A population of more than six 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.

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well‐controlled cell model systems. We have developed a first nonintegration‐reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high‐resolution whole genome CGH‐array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high‐content microscopic analysis. Early differentiation shows an imbalance of the lineage‐specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC‐derived neurons show increased production of amyloid peptide‐containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21‐derived neurons show significantly higher number of DNA double‐strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21. Stem Cells 2015;33:2077–2084

Children with Down syndrome (DS) and acute lymphoblastic leukaemia (ALL) have poorer survival and more relapses than non-DS children with ALL, highlighting an urgent need for deeper mechanistic understanding of DS-ALL. Here, using full-exome or cancer genestargeted sequencing of 42 ALL samples from 39 DS patients, we uncover driver mutations in RAS, (KRAS and NRAS) recurring to a similar extent (15/42) as JAK2 (12/42) mutations or P2RY8-CRLF2 fusions (14/42). RAS mutations are almost completely mutually exclusive with JAK2 mutations (P ¼ 0.016), driving a combined total of two-thirds of analysed cases. Clonal architecture analysis reveals that both RAS and JAK2 drove sub-clonal expansions primarily initiated by CRLF2 rearrangements, and/or mutations in chromatin remodellers and lymphocyte differentiation factors. Remarkably, in 2/3 relapsed cases, there is a switch from a primary JAK2-or PTPN11-mutated sub-clone to a RAS-mutated sub-clone in relapse. These results provide important new insights informing the patient stratification strategies for targeted therapeutic approaches for DS-ALL.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have 4 a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 5 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or 6 interference (CRISPRi). However, the safety concerns associated with viral vectors and the 7 possible off-target issues of systemic administration remain huge concerns to be safe delivery 8 methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide 9(SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 10 systems. Specifically, an amphiphilic negatively charged SAPis first coated onto PCL 11 nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively 12 charged SAP + -RGD are then absorbed via electrostatic interactions. The SAP-coated scaffolds 13 facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell 14 adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF 15 expression in mammalian cells, and the secreted GDNF subsequently promotes neurite 16 outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide 17 coated nanofibers can be a new route to establish a bioactive interface, which provides a simple 18 and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.

Scaffold-mediated RE-1 silencing factor (REST) knockdown enhanced neuronal differentiation from human iPSC-derived neural progenitor cells after transplantation to the injured spinal cord tissues.

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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 T21organoids 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 crossinhibited 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.These studies uncovered that BACE2 can cleave the product of β-secretase (APP β-CTF) between aa19 and aa20, generating a 1-19 fragment [13][14][15] , thereby potentially preventing the formation of amyloidogenic Aβ, and degrading the β-CTF that has been implicated in neuronal toxicity, and impairment of several neuronal functions, such as axonal transport and autophagy 16 .When offered synthetic Aβ40/42 peptides in solution, purified BACE2 protein can rapidly degrade them by cutting after aa20 and aa34, to generate the 1-20 and 1-34 peptide products, but only at very acidic pH (3.5-4). In this reaction, BACE2 is 150-fold more efficient than BACE1, which is also capable of this cleavage, upon conditions of increased enzyme concentration/time 12,14 . Neither of these two putative anti-amyloidogenic actions of BACE2 (the θ-secretase activity, generating aa1-19, or the Aβ-degrading-protease activity (AβDP or Aβ clearance) generating aa1-20 and aa1-34), have yet been demonstrated to be the functional role of BACE2 under physiologically fluctuating gene doses in vivo in the human brain. A naturally occurring form of gene overdose for both APP and BACE2 is DS, caused by the trisomy of human chromosome 21 (T21) that harbours both APP and BACE2 genes. As increased levels of soluble Aβ were observed already in foetal brains in DS 17 , we examined cerebral organoids grown from induced pluripotent stem cell (iPSC) generated by non-integr...