So-called Side Population Research Articles

Influence of high-molecular hyaluronic acid on MCF-7 breast cancer stem cells

In recent years, more and more data have been accumulating on the high radio- and chemo-resistance of tumor stem cells (CSCs), confirming the assumption that these cells play an important role in the recurrence of the tumor process in some cancer patients. Therefore it is of considerable interest to elucidate the patterns and mechanisms of the formation of the CSC pool under the influence of various factors, including one of the main components of the extracellular matrix, hyaluronic acid (HA). The purpose of the study was to elucidate the effects of HA with a molecular weight of (1-3) x 106 Dа on the amount of CSCs in MCF-7 breast cancer cell culture in vitro. Material and methods. Using the MTT test, the viability of MCF-7 cancer cells was determined after incubation with HA at a concentration of 0.3-5.0 mg/ml. Using flow cytometry, we evaluated the change in the relative and absolute amount of CSCs under the influence of HA (0.6 mg/ml). CSCs were detected by CD44+CD24-/low immunophenotype, as well as by their ability to efficiently efflux Hoechst 33342 fluorescent dye from cells and form so-called side population (SP). Results. Viability of cancer cells in the general population decreased with increasing concentration of HA (R=-0.94, p=0.006). At the same time the incubation of cells with HA led to an increase in both the relative and absolute amounts of CSCs (SP) compared with the control. Thus, the relative number of SP cells increased by 2.0 times (p = 0.002), CD44 + CD24- / low cells - by 6.1 times (p = 0.005). The absolute number of these cells changed to a lesser extent compared to the control: the increase was 1.6 (p = 0.004) and 2.9 times (p = 0.02), respectively. Conclusion. Thus, the effect of high-molecular weight HA on breast cancer cells of MCF-7 line leads to an increase in amount of CSCs, while the viability of non-stem cancer cells decreases under the same conditions.

Integrated glycomic analysis of ovarian cancer side population cells.

BackgroundOvarian cancer is the most lethal gynecological malignancy due to its frequent recurrence and drug resistance even after successful initial treatment. Accumulating scientific evidence indicates that subpopulations of cancer cells with stem cell-like properties, such as so-called side population (SP) cells, are primarily responsible for these recurrences. A better understanding of SP cells may provide new clues for detecting and targeting these cancer-initiating cells and ultimately help to eradicate cancer. Changes in glycosylation patterns are remarkable features of SP cells. Here, we isolated SP cells from ovarian cancer cell lines and analyzed their glycosylation patterns using multiple glycomic strategies.MethodsSix high-grade serous ovarian cancer cell lines were used for SP cell isolation. Among them, HO8910 pm, which contained the highest proportion of SP cells, was used for glycomic analysis of SP cells. Cell lysate of SP cells and main population cells was applied to lectin microarray and mass spectrometry for glycan profiling. Differently expressed glycan structures were further verified by lectin blot, flow cytometry, and real-time PCR analysis of their relevant enzymes.ResultsExpression of core fucosylated N-glycan and tumor-associated Tn, T and sT antigens were increased in SP cells. By contrast, SP cells exhibited decreased hybrid glycan, α2,3-linked sialic glycan and multivalent sialyl-glycan.ConclusionsGlycan structures, such as Tn, T, sT antigens, and core fucosylation may serve as biomarkers of ovarian cancer stem cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9131-z) contains supplementary material, which is available to authorized users.

ABCG2 is a selectable marker for enhanced multilineage differentiation potential in periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth.

Hedgehog and Notch Signaling Regulate Self-Renewal of Undifferentiated Pleomorphic Sarcomas

Like many solid tumors, sarcomas are heterogeneous and include a small fraction of the so-called side population (SP) cells with stem-like tumor-initiating potential. Here, we report that SP cells from a soft tissue tumor of enigmatic origin termed undifferentiated pleomorphic sarcoma (also known as malignant fibrous histiocytoma or MFH sarcoma) display activation of both the Hedgehog and Notch pathways. Blockade to these pathways in murine xenograft models, this human cancer decreased the proportion of SP cells present and suppressed tumor self-renewal, as illustrated by the striking inability of xenograft tumors subjected to pathway blockade to be serially transplanted to new hosts. In contrast, conventional chemotherapies increased the proportion of SP cells present in tumor xenografts and did not affect their ability to be serially transplanted. SP cells from these tumors displayed an unexpectedly high proliferation rate which was selectively inhibited by Hedgehog and Notch blockade compared with conventional chemotherapies. Together, our findings deepen the concept that Hedgehog and Notch signaling are fundamental drivers of tumor self-renewal, acting in a small population of tumor-initiating cells present in tumors. Furthermore, our results suggest not only novel treatment strategies for deadly recurrent unresectable forms of this soft tumor subtype, but also potential insights into its etiology which has been historically controversial.

Detektion der Side Population zur Identifizierung von Stammzellen

The quantification of the cellular transport capacity for the DNA staining dye Hoechst 33342 out of the cell is a widely accepted marker for recognition of stem cells. Cells with such an increased level of dye efflux (the so-called side population) can be detected on a flow cytometer equipped with a UV laser. Active transport mechanisms of this type play an important role for the elimination of anti-proliferative agents by cancer cells. A staining method for cells with high dye efflux is decribed.

Global Profiling of Signaling Networks: Study of Breast Cancer Stem Cells and Potential Regulation

There is overwhelming evidence that breast cancer may be driven by a small subset of breast cancer stem cells (BCSCs) that display stem/progenitor cell properties. In the present study, we identified the rare population of BCSCs, the so-called side population (SP) cells, using flow cytometry. Then, we used microarray analysis to study the differential gene expression profiles between SP and non-SP cells. Sixty-three probe sets showed a more than fourfold difference. Next, we compared the levels of proteins with Pathway Array using 154 antibodies, focusing on the proteins and phosphorylation sites that differed among SP cells, malignant mammary cells, and breast cancer tissues. Our results revealed that 40 proteins and phosphorylation sites were more than 1.5-fold different in SP cells than in non-SP cells. By comparing SP cells, MCF7 cells, and nontumorigenic MCF10A cells, we found 12 proteins that were significantly upregulated in SP cells; these proteins-cAMP-response element binding protein (CREB), cyclic AMP-dependent transcription factor 1, mesothelin, thyroid transcription factor 1, phosphorylated (p)-focal adhesion kinase, p38, Bad, p-CREB, p-protein kinase C (PKC)δ, Wee1, cell division cycle 42, and Twist-were more likely to play important roles in the signaling regulation of BCSCs. Further, 16 proteins and phosphoproteins showed differential expression in SP cells and tumor tissues. β-catenin, p-PKCα, and p-CREB were upregulated in both SP cells and breast tumors. Finally, we filtered the differential expression proteins, summarized the pathway interactions of these proteins, and rebuilt Path-Net in order to determine molecular mechanisms and core regulators. This process will allow us to identify signature patterns and mechanisms of signaling networks in BCSCs.

Cancer stem cells and escape from drug-induced premature senescence in human lung tumor cells: Implications for drug resistance and in vitro drug screening models

In this study, using an in vitro human tumor model, we show that non-small lung adenocarcinoma A549 cells after treatment with DNA damaging antitumor drugs become permanently growth-arrested as a result of so-called drug-induced premature senescence (pseudo-senescence). However, a small fraction of drug-treated cells escapes pseudo-senescence that leads to re-growth of tumor cell population after drug treatment. We show that this re-growth is associated with the presence of cancer stem cells (CSCs) in lung tumor cell population. We also document that re-growth of CSCs can be greatly delayed if lung tumor cells are treated with drug/caffeine combination that leads to the inhibition of the ATM/ATR pathway and decreased phosphorylation of PKB/Akt at Ser473. We show that in non-treated A549 cells caffeine by itself induces a reversible growth arrest that is associated with increased fraction of so-called side population cells, containing CSCs. These results point to the existence of an unknown, caffeine-sensitive mechanism that controls the number of CSCs in lung tumor cell population. Full characterization of this mechanism may lead to the development of innovative cancer therapies which are based on small molecular weight inhibitors of CSC differentiation and self-renewal, that mimic caffeine action. Our results have also important implications for drug screening tumor models in vitro.

Spotlight

Lee et al., pp. 2755–2765 Heparin's powerful anticoagulant properties are clinically widely used. Other biological activities of heparin, however, remain therapeutically unexploited because of the increased risk of bleeding. Positive side effects of heparin include suppression of angiogenesis, tumor metastasis and cell proliferation. Lee and colleagues generated new bile acid-acylated heparin derivatives to study heparin's anticancer effects. Heparin is a highly sulfated glycosaminoglycan. The sulfation pattern is an important determinant of binding to antithrombin III or angiogenic growth factors, which suppresses blood coagulation or angiogenesis, respectively. The authors identified one compound (LHT7) which was fully saturated with taurocholate moieties and exerted potent anticancer and weak anticoagulant effects. LHT7 is oversulfated and binds with high affinity to vascular endothelial growth factor 165 (VEGF165), an important mediator of angiogenesis. The strength of this binding is comparable to the interaction of antithrombin III with regular heparin and potently suppresses VEGF165-dependent angiogenesis. In addition, LHT7 administration slowed tumor growth and prolonged survival in mice. Tumors induced by subcutaneous injection of squamous cell carcinoma (SCC7) cells were reduced in size by ∼70% when mice were treated systemically with LHT7. An ∼90% reduction in tumor growth was seen in mice inoculated with human breast cancer cells (MDA-MB231). The authors propose that the potent antiangiogenic properties of LHT7 could successfully supplement existing anticancer treatment regimens. LHT7 inhibits angiogenesis induced by VEGF. Hematoxylin and eosin stain of Matrigel plugs containing the indicated reagents. LMWH=low molecular heparin. Red arrows mark red blood cells, blue arrows blood vessel walls, and black arrows Matrigel background. Wong et al., pp. 2766–2770 Cytokine profiling was performed in individuals chronically infected with hepatitis B virus (HBV) in search of new biomarkers that predict development of hepatocellular carcinoma. Chronic HBV infection is the most common cause of hepatocellular carcinoma in Asia. Measurements of alpha fetal protein are commonly used to monitor HBV-infected patients, but the sensitivity for hepatocellular carcinoma is low (40–66 %). Wong and colleagues aimed to find new tumor markers in a prospective nested case control study of 37 HBV+ patients who eventually developed liver cancer and control patients with chronic HBV infection but no cancer. They identified high serum interleukin-6 (IL-6) levels as a new predictor of hepatocellular carcinoma development. Patients with IL-6 levels above 7 pg/ml were more likely to develop liver cancer or die over time (unadjusted hazard ratio 3.2; 95%CI 1.6, 6.3, p = 0.001). This was observed when alanine aminotransferase levels were highest, indicating active hepatic inflammation, but was independent of other risk factors such as age, male gender, cirrhosis and viral load. While temporary IL-6 treatment appears to be protective in mice receiving liver transplantations from a steatotic graft, ablation of IL-6 is known to abolish hepatic carcinogenesis. It is therefore believed that short-term IL-6 production is a natural response to liver injury, which produces a protective effect; in contrast, prolonged IL-6 exposure may increase liver injury and promote carcinogenesis. Monitoring serum IL-6 levels in patients chronically infected with HBV may assist in identifying individuals at high risk for developing hepatocellular carcinoma. Kabashima et al., pp. 2771–2779 There is increasing evidence that malignant tumors contain a small population of undifferentiated cancer stem cells. These cells play pivotal roles in tumor mass formation, resistance to chemotherapy, local invasion and distant metastasis. In some tumors, cancer stem cells are concentrated in a so-called side population (SP) of cells. These cells characteristically expel Hoechst 33342 dye through active membrane transport and can thereby be distinguished from main population (MP) cancer cells. Kabashima and colleagues focused on SP cells from pancreatic cancer cell lines. While Capan-2, PANC-1 and KP-1NL cell lines included substantial amounts of SP cells (8– 20 %), no SP cells were detected in MIAPaCa2 cell cultures. Isolated pancreatic SP cells caused enhanced liver metastasis as compared to MP cells and their invasion activities were notably increased after treatment with transforming growth factor beta (TGF-β). TGF-β plays a pivotal role in the so-called epithelial to mesenchymal transition (EMT), a process critical during embryonic development and cancer progression. The loss of the epithelial and gain of mesenchymal phenotype leads to enhanced motility mediated by downregulation of cell-to-cell adhesion proteins such as E-cadherin. The authors observed that TGF-β responsiveness was greater in pancreatic SP cells than in MP cells, resulting in enhanced induction of EMT and invasiveness. SP cells were also responsive to TGF-β removal, as seen in morphological changes and E-cadherin restoration. Because switches between epithelial and mesenchymal stages are critical for pancreatic cancer development, this study underlines the importance of SP cell-targeting strategies in the prevention and treatment of pancreatic cancer.

MCF7 Side Population Cells with Characteristics of Cancer Stem/Progenitor Cells Express the Tumor Antigen MUC1

Chemotherapy, radiation, and growth inhibitory drugs preferentially eliminate actively growing cancer cells. Cancer recurrence is currently thought to be due to nondividing cancer stem/progenitor cells that are resistant to these therapies. Different therapeutic approaches need to be considered for the elimination of the cancer stem cell population. Immunotherapy is one such approach. In addition to specificity and lack of toxicity, immunotherapy targets cancer cells irrespective of their state of proliferation, as long as they express particular tumor antigens. For that reason, it is important to examine if the tumor antigens that are currently being tested as immunotherapeutic agents are also present on cancer stem cells. This study aimed to determine if one well-known tumor antigen, MUC1, which is being tested as an immunotherapy target on tumor cells, is also expressed on the quiescent cancer stem/progenitor cells. We used the so-called side population (SP) cells found in the MCF7 breast cancer cell line, which we first confirmed by cell surface markers and gene profiling to be highly enriched in cells that fulfill specific functional, phenotypic, and molecular criteria for being tumor stem/progenitor cells. We show that these cells express MUC1 and give rise to MUC1(+) tumors in vivo, which maintain the MUC1(+) SP population. MUC1 on SP cells is hypoglycosylated and heavily sialylated; the characteristics of the tumor-specific form were expressed on mature cancer cells and recognized by tumor-specific T cells and antibodies. This suggests that stem/progenitor cells, like mature tumor cells, would be targets of MUC1-directed immunotherapy.

Side Population Does Not Define Stem Cell-Like Cancer Cells in the Adrenocortical Carcinoma Cell Line NCI h295R

Recent evidence suggests the existence of a stem cell-like subpopulation of cells in hematological and solid tumor entities, which determine the malignant phenotype of a given tumor through their proliferative potential and chemotherapy resistance. A recently used technique for the isolation of this cell population is through exclusion of the vital dye Hoechst 33342, which defines the so-called side population (SP). Herein we demonstrate the presence of SP cells in a variety of adrenal specimens, including primary cultures of human adrenocortical tumors and normal adrenal glands as well as established human and murine adrenocortical cancer cell lines by fluorescence-activated cell sorter analysis and confocal microscopy. On a functional level, SP cells from the human adrenocortical tumor cell line NCI h295R revealed an expression pattern consistent with a less differentiated phenotype, including lower expression of steroidogenic enzymes such as steroid acute regulatory protein (StAR) and side-chain cleavage enzyme (P450scc) in comparison with non-SP cells. However, proliferation between SP and non-SP cells did not differ (105.6 +/- 18.1 vs. 100.0 +/- 3.5%). Furthermore, re-sorting and tracing experiments revealed the capacity for both cell types to give rise to the original SP- and non-SP-containing cell population. Similarly to the baseline growth kinetics, no survival benefit was evident in SP cells after treatment with cytotoxic agents commonly used in adrenocortical carcinomas. Taken together, these findings provide evidence that Hoechst dye exclusion, in contrast to what has been reported for other tumor entities, is not a major tumor stem cell defining marker in adrenocortical NCI h295R tumor cells.

Identification of a Small Subpopulation of Candidate Leukemia Initiating Cells within the Side Population (SP) of Patients with Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) likely originates from the CD34+CD38- hematopoietic stem cell (HSC). The so-called side population (SP), defined by Hoechst 33342 dye, might offer an alternative/supplementary stem cell compartment. The relationship between both compartments is largely unknown. We found that the CD34+CD38- compartment can be subdivided in an AML and normal compartment, based on expression of AML stem cell specific antigen CLL-1: CLL-1 positive CD34+CD38- cells carry AML specific cytogenetic aberrations and initiate leukemia in NOD/SCID mice (van Rhenen, Blood 2007, in press). Lineage aberrancies including CD7, CD19, CD56 and aberrant myeloid aberrancies, enabled to further define AML and normal CD34+CD38- sub-compartments (van Rhenen et al., Leukemia 21:1700, 2007). This led us to investigate whether SP too have aberrancies and whether these define primitive stem cells. SP cells were detected in 40 of 47 AML patients with median frequency of 0.02% (range 0.002–7.6%). In the majority of cases there was also a CD34+CD38- compartment with a median frequency of 0.44% (range 0–27%), which is 22 fold higher (in all individual cases >1-fold) than SP frequency. The median frequency of CD34+CD38- cells within SP compartment was only 2.5% (range: 0–49%). SP cells were partly or completely positive for CLL-1 (median 53%), CD123 (27%), CD7 (35%), CD19 (20%) and CD56 (53%). SP cells in NBM (n=12; median frequency 0.12%, range 0.008–4.1%) were completely negative for CLL-1 and lineage markers (median 0%, ranges 0–4% at maximum), but not for CD123 (median expression 27%, range 1–82%). These results strongly suggest that considerable part of SP cells are malignant, which was confirmed by FISH analysis. The whole SP fraction was remarkably heterogeneous with at least 4 different subpopulations present:1.3 with lymphoid characteristics, ie CD7+ (median 7% of total SP), CD19+ (2%) and CD56+ (4%), all 3 CD45high and CD48+;2.a myeloid population (median 54% of SP population; range 4–91%), CD45low and CD48-, with relatively high forward and sideward scatter (FSChigh/SSChigh) and high CD38 expression (median 82%) and usually with aberrant marker expression;3.a low-frequency FSClow/SSClow myeloid fraction, CD45low and CD48-, with lower CD38 expression (median 48%), and negative for aberrant markers and4.a similar population but with aberrant markers present. The latter, presumably primitive, malignant population had median frequency (of WBC) of 0.0018% (range 0.00016–0.0056%).The CD34+CD38- content herein was 20% at maximum. NBM too had the 3 lymphoid populations (median 8%, 2% and 5%, resp), the FSChigh/SSChigh myeloid population (in 9/12 cases) and the FSClow/SSClow myeloid population (12/12 cases). The putative primitive character of the AML FSClow/SSClow SP subpopulation was substantiated by suspension culture for 5 weeks with subsequent CFU assay (14 days): FSClow/SSClow cells had >200 fold clonogenic ability compared to FSChigh/SSChigh cells. In conclusion, the AML SP compartment is highly heterogeneous and contains a low-frequency (median 1:50,000) subpopulation, defined by aberrant markers and with primitive characteristics, as a likely candidate stem cell population. In a stem cell concept integrating the CD34+CD38- and SP compartment, the presumed AML stem cell frequency would be in the order of 1:250,000. which probably is close to the presumed frequency of leukemia initiating cells in diagnosis AML.

Mechanisms of Aging in Bone Marrow Stem Cells

Mechanisms of Aging in Bone Marrow Stem Cells

Identification of Primitive Subpopulations of Acute Myeloid Leukemia Side Population (SP) Stem Cells Defined by Differentiation Status and Malignant Character.

Acute myeloid leukemia (AML) is generally regarded as a stem cells disease. A large proportion of remission patients ultimately relapses, indicating ineffectiveness of current therapies in eradicating leukemia stem cells (LSCs). Additional to the CD34+CD38− compartment, the so-called side population (SP) exists. In order to trace residual SP stem cells under remission conditions, allowing adapted risk stratification or more accurate prediction of relapses, we sought for leukemia stem cell specific immunophenotypic markers i.e. not staining the normal SP stem cells. We used C-type Lectin-like molecule-1 (CLL-1, van Rhenen, Blood 106: 6a, 2005) and IL-3 receptor α-chain CD123, previously shown to be stem cell markers. Lastly, several markers making up the so-called leukemia associated phenotypes (LAP), used for MRD detection (Feller, Leukemia 18:1380, 2004), were tested for their ability to stain the AML SP stem cell as well. Using Hoechst 33342 dye, PI and monoclonal antibodies against CD7, CD19, CD56, CD123 and CLL-1, SP immunophenotyping using FACS analysis was performed on bone marrow (BM) samples of 17 AML patients at diagnosis, 8 healthy donors (nBM) and 4 relevant donors with BM regenerating after chemotherapy (rBM). SP cells were detected in 13 of 17 AML patients with median frequency of 0.01% (% of WBC, range 0.00002-0.17%). In all 13 cases, SP cells were partly or completely positive for CLL-1 and/or CD123. In 10/13 cases SP cells were partly or completely positive for LAP markers These results strongly suggest that the majority of SP cells are malignant, which was confirmed by FISH analysis [n=2; t(8;21)]. Marker positive SP cells as a fraction of total SP was 0.66 (range 0.22–1.0). Furthermore, SP cells from control nBM and rBM were completely negative for CLL-1 and LAP markers, but not for CD123 (40%-82%). Remarkably, 11/13 AML samples revealed two subpopulations within the SP, in terms of sideward scatter (SSC): high side scatter (HSSC) SP cells, with median frequency of 46 % (% of whole SP compartment, range 21–86%) and low side scatter (LSSC) SP cells (median frequency 54 %, range 14–79%). In the 2 remaining cases only LSSC SP was present. HSSC and LSSC populations were also seen in 5/8 nBM SP cells, with only LSSC SP present in the remaining 3 cases. HSSC AML SP cells had a more differentiated phenotype (high SSC, high CD38 expression) and were all malignant in terms of LAP marker positivity and CLL-1 positivity. LSSC SP cells were more primitive (low SSC, mostly CD38 negative or low), only partly expressing CLL-1 or LAP. FISH analysis for a patient with t(8;21) confirmed the malignant nature of both the HSSC and LSSC LAP marker (CD19 in this case) positive cells and the normal character of the CD19 negative LSSC cells. Marker positive, primitive LSSC SP cells (n=13) had median frequency of 14.2% of total SP (range 4.6–48.9%). The presumed median frequency of these LSSC SP cells at diagnosis is thereby close to 1: 105, (% of WBC) which is close to the presumed AML stem cell frequency in diagnosis AML. In conclusion, diagnosis AML SP cells can be discriminated from normal SP cells based on expression of CLL-1 and LAP markers. SP cells show a primitive low-frequency sub fraction as a likely candidate to contain the leukemia initiating cell. Future studies include functional characterization of this LSSC SP subfraction for purposes of risk stratification and therapeutic intervention.

Non-side-population hematopoietic stem cells in mouse bone marrow

AbstractMost hematopoietic stem cells (HSCs) are assumed to reside in the so-called side population (SP) in adult mouse bone marrow (BM). We report the coexistence of non-SP HSCs that do not significantly differ from SP HSCs in numbers, capacities, and cell-cycle states. When stained with Hoechst 33342 dye, the CD34-/low c-Kit+Sca-1+lineage marker- (CD34-KSL) cell population, highly enriched in mouse HSCs, was almost equally divided into the SP and the main population (MP) that represents non-SP cells. Competitive repopulation assays with single or 30 SP- or MP-CD34-KSL cells found similar degrees of repopulating activity and frequencies of repopulating cells for these populations. Secondary transplantation detected self-renewal capacity in both populations. SP analysis of BM cells from primary recipient mice suggested that the SP and MP phenotypes are interconvertible. Cell-cycle analyses revealed that CD34-KSL cells were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or MP. Bcrp-1 expression was similarly detected in SP- and MP-CD34-KSL cells, suggesting that the SP phenotype is regulated not only by Bcrp-1, but also by other factors. The SP phenotype does not specify all HSCs; its identity with stem cell function thus is unlikely.

Cell-Based Cardiac Repair

Received February 28, 2005; revision received April 25, 2005; accepted April 29, 2005. It has now been more than a decade since the first experiments were performed using cell transplantation for the prevention and treatment of heart failure.1–3 Although the biomedical community was initially somewhat skeptical of this approach, a large body of experimental evidence was amassed showing that injected cells could create new tissue and improve function of the failing heart. This evidence, coupled with the recognized limitations of heart failure treatments and the intuitively appealing concept of “regenerative medicine,” has contributed to a crescendo of activity in cell-based cardiac repair. Given the flurry of clinical trials that are currently under way, we think it is timely to review progress over the past 10 years and provide a critical assessment of where the field stands and where it appears to be headed. Cell-based cardiac repair began with studies of skeletal myoblasts derived from skeletal muscle satellite cells.1–3 Myoblasts were the initial choice because of their availability from autologous or syngeneic sources, their ability to proliferate, and their ability to withstand ischemia better than many cell types. Although it was originally hoped that these cells would transdifferentiate into cardiomyocytes, it is now clear that myoblasts remain stubbornly committed to form mature skeletal muscle in the heart3–5 (with the exception of rare cell fusion events at the graft–host interface6). Skeletal muscle is one of the few cell types in the body that does not normally express gap junction proteins, and hence, structural and physiological studies indicate that skeletal muscle cells do not form electromechanical junctions with cardiomyocytes when engrafted into the heart.7,8 Despite this, numerous studies have shown beneficial effects of skeletal myoblast grafting into the infarcted heart in rodents and large animals.8–13 Cardiomyocytes would …

Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)

This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so-called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA-binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi-drug-like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.

Side-population cells from different precursor compartments.

The rapid efflux of the fluorescent DNA-binding dye Hoechst 33342 identifies a rare, so-called side population (SP), which rapidly expels the dye, can reconstitute the bone marrow (BM) of lethally irradiated mice, and has proven negative for most lineage markers including CD34. Because SP cells from human cell sources, such as mobilized peripheral blood [apheresis products (AP)], cord blood (CB), or BM have not been extensively characterized to date, we sought to analyze SP cells from various cell sources. We detected murine SP cells with a median frequency of 0.04% (n = 23) and a 52-fold colony-forming units (CFU) increase compared to unsorted cells (p = 0.028). The median frequency of human SP cells was 0.02% (n = 90), with highest numbers in donor AP, and lower in CB and BM. Human SP cells were mostly CD34(-) and lineage marker-negative. These showed no enrichment in CFU before expansion; however, they displayed a CFU increase after 5-7 days of cytokine-supported suspension culture (10.7-fold at day 5, 7.2-fold at day 7; n = 17) that was significant compared to both input (day 0) SP and to non-SP cells before and after expansion (p < 0.05). SP cells demonstrated a significant long-term culture-initiating cell (LTC-IC) increase of 167-fold (n = 17) as compared to non-SP cells (p = 0.002), with the highest numbers from AP specimens. We conclude that human primitive hematopoietic cells can be isolated via Hoechst staining and that SP cells of various human sources show substantial differences and represent a rare CD34(-) population with stem cell potential.

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells.

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.