MicroRNA Profiling of Individual Hematopoietic Stem Cells

Single cell studies are increasingly important to stem cell biology as rare cell subsets are found to play a critical role in the maintenance of processes such as hematopoiesis. MicroRNAs are regulated during hematopoiesis, and the ability to study the expression levels of these genes in single cells is crucial to understanding the role microRNAs may play in hematopoietic stem cell function. Purified hematopoietic stem cell populations are heterogeneous, making it challenging to study these rare cells when data are averaged over an entire population. This report presents the use of microfluidics to assemble and conduct thousands of small-volume microRNA expression assays, using individual cells from different hematopoietic populations, including highly purified stem cells. Multiple miRNAs are assayed from single cell lysates using quantitative real-time PCR, and the resulting data have the accuracy and precision required to detect biological variability in expression levels among individual cells.

Introduction

Understanding the function of individual cells demands new experimental approaches capable of analysing minute amounts of material. An ideal model for single cell applications is hematopoiesis, the continual process of blood cell production via

differentiation of hematopoietic stem cells (HSCs) into mature blood cell lineages (Figure 1). HSCs represent a small fraction of the total cells present in bone marrow and in the circulating blood, and so cannot be studied in tissue samples or large pools of cells. Fluorescence activated cell sorting (FACS) can be used to select various hematopoietic cell types based on expression of cell surface markers. However, known stem cell markers yield populations of limited purity, so even carefully sorted populations remain heterogeneous. Averaging data from these populations results in the substantial loss of information specific to stem cells, since signal from minority stem cells is swamped by the contributions of more abundant progenitor cell types. Furthermore, stem cell populations may contain sub-populations with subtle phenotypic variations that can only be detected with single cell assays.