280 ISOLATION, PRESERVATION, AND CHARACTERIZATION OF EQUINE UMBILICAL CORD BLOOD STEM CELLS

Abstract

The isolation, preservation, and identification of hematopoietic and mesenchymal stem cells from fresh umbilical cord blood (UCB) has been extensively reported in humans. Although both types of stem cells may be of therapeutic interest in horses, data on equine UCB cells are scarce. In the present study, two separation methods to isolate stem and progenitor cells from equine UCB and two cryoprotectant solutions for their subsequent freezing were compared. Characterization of the isolated cells was evaluated flow cytometrically, based on the presence of the cytosolic enzyme aldehyde dehydrogenase (ALDH), which has been shown to be highly expressed in primitive hematopoietic cells in a number of species. Cord blood was collected from 15 foals immediately after birth. While the placenta was still in utero, the umbilical cord was clamped and disinfected. A sterile blood bag collection system containing citrate-phosphate-dextrose-adenine anticoagulant was used to collect the UCB by gravity. The UCB units were stored at 4°C and processed within 36 h. Percoll density gradient separation and rouleaux formation induced by hydroxyethyl starch (HES) were tested in parallel on equal volumes of each UCB unit. The enriched progenitor cell fraction was cryopreserved at 10 × 10⁶ nucleated cells mL^–1 using two cryoprotectant solutions based on plasma or RPMI 1640, and both containing 10% DMSO and DNase I (20 IU mL^–1). Before and after thawing, cells were labeled using a fluorescent ALDH substrate (Aldefluor^®, StemCell Technologies SARL, Grenoble, France) including a negative control. Cell viability was simultaneously evaluated by means of exclusion of propidium iodide. Cryopreservation was performed using a programmable freezer (–1°C/min^–1 until –70°C, then –10°C/min^–1 until –140°C) prior to storage in liquid nitrogen. Results were analyzed statistically with a nonparametric Mann-Whitney test. The concentration of the isolated UCB cells ranged from 0.3 to 4 × 10⁶ cells mL^–1 for Percoll and from 0.4 to 7.3 × 10⁶ cells mL^–1 for HES. The average viability before and after freezing was 94% and 93% for Percoll-, and 93% and 94% for HES-separated cells, respectively. No significant differences in concentration or in viability were observed between both isolation procedures and both cryoprotectant solutions. Before freezing, the proportion of Aldefluor^®-positive cells after Percoll and HES isolation ranged between 0.5 and 38% and between 1 and 60.5%, respectively. No significant differences were found. In conclusion, the percentage of ALDH-positive cells as determined by flow cytometry was highly variable between foals, but was independent of the isolation procedures used. Whether the isolated cells represent true progenitor cells remains to be confirmed. Ongoing, flow cytometrical experiments showed that the isolated cells are CD29+ and CD44+, which may be indicative for their mesenchymal origin.