scholarly journals Evaluation of a side population of canine lymphoma cells using Hoechst 33342 dye

2013 ◽  
Vol 14 (4) ◽  
pp. 481 ◽  
Author(s):  
Myung-Chul Kim ◽  
Susan D'Costa ◽  
Steven Suter ◽  
Yongbaek Kim
Keyword(s):  
Side Population ◽  
Lymphoma Cells ◽  
Hoechst 33342 ◽  
Canine Lymphoma

Most of the Short Term Repopulating Cells in Human Mobilized Peripheral Blood Do Not Have a Side Population (SP) Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2867-2867
Author(s):  
M. Fischer ◽  
M. Schmidt ◽  
S. Klingenberg ◽  
C. Eaves ◽  
C. von Kalle4 ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Side Population ◽  
Isolation Procedure ◽  
Lymphoid Cells ◽  
Short Term ◽  
Hoechst 33342 ◽  
Hoechst Staining ◽  
Mobilized Peripheral Blood

Abstract The multidrug resistance transporter, ABCG2, is expressed in primitive hematopoietic stem cells from a variety of sources. These cells are detected in dual wave-length fluorescent FACS profiles as a “side population” (SP cells) on the basis of their ability to efflux the fluorescent dye, Hoechst 33342. We have previously shown that 2 types of human short term repopulating cells (STRC) can be enumerated by limiting dilution analysis of their efficient ability to regenerate exclusively myeloid cells after 3 weeks (STRC-Ms), or both myeloid and lymphoid cells after 6–12 weeks (STRC-MLs) in NOD/SCID-b2microglobulin-/- (b2m-/-) mice. Previous findings also implicated these STRCs as determinants of the rapidity of early hematologic recovery in patients transplanted with cultured mobilized peripheral blood (mPB) cells. Here we asked whether any human STRCs have an SP phenotype and hence whether the isolation of SP cells would retain the rapid repopulating activity of a clinical transplant. CD3- SP and non-SP cells were isolated by FACS from low-density (LD) mPB cells after Hoechst staining and transplanted at limiting dilutions into 117 sublethally irradiated b2m-/- mice. The numbers and types of human hematopoietic cells present in the bone marrow of these mice were subsequently monitored by FACS analysis of bone marrow cells aspirated serially, 3, 8 and 12 wks post-transplant. A verapamil-sensitive SP population was reproducibly detected in all 5 patients’ samples studied (0.039 ± 0.012% of the CD3- LD cells). The in vivo assays failed to detect either STRC-Ms or STRC-MLs in the SP fraction and all these activities were obtained from the non-SP cells. If even a single recipient of the largest dose of SP cells transplanted had been positive, this would have detected 10% of the STRCs present. Thus, >90% of all STRC-M and STRC-ML in mPB are non-SP cells. However, 4 of 40 mice transplanted with SP mPB cells produced some B-lymphoid cells only starting 12 wks post-transplant. However, this result is difficult to interpret since subjecting the STRC-Ms to the Hoechst 33342 staining and FACS isolation procedure alone eliminated their ability to generate megakaryocytic progeny in vivo, although this did not occur when these cells were just stained for CD34 and then isolated by FACS. In addition, the differentiation behaviour of STRC-MLs was not affected by the Hoechst staining and subsequent FACS isolation procedure. In summary, we demonstrate that purification of SP cells depletes human mPB transplants of STRCs, thereby raising serious concerns about the safety of any clinical use of SP cell-enriched transplants as stem cell support after myeloablation. Our results also suggest that the staining and enrichment procedure for isolating SP human cells may differentially affect the lineage potential of some types of STRCs, including those whose activity may be indispensable for rapid and multi-lineage hematologic recovery.

Age Related Changes in Hoechst 33342 Efflux Dynamics and Side Population Phenotype in Murine Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3220-3220
Author(s):  
Matthew J. Greenwood ◽  
Peter M. Lansdorp
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Side Population ◽  
Hoechst 33342 ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Old Mice ◽  
Young Mice ◽  
In Cells

Abstract The mechanisms underlying the aging of the hematopoietic stem cell (HSC) compartment remain poorly understood. The ATP-binding cassette cell surface transport protein, ABCG2, has been identified as the transporter responsible for Hoechst 33342 (Hst) efflux in primitive stem cells and its expression is associated with the side population (SP) phenotype in both murine and human bone marrow (BM). ABCG2 expression and Hst efflux activity is highest in those cells with the greatest repopulating potential and is progressively downregulated during differentiation. The substrate profile of ABCG2, which includes a number of antineoplastic drugs, protoporphyrin IX and the chlorophyll breakdown product pheophorbide, suggest that ABCG2 transporters may function to protect stem cells from cytotoxic insults, a function which may be of great importance in stem cell maintenance. Amongst laboratory mice, the C57BL/6 strain is the longest lived and appears to accumulate HSC’s with age as assessed by both phenotype and colony forming assays. While the phenotypic features of the SP profile have been well characterized in both humans and young mice, little is known of the Hst efflux dynamics or phenotype of the SP profile in old and very old C57BL/6 mice. In order to further characterize the SP profile in old mice, whole BM was extracted from the femurs, tibiae, pelvis and thoracolumbar vertebral bodies of young (9–13 week) and old (95–108 week) C57BL/6 (Ly5.1) mice. Cells were stained with 5μg/ml Hst followed by staining with a combination of CD45.1 FITC, Sca1 PE, c-kit APC, CD34 FITC, biotinylated CD34 and lineage markers and strep PE-Texas Red. In addition, serial sampling of Hst incubated cells was performed to assess Hst efflux activity at 20 mins incubation through to 100mins. Six-color flow analysis was performed on a FACS Vantage™ (BD) cytometer and data analyzed using FlowJo™ software. Results show a marked increase in cells with an SP phenotype in old vs young mice (mean±SD 1.85%±0.88 vs 0.15%±0.09) which were more highly enriched for CD34-Sca1+ckit+ (22.2%±8.65 vs 8.89%±6.7) cells. Subdividing the SP profile into four regions (R1 to R4) from highest to lowest Hst efflux activity revealed that in old mice, SP cells with the highest Hst efflux activity were almost exclusively of a CD34-Sca1+ckit+ phenotype (82.3%±14.0 vs 11.5%±7.8), with a decreasing proportion of these cells represented throughout the remaining SP tail, though a significant proportion of cells within R4 remain CD34-Sca1+ckit+ (15.3%±7.83 vs 4.19±3.01). Similar patterns have been observed in both whole and lineage depleted BM. In addition, BM cells from old C57BL/6 mice show prolonged Hst efflux activity with an increase in cells in the SP gate at 100 mins (1.51%±0.50 vs 0.10%±0.06). We conclude that in old C57Bl/6 mice, cells accumulate which have the capacity to efflux Hst in agreement with previous reports of an increase in HSC number with age in this mouse strain.

scholarly journals Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells

2008 ◽  
Vol 295 (3) ◽  
pp. F680-F687 ◽  
Author(s):  
Sanjai K. Addla ◽  
Mick D. Brown ◽  
Claire A. Hart ◽  
Vijay A. C. Ramani ◽  
Noel W. Clarke
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Side Population ◽  
Kidney Tissue ◽  
Human Kidney ◽  
Proliferative Potential ◽  
Stem Cell Markers ◽  
Hoechst 33342 ◽  
Tissue Samples

The fundamental changes which predispose for renal cell carcinoma (RCC) are poorly characterized. It is hypothesized that “cancer stem cells” may be influential in carcinogenesis, and the epithelial side population (SP) is enriched for stemlike cells in other epithelial cancers. In this study, we have isolated and characterized the SP and non-SP (NSP) populations from normal (NK) and malignant (RCC) human kidney tissue. NK specimens were taken from patients undergoing non-renal cancer surgery and paired malignant and macroscopically normal tissue samples were taken from patients undergoing surgery for RCC. The Hoechst 33342 dye efflux technique was used to isolate epithelial SP and NSP from normal and malignant human renal tissue. Cellular subpopulations were phenotyped for lineage, cell cycle, and putative stem cell markers, and functionally characterized using in vitro colony-forming and proliferation assays. The SP constituted 3.8 ± 0.4 and 5.9 ± 0.9% of epithelial cells in NK and RCC, respectively, of which 14.1 ± 3.5 and 13.2 ± 3.6% were shown to be in G0. SP cells demonstrated greater proliferative potential in colony-forming efficiency, long-term culture, and spheroids assays and were shown to be maintained upon tissue culture passage. We have shown that the renal SP is enriched for quiescent cells, with a high proliferative capacity and stemlike properties. The population is, however, heterogeneous, confirming that the terms “SP cell” and “stem cell” cannot be used interchangeably.

Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34+CD38− progenitor cells from patients with acute myeloid leukemia

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3882-3889 ◽  
Author(s):  
Michaela Feuring-Buske ◽  
Donna E. Hogge
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Side Population ◽  
Severe Combined Immunodeficiency ◽  
Hoechst 33342 ◽  
Nonobese Diabetic ◽  
Abnormal Cells ◽  
Hoechst Dye ◽  
Mdr 1 ◽  
Acute Myeloid

Efflux of Hoechst 33342 from normal hematopoietic cells identifies a “side population” (SP+) of negatively staining cells that, in the mouse, are largely CD34− and are enriched for primitive progenitors. To further characterize human SP+cells, blood or bone marrow from 16 patients with acute myeloid leukemia (AML) was analyzed for their presence, immunophenotype, and cytogenetic and functional properties, and for the relation between SP phenotype and multidrug resistance-1 (MDR-1) expression. The mean percentages of SP+ and MDR+ cells was 8.1% (range, 0.5%-29.9%) and 12.8% (range, 0%-54.8%), respectively, with no correlation between the 2 values. The percentages of SP+ cells that were CD34+CD38−, CD34+CD38+, or CD34− were 12% (range, 0.4%-50%), 25% (range, 0.5%-96%), and 63% (range, 4%-99%). Cytogenetically abnormal cells were always detected in the SP−CD34+CD38− and SP+CD34− fractions, and abnormal colonies (CFC), long-term culture-initiating cells (LTC-IC), and nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mouse leukemia–IC were detected in the former fraction. No progenitors were detected among SP+CD34− cells in any of these assays from 9 of 10 samples. In contrast, exclusively normal cells were detected in the SP+CD34+CD38−fraction from 9 of 15 samples, and CFC, LTC-IC, and multilineage engraftment in NOD/SCID mice from this subpopulation were also cytogenetically normal in 6 of 8, 6 of 7, and 2 of 2 cases studied, respectively. In contrast to murine studies, primitive progenitors are enriched among SP+CD34+CD38− cells from patients with AML. The molecular basis for Hoechst dye efflux is uncertain because it does not appear to be related to MDR-1 expression.

scholarly journals Clinical significance of cancer stem cell markers in lung carcinoma

2021 ◽  
Author(s):  
Liang Zhang ◽  
Lianglan Shen ◽  
Dijun Wu
Keyword(s):  
Stem Cell ◽  
Lung Adenocarcinoma ◽  
Tumor Invasion ◽  
Side Population ◽  
Stem Cell Markers ◽  
Hoechst 33342 ◽  
Cancer Stem Cell Markers ◽  
Transporter Protein ◽  
Cancer Studies

The identification of side population (SP) cells in several cancer studies has been proved to be involved in the treatment failure (chemotherapy) and tumor relapse. Here we have sorted 7% of side population (SP) cells from lung adenocarcinoma by Hoechst 33342 dye expulsion method. Further, the characterization of sorted SP cells showed cancer stem like properties such as transcriptional upregulation of stemness genes (OCT-4, SOX2 and NANOG), ATP binding cassette (ABC) transporter protein (ABCG2) and enhanced level of stem cell surface markers such as CD133 and CD44. Therefore, the aforesaid properties of lung adenocarcinoma SP cells play a significant and functional role in tumor invasion, metastasis, chemotherapeutic drug resistance and tumor recurrence in lung cancer.

ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4487-4495 ◽  
Author(s):  
Naoyuki Uchida ◽  
Brad Dykstra ◽  
Kristin Lyons ◽  
Frank Leung ◽  
Merete Kristiansen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Side Population ◽  
Rhodamine 123 ◽  
Hoechst 33342 ◽  
Hematopoietic Stem ◽  
Activation Status

Abstract Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver, very few HSCs efflux Hoechst 33342 efficiently, and they are thus not detected as “side population” (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment, or in vitro with cytokines, induces variable losses in Rhodamine-123 and Hoechst 33342 efflux activities, and HSCs from mdr-1a/1b-/- mice show a dramatic decrease in Rhodamine-123 efflux ability. Thus, the Rhodamine-123 and Hoechst 33342 efflux properties of murine HSCs fluctuate in the same fashion as a number of other HSC markers, suggesting these are regulated by a common control mechanism that operates independently of that regulating the regenerative function of HSCs. (Blood. 2004;103:4487-4495)

Phenotypic Examination of Side Population (SP) Cells in Highly Enriched Human CD34+ Cells Obtained from Peripheral Blood Progenitor Cells (PBPC) and Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4140-4140
Author(s):  
Dag Josefsen ◽  
Leiv S. Rusten ◽  
Trond Stokke ◽  
Lise Forfang ◽  
Erlend B. Smeland ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Cell Population ◽  
Peripheral Blood ◽  
Side Population ◽  
Hoechst 33342 ◽  
Hematopoietic Stem ◽  
Immature Cell ◽  
Cd38 Expression ◽  
Cd34 Cells

Abstract CD34+ cells isolated from bone marrow include hematopoietic stem cells (HSC) as well as more lineage committed hematopoietic progenitor cells (HPC), demonstrating that CD34+ cells are a relatively heterogeneous cell population. Highly enriched CD34+ cells isolated from peripheral blood (PBPC) after mobilization shows a more immature profile with less expression of lineage restricted markers indicating that CD34+ cells from PBPC are a more homogenous immature cell population than CD34+ cells obtained from bone marrow. By using Hoechst 33342-dye efflux assay, which identifies a population of immature HPC, termed side population (SP) cells we have examined the phenotypical profile of SP+CD34+ cells obtained from bone marrow and SP+CD34+ cells isolated from PBPC. Highly enriched CD34+ cells were isolated from PBPC obtained from patients with Hodgkin lymphoma, and bone marrow was obtained from healthy volunteer donors by iliac crest aspiration after informed consent. To identify the SP+ cells, enriched CD34+ cells were stained with Hoechst 33342 dye. Using flowcytometric techniques (FACStar+, FACSDiva, Becton Dickinson, San Jose, CA) we were able to visualize the dye efflux in SP+ cells. SP+ cells were functionally confirmed using Verapamil staining. The frequenzy of LTC-IC was markedly increased in SP+CD34+ cells compared to SP−CD34+ cells (n=5), in line with previous reports. The percentage of SP+CD34+ cells varied from 0,4 to 18% of the total CD34+ cell population obtained from PBPC (n= 16), whereas the level of SP+CD34+ cells obtained from bone marrow varied between 4–7% of the total CD34+ cell population (n=4). Expression of lineage committed markers, including CD10, CD15 and CD19 was less then 10% of the whole CD34+ cell population obtained from PBPC, whereas we found a higher level of expression of these markers in CD34+ cells isolated from bone marrow. However, when we examined the SP+CD34+ cells from either PBPC or bone marrow, we observed that the phenotypical profile of these cells were similar with almost no expression of lineage markers. Thus, the more lineage-committed cells in the CD34+ cell population obtained from bone marrow seems to be restricted to the SP−CD34+ cell fraction. Examination of CD90 and CD133 expression revealed a higher level in the SP+ CD34+ cell fractions compared to the SP− fractions. Furthermore, we investigated the level of CD38 expression. Previous studies have demonstrated that lack of CD38 expression in CD34+ cells identifies a more immature cell population. Surprisingly, we observed that 30–40% of SP+CD34+ cells obtained from bone marrow were CD38 negative, whereas the level of SP+CD34+CD38− cells from PBPC was 2–5%, which is similar to the level of CD38− cells in the CD34+ cell population isolated from both PBPC and bone marrow. Currently, we are exploring the frequency of LTC-IC in SP+CD34+CD38− cells from bone marrow, and we are also planning cell sorting of these cells for functional analyses. In conclusion, we find that the level of CD38 negative cells in SP+CD34+ subpopulation of CD34+ bone marrow cells are higher than what observed in SP+CD34+ and SP−CD34+ from PBPC as well as in SP−CD34+ from bone marrow. Our ongoing studies will clarify if these results define SP+CD34+CD38− cells from bone marrow as a source of highly enriched primitive HPC.

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