- Nov 2016
Y. Yan, S. Shin, B. S. Jha, Q. Liu, J. Sheng, F. Li, M. Zhan, J. Davis, K. Bharti, X. Zeng, M. Rao, N. Malik, M. C. Vemuri, Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells. Stem Cells Transl. Med. 2, 862–870 (2013).
This study presents an efficient method to produce neural stem cells from human pluripotent stem cells.
The system presented in this study enables the creation of NSC banks, increasing cell therapy applications.
Z. W. Naing, G. M. Scott, A. Shand, S. T. Hamilton, W. J. van Zuylen, J. Basha, B. Hall, M. E. Craig, W. D. Rawlinson, Congenital cytomegalovirus infection in pregnancy: A review of prevalence, clinical features, diagnosis and prevention. Aust. N. Z. J. Obstet. Gynaecol. 56, 9–18 (2016).
This study examines the effects on the developing fetus of congenital cytomegalovirus infection.
C. Grief, R. Galler, L. M. C. Côrtes, O. M. Barth, Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation. Arch. Virol. 142, 2347–2357 (1997).
In this study, Grief et al used electron microscopy to localize dengue virus in infected mosquito cells.
They concluded that the smooth membrane structures are an important site for the production of virus particles.
H. Tang, C. Hammack, S. C. Ogden, Z. Wen, X. Qian, Y. Li, B. Yao, J. Shin, F. Zhang, E. M. Lee, K. M. Christian, R. A. Didier, P. Jin, H. Song, G. L. Ming, Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell 18, 1–4 (2016).
Tang’s article highlights the impact of ZIKV infection on both cell death and dysregulation of the cell cycle.
G. Calvet, R. S. Aguiar, A. S. Melo, S. A. Sampaio, I. de Filippis, A. Fabri, E. S. Araujo, P. C. de Sequeira, M. C. de Mendonça, L. de Oliveira, D. A. Tschoeke, C. G. Schrago, F. L. Thompson, P. Brasil, F. B. Dos Santos, R. M. Nogueira, A. Tanuri, A. M. de Filippis, Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: A case study. Lancet Infect. Dis. (2016).
In this article, the authors were able to detect the Brazilian Zika virus in amniotic fluid and compare its genome to other Zika strains and flaviviruses. In doing so, they hoped to find out if there had been recombination events between them.
The authors collected amniotic fluid samples from women whose fetuses were diagnosed with microcephaly, extracted DNA purified virus particles, and analyzed the samples with qRT-PCR.
They found that the different viruses share 97–100% of their genomes and that there had been no recombination events.
C. G. Woods, J. Bond, W. Enard, Autosomal recessive primary microcephaly (MCPH): A review of clinical, molecular, and evolutionary findings. Am. J. Hum. Genet. 76, 717–728 (2005).
Woods et al. discuss some clinical aspects of microcephaly but mainly focus on molecular and evolutionary factors.
From an evolutionary point of view, changes in genes linked to a microcephalic phenotype might have been responsible for the evolution of the human brain size.
Woods et al. also note that microcephaly is the consequence of a mitotic deficiency in neural precursors.
E. C. Gilmore, C. A. Walsh, Genetic causes of microcephaly and lessons for neuronal development. WIREs Dev. Biol. 2, 461–478 (2013).
Microcephaly is caused by abnormal cell growth in the brain leading to a reduced brain size.
Mutations in genes involved in the cell cycle could be one factor causing this phenomenon.
Here, the authors showed that variations in brain size is more related to the number of connections between neurons.
Our results, together with recent reports showing brain calcification in microcephalic fetuses and newborns infected with ZIKV (10, 14) reinforce the growing body of evidence connecting congenital ZIKV outbreak to the increased number of reports of brain malformations in Brazil.
The authors report that their results confirm previous evidence connecting the ZIKV outbreak in Brazil to an increase in cases of microcephaly.
further characterize the consequences of ZIKV infection during different stages of fetal development
The authors used models that allowed them to study early stages of brain development. They suggest there is more work to be done to determine the effects of ZIKV infection on later stages of fetal development.
Our results demonstrate that ZIKV induces cell death in human iPS-derived neural stem cells, disrupts the formation of neurospheres and reduces the growth of organoids (fig. S2), indicating that ZIKV infection in models that mimics the first trimester of brain development may result in severe damage.
Summarizing the results, the authors conclude that in their model of early brain development, ZIKV causes severe damage.
Development of the layers of the brain.
brain organoids recapitulate the orchestrated cellular and molecular early events comparable to the first trimester fetal neocortex
Neurospheres are useful for modeling very early (embryonic) development, while organoids are used to study later stages of development.
These results suggest that the deleterious consequences of ZIKV infection in human NSCs, neurospheres and brain organoids are not a general feature of the flavivirus family.
Because DENV2 did not reduce cell growth or affect morphology, the researchers concluded that those effects are unique to ZIKV and not characteristic of the flavivirus family (to which both DENV2 and ZIKV belong).
similarly to the results described by Tang and colleagues
In Tang’s article, ZIKV infection led to a significantly higher caspase-3 activation in human NPCs.
ZIKV infection of hNPCs resulted in reduced growth, which led Tang to suggest that it might be due to both increased cell death and an interrupted cell-cycle.
ZIKV induced caspase 3/7 mediated cell death in NSCs
Zika virus induces the expression of caspase 3/7, indicating a cell is preparing to die.
Dengue virus 2, on the other hand, did not increase caspase 3/7.
Caspases are endoproteases (a type of enzyme that breaks down proteins) that play a critical role in both inflammation and cell death.
The presence of caspase 3 and 7 can be used as a sign that cells are preparing to die.
In addition to MOCK infection, we used dengue virus 2 (DENV2), a flavivirus with genetic similarities to ZIKV (11, 19), as an additional control group.
The authors also compared ZIKV infection to dengue virus 2 (DENV2) infection. DENV2 is similar to ZIKV.
Fig. 4. ZIKV reduces the growth rate of human brain organoids.
Objective To examine the effects of infection with Zika virus on neurogenesis at a larger scale, i.e., with organoids
Human iPS-derived brain organoids were exposed with ZIKV and followed for 11 days in vitro
Panels A and B ZIKV infected organoids in (B) show a reduction in their size when compared with MOCK organoids in (A).
Arrows show cells detaching from the brain organoid.
Panels C-E (C) and (D) show a comparison of organoid area before and after the experiment. The average area of MOCK organoids (C) was higher than ZIKV-infected organoids (D)
(E) shows a comparison of the two conditions after 46 days. The trend is the same.
Conclusion The average growth area of the organoids infected with ZIKV was reduced by 40% when compared to brain organoids in mock conditions.
reduced by 40% when compared to brain organoids under mock conditions
Brain organoids infected with Zika virus were, on average, 40% smaller
The growth rate of 12 individual organoids (6 per condition) was measured during this period
Both infected and uninfected organoids were immersed in a fixative solution to "freeze" them and allow them to be visualized.
It was then possible to use an electron microscope to compare the infrastructure of infected cells and uninfected cells
A nucleus whose chromatin has condensed in preparation for apoptosis (programmed cell death)
Fig. 3. ZIKV induces death in human neurospheres.
Objective To highlight the effects of ZIKV on the internal cellular structure.
Panels A and B (A) is a control, showing the Mock-infected neurosphere
(B) shows a ZIKV-infected neurosphere with several signs of cell death, including apoptotic nuclei, swollen mitochondria, and viral envelopes (arrow) bound to the smooth membrane structures
(C) and (D) Arrows point to ZIKV bound to cell surfaces (D) and inside mitochondria (C)
(E) and (F) Arrows point to viral envelopes close to smooth structures, similar to those previously described in cells infected with dengue virus.
Conclusion These results suggest that ZIKV induces cell death in human neural stem cells and thus impairs the formation of neurospheres.
ZIKV-infected cells in neurospheres presented smooth membrane structures (SMS) (Fig. 3, B and F), similarly to those previously described in other cell types infected with dengue virus (17).
Using in situ hybridization (labeling nucleic acids with probes) on sections of dengue-2 infected mosquito cells, Grief showed that in dengue-2 infected mosquito cells, the smooth membrane structures contained both viral RNA and virus particles.
This suggests that the smooth membrane structures are important sites for the concentration of viral RNA and possibly for formation of the viral envelope.
A nucleus that has started to prepare for programmed cell death (apoptosis)
Cells located in the central nervous system which protect and support neurons in their function.
Glial cells differ from neurons since they do not participate in electrical signaling.
Smaller than what can be seen with a light microscope
Fig. 2. ZIKV alters morphology and halts the growth of human neurospheres.
Major Question: What are the effects that ZIKV has on neural differentiation?
Panels A and B: A comparison at 3 days between a neurosphere made up of mock cells (A) and those that were (B). Mock cells show no abnormalities, but cells that were infected produced malformed neurospheres.
Panels C and D At 6 days, the mock neurosphere culture shows hundreds of live cells (C), while the ZIKV-infected plate contains only a few (D).
Panel E: The graph shows the number of neurospheres in the mock reaction as compared to various Zika MOI.
Conclusions: ZIKV infects human neural stem cells.
ZIKV infection resulted in both a decrease in cell viability and in morphological abnormalities
In a controlled experimental environment.
morphological abnormalities and cell detachment
Neurospheres that contained cells infected with Zika virus were oddly shaped, and some cells broke away.
Mock NSCs were not infected with Zika.
Real-time polymerase chain reaction, a variant of PCR. It allows for real-time monitoring of DNA amplification and quantification of the product.
Fig. 1. ZIKV infects human neural stem cells.
Major Question: What are the consequences of ZIKV infection in iPS-derived NSCs ?
Confocal microscopy is an imaging technique used here to generate the images of iPS-derived NSCs double stained for:
(A) Red stains reveal the presence of ZIKAV, confirming it can infect the cells.
(B) Stains display the expression of the protein SOX2, which is involved in expression of genes for embryonic development (making it vital for the pluripotency of embryonic stem cells). This shows that the cells are still undifferentiated.
(C) DAPI staining is a technique of labeling the DNA on A-T rich regions. It can penetrate the membrane of both live & fixed cells, but it stains live cells less efficiently.
(D) All the 3 pictures are mixed together to show the localization of ZIKV.
Panel E: Both concentrations of the virus (0.25 and 0.025 MOI) were effective (symbolized by the *) at infecting neural stem cells.
Panel F: RT-PCR data shows the increase of virus production at 2 and 3 days.
The "multiplicity of infection," which is the average number of virus particles that infect a cell.
neural stem cells
Undifferentiated cells in the nervous system that have the potential to develop into any type of cell.
induced pluripotent stem (iPS)
These are differentiated cells which have been reprogrammed into pluripotent ones. This means that they have the ability to develop into any type of cell.
to explore the consequences of ZIKV infection during neurogenesis and growth
In order to obtain neural stem cells from human iPS, researchers cultured iPS in a special medium.
To create neurospheres and organoids, neural stem cells were divided and again cultured in a special medium.
Finally, ZIKV was diluted and added to the different types of culture for 2 hours.
here is direct evidence that ZIKV is able to infect and cause death of neural stem cells (15)
Tang et al. obtained human neural progenitor cells (hNPCs) from stem cells. They used a particular ZIKV strain that successfully infected hNPCs, and found that the infected cells released ZIKV particles.
The growth of hNPCs was stunted, and an analysis of DNA content suggested that this attenuation might have been due to a disturbance in the cell cycle.
ZIKV has been described
In several case studies of pregnant women diagnosed with fetal microcephaly, the women suffered from symptoms of infection with Zika virus.
After miscarrying, ZIKAV RNA and antigens were detected in the placental tissues and the amniotic fluid of the microcephalic fetuses. The sequencing analysis of the virus genotype revealed a genotype of Asian origin.
Read more case studies that made headlines:
ZIKV had also been detected within the brain of a microcephalic fetus (
Zika virus has also been detected in microcephalic fetuses.
The Brazilian strain of the virus has been traced to an Asian strain.
The liquid that surrounds the fetus for its protection, keeping a constant temperature and environment.
An organ that develops only during pregnancy to provide oxygen and nutrients needed for the growth of the baby.
A type of viruses usually spread through mosquito and tick bites. They include West Nile and dengue virus.
In the womb.
The infection of a baby by a virus during a pregnancy.
Such a transmission between the baby and the mother is possible either through the placenta or the birth canal.
Syphilis is a sexually transmitted infection caused by a bacteria known as Treponema pallidum.
Herpes virus infections take place around mouth, lips, genitals, or rectum.
CMV infections spread through contact with body fluids, and often occur in those with weak immune systems.
Rubella is an RNA virus that is normally spread through the air by coughing or breathing.
A disease caused by a parasite called Toxoplasma gondiiand.
It is usually transmitted by eating uncooked food that contains cysts or by exposure to infected cat feces.
What are TORCHS factors? Learn more by reading the following links:
The cause of a disease or disorder.
Microcephaly is associated with decreased neuronal production as a consequence of proliferative defects and death of cortical progenitor cells
The cerebral cortex (the outer layer of the brain) shows the most severe reduction in microcephaly. This might be explained by reduced division in the cells that neurons come from, resulting in fewer neurons. This, in turn, leads to a smaller cerebral cortex.
A technique that uses a beam of electrons as a light source and has a magnification of up to 1,000,000x (a light microscope's magnification power is 1,500x).
A microscopy technique for seeing cellular components by targeting them in tissue samples.
An organ bud (miniature organ) that is anatomically similar to the organ it models. Organoids are used to study organ development and function.
A three-dimensional culture system made up of free-floating clusters of neural stem cells. They are used to study neural precursor cells in vitro.
An abnormally small head due to failure of the brain to grow sufficiently. It is associated with mental disability.
The growth of the brain can be impaired by many genetic and environmental factors, including infections by viruses and genetic syndromes.
Zika virus (ZIKV)
An RNA virus transmitted by mosquitos and sexual interaction with a carrier.
It was first isolated from the Zika Forest of Uganda in 1947. It was previously only known to occur in a narrow range in Africa and Asia. However, in 2015 there was a Zika outbreak in Brazil.
Zika virus impairs growth in human neurospheres and brain organoids. Garcez et al.
Zika: growing infection, shrinking neurons
The Zika virus is a contagious virus that can spread from a pregnant mother to her fetus, leading to a reduction in the size of the brain called microcephaly. This can cause mental disabilities in the child. The present study shows the effects of the Zika virus on the formation of neurons. The authors found a relationship between infection by the virus and reduced growth of neurons. It still remains to discover the consequences of Zika infection for each stage of fetal development.
- Sep 2016
Student’s t test,
A statistical test that is used to determine if two sets of data are significantly different from each other.