NBB-Psy data and tissue

Detailed clinical and pathological information of NBB-Psy donors

All donors with psychiatric disorders are asked to complete a medical questionnaire when they register. In addition, they are asked to complete a web-based questionnaire to generate five-yearly updates on relevant clinical parameters. Researchers can request clinical information with the tissue, such as  the diagnoses as well as clinical and medication use. The information contains the clinical diagnosis, such as Major Depressive Disorder but also a clinical history of symptoms like apathy, hallucinations or disorientation. This is supported by additional post-mortem neuropathological characterisation, confirming, for example the absence of Alzheimer’s Disease by Braak staging, the size of the amygdala or different protein aggregations (e.g. amyloid beta). In addition, a large selection of standardised parameters will also become available through the Netherlands Neurogenetics Database.

 

NBB-Psy tissue Up to 200 samples from different brain regions Fresh frozen and fixed tissue blocks Fresh tissue (prospective collection) Glass mounted FFPE slides Biofluids: CSF, buffy coat & plasma iPSCs and microglia 5-yearly medical questionnaire Detailed neuropathological characterization

Large collection of frozen and fixed NBB-Psy tissue

Normally, the NBB obtains for all donors approximately 90 different samples. This includes tissue from different brain regions, such as hippocampus, amygdala and cortical areas as well as cerebrospinal fluid (CSF), buffy coat and plasma. Brain tissue from the left hemisphere is stored as frozen tissue, tissue from the right hemisphere is formalin fixed paraffin embedded (FFPE).In order to optimally support researchers who investigate psychiatric disorders, approximately 200 samples are collected for NBB-Psy, including frozen en fixed tissue from both hemispheres to investigate lateralisation as well as extra samples from the same region. 

Primary microglia and iPSCs supporting downstream molecular analyses

NBB-Psy has used novel methods to enrich the brain material (see also e-NBB). We generated primary microglia and fibroblast-derived human induced pluripotent stem cells (iPSC) of a subset of the NBB-Psy donors. This allows for research into unravelling genetic and environmental factors contributing to development of psychiatric disorders. Due to the long-standing experience of the NBB with postmortem tissue and optimising conditions such as the post mortem delay (average 6.5 h), the tissue is of excellent quality (e.g. optimal RNA integrity as described in a recent publication[1]).  This supports the use of the NBB-Psy collection for state of the art molecular techniques, including: single cell or single nucleus RNA expression, sequencing and protein profiling in selected tissues. Additionally, the blood and CSF samples enable translational research including biomarker identification and validation. For an overview of the high-quality data obtained with NBB-Psy tissue, see publications.

• Isolation of primary microglia

Pure microglia were isolated from white matter and occipital cortex using rapid procedure based on Percoll density gradient centrifugation followed by CD11b-positive selection . Isolated microglia are phenotyped using flow cytometric analysis and stored as lysates in lysisbuffer Trisure in -80°C or cryogenically frozen in 20% DMSO in liquid nitrogen.

• Human induced pluripotent stem cells (iPSC’s)

Fibroblasts have been isolated (from skin fibroblasts) of major depression, schizophrenia, bipolar disorder, autism spectrum disorder, post-traumatic stress disorder and control donors. These fibroblast cultures have been reprogrammed into pluripotent stem cell lines and will be made available to researchers as cryogenically frozen samples. Through published protocols, researchers can obtain neural stem cells, and subsequently acquire donor-specific glial cells and neurons. A list of iPSC lines and donor characteristics can also be found in the NBB iPSC document.

iPSC collection procedure

The iPSCs are derived at the Erasmus Medical Centre IPS core facility using a fast epigenetic silencing lentiviral vector with a multicistronic reprogramming vector carrying Oct4, Klf4, Sox2 and c-Myc (Warlich et.al., 2011)[1]. It also carries a red fluorescent marker to monitor silencing of reprogramming factors and the selection of thoroughly reprogrammed iPS clones. These lines are derived and cryopreserved on irradiated mouse embryonic fibroblasts in Human ES medium. From each line several validated clones are banked. Each clone has an associated document providing information about culture, cryopreservation and quality controls. Clones can be provided in common feeder free media at additional cost. For feeder free media the options are mTeSR1, mTeSRplus or Stemflex in combination with a Geltrex, Matrigel or UltiMatrix coating.