Isabella Rodrigues Fernandes

The Brazilian Zika virus strain causes birth defects in experimental models

Summary Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (Family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys1. Until the 20th century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the island of Yap in Micronesia2. Patients experienced fever, skin rash, arthralgia and conjunctivitis2. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America3. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome4,5. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKVBR) strain causes birth defects remains missing6. Here we demonstrate that the ZIKVBR infects fetuses, causing intra-uterine growth restriction (IUGR), including signs of microcephaly in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. Finally, we observed that the infection of human brain organoids resulted in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKVBR crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKVBR outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKVBR in human neurodevelopment.

Induced pluripotent stem cells for modeling neurological disorders

Several diseases have been successfully modeled since the development of induced pluripotent stem cell (iPSC) technology in 2006. Since then, methods for increased reprogramming efficiency and cell culture maintenance have been optimized and many protocols for differentiating stem cell lines have been successfully developed, allowing the generation of several cellular subtypes in vitro. Gene editing technologies have also greatly advanced lately, enhancing disease-specific phenotypes by creating isogenic cell lines, allowing mutations to be corrected in affected samples or inserted in control lines. Neurological disorders have benefited the most from iPSC-disease modeling for its capability for generating disease-relevant cell types in vitro from the central nervous system, such as neurons and glial cells, otherwise only available from post-mortem samples. Patient-specific iPSC-derived neural cells can recapitulate the phenotypes of these diseases and therefore, considerably enrich our understanding of pathogenesis, disease mechanism and facilitate the development of drug screening platforms for novel therapeutic targets. Here, we review the accomplishments and the current progress in human neurological disorders by using iPSC modeling for Alzheimers disease, Parkinsons disease, Huntingtons disease, spinal muscular atrophy, amyotrophic lateral sclerosis, duchenne muscular dystrophy, schizophrenia and autism spectrum disorders, which include Timothy syndrome, Fragile X syndrome, Angelman syndrome, Prader-Willi syndrome, Phelan-McDermid, Rett syndrome as well as Nonsyndromic Autism.

In-a-Dish: Induced Pluripotent Stem Cells as a Novel Model for Human Diseases

Human pluripotent stem cells bring promise in regenerative medicine due to their self‐renewing ability and the potential to become any cell type in the body. Moreover, pluripotent stem cells can produce specialized cell types that are affected in certain diseases, generating a new way to study cellular and molecular mechanisms involved in the disease pathology under the controlled conditions of a scientific laboratory. Thus, induced pluripotent stem cells (iPSC) are already being used to gain insights into the biological mechanisms of several human disorders. Here we review the use of iPSC as a novel tool for disease modeling in the lab.

Repurposing of the anti-malaria drug chloroquine for Zika Virus treatment and prophylaxis

One of the major challenges of the current Zika virus (ZIKV) epidemic is to prevent congenital foetal abnormalities, including microcephaly, following ZIKV infection of pregnant women. Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appears to be a viable and immediate solution. We demonstrate that the common anti-malaria drug chloroquine (CQ) extends the lifespan of ZIKV-infected interferon signalling-deficient AG129 mice. However, the severity of ZIKV infection in these mice precludes the study of foetal (vertical) viral transmission. Here, we show that interferon signalling-competent SJL mice support chronic ZIKV infection. Infected dams and sires are both able to transmit ZIKV to the offspring, making this an ideal model for in vivo validation of compounds shown to suppress ZIKV in cell culture. Administration of CQ to ZIKV-infected pregnant SJL mice during mid-late gestation significantly attenuated vertical transmission, reducing the ZIKV load in the foetal brain more than 20-fold. Given the limited side effects of CQ, its lack of contraindications in pregnant women, and its worldwide availability and low cost, we suggest that CQ could be considered for the treatment and prophylaxis of ZIKV.

The term basal plate of the human placenta as a source of functional extravillous trophoblast cells

BackgroundExtravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta.MethodsThe basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained.ResultsIsolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator.ConclusionsTerm basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

Blocking Zika virus vertical transmission

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

Modeling neuro-immune interactions during Zika virus infection

Although Zika virus (ZIKV) infection is often asymptomatic, in some cases, it can lead to birth defects in newborns or serious neurologic complications in adults. However, little is known about the interplay between immune and neural cells that could contribute to the ZIKV pathology. To understand the mechanisms at play during infection and the antiviral immune response, we focused on neural precursor cells (NPCs)-microglia interactions. Our data indicate that human microglia infected with the current circulating Brazilian ZIKV induces a similar pro-inflammatory response found in ZIKV-infected human tissues. Importantly, using our model, we show that microglia interact with ZIKV-infected NPCs and further spread the virus. Finally, we show that Sofosbuvir, an FDA-approved drug for Hepatitis C, blocked viral infection in NPCs and therefore the transmission of the virus from microglia to NPCs. Thus, our model provides a new tool for studying neuro-immune interactions and a platform to test new therapeutic drugs.

Serum amyloid A in the placenta and its role in trophoblast invasion.

The serum amyloid A (SAA) protein is known to function in the acute phase response and immunoregulation. Recently, SAA has been shown to be involved in cell proliferation, differentiation and migratory behavior in different cell types. Here, we evaluated whether exogenous SAA could influence trophoblast invasion and differentiation using both the trophoblast-like BeWo cell line and fully differentiated human extravillous trophoblast cells (EVT) isolated from term placentae. SAA stimulated BeWo cell invasion, as measured in Matrigel invasion assays, and induced metalloprotease mRNA expression and activity. Given that BeWo cells express Toll-like receptor 4 (TLR4), a known receptor for SAA, we examined the role of TLR4 in SAA-induced invasion using a TLR4 neutralizing antibody. We also tested whether SAA could affect markers of trophoblast syncytialization in BeWo cells. We observed that SAA decreased βhCG secretion and did not influence trophoblast syncytialization. Using EVT cells isolated from human term basal plates, we confirmed that SAA at 1 and 10 µg/mL doubled EVT invasion in a TLR4-dependent manner, but at 20 µg/mL inhibited EVT cells invasiveness. In addition, we observed that SAA was expressed in both BeWo cells and human term placentae, specifically in the syncytiotrophoblast, decidual cells and EVT. In conclusion, SAA was identified as a molecule that functions in the placental microenvironment to regulate metalloprotease activity and trophoblast invasion, which are key processes in placentation and placental homeostasis.

Modeling the interplay between neurons and astrocytes in autism using human induced pluripotent stem cells

BACKGROUND Autism spectrum disorder (ASD) is a neurodevelopmental disorder with unclear etiology and imprecise genetic causes. The main goal of this work was to investigate neuronal connectivity and the interplay between neurons and astrocytes from individuals with nonsyndromic ASD using induced pluripotent stem cells. METHODS Induced pluripotent stem cells were derived from a clinically well-characterized cohort of three individuals with nonsyndromic ASD sharing common behaviors and three control subjects, two clones each. We generated mixed neural cultures analyzing synaptogenesis and neuronal activity using a multielectrode array platform. Furthermore, using an enriched astrocyte population, we investigated their role in neuronal maintenance. RESULTS ASD-derived neurons had a significant decrease in synaptic gene expression and protein levels, glutamate neurotransmitter release, and, consequently, reduced spontaneous firing rate. Based on co-culture experiments, we observed that ASD-derived astrocytes interfered with proper neuronal development. In contrast, control-derived astrocytes rescued the morphological neuronal phenotype and synaptogenesis defects from ASD neuronal co-cultures. Furthermore, after identifying interleukin-6 secretion from astrocytes in individuals with ASD as a possible culprit for neural defects, we were able to increase synaptogenesis by blocking interleukin-6 levels. CONCLUSIONS Our findings reveal the contribution of astrocytes to neuronal phenotype and confirm previous studies linking interleukin-6 and autism, suggesting potential novel therapeutic pathways for a subtype of individuals with ASD. This is the first report demonstrating that glial dysfunctions could contribute to nonsyndromic autism pathophysiology using induced pluripotent stem cells modeling disease technology.

Fibroblast sources: Where can we get them?

Fibroblasts are cells widely used in cell culture, both for transient primary cell culture or permanent as transformed cell lines. Lately, fibroblasts become cell sources for use in disease modeling after cell reprogramming because it is easily accessible in the body. Fibroblasts in patients will maintain all genetic background during reprogramming into induced pluripotent stem cells. In spite of their large use, fibroblasts are obtained after an invasive procedure, a superficial punch skin biopsy, collected under patient’s local anesthesia. Taking into consideration the minimum patient’s discomfort during and after the biopsy procedure, as well as the aesthetics aspect, it is essential to reflect on the best site of the body for the biopsy procedure combined with the success of getting robust fibroblast cultures in the lab. For this purpose, we compared the efficiency of four biopsy sites of the body (skin from eyelid, back of the ear, abdominal cesarean scar and groin). Cell proliferation assays and viability after cryopreservation were measured. Our results revealed that scar tissue provided fibroblasts with higher proliferative rates. Also, fibroblasts from scar tissues presented a higher viability after the thawing process.