LIN28 Family in Testis: Control of Cell Renewal,Maturation, Fertility and Aging

Male reproductive development starts early in the embryogenesis with somatic and germ cell differentiation in the testis. The LIN28 family of RNA-binding proteins promoting pluripotency has two members—LIN28A and LIN28B. Their function in the testis has been investigated but many questions about their exact role based on the expression patterns remain unclear. LIN28 expression is detected in the gonocytes and the migrating, mitotically active germ cells of the fetal testis. Postnatal expression of LIN28 A and B showed differential expression, with LIN28A expressed in the undifferentiated spermatogonia and LIN28B in the elongating spermatids and Leydig cells. LIN28 interferes with many signaling pathways, leading to cell proliferation, and it is involved in important testicular physiological processes, such as cell renewal, maturation, fertility, and aging. In addition, aberrant LIN28 expression is associated with testicular cancer and testicular disorders, such as hypogonadotropic hypogonadism and Klinefelter’s syndrome. This comprehensive review encompasses current knowledge of the function of LIN28 paralogs in testis and other tissues and cells because many studies suggest LIN28AB as a promising target for developing novel therapeutic agents.     

Genetic Variants of GPER/GPR30, a Novel Estrogen-Related G Protein Receptor,Are Associated with Human Seminoma

Testicular germ cell tumors (TGCTs) are the most common solid cancers in young men, with an increasing incidence over several years. However, their pathogenesis remains a matter of debate. Some epidemiological data suggest the involvement of both environmental and genetic factors. We reported two distinct effects of estrogens and/or xeno-estrogens on in vitro human seminoma-derived cells proliferation: (1) an antiproliferative effect via a classical estrogen receptor beta-dependent pathway, and (2) a promotive effect via a non-classical membrane G-protein-coupled receptor, GPR30/GPER, which is only overexpressed in seminomas, the most common TGCT. In order to explain this overexpression, we investigated the possible association of polymorphisms in the GPER gene by using allele-specific tetra-primer polymerase chain reaction performed on tissue samples from 150 paraffin-embedded TGCT specimens (131 seminomas, 19 non seminomas). Compared to control population, loss of homozygous ancestral genotype GG in two polymorphisms located in the promoter region of GPER (rs3808350 and rs3808351) was more frequent in seminomas but not in non-seminomas (respectively, OR = 1.960 (1.172–3.277) and 7.000 (2.747–17.840); p < 0.01). These polymorphisms may explain GPER overexpression and represent a genetic factor of susceptibility supporting the contribution of environmental GPER ligands in testicular carcinogenesis.     

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.     

SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.     

Wild-Type and Mutant FUS Expression Reduce Proliferation and Neuronal Differentiation Properties of Neural Stem Progenitor Cells

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.     

Actions and Roles of FSH in Germinative Cells

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic–pituitary–gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and β. The FSH β-subunit (FSHβ) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH β-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.     

Modified Industrial Three-Dimensional Polylactic Acid Scaffold Cell Chip Promotes the Proliferation and Differentiation of Human Neural Stem Cells

In this study, we fabricated a three-dimensional (3D) scaffold using industrial polylactic acid (PLA), which promoted the proliferation and differentiation of human neural stem cells. An industrial PLA 3D scaffold (IPTS) cell chip with a square-shaped pattern was fabricated via computer-aided design and printed using a fused deposition modeling technique. To improve cell adhesion and cell differentiation, we coated the IPTS cell chip with gold nanoparticles (Au-NPs), nerve growth factor (NGF) protein, an NGF peptide fragment, and sonic hedgehog (SHH) protein. The proliferation of F3.Olig2 neural stem cells was increased in the IPTS cell chips coated with Au-NPs and NGF peptide fragments when compared with that of the cells cultured on non-coated IPTS cell chips. Cells cultured on the IPTS-SHH cell chip also showed high expression of motor neuron cell-specific markers, such as HB9 and TUJ-1. Therefore, we suggest that the newly engineered industrial PLA scaffold is an innovative tool for cell proliferation and motor neuron differentiation.     

Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells

We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7⁺hSMSC)-derived osteoblast-like (α7⁺hSMSC-OB) cells, and found that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.     

DNA microarray expression analysis of baicalin-induced differentiation of C17.2 neural stem cells

Identifying baicalin-regulated genes for neuronal differentiation: Baicalin is a potent neuronal-differentiation-inducing compound. This study explored the gene expression regulated through baicalin-induced differentiation of C17.2 neural stem cells by using a DNA microarray followed by qPCR validation. The expression of 15 genes was significantly regulated among the 58 differentially expressed genes important for nervous system development and function.     

Single-Cell Atlas of Adult Testis in Protogynous Hermaphroditic Orange-Spotted Grouper,Epinephelus coioides

Spermatogenesis is a process of self-renewal and differentiation in spermatogonial stem cells. During this process, germ cells and somatic cells interact intricately to ensure long-term fertility and accurate genome propagation. Spermatogenesis has been intensely investigated in mammals but remains poorly understood with regard to teleosts. Here, we performed single-cell RNA sequencing of ~9500 testicular cells from the male, orange-spotted grouper. In the adult testis, we divided the cells into nine clusters and defined ten cell types, as compared with human testis data, including cell populations with characteristics of male germ cells and somatic cells, each of which expressed specific marker genes. We also identified and profiled the expression patterns of four marker genes (calr, eef1a, s100a1, vasa) in both the ovary and adult testis. Our data provide a blueprint of male germ cells and supporting somatic cells. Moreover, the cell markers are candidates that could be used for further cell identification.     

红系造血相关转录因子在红系诱导分化中表达的变化

目的探讨红系细胞分化中细胞生长及相关转录因子表达的变化。方法利用红白血病细胞株HB60-5的生长特性,通过改变培养条件,促使细胞向成熟细胞分化。绘制细胞生长曲线,应用Northern blot方法检测诱导分化过程中红系造血相关基因表达变化。结果HB60-5细胞随着诱导分化时间的延长,生长减慢,红系分化标记α-珠蛋白表达逐渐升高,同时伴有GATA-1、p45NF-E2、EDRF等基因表达上调,Fli-1基因表达下调。结论红系分化过程中,细胞增殖受到抑制,红系细胞分化的基因表达上调,红系细胞增殖的基因表达下调。     

Epigenetic Regulation of Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells

With the intent to achieve the best modalities for myocardial cell therapy, different cell types are being evaluated as potent sources for differentiation into cardiomyocytes. Embryonic stem cells and induced pluripotent stem cells have great potential for future progress in the treatment of myocardial diseases. We reviewed aspects of epigenetic mechanisms that play a role in the differentiation of these cells into cardiomyocytes. Cardiomyocytes proliferate during fetal life, and after birth, they undergo permanent terminal differentiation. Upregulation of cardiac-specific genes in adults induces hypertrophy due to terminal differentiation. The repression or expression of these genes is controlled by chromatin structural and epigenetic changes. However, few studies have reviewed and analyzed the epigenetic aspects of the differentiation of embryonic stem cells and induced pluripotent stem cells into cardiac lineage cells. In this review, we focus on the current knowledge of epigenetic regulation of cardiomyocyte proliferation and differentiation from embryonic and induced pluripotent stem cells through histone modification and microRNAs, the maintenance of pluripotency, and its alteration during cardiac lineage differentiation.     

The Homeobox Only Protein Homeobox (HOPX) and Colorectal Cancer

The HOP (homeobox only protein) homeobox (HOPX) is most closely related to the homeobox protein that contains a homeobox-like domain but lacks certain conserved residues required for DNA binding. Here, we review the current understanding of HOPX in the progression of colorectal cancer (CRC). HOPX was initially reported as a differentiation marker and is expressed in various normal tissues. In the colon, HOPX is expressed uniquely in the quiescent stem cell, +4, and in differentiated mucosal cells of the colon. HOPX expression is markedly suppressed in a subset of cancers, mainly in an epigenetic manner. CRC may include separate entities which are differentially characterized by HOPX expression from a prognostic point of view. HOPX itself can regulate epigenetics, and defective expression of HOPX can result in loss of tumor suppressive function and differentiation phenotype. These findings indicate that HOPX may be both a central regulator of epigenetic dynamics and a critical determinant for differentiation in human cells. HOPX downstream targets were identified in CRC cell lines and hold promise as candidates for therapeutic targets of CRC, such as EphA2 or AP-1. Further analysis will elucidate and confirm the precise role of such proteins in CRC progression.     

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers

Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. Methods: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). Results: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. Conclusion: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.     

BAF-L Modulates Histone-to-Protamine Transition during Spermiogenesis

Maturing male germ cells undergo a unique developmental process in spermiogenesis that replaces nucleosomal histones with protamines, the process of which is critical for testicular development and male fertility. The progress of this exchange is regulated by complex mechanisms that are not well understood. Now, with mouse genetic models, we show that barrier-to-autointegration factor-like protein (BAF-L) plays an important role in spermiogenesis and spermatozoal function. BAF-L is a male germ cell marker, whose expression is highly associated with the maturation of male germ cells. The genetic deletion of BAF-L in mice impairs the progress of spermiogenesis and thus male fertility. This effect on male fertility is a consequence of the disturbed homeostasis of histones and protamines in maturing male germ cells, in which the interactions between BAF-L and histones/protamines are implicated. Finally, we show that reduced testicular expression of BAF-L represents a risk factor of human male infertility.