Single-cell qRT-PCR Research Articles

Identification of Novel Adult Mesenchymal Progenitors That Can Contribute to HSC Niche

AbstractAbstract 3467The microenvironment (niche) cues hematopoietic stem cell (HSC) receives play an important role in the regulation of their decisions between self-renewal and differentiation. However, the cellular constitution of niches remains poorly understood. We identified three adult progenitor populations near the endosteum based on differential expression of cell surface markers, including CD166, CD146 and Sca1. Upon co-transplantation with fetal skeletal progenitors, Sca1+ progenitors can give rise to CD146+ and CD166+ stroma and phenotypically CXCL12 abundant reticular (CAR) cells in marrow. CD146+ and CD166+ progenitors, on the other hand, form bone without marrow cavity. Multiplex single cell qRT-PCR reveals all three progenitors expressed high levels of genes involved in HSC maintenance. In vitro co-culture assay demonstrated that all three progenitors could preserve HSC long-term multi-lineages reconstitution capability. Furthermore, disruption of stem cell factor (SCF) production in Sca1+ progenitor severely limits its ability to support HSC both in culture condition and after transplantation. Our results suggested that Sca1+, CD146+, and CD166+ mesenchymal progenitors and their progeny collaborate to provide supportive environment for hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns

Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.

Requirement of New Protein Synthesis of a Transcription Factor for Memory Consolidation: Paradoxical Changes in mRNA and Protein Levels of C/EBP

Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L.stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation.