Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

Abstract

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here, we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single cell transplantation studies, but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggests that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse post irradiation. This technique can be applied to any viral accessible cell type for both in vitro and in vivo processes.

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells

“…These include cells that have distinctive behaviors in terms of cell production and lineage bias (Dykstra et al, 2007;Picelli et al, 2013). Hematopoietic stem cells have been demonstrated to exhibit bias toward myeloid, lymphoid, or megakaryocytic lineage upon transplantation of single cells (Dykstra et al, 2007(Dykstra et al, , 2011Morita et al, 2010), on ex vivo barcoding and transplantation of populations (Aiuti et al, 2013;Gerrits et al, 2010;Jordan and Lemischka, 1990;Lemischka, 1993;Lemischka et al, 1986;Lu et al, 2011;Mazurier et al, 2004;Shi et al, 2002;Snodgrass and Keller, 1987), or by retrotransposon tagging of endogenous cells (Sun et al, 2014b). Further, single-cell transplant data have been coupled with single-cell gene expression analysis on different cells to resolve subpopulations with corresponding gene expression and repopulation potential (Wilson et al, 2015).…”

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

“…Recent efforts have applied a variety of techniques, including genetic DNA barcoding, 3 transposon-based mutagenesis, 4 fluorescentlabeling approaches, or combinations thereof. [5][6][7] Although genetic DNA barcoding allows labeling of highly complex subpopulations, it is largely restricted to the analysis of lysed cells and requires multiple time-consuming and costly steps, including next-generation sequencing and biostatistical analysis, before the different clonal populations can be quantified. 6 Cell marking with fluorescent proteins, on the other hand, enables tracking of live cells and their quick analysis in and ex vivo.…”

“…[5][6][7] Although genetic DNA barcoding allows labeling of highly complex subpopulations, it is largely restricted to the analysis of lysed cells and requires multiple time-consuming and costly steps, including next-generation sequencing and biostatistical analysis, before the different clonal populations can be quantified. 6 Cell marking with fluorescent proteins, on the other hand, enables tracking of live cells and their quick analysis in and ex vivo. 8,9 The number of exploitable fluorescent colors can be significantly increased by applying combinatorial labeling strategies such as red-green-blue (RGB) marking.…”

A generic, cost-effective, and scalable cell lineage analysis platform

“…Moreover, unraveling the dynamics of diseased cells, which depend on the specific cellular microenvironment and stochastic events, through their cell lineage tree can help in selecting the appropriate treatment, thus facilitating the advancement of personalized medicine. Since the landmark mapping of the complete cell lineage tree of Caenorhabditis elegans, a 1000-cell nematode, methodologies which are based on cellular labeling by reporters (e.g., dyes, fluorescent genes [Kretzschmar and Watt 2012]), and DNA barcodes [Lu et al 2011]) were developed to enable lineage tracing in higher model organisms including mammals. However, being invasive, they cannot be applied to human research.…”