CD14+CD34low Cells With Stem Cell Phenotypic and Functional Features Are the Major Source of Circulating Endothelial Progenitors
Endothelial progenitor cells (EPCs) seem to be a promising tool for cell therapy of acute myocardial infarction, but their nature is still unclear. We show here that EPCs obtainable from peripheral blood (PB) derive from the adhesion-related selection in culture of a subset of CD14+ cells, which, when assessed by the highly-sensitive antibody-conjugated magnetofluorescent liposomes (ACMFL) technique, were found to express CD34. These CD14+CD34low cells represented a variable proportion at individual level of CD14+ cells, ranging from 0.6% to 8.5% of all peripheral-blood leukocytes, and constituted the dominant population among circulating KDR+ cells. By using the ACMFL technique, virtually all CD14+ cells present in the bone marrow were found to be CD14+CD34low double-positive cells. EPCs, as well as purified circulating CD14+CD34low cells, exhibited high expression of embryonic stem cell (SC) markers Nanog and Oct-4, which were downregulated in a STAT3-independent manner when they differentiated into endothelial cells (ECs). Moreover, circulating CD14+CD34low cells, but not CD14+CD34− cells, proliferated in response to SC growth factors, and exhibited clonogenicity and multipotency, as shown by their ability to differentiate not only into ECs, but also into osteoblasts, adipocytes, or neural cells. The results of this study may reconcile apparently contradictory data of the literature, showing the generation of PB-derived EPCs from either CD34+ or CD14+ cells. We suggest that the use of this previously unrecognized population of circulating CD14+CD34low cells, which exhibit both phenotypic and functional features of SCs, may be useful in improving cell-based therapies of vascular and tissue damage.
Infusion of endothelial progenitor cells (EPCs) augments neovascularization of tissue after ischemia providing a novel therapeutic option.1–3 Indeed, these cells have been successfully used to repair tissue damage in both murine experimental models3–5 and in preliminary trials performed in humans experiencing acute myocardial infarction.4,5 EPC populations are usually derived from peripheral blood mononuclear cells (PBMNCs) cultured in presence of vascular endothelial growth factor (VEGF), and identified as a population of adherent cells expressing both acLDL and Ulex-lectin. However, controversy exists with respect to the true nature and origin of circulating EPCs. Although it is well known that CD34+, or CD133+ progenitors, purified by the immunomagnetic technique and cultured in the presence of VEGF, can generate endothelial cells (ECs) and exhibit revascularization properties in vivo,1–7 these cells represent a very small subset of PBMNCs, ranging from 0.02% to 0.1%. Thus, the recovery of a sufficient number of these cells for therapeutic treatments usually requires the mobilization of bone marrow (BM)-derived CD34+ or CD133+ populations by administration of granulocyte colony-stimulating factor (G-CSF);5,7 however, this treatment has been questioned, because of the high risk of adverse events in subjects with vascular disorders.8 On the other hand, adherence-related selection of cultured PBMNCs allows for the recovery of a number of EPCs sufficient for therapeutic treatments, suggesting that EPCs can also originate from circulating populations other than CD34+ or CD133+ progenitors.1–5 Accordingly, conventional cytofluorimetric techniques of PBMNCs-derived EPCs have shown that these cells consist of a population sharing monocytic and EC markers, but not the classic stem cell (SC) markers CD34 and CD133.1–5,9,10 Thus, the precise nature of cells effective in clinical trials4,5 remains unclear.
In this study, we demonstrate that PBMNC-derived EPCs appear to be CD14+ by using the conventional cytofluorimetric technique, but virtually all of them were also found to express low levels of surface CD34, when assessed by highly-sensitive flow cytometric techniques, such as the antibody-conjugated magnetofluorescent liposomes (ACMFL)11 and the fluorescence amplification by sequential employment of reagents (FASER). PBMNC-derived EPCs resulted from the adherence-related selection in culture of a subset of double-positive CD14+CD34low cells preexisting, even if highly diluted, in the circulation. CD14+CD34low cells constituted the dominant population among circulating KDR+ cells and exhibited high expression of mRNA for embryonic SC (ESC) markers, such as Nanog12,13 and Oct-4,14 as well as for a marker of adult SCs, Bmi-1.15 The expression of stemness markers was strongly downregulated in a STAT-3–independent manner after in vitro differentiation of these cells into mature ECs. Finally, circulating double-positive CD14+CD34low cells exhibited clonogenicity in response to SC growth factors and gave rise not only to ECs, but also to osteoblasts, adipocytes, or neural cells.
These data indicate that the major source of EPCs obtainable from peripheral blood (PB) is a subset of double-positive CD14+CD34low cells, showing phenotypic and functional features of multipotent SCs. These properties distinguish them from truly differentiated monocytes, allowing their recovery from PB for cell-based therapies of vascular damages, as well as other tissue damage.
Materials and Methods
See online data supplement available at http://circres.ahajournals.org.
Tissues and Cells
Primary cell cultures were obtained as previously described.16,17 See online data supplement.
Immunomagnetic Isolation of Circulating Cells
Isolation of CD14+ monocytes, CD34+ HSCs, or KDR+ cells was performed by using the MACS system, as described elsewhere.18
PBMNCs (8×106 cells per well), CD14+, CD14+CD34−, and CD14+CD34low cells were cultured as previously described.1–2 See online data supplement.
Flow Cytometry Cell Analysis and Sorting
Flow cytometry analysis of cell surface molecules was performed as detailed elsewhere.18 CD14+CD34low and CD14+CD34− as well as CD133+CD34+ or CD133−CD34+ cells were sorted by using a FACS ARIA with the Diva.
ACMFL and FASER Techniques
The ACMFL technique was performed as detailed elsewhere.11 See online data supplement.
STAT3 phosphorylation was detected in CD14+CD34low cells by Western blotting (see online data supplement), as detailed elsewhere.19
Real-Time Quantitative RT-PCR (TaqManTM)
Taq-Man RT-PCR was performed as described elsewhere.17 See online data supplement.
Confocal microscopy was performed as previously described.20 See online data supplement.
Labeling With CFDA-SE
Labeling of circulating CD14+CD34low or CD14+CD34− sorted cells with CFDA-SE was performed as described previously.18
Colony Formation and In Vitro Multidifferentiation
Generation of clonal cell lines and in vitro differentiation into osteoblasts, adipocytes or neural cells from sorted circulating CD14+CD34low cells was preformed as described elsewhere.21,22 See online data supplement.
Statistical analysis were performed using SPSS software (SPSS, Inc). See online data supplement.
EPCs Derived From In Vitro Cultured Human Adult PBMNCs Are Double-Positive CD14+CD34low Cells
Culturing human adult PBMNCs for 5 days in EGM-MV supplemented with VEGF resulted in an adherent population consisting mainly of acLDL+ Ulex-lectin+ cells (≈90%), as assessed by confocal microscopy (Figure 1A), thus matching the previously described EPC phenotype.1–5 In agreement with the described features of PBMNC-derived EPCs,1,2 virtually all adherent cells examined on day 5 of culture expressed CD14 together with other markers (CD11c, CD16, CD31, CD86, CD105, HLA-DR), in the apparent absence of SC (CD34, CD133) markers (online Table I).
These findings were confirmed at RNA level by using Real time RT-PCR (Figure 1B and data not shown), the only exception being the high expression in PBMNC-derived EPCs of CD34 mRNA (Figure 1B), which was in contrast with the lack of surface CD34 protein (online Table I). The marked dissociation in CD14+ EPCs between mRNA and protein CD34 expression allowed the hypothesis that these cells could display CD34 on their surface at levels below detection sensitivity of the classic flow cytometry. To verify this possibility, we assessed the presence of CD34 on the same cells by a highly sensitive technique, such as ACMFL, which can increase fluorescence signal intensity 100- to 1000-fold compared with conventional methods.11 By using this technique, virtually all EPCs were found to express on their surface not only CD14, but also CD34 (Figure 1C).
EPCs Derived From In Vitro Cultured PBMNCs Result From the Adhesion-Related Selection Of Circulating Double-Positive CD14+CD34low Cells
To establish whether CD14+CD34low EPCs found on day 5 of culture derived from circulating CD14−CD34+ hematopoietic SCs (HSCs) or CD14+ MNCs, CD14+ cells were first purified from PBMNC suspensions by the immunomagnetic technique. The few CD14−CD34+ HSCs (0.012% to 0.2%) were then isolated from the CD14− fraction by the same technique, and the degree of purification was assessed by flow cytometry. CD14+ cells were then stained with CFDA-SE (green) and CD34+ HSCs with PKH26 (red), and both populations were mixed with the unstained CD14−CD34− cells in proportions corresponding to those present in the starting PBMNC population. On day 5 of culture, ie, 1 day after the removal of nonadherent cells, the proportions of green- or red-stained adherent cells were evaluated. Adherent cells consisted mainly of green (CD14+) cells (≈90%), whereas the proportions of red (CD34+) cells never exceeded 1% of the adhering population (Figure 2B), suggesting that, at least at this culture time, CD14−CD34+ HSCs had not represented a relevant source of EPCs, whereas the great majority of them originated from CD14+ cells. To exclude the possibility that preselection of CD14+ cells results in a bias for the generation of EPCs from CD34+ HSCs, in subsequent experiments CD34+ HSCs were first selected by the immunomagnetic technique and stained with PKH26 (red), whereas CD14+ cells were subsequently isolated by the same technique from the remaining CD34− fraction and stained with CFDA-SE (green). The 2 stained populations were mixed together with the unstained CD14-CD34− cells, cultured under the conditions described above, and the proportions of green- or red-stained adherent cells present on day 5 were evaluated. Again, adherent cells consisted mainly of green (CD14+) cells (>90%), whereas the proportions of red (CD34+) cells were never higher than 1% (online Figure I), supporting the concept that independently of which cell fraction was first selected, virtually all cells giving rise to EPCs were contained in the CD14+ population. We therefore asked whether EPCs were derived from CD14+ cells capable of acquiring CD34 after their culturing or from the selection of a preexisting circulating subset of CD14+CD34low cells. To this end, CD14+ cells were purified from PBMNCs by the immunomagnetic technique and then assessed for both CD14 and CD34 expression. As expected, by using conventional flow cytometry, virtually all purified CD14+ cells expressed CD14 but not CD34 (Figure 3A). However, when the highly sensitive ACMFL technique was used, a high proportion of CD14+ cells (range 12% to 75%; mean value: 47%±16%) were found to coexpress CD34 (Figure 3D). Subsequent isolation by the conventional technique from the remaining PBMNCs of CD34+ HSCs (Figure 3B) and their assessment by ACMFL, demonstrated that ≈100× higher levels of CD34 protein were present on CD34+ HSCs than on CD14+CD34low cells (Figure 3E). By contrast, no CD34 expression was observed on the remaining CD14−CD34− PBMNCs even with the ACMFL technique (Figure 3C and 3F). Similar results were obtained by another highly sensitive technique, such as FASER (Figure 3G, 3H, and 3I). No CD133 expression was observed on total CD14+ or CD14−CD34− cells by either classic flow cytometry, ACMFL, or FASER (data not shown). The proportions of CD14+CD34low cells were then evaluated by ACMFL in PBMNCs from a total number of 20 healthy subjects, aged between 24 and 40 years. Percentages of CD14+CD34low cells ranged from 0.6% to 8.5% of all PB leukocytes, the mean value being 4.0%±2.7%.
Previous work has shown that EPCs are contained in the circulating KDR+ population.23 To establish whether CD14+CD34low cells are contained in the same population, KDR-expressing cells were purified from PBMNCs by the immunomagnetic technique and then assessed for CD14 and CD34 expression by conventional flow cytometry. More than 90% of these cells were CD14+, whereas proportions of CD34+ cells were never higher than 1% (Figure 4A). However, when CD34 expression was assessed by ACMFL, the great majority of KDR-purified cells were found to be CD14+CD34low cells (Figure 4B).
Finally, the possibility that CD14+CD34low cells could exist also in the BM was investigated. As expected, by using conventional flow cytometry substantial numbers of both single positive CD14+ or CD34+ cells could be detected in the BM (Figure 4C). Surprisingly, however, when CD14+ BM cells were examined for the expression of CD34 by the ACMFL technique, virtually all them appeared to be CD14+CD34low cells (Figure 4D).
Circulating CD14+CD34low Cells Exhibit Phenotypic Markers of SCs
We then asked whether circulating CD14+CD34low possess phenotypic markers of SCs. To this end, the expression of Nanog, a transcription factor that plays key roles in self-renewal and maintenance of pluripotency in ESCs,12,13 was quantitatively assessed in several adult human tissues, as well as in freshly derived circulating cell populations or primary cultures of different human cell types by real time quantitative RT-PCR (Figure 5A). High Nanog mRNA expression was detectable in human teratocarcinoma, whereas normal human BM, heart, kidney, prostate, skeletal muscle, and spleen exhibited very low or undetectable Nanog mRNA levels, slightly higher levels being found only in adult testis (Figure 5B). Very low or undetectable Nanog mRNA levels were also observed in primary cultures of keratinocytes, human renal proximal tubular epithelial cells, human microvascular endothelial cells, human aortic smooth muscle cells, human dermal fibroblasts, or PBMNCs. By contrast, substantial amounts of Nanog mRNA levels were detectable in purified CD133+CD34+(CD14−) or CD133−CD34+(CD14−) and, even if at lower levels, in purified CD14+ PBMNCs (Figure 5C). However, when purified CD14+ cells were sorted into CD14+CD34low and CD14+CD34− cells by the ACMFL technique, Nanog expression appeared to be enriched in CD14+CD34low cells and was negligible in the CD14+CD34− cell fraction (Figure 5C).
To further support the SC nature of CD14+CD34low PBMNCs and to distinguish them from fully differentiated CD14+CD34− monocytes, CD14+CD34low and CD14+C34− cells sorted by the ACMFL technique (Figure 6A) were cultured in EGM-MV supplemented with VEGF. Nanog, as well as another ESC marker (Oct-4)14 and an adult SC marker (Bmi-1),15 were assessed before culturing, as well as on day 5 and on day 11 of culture. Low levels of these markers were found in CD14+CD34− cells at all times of culture (Figure 6B), and these cells did not acquire phenotypic markers of mature ECs (Figure 6C). By contrast, the 3 SC markers were expressed by fresh derived CD14+CD34low PBMNCs and were maintained at high levels until day 5 of culture, then declining to became virtually undetectable on day 11, when the cells acquired the phenotypic markers of ECs (Figure 6B and 6C). Of note, Nanog protein could also be detected in both fresh and 5-day-cultured CD14+CD34low cells by using confocal microscopy (online Figure II). Similar results were obtained when Oct-4 and Bmi-1 expression was assessed (data not shown).
It has been shown that Nanog expression in human ESCs is downregulated when these cells are induced to differentiate and that, unlike in mouse ESCs, this occurs in a STAT-3–independent manners.24 We therefore assessed whether CD14+CD34low cells exhibited a similar behavior. Indeed, although both fresh CD14+CD34low cells and EPCs were found to express gp130 receptors (Figure 7A), and to respond to leukemia inhibitory factor (LIF) with STAT-3 phosphorylation (Figure 7B), their undifferentiated state was not maintained, as shown by the downregulation of Nanog (Figure 7C) and Oct-4 (data not shown) and the upregulation of ECs markers (Figure 7D).
Circulating CD14+CD34low Cells Possess Clonogenicity and Multidifferentiation Capacity
To provide direct evidence that circulating CD14+CD34low cells also possess the functional features of SCs, CD14+CD34low were separated from CD14+CD34− cells, and both populations assessed for their ability to proliferate in response to stem cell factor (SCF), fms-like tyrosine kinase 3-ligand (Flt3-L), and thrombopoietin. Only CD14+CD34low cells proliferated in response to SC growth factors, as detected by the CFDA-SE technique (Figure 8A). Moreover, under the same conditions, CD14+CD34low cells showed the ability to generate clones, which consisted of at least 30 to 300 cells each (Figure 8B), with a clonal efficiency equal to 27%±6%, although the clonal efficiency of CD14+CD34− was irrelevant (0.8%±0.3%). Finally, sorted CD14+CD34low cells cultured under appropriate conditions were able to differentiate not only into mature ECs (Figure 6D), but could also originate osteoblasts (Figure 8C), adipocytes (Figure 8D), or neural cells (Figure 8E). Differentiation into osteoblasts was demonstrated by the ability of almost every adherent cell to stain for alkaline phosphatase and to form calcium deposits, which stained with Alizarin Red (Figure 8C). Moreover, undifferentiated CD14+CD34low cells did not express the bone-specific transcription factors Osterix and Runx2, but their expression was markedly upregulated after the induction treatment (Figure 8C). Differentiation into adipogenic phenotypes was confirmed by characteristic cell morphology and oil red O staining of lipid vacuoles, that was completely absent from undifferentiated cells (Figure 8D). Furthermore, real-time RT-PCR demonstrated the appearance of very high levels of AP-2 and PPARγ mRNA, two adipocyte-specific transcription factors, that were completely absent in undifferentiated CD14+CD34low cells (Figure 8D). Finally, undifferentiated CD14+CD34low cells did not express glial fibrillary acidic protein (GFAP), neurofilament 200, or the mRNA for neuron specific enolase, although the expression of all these markers was markedly upregulated after the induction treatment (Figure 8E). The same multidifferential potential was observed when differentiation media were added to CD14+CD34low-derived EPCs after 5 days of culture in EGM-MV supplemented with VEGF (data not shown).
Although it has been demonstrated that CD14+ cells can generate EPCs and exhibit revascularizing properties,1–5 they are considered as terminally differentiated cells and there is still no proof of their possible “stemness,” or of their relationships with angioblasts derived from CD34+ cells. Indeed, some authors suggested that the angiogenic effects of EPCs derived from circulating monocytes might be mediated by growth factor secretion.9 Recently, however, Kuwana et al25 described a population of CD14+ monocytes that could differentiate into several distinct mesenchymal cell lineages. These cells, named as monocyte-derived mesenchymal progenitors (MOMPs), were obtained from circulating MNCs cultured on fibronectin for 7 days and had a unique molecular phenotype-CD14+CD45+CD34+.25 MOMPs could be obtained from PB even if deprived of CD34+ cells, but their source, as well as their SC nature, was not clearly defined. Furthermore, the possible relationship between MOMPs and EPCs was not investigated.
In this study, we demonstrate that cells which are usually defined as PBMNC-derived EPCs are CD14+ cells characterized by CD34 surface expression at levels below the detection limits of the classic flow cytometry. Indeed, by using techniques ≈100- to 1000-fold more sensitive, such as ACMFL and FASER, we could demonstrate that virtually all adhering EPCs were double-positive CD14+CD34low cells. These cells originated from the adherence-related selection of a subset of CD14+CD34low cells, representing proportions between 12% and 75% of circulating CD14+ cells, depending of the subject analyzed. The existence of a circulating double-positive CD14+CD34low population may reconcile apparently contradictory data of the literature, some showing the generation of EPCs from CD14+,9,10 and others from CD34+6,7 cells, when recovered by the classic immunomagnetic technique with anti-CD14 or anti-CD34 antibodies, respectively.1–7,9–10 It can also explain the variability in the numbers of EPCs isolated in previous studies from the PB of human adults.1–5 The low levels of CD34 and the higher levels of CD14 expression on these cells is consistent with the observation that more than 95% of this population is usually recovered together with CD14+ cells, and only an irrelevant percentage can be recovered together with CD34+ HSCs, thus also explaining why PBMNC-derived EPCs appear as CD14+ cells if analyzed after their adhesion in culture.
To provide further evidence that CD14+CD34low cells are the major source of PBMNC-derived EPCs, we purified KDR+ cells from PB by the immunomagnetic technique and assessed the expression of CD14 and CD34 by conventional flow cytometry or ACMFL. Indeed, KDR is expressed by ECs but is also considered as a critical marker of EPCs,1–5,7,23 and its expression on CD34+ cells has been reported to identify true SCs and allow to distinguish them from lineage committed progenitors.26 Only ≈1% of KDR+ cells were represented by CD34+CD14− cells when assessed by conventional flow cytometry, although ≈80% of KDR+ cells consisted of CD14+CD34low cells. These findings strongly support the concept that CD14+CD34low cells are the major source of EPCs obtainable from PB and probably constitute a subset of pluripotent circulating SCs. Of note, virtually all BM CD14+ cells, when assessed with ACMFL, also appeared to express CD34, suggesting that circulating CD14+CD34low cells probably result from the migration of a population already present in the BM.
A major difficulty in the identification of the subset that represents the source of PBMNC-derived EPCs has been represented by the absence of an undoubt stemness marker. The results of this study demonstrate that purified CD14+CD34low cells express Nanog, presently known as the most important marker of stemness in mouse and human ESCs,12,13,27 at both mRNA and protein level. By contrast, Nanog was virtually absent from differentiated human adult cells and strongly downregulated when EPCs differentiated into ECs. The stemness phenotype of circulating CD14+CD34low cells was then confirmed by the detection of Oct-4, another marker of ESCs14 and of Bmi-1, an adult SC marker which plays a central role in self-renewal.15 These findings not only provide the first demonstration that Nanog can be a useful marker for the identification of human adult SCs, but also suggest the possibility that it plays a crucial role in maintaining their undifferentiated state. This possibility is only apparently in contrast with the expression by EPCs of acLDL and Ulex-lectin, which are considered as markers of committed endothelial progenitors. Indeed, acLDL and Ulex-lectin expression has also been detected in different BM cell types, including SCs.3 It has been shown that Nanog expression in mouse ESCs is maintained through a signal transduction pathway involving the gp130 receptors and STAT-3 activation.24 By contrast, stimulation of human ESCs by gp130 cytokines was not sufficient to maintain these cells in an undifferentiated state.24 Likewise, despite the expression of gp130 receptors, the addition of LIF to VEGF-containing cultures induced STAT-3 phosphorylation, but it was unable to maintain their undifferentiated state, as shown by the downregulation of both Nanog and Oct-4 and the appearance of markers of ECs. These findings demonstrate that signaling through the gp130/STAT3 pathway is insufficient to prevent the onset of differentiation at least in this type of adult human SCs. Very recently, the ability of activin A to maintain high Nanog and Oct-4 levels, as well as pluripotency, in human ESCs has been reported.28 Whether activin A is able to play the same role in double-positive CD14+CD34low cells remains to be established.
The expression of Nanog and Oct-4 strongly suggested that circulating CD14+CD34low cells might represent multipotent SCs. Accordingly, these cells exhibited proliferative response to SC growth factors, clonogenicity, and multidifferentiation potential, as shown by their ability to give rise not only to ECs, but also to osteoblasts, adipocytes, or neural cells. By contrast, CD14+CD34− cells neither exhibited clonogenicity nor multidifferentiation capacity, suggesting their nature of fully differentiated monocytes. Taken together, these data provide the first evidence that a relevant, even if variable, percentage of CD14+ cells consist of double-positive CD14+CD34low cells showing phenotypic and functional features of multipotent SCs. They also suggest the possibility that the purification of these cells may provide a useful tool to get high numbers of purified EPCs for possible practical use in neovascularization and for the repair of tissue damages.
This work was supported by the Ministery of Health of Tuscany (Italy).
↵*Both authors contributed equally to this work.
Original received December 15, 2004; resubmission received April 28, 2005; revised resubmission received July 5, 2005; accepted July 6, 2005.
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