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Summary. Fetal haemopoietic cells continually circulate and migrate into tissues, and thus may have specialized homing capabilities. In this study we investigated the in vitro features of haemopoietic cells in fetal blood and liver which are relevant to homing and engraftment. Fetal cells were examined for long-term culture-initiating cell (LTC-IC) and progenitor content, adhesion molecule expression, cell cycle behaviour and transendothelial migratory activity. The LTC-IC content of fetal CD34+ cells is similar to that of CD34+ cells from cord and adult mobilized blood. In contrast to adult and cord blood CD34+ cells, fetal CD34+ cells were actively cycling (11·0 ± 1·7% and 28 ± 1·1% of fetal blood and liver CD34+ cells, respectively, in S+G2M, P < 0·001, compared with cord and adult cells). The striking finding was that fetal haemopoietic cells (both LTC-ICs and committed progenitors) displayed significantly higher levels of migration across endothelium (P < 0·05 compared with cord, P < 0·01 compared with adult blood and bone marrow CD34+ cells), which were further increased by chemokines and growth factors. The superior migratory activity of fetal haemopoietic cells may underlie a more efficient homing ability, in keeping with their physiological role.
The use of primitive haemopoietic cells for stem cell transplantation is now an established treatment modality for many malignant and inherited disorders; however, our understanding of the molecular basis for stem cell engraftment remains fragmentary. Infused stem cells leave the circulation by migrating across vascular endothelium; this is an early but critical step in the homing of these cells into extravascular haemopoietic tissue. Tracking of labelled stem cells suggests that this occurs within a few hours of transplantation (Hendrikx et al, 1996); thus, cells which fail to transmigrate successfully are eliminated, probably through uptake by the reticuloendothelial system. We have previously shown that the transendothelial migration of human mobilized adult blood progenitor cells is mediated by adhesion molecules such as CD18 and platelet endothelial cell adhesion molecule (PECAM)-1, and is regulated differently from the transmigration of mature inflammatory cells (Yong et al, 1998). Our studies also indicate that, while adult progenitor cells require growth factor stimulation in order to transmigrate, migration occurs preferentially in cells which are in G0/G1 phase of the cell cycle (Yong et al, 1999a). Thus, cell cycle behaviour can impose constraints on the migratory, and hence homing, ability of haemopoietic cells, with optimal homing occurring in cells residing in G0/G1. This may account for the observation made by Gothot et al (1998) that CD34+ cells engraft best when in G0/G1 phase of the cell cycle, and similar observations that prior exposure to cytokines in vitro reduces the engraftment capability of haemopoietic cells (Peters et al, 1996).
The homing of transplanted cells to haemopoietic tissue and their subsequent engraftment recapitulates the physiological process in fetal life, whereby haemopoiesis is established sequentially and simultaneously in different tissues by the seeding of circulating haemopoietic stem cells. Intriguingly, the cell cycle constraints on engraftment by adult cells may not apply to fetal repopulating cells. Recently, two groups reported preliminary results indicating that fetal cells in S+G2/M are able to engraft, albeit to a lesser degree than their counterparts in G0/G1 (Bhatia et al, 1999; Wilpshaar et al, 1999). Hence, fetal haemopoietic cells may be intrinsically different from their adult counterparts, particularly with respect to engraftment behaviour. Fetal haemopoietic cells, by virtue of their ontogeny and function, represent a potentially valuable source of primitive haemopoietic cells, not only for use in stem cell transplantation and gene therapy, but also for studying in detail the biological processes involved in stem cell homing and engraftment.
The aim of this study was to conduct a detailed analysis of the features and properties of fetal cells which are directly relevant to homing and engraftment. Thus, we have examined the stem cell and progenitor content, adhesion molecule expression, cell cycle profile and transendothelial migratory ability of first trimester fetal blood and liver cells, and compared these with our observations on cord and adult mobilized CD34+ cells.
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The establishment of haemopoiesis in diverse anatomical locations during embryonic and fetal life means that multipotent haemopoietic cells must circulate and continually migrate into extravascular fetal tissues. Early studies have reported the presence of haemopoietic progenitors in fetal blood, the numbers correlating inversely with gestational age, as would be expected (Hann et al, 1982; Linch et al, 1982). Such cells, in keeping with their physiological role and ontogenic status, may possess superior homing and migratory properties. Our findings confirm the presence of circulating haemopoietic cells in the fetus and also indicate that, at least in the liver, the percentage of CD45+ cells co-expressing CD34 is highest in early gestation. When normalized for CD34+ content, fetal haemopoietic cells had LTC-IC and progenitor frequencies which were similar to cord and adult haemopoietic cells. The striking difference between fetal and cord/adult CD34+ cells lies in their migratory and cell cycle behaviour. Fetal LTC-IC and progenitors demonstrated significantly higher levels of migration across endothelium, which were augmented further by prior growth factor stimulation, and the presence of chemokines (SDF-1 and MIP-3β). In addition, fetal blood CD34+ cells were actively in cycle, again in contrast to cord and adult blood haemopoietic cells.
A number of previous reports have characterized the haemopoietic cells obtained from fetal liver; however, in these studies, fetal cells were obtained from pregnancies in late second trimester (Huang et al, 1998; Roy & Verfaillie, 1999). Our report has focused on fetal blood as well as liver cells obtained largely from the first trimester of pregnancy, and hence provides unique insights into the behaviour and function of haemopoietic cells found very early in ontogeny. The CD34+ content of CD45-positive cells in fetal blood reported here was similar to that in previous studies, one of which used a lymphoid gate on flow cytometric forward and side scatter plots (Thilaganathan et al, 1994; Roy & Verfaillie, 1999). Clonogenic cell frequencies obtained in this present study also correlated well with an earlier report from our group (Linch et al, 1982). Fetal liver cells generated a greater proportion of erythroid colonies than adult cells, hence the total clonogenic output per CD34+ cell was also higher (Table II).
The increased numbers of cycling CD34+ cells in fetal blood and liver may simply reflect the enhanced proliferative status and shorter cell cycle transit time of fetal haemopoiesis in general. The higher proportion of cycling CD34+ cells in extravascular fetal haemopoietic tissue, when compared with circulating CD34+ cells, is reminiscent of the situation in the adult in which the vast majority of mobilized peripheral blood CD34+ cells are in G0/G1 phase of the cell cycle, while up to 15% of bone marrow CD34+ cells are cycling (in S+G2/M) (our data and Fruehauf et al, 1998). The effect of tissue localization on cell cycle status may be related to the absence of proliferative stimuli in the circulation. The precise relationship between cell cycling and migration is unclear, and may differ between ontogenically distinct sources of haemopoietic cells. Fetal blood progenitors, which contain a lesser proportion of cells in S+G2/M, nevertheless migrated more efficiently than their fetal liver counterparts. In the adult, bone marrow CD34+ cells were actively cycling but were no more migratory than peripheral blood CD34+ which were largely quiescent.
The superior migratory activity of fetal haemopoietic cells was evident in both LTC-IC and progenitors, indicating that enhanced migration is an intrinsic property of fetal haemopoietic cells at all maturational stages. Our observation that spontaneous levels of transmigration were highest in the most ontogenically primitive cells is in keeping with the physiology of these cells which must continually seed and establish haemopoiesis in different tissues, particularly in early fetal life. It is interesting that the migration capacity was higher in fetal blood progenitors, an observation which may simply reflect the preferential extravasation of more migratory cells into the vascular compartment. Similarly, in the adult, G-CSF-mobilized CD34+ cells were found to migrate more readily across Matrigel membranes than bone marrow CD34+ cells (Janowska-Wieczorek et al, 1999). The high levels of migration in response to SDF-1 may also reflect an intrinsically greater migratory potential, rather than an enhanced chemokine responsiveness. Indeed the levels of CXCR4 expression on fetal CD34+ cells are similar to those on adult CD34+ cells and both were lower than levels on cord blood CD34+ cells. The high expression of chemokine receptors on cord blood cells has recently been confirmed by another group (Rosu-Myles et al, 1999) and may be related to stimulation by cytokines released during labour and delivery. A recent report from another group confirmed that expression of CXCR4 on fetal and adult CD34+ cells was similar (Aiuti et al, 1999).
Transendothelial migration is mediated by cell surface adhesion molecules, although the relative importance of different receptor–ligand pairs may depend upon the particular assay conditions used. We have previously shown that the transendothelial migration of haemopoietic cells was mediated by the adhesion molecules CD11a/CD18 (LFA-1) and CD31 (PECAM-1) (Yong et al, 1998), while others have reported a role for E-selectin/ESL-1 ligand (Naiyer et al, 1999). In this latter study, the reported levels of adult LTC-IC migration were similar to our data presented here. High expression of VLA-4 on fetal CD34+ cells may be relevant to their migratory and engraftment properties. Although there is evidence from animal models that VLA-4 plays a part in the engraftment of both murine and human progenitors (Papayannopoulou et al, 1995; Peled et al, 2000), in vitro studies have yielded conflicting reports on the role of VLA-4 in the transendothelial migration of CD34+ cells (Yong et al, 1998; Naiyer et al, 1999). LFA-1 expression was lower on fetal CD34+ cells, a finding which may reflect a more primitive state, as LFA-1 levels increase with haemopoietic cell maturation (Campana et al, 1986). Our data on LFA-1 expression on fetal cells are in accordance with a recent study on fetal liver cells (Roy & Verfaillie, 1999); however, those authors also reported that fetal liver CD34+ cells expressed higher levels of l-selectin, a finding which we did not confirm. Roy & Verfaillie (1999) also examined the differential adhesive behaviour of fetal liver, cord blood and adult BM CFC, with the notable finding that fetal CFC adhered significantly more to collagen IV, and that this was mediated by VLA-2. It is not clear, however, from the results of either study, that any specific pattern of adhesion molecule expression or function can account for the distinct migratory features of fetal haemopoietic cells reported here.
In conclusion, we have demonstrated that, when compared with cord or adult cells, fetal haemopoietic cells in blood and liver display superior transmigratory activity which is not directly attributable to adhesion molecule phenotype, or to chemokine receptor expression. The enhanced migratory activity of fetal haemopoietic cells may be a prerequisite for the sequential establishment of haemopoiesis in separate organs and tissues throughout fetal life.