Although there is ample evidence that ADSC are bona fide adult stem cells, the location of these cells in adipose tissue remained unknown. Recently, Yamamoto et al. [6
] used IF staining on mouse adipose tissue to locate cells positive for CD90, CD105, Sca-1, and/or p75NTR. Their results showed widespread distribution of each of these four markers, thus casting doubts on the suitability of these markers for the identification of ADSC. Another study by Zannettino et al. [7
] attempted to identify ADSC in human adipose tissue by employing IF staining for cellular markers 1A6.12, 1B5, STRO-1, CD146, and 3G5. In the histology images presented, each of the two large blood vessels was shown without any reference landmarks, thus making it impossible to know their relationship with the rest of the adipose tissue. Furthermore, although the images show that all of the tested markers localized to the vessel wall, they do not display sufficient details as to tell whether the markers are located in the intima or in the adventitia. Thus, this latter study does not provide us a better understanding of the identity of ADSC within adipose tissue.
Another study [20
], which was published online while this manuscript was in preparation, focused on CD34+ cells in adipose tissue. The results showed that CD34+ cells are widely distributed among adipocytes and predominantly associated with vascular structures. Although these observations appear to be similar to ours, their histological analyses show only the longitudinal aspect of two blood vessels of unknown sizes and thus are of limited usefulness in terms of delineating the cellular composition of the adipose vasculature. In spite of these limitations, the study further showed that the majority of the CD34+ cells expressed pericytic markers, and this appears to suggest that ADSC are CD34+ pericytes. Whether CD34+ cells are ADSC and whether ADSC are CD34+ will be discussed below.
As pointed out by Zannettino et al. [7
], mesenchymal stem cells, including ADSC, likely reside in perivascular niches. The fact that ADSC are isolated from adipose SVF further points to a close relationship between ADSC and blood vessels. Thus, in the present study, we sought to locate ADSC by employing vascular smooth muscle marker α-SMA, endothelial marker CD31, hematopoietic marker CD34, niche marker Wnt5a, and stem cell markers OCT4, telomerase, SSEA1, and STRO-1. Because IH staining provides optimal histology whereas IF enables colocalization (with double or triple staining), both techniques were used throughout this study to localize these markers except for OCT4, telomerase, and SSEA1 whose antibodies produced poor IF staining.
Due to their specificity for smooth muscle and the endothelium, respectively, anti-α-SMA and anti-CD31 antibodies enabled us to visualize the distribution of blood vessels in the adipose tissue specimens. Anti-CD31 antibody was not expected to stain ADSC as it is generally agreed that ADSC lack CD31 expression [1
]. Anti-α-SMA antibody clearly stained cells in capillaries (), thus implicating these cells as pericytes. However, these cells were invariably CD34−, suggesting that pericytes in the adipose vasculature do not express CD34 and thus contradicting the findings by Traktuev et al. [20
]. Despite of this disagreement, our results do not exclude the possibility that ADSC are pericytes and vice versa.
Wnt signaling has been shown to regulate the self-renewal and differentiation of both hematopoietic and BMSC, and importantly Wnt5a has been localized to the bone marrow niche environment [23
]. In the present study, Wnt5a localized to the smooth muscle of small blood vessels, arterioles, and venules. It does not appear that Wnt5 was expressed in the endothelium, based on the lack of colocalization with CD34 (). In capillaries Wnt5a staining was still evident (), and which again was distinct from CD34 staining. Therefore, it appears that, similar to α-SMA, Wnt5a was expressed in smooth muscle cells of small vessels and in pericytes of capillaries, and neither cell types expressed CD34. This expression pattern suggests that neither vascular smooth muscle cells nor pericytes are ADSC but rather niche cells of ADSC. However, since “stem cell niche” is still a poorly defined biological entity, this Wnt-niche-ADSC hypothesis can only be substantiated in future studies. Interestingly, a recent study showed that recombinant Wnt5a protein could induce the differentiation of ADSC into beating cardiomyocyte colonies in a dose-dependent manner [25
CD34 has long been regarded a reliable marker for hematopoiectic stem cells (HSC), but recent studies have demonstrated the existence of CD34-negative HSC and that the two populations of HSC (CD34+ and CD34−) can differentiate into one another [26
]. Several papers, including ours, have shown that CD34 is highly expressed in freshly isolated ADSC (SVF cells), but its expression is quickly lost in cultured ADSC within the first few (<3) passages [1
]. This loss of expression is probably due to downregulation of CD34 expression rather than death of CD34+ cells (unpublished observation). In any event, the abundance of CD34 expression in SVF cells can perhaps be explained by the abundance of CD34+ cells in adipose tissue, as reported both in the present study and in a recent study [20
]. Although CD34 localized to the endothelium, its staining was homogeneous while that of CD31 was more intense at intercellular junctions (–). This differential staining of the endothelium by anti-CD31 and anti-CD34 antibodies has been previously observed in blood and lymphatic vessels [27
]. In addition, CD34 staining was different from that of CD31 in that it was visible in the adventitia of all blood vessels; thus, the cross section of these CD34-stained vessels had the appearance of two concentric circles (endothelium and adventitia) sandwiching the unstained smooth muscle layer ( and ). A similar staining pattern in subcutaneous tissue has been previously reported [30
Although the CD34+CD31+ inner circle of blood vessels is undoubtedly the endothelium, the CD34+CD31− outer circle is an entity of uncertain cellular identity. The outer circle, being outside of the muscle layer, is by definition the adventitia. Although the identification of CD34+CD31− cells in the adventitia of blood vessels in adipose tissue is a novel contribution by the present study, similar cells have in fact been observed previously in the aortic adventitia, and, interestingly, these cells have been shown to be vascular progenitor cells [31
]. In regard to the CD34+CD31− cells in adipose tissue, two previous studies have shown that this particular cell population possess both endothelial and adipocytic differentiation potentials [16
]. With a focus on in vitro differentiation, these studies presented no data on the localization of the CD34+ CD31− cells in adipose tissue. More recently, as mentioned above, a recent study [20
] showed that CD34+CD31− cells in adipose tissue are pericytes that also express α-SMA, and the authors went on to suggest that these CD34+CD31−α-SMA+ cells are ADSC. However, our staining data clearly showed that, while CD34+CD31− cells resided in the adventitia, the α-SMA+ cells were found exclusively in the media of arteries and arterioles ( and ). Thus, while we agree that ADSC are generally CD34+CD31−, we disagree that ADSC are CD34+CD31−α-SMA+. We also dispute the contention that pericytes are CD34+, as we clearly show that CD34 did not colocalize with CD140/PDGFRβ (). However, as pericytes remain an ill-defined entity, we cannot exclude the possibility that ADSC are certain types of pericytes.
OCT4 is a widely accepted marker of embryonic stem cells, but its expression in adult stem cells is less certain [33
]. To our knowledge, only one paper has provided experimental data concerning OCT4 expression in ADSC and it showed that OCT4 was abundantly expressed in cultured ADSC [34
]. Similar to this study, we performed RT-PCR and western blot analysis on OCT4 expression in human adipose tissue and in cultured human ADSC, but our results were different from the above study in that, while clearly detectable, OCT4 expression was low in all tested tissue and cell samples (data not shown). In the present study, histological and flow cytometric analyses again respectively showed that OCT4+ cells were rarely detectable in the adipose tissue and cultured ADSC. These results are consistent with the well-known fact that OCT4 is an embryonic transcription factor whose expression persists in only a limited number of cells in adult tissues [33
Similar to OCT4, telomerase is better accepted as a stem cell marker for ESC than for adult stem cells [35
]. In cultured ADSC, telomerase was reported to be present [36
] or absent [40
]. In adipose tissue, our present study appears to be the first to examine telomerase expression, and which was detectable in two capillary cells. Their location and morphology suggest they are undifferentiated cells.
SSEA1 is a marker for ESC and BMSC [41
]. Its expression was reported to be negative in cultured human ADSC by flow cytometric analysis [38
]. However, in the present study, both cytometric and histological analyses showed that SSEA1 was expressed at rather high levels. In adipose tissue SSEA1 was expressed at a lower level than CD34 (), but in cultured ADSC of passage 0, it was expressed at a higher level than CD34 (25.7% vs. 22.5%, ). This is most likely due to the rapid downregulation of CD34 in cultured ADSC.
STRO-1 is by far the best-known MSC marker [43
] but whether or not it is expressed in ADSC is controversial [1
]. Although it was reported to be negative by Gronthos et al. in 2001 [44
], it was nevertheless used in the author's recent paper as an ADSC marker [7
]. In our previous and present studies, we showed that STRO-1 was detectable both in cultured ADSC [22
] and in adipose tissue. However, its expression pattern in adipose tissue was surprising—highly specific for endothelial cells in arterioles and capillaries but not in arteries. In capillaries, some of the positively stained cells were also stained positive for α-SMA (), suggesting the existence of vascular progenitor cells. Positive staining of endothelial cells by anti-STRO-1 antibody has been reported before, but other cell types were also positively stained [45
]. One of the reasons why immunostaining with the STRO-1 antibody produced conflicting results is that, despite its widespread use in hundreds of published studies, this antibody remains an orphan with no known antigen. In our current research, we have found that the STRO-1 antibody produced rather complicated staining patterns in various tissues. Thus, prolonged efforts will be required to solve the problems.
Based on our experimental results and the above-discussed evidence, we believe that ADSC exist in adipose tissue as a mixed population of “vascular stem cells (VSC)”, as opposed to “fat stem cells”, which is casually but incorrectly used as a synonym for ADSC. This “vascular stem cell theory” suggests (1) ADSC and mesenchymal stem cells in general are VSC, (2) the differentiation potential of VSC is proportional to the angiogenic potential of the vasculature and ranges from pluri to multi to uni in a continuous rather than discrete fashion, (3) in addition to vascular smooth muscle and endothelial cells, VSC are capable of differentiating in situ into host tissue-specific cell types (e.g., adipocytes in adipose tissue), and (4) depending on its differentiation potential at the time of isolation from the host tissue, an individual VSC can be experimentally induced to differentiate into various cell types. The theory thus helps explain many perplexing issues in regard to ADSC's cellular properties, such as (1) why ADSC and BMSC are virtually identical, (2) why conflicting evidence persists in regard to ADSC surface marker expression and differentiation potential, and (3) why ADSC exhibit various differentiation potentials in a clonal fashion. Furthermore, by equating ADSC to VSC, the theory helps explain why the yield of ADSC is much greater than BMSC [5
] as adipose tissue is known to be highly vascularized and have angiogenic properties [9
]. Specifically, the extensive capillary network that surrounds each adipocyte [14
] and the angiogenic (differentiation) potential are additional attributes of the adipose tissue, which has been known to possess many advantages over the other tissues (see Introduction
), as an optimal source of adult stem cells. Thus, by examining the stem cell characteristics of adipose tissue and proposing the concept of “vascular stem cells”, the present study helps clarify several perplexing issues surrounding ADSC research and further elevates the status of ADSC as a legitimate and optimal stem cell source.