Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture

Abstract

Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45-CD34+CD105+ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31+ cells, CD31- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31+ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal +CD105+CD31- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.

Figure 1.

Phenotypic characterization of CD45^- cells from human adipose tissue. Expression is shown for CD34 versus CD31 (A), CD105 (endoglin, using the SH2 MAb) versus CD31 (B), and HLA DR versus CD31 (C). Using intracellular staining, the expression of von Willebrand factor versus CD31 is shown in E. The result using medium in stead of vWf MAb is shown in D.

Figure 2.

Photomicrograph of CD31⁺ (A) and CD31^- (B) cells ∼1 h after establishment in cell culture (×400). (C) Typical ADASC at passage 4 (×100).

Figure 3.

Staining of polyclonal cultures of ADASC after differentiation in adipogenic direction (Oil-Red O, A), osteogenic direction (Alzarin Red, B), chondrogenic direction (Toluidine blue, C) and neurogenic direction (NF-200, D). Differentiated cells are shown in the left-hand panels and undifferentiated cells in the righthand panels. In D, images obtained in fluorescent light is shown in the top panels, and images obtained in white light are shown in the bottom panels. All images (A–D): magnification ×200.

Figure 4.

Staining of differentiated clonal cells. Four different clones obtained from two donors are shown to illustrate differences observed in differentiation between different clones. Staining is for adipogenic differentiation (Oil-Red O, left panels), osteogenic differentiation (Alzarin Red, middle panels), and neurogenic differentiation (anti-NF-200, right panels). Magnification, ×200.