Bone Marrow Side Population Cells Research Articles

Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters

Side population (SP) cells isolated from bone marrow, skeletal muscle, and skin have been shown to engraft in dystrophic muscle. However, there have been questions on the phenotypical heterogeneity, tissue of origin, and relationships among SP cell populations extracted from different tissues. Studies on bone marrow SP cells have followed a consistent protocol for their isolation and results obtained are concordant. In contrast, protocols for the isolation of muscle SP cells vary greatly, and consequently reports on their phenotype, differentiation potential and origin have been inconsistent. To address this controversy, we demonstrate that isolation parameters, such as tissue dissociation, cell counting, Hoechst concentration, and stringency in the selection of SP cells, have an effect on the yield, viability, and homogeneity of SP cells derived from bone marrow, skeletal muscle, and skin. In this paper, we demonstrate that SP cells isolated from the bone marrow are distinct from SP cells extracted from skeletal muscle and skin tissues. This study offers an explanation for the controversy surrounding muscle SP cells, provides a detailed standardized protocol for their isolation, and highlights basic guidelines for reproducible and reliable isolation of SP cells from any tissue.

Translational physiology: origin and phenotype of lung side population cells.

Side population (SP) cells, a rare cell type identified by their ability to efflux the vital dye Hoechst 33342, are highly enriched for stem cell activity. Bone marrow (BM) SP cells uniformly express the pan-hematopoietic marker CD45, whereas tissue SP cells are heterogeneous in CD45 expression. In previous studies, we found that CD45 is expressed on 75% of lung SP cells. By performing whole BM transplantations, we determined that CD45-positive and CD45-negative lung SP cells are marrow derived. Transplantation of 200 highly purified BM SP cells indicated that both lung SP cell subtypes are derived from this marrow cell type. Morphologically, CD45-positive lung and BM SP cells possess similar features. They are small, round, and contain scant cytoplasm. CD45-negative lung SP cells are larger and contain abundant granular cytoplasm. Gene expression patterns for hematopoietic transcription factors GATA-1, GATA-2, and PU.1 further differentiated SP marrow and lung subtypes. By immunostaining for alpha-smooth muscle actin and cytokeratin, we found significant differences in the relative expression patterns of these markers in lung and marrow SP cell subtypes. In summary, these findings demonstrate that lung SP cells are derived from the BM and that CD45-positive and -negative subtypes can be distinguished by morphological differences and gene expression patterns.

Side Population Keratinocytes Resembling Bone Marrow Side Population Stem Cells Are Distinct From Label-Retaining Keratinocyte Stem Cells

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.

Lung cells transplanted to irradiated recipients generate lymphohematopoietic progeny.

Bone marrow (stem) cells can differentiate into cells in multiple tissues, including lung. Conversely, there are reports that cells of nonhematopoietic tissues (brain, muscle) can give rise to lymphohematopoietic cells. Here we show that the lung contains cells capable of giving rise to lymphohematopoietic cells when transplanted to irradiated recipients. Whole lung cell suspensions, lung side population (SP) cells, and CD45(+/-) lung cells obtained from male transgenic enhanced green fluorescent protein-expressing mice were transplanted intravenously to total body irradiated female mice. Green fluorescent cells were recovered from the circulation and phenotyped for their expression of lymphohematopoietic markers (CD3, CD4, CD8, B220, Gr-1, and Mac-1). Lung SP cells were composed of heterogeneous populations and had less ability to give rise to lymphohematopoietic cells than did bone marrow SP cells. Furthermore, the ability of cells from the lung of aged mice to generate lymphohematopoietic progeny was equivalent to that of cells from young mice. Cells from lung with radioprotective and lymphohematopoietic reconstituting abilities were CD45(+). CD45(+) cells in the lung cells have lymphohematopoietic stem/progenitor cell characteristics, and this has implications for cell or gene therapy applications.

Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration.

Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration.

Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration

Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration.

Lineage-negative side-population (SP) cells with restricted hematopoietic capacity circulate in normal human adult blood: immunophenotypic and functional characterization.

Side-population (SP) cells are a recently described rare cell population detected in selected tissues of various mammalian species, but not yet described in the peripheral circulation. In the present study, we have identified for the first time SP cells in lineage-negative adult human blood and have provided an extensive functional and immunophenotypic characterization of these cells. Adult peripheral blood was depleted of mature leukocyte cell types by density gradient and immunomagnetic separation. The SP cell population was identified by its characteristic Hoechst 33342 profile. Immunophenotypic analysis revealed that blood SP cells expressed high levels of CD45, CD59, CD43, CD49d, CD31, and integrin markers and lacked CD34. Highly purified SP cells were put into cobblestone area-forming cell (CAFC), long-term culture-initiating cell (LTC-IC), and liquid cell culture assays; repopulating assays were performed utilizing nonobese diabetic/severe combined immunodeficient mice. Circulating SP cells were shown to exhibit verapamil sensitivity and a low growth rate. LTC-IC, CAFC, and engraftment assays indicated that circulating SP cells had lost the multipotentiality described in murine bone marrow SP cells. However, outgrowth of mature cell types from liquid cell culture suggests the presence of common lymphoid (T and natural killer) and dendritic cell precursor(s) within circulating SP cell populations. The absence of SP cell growth in the LTC-IC, CAFC, and repopulating assays might be intrinsic to the tissue source (marrow versus blood) or species (mouse versus human) tested. Thus, human blood SP cells, although rare, may serve as a source of selected leukocyte progenitor cells. The immunophenotype of circulating SP cells may provide clues to their seeding and homing capacity.