Abstract

MicroRNAs (miRNAs) have been shown to be developmental regulators in various organisms and tissues such as the hematopoietic system. miRNA profiling studies have been primarily performed on specific aspects of hematopoiesis like lymphocyte or red blood cell development. However, a comprehensive study including rare hematopoietic stem cell populations and various lineages has yet to be published. MiRNA expression profiling within the hematopoietic tree is challenging due to difficulties in obtaining highly purified samples of stem and progenitor cell populations as well as the high cost and labour associated with global profiling approaches. The combined requirements of high sensitivity, dynamic range and efficient throughput pose serious obstacles to the use of established methods including Northern Blot, cloning, miRNA microarrays, and deep sequencing. Real time PCR offers the requisite dynamic range and sensitivity, but is labour intensive and prohibitive in cost using conventional formats. To overcome these limitations we combined new high throughput microfluidic technologies with a 288-plex real time PCR approach to quantify miRNAs in hematopoietic stem cells and lineage positive cells. This approach allowed us the simultaneous detection of 288 miRNAs in small numbers (≤3000) of cells across multiple subpopulations of the murine hematopoietic tree. Twenty unique murine hematopoietic cell populations were isolated through current FACS sorting strategies, including hematopoietic stem cells (HSCs) based on SLAM and LSK markers, myeloid and lymphoid progenitor cells as well as mature populations of all lineages. The cells were immediately lysed after sorting and reverse transcribed using 3 pools of 96 stemloop RT-primers, followed by a PCR pre-amplification step. In order to detect individual amplified products, we used BioMark 48.48 Dynamic Arrays (Fluidigm Corp, San Francisco, USA) and miRNA specific TaqMan probes. For each miRNA, a series of synthetic miRNA dilutions was used as a standard to determine the absolute number of miRNA molecules per cell. This analysis further revealed systematic and miRNA-specific variations in the sensitivities of Taqman assays, highlighting that RT-PCR analysis without the inclusion of an absolute standard may be misrepresentative of the true molecular abundance. Hierarchical clustering analysis and comparison between hematopoietic stem cell (HSC) populations and mature populations revealed miRNAs that are critical for hematopoietic development and maturation. In general, miRNAs detected at the highest abundance were miR-706 and miR-720, which is likely due to highly reactive Taqman assays for these targets. Of the tested 288 miRNAs, only 133 were detected across all cell populations. Most of these miRNAs exhibited a mixed expression profile, with expression peaks in the differentiated populations. Consistent with previous results, we detected a strong increase of miR-223 within myeloid differentiated populations. The highest levels were detected in neutrophils and monocytes, but surprisingly low levels were found in mast cells, supporting the specific role of miR-223 in myeloid differentiation. Other miRNAs highly enriched in differentiated cells were miR-142 and miR-16. Clustering revealed a distinct miRNA expression pattern for the profiled HSC populations including miRNAs located in the Hox cluster and the miR-181 family. In conclusion, we applied a novel technical approach to quantify a broad range of miRNAs in rare cell populations. With this approach we can further define the expression patterns of miRNAs from hematopoietic stem cells to mature lineages.

Disclosures: No relevant conflicts of interest to declare.

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