embryo-transfer-surgery-via-laparotomy-in-gilts

Embryo Transfer Surgery via Laparotomy in Gilts

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of ...

Universidade de São Paulo

Several methods have been used to study the neuropathogenesis of Down syndrome (DS), such as mouse aneuploidies, post mortem human brains, and in vitro cell culture of neural progenitor cells. More recently, induced pluripotent stem cell (iPSC) technology has offered new approaches in investigation, providing a valuable tool for studying specific cell types affected by DS, especially neurons and astrocytes. Here, we investigated the role of astrocytes in DS developmental disease and the impact of the astrocyte secretome in neuron mTOR signaling and synapse formation using iPSC derived from DS and wild-type (WT) subjects. We demonstrated for the first time that DS neurons derived from hiPSC recapitulate the hyperactivation of the Akt/mTOR axis observed in DS brains and that DS astrocytes may play a key role in this dysfunction. Our results bear out that 21 trisomy in astrocytes contributes to neuronal abnormalities in addition to cell autonomous dysfunctions caused by 21 trisomy in neurons. Further research in this direction will likely yield additional insights, thereby improving our understanding of DS and potentially facilitating the development of new therapeutic approaches.

Down Syndrome iPSC-Derived Astrocytes Impair Neuronal Synaptogenesis and the mTOR Pathway In Vitro.

Zika virus (ZIKV) causes congenital Zika syndrome (CZS), which is characterized by fetal demise, microcephaly and other abnormalities. ZIKV in the pregnant woman circulation must cross the placental barrier that includes fetal endothelial cells and trophoblasts, in order to reach the fetus. CZS occurs in ~1-40% of cases of pregnant women infected by ZIKV, suggesting that mothers' infection by ZIKV during pregnancy is not deterministic for CZS phenotype in the fetus. Therefore, other susceptibility factors might be involved, including the host genetic background. We have previously shown that in three pairs of dizygotic twins discordant for CZS, neural progenitor cells (NPCs) from the CZS-affected twins presented differential in vitro ZIKV susceptibility compared with NPCs from the non-affected. Here, we analyzed human-induced-pluripotent-stem-cell-derived (hiPSC-derived) trophoblasts from these twins and compared by RNA-Seq the trophoblasts from CZS-affected and non-affected twins. Following in vitro exposure to a Brazilian ZIKV strain (ZIKVBR), trophoblasts from CZS-affected twins were significantly more susceptible to ZIKVBR infection when compared with trophoblasts from the non-affected. Transcriptome profiling revealed no differences in gene expression levels of ZIKV candidate attachment factors, IFN receptors and IFN in the trophoblasts, either before or after ZIKVBR infection. Most importantly, ZIKVBR infection caused, only in the trophoblasts from CZS-affected twins, the downregulation of genes related to extracellular matrix organization and to leukocyte activation, which are important for trophoblast adhesion and immune response activation. In addition, only trophoblasts from non-affected twins secreted significantly increased amounts of chemokines RANTES/CCL5 and IP10 after infection with ZIKVBR. Overall, our results showed that trophoblasts from non-affected twins have the ability to more efficiently activate genes that are known to play important roles in cell adhesion and in triggering the immune response to ZIKV infection in the placenta, and this may contribute to predict protection from ZIKV dissemination into fetuses' tissues.

Differential gene expression elicited by ZIKV infection in trophoblasts from congenital Zika syndrome discordant twins.

Neural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells.

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization.

The shortage of viable lungs is still a major obstacle for transplantation. Trauma victims who represent potential lung donors commonly present hypovolemic shock leading to pulmonary inflammation and deterioration and rejection after transplantation. Seeking to improve lung graft, new approaches to donor treatment have been tested. This study focuses on treatment with mesenchymal stem cells (MSCs) or soluble factors produced by MSCs (FS-MSC) using a rat model for lung donors after hemorrhagic shock.

Soluble factors of mesenchimal stem cells (FS-MSC) as a potential tool to reduce inflammation in donor's lungs after hypovolemic shock.

Currently, the only feasible option for patients with progressive and/or end-stage organ degeneration is to undergo transplantation. Due to the growing unmatched demand of available organ donors and, as a consequence, the continuous growth of patients' waiting lists, the development of new tissue engineering technologies is a relevant need. In this chapter, we will focus on the liver as a model organ to discuss contemporary tissue engineering strategies. Induced pluripotent cells are an attractive alternative to serve as a cell source for tissue engineering applications due to their pluripotency, the potentiality to generate autologous transplantation, and for their high proliferation rate. Among the main liver tissue engineering technologies, 3D bioprinting, hepatic organoids, and decellularization/recellularization of biological matrixes have grown much attention as alternatives to derive functional liver grafts. Thus, this chapter will discuss how recent publications have demonstrated the use of induced pluripotent cells in the development of the aforementioned technologies. Bioprinting is an additive manufacturing biofabrication process where cells are dispersed within a matrix formulation (i.e., bioink) and extruded in a modified 3D-printer. Polymers within bioink can be cross-linked to increase stiffness. Hepatic spheroids showed greater viability and liver function, due to preserved epithelial phenotype over time. Organoid is multi-lineage tissue constructs derived from a stem cell that recapitulates the early stages of organogenesis. The influence of cellular composition of non-parenchymal cells using induced pluripotent-derived cells or primary adult cells for hepatic organoid formation was recently tested. Decellularization is a process where harvested tissues or organs are washed with a detergent-based solution, to lyse and remove all cellular components. The final product is an extracellular scaffold with preserved tissue vasculature and ultra-structure, which can be used for subsequent recellularization with recipient cells. This chapter sheds light on recent works on the use of induced pluripotent-derived cells for liver tissue engineering approaches and on how such technologies could potentially generate therapeutic alternatives for patients on waiting lists for liver transplantation.

A Review of Stem Cell Technology Targeting Hepatocyte Growth as an Alternative to Organ Transplantation.

For decades, scientists and physicians aim to generate functional human tissues in the laboratory using stem cells. In this section, we will describe a novel method that combines state-of-the-art stem cell culture with 3D bioprinting to generate potentially transplantable tissue grafts. For this purpose, we will use the liver as a model. The liver is an important metabolic hub and responsible to perform many endocrine and exocrine vital functions. It is estimated that two million deaths/year are associated with liver disease. This illustrates the need for developing new efficient alternatives for liver transplantation. Modern 3D bioprinting technologies in combination with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end-stage liver disease. Here, we described a novel method for 3D bioprinting functional and stable liver grafts using human iPS-derived cells. The novel method described in this section uses the hepatocyte-like cells in a spheroid culture format (i.e., obtained from three-dimensional cell culture), in combination or not with non-parenchymal cells (e.g., mesenchymal and endothelial cells), which generates the final liver graft. This method has proved to sustain epithelial phenotype and to increase the stability and functionality of the constructs for prolonged periods.

Stem Cell Technology in Organ Transplantation: A Novel Method for 3D Bioprinting Functional and Stable Liver Grafts Using Human iPS Cells Derived Cells.

Ernesto Goulart

Centro de Estudos sobre o Genoma Humano e Células-Tronco,
Instituto de Biociências,
Centro de Estudos sobre o Genoma Humano e Células-Tronco, Instituto de Biociências

Universidade de São Paulo

Embryo Transfer Surgery via Laparotomy in Gilts

Ernesto Goulart

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Centro de Estudos sobre o Genoma Humano e Células-Tronco,Instituto de Biociências,Centro de Estudos sobre o Genoma Humano e Células-Tronco, Instituto de Biociências

Universidade de São Paulo

Embryo Transfer Surgery via Laparotomy in Gilts

Centro de Estudos sobre o Genoma Humano e Células-Tronco,
Instituto de Biociências,
Centro de Estudos sobre o Genoma Humano e Células-Tronco, Instituto de Biociências