Tracking stem cell differentiation in the setting of automated optogenetic stimulation.

Membrane depolarization has been shown to play an important role in the neural differentiation of stem cells and in the survival and function of mature neurons. Here, we introduce a microbial opsin into ESCs and develop optogenetic technology for stem cell engineering applications, with an automated system for noninvasive modulation of ESC differentiation employing fast optogenetic control of ion flux. Mouse ESCs were stably transduced with channelrhodopsin-2 (ChR2)-yellow fluorescent protein and purified by fluorescence activated cell sorting (FACS). Illumination of resulting ChR2-ESCs with pulses of blue light triggered inward currents. These labeled ESCs retained the capability to differentiate into functional mature neurons, assessed by the presence of voltage-gated sodium currents, action potentials, fast excitatory synaptic transmission, and expression of mature neuronal proteins and neuronal morphology. We designed and tested an apparatus for optically stimulating ChR2-ESCs during chronic neuronal differentiation, with high-speed optical switching on a custom robotic stage with environmental chamber for automated stimulation and imaging over days, with tracking for increased expression of neural and neuronal markers. These data point to potential uses of ChR2 technology for chronic and temporally precise noninvasive optical control of ESCs both in vitro and in vivo, ranging from noninvasive control of stem cell differentiation to causal assessment of the specific contribution of transplanted cells to tissue and network function.

A Safe and Effective Magnetic Labeling Protocol for MRI-Based Tracking of Human Adult Neural Stem Cells.

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 × 103 haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 × 104 cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method.

Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of Parkinson disease.

Magnetic resonance imaging (MRI) of magnetically labeled stem cells has become a valuable tool in the understanding and evaluation of experimental stem cell-based therapies of degenerative central nervous system disorders. This comprehensive study assesses the impact of magnetic labeling of both human and rodent stem cell-containing populations on multiple biologic parameters as maintenance of stemness and oxidative stress levels. Cells were efficiently magnetically labeled with very small superparamagnetic iron oxide particles. Only under the condition of tailored labeling strategies can the impact of magnetic labeling on vitality, proliferation, pluripotency, and oxidative stress levels be minimized. In a rat model of Parkinson disease, magnetically labeled mouse embryonic stem cells were tracked by high-field MRI for 6 months. Significant interindividual differences concerning the spatial distribution of cells became evident. Histologically, transplanted green fluorescent protein-positive iron oxide-labeled cells were clearly identified. No significant increase in oxidative stress levels at the implantation site and no secondary uptake of magnetic label by host phagocytotic cells were observed. Our study strongly suggests that molecular MRI approaches must be carefully tailored to the respective cell population to exert minimal physiologic impact, ensuring the feasibility of this imaging approach for clinical applications.