CD166+CD34+ Cells Exhibit Marked Functional Differences During Fetal and Adult Life

ALCAM/CD166 is expressed from the onset of hematopoiesis in the yolk sac and in a variety of hematopoietic tissues throughout ontogeny. Both hematopoietic and stromal cells in the AGM region, fetal liver, and fetal and adult marrow express this molecule. CD166 double knockout mice are viable and fertile, without any major blood defects, but their microenvironment and hematopoietic stem cells (HSC) exhibit deficiencies in their ability to support and engraft long-term, respectively. In order to further study the role of CD166 in hematopoiesis, we characterized, during ontogeny, the origin, function, and sub-populations of CD166⁺ cells in different hematopoietic organs. To this end, we used flow cytometry, confocal microscopy, and colony-forming assays to analyze human fetal liver (FL) at 18 and 20 gestational weeks (gw), bone marrow (BM) from 10 to 20gw, and adult BM. Flow cytometric analysis of FL at 18 and 20gw demonstrated that although 3±1% of liver cells at this age were CD166+, less than 1% were endothelial CD166+CD34+ cells, and no hematopoietic CD166+CD34+CD45+ cells were detected. In fetal BM, CD166+ cells emerged after 15gw, expressed Flk-1 and CD34, and their percentage increased progressively with gestational age. Flow cytometric analysis at 20gw showed that 1.5±1% of cells were CD166+CD34+, of which 93±0.5% were CD45+. Human adult BM contained 2±0.5% of CD166+CD34+ cells, of which only 66±0.6% were CD45+. In order to functionally characterize CD166+CD34+ cells from adult and fetal BM (20gw), we plated these cells in mesenchymal cell growth medium (MSCGM), endothelial growth medium (EGM-2), and complete methylcellulose (MC). MSCGM and EGM-2 did not support growth and expansion of fetal or adult CD166+CD34+ cells. Quantification of the hematopoietic colony-forming potential of these cells demonstrated that fetal CD166+CD34+ generated/1000 cells: 8±1 Blast; 27±2 CFU-Mix; 51±10 CFU-GM; and 0 BFU-E, while adult CD166+CD34+ gave rise to 7 Blast; 17 CFU-Mix; 36 CFU-GM; and 10 BFU, demonstrating differences in the hematopoietic potential of these cells. Furthermore, at day 12 of MC culture, adherent stromal cells were detected underneath MC, but only in cultures from fetal BM. Characterization of these cells by flow cytometry showed that more than 90±2% of these cells were CD166+CD9+, and 30±5% were CD146+. Furthermore, these stromal CD166⁺ cells did not express CD34, CD45, CD31, CD209, or CD6. Immunostaining demonstrated that the CD166+CD146+ cells expressed osteopontin and Stro-1. A CD41+CD68+ population of cells was also found.

In conclusion, we found that, during ontogeny, expression of CD166 in FL is not associated with hematopoietic cells. In the BM, expression of CD166 is associated with CD34 and Flk2, and its expression on HSC commences later in gestation, suggesting that these cells either arise in the BM, or that CD166 expression is triggered at a certain time point in gestation, probably associated with rapid proliferation of HSC during this time period. Furthermore, we demonstrated that CD34+CD166+ cells from 20gw fetal BM contain hematopoietic and stromal cell populations, while adult BM-derived CD34⁺ CD166⁺ cells are exclusively hematopoietic.