It has been proposed that the clinical mobilization of hematopoietic stem cells mimics mechanisms that naturally occur during the migration and engraftment of HSC in the fetus and adult [8
]. However, the molecular profile of adult and fetal HSC differs significantly. In studies comparing extracellular matrix and adhesion molecule gene expression, LSK from FL14.5 and adult bone marrow differed in more than 20% of the genes studied [34
]. This study analyzed only fetal liver LKS cells at 14.5 dpc and not other developmental stages. A more inclusive quantitative analysis, including different fetal developmental stages and microenvironments, is necessary to evaluate differences between the adult and fetal LSK molecular phenotype. Here, we have compared the extracellular matrix, adhesion and chemokine gene expression pattern of LSK populations from FL14.5, FL17.5 as well as FBM17.5 to adult bone marrow LSK in order to identify genes that modulate during development.
We initiated our study by analyzing the frequency and absolute cell numbers of the LSK population as well as LT-HSC in the aforementioned tissues. During ontogeny, the increase in fetal liver HSC numbers takes place after 12.5 dpc, which agrees with our observed HSC increase from 14.5 dpc to 17.5 dpc. However, the number of fetal liver HSCs attains its maximum by 15.5 to 16.5 dpc, reaching a plateau and then declining [8
]. The migration of HSCs to the blood, spleen, and bone marrow by 17.5 dpc [8
] could account for the reduction of HSC in the fetal liver at this developmental time, and is consistent with the lack of significant differences observed in the frequencies and absolute cell numbers of LSK and LT-HSCs between the fetal tissues studied. Moreover, when adult bone marrow was analyzed, significant increases were observed in LSK and LT-HSCs cells, suggesting a definitive expansion of the HSCs pool in this tissue.
We sorted the LSK and LT-HSC populations from different locations during development in order to evaluate their molecular profiles. We found that the absolute number of LT-HSCs from these different embryonic sites was very low; especially in the FBM17.5, where an average of five LT-HSCs could be isolated per embryo. This low number of cells precluded us from further analyzing LT-HSC by quantitative PCR array. Therefore, we decided to quantify only the molecular profile of LSK cells, since this population includes LT-HSCs [34
]. LSK cells from FL14.5 showed a different gene expression profile than adult BM, with significant differences in 44% of the extracellular matrix and adhesion molecules genes analyzed. This difference is greater than what was previously reported by Ivanova et al. using Affymetrix oligonucleotide arrays [34
]. In general, the gene expression of extracellular, adhesion and chemokine molecules in FL14.5 LSK cells was lower than in the rest of the tissues studied. This lower expression could result from the downregulation of migration related molecules at this stage, when LSKs are in a process of vigorous cellular expansion [6
]. At later stages of development, no significant differences in gene expression were found between LSK cells from FL17.5 and FBM17.5, or between FBM17.5 cells and adult BM LSK. The lack of difference among these LSK samples suggests a similar extracellular matrix and adhesion molecule genetic program throughout these different microenvironments. Few genes were differentially expressed when the chemokines and chemokine receptor profile was compared in LSK populations at 17.5 dpc and adult BM. Overall, the genetic program in LSK populations was more similar the closer they were in temporal developmental stage. However, there are some genes that remain significantly different between all the stages of development studied and these merit further discussion.
One of these genes is Col4a1
(Collagen, type IV, alpha 1), a major constituent of basement membranes [37
]. Its expression was significantly different midst LSK populations at all the stages of development, with the exception of FL17.5 and FBM17.5. We hypothesize that LSK cells may contribute to the architecture of its immediate microenvironment by secreting collagen IV onto the extracellular matrix [31
]. Matrix metalloproteinase 2 (Mmp2
) was significantly upregulated in the adult BM when compared with either the FL14.5 or FL17.5 sample. No differences were detected when either FL tissues were compared to FBM17.5, due to its large gene expression variation. MMP2 degrades collagen IV and it could help in LSK motility by releasing them from the extracellular matrix [31
]. However, increased expression of Mmp2
can mediate also the inactivation of CXCL12, impairing the homing of the HSC to the bone marrow [38
]. On the other hand, multipotent progenitors (MPP) have been described to upregulate Mmp2
expression in adult bone marrow [31
]. Whether our observed increase of Mmp2
expression is due to LT-HSCs or MPPs still needs to be clarified. Interestingly, the tissue inhibitor of metalloproteinase 2 (Timp2
), whose main target is MMP2 [39
], showed a pattern of expression similar to Mmp2
. However, it is also significantly increased in FBM17.5 LSK cells when compared to FL14.5 LSKs. TIMP2 can be upregulated in order to modulate the proteolytic activity of MMP2, which might be required to help LSK cells leave or remodel their microenvironment. Multipotent progenitors downregulate Timp2
expression in the adult bone marrow [31
] suggesting that its increased expression could reside within the LT-HSCs or ST-HSC populations.
The expression of transforming growth factor beta-induced (Tgfbi
) increased significantly during development, with no difference between the FL and FBM at 17.5 dpc. TGFBI is a secreted extracellular matrix adaptor protein whose physiological function involves cell-matrix interactions and cell migration. It binds to type I, type II and type IV collagens and it may be involved in endochondral bone formation in cartilage [40
]. TGF-β upregulates the expression of Tgfbi
] and its expression is higher on HSC adherent to mesenchymal stem cells [42
]. Similarly to Tgfbi
, the expression of Sell
) increased at the different stages of development studied without significant difference between the FL14.5 and FBM17.5 sample, due to the large gene expression variability in the FBM17.5 sample. Selectin L is a membrane-bound C-type lectin that binds to cell-surface glycosylated ligands [14
] and its role in the migration of hematopoietic stem cells is controversial. Sell
is highly expressed in mobilized CD34+
], but it does not appear to contribute to the interaction of HSCs with bone marrow microvessels [44
]. Furthermore, our data show that the expression of ectonucleoside triphosphate diphosphohydrolase 1 (Entpd1
) increased also at the different developmental stages studied, following a pattern similar to Sell
. CD39 hydrolyzes NTP and NDP to NMP. Extracellular nucleotides enhance the stimulatory activity of several cytokines and can expand the number of human HSCs repopulating the bone marrow [45
]. Taken together, the upregulation of these extracellular matrix and adhesion molecules suggest that they might play a role in the homing and migration of LSK cells between fetal microenvironments during development.
Our results also showed differences in the expression of chemokines and their receptors during development. The expression of the chemokine (C-C motif) ligand 4 (Ccl4
or macrophage inflammatory protein-1β, MIP-1β
) increased significantly in the LSK population from different microenvironments. CCL4 could be involved in migration processes of LSK cells since it has chemoattractant properties and it is inducible in most mature hematopoietic cells [46
]. Similarly, chemokine (C-C) motif ligand 9 (Ccl9
, or macrophage inflammatory protein-1γ MIP-1γ
), showed a significant upregulation along development in all the tissues studied. It has been shown that rat CCL9 induces the migration of adult bone marrow cells, although the population responsive to CCL9 was not characterized [47
]. However, in adult bone marrow LSK, Ccl4
expression is upregulated in multipotent progenitors [31
]. The expression of Interleukin-18 (Il18
), a pro-inflammatory cytokine produced by macrophages and other cells, increased also significantly in all the populations studied. The involvement of Il18
in hematopoietic stem cell migration is unclear. It will be necessary to discern if the observed increase in the expression of these chemokines at the different microenvironments studied occurs in the MMP or the LT-HSC population.
The expression of the chemokine (C-X-C motif) receptor 4 (Cxcr4
) augmented significantly in the adult bone marrow when compared to the fetal samples studied. CXCR4 has been described extensively as the key chemokine receptor involved in the migration of HSCs [6
]. However, few of these studies evaluated the role of CXCR4 in the migration of fetal liver HSCs. Christensen et al. observed that 14.5 dpc fetal liver HSCs did not migrate at the same level as adult HSCs upon stimulation with SDF-1α [8
]. The expression of Cxcr4
in fetal liver LSK is significantly lower than in adult or fetal bone marrow LSK, which may explain the reduced migration observed in their studies. In summary, the upregulation of the aforementioned chemokines and CXCR4 indicates that they might govern the migration of LSK cells between fetal microenvironments during development.
Surprisingly, adhesion molecules described as relevant for the homing and migration of adult hematopoietic stem cells, such as Vla4
] and Cdh2
] did not show significant differences among the populations studied. Similarly, no significant differences were observed with chemokines receptors expressed in adult HSCs, such as Ccr9
]. However, molecules whose expression does not show significant variance might still play an important role in LSK migration during development.
Our study shows that LSK cells upregulate the expression of several extracellular matrix, adhesion, and chemokine molecules, shown to be components of the bone marrow niche [1
]. These data suggest that LSK might contribute to the architecture of their niche [31
]. However, cells other than LSK might contribute to a greater extent to the architecture of the stem cell niche, as LSK are extremely rare cells. The final architecture of the stem cell niche might result from the combined molecular and cellular contributions of the stem cells as well as niche cells.