Marrow Side Population Cells Research Articles

Growth of bone marrow and skeletal muscle side population stem cells in suspension culture.

The ability to efficiently isolate and expand various stem cell populations in vitro is crucial for successful translation of cell-based therapies to the clinical setting. One such heterogeneous population that possesses a remarkable potential for the development of cell-based treatments for a variety of degenerative diseases and disorders is called the Side Population (SP). For many years, investigators have isolated these primitive cells based upon their ability to efflux the fluorophore Hoechst 33342. This attribute enabled separation of SP cells derived from multiple tissue sources from other endogenous cell populations using fluorescence-activated cell sorting (FACS). While all tissue-specific SP fractions appear to contain cells with multi-potent stem cell activity, the therapeutic utility of these cells has yet to be fully realized because of the scarcity of this fraction in vivo. In view of that, we developed a method to expand adult murine bone marrow and skeletal muscle-derived SP cells in vitro. Here, we describe a spinner-flask culture system that supports the growth of SP cells in suspension when they are combined with feeder cells cultured on spherical microcarriers. In this way, their distinguishing biological characteristics can be maintained, attachment-stimulated differentiation is avoided, and therapeutically relevant quantities of SP cells are generated. Modification of the described procedure may permit expansion of the SP from other relevant tissue sources and our method is amenable to establishing compliance with current good manufacturing practices.

Bone marrow side population cells are enriched for progenitors capable of myogenic differentiation

Although the contribution of bone marrow-derived cells to regenerating skeletal muscle has been repeatedly documented, there remains considerable debate as to whether this incorporation is exclusively a result of inflammatory cell fusion to regenerating myofibers or whether certain populations of bone marrow-derived cells have the capacity to differentiate into muscle. The present study uses a dual-marker approach in which GFP(+) cells were intravenously transplanted into lethally irradiated beta-galactosidase(+) recipients to allow for simple determination of donor and host contribution to the muscle. FACS analysis of cardiotoxin-damaged muscle revealed that CD45(+) bone-marrow side-population (SP) cells, a group enriched in hematopoietic stem cells, can give rise to CD45(-)/Sca-1(+)/desmin(+) cells capable of myogenic differentiation. Moreover, after immunohistochemical examination of the muscles of both SP- and whole bone marrow-transplanted animals, we noted the presence of myofibers composed only of bone marrow-derived cells. Our findings suggest that a subpopulation of bone marrow SP cells contains precursor cells whose progeny have the potential to differentiate towards a muscle lineage and are capable of de novo myogenesis following transplantation and initiation of muscle repair via chemical damage.

Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo

Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.

Kidney Side Population Reveals Multilineage Potential and Renal Functional Capacity but also Cellular Heterogeneity

Side population (SP) cells in the adult kidney are proposed to represent a progenitor population. However, the size, origin, phenotype, and potential of the kidney SP has been controversial. In this study, the SP fraction of embryonic and adult kidneys represented 0.1 to 0.2% of the total viable cell population. The immunophenotype and the expression profile of kidney SP cells was distinct from that of bone marrow SP cells, suggesting that they are a resident nonhematopoietic cell population. Affymetrix expression profiling implicated a role for Notch signaling in kidney SP cells and was used to identify markers of kidney SP. Localization by in situ hybridization confirmed a primarily proximal tubule location, supporting the existence of a tubular "niche," but also revealed considerable heterogeneity, including the presence of renal macrophages. Adult kidney SP cells demonstrated multilineage differentiation in vitro, whereas microinjection into mouse metanephroi showed that SP cells had a 3.5- to 13-fold greater potential to contribute to developing kidney than non-SP main population cells. However, although reintroduction of SP cells into an Adriamycin-nephropathy model reduced albuminuria:creatinine ratios, this was without significant tubular integration, suggesting a humoral role for SP cells in renal repair. The heterogeneity of the renal SP highlights the need for further fractionation to distinguish the cellular subpopulations that are responsible for the observed multilineage capacity and transdifferentiative and humoral activities.

Out of the blue: A comparison of Hoechst side population (SP) analysis of murine bone marrow using 325, 363 and 407 nm excitation sources

Background The discrimination of side population (SP) cells is a useful flow cytometric tool for the study of putative stem cells in many tissue types. Previous studies on murine bone marrow (BM) SP cells and concurrent surface immunophenotyping provide a good model for comparing Hoechst bivariate profiles. Here we compare the results of SP cells from the same specimens generated on three different instruments. Methods Concurrent SP analysis and immunophenotyping was performed on murine BM. The data acquisition was performed on a DakoCytomation MoFlo, a Becton Dickinson LSR and a Becton Dickinson FACSAria to compare the ability of each instrument to discriminate putative stem cells. Further experiments examined the effect of apoptotic changes, assessed by Annexin V binding, and the presence of nucleated erythroid precursors on the Hoechst profile. Results Identifiable and equivalent side populations were generated on all three instruments despite differences in appearance of the Hoechst profiles. Differences in laser type, collection filter combinations and the presence of erythroid precursors and apoptotic cells all contributed to this effect. Conclusions In our hands, murine SP cells with the expected surface immunophenotype can be identified on a MoFlo, LSR and a factory standard FACSAria.

Relationship between ABCG2 Expression and Side Population Stem Cell Phenotype in Murine Hematopoietic System Revealed by an Abcg2/GFP Knock-In Mouse Model.

Stem cells from a variety of tissues could be identified by a side population (SP) phenotype. We have identified an association between expression of the ABCG2 transporter and the SP phenotype, although the exact relationship between these two cellular characteristics is still not well understood. To further explore the relationship between Abcg2 expression and SP phenotype, we have generated a Abcg2/GFP reporter mouse model in which an IRES-GFP expression cassette was inserted down-stream of the stop codon of Abcg2 gene by homologous recombination, so that the expression of GFP is under the control of Abcg2 locus but endogenous Abcg2 expression was unperturbed. Immunohistochemistry of tissue sections using an anti-GFP antibody confirmed expression of GFP in tissues that normally express Abcg2, such as renal proximal tubules and intestinal epithelium. Flow cytometry analysis showed that about 10% of total bone marrow mononuclear cells expressed the Abcg2/GFP allele. Within this total GFP+ population, greater than 99% of the cells fall outside the SP region. Staining with lineage specific antibodies showed that 77% of the non-SP/GFP+ cells are Ter119+ erythroid cells. In contrast, only 0.5% of the total GFP+ population falls within the SP region. Approximately 90% of these bone marrow SP cells expressed the Abcg2/GFP allele. Transplantation studies with bone marrow cells that were depleted of lineage positive cells were then performed. All 5 mice transplanted with 100 Lin−, SP+, GFP+ cells were reconstituted in both myeloid and lymphoid lineages. In contrast, no repopulating activity was detected with up to 10,000 Lin−, non-SP, GFP− cells. When Lin−, non-SP, GFP+ cells were transplanted, only 2 of 5 animals were reconstituted with 5000 cells, and no repopulation was seen with lower cell doses. These results show a complex relationship between Abcg2 expression and the SP phenotype. Virtually all SP cells express the Abcg2/GFP allele, and these cells are highly enriched for repopulating activity in transplant assays. However, the majority of bone marrow cells that expressed the Abcg2/GFP allele were Ter119+ erythroid cells that do not bear the SP phenotype. In addition, there are non-SP, GFP+ cells that lack expression of Ter119 and other mature lineage markers. These Lin−, non-SP, GFP+ cells have relatively lower repopulating activity compared to SP cells.

Cardiac Progenitor Cells

See related article, pages 1090–1092 In the last few years we have witnessed one of most extraordinary revolutions in cardiovascular medicine, namely, an explosion of basic and clinical studies that support the notion that the diseased heart can be repaired by administration of stem cells, resulting in formation of functional new myocytes and vessels. Although the mechanism by which cell therapy improves cardiac function and anatomy remains uncertain, translation of basic findings to the clinical setting is proceeding at a feverish pace.1 A multitude of small, mostly nonrandomized clinical studies have reported improvement in cardiac perfusion and function after therapy with various cell types in patients with acute myocardial infarction or chronic ischemic cardiomyopathy.1 Larger, randomized, double-blinded studies will be reported soon; if they confirm the salubrious effects of cell-based therapies observed in the initial trials, our management of acute myocardial infarction and heart failure will change dramatically. One of the most important and unresolved issues in this scenario is the identity of the ideal cell for myocardial reconstitution. Although most of the clinical studies reported to date have used bone marrow– or skeletal muscle–derived cells, a host of other cells are being investigated in the experimental laboratory. Among these, resident cardiac stem cells (CSCs), discovered by Anversa’s group in 2003,2 hold great promise. In their initial report,2 Beltrami et al identified a lin−/c-kit+ population of primitive cells that can be clonally expanded, differentiates into cardiac myocytes, smooth muscle cells, and endothelial cells in vitro, and is able to reconstitute infarcted myocardium in vivo. It is of translational interest that these cells can effect cardiac repair when delivered via the intravascular route.3 CSCs have also been shown to be present in the human heart, where they give rise to new myocytes in patients with …

P57 Kip2 is expressed in quiescent mouse bone marrow side population cells

Hematopoietic stem cells can be accurately identified by the side population (SP) phenotype. It has been previously shown that hematopoietic stem cells are cell cycle arrested, but the mechanisms involved are currently poorly understood. In the present study, results from quantitative real-time RT-PCR show that while SP cells have increased expression of various cyclins and cyclin-dependent kinases, the increased expression of cyclin-dependent kinase inhibitors, in particular p57 Kip2, is responsible for the observed cell cycle arrest. In addition, gene expression analysis of c-kit +//Sca-1 +/Lineage − SP (KSL-SP) cells demonstrates that only p57 Kip2 shows both higher expression compared to both SP and non-SP cells. Furthermore, immunostaining also demonstrates significantly higher protein expression in KSL-SP cells. These results demonstrate that the maintenance of bone marrow SP cells in G0/G1 may be carefully controlled by p57 Kip2.

Murine Side Population Cells Contain Cobblestone Area-forming Cell Activity in Mobilized Blood

Primitive hematopoietic stem cells (HSCs) can be purified from murine bone marrow by sorting Hoechst 33342-effluxing side population (SP) cells. The aim of this study was to establish whether SP cells from peripheral blood contain primitive HSCs and whether this is altered in mice following mobilization. SP cells were analyzed and isolated from bone marrow and blood of mice after mobilization; the HSC content of isolated SP cells was determined through surrogate cobblestone area-forming cell (CAFC) assays. SP cells in normal blood were not found in the high Hoechst dye effluxing portion of the SP tail, did not express the stem cell markers c-Kit and CD34, and did not have measurable CAFC activity. In contrast, SP cells in mobilized blood expressed both stem cell markers, contained cells in the high dye efflux portion of the SP tail, and displayed significant day- 28 to day-35 CAFC activity with 165- to 334-fold enrichment. In comparison to mobilized blood SP cells, normal marrow SP cells contained a higher proportion of cells expressing c-Kit and CD34 and had a greater percentage of cells in the high Hoechst dye-effluxing portion of the SP tail. Analysis of SP cells in the bone marrow after mobilization revealed a decrease in the frequency of SP cells, in expression of c-Kit and Sca+ CD34(+)/CD34(-), and in day-7 to day-35 CAFC activity, consistent with mobilization into blood. We conclude that murine SP cells mobilized into blood contain primitive hematopoietic stem cell activity (day-28 to day-35 CAFC activity). This model offers a means to study the mechanisms of mobilization of primitive stem cells directly in a murine model.

CD31 − but Not CD31 + Cardiac Side Population Cells Exhibit Functional Cardiomyogenic Differentiation

Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlying the development of cardiac dysfunction is a decrease in the number of viable cardiomyocytes. Recent observations have suggested that the adult heart may contain a progenitor cell population. Side population (SP) cells, characterized by a distinct Hoechst dye efflux pattern, have been shown to exist in multiple tissues and are capable of tissue-specific differentiation. In this report, we confirm the existence of a cardiac SP cell population, immunophenotypically distinct from bone marrow SP cells. Moreover, we demonstrate that among cardiac SP cells, the greatest potential for cardiomyogenic differentiation is restricted to cells negative for CD31 expression and positive for stem cell antigen 1 (Sca1) expression (CD31-/Sca1+). Furthermore, we determine that CD31-/Sca1+ cardiac SP cells are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, with expression of cardiomyocyte-specific transcription factors and contractile proteins, as well as stimulated cellular contraction and intracellular calcium transients indistinguishable from adult cardiomyocytes. We also determine the necessity of cell-extrinsic signaling through coupling, although not fusion, with adult cardiomyocytes in regulating cardiomyogenic differentiation of cardiac SP cells. We, therefore, conclude that CD31-/Sca1+ cardiac SP cells represent a distinct cardiac progenitor cell population, capable of cardiomyogenic differentiation into mature cardiomyocytes through a process mediated by cellular coupling with adult cardiomyocytes.

Long-Term Transgene Expression and Survival of Transgene-Expressing Grafts Following Lentivirus Transduction of Bone Marrow Side Population Cells

Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.

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.

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.

Skeletal muscle engraftment potential of adult mouse skin side population cells.

Adult bone marrow and skeletal muscle have been shown to contain a subpopulation of cells, called side population (SP) cells, that can be isolated with the fluorescence-activated cell sorter. We used a similar method to identify SP cells in the skin of adult mice. These cells express surface markers similar to SP cells isolated from skeletal muscle, but differ from bone marrow SP cells and do not express hematopoietic markers. When transplanted into nonirradiated mdx mice, nuclei from donor skin SP cells are found within myofibers that express dystrophin. Thus, adult skin SP cells can engraft in dystrophic skeletal muscle even in the absence of total body irradiation.

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.