Resolving the hematopoietic stem cell state by linking functional and molecular assays

Abstract

One of the most challenging aspects of stem cell research is the reliance on retrospective assays for ascribing function. This is especially problematic for hematopoietic stem cell (HSC) research in which the current functional assay that formally establishes its HSC identity involves long-term serial transplantation assays that necessitate the destruction of the initial cell state many months before knowing that it was, in fact, an HSC. In combination with the explosion of equally destructive single-cell molecular assays, the paradox facing researchers is how to determine the molecular state of a functional HSC when you cannot concomitantly assess its functional and molecular properties. In this review, we will give a historical overview of the functional and molecular assays in the field, identify new tools that combine molecular and functional readouts in populations of HSCs, and imagine the next generation of computational and molecular profiling tools that may help us better link cell function with molecular state.

Graphical abstract

Figure 1.

The fundamental challenge of retrospective assays. The vast majority of HSC assays require destruction of the initial cell state to perform either a functional or a molecular assay. Establishing that a cell population, being the “soil,” contains an HSC, the “seed,” necessitates lengthy functional assays but also destroys the seed in the process. Once the presence of an HSC has been established, the cell is no longer present, and the molecular program of similar cells isolated at the same time cannot easily be distinguished from other non-HSCs profiled at the same time.

Figure 2.

A broad timeline of single-cell functional, molecular, and computational techniques. Although clonal functional assays have developed slowly over many decades, global single-cell molecular profiling (and its associated analytical tools) have been largely developed in the last 10 years. The pace of these developments and the breadth of competing/complementary tools makes it more challenging to navigate. The Venn diagram (bottom-left) depicts each tool and identifies the aspects of HSC biology that can be assessed via each technique (cellular function, molecular profile, or clonal tracking). In addition to these individual limitations, it is important to note that most multiomic techniques are not as robust as singleomic techniques optimized to capture 1 molecular aspect, and these combinatorial technologies should be used with great caution. ATAC-seq, assay for transposase-accessible chromatin sequencing; G&T-seq, genome and transcriptome sequencing; NEAT-seq, sequencing of nuclear protein epitope abundance, chromatin accessibility and the transcriptome in single cells; qPCR, quantitative polymerase chain reaction; sc-assays, single-cell assays; sci-CAR, single-cell combinatorial indexing-based coassay that jointly profiles chromatin accessibility and mRNA; scM&T-seq, single-cell methylome and transcriptome sequencing; scNMT-seq, single-cell nucleosome, methylation and transcription sequencing.