212. Differential, Transient Transduction of Defined Human Stem Cell Subsets Using Nanoparticles in a Clinically Applicable Ex Vivo Protocol
Authors: Jesper Bonde, Dustin J. Maxwell, David A. Hess, Ryan Lahey, David Piwnica-Worms, Jan A. Nolta
Journal: Molecular Therapy (2005)

The use of novel nano-sized iron particles and magnetic imaging techniques are ideal for studies of homing and trafficking of long-term repopulating, human hematopoietic stem cells (HSC). However, defining ex vivo cultivation strategies for the transduction of nano-particles into the HSC involves the use of strictly defined in vitro culture conditions with the aim of maintaining viability without inducing terminal differentiation. Here we present data on in vitro uptake and retainment of nanoparticles in defined human stem cell subsets. We have chosen to use protamine sulphate-complexed Ferridex nano-particles conjugated to an Alexa 647 dye (FE-PRO[647]) to load the cells ex vivo prior to analysis, as these particles are biodegradable and, eventually, will be metabolized by the cells. This makes these particles very well suited for loading of primary cells for transplantation and subsequent imaging of the transplanted cells by MRI. Whereas most cell line studies performed show a uniform 60-75% uptake of the FE-PRO within the first 24 hours in vitro, our study of primary human cord blood (CB) lineage depleted (lin-) cells shows that the overall load of Fe-Pro [647] particles declines from 61.4+/|[minus]|0.9 (n=2) after 24hrs to a level of 39.7+/|[minus]|0.4 (n=2) after 72 hours in vitro. This indicates that the cells ability to uptake the FE-Pro [647] is declining with a prolonged ex vivo cultivation. Evaluating the FE-Pro [647] uptake based on the primary stem cell markers CD34 and CD133, however, reveals a different picture. Most notably, after 72 hours in culture the two stem cell populations have a very uniform level of the G0 determinant p27kip1, indicating that the cells are highly quiescent, but only 8.3+/|[minus]|3.7% CD34 and 5.1+/|[minus]|4.5% (n=2) CD133 cells contain the FE-Pro [647]. This points towards the idea that quiescent stem cells after subjection to ex vivo cultivation might not easily uptake the particles, possibly due to their low metabolic activity. Based on these data we next reduced the ex vivo protocol to a single 24 hr. exposure of the cells to the FE-PRO [647] particles. Using this modified ex vivo protocol we have tested a number of defined stem cell subsets for their ability to uptake and retain FE-PRO [647]. FE-PRO [647] uptake in CB CD34 was 17%, with only 15.1% of these displaying the p27kip1 high quiescent phenotype, indicating that the majority of the transduced cells were actively cycling ex vivo. Currently in vivo data are being collected from cohorts of beta-2 NOD/SCID mice. Using the clinically interesting cell sources of AMD3100 and G-CSF mobilized CD34 cells, we found FE-PRO [647] uptake over 24 hrs. ex vivo to be 7% (G-CSF) and 10.3% (AMD3100), indicating that in these cell sources, targeting of the nanoparticles to the quiescent repopulating stem cells might be even more challenging. Overall our data shows that the use of nanoparticles to transiently transduce human HSC is indeed feasible, using a clinically applicable protocol.