gms | German Medical Science

Objectives: Currently, there is an increasing focus on mesenchymal stromal cells (MSCs) as therapeutic option in bone pathologies and tissue engineering. The limited biological resources in bone regeneration make MSCs a promising cell population as they provide regenerative potential and therapeutic option. Instant specific functional assessment of MSCs is critical for quality control in cellular therapy and would contribute to the understanding of the mechanisms behind pathological conditions. Imaging of intracellular messenger RNAs (mRNAs) not only helps us to better understand the formation and transcription of mRNAs, but also provides vital information for disease diagnosis and treatment. The aims of the current study are to design, synthesize and applyNanoflares (NFs) on towards osteogenic differentiating MSCs for live tracking expression of osteogenic early and late gene markers.

Methods: Femur head-derived MSCs were differentiated towards the osteogenic lineage with osteogenic induction growth factors. Typical osteoblast early and late markers that verify the osteoprogenitor maturation of MSCs were designed, synthesized and validated for their performance in reflecting cellular expression of the respective mRNAs. Specifically, single-stranded DNAs (ssDNA) which are complementary to the early osteogenic marker and late osteogenic markers mRNA sequences were specifically designed and functionalized with thiol ending. NFs recognition sequences were then pre-hybridized with short oligonucleotides containing the fluorophore. MSCs were then incubated with NFs for 4 hours and the signal intensity was expected to specifically reflect mRNA expression. The target mRNA expression level was analyzed by quantifying the fluorescence value through live imaging and compared with RT-PCR results.

Results: MSCs showed solid mineralization when induced towards the osteogenic lineage compared to the respective controls. All synthesized NFs were successfully validated via sensitivity tests and MSC viability was not impaired when incubated with NFs. mRNA expression level in towards osteogenic lineage induced MSCs showed 2.5-fold increase after fluorescence signals quantification through live imaging compared to the respective control. Additionally, a quick screening using plate reader measurement of the florescence signals also indicated a steady increase of ALPL mRNA expression at different time intervals. Moreover, mRNA expressions of the analyzed genes showed the same expression tendencies using RT-PCR.

Conclusion: FluorescenceNFs are promising candidates to realize the continuous, non-disruptive and real-time reporting and quantification of the gene biomarkers without the need of genetic engineering. NFs take advantage of the highly-efficient fluorescence quenching properties of gold, the effective cellular uptake of NFsthat occurs without the use of any transfection agents, and the stability of such constructs to report the presence of intracellular target mRNA.