The non-coding genome in early human development-Recent advancements

Not that long ago, the human genome was discovered to be mainly non-coding, that is comprised of DNA se-quences that do not code for proteins. The initial paradigm that non-coding is also non-functional was soon overturned and today the work to uncover the functions of non-coding DNA and RNA in human early embryogenesis has commenced. Early human development is characterized by large-scale changes in genomic activity and the transcriptome that are partly driven by the coordinated activation and repression of repetitive DNA elements scattered across the genome. Here we provide examples of recent novel discoveries of non-coding DNA and RNA interactions and mechanisms that ensure accurate non-coding activity during human maternal-to -zygotic transition and lineage segregation. These include studies on small and long non-coding RNAs, trans-posable element regulation, and RNA tailing in human oocytes and early embryos. High-throughput approaches to dissect the non-coding regulatory networks governing early human development are a foundation for func-tional studies of specific genomic elements and molecules that has only begun and will provide a wider un-derstanding of early human embryogenesis and causes of infertility.Peer reviewe

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.Peer reviewe

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix

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Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells

Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.Peer reviewe

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

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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.Peer reviewe

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

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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.Peer reviewe

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.Peer reviewe

Ihomelanooman onkologinen hoito päivittyi

Suomen Melanoomaryhmä ry päivitti suosituksensa ihomelanooman onkologisesta hoidosta. Vuoden kestävää liitännäislääkehoitoa PD-1-vasta-aineella eli immuuniaktivaation vapauttajalla (nivolumabi tai pembrolitsumabi) tai dabrafenibilla ja trametinibilla harkitaan leikatuille, levinneisyysasteen IIIB-IV melanoomapotilaille melanooman uusiutumisriskin pienentämiseksi. Päätös liitännäishoidosta tehdään yhdessä potilaan kanssa hyödyt ja haitat huomioiden. Levinneen melanooman ensilinjan hoitovaihtoehtona ovat PD-1-vasta-aineet, joiden lisäksi BRAFV600-mutaatiopositiivisille potilaille soveltuvat hoidot BRAF:n ja MEK:n estäjillä. Immunologista yhdistelmähoitoa CTLA-4- ja PD-1-vasta-aineilla (ipilimumabi ja nivolumabi) harkitaan huonoennusteisille hyväkuntoisille potilaille. Muita edenneen melanooman lääkehoitovaihtoehtoja ovat onkolyyttinen virushoito (TVEC) pinnallisen metastasoinnin ja isoloitu raajaperfuusio- eli ILP-hoito raajaan rajoittuvan metastasoinnin yhteydessä sekä solunsalpaajahoidot. Nopeasti kehittyvän hoidon vuoksi melanoomapotilaat pyritään rekrytoimaan kliinisiin hoitotutkimuksiin aina kun se on mahdollista