Historically, MSCs were isolated on their capacity to selectively proliferate in cultures of total bone marrow and to differentiate in mesodermal cell lineages. Some authors have also described MSC differentiation into nonmesodermal cell lineages, although this is still a matter of debate [30
]. Remarkably though, MSC did not benefit the theoretical and technologic progress that permitted the prospective identification of hematopoietic and other stem cells. The instrumentalization of MSC for tissue-engineering/cell therapy eclipsed latent questions regarding the identity, origin, and native distribution of these adult stem cells. Only recently were similarities noticed to exist between MSC and perivascular cells [10
]. Indeed, vascular pericytes purified to homogeneity from multiple organs express MSC markers, can differentiate into mesoderm lineage cells, and give rise in culture to genuine MSC [15
]. Are all MSC derived from pericytes, which are exclusively associated with capillaries and microvessels [16
]? The current work addressed this issue along a substractive approach, by investigating the ability of FACS selected nonpericyte cells, within the stromal vascular fraction of hWAT, to give rise to MSC in culture. Our general conclusion is that CD34+CD31-CD146-CD45- cells residing in the outer layer (tunica adventitia
) of arteries and veins yield in culture multipotent progenitors functionally and antigenically similar to MSC. Coincidentally, the tunica adventitia
is believed to play a role in vascular remodeling and be involved in the development of atherosclerosis [17
]. Adventitial cells indeed proliferate, differentiate into myofibroblasts, and migrate into the inner layer of blood vessels in response to injury or stress [21
]. CD34+CD31- cells located in the “vasculogenic zone” of the thoracic artery, between the tunica media
and tunica adventitia
, can also give rise to endothelium; hence, they could represent a resident pool of endothelial progenitor cells for postnatal vasculogenesis [26
]. Multipotent progenitors can originate from larger vessels not surrounded by typical pericytes, such as the human pulmonary artery, fetal aorta wall, and vena saphena [27
], thus further supporting our present conclusions.
The presence in hWAT of CD34+ MSC progenitors has been reported, although the native identity of these cells has been controversial so far. Traktuev et al. described in adipose tissue CD34+CD31- perivascular cells that, once enriched by plastic adherence and briefly cultured, expressed markers of pericytes (PDGFR-β), MSC (CD90), and smooth muscle cells (α-SMA) [32
]. In 2 other studies, CD34+CD31- cells were clearly identified in human adipose tissue as adventitial cells distinct from α-SMA+ smooth muscle cells and PDGFR-β+ or CD146+ pericytes, all of which are invariably CD34 negative [33
]. Adventitial cells and pericytes from fat, therefore, include 2 anatomically and phenotypically distinct perivascular primary MSC, an observation we have extended to human fetal lung, fetal and adult pancreas, and adult muscle. Importantly, no potential to generate MSC was observed, in any tissue analyzed, outside these 2 perivascular cell compartments. Adventitial fibroblasts can differentiate into α-SMA+myofibroblasts under hypoxia, or when exposed to growth factors involved in vascular remodeling and angiogenesis, TGF-β or angiotensin II [35
]. Consistently, adventitia-derived MSC treated with angiotensin II upregulate the expression of α-SMA, which typifies fibroblasts, pericytes, smooth muscle cells, and myofibroblasts. We also show for the first time that adventitial cells cultured in the presence of angiotensin II upregulates other pericyte markers: CD146, PDGFR-β, and NG2. The same outcome was observed when adventitial cells were treated with angiopoietin-2, a growth factor produced at the site of vascular remodeling that disrupts interactions between smooth muscle cells and endothelial cells, thus leading to angiogenesis in the presence of VEGF [38
]. Altogether, our results indicate the ability of adventitial cells to differentiate into pericytes-like cells under appropriate signaling, thus supporting the previously hypothesized existence of CD34+CD31- pericyte progenitors [39
]. Differentiation into myofibroblasts suggests that adventitial cells are precursors of smooth muscle cells in homeostasis and disease, in agreement with the emerging theory of vascular remodeling “from the outside-in,” as the adventitia being the sensor of vascular injury or other environmental stress [41
]. Given the observed capacity of adventitial cells to differentiate into MSC and pericyte-like cells, here we propose a hypothesis: the centrifugal migration of multipotent adventitial progenitors to surrounding regenerating tissues, in particular at the developing blood vessels.
In final conclusion, we have demonstrated that human blood vessels, depending on their size, are associated with 2 distinct peripheral MSC progenitors, namely pericytes encircling capillaries and microvessels [15
], and adventitial cells surrounding larger arteries and veins. This hypothesis of a dual origin for MSC is also supported by the presence of both CD146+ cells (40%–60%) and CD146- cells within MSC conventionally derived in culture [13
]. We propose that, in hWAT and presumably all organs, vascular progenitors of MSC are hierarchically organized with adventitial cells being precursors of pericytes, based on their higher proliferation rate and potential to differentiate into pericytes on proper stimulation.