Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1−/CD34+/CD24+ Adipose Stem/Progenitor Cells

Abstract

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34−, DLK1+/CD34dim and DLK1−/CD34+ cells. We demonstrate that DLK1−/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPβ are highly expressed. Further sorting of ASCs into DLK1−/CD34+/CD24− and DLK1−/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1−/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1−/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1−/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPβ but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1−/CD34+/CD24+ relative to DLK1−/CD34+/CD24− subpopulations.

Induction of the CD24 Surface Antigen in Primary Undifferentiated Human Adipose Progenitor Cells by the Hedgehog Signaling Pathway

“…Recently, Florian M. Hatzmann et al found that approximately 3% of human SVF ASCs were positive for CD24. Furthermore, they could induce CD24 expression in hASCs using a serum-free medium without apparently activating the Hh signaling pathway [33]. These two studies indicate that CD24 may also be upregulated by other, currently non-characterized, signal transduction circuits.…”

“…Indeed, in mice, only less than 1% of the SVF cells that express CD24 also display the complete cell surface marker profile of adipocyte progenitors [19]. In the human SVF, CD24-positive ASCs are also rare [33]. The properties and functions attributed to this surface protein definitively promote it as a marker of hASCs and other types of stem cells.…”

“…Two studies suggest that CD24 may also be upregulated by other, currently noncharacterized, signal transduction circuits different from the Hh signaling pathway [33,72]. It is proposed that CD24 may act as a membrane-organizing factor that regulates the association and localization of cell surface receptors with their canonical ligands [30].…”

“…Florian M. Hatzmann et al reported that approximately 3% of human SVF ASCs were positive for CD24. Furthermore, their data suggested that the stemness of human CD24 positive ASCs was higher than that of CD24 negative ASCs and that this protein was necessary for adequate ASC proliferation and adipogenesis [33]. Although CD24 is not yet part of the panel suggested by IFATS and ISCT to characterize ASC, all these data cannot be ignored and elect CD24 as a very useful and exciting marker for studying the biology of subcutaneous adipose tissue stem cell in mice and man.…”

Emerging Roles of DLK1 in the Stem Cell Niche and Cancer Stemness

“…48,79 At last, in different in vitro models of glucocorticoids and insulin-induced adipocyte differentiation, a quick temporary increase in the levels of the membranebound variant of Dlk1 was observed just after induction of differentiation, while the full-length, cleavable Dlk1 started to decrease just after induction and both became undetectable by the end of the differentiation process. [80][81][82][83] Both the initial overexpression of the membrane-bound DLK1 and the drop in the levels of secreted DLK1 play a role in adipocyte differentiation, and the switch between the two forms of DLK1 is necessary to induce a permissive state for differentiation in response to extracellular stimuli (Fig. 1B).…”