Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease.

BACKGROUND: The function of the blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the associated molecular mechanisms, particularly with respect to the high-risk APOE4/4 genotype, are not well understood. For this purpose, we developed a multicellular isogenic model of the neurovascular unit (NVU) based on human induced pluripotent stem cells. METHODS: The human NVU was modeled in vitro using isogenic co-cultures of astrocytes, brain capillary endothelial-like cells (BCECs), microglia-like cells, neural stem cells (NSCs), and pericytes. Physiological and pathophysiological properties were investigated as well as the influence of each single cell type on the characteristics and function of BCECs. The barriers established by BCECs were analyzed for specific gene transcription using high-throughput quantitative PCR. RESULTS: Co-cultures were found to tighten the barrier of BCECs and alter its transcriptomic profile under both healthy and disease conditions. In vitro differentiation of brain cell types that constitute the NVU was not affected by the LOAD background. The supportive effect of NSCs on the barrier established by BCECs was diminished under LOAD conditions. Transcriptomes of LOAD BCECs were modulated by different brain cell types. NSCs were found to have the strongest effect on BCEC gene regulation and maintenance of the BBB. Co-cultures showed cell type-specific functional contributions to BBB integrity under healthy and LOAD conditions. CONCLUSIONS: Cell type-dependent transcriptional effects on LOAD BCECs were identified. Our study suggests that different brain cell types of the NVU have unique roles in maintaining barrier integrity that vary under healthy and LOAD conditions. .

Generation of a set of induced pluripotent stem cell lines from two Alzheimer disease patients carrying APOE4 (MLUi007-J; MLUi008-A) and healthy old donors carrying APOE3 (MLUi009-A; MLUi010-B) to study APOE in aging and disease.

Late-onset Alzheimer disease (LOAD) is the most frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for LOAD. Four human induced pluripotent stem cell (iPSC) lines MLUi007-J, MLUi008-B, MLUi009-A, and MLUi010-B were generated from LOAD patients and healthy matched donors by reprogramming of B-lymphoblastoid cells (B-LCLs) with episomal plasmids. The application of B-LCLs holds a great promise to model LOAD and other diseases because they can easily be generated from primary peripheral blood mononuclear cells (PBMCs) by infection with the Epstein-Barr virus (EBV).

Analysis of genomic alterations in cancer associated human pancreatic stellate cells.

Pancreatic stellate cells (PSCs) constitute important cells of the pancreatic microenvironment and their close interaction with cancer cells is important in pancreatic cancer. It is currently not known whether PSCs accumulate genetic alterations that contribute to tumor biology. Our aim was to analyze genetic alterations in cancer associated PSCs. PSC DNA was matched to DNA isolated from pancreatic cancer patients' blood (n = 5) and analyzed by Next-Generation Sequencing (NGS). Bioinformatic analysis was performed using the GATK software and pathogenicity prediction scores. Sanger sequencing was carried out to verify specific genetic alterations in a larger panel of PSCs (n = 50). NGS and GATK analysis identified on average 26 single nucleotide variants in PSC DNA as compared to the matched blood DNA that could be visualized with the Integrative Genomics Viewer. The absence of PDAC driver mutations (KRAS, p53, p16/INK4a, SMAD4) confirmed that PSC isolations were not contaminated with cancer cells. After filtering the variants, using different pathogenicity scores, ten genes were identified (SERPINB2, CNTNAP4, DENND4B, DPP4, FGFBP2, MIGA2, POLE, SNRNP40, TOP2B, and ZDHHC18) in single samples and confirmed by Sanger sequencing. As a proof of concept, functional analysis using control and SERPINB2 knock-out fibroblasts revealed functional effects on growth, migration, and collagen contraction. In conclusion, PSC DNA exhibit a substantial amount of single nucleotide variants that might have functional effects potentially contributing to tumor aggressiveness.

Hypoxia supports reprogramming of mesenchymal stromal cells via induction of embryonic stem cell-specific microRNA-302 cluster and pluripotency-associated genes.

Pluripotency is characterized by specific transcription factors such as OCT4, NANOG, and SOX2, but also by pluripotency-associated microRNAs (miRs). Somatic cells can be reprogrammed by forced expression of these factors leading to induced pluripotent stem cells (iPSCs) with characteristics similar to embryonic stem cells (ESCs). However, current reprogramming strategies are commonly based on viral delivery of the pluripotency-associated factors, which affects the integrity of the genome and impedes the use of such cells in any clinical application. In an effort to establish nonviral, nonintegrating reprogramming strategies, we examined the influence of hypoxia on the expression of pluripotency-associated factors and the ESC-specific miR-302 cluster in primary and immortalized mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) treatments led to the induction of OCT4 and NANOG in an immortalized cell line L87 and primary MSCs, accompanied with increased doubling rates and decreased senescence. Most importantly, the endogenous ECS-specific cluster miR-302 was induced upon hypoxic culture and FGF2 supplementation. Hypoxia also improved reprogramming of MSCs via episomal expression of pluripotency factors. Thus, our data illustrate that hypoxia in combination with FGF2 supplementation efficiently facilitates reprogramming of MSCs.