellarium, and ovariole stalk. The germarium houses developing germ cell clusters and somatic prefollicular and follicular cells. The significantly elongated vitellarium contains linearly arranged ovarian follicles in successive stages of oogenesis (previtellogenesis, vitellogenesis, and choriogenesis). Each follicle consists of an oocyte and seven nurse cells surrounded by follicular epithelium. During oogenesis, follicular cells diversify into five morphologically and functionally distinct subpopulations: (1) main body follicular cells (mbFC), (2) stretched cells (stFC), (3) posterior terminal cells (pFC), (4) centripetal cells (cpFC), and (5) interfollicular stalk cells (IFS). Centripetal cells are migratorily active and finally form the micropyle. Interfollicular stalk cells derive from mbFC as a result of mbFC intercalation. Differentiation and diversification of follicular cells in Pieris significantly differ from those described in Drosophila in the number of subpopulations and their origin and function during oogenesis.

Aire deficient dendritic cells promote the T follicular helper cells differentiation

Autoimmune regulator (Aire), primarily expressed in medullary thymic epithelial cells (mTECs), maintains central immune tolerance through the clearance of self-reactive T cells. Aire can also be expressed in dendritic cells (DCs), and DCs can mediate T follicular helper (TFH) cell differentiation and self-reactive B cell activation through inducible costimulator molecule ligand (ICOSL) and interleukin 6 (IL-6), which can cause autoimmune diseases. To confirm whether Aire in DCs affects TFH cell differentiation and to determine the role of Aire in the maintenance of peripheral immune tolerance, this study observed the effects of Aire deficiency on TFH cells using Aire knockout mice. The results showed that Aire deficiency caused increased number of TFH cells, both in vivo and in vitro. Further studies showed that Aire deficiency promoted TFH differentiation through the upregulation of ICOSL and IL-6 in DCs. Thus Aire could suppress the expression of ICOSL and IL-6 to inhibit TFH cell differentiation.

Therapeutic effects of baicalin on lupus nephritis in a lupus-prone mouse model and its regulatory role in follicular helper T cell differentiation

Objective To evaluate the therapeutic effect of baicalin on lupus nephritis in a lupus-prone mouse model, and to investigate its regulatory role in the differentiation of follicular helper T (Tfh) cells. Methods Eight 12-week-old female MRL/lpr lupus-prone mice were randomly and equally divided into two groups by a random number table i.e., baicalin group and control group intraperitoneally injected with 200 mg/kg baicalin sodium and chloride physiological solution, respectively, once every day for 4 weeks. After the end of treatment, Coomassie brilliant blue staining was performed to detect the level of 24-hour urine protein. Then, the mice were sacrificed, and the spleens were resected and weighed. Mononuclear cells were isolated from these spleens, and flow cytometry was conducted to determine the proportion of Tfh cells. Additionally, the kidneys were resected and subjected to hematoxylin and eosin (HE) staining for the evaluation of kidney impairment. Moreover, some other mononuclear cells were isolated from the spleens of the lupus-prone mice in the control group, and magnetic activated cell sorting (MACS) was performed to isolate naive CD4+ T cells, which were divided into 3 groups: blank control group receiving no treatment, induction group treated with 10 μg/L anti-interleukin (IL) -21 and anti-IL-6 antibodies and 3 μg/L anti-CD3 and anti-CD28 antibodies for 5 days, and intervention group additionally treated with 40 μmol/L baicalin for 5 days besides the above treatment. Then, 50 μg/L phorbol ester, 750 μg/L ionomycin and 20 mg/L brefeldin A were used to stimulate some cultured naive CD4+ T cells in the above groups. Flow cytometry was conducted to determine the proportion of CD4+CXCR5+PD-1+ cells and CD4+IL-21+ cells. Statistical analysis was carried out with SPSS20.0 software by using one-way analysis of variance (ANOVA) and student t test for the comparison of quantitative data between groups. Results The baicalin treatment could effectively improve the kidney impairment in the lupus-prone mice. Compared with the control group, the baicalin group showed significantly decreased 24-hour urine protein level ([1 416 ± 171] vs. [2 623 ± 278] μg/24 h, P = 0.022) , and significantly decreased proportion of Tfh cells in the spleen (12.6% ± 2.3% vs. 40.2% ± 1.1%, P = 0.005) . In vitro baicalin could further inhibit the differentiation of Tfh cells. Compared with the induction group, the intervention group showed significantly decreased proportion of CD4+CXCR5+PD-1+ Tfh cells (13.3% ± 0.8% vs. 17.6% ± 0.9%, P = 0.04) and CD4+IL-21+ cells (1.0% ± 0.4% vs. 2.7% ± 0.2%, P < 0.01) . Conclusion Baicalin can effectively ameliorate lupus nephritis, which may be associated with the inhibition of Tfh cell differentiation. Key words: Lupus erythematosus, systemic; Lupus nephritis; Baicalin; Disease models, animal; Follicular helper T cells; Interleukin-21

Tissue resident and follicular Treg cell differentiation is regulated by CRAC channels

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

The Highly Expressed FAM83F Protein in Papillary Thyroid Cancer Exerts a Pro-Oncogenic Role in Thyroid Follicular Cells.

Thyroid cancer is the most common endocrine cancer with predominant prevalence of papillary thyroid cancer (PTC) histotype. MAPK signaling genetic alterations are frequent in PTC, affecting more than 80% of cases. These alterations constitutively activate MAPK signaling cross-regulating different pro-oncogenic pathways. However, additional molecular alterations associated with thyroid cancer are not completely understood. In this extent, the new family of proteins named FAM83 (FAMily with sequence similarity 83) was recently identified as mediator of oncogenic signaling in different types of cancer. Here we report FAM83F as a novel highly expressed protein in PTC. We evaluated FAM83F levels in 106 PTC specimens, 34 goiter, and 41 adjacent non-tumoral human thyroid, and observed FAM83F cytoplasmic overexpression in 71% of PTC (76 of 106) while goiter tissues showed nuclear positivity and normal thyroid showed no staining by immunohistochemistry. Moreover, TSH-induced goiter and BRAFT1799A-induced PTC animal models also showed FAM83F activation. In vitro, we generated a stable thyroid cell line PCCL3 with FAM83F overexpression and observed that FAM83F deregulates thyroid follicular cell biology leading to loss of thyroid differentiation genes such as Sodium-Iodide Symporter (NIS), reactivation of stem cell markers such as LIN28B and SOX2, induction of cell migration and resistance to doxorubicin-induced apoptosis. Moreover, FAM83F activates MAPK signaling through interaction with BRAF and RAF while impairs TGFβ antiproliferative signaling transduction. In this study, we showed FAM83F as a new pro-oncogenic protein overexpressed in thyroid cancer that modulates thyroid follicular cell biology and differentiation through cross-regulation of MAPK and TGFβ signaling.

Regulation of Foxe1 by Thyrotropin and Transforming Growth Factor Beta Depends on the Interplay Between Thyroid-Specific, CREB and SMAD Transcription Factors.

Background: Thyroid follicular cells are characterized by the expression of a specific set of genes necessary for the synthesis and secretion of thyroid hormones, which are in turn regulated by the transcription factors Nkx2-1, Pax8, and Foxe1. Thyroid differentiation is finely tuned by the balance between positive regulatory signals, including thyrotropin (TSH), and by negative regulatory signals, such as transforming growth factor beta (TGF-β), which counteracts the action of TSH. A role for Foxe1 as a mediator of hormonal and growth-factor control of thyroid differentiation has been previously suggested. Therefore, the aim of this work was to study the mechanisms governing Foxe1 expression to define the ligands and signals that regulate one of the important factors in thyroid differentiation. Methods: Expression of Foxe1 was evaluated in rat PCCl3 thyroid follicular cells under different treatments. The mouse Foxe1 promoter was cloned, and site-directed mutagenesis was undertaken to study its transcriptional regulation and to identify response elements. Protein/DNA binding assays were performed to evaluate the binding of different transcription factors, and gene-silencing approaches were used to elucidate their functional roles. Results:In silico analysis of the Foxe1 promoter identified binding sites for Nkx2-1, Pax8, Foxe1, and Smad proteins, as well as cAMP-response element (CRE) sites. It was found that both CRE-binding protein and CRE modulator were necessary for the TSH-mediated induction of Foxe1 expression via the cAMP/PKA signaling pathway. Moreover, transcription of Foxe1 was regulated by Nkx2-1 and Pax8 and by itself, suggesting an autoregulatory mechanism of activation and an important role for thyroid transcription factors. Finally, TGF-β, through Smad proteins, inhibited the TSH-induced Foxe1 expression. Conclusions: This study shows that Foxe1 is the final target of TSH/cAMP and TGF-β regulation that mediates expression of thyroid differentiation genes, and provides evidence of an interplay between CRE-binding proteins, thyroid transcription factors, and Smad proteins in its regulation. Thus, Foxe1 plays an important role in the complex transcriptional network that regulates thyroid follicular cell differentiation.

The role of glucocorticoids in ovarian development of sleep deprived rats

ObjectiveSleep deprivation (SD) adversely affects female reproductive function. In this study, we investigated the role of glucocorticoids in ovarian development in sleep deprived rats. Materials and methodsFemale rats were subjected to SD for 1–4 days. Concentrations of serum estradiol and corticosterone were assessed. Betamethasone (BET) and/or recombinant human follicle-stimulating hormone (FSH) were administered to 21-day-old female rats for 2 days to evaluate ovarian status for follicular development. Intact preantral follicles were mechanically dissected from the rat's ovaries and cultured for 72 h with or without FSH in the presence or absence of BET to evaluate follicular development. ResultsSD led to a significant difference in serum estradiol concentrations between the sham and SD groups, and corticosterone concentrations were significantly elevated in groups with more than 2 days of SD (P < 0.05). FSH stimulated ovarian growth in immature rats, whereas BET inhibited ovarian development caused by the FSH treatment. Treatment of the preantral follicles with FSH induced an increase in both follicle size and follicular cell number, while follicular cell differentiation was accompanied by enhanced inhibin-α and connexin 43 expression. Inhibition of FSH-stimulated follicular growth through BET treatment exhibited a dose-dependent reciprocal trend; as the BET dose increased (0.001–1 μg/mL), preantral follicular growth decreased. This decrease was associated with a decrease in follicular cell numbers and suppression of a proliferating cell nuclear antigen, inhibin-α, and connexin 43 expression. ConclusionThe findings suggest that the adverse effects of SD may inhibit follicular development during ovarian hyperstimulation by corticosterone elevation in rat.

CTLA-4 regulates T follicular regulatory cell differentiation and participates in intestinal damage caused by spontaneous autoimmunity

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is a co-inhibitory molecule expressed by T cells and is required for immune regulation and inflammation prevention. In clinical patients, the CTLA-4 mutation causes spontaneous immune-related early-onset Crohn's disease; however, its potential mechanism is still unknown. In the current study, we found that defects in CTLA-4 in CD4 cells lead to limited differentiation of T follicular regulatory (Tfr) cells and relatively increased T follicular helper (Tfh) cells and spontaneous B cell germinal centres (GCs) responses that trigger the accumulation of autoantibodies in intestinal epithelial cells. In addition, the deficiency of Tfr cells caused by defects in CTLA-4 causes these cells to lose their function of inhibiting the non-specific immune response produced during the specific humoural immune response induced by MCMV (mouse cytomegalovirus), resulting in acute intestinal injury and death in mice. The lack of Tfr cells may be responsible for the immunosuppressive disorder of inflammatory bowel disease caused by CTLA-4 deficiency. In conclusion, we verified that CTLA-4 may be required for Tfr cell differentiation and production. Tfr cells inhibit B cell responses and prevent humoural autoimmune-mediated intestinal damage by regulating Tfh-dependent GC responses.

HCV-associated exosomes promote myeloid-derived suppressor cell expansion via inhibiting miR-124 to regulate T follicular cell differentiation and function

Virus-infected cells can regulate non-permissive bystander cells, but the precise mechanisms remain incompletely understood. Here we report that this process can be mediated by transfer of viral RNA-loaded exosomes shed from infected cells to myeloid-derived suppressor cells (MDSCs), which in turn regulate the differentiation and function of T cells during viral infection. Specifically, we demonstrated that patients with chronic hepatitis C virus (HCV) infection exhibited significant increases in T follicular regulatory (TFR) cells and decreases in T follicular helper (TFH) cells. These MDSC-mediated T-cell dysregulations resulted in an increased ratio of TFR/TFH and IL-10 production in peripheral blood. Specifically, co-culture of MDSCs derived from HCV patients with healthy peripheral blood mononuclear cells (PBMCs) induced expansion of TFR, whereas depletion of MDSCs from PBMCs of HCV patients reduced the increases in TFR frequency and IL-10 production, and promoted the differentiation of IFN-γ-producing TFH cells. Importantly, we found that exosomes isolated from the plasma of HCV patients and supernatant of HCV-infected hepatocytes could drive monocytic myeloid cell differentiation into MDSCs. These exosomes were enriched in tetraspanins, such as CD63 and CD81, and contained HCV RNA, but exosomes isolated from patients with antiviral treatment contained no HCV RNA and could not induce MDSC differentiation. Notably, these HCV RNA-containing exosomes (HCV-Exo) were sufficient to induce MDSCs. Furthermore, incubation of healthy myeloid cells with these HCV-Exo inhibited the expression of miR−124, whereas reconstitution of PBMCs with miR−124 abolished the effects of HCV−Exo on MDSC induction. Taken together, these results indicate that HCV-associated exosomes can transfer immunomodulatory viral RNA from infected cells to neighboring immune cells and trigger MDSC expansion, which subsequently promotes TFR differentiation and inhibits TFH function. This study reveals a previously unrecognized path that represents a novel mechanism of immune dysregulation during chronic viral infection.

GLIS3 is indispensable for TSH/TSHR-dependent thyroid hormone biosynthesis and follicular cell proliferation

Mutations in the Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) have first been associated with a syndrome combining two rare genetic endocrine diseases, namely neonatal diabetes and congenital hypothyroidism (OMIM #610199)1. Since then, 12 patients have been reported so far with a broad spectrum of a multi-organ disease including growth delay, renal cystic dysplasia, liver fibrosis, congenital glaucoma and osteopenia, besides neonatal diabetes and congenital hypothyroidism. In a large cohort of Chinese patients with congenital hypothyroidism without further multi-organ disease, the prevalence of GLIS3 mutations was 0.3%2. The role of GLIS3 has been extensively described for beta cell differentiation. However, the role of GLIS3 for thyroid follicular cell differentiation and function remained unknown.

ERRATUM FOR "Ovarian Follicular Theca Cell Recruitment, Differentiation, and Impact on Fertility: 2017 Update".

ERRATUM FOR "Ovarian Follicular Theca Cell Recruitment, Differentiation, and Impact on Fertility: 2017 Update".

MiR-192 suppresses T follicular helper cell differentiation by targeting CXCR5 in childhood asthma

The aim of this study was to investigate the role of miR-192 in differentiation of T follicular helper cells in childhood asthma. Blood samples were taken from eighteen children with acute asthma attacks and fifteen healthy children (HC). Quantitative real-time PCR and Western blotting were used to detect the expression levels of miR-192, C-X-C chemokine receptor type 5 (CXCR5), B-cell lymphoma 6 (BCL-6) and inducible T-cell costimulator (ICOS). The flow cytometry was performed to detect the proportion of CD4 + CXCR5+ Tfh cells on CD4 + T lymphocytes. The enzyme-linked immunosorbent assay (ELISA) was carried out to determine the plasma concentrations of total IgE and IL-21. The effect of miR-192 on the T follicular helper cells differentiation by targeting CXCR5 was determined by dual-luciferase reporter assay. Children with asthma had lower levels of miR-192 than HC. The proportion of CD4 + CXCR + Tfh cells was significantly higher in the acute asthma group than HC. Similarly, the plasma concentration of total IgE and IL-21 in the acute group markedly increased compared with the HC, and IgE concentration was positively correlated with the proportion of CD4 + CXCR5 + Tfh cells. Furthermore, the expression levels of CXCR5, Bcl-6 and ICOS were significantly higher in the acute group than in the HC. While the proportion of CD4 + CXCR5 + Tfh cells, IL-21, CXCR5, Bcl-6 and ICOS were obviously lower in the CD4 + T cells transfected with miR-192 plasmid than that in miR-192 + CXCR5 group and control group. In conclusion, miR-192 blocks the activation pathway of Tfh cells by targeting CXCR5, which is a reasonable cellular target for therapeutic intervention.

Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage

BackgroundThe LH-induced folliculo-luteal transformation is connected with alterations of the gene expression profile in cells of the granulosa layer. It has been described that hypoxic conditions occur during luteinization, thus favoring the formation of L-lactate within the follicle. Despite being a product of anaerobic respiration, L-lactate has been shown to act as a signaling molecule affecting gene expression in neuronal cells. During the present study, we tested the hypothesis that L-lactate may influence differentiation of follicular granulosa cells (GC).MethodsIn a bovine granulosa cell culture model effects of L- and D-lactate, of increased glucose concentrations and of the lactate transport inhibitor UK5099 were analyzed. Steroid hormone production was analyzed by RIA and the abundance of key transcripts was determined by quantitative real-time RT-PCR.ResultsL-lactate decreased the production of estradiol and significantly affected selected genes of the folliculo-luteal transition as well as genes of the lactate metabolism. CYP19A1, FSHR, LHCGR were down-regulated, whereas RGS2, VNN2, PTX3, LDHA and lactate transporters were up-regulated. These effects could be partly or completely reversed by pre-treatment of the cells with UK5099. The non-metabolized enantiomer D-lactate had even more pronounced effects on gene expression, whereas increased glucose concentrations did not affect transcript abundance.ConclusionsIn summary, our data suggest that L-lactate specifically alters physiological and molecular characteristics of GC. These effects critically depend on L-lactate uptake, but are not triggered by increased energy supply. Further, we could show that L-lactate has a positive feedback on the lactate metabolism. Therefore, we hypothesize that L-lactate acts as a signaling molecule in bovine and possibly other monovular species supporting differentiation during the folliculo-luteal transformation.

Genome-wide mapping of estrogen receptor α binding sites by ChIP-seq to identify genes related to sexual maturity in hens

In ovarian follicle development, estrogen acts as a regulatory molecule to mediate proliferation and differentiation of follicular cells. ERα (estrogen receptor α) exerts regulatory function classically by binding directly to the estrogen response element, recruiting co-factors and activating or repressing transcription in response to E2. In this study, we used ChIP-seq to map ERα-binding sites in ovaries of Hy-line Brown commercial hens at 45d, 90d and 160d. In total, 24,886, 21,680 and 23,348 binding sites were identified in the ovaries of hens at 45d, 90d and 160d, which are linked to 86, 83 and 74 genes, respectively. The PPI network contains 47 protein nodes and 164 interaction edges, among which, AKT1 (V-Akt Murine Thymoma Viral Oncogene Homolog 1) and ACTN2 (Actinin Alpha 2) with the highest weight in the network, followed by CREB1 (CAMP Responsive Element Binding Protein 1), and EPHA5 (EPH Receptor A5) were identified. These genes are likely related to sexual maturity in hens. This study also provides insight into the regulation of the ERα target gene networks and a reference for understanding ERα-regulated transcription.

Ovarian Follicular Theca Cell Recruitment, Differentiation, and Impact on Fertility: 2017 Update.

The major goal of this review is to summarize recent exciting findings that have been published within the past 10 years that, to our knowledge, have not been presented in detail in previous reviews and that may impact altered follicular development in polycystic ovarian syndrome (PCOS) and premature ovarian failure in women. Specifically, we will cover the following: (1) mouse models that have led to discovery of the derivation of two precursor populations of theca cells in the embryonic gonad; (2) the key roles of the oocyte-derived factor growth differentiation factor 9 on the hedgehog (HH) signaling pathway and theca cell functions; and (3) the impact of the HH pathway on both the specification of theca endocrine cells and theca fibroblast and smooth muscle cells in developing follicles. We will also discuss the following: (1) other signaling pathways that impact the differentiation of theca cells, not only luteinizing hormone but also insulinlike 3, bone morphogenic proteins, the circadian clock genes, androgens, and estrogens; and (2) theca-associated vascular, immune, and fibroblast cells, as well as the cytokines and matrix factors that play key roles in follicle growth. Lastly, we will integrate what is known about theca cells from mouse models, human-derived theca cell lines from patients who have PCOS and patients who do not have PCOS, and microarray analyses of human and bovine theca to understand what pathways and factors contribute to follicle growth as well as to the abnormal function of theca.

Functional Thyroid Follicular Cells Differentiation from Human-Induced Pluripotent Stem Cells in Suspension Culture.

The replacement of regenerated thyroid follicular cells (TFCs) is a promising therapeutic strategy for patients with hypothyroidism. Here, we have succeeded in inducing functional TFCs from human-induced pluripotent stem cells (iPSCs) in scalable suspension culture. Differentiation of iPSCs with Activin A treatment produced Sox17- and FoxA2-expressing definitive endodermal cells that also expressed thyroid transcription factors Pax8 and Nkx2-1. Further treatment with thyroid-stimulating hormone (TSH) induced TFCs expressing various types of thyroid proteins including TSH receptor, sodium–iodide symporter, thyroglobulin, and thyroid peroxidase. Interestingly, differentiated cells secreted free thyroxine in vitro. These results indicate successful differentiation of human iPSCs to functional TFCs that may enable us to fabricate thyroid tissues for regenerative medicine and disease models.

Transcriptomic analysis of gene cascades involved in protein kinase A and C signaling in the KGN line of human ovarian granulosa tumor cells†.

The developmental competence of an oocyte is its capacity to resume maturation, undergo successful fertilization, and reach the blastocyst stage. This competence is acquired through interaction with somatic cells of the follicle. Cumulus and granulosa cells support oocyte development, while the oocyte influences follicular cell growth and differentiation. Studies suggest that follicle-stimulating hormone and luteinizing hormone play an essential role in oocyte competence acquisition through signaling initiated by protein kinases A and C (PKA and PKC) in granulosa cells. Using a microarray and RT-qPCR, the transcriptome of human granulosa-like tumor cells (KGN) treated for 24 h with forskolin (FSK) or phorbol 12-myristate 13-acetate (PMA) was analyzed to determine the effects of PKA and PKC stimulation on gene expression. Protein-kinase-driven signaling appeared to involve five major upstream regulators, namely epidermal growth factor (EGF), transforming growth factor beta 1 (TGFβ1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), and hepatocyte growth factor (HGF). Gene associations with seven major ovarian functions were identified: Prostaglandin- endoperoxide synthase 2 (PTGS2), interleukin 8 (IL8), and interleukin 6 (IL6) with inflammation; Steroidogenic acute regulatory protein (STAR), cytochrome P450scc (CYP11A1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) with steroidogenesis; Vascular endothelial growth factor C (VEGFC), Vascular endothelial growth factor A (VEGFA), and C-X-C chemokine receptor type 4 (CXCR4) with angiogenesis; Amphiregulin (AREG), epidermal growth factor receptor (EGFR), and sprouty RTK signaling antagonist 2 (SPRY2) with differentiation, BCL2 associated X (BAX), BCL2 like 12 (BCL2L12), and caspase 1(CASP1) with apoptosis, Cyclin D1 (CCND1), cyclin B1 (CCNB1), and cyclin B2 (CCNB2) with division; and Matrix metalloproteinase-1 (MMP1), Matrix metallopeptidase 9 (MMP9), and TIMP metallopeptidase inhibitor 1 (TIMP1) with ovulation. Overall, these results indicate that signaling via both PKA and PKC potentiates gene regulation of functions such as inflammation and apoptosis, while functions such as differentiation, ovulation and angiogenesis are partial to one kinase or the other. These results improve understanding of the pathways underlying the most important changes that occur in the follicle prior to ovulation.

TAZ Induction Directs Differentiation of Thyroid Follicular Cells from Human Embryonic Stem Cells.

The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors, including PAX8 and NKX2-1, which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells, and TAZ interacts directly with both PAX8 and NKX2-1, leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. The use of a small molecule, ethacridine, recently identified as a TAZ activator, in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First, endodermal cells were derived from hES cells using Activin A, followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH, the thyroid-specific genes TG, TPO, TSHR, and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes, thyroid follicle formation and abundant TG protein expression were observed. Furthermore, such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine, a TAZ activator, which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes, without any gene transfection or complex culture conditions, by directly manipulating the transcriptional machinery without interfering with intermediate signaling events.