Multiplexed Division Tracking Dyes for Proliferation-Based Clonal Lineage Tracing

Abstract

The generation of cellular heterogeneity is an essential feature of immune responses. Understanding the heritability and asymmetry of phenotypic changes throughout this process requires determination of clonal-level contributions to fate selection. Evaluating intraclonal and interclonal heterogeneity and the influence of distinct fate determinants in large numbers of cell lineages, however, is usually laborious, requiring familial tracing and fate mapping. In this study, we introduce a novel, accessible, high-throughput method for measuring familial fate changes with accompanying statistical tools for testing hypotheses. The method combines multiplexing of division tracking dyes with detection of phenotypic markers to reveal clonal lineage properties. We illustrate the method by studying in vitro-activated mouse CD8 T cell cultures, reporting division and phenotypic changes at the level of families. This approach has broad utility as it is flexible and adaptable to many cell types and to modifications of in vitro, and potentially in vivo, fate monitoring systems.

Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

“…Defining a family as all descendants from a marked ancestor cell, the experimental method employed here is based on the observation that by labeling cells with distinct combinations of division diluting dyes, such as 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and CellTrace Violet (CTV), through the use of flow cytometry it is possible to simultaneously determine each cell's family membership, generation (i.e. number of cell divisions), and phenotype [23,24] (Figure 1). In the present application, by index sorting labeled cells, we could also relate down-stream familial fate to ancestral cell surface expression.…”

“…Tackling it requires an experimental system that enables the simultaneous identification of cells that are descendent from a common ancestor, the number of divisions that has led to each of them, and their differentiation status. Towards that end, we further developed a recently published divisiondye multiplex system that was introduced for the study of lymphocytes [23,24], making it suitable for the study of hematopoietic system. Amongst other findings, the data from our study revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and there were significant familial effects on differentiation.…”

Modifying clonal selection theory with a probabilistic cell

“…In other situations, recently discovered, division will have no effect on the time to stop dividing or the time to die . This latter rule should lead to families that share timed events (such as division cessation or death) in the same generation, as is frequently observed by experiment . Given the mechanics and rules of interaction between components, and the effects to be altered over time, a calculation scheme to model the population cell dynamics in number and type is possible.…”

“…59 This latter rule should lead to families that share timed events (such as division cessation or death) in the same generation, 59 as is frequently observed by experiment. 50,60,61 Given the mechanics and rules of interaction between components, and the effects to be altered over time, a calculation scheme to model the population cell dynamics in number and type is possible. As processes are operating and dictating cell changes over time and require signal integration and affect differentiation, it is reminiscent of the calculus, and with Amanda Gett, we labeled these operations as the Cellular Calculus.…”

“…Evidence that these molecular "construction" differences are significant is highlighted by the finding of remarkable fate similarities between siblings and families in vitro and even in vivo. [50][51][52]60,61,83,84 It appears if cells are molecular "clones" they behave almost identically, and that the large amount of diversity between similar cells might presumably be traced to a set of molecular expression differences.…”