The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency.

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

Post-Thaw Day 5 Blastocyst Culture Time Prior to Transfer Does Not Affect Assisted Reproduction Technology (ART) Outcomes in Frozen-Thawed Embryo Transfer Cycles.

The frozen embryo transfer (FET) technique has been progressively used more worldwide due to improved culture conditions, as well as enhanced survival rates after vitrification. However, little is known about the effect of the post-thaw blastocyst culture duration prior to transfer on live birth rate in FET cycles. In this retrospective observational study, we evaluated the influence of two distinct post-thaw blastocyst culture spans (2-4 h versus 20-22 h) on clinical pregnancy and live birth rate. A total of n = 1927 frozen-warmed cycles were included in the analysis. Among those, n = 885 warmed blastocysts were cultured for 2-4 h, and n = 1029 were kept in culture for 20-22 h prior to transfer; the remaining blastocysts did not survive the warming protocol. We observed no significant differences in live birth and clinical pregnancy rates between the two groups. The blastocyst morphological evaluation at transfer improved following the longer culture time. No differences between the two groups were found also for gestational and neonatal outcomes. This work shows that different post-thaw embryo culture timings do not negatively impact pregnancy outcomes. Overall, these results are important in the context of the embryological laboratory in order to better organize the workflow and avoid unnecessary timing-related workload.

Opportunities and Limits of Conventional IVF versus ICSI: It Is Time to Come off the Fence.

Conventional IVF (c-IVF) is one of the most practiced assisted reproductive technology (ART) approaches used worldwide. However, in the last years, the number of c-IVF procedures has dropped dramatically in favor of intracytoplasmic sperm injection (ICSI) in cases of non-male-related infertility. In this review, we have outlined advantages and disadvantages associated with c-IVF, highlighting the essential steps governing its success, its limitations, the methodology differences among laboratories and the technical progress. In addition, we have debated recent insights into fundamental questions, including indications regarding maternal age, decreased ovarian reserve, endometriosis, autoimmunity, single oocyte retrieval-cases as well as preimplantation genetic testing cycles. The "overuse" of ICSI procedures in several clinical situations of ART has been critically discussed. These insights will provide a framework for a better understanding of opportunities associated with human c-IVF and for best practice guidelines applicability in the reproductive medicine field.

Keep the fate: how chromatin regulators safeguard embryonic stem cell identity.

Segregation of cells that form the embryo from those that produce the surrounding extra-embryonic tissues is critical for early mammalian development, but the regulatory layers governing these first cell fate decisions remain poorly understood. Recent work in The EMBO Journal identifies two chromatin regulators, Hdac3 and Dax1, that synergistically restrict the developmental potential of mouse embryonic stem cells and act as a lineage barrier to primitive endoderm formation.

Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration.

Defective cell migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective and affordable treatment able to improve wound healing capacity. However, the precise molecular mechanism through which AMG exhibits its beneficial effects remains unrevealed. Herein we show that AMG improves skin re-epithelialization by accelerating the migration of fibroblasts and keratinocytes. More specifically, AMG-treated wounds showed improvement of indispensable events associated with successful wound healing such as granulation tissue formation, organized collagen content, and newly formed blood vessels. We demonstrate that AMG is enriched with a pool of WH-associated growth factors that may provide the starting signal for a faster endogenous wound healing response. This work links the increased cell migration rate to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, which is followed by an increase in matrix metalloproteinase expression and their extracellular enzymatic activity. Overall we reveal the AMG-mediated wound healing transcriptional signature and shed light on the AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process. In this way, we present a framework for future improvements in AMG therapy for skin tissue regeneration applications.

Activator Protein-1 Transcriptional Activity Drives Soluble Micrograft-Mediated Cell Migration and Promotes the Matrix Remodeling Machinery.

Impaired wound healing and tissue regeneration have severe consequences on the patient's quality of life. Micrograft therapies are emerging as promising and affordable alternatives to improve skin regeneration by enhancing the endogenous wound repair processes. However, the molecular mechanisms underpinning the beneficial effects of the micrograft treatments remain largely unknown. In this study, we identified the active protein-1 (AP-1) member Fos-related antigen-1 (Fra-1) to play a central role in the extracellular signal-regulated kinase- (ERK-) mediated enhanced cell migratory capacity of soluble micrograft-treated mouse adult fibroblasts and in the human keratinocyte cell model. Accordingly, we show that increased micrograft-dependent in vitro cell migration and matrix metalloprotease activity is abolished upon inhibition of AP-1. Furthermore, soluble micrograft treatment leads to increased expression and posttranslational phosphorylation of Fra-1 and c-Jun, resulting in the upregulation of wound healing-associated genes mainly involved in the regulation of cell migration. Collectively, our work provides insights into the molecular mechanisms behind the cell-free micrograft treatment, which might contribute to future advances in wound repair therapies.

The effect of pulsed electromagnetic field exposure on osteoinduction of human mesenchymal stem cells cultured on nano-TiO2 surfaces.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with TiO2 nanostructured surface might be a great option in bone tissue engineering applications.

In Vitro and In Vivo Studies of Alar-Nasal Cartilage Using Autologous Micro-Grafts: The Use of the Rigenera® Protocol in the Treatment of an Osteochondral Lesion of the Nose.

Cartilage defects represent a serious problem due to the poor regenerative properties of this tissue. Regarding the nose, nasal valve collapse is associated with nasal blockage and persistent airway obstruction associated with a significant drop in the quality of life for patients. In addition to surgical techniques, several cell-based tissue-engineering strategies are studied to improve cartilage support in the nasal wall, that is, to ameliorate wall insufficiency. Nevertheless, there are no congruent data available on the benefit for patients during the follow-up time. In this manuscript, we propose an innovative approach in the treatment of cartilage defects in the nose (nasal valve collapse) based on autologous micro-grafts obtained by mechanical disaggregation of a small portion of cartilage tissue (Rigenera® protocol). In particular, we first analyzed in vitro murine and human cartilage micro-grafts; secondly, we analyzed the clinical results of a patient with pinched nose deformity treated with autologous micro-grafts of chondrocytes obtained by Rigenera® protocol. The use of autologous micro-graft produced promising results in surgery treatment of cartilage injuries and could be safely and easily administrated to patients with cartilage tissue defects.

Exploring Wound-Healing Genomic Machinery with a Network-Based Approach.

The molecular mechanisms underlying tissue regeneration and wound healing are still poorly understood despite their importance. In this paper we develop a bioinformatics approach, combining biology and network theory to drive experiments for better understanding the genetic underpinnings of wound healing mechanisms and for selecting potential drug targets. We start by selecting literature-relevant genes in murine wound healing, and inferring from them a Protein-Protein Interaction (PPI) network. Then, we analyze the network to rank wound healing-related genes according to their topological properties. Lastly, we perform a procedure for in-silico simulation of a treatment action in a biological pathway. The findings obtained by applying the developed pipeline, including gene expression analysis, confirms how a network-based bioinformatics method is able to prioritize candidate genes for in vitro analysis, thus speeding up the understanding of molecular mechanisms and supporting the discovery of potential drug targets.

Met-Activating Genetically Improved Chimeric Factor-1 Promotes Angiogenesis and Hypertrophy in Adult Myogenesis.

BACKGROUND: Myogenic progenitor cells (activated satellite cells) are able to express both HGF and its receptor cMet. After muscle injury, HGF-Met stimulation promotes activation and primary division of satellite cells. MAGIC-F1 (Met-Activating Genetically Improved Chimeric Factor-1) is an engineered protein that contains two human Met-binding domains that promotes muscle hypertrophy. MAGIC-F1 protects myogenic precursors against apoptosis and increases their fusion ability enhancing muscle differentiation. Hemizygous and homozygous Magic-F1 transgenic mice displayed constitutive muscle hypertrophy. METHODS: Here we describe microarray analysis on Magic-F1 myogenic progenitor cells showing an altered gene signatures on muscular hypertrophy and angiogenesis compared to wild-type cells. In addition, we performed a functional analysis on Magic-F1+/+ transgenic mice versus controls using treadmill test. RESULTS: We demonstrated that Magic-F1+/+ mice display an increase in muscle mass and cross-sectional area leading to an improvement in running performance. Moreover, the presence of MAGIC-F1 affected positively the vascular network, increasing the vessel number in fast twitch fibers. Finally, the gene expression profile analysis of Magic-F1+/+ satellite cells evidenced transcriptomic changes in genes involved in the control of muscle growth, development and vascularisation. CONCLUSION: We showed that MAGIC-F1-induced muscle hypertrophy affects positively vascular network, increasing vessel number in fast twitch fibers. This was due to unique features of mammalian skeletal muscle and its remarkable ability to adapt promptly to different physiological demands by modulating the gene expression profile in myogenic progenitors.

Nanostructured TiO2 Surfaces Promote Human Bone Marrow Mesenchymal Stem Cells Differentiation to Osteoblasts.

Micro- and nano-patterning/modification are emerging strategies to improve surfaces properties that may influence critically cells adherence and differentiation. Aim of this work was to study the in vitro biological reactivity of human bone marrow mesenchymal stem cells (hBMSCs) to a nanostructured titanium dioxide (TiO2) surface in comparison to a coverglass (Glass) in two different culture conditions: with (osteogenic medium (OM)) and without (proliferative medium (PM)) osteogenic factors. To evaluate cell adhesion, hBMSCs phosphorylated focal adhesion kinase (pFAK) foci were analyzed by confocal laser scanning microscopy (CLSM) at 24 h: the TiO2 surface showed a higher number of pFAK foci with respect to Glass. The hBMSCs differentiation to osteoblasts was evaluated in both PM and OM culture conditions by enzyme-linked immunosorbent assay (ELISA), CLSM and real-time quantitative reverse transcription PCR (qRT-PCR) at 28 days. In comparison with Glass, TiO2 surface in combination with OM conditions increased the content of extracellular bone proteins, calcium deposition and alkaline phosphatase activity. The qRT-PCR analysis revealed, both in PM and OM, that TiO2 surface increased at seven and 28 days the expression of osteogenic genes. All together, these results demonstrate the capability of TiO2 nanostructured surface to promote hBMSCs osteoblast differentiation and its potentiality in biomedical applications.