Aug 2017     Issue 5
Research
A Novel Nanometer-scale Probe Enables a Better Understanding of Cells

Prof. Liming Bian, Department of Biomedical Engineering

Human mesenchymal stem cells (hMSCs) serve as a very promising cell source for tissue engineering and regenerative medicine, owing to their ease of isolation and multipotency.1 The determination of the differentiation status of hMSCs (e.g. differentiating versus undifferentiated) is critical to the application of hMSCs to stem-cell-based therapies.2,3 In this study, we report a novel hairpin-DNA-based nanoprobe for detecting specific microRNAs in living hMSCs. This nanoprobe possesses a core-shell structure formed by depositing a layer of polydopamine (PDA) on the surface of a gold nanoparticle (AuNP) core via in situ polymerization under alkaline conditions. Such gold-PDA core-shell nanoparticles (Au@PDA NPs) are amenable to subsequent immobilization of fluorescently-labeled hairpin DNA strands (hpDNAs) onto the PDA shell simply by π-π interactions (Fig. 1).

 

MicroRNAs (miRNAs) are single-stranded non-coding RNAs with a typical short length of 21–23 nucleotides.4 They play an important role in controlling the expression of target proteins via either the repression of mRNAs or the inhibition of mRNA translation in a sequence-specific manner, thereby providing an additional level of gene regulation.5,6 In  stem cell studies, miRNAs have newly emerged as a mediator of various stem cell behaviors, including differentiation.7-10 miR-29b11 and miR-3112 are two distinct miRNA markers for the osteogenesis of hMSCs. Profiling studies18,21,13 show that these specific miRNAs are significantly up-regulated in stem cells following osteogenic induction. The dynamic nature of these specific miRNA expression profiles mediated by osteogenic differentiation indicates that miRNAs function as viable biomarkers for monitoring the differentiation progress of stem cells. Although much effort has been devoted to tracking intracellular mRNAs,14-17 very few attempts have been reported to detect cancer-related miRNAs in cancerous cell lines.18-19 We now demonstrate that a long-term monitoring (beyond 24 h) of miRNA levels in living stem cells is possible by using the newly designed probe.

 

Existing techniques

Conventionally, to determine the differentiation status of hMSCs, end-point methods such as qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot are used. Although these analytical methods are reliable, a large number of cell samples and lysis of cells are required for the analysis, which leads to the unavoidable loss of cell sources. Immunofluorescence and chemical staining are two other common methods to examine the differentiation status of fixed stem cells. However, they are not sensitive enough to monitor the early differentiation stage of stem cells and also preclude real-time monitoring of intracellular activities. Newer techniques including fluorescence-activated cell sorting20,21 (FACS) and surface-enhanced Raman spectroscopy22 (SERS) offer a non-destructive alternative to sort or distinguish between differentiated and undifferentiated stem cells via examination of changes in membranous features in living stem cells. Nevertheless, these techniques generally require expensive staining reagents or specialized instruments and they are not suitable for detecting intracellular biomarkers.
NanoFlare, a polyvalent oligonucleotide functionalized nanoconstruct, has been applied to detection of intracellular messenger RNA (mRNA) levels in live cells either in culture14-15 or from human blood.17 Despite the unique properties of the NanoFlare, detection of cellular components in cell types with low transfection efficiency such as stem cells23 has not been reported using the system. Furthermore, it remains unclear whether the NanoFlare can allow long-term tracking of intracellular targets in living cells (beyond 24 h) upon a single administration due to rapid disassembly.

 

Hence, developing a facile and non-invasive way either to monitor the differentiation process or to distinguish the differentiation status of living stem cells was highly desirable.

 

Comparison to exiting techniques

Unlike the existing intracellular detection platforms such as Molecular Beacons (MBs), our resultant Au@PDA−hpDNA NPs can naturally enter stem cells without the need of transfection. And different from the NanoFlare, fabrication of our nanoprobes requires only one single type of DNA sequence that can recognize the target miRNA. Due to the close proximity of hpDNAs and the AuNP core (<5 nm)24 and compounded by the intrinsic quenching property of the PDA shell,25 the immobilized hpDNAs on the nanoprobes do not fluoresce appreciably. The presence of two quenching entities (i.e. AuNP core and the PDA shell) makes our nanoprobes unique from the existing nanoparticle-based detection probes that typically possess only a single quenching entity such as the AuNP core of the NanoFlare.

In the presence of miRNA targets with a sequence complementary to the recognition region of the immobilized hpDNAs, we show in buffers that the specific binding between the hpDNAs and the target miRNAs triggers the dissociation of the hpDNAs from Au@PDA NPs, and thereafter generates detectable fluorescent signals (Fig. 2). Using these nanoprobes, we demonstrated the specific and long-term detection of two important osteogenic marker miRNAs, namely miR-29b and miR-31, in living hMSCs undergoing osteogenic differentiation as well as living primary osteoblasts (Fig. 2).

 

Summary

To summarize, we have demonstrated that our nanoprobes outperform the conventional methods (e.g. qRT-PCR analysis and staining) and the existing commercial intracellular RNA detection probe (e.g. SmartFlareTM) in the context of long-term intracellular detection of miRNAs. More significantly, we have not only established an approach to distinguishing differentiating stem cells from undifferentiated stem cells, but also demonstrated the time-dependent and dynamic expression of specific miRNAs in differentiating stem cells. The capability of our nanoprobes of the multiplexed detection of miRNAs allows enhanced monitoring of cellular events (e.g. differentiation) in living stem cells. More importantly, our nanoprobes afford long-term tracking of intracellular miRNAs in living stem cells which cannot be achieved by commercially available RNA detection probes such as the SmartFlare. The modular design of our nanoprobes offers facile switching of customized hairpin DNA probes, thus opening up an avenue for detecting other biomarkers such as mRNAs in living stem cells. We believe that our Au@PDA−hpDNA nanoprobes show great promise in the investigation of the dynamics of stem cell differentiation, the identification and isolation of specific cell types, and the high-throughput drug screening.

  

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		Fig. 1. (A) Preparation of polydopamine-coated gold nanoparticles (Au@PDA NPs) and hairpin-DNA-based (hpDNA) nanoprobes; (B) Intracellular detection of miRNAs in living human mesenchymal stem cells (hMSCs)
Fig. 1. (A) Preparation of polydopamine-coated gold nanoparticles (Au@PDA NPs) and hairpin-DNA-based (hpDNA) nanoprobes; (B) Intracellular detection of miRNAs in living human mesenchymal stem cells (hMSCs)
  
		Fig. 2. Monitoring of differentiation progress of hMSCs via intracellular detection of miRNAs. (A) Confocal images of hMSCs treated with nanoprobes targeting miR-29b (green). Scale bar is 100 μm. Inset: High-magnification images of the boxed area. Scale bar is 25 μm; (B) Confocal images of hMSCs treated with nanoprobes targeting miR-31 (red). Scale bar is 100 μm. Results show that hMSCs express detectable levels of miR-29b and miR-31 in a time-dependent manner only when they undergo osteogenic differentiati
Fig. 2. Monitoring of differentiation progress of hMSCs via intracellular detection of miRNAs. (A) Confocal images of hMSCs treated with nanoprobes targeting miR-29b (green). Scale bar is 100 μm. Inset: High-magnification images of the boxed area. Scale bar is 25 μm; (B) Confocal images of hMSCs treated with nanoprobes targeting miR-31 (red). Scale bar is 100 μm. Results show that hMSCs express detectable levels of miR-29b and miR-31 in a time-dependent manner only when they undergo osteogenic differentiati
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