Preterm delivery is one of the most important factors of neonatal mortality and morbidity throughout the world. Recently, the incidence of perinatal death has considerably decreased as neonatological care has improved. However, managing morbidity after preterm labour is still a critical issue
. One of the current major issues in perinatal medicine is the search of valuable and early indicators of prematurity complications onset. Several recent retrospective studies were settled to find relationship between the blood characteristic markers or abnormalities and specific prematurity complication or an inflammatory process, however, not always giving the accurate results
During prenatal development, various types of stem cells migrate, proliferate and differentiate to form tissues and organs. Tissue and peripheral blood SPCs pools are in dynamic equilibrium with each other, allowing stem cells to migrate from extravascular sites or marginal pools into the circulation and vice versa
. Whereas 37–42 weeks of gestation provide an optimal period of time for an infant’s maturation to extrauterine life, preterm birth deeply disturbs normal development. This prospective study was performed to elucidate the potential cause and effect relationship between the circulating SC populations and evolution of premature birth complications.
The absolute numbers of circulating non-HSCs/VSELs in PB are remarkably low (1–2 cells in 1 μL of blood under steady-state conditions). Similarly, circulating HSCs represent a very small fraction of PB cells, and thus special flow cytometry protocols have to be applied for identification of those highly restrictive SC populations
. We employed nuclear staining for detection of NCs present in blood samples and for exclusion of anucleated cellular debris from further analysis. Such a defined fraction of small NCs was further analyzed for CD45 and CD184 antigen expression, therefore two subpopulations, i.e. CD45–lin–CD184+ non-HSCs/VSELs and CD45+lin–CD184+ HSCs, could be distinguished
. Receptor CXCR4 (CD184) plays an important role in the mechanisms of HSCs migration and repopulation, in regard to the observation, that murine fetuses lacking this receptor (CXCR4-null model) have multiple defects that are lethal, including impaired BM hematopoiesis
. Recently, we were able, not only to confirm the presence of such highly restrictive SCs populations among blood leukocytes, but also to quantitatively determine the absolute numbers of these infrequent cells circulating in the blood of patients with various tissue/organ injuries and disorders
[5, 6, 17]. Of note, the defined CD45–lin–CD184+ and CD45+lin–CD184+ SC populations have not been previously evaluated in relatively long-term prospective observation in PB of preterm infants.
This study demonstrated for the first time that the number of primitive non-HSCs circulating in CB is inversely associated with the birth weight of premature infants. Our observations imply that during the fetal stage of human life, primitive, undifferentiated stem cells circulate in the blood in a large number and contribute to organ/tissue formation. Gradually, the number of non-HSCs decreases and stabilizes along with the infant’s maturation. The characteristics of the actually analyzed non-HSCs correspond with very small embryonic-like SCs (VSELs) expressing pluripotent SC markers, described recently in CB by Kucia et al.
, with a contribution by our research team. It has been demonstrated in mice that VSELs reveal pluripotency and are able to form all types of mature cells
[30, 31]. Furthermore, after the number of molecular analyses and in vivo experiments performed by Ratajczak et al., it has been recently proposed that human CB-derived VSELs correspond to the population of the most primitive HSCs circulating in the PB
. Therefore, if a small premature infant loses a substantial number of circulating non-HSCs, enriched in VSELs, together with secundines, this might well have negative clinical consequences for the infant’s development in the long-term. As we and others have recently reported, VSELs are mobilized in the PB of patients with ischemic stroke, myocardial infarction, or heavy burns, and this increase in the number of circulating cells is accompanied by elevated plasma levels of SDF-1
[5–7]. It has been hypothesized that these cells could attempt to regenerate the damaged tissue. Although we did not observe strict evidence of an analogous phenomenon in this study, however the number of non-HSCs/VSELs circulating in PB of premature infants was significantly greater than in full-term babies two weeks after delivery. At the same time point, SDF-1 plasma levels in preterm infants were considerably higher compared to the initial concentration of this chemokine at premature birth. Noteworthy, we noticed a strong correlation between CB SDF-1 levels and the number of non-HSCs/VSELs two weeks after delivery. Together, these observations may suggest that similar pathophysiological responses observed in stroke patients are noticed in small premature infants in an attempt to maintain PB-derived non-HSCs in relatively high concentrations. In order to clarify whether or not and how the undifferentiated non-HSCs/VSELs actively support regeneration in the first few weeks of human life after birth, or whether they remain largely in a dormant state, further studies are necessary. However, the differentiation of VSEL-SCs into human tissue-specific SCs (e.g. hematopoietic or neural) is not entirely elucidated and it is postulated that this process requires a relatively long period of time
Clearly, the question arises as to whether or not a number of SCs circulating in CB is associated with the development of premature birth complications. As a first step towards addressing this issue, we sought to identify the highly purified populations of SCs with either durable or limited cause-and-effect relationships. Our results provide for the first time evidence that the number of HSCs circulating in CB is the independent predictor inversely associated with the development of premature birth complications. These include infections, anemia, IVH, and RDS, complications associated with blood and vascular systems origin, the development of which appears to be conditioned either directly (anemia, infections) or indirectly (other complications) by HSCs activity. Hematopoiesis is a complex, hierarchical process which involves the expansion and differentiation of a limited number of HSCs into multipotential and lineage-restricted progenitors, leading to the production of mature and functional blood cells
. HSCs, and their downstream precursors such as common lymphoid or myeloid progenitors that possess substantial clonogenic properties, are able to repopulate the whole medullary and extramedullary hematopoiesis in case of blood loss that, for example, may occur in case of IVH in preterm neonates
. Similarly, rapid rate of neonatal growth is one of the causes of anemia of prematurity that usually occurs during the first or second week of life in very small preemies. On the other side, iatrogenic blood loss secondary to sampling of blood for laboratory tests is nowadays the commonest cause of anemia of prematurity
. Relative but chronic deficiency of RBCs must be quickly compensated by increased proliferative activity of HSCs to reduce the potential biological effects of the RBCs scarcity in the body that might result in or might exaggerate the hypoxia-related complications in preterm newborns. Similarly, HSCs and their progenies are responsible for proper homeostasis maintenance of immune cells of myeloid and lymphoid lineage that protect the newborns against infections during the early stages of post-natal development. Thus, taken together, the number of active HSCs determines blood morphology and functions to effectively prevent both anemia and infections. However, it should be noted that association of HSCs with anemia lost significance in multivariate model and severe confounding by gestational age was observed. Nevertheless, there was no proof for other such confounding effects regarding associations of other complications with HSCs, since the associations retained statistical significance in multivariate analyses and respective OR values were similar in the uni- and multivariate logistic regression models.
What is more, HSCs also influence vascular homeostasis since the endothelial progenitor/precursor cell types are thought to be derived from the hematopoietic and vascular systems
. The concurrent emergence of hematopoietic and endothelial precursors in the embryonic yolk sac, as well as their overlapping patterns of gene expression, provides circumstantial evidence for the derivation of these cell types through a common progenitor, called hemangioblast, capable of giving rise to both endothelial and hematopoietic SCs at the earliest stages of hemato-endothelial differentiation
. The multifaceted modality of hemangioblast progenies, up to some extent, might be responsible for the fact, that circulating HSCs derived from bone marrow have been shown to participate in the normal and pathologic postnatal angiogenesis. Moreover, they were also able to induce neovascularization when transplanted into ischemic tissues
. Since, in such indirect way, HSCs could also correspond to the development of IVH or RDS in premature infants. As there is at least indirect association between vascular dysfunction and pathogenesis of RDS and IVH, these pathologies might be defined as ‘vascular-associated’ disorders
Furthermore, we showed that the number of HSCs remains stable for six weeks after birth, the period when most of the abovementioned complications develop. This, in turn, at least partly explains the protective effect of HSCs against prematurity complications, since their low pool in CB at birth is associated with a long-term HSC deficit as evidenced by strong correlations between HSC percentages at birth and six weeks after birth. Of note, the number of circulating HSCs, unlike the non-HSCs/VSELs, did not increase in parallel with increased SDF-1 plasma level, detected two weeks after birth, in preterm infants. Our finding is similar to the observation described recently in patients with heavy burns
. In the light of recent reports, the involvement of other factors including small bioactive lipids that may direct mobilization and trafficking of SCs, should be also considered to determine precisely the conditions responsible for egress and trafficking of CXCR-4 positive HSCs
. Indeed, Bowie et al.
 have recently reported that up to ~4 weeks post-partum BM-derived HSCs have higher cell-cycling rates than those from adult BM. They provide evidence that the chronologically younger HSCs produce considerable amounts of SDF-1, which stimulates these cells into cycle, and this may reflect the need for HSCs proliferation or self-renewal during periods of rapid body growth. Probably, the above observation made by Bowie et al.
 would also explain the lack of direct association between circulating HSCs counts and post-natal SDF-1 levels in full-term and preterm infants found in our study. It is postulated that secretion of considerable amounts of SDF-1, in an autocrine manner by cycling post-natal HSCs, might interfere with HSC ability to respond to a chemotactic SDF-1 gradient, and so impede their trans-marrow trafficking, lodgment and retention in BM niches
To the best of our knowledge, this is the first report clearly demonstrating that the human HSC population is associated with certain prematurity complications. Our results are in accordance with other clinical studies and suggest an important role of different circulating SC subpopulations in the development of preterm birth complications. Likewise, recent findings suggest a protective role of circulating progenitor cells in respiratory system disorder found in neonates born prematurely. The group of Qi et al. observed that RDS survivors had higher counts of CD34+ progenitor cells compared to non-survivors. Moreover, they observed that low concentrations of circulating CD34+ cells were correlated with a prolonged duration of mechanical ventilation of neonates with RDS, which can indicate the disease progression
. In a similar manner, it has recently been suggested that circulating hematopoietic cells may play an important vital role in repairing injured tissues and in disease progression, and thus differential hematopoietic cell populations may reveal a special paradigm as predictors for human morbidity. Great attention has recently been given to selected subsets of circulating CD34+ cells in patients with ischemic heart disease, as these cell populations might include those involved directly or indirectly in vascular repair. Likewise, it has been recently demonstrated that some cells, defined according to the International Society of Hematotherapy and Graft Engineering (ISHAGE) criteria as circulating hematopoietic progenitor cells (HPCs; CD34+CD45dimVEGFR2- and CD34+CD45dimCD133+VEGFR2-), were reduced in patients presenting clinical signs of endothelial dysfunction, and thus it was postulated that the number of HPCs might be an independent predictor of morbidity from cardiovascular diseases and a marker of atherosclerotic disease progression
. Interestingly, these results are, to some extent, analogous to our data, in that the number of highly selected circulating HSCs is inversely correlated with the risk of the development of prematurity complications. Altogether, based on the above-mentioned data, we postulate that analysis of circulating HSC counts may be a valuable predictor for preterm birth-related morbidity.
A number of different circulating hematopoietic cell subsets described as CD34-positive cells were recently shown to be altered in neonates with prematurity complications, but without discrimination between more specific cell phenotype characteristics such as Lin- or CD184+CD45+ and CD184+CD45-[43, 45]. These observations were confirmed and extended in the present study. Especially, Lin-CD184+ cells were suggested to be more immature precursor cells of CD34-positive cells with a higher potency regarding peripheral tissue repair mechanisms
[46, 47]. Finally, in some instances, HSCs have been shown to contribute to the regeneration of chronically injured non-hematopoietic tissues. It is interesting to note that, according to recent critical reports, HSCs neither undergo transdifferentiation into cell types other than hematopoietic lineage cells nor structurally contribute to non-hematopoietic tissue regeneration on a significant scale
[48, 49]. However, after incorporation into injured tissue, HSCs might well produce and secrete humoral factors, creating a conducive microenvironment which promotes cell chemoattraction, survival and proliferation
. Likewise, circulating CB and early post-natal bone marrow HSCs have been previously reported not to home to the BM niches and, within the HSCs engrafting populations, ~95% were in the G0 phase (see
 and references therein). These observations suggest that mobilized HSCs migrate from BM to the peripheral circulation where they indirectly contribute to organ regeneration and help to restore the integrity of extra-marrow tissues. It is worth noting that HSC transplantation has recently been used extensively for the treatment of numerous metabolic diseases in children
. Because of the variety of functions of HSCs in the human organism, their precise role in each disease condition may vary and should be carefully examined.
Previous studies have shown that hematopoietic progenitor cells express the CD34 surface antigen
. Likewise, CD34-positive cells represent a functionally primitive population of progenitor cells that seem to have a higher cloning efficiency and a very rapid proliferative response to cytokine stimulation
. Moreover, recent studies in bone marrow have also identified a CD133+ cell population, which is rare, undergoes self-renewal and differentiation and might represent stem/progenitor cell population
. Thus, we evaluated cell surface expression of CD34 and CD133 antigens, aiming to characterize the highly heterogenic circulating progenitor cell compartment in the blood of preterm infants and their relation with preterm morbidity. Our results showed that the percentage of CSPCs detectable in CB was significantly higher in preterm newborns compared with full-term fetuses. These results are in accordance with those of Haneline et al.
 and Opie et al.
, who reported significantly higher numbers of circulating progenitors in the CB of surviving preterm infants (23–32 wks of GA) and stillborn fetuses (< 24 wks) compared with the CB of mature newborns. In addition, we found that the number of CB-derived CD133+CD34+ and CD133-CD34+ cells was higher in preterm infants who developed prematurity complications such as RDS, BPD and NEC. Besides, anemia was associated only with a higher number of CD133-CD34+ CSPCs. However, a statistically significant correlation between the number of CSPCs and GA was also noticed and the associations of CSPCs with prematurity complications lost significance in multivariate analysis adjusted for gestational age.
In the present study, to identify hematopoiesis-related cells among SPCs circulating in cord blood, clonogenic assays were employed. Here, we found that the proliferative potential of BFU-E and CFU-GM was significantly higher in preterm infants than in full-term infants and colony numbers positively correlated with the number of CSPCs in CB. Our findings seem to reproduce the studies of Opie et al.
, showing a decreased frequency of clonogenic precursors with advancing gestational age. This strongly supports the notion that detected CD133+CD34+ and CD133-CD34+ cells, circulating in CB, determines a strong clonogenic potential of hematopoietic origin, whereas other examined populations such as CD45-lin-CD184+ and CD45+lin-CD184+ cells are not directly associated with clonogenicity, what might indicate their stem derivation and more quiescent state. Finally, the differences in BFU-E and CFU-GM numbers between preterm and full-term infants were consistent with differences in the number of CD133+CD34+ and CD133-CD34+ CB cells between these groups (Table
2). The results obtained strongly indicate that analyzed subpopulations represent CB-derived hematopoietic progenitors.
Furthermore, our data seemed to show significant changes in the numbers of CSPCs in blood after birth. The number of CD133+CD34+ and CD133-CD34+ cells in PB gradually decreased during the first six weeks after birth until the quantities were similar to those detected in full-term infants. As the observed decrease in the number of CSPCs runs in parallel with advancing GA only in the premature infants group, this could indicate that the quantities of hematopoietic progenitors decrease physiologically until the 36th week of GA, and are stable thereafter.