Morphological Analysis of Neurons and Glia Using Mosaic Analysis with Double Markers.

Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS). MADM for any candidate gene can easily incorporate morphological analysis within the experimental workflow. Here, we describe the methods of morphological cell analysis which we developed in the course of diverse recent MADM studies. This chapter will specifically focus on methods to quantify aspects of the morphology of neurons and astrocytes within the CNS, but these methods can broadly be applied to any MADM-labeled cells throughout the entire organism. We will cover two analyses-soma volume and dendrite characterization-of physical characteristics of pyramidal neurons in the somatosensory cortex, and two analyses-volume and Sholl analysis-of astrocyte morphology.

TGFß superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor ß (TGFß) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFß-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Identification of neural oscillations and epileptiform changes in human brain organoids.

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.

Prevalence and characterization of carbapenem-resistant bacteria in water bodies in the Los Angeles-Southern California area.

Carbapenems are ß-lactam antibiotics used in healthcare settings as last resort drugs to treat infections caused by antibiotic-resistant bacteria. Carbapenem-resistant bacteria are increasingly being isolated from healthcare facilities; however, little is known about their distribution or prevalence in the environment, especially in the United States, where their distribution in water environments from the West Coast has not been studied before. The aim of this study was to determine the prevalence of carbapenem-resistant bacteria and carbapenemase genes in water bodies from the Los Angeles area (California, USA). All samples that were analyzed contained carbapenem-resistant bacteria with a frequency of between 0.1 and 324 carbapenem-resistant cfu per 100 mls of water. We identified 76 carbapenem-resistant or -intermediate isolates, most of which were also resistant to noncarbapenem antibiotics, as different strains of Enterobacter asburiae, Aeromonas veronii, Cupriavidus gilardii, Pseudomonas, and Stenotrophomonas species. Of them, 52 isolates were carbapenemase-producers. Furthermore, PCR and sequence analysis to identify the carbapenemase gene of these carbapenemase-producing isolates revealed that all Enterobacter asburiae isolates had a blaIMI-2 gene 100% identical to the reference sequence, and all Stenotrophomonas maltophlia isolates had a blaL1 gene 83%-99% identical to the reference blaL1 . Our findings indicate that water environments in Southern California are an important reservoir of bacteria-resistant to carbapenems and other antibiotics, including bacteria carrying intrinsic and acquired carbapenemase genes.