Using BrdU-labeling methodology followed by immunohistochemical, immunofluorescent, and flow cytometric analyses, we demonstrate the following: (1) LRC are detected in iWAT, eWAT and BAT up to 52 days of age, (2) LRC are found in two areas—single positive cells distributed among adipocytes and those closely adjacent to the blood vessels wall, and (3) the expression of the stem cell associated surface antigen, Sca-1, co-localizes with LRC.
To our knowledge this study is the first attempt to label cells within fat tissues with BrdU at a very early postnatal life and follow the retention of the labeled cells into adulthood. The seven sequential BrdU-injections into 3-day-old animals resulted in the labeling of 27% of iWAT, 65% of BAT (at Day 10), and 26% (at Day 28) of eWAT cells. Studies of others showed initially higher percentages of BrdU-labeled cells in the epithelial compartment of colon—77% [14
], endometrium—72% [11
], and bladder—90% [10
]. It is possible that the difference between our studies and those of others can be attributed to the slower turnover of mesenchymal cells relative to epithelial cells as has been noted for bladder epithelial and stromal cells [10
]. During the chase period of 52 days we observed a rapid reduction in the number of BrdU-labeled cells from Day 10 to Day 28 (13-fold) in iWAT and a slower decline in BAT (3-fold). However, at Day 52 all fat depots examined showed a low but clearly detectable level of LRC with the lowest observed in eWAT depot (0.53%).
Analysis of immunohistological samples consistently showed that BrdU-cells were confine to two regions within fat tissues: the perivascular wall and sparsely arranged among adipocytes regardless of fat depot or chase time point. Localization of a major group of BrdU-labeled cells to the perivasculature is consistent with the recent studies on human fat tissues that have attempted to localize the ASC niche. Traktuev et al. demonstrated that stromal cells of subcutaneous adipose tissue with properties of ASC serve structurally and functionally as pericytes [19
]. A similar study by Zannettino et al. revealed that multipotential stem cell populations within human adipose tissues are intimately associated with perivascular cells [20
]. In those studies [19
], flow cytometric methods were applied to phenotype ASC in human fat tissues. We detected two different niches of LRC/ASC in mice fat depots with immunohistochemical and immunofluorescence techniques that are standard methods for the detection and quantification of molecules of interest in adipocyte tissues [21
In our second experiment (BrdU labeling of fat depot in adult animals) we used flow cytometry to quantify and molecularly phenotype BrdU-labeled cells. The analysis showed that 1.7% of iWAT and 6% of eWAT stromal-vascular fractions of cells are BrdU-positive. These data correlate well with morphometrically quantified 2.9% of BrdU-labeled cells in retroperitoneal fat depot collected from mature rats that were given intraperitoneal injection of BrdU [21
]. Earlier studies that used [3
H]thymidine labeling showed that populations of cells in stromal-vascular fractions of adipose tissue can take up this DNA precursor suggesting the presence of proliferating cells within tissue [2
]. Hirsh and co-workers in their studies of de novo
production of adipocytes in adult rats used biochemical and radioautographic techniques for detection of incorporated radiolabeled thymidine into fat DNA [2
]. They showed that a small population of stromal-vascular fraction of epididymal (0.1% of total cells) and retroperitoneal (0.3% of total cells) fat depots, but not fat cells incorporate [3
H]thymidine after IP injection into adult rats [2
]. Although not substantial, discrepancies between Hirsh's and our own data can be attributed to the distinct methods of labeling followed by different detection assays and/or differences between species (rats vs. mouse model).
To further characterize LRC we analyzed the expression of stem cell antigen-1 (Sca-1). Sca-1 has been used as a candidate marker in the search for tissue-resident stem cells [22
]. Recent studies have associated Sca-1 antigenicity with adipocyte progenitors in fat depots [23
] and our recent studies suggest that Sca-1 is a biomarker for ear mesenchymal stem cells (EMSC) that have the potential to become functionally active adipocytes [17
]. Present FACS analysis of BrdU-labeled stromal-vascular fraction cells revealed that more than half of iWAT and eWAT fractions are Sca-1 positive. Simultaneous analysis of LRC for markers of the hematopoietic lineage (CD45 and CD4) showed that approximately 70% of inguinal and epididymal cells are positive. We conclude that LRC in stromal-vascular fractions of fat depots include the following immunophenotypes: Sca-1+
; and Sca-1−
. Further analyses of the properties of each of these sub-populations are currently underway in our laboratory.