Surface protein characterization of human adipose tissue-derived stromal cells.

Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue-derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue-derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49(d), CD 49(e), CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow-derived stromal cells, we did not detect the STRO-1 antigen on human adipose tissue-derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPalpha and PPARdelta, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue-derived and human bone marrow-derived stromal cells could have broad implications for human tissue engineering.

Hoechst dye efflux reveals a novel CD7(+)CD34(-) lymphoid progenitor in human umbilical cord blood.

A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34(-) cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34(+) cells but also contained a large population of CD34(-) cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin(-) UCB) contained CD34(+) and CD34(-) cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34(+)Lin(-) SP cells were CD38(dim)HLA-DR(dim)Thy-1(dim)CD45RA(-)CD71(-) and were enriched for myelo-erythroid precursors. In contrast, the CD34(-)Lin(-) SP cells were CD38(-)HLA-DR(-)Thy-1(-)CD71(-) and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7(+)CD11b(+)CD45RA(+), as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7(+)CD34(-)Lin(-) UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin(-) UCB SP contains both CD34(+) multipotential stem cells and a novel CD7(+)CD34(-)Lin(-) lymphoid progenitor. This observation adds to the growing body of evidence that CD34(-) progenitors exist in humans.

Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity, we developed a fluorescent substrate for ALDH, termed BODIPY aminoacetaldehyde (BAAA), and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells, 40-90% pure for CD34(+)CD38(lo/-) cells, and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together, these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well.

Alterations within pp59v-rel-containing protein complexes following the stimulation of REV-T-transformed lymphoid cells with zinc.

pp59v-rel exists in association with specific cellular proteins within lymphoid cells transformed by reticuloendotheliosis virus (REV-T). These include the cellular rel homolog (p75c-rel) as well as a 40-kDa avian homolog to I kappa B. The brief exposure of REV-T-transformed lymphoid cells to micromolar concentrations of ZnSO4 induces profound alterations within these protein complexes. Most of the constituents of the rel protein complexes (to include pp59v-rel, p75c-rel, and p115) translocate from the cytosol to the nucleus. This system has been used to characterize the molecular events that accompany the activation of rel protein complexes. The level of phosphorylation increases on three proteins within these complexes: pp59v-rel, p75-c-rel, and pp40. The degree of phosphorylation on pp59v-rel is such that its relative mass increases 3 to 6 kDa when resolved by SDS-polyacrylamide gel electrophoresis. pp59v-rel is phosphorylated on serine and threonine residues predominantly within a single domain of 17.5 kDa. Similarly, p75c-rel exhibits a corresponding increase in its relative mass with increased phosphorylation. The increased phosphorylation of pp40 is accompanied by its dissociation from the cytosolic rel protein complexes. These observations draw parallels with the induction of the NF-kappa B trans-activating factor.

Oncogenes, protooncogenes, and signal transduction: toward a unified theory?

This paper has reviewed, in a broad sense, the potential involvement of the oncogenes and their progenitors, the protooncogenes, in signal transduction pathways. The membrane-associated oncogene products appear to be connected with the generation and/or regulation of secondary messengers, particularly those associated with Ca2+/phospholipid-dependent activation of the serine/threonine kinase protein kinase C. Activation of transmembrane receptors, either through binding their native ligand or through point mutations that lead to constitutive expression, results in the expression of their intrinsic tyrosine-specific protein kinases. In PDGF-stimulated cells, this results in the increased turnover of phosphatidylinositols and the subsequent release of IP3 (Habenicht et al., 1981; Berridge et al., 1984). This coincides with activation of a PI kinase activity (Kaplan et al., 1987). Likewise, the fms product, which is the receptor for CSF-1, induces a guanine nucleotide-dependent activation of phospholipase C (Jackowski et al., 1986). Receptor functions are potentially regulated through differential binding of ligands (as proposed with PDGF), through interactions with other receptors, and through the "feedback" regulation mediated by protein kinase C. PDGF stimulation leads to modulation of the EGF receptor through protein kinase C (Bowen-Pope et al., 1983; Collins et al., 1983; Davis and Czech, 1985). Similarly, the neu product becomes phosphorylated on tyrosine residues following treatment of cells with EGF, although the neu protein does not bind EGF itself (King et al., 1988; Stern and Kamps, 1988). The tyrosine kinases of the src family are not receptors themselves, although they may mediate specific receptor-generated signals. The clck product is physically and functionally associated with the T-cell receptors CD4 and CD8, and becomes active upon specific stimulation of cells expressing those markers (Veillette et al., 1988a,b). The precise physiological role of the src family products has not been established, but their kinase activity is intrinsic to that function. The v- and c-src products are hyperphosphorylated during mitosis (Chackalaparampil and Shalloway, 1988), which correlates with periods of reduced cell-to-cell adhesion and communication (Warren and Nelson, 1987; Azarnia et al., 1988). Furthermore, pp60c-src is associated with a PI kinase activity when complexed with MTAg of polyoma virus, suggesting a function in stimulating increased turnover of the phosphatidylinositols (Heber and Courtneidge, 1987; Kaplan et al., 1987).(ABSTRACT TRUNCATED AT 400 WORDS)