Human adipose tissue is a source of multipotent stem cells

Abstract

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

Figures

Figure 1

PLA cells express a unique set of CD markers. (A) PLA cells and MSCs were processed by immunofluorescence for expression of multiple CD antigens (green). Cells were costained with 4,6-diamidino-2-phenylindole to visualize nuclei (blue) and the fluorescent images combined. The differential expression of CD49d and CD106 between PLA cells and MSCs is shown (Figure S1 for remaining CD antigens). (B) Flow cytometric analysis on PLA cells and MSCs for the expression of CD49d and CD106 was performed (red). Cells stained with a fluorochrome-conjugated nonspecific IgG were examined as a control (γPE, green). The geometric mean and median values for CD49d and Cd106 are shown below. Significant differences are shown in bold.

Figure 2

Adipogenic PLA cells express several genes and proteins consistent with adipogenic differentiation. (A) Triplicate samples of PLA cells and 3T3-L1 controls were induced for up to 5 wk in AM (PLA-AM and 3T3-AM, respectively) and assayed for GPDH activity (GPDH per microgram). Non-induced PLA cells were analyzed as a negative control (PLA-control). Values were expressed as mean ± SD. (B) PLA cells were induced in AM (PLA-AM) or maintained in non-inductive control medium (PLA-control) for 14 d. Cells were examined for the expression of GLUT4 and leptin by indirect immunofluorescence. (C) PLA cells were induced in AM or maintained in non-inductive control medium for up to 5 wk. Samples were analyzed by RT-PCR for the indicated genes. 3T3-L1 cells maintained for 2 wk in AM were analyzed as a positive control. (D) Expression of the gene LPL was quantitated by real-time PCR in PLA cells induced in control medium and AM for up to 5 wk. LPL expression levels were normalized with respect to endogenous GAPDH. LPL expression in PLA cells induced for 3 (D21) and 5 wk (D35) in AM were expressed relative to 1-wk levels.

Figure 3

Osteo-induced PLA cells express several osteogenic genes and proteins. (A) PLA cells and MSCs were induced for up to 6 wk in OM. Cells were assayed for AP activity and total calcium and normalized with respect to protein. Non-induced PLA cells (control) were analyzed as a negative control. Values were expressed as the mean ± SD. (B) PLA cells were cultured in OM or noninductive control medium for up to 6 wk and analyzed by RT-PCR for the indicated genes. NHOst cells maintained in control medium (Con) or OM for 4 wk (28 d) were analyzed as a positive control. (C) Expression of the genes CBFA-1 and AP was quantitated by real-time PCR in PLA cells induced in control medium and OM for up to 4 wk. Gene expression levels were normalized with respect to endogenous GAPDH and expressed relative to non-induced control levels. (D) PLA cells were cultured in OM or control medium for 7 and 28 d and analyzed by Western blotting for the expression of OP, ON, AP, RARα, the VDR, and CNI. Expression of the TfR and α-actin was assessed as internal controls.

Figure 4

PLA cells induced toward the chondrogenic lineage synthesize a cartilagenous matrix and express genes consistent with the chondrogenic lineage. (A) PLA cells were induced in CM under high-density conditions for 14 d. Nodules were sectioned and stained with AB, in addition to antibodies to CNII, keratan sulfate (KS), and chondroitin-4-sulfate (CS). (B) PLA cells were induced for up to 3 wk in CM (PLA-CM). Sulfated proteoglycan levels were determined and normalized with respect to protein per microgram (PG). Non-induced PLA cells (PLA-control) were analyzed as a negative control. Values were expressed as the mean ± SD. (C) PLA nodules were induced in CM for up to 14 d or maintained in non-inductive control medium for 10 d (PLA-Con). Samples were analyzed by RT-PCR for the indicated genes. NHCK cells induced for 2 wk in CM were analyzed as a positive control. Dec, decorin.

Figure 5

PLA cells induced toward the myogenic lineage express several myogenic genes. PLA cells induced in MM for up to 6 wk or maintained in control medium were analyzed by RT-PCR for the expression of the indicated myogenic genes. Total RNA prepared from human skeletal muscle (SKM) was analyzed as a positive control. DES, desmin; MD1, myod1; MG, myogenin; Myf, myogenic regulatory factor; MYS, myosin heavy chain.

Figure 6

PLA cells exhibit neurogenic capacity in vitro. (A) PLA cells were maintained in NM or control medium for 5 h (PLA-NM and PLA-control, respectively) and analyzed for expression of neural (NSE and NeuN), astrocytic (GFAP), and oligodendritic (GalC) markers. (B) PLA cells were induced in 1) NM for 9 h or 2) NM for 9 h and maintained for 1 wk in NPMM or 3) control medium supplemented with IIM for 1 wk. Samples were analyzed by RT-PCR for the indicated genes. Non-induced PLA cells (con) were analyzed as a negative control. Total RNA prepared from human brain (brain) was examined as a positive control. ChaT, choline acetyltransferase.

Figure 7

PLA clones possess multi-lineage potential. (A) PLA clonal isolates were analyzed for osteogenic (alkaline phosphatase), adipogenic (Oil Red O), and chondrogenic (Alcian blue) capacity. (B) Tri-lineage clones (osteogenic, adipogenic, and chondrogenic), or ADSCs, were cultured in either OM (ADSC-bone), AM (ADSC-fat), or CM (ADSC-cartilage), in addition to control medium (ADSC-control). ADSCs were analyzed by RT-PCR for the indicated lineage-specific genes.