Diferencijacija stanica u primarnoj kulturi neurona iz dva soja transgeničnih miševa

Matične stanice mogu se diferencirati u različite vrste stanica i nailaze na široku eksperimentalnu primjenu. Iz tog su razloga istraživanja matičnih stanica u posljednjih petnaestak godina u velikom porastu. Cilj je ovog istraživanja usporedba diferencijacije neurona podrijetlom iz dvaju različitih transgeničnih sojeva miša u svrhu prikaza njihovih osobitosti za daljnju primjenu u istraživanjima matičnih stanica. U ovom je radu uzgojena primarna kultura neurona iz dvaju mišjih sojeva, ubikvitarnog GFP-LUC i Thy1-YFP. Uzgojeni neuroni analizirani su 7. i 14. dan, opisan je uzorak izražaja bjelančevina te su međusobno uspoređeni neuroni podrijetlom iz dvaju navedenih mišjih sojeva. Stanice mišjega soja GFP-LUC tijekom 7. dana diferencirale su se u potpunosti u zrele neurone i astrocite. U 100 % stanica pozitivan je GFP. U ovoj su skupini stanica biljezi neurona (MAP2, ß3-tubulin i NeuN) pozitivni u 90 % diferenciranih stanica, a biljeg astrocita (GFAP) samo u 10 % stanica. Tijekom 14. dana stanice su razvijenije, na što upućuju deblji nastavci koji su prisutni u većem broju. Biljezi neurona pozitivni su u 80 % diferenciranih stanica, a biljeg astrocita pozitivan je u 20 % stanica. Stanice mišjega soja Thy1-YFP tijekom 7. dana također su se diferencirale u zrele neu- rone i astrocite, međutim samo je 20 % neurona YFP-pozitivno. Kao i u GFP-LUC mišjeg soja, 7. dan diferencijacije prisutno je 90 % neurona i 10 % astrocita, a 14. dan 80 % neurona i 20 % astrocita

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain

A population of >6 million people worldwide at high risk of Alzheimer’s disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of β-amyloid-(Aβ)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aβ deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome-21-gene BACE2, but prevented by combined chemical β and γ-secretase inhibition. We found that T21-organoids secrete increased proportions of Aβ-preventing (Aβ1-19) and Aβ-degradation products (Aβ1-20 and Aβ1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1-inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases

Cerebral organoids with chromosome 21 trisomy secrete Alzheimer's disease-related soluble aggregates detectable by single-molecule-fluorescence and super-resolution microscopy

Understanding the role of small, soluble aggregates of beta-amyloid (Aβ) and tau in Alzheimer's disease (AD) is of great importance for the rational design of preventative therapies. Here we report a set of methods for the detection, quantification, and characterisation of soluble aggregates in conditioned media of cerebral organoids derived from human iPSCs with trisomy 21, thus containing an extra copy of the amyloid precursor protein (APP) gene. We detected soluble beta-amyloid (Aβ) and tau aggregates secreted by cerebral organoids from both control and the isogenic trisomy 21 (T21) genotype. We developed a novel method to normalise measurements to the number of live neurons within organoid-conditioned media based on glucose consumption. Thus normalised, T21 organoids produced 2.5-fold more Aβ aggregates with a higher proportion of larger (300-2000 nm2) and more fibrillary-shaped aggregates than controls, along with 1.3-fold more soluble phosphorylated tau (pTau) aggregates, increased inflammasome ASC-specks, and a higher level of oxidative stress inducing thioredoxin-interacting protein (TXNIP). Importantly, all this was detectable prior to the appearance of histological amyloid plaques or intraneuronal tau-pathology in organoid slices, demonstrating the feasibility to model the initial pathogenic mechanisms for AD in-vitro using cells from live genetically pre-disposed donors before the onset of clinical disease. Then, using different iPSC clones generated from the same donor at different times in two independent experiments, we tested the reproducibility of findings in organoids. While there were differences in rates of disease progression between the experiments, the disease mechanisms were conserved. Overall, our results show that it is possible to non-invasively follow the development of pathology in organoid models of AD over time, by monitoring changes in the aggregates and proteins in the conditioned media, and open possibilities to study the time-course of the key pathogenic processes taking place

Dose imbalance of DYRK1A kinase causes systemic progeroid status in Down syndrome by increasing the un-repaired DNA damage and reducing LaminB1 levels.

BACKGROUND: People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down. METHODS: Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n = 246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n = 256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids. FINDINGS: Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage. INTERPRETATION: Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies. FUNDING: Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements"