Since the discovery of these cells Ingram et al

Since the discovery of these Leupeptin (Ingram et al., 2004), secondary colonies have been generated in several studies to assess the immaturity of ECFCs from blood or vascular wall and hierarchize them (Alphonse et al., 2015; Ingram et al., 2005; Patel et al., 2016). The aim of this work was to identify relevant information directly from the initial number of colonies obtained at the first isolation step of CB-ECFCs and AB-ECFCs.
Materials and methods
Results
Discussion Based on the substantial size of the studied populations, we were able to determine the clonogenic profile of ECFCs from umbilical cord blood and peripheral adult blood (65 and 74 samples, respectively). We identified three groups based on their initial clonogenicity: highly (>100 colonies), intermediate (10–50 colonies) and low (<10 colonies) clonogenic samples. Two proliferative populations of CB-ECFCs were distinguished based on a threshold of 10 colonies. Beyond this clonogenic limit, CB-ECFCs showed a comparable proliferative potential (a maximum of 50 CPD, PD in 1day at early passages and in 2–3days at late passages), a low accumulation of senescent cells even after P15 and a higher angiogenic potential in vitro. Thus, in addition to the higher initial number of cells conferred by a high clonogenicity, this parameter appeared as the earliest accessible predictive marker for the further functionality of CB-ECFCs. However, the parameters related to an intermediate or high clonogenicity, before MNC isolation, remain to be determined. Despite the generally accepted higher yield of CB-ECFCs compared to AB-ECFCs also confirmed here, in this study, the CB samples mainly appeared as non- (24.6%) or poorly (44.6%) clonogenic. Nevertheless, it should be noted that the blood units for research purpose are actually samples withdrawn from the therapeutic circuit of cord blood bank, due to a too small volume or low concentration of CD34+ cells. Thus, this yield could be possibly increased with a better donor selection. Moreover, these results are independent of the operator isolating ECFCs and we have used for several years the canonical protocol described in the literature based on the works of David Ingram and Mervin Yoder. Additionally, our provider, the French cord blood bank of St Louis Hospital, centralizes samples collected by different midwives from several maternities in Paris area. Due to the high sample variety, we cannot exclude a geographic bias of our results. Nevertheless, the cord blood clonogenicity is unlikely related to the blood collection process. Only the concentration of MNC and the age of the mother appeared as determinant parameters. However, none of the analyzed factors enabled to early completely exclude poorly and non-clonogenic samples. Interestingly, >90% of intermediate clonogenic CB-ECFCs were from mothers aged under 35 (mother mean age: 32years) whereas highly clonogenic CB-ECFCs were from older mothers (mother mean age: 38years). We were not informed whether the mothers were multiparous or if principle of segregation received a particular hormonal treatment which could have influenced the fetal concentration of CB-ECFCs. The impact of other clinical features of the mothers has been recently described such as the pre-pregnancy body mass index which positively correlates with the number of ECFCs (Moreno-Luna et al., 2014). Regarding peripheral adult blood, our results were opposed to those described in a previous study on aging conducted in monkeys (Shelley et al., 2012). Indeed, we found that ECFC clonogenicity and proliferation did not correlate with the donor age and did not decrease with aging. Nevertheless, our data only involved adults over 18 and ECFC yield in childhood should also be informative. Interestingly, the non-clonogenic fraction was reduced in adults over 50. According to some studies, the number of ECFCs increases in patients with cardiovascular diseases (Güven et al., 2006; Massa et al., 2009; Tagawa et al., 2015) and the incidence of such diseases correlate with age. Although our samples were from healthy donors, we should take into account that the exclusion from blood donation in France is essentially limited to infectious risks. Cardiovascular or cancer risk factors such as smoking or physical inactivity are not taken into account, thus the good health of donors is assumed but relative.