Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate

This suite of studies represents a landmark achievement in illuminating the complexity of the human brain at the cellular level," said John Ngai, Ph.D., director of the NIH BRAIN Initiative. "The scientific collaborations forged through BICCN are propelling the field forward at an exponential pace; the progress -- and possibilities -- have been simply breathtaking." Differentially expressed genes were identified by using generalized linear models that incorporate empirical Bayes methods to shrink the standard errors towards a common value, thereby improving testing power 36, 40. Genes were defined as significantly changed when FDR< 0.05. Venn diagrams, showing the differentially expressed genes were made using Venny 2.1 41. The top 500 genes with the most variation were selected by using the interquartile range (IQR) as a measure, which is the difference between the 25th and 75th percentile. The coefficient of variation, which measures how much the values differ within a treatment, was set to 0.5 to select for differential gene expression that was related to the treatment instead of at random. Data was standardized by converting values to a z-score which sets the average to 0 and scales the other values from – 2 to 2. A principle component analysis (PCA) was performed using the library PCAtools on the top 500 differentially expressed genes (see selection process of the genes below) 42. Subsequently, all individual samples were hierarchically clustered based on of the top 500 differentially expressed genes and presented in a heatmap using heatmapper 43. Characterization of cellular makeup of IEC layers The epithelial cells that make up the nose are classified into three types: basal, supporting, and olfactory receptor cells. The basal cell (seen in the image below) is a stem cell that gives rise to the olfactory receptor cells. There are several theories about the origin of small molecules that led to life on the early Earth. They may have been carried to Earth on meteorites (see Murchison meteorite), created at deep-sea vents, or synthesized by lightning in a reducing atmosphere (see Miller–Urey experiment). There is little experimental data defining what the first self-replicating forms were. RNA is thought to be the earliest self-replicating molecule, as it is capable of both storing genetic information and catalyzing chemical reactions (see RNA world hypothesis), but some other entity with the potential to self-replicate could have preceded RNA, such as clay or peptide nucleic acid. [41] Gabaldón, T. (October 2021). "Origin and Early Evolution of the Eukaryotic Cell". Annual Review of Microbiology. 75 (1): 631–647. doi: 10.1146/annurev-micro-090817-062213. PMID 34343017. S2CID 236916203.The bud is made up of two types of cells: supporting cells and gustatory cells. The supporting (sustentacular) cells, which are commonly arranged in the same way that cask staves do, serve as an outer envelope for the bud. However, a few of these are found within the bud’s interior between its gustatory cells. The Anatomy Of A Taste Bud Genes contain the instructions to produce specific proteins within cells. However, some genes undergo a process called alternative splicing, where cells selectively combine gene fragments to generate multiple protein variants. This mechanism enhances the complexity and adaptability of our bodies, playing a crucial role in the functioning of living organisms.

Multicellularity has evolved independently at least 25 times, [37] including in some prokaryotes, like cyanobacteria, myxobacteria, actinomycetes, Magnetoglobus multicellularis, or Methanosarcina. However, complex multicellular organisms evolved only in six eukaryotic groups: animals, fungi, brown algae, red algae, green algae, and plants. [38] It evolved repeatedly for plants ( Chloroplastida), once or twice for animals, once for brown algae, and perhaps several times for fungi, slime molds, and red algae. [39] Multicellularity may have evolved from colonies of interdependent organisms, from cellularization, or from organisms in symbiotic relationships.

Several human intestinal cell models have been used to study the mode of action of SCFAs. Initially, well-characterized conventional cell-line based models like Caco-2 cells and their coculture variants (i.e. Caco-2 and HT29 cells) were exposed to SCFAs to study their effects 18, 19. However, these are cancer cells and the effects of SCFAs on these cells cannot be easily extrapolated to human in vivo conditions as cancer cells do not use butyrate as their energy source. This leads to a build-up of butyrate and is referred to as the butyrate paradox 20, 21, 22, 23. Novel directions in research have been made possible after the hallmark success of culturing primary intestinal tissues and adult stem cell-based 3D human intestinal organoids (HIOs) in vitro. Subsequently, induced pluripotent stem cell (iPSC) based 3D HIOs were developed 24. iPSCs can be differentiated into a large variety of cells in the human body by applying growth factors during their development. Using a protocol for differentiation into HIOs resulted in a microarchitecture and cell type composition similar to the human intestine. iPSC derived HIOs consist of enterocytes, goblet cells, mesenchymal and enteroendocrine cells 25, 26, 27, 28. Furthermore, iPSC derived HIOs have shown improved cytochrome P450 and transporter expression in comparison with Caco-2 cells 29, 30. More recently, iPSCs have been used to create 2D intestinal epithelial cell (IEC) layers that emulate the full cellular complexity of the 3D HIOs, but have an easily accessible apical and basolateral side in comparison with their 3D HIO variants which are closed spheres. This difference makes it easier to perform relevant experiments as compounds can be added to the apical side which represents the lumen, there is no build-up of cellular debris near the cells, cells are not encompassed in extracellular matrix providing free access and samples opportunities. The interaction between mitochondria and the endoplasmic reticulum is also altered in syndromes presenting insulin resistance, such as diabetes and obesity. Therefore, this finding presents a potential therapeutic strategy worth exploring," explains Dr. Zorzano, who is also a Professor at the Faculty of Biology of the University of Barcelona (UB) and a member of CIBERDEM. Both the PCA analysis and the hierarchical clustering indicated a distinct effect on gene expression profiles following exposure to 10 mM butyrate compared with all other exposures, while the gene expression profiles following butyrate 1 mM and propionate 10 mM clustered together in both the PCA and the hierarchical clustering. The remaining exposure conditions (i.e. 1 and 10 mM acetate, 1 mM propionate) clustered together with the control exposure group in both the PCA and the hierarchical clustering indicating little effect of the exposure. Pathway analysis (i.e. GSEA) on the data from the exposure groups with a high number of differentially expressed genes (i.e. propionate 10 mM, butyrate 1 mM and butyrate 10 mM) revealed a clear concentration-dependent switch in the differential gene expression in the butyrate exposure groups from mostly upregulated pathways at 1 mM (8 down- and 72 upregulated pathways) to mostly downregulated pathways at 10 mM (89 down- and 18 upregulated pathways). Exposure to the low butyrate concentration appeared to induce metabolism and absorption processes as indicated by the upregulated pathways (7 pathways out of the top 10). Pathways related to genetic information processing were mainly downregulated (5 pathways out of the top 7) and were related to RNA transcription, suggesting that low butyrate concentrations could lead to a downregulation of protein synthesis. Six of the top 10 induced pathways, following exposure to 10 mM butyrate, were related to genetic information processing and could be connected to DNA replication and repair, suggesting that high concentrations can induce DNA damage, but there were no indications of induced apoptosis or cell death. Four of the top 10 downregulated pathways, following exposure to butyrate 10 mM, were related to signal transduction. Propionate 10 mM had overall less affected pathways in comparison with butyrate exposure, 47 pathways in total, and the numbers were less skewed with 16 down- and 31 upregulated pathways. It appears that propionate 10 mM exposure induced absorption and metabolism processes as 7 of the top 10 upregulated pathways were connected to these processes. In the downregulated pathways 5 out of 10 pathways were in the genetic information processing category and related mostly to RNA and protein processing processes. Interestingly, many of the up- and downregulated pathways in the top 10 were similar to those of the butyrate 1 mM exposure, indicating that these treatments seem to have similar effects.