Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discoverySG was supported by the Roche Postdoctoral Fellowship (RPF) program and IP by the Roche Internships for Scientific Exchange (RiSE) progra

Evaluation of bone mineral density and bone turnover in children on anticoagulation

BackgroundChildhood and adolescence are critical periods of bone mineral acquisition. Children on anticoagulation (AC) might have an increased risk for reduced bone mineral density (BMD). Risk factors for impaired bone accumulation include chronic diseases, immobility, and medication. Vitamin K (VK) deficiency reflected by undercarboxylated osteocalcin levels (ucOC) has been identified as a predictor of osteoporosis and fractures. Data on bone health in children under AC are sparse.AimsTo evaluate BMD in children on AC and characterize the risk factors of low BMD, including VK and Vitamin D (VD) status.MethodsSingle-center cross-sectional study of clinical, biochemical, and densitometric parameters. Assessment of VK surrogate parameters included ucOC and matrix gla protein (MGP).ResultsA total of 39 children (4–18 years; 12 females) receiving AC were included, 31 (79%) on VK antagonists and 8 (21%) on direct oral anticoagulants. Overall, BMD was decreased for both the lumbar spine (LS; −0.7SDS) and total body less head (TBLH; −1.32SDS) compared with pediatric reference data. Significant associations were found between early pubertal development and TBLH-BMD, and between BMI and LS-BMD. VK surrogate parameters were highly related to patients’ age and pubertal development. Neither serum parameters nor AC-related factors predicted BMD. VD was detected in 10/39 patients with lower values during puberty.ConclusionOur data indicate BMD reduction in pediatric patients on AC. Although AC-related factors did not predict reduced BMD, low BMI and pubertal stages represented important risk factors. Awareness of risk factors for low BMD and high prevalence of VD deficiency during puberty could contribute to the improvement of bone health in this vulnerable patient group

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.Initiative Joint Undertaking under grant agreement no. 115439, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. A.H., S.C., and M.Z.C. were also funded by the NIHR (Oxford BRC). K.M. and A.B. were also supported by the NIHR GOSH BRC

Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells.

Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells

Generation of a homozygous GBA deletion human embryonic stem cell line

We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to a complete loss of glucocerebrosidase (GCase) protein expression