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Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

ABSTRACT: Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.

SUBMITTER: Azimi SM

PROVIDER: S-EPMC5959718 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

Azimi Sayyed M SM Sheridan Steven D SD Ghannad-Rezaie Mostafa M Eimon Peter M PM Yanik Mehmet Fatih MF

eLife 20180501

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved deli ...[more]

PMID: 29714688

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Project description:The objective of this proof-of-concept study was to demonstrate the targeted delivery of erythropoietin (EPO) using magnetically guided magnetic nanoparticles (MNPs).MNPs consisting of a ferric-ferrous mixture (FeCl3·6H2O and FeCl2·4H2O) were prepared using a co-precipitation method. The drug delivery system (DDS) was manufactured via the spray-drying technique using a nanospray-dryer. The DDS comprised 7.5 mg sodium alginate, 150 mg MNPs, and 1000 IU EPO.Scanning electron microscopy revealed DDS particles no more than 500 nm in size. Tiny particles on the rough surfaces of the DDS particles were composed of MNPs and/or EPO, unlike the smooth surfaces of the only alginate particles. Transmission electron microscopy showed the tiny particles from 5 to 20 nm in diameter. Fourier-transform infrared spectroscopy revealed DDS peaks characteristic of MNPs as well as of alginate. Thermal gravimetric analysis presented that 50% of DDS weight was lost in a single step around 500°C. The mode size of the DDS particles was approximately 850 nm under in vivo conditions. Standard soft lithography was applied to DDS particles prepared with fluorescent beads using a microchannel fabricated to have one inlet and two outlets in a Y-shape. The fluorescent DDS particles reached only one outlet reservoir in the presence of a neodymium magnet. The neurotoxicity was evaluated by treating SH-SY5Y cells in 48-well plates (1 × 10 cells/well) with 2 μL of a solution containing sodium alginate (0.075 mg/mL), MNPs (1.5 mg/mL), or sodium alginate + MNPs. A cell viability assay kit was used to identify a 93% cell viability after MNP treatment and a 94% viability after sodium alginate + MNP treatment, compared with the control. As for the DDS particle neurotoxicity, a 95% cell viability was noticed after alginate-encapsulated MNPs treatment and a 93% cell viability after DDS treatment, compared with the control.The DDS-EPO construct developed here can be small under in vivo conditions enough to pass through the lung capillaries with showing the high coating efficiency. It can be guided using magnetic control without displaying significant neurotoxicity in the form of solution or particles.

Project description:Pulsatile chemotherapeutic delivery profiles may provide a number advantages by maximizing the anticancer toxicity of chemotherapeutics, reducing off-target side effects, and combating adaptive resistance. While these temporally dynamic deliveries have shown some promise, they have yet to be clinically deployed from implantable hydrogels, whose localized deliveries could further enhance therapeutic outcomes. Here, several pulsatile chemotherapeutic delivery profiles were tested on melanoma cell survival in vitro and compared to constant (flatline) delivery profiles of the same integrated dose. Results indicated that pulsatile delivery profiles were more efficient at killing melanoma cells than flatline deliveries. Furthermore, results suggested that parameters like the duration of drug "on" periods (pulse width), delivery rates during those periods (pulse heights), and the number/frequency of pulses could be used to optimize delivery profiles. Optimization of pulsatile profiles at tumor sites in vivo would require hydrogel materials capable of producing a wide variety of pulsatile profiles (e.g., of different pulse heights, pulse widths, and pulse numbers). This work goes on to demonstrate that magnetically responsive, biphasic ferrogels are capable of producing pulsatile mitoxantrone delivery profiles similar to those tested in vitro. Pulse parameters such as the timing and rate of delivery during "on" periods could be remotely regulated through the use of simple, hand-held magnets. The timing of pulses was controlled simply by deciding when and for how long to magnetically stimulate. The rate of release during pulse "on" periods was a function of the magnetic stimulation frequency. These findings add to the growing evidence that pulsatile chemotherapeutic delivery profiles may be therapeutically beneficial and suggest that magnetically responsive hydrogels could provide useful tools for optimizing and clinically deploying pulsatile chemotherapeutic delivery profiles.

Dataset Information

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

Publications

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

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