Jerry I. Huang

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

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.

PURPOSE: We have isolated pluripotent mesenchymal progenitor cells in large numbers from liposuction aspirates (processed lipoaspirate cells or PLAs). This study examines the osteogenic potential of PLAs and bone marrow aspirate cells (BMAs), when exposed to either recombinant human bone morphogenetic protein (BMP)-2 (rh-BMP-2) or adenovirus containing BMP-2 cDNA (Ad-BMP-2). METHODS: Liposuction aspirates underwent proteolytic digestion to obtain PLAs. After exposure to exogenous rh-BMP-2 or Ad-BMP-2 for four or seven days, PLAs and BMAs were assessed by histochemistry, spectrophotometry and RT-PCR. Western blotting and ELISA confirmed BMP gene transduction. Results were compared to osteoblasts and cells in osteogenic media only. PLA-Ad-BMP-2 cells were seeded on matrices and implanted in the hind limbs of SCID mice. RESULTS: Analysis of quantified bone precursor assays including extracellular ALP histomorphometry, intracellular ALP spectrophotometry, and calcified extracellular matrix (von Kossa) histomorphometry revealed that PLAs treated with exogenous rh-BMP-2 or transduced with a BMP-2 containing adenovirus (PLA-Ad-BMP-2) produced more bone precursors than osteoblasts (p=0.001). PLAs treated with exogenous rh-BMP-2 or PLA-Ad-BMP-2 also produced more bone precursors than BMAs (p=0.001), except for day 7 ALP histomorphometry (p=0.343). ELISA confirmed successful BMP-2 production by both progenitor cell groups transduced with Ad-BMP-2. H&E sections from collagen I matrices seeded with PLA-Ad-BMP-2 cells confirmed bone formation at six weeks. CONCLUSIONS: Liposuction aspirates contain PLAs that can be transfected with the BMP-2 gene, with rapid induction into the osteoblast phenotype at a rate comparable to rh-BMP-2 and osteoblast groups. Transduced PLAs produce more bone precursors with faster onset of calcified extracellular matrix than transduced BMAs. PLAs may be an ideal source of mesenchyme-lineage stem cells for gene therapy and tissue engineering.