Uptake of Protamine Sulphate Complexed Fluorescent Nano-Particles Is Defined by Cell Cycle Status in Primary Human CD34+ Cells: Use of a Multi-Color p27 kip1 Based Flow Cytometric Assay.
Authors: Lahey Ryan , Bonde Jesper , Nolta Jan A. 
Published: April 2020

Abstract
The use of iron based nano-particles for multi-modal imaging is gaining interest, since it allows high resolution non-invasive in vivo imaging of human hematopoietic homing and engraftment events in xenograft models. The uptake of ferridex nano-particles complexed to cationic protamine sulphate is believed to be non-specific through mechanisms like endocytosis, but this has not been well defined for hematopoietic stem cells (HSC). In defining ex vivo cultivation strategies for manipulation of human HSC, a key factor is the responsiveness of the most primitive cells to the in vitro conditions, with the aim of maintaining viability without inducing terminal differentiation. Here, we present a novel flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable ex vivo protocol, aimed at facilitating labeling of human stem/progenitor cells using protamine sulphate complexed nano-particles for subsequent in vivo imaging. We used intracellular staining for the cell cycle inhibitor p27kip1, which is present in the highest levels in non-cycling cells, as the primary flow cytometric marker in combination with CD34, CD133 and Alexa 488, 647 and 750 conjugated ferridex nano-particles and the membrane dye PKH26. An assay was developed to simultaneously assess the molecular events occurring in individual human cord blood Lin− or CD34+ cells while they were cultured for up to 72 hours in X-Vivo 15 serum free medium supplemented with Flt3, SCF and TPO on Retronectin (RN) coated plates with or without nano-particles. Co-expression of p27kip1, CD34 or CD133 in the cultured cells slowly decreases from 86.1% CD34+p27kip1 (T=0) to 76.7%+/−12.2% (T=72) and from 89.6% CD133+p27kip1+ (T=0) to 54.1%+/−10.4% (T=72). We suggest that this slow decrease represents cells dividing and potentially differentiating over the time course of the ex vivo cultivation period. Assessing uptake of fluorescent conjugated nano-particles over a 72 hr period showed that the uptake of particles in CD34+ and CD133+ cells declined significantly after the first 24 hrs., from 32.5+/−3.7% nano-positive CD34+ cells to 19.2+/−2.9% at 48 hours ex vivo with a more significant decline to only 8.3+/−3.7% nano positive CD34+ cells in the culture after 72 hours ex vivo. The same decline in uptake over time was observed in cultured human CB cells that were positive for CD133. PKH26 co-staining demonstrated that the majority of cells that undergo cell division within the first 24 hours of ex vivo culture are the most likely to uptake the nano-particles. In summary, using a multi color p27kip1 based flow-cytometry assay, we found that human Lin−, CD133+, and CD34+ cells uptake Fe-Pro in a fashion which is not entirely cell cycle independent as previously suggested. These data indicate that cell cycle or metabolic status may influence the ability of human hematopoietic stem and progenitor subsets to uptake the protamine sulphate-complexed nano-particles. These findings emphasize the need to carefully develop ex vivo conditions for nano-particle labeling of primary human stem cells in order to perform accurate in vivo imaging of the most primitive human hematopoietic stem and progenitor cells.