Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

University of Cologne

Labeled nitrogen ((15)N) was applied to a soil-based substrate in order to study the uptake of N by Glomus intraradices extraradical mycelium (ERM) from different mineral N (NO(3)(-) vs. NH(4)(+)) sources and the subsequent transfer to cowpea plants. Fungal compartments (FCs) were placed within the plant growth substrate to simulate soil patches containing root-inaccessible, but mycorrhiza-accessible, N. The fungus was able to take up both N-forms, NO(3)(-) and NH(4)(+). However, the amount of N transferred from the FC to the plant was higher when NO(3)(-) was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher (15)N enrichment when the FC was supplied with (15)NH(4)(+) compared with (15)NO(3)(-). The (15)N shoot/root ratio of plants supplied with (15)NO(3)(-) was much higher than that of plants supplied with (15)NH(4)(+), indicative of a faster transfer of (15)NO(3)(-) from the root to the shoot and a higher accumulation of (15)NH (4)(+) in the root and/or intraradical mycelium. It is concluded that hyphae of the arbuscular mycorrhizal fungus may absorb NH(4)(+) preferentially over NO(3)(-) but that export of N from the hyphae to the root and shoot may be greater following NO(3)(-) uptake. The need for NH(4)(+) to be assimilated into organically bound N prior to transport into the plant is discussed.

Influence of different mineral nitrogen sources (NO3(-)-N vs. NH4(+)-N) on arbuscular mycorrhiza development and N transfer in a Glomus intraradices-cowpea symbiosis.

Biomass estimation of arbuscular mycorrhiza (AM) fungi, widespread plant root symbionts, commonly employs lipid biomarkers, predominantly the fatty acid 16:1ω5. We briefly reviewed the application of this signature fatty acid, followed by a case study comparing biochemical markers with microscopic techniques in an arable soil following a change to AM non-host plants after 27 years of continuous host crops, that is, two successive cropping seasons with wheat followed by amaranth. After switching to the non-host amaranth, spore biomass estimated by the neutral lipid fatty acid (NLFA) 16:1ω5 decreased to almost nil, whereas microscopic spore counts decreased by about 50% only. In contrast, AM hyphal biomass assessed by the phospholipid (PLFA) 16:1ω5 was greater under amaranth than wheat. The application of PLFA 16:1ω5 as biomarker was hampered by background level derived from bacteria, and further enhanced by its incorporation from degrading spores used as microbial resource. Meanwhile, biochemical and morphological assessments showed negative correlation for spores and none for hyphal biomass. In conclusion, the NLFA 16:1ω5 appears to be a feasible indicator for AM fungi of the Glomales group in the complex field soils, whereas the use of PLFA 16:1ω5 for hyphae is unsuitable and should be restricted to controlled laboratory studies.

Use of the signature Fatty Acid 16:1ω5 as a tool to determine the distribution of arbuscular mycorrhizal fungi in soil.

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th β-strands (β9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis.

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.

CPAP promotes timely cilium disassembly to maintain neural progenitor pool.

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.

Elke Gabriel

Center for Molecular Medicine Cologne

University of Cologne

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Elke Gabriel

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University of Cologne

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Center for Molecular Medicine Cologne