Identical Cells, Different Responses

Cellular heterogeneity is a biological phenomenon in which genetically identical cells respond differently to the same stimuli. This diversity has significant implications in medical contexts. Heterogeneity directly influences essential processes such as growth, stress response, and cell survival. For example, in cancer, some tumour cells survive treatment while others perish; similarly, in bacterial infections, heterogeneity helps explain how antibiotic resistance emerges. Although we can observe and quantify these differences, the molecular mechanisms underlying this diversity remain largely unknown, representing a major challenge in modern biology.

The development of single-cell analytical techniques has transformed our capacity to study heterogeneity. RNA sequencing of individual cells has allowed the classification of variation in cell populations at multiple levels. However, to advance toward a causal understanding, a system that combines genetic and environmental perturbations at a genomic scale is needed.

To this end, a team of researchers led by Dr. Francesc Posas and Dr. Laia de Nadal at IRB Barcelona have developed a tool for the high-throughput analysis of the relationship between genes and cell “behaviour”. This tool provides key data on how gene expression heterogeneity affects processes such as cell differentiation and ageing. Published in the journal Nature Communications, the work presents a library of RNA “barcodes”, designed to simultaneously track the genotype and transcriptome of each cell in high-throughput sequencing experiments.

“Thanks to this advancement, we have developed a novel transcriptional map of a cell population, as well as for more than 4,000 mutants, that reveals the heterogeneity of the population and the phenotypic consequences of the same,” explains Dr. Posas.

A new version of the yeast knockout collection

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To this end, the team “reconfigured” the standard mutant yeast collection (YKOC) so that each mutant was uniquely identified. Thus, RNA analysis of the cell population using sequencing techniques allowed them to easily identify the mutant that corresponded to each cell. Thanks to the implementation of a microwell platform, the technique significantly reduces the cost and improves efficiency with respect to traditional methods.

“Our main aim was to discover how, in response to the same signal, cells activate certain genes while others do not. This variability is difficult to study in global experiments and single-cell tools are needed,” says Dr. Mariona Nadal, first author of the study.

The states of cell diversity

The tool developed by the team has allowed the profiling of more than a million cells from 3,500 mutants under control and stress conditions. The findings of the study reveal that, although most of the mutants share a pattern of “nucleus”  or “core” cellular states resistant to perturbations, 10% deviate from these and “stall” in specific states. Related to functions such as iron metabolism and hypoxic response, these states can affect the behaviour of cells and their capacity to adapt.

Among the key conclusions of the study is evidence that transcriptional diversity, even in a single-cell organism like yeast, is organized in a continuum of cellular states. This finding may have important implications for understanding processes like ageing, treatment resistance, and response to adverse environmental conditions, in more complex organisms.

Related article:
A single-cell resolved genotype-phenotype map using genome-wide genetic and environmental perturbations
Mariona Nadal-Ribelles, Carme Solé, Anna Diez-Villanueva, Camille Stephan-Otto Attolini, Yaima Matas, Lars Steinmetz, Eulalia de Nadal, and Francesc Posas
Nature Communications  (2025) DOI: 10.1038/s41467-025-57600-4