Flow Cytometry Sorting Research Articles

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Isolation of cardiomyocyte nuclei from post-mortem tissue.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins(1). Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions(2). Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating(3), cell-cycle analysis(4), visualization of thymidine analogues (e.g. BrdU and IdU)(4), transcriptome and epigenetic analysis.

Toxin content differs between life stages of Alexandrium fundyense (Dinophyceae)

Different life stages of two mating-compatible clones of the paralytic shellfish toxin (PST)-producing dinoflagellate Alexandrium fundyense Balech were separated using a combination of techniques; culturing and sampling methods were used to separate vegetative cells and gametes, and sorting flow cytometry was used to separate zygotes. PST profiles were significantly different between life stages; the two gonyautoxins GTX1 and 2 were present in vegetative and senescent cells, but disappeared from gametes and zygotes. Toxin-profile changes were shown to occur very quickly in both strains when pellicle cyst formation was induced by shaking (four minutes) followed by rinsing on a screen. These pellicle cysts produced from exponentially-growing, vegetative cells lost GTX1 and 2 completely. Rapid toxin epimerization of GTX1 to GTX4 and GTX2 to GTX3 is one possible explanation, although the biological advantage of this remains unclear. Another possible explanation is that during the mating phase of a bloom or when cells are disturbed, GTX1 and GTX2 are released into the surrounding water. It may be advantageous for a dinoflagellate bloom to be surrounded by free toxins in the water.

Diamagnetic particle focusing using ferromicrofluidics with a single magnet

Focusing particles into a tight stream is critical to many applications such as microfluidic flow cytometry and particle sorting. Current magnetic field-induced particle focusing techniques rely on the use of a pair of repulsive magnets, which makes the device integration and operation difficult. We develop herein a new approach to focusing nonmagnetic particles in ferrofluid flow through a T-microchannel using a single permanent magnet. Particles are deflected across the suspending ferrofluid by negative magnetophoresis and confined by a water flow to the center plane of the microchannel, leading to a focused particle stream flowing near the bottom channel wall. Such three-dimensional diamagnetic particle focusing is demonstrated in a sufficiently diluted ferrofluid through both the top and side views of the microchannel. As the suspended particles can be visualized in bright field, this magnetic focusing method is expected to find applications to label-free (i.e., no magnetic or fluorescent labeling) cellular focusing in lab-on-a-chip devices.

Extensive p‐Tau Pathology and SDS‐Stable p‐Tau Oligomers in Alzheimer's Cortical Synapses

Like amyloid beta (Aβ) oligomers, tau aggregates are increasingly recognized as potential key toxic intermediates in Alzheimer's disease (AD) and as therapeutic targets. P-tau co-localizes with Aβ in cortical AD synapses and may contribute to synapse dysfunction and loss. Flow cytometry analysis of synaptosomes from AD compared with aged cognitively normal cortex demonstrates increased immunolabeling for three p-tau antibodies (AT8, PHF-1 and pS422), indicating phosphorylation at multiple tau epitopes. Sequential extraction experiments show increased soluble p-tau in AD synapses, but a sizable pool of p-tau requires detergent solubilization, suggesting endosomal/lysosomal localization. P-tau is co-localized with Aβ in individual synaptosomes in dual labeling experiments, and flow cytometry sorting of Aβ-positive synaptosomes from an AD case reveals a marked enrichment of p-tau aggregates. The p-tau enrichment, a 76-fold increase over the initial homogenate, is consistent with sequestration of p-tau in internal synaptic compartments. Western analysis of a series of AD and normal cases shows SDS-stable tau oligomers in the dimer/trimer size range in AD samples. These results indicate that widespread synaptic p-tau pathology accompanies Aβ accumulations in surviving synaptic terminals, particularly in late-stage AD.

Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples

BackgroundFlow cytometry and cell sorting are powerful tools enabling the selection of particular cell types within heterogeneous cell mixtures. These techniques, combined with whole genome amplification that non-specifically amplify small amounts of starting DNA, offer exciting new opportunities for the study of malaria genetics. Among them, two are tested in this paper: (1) single cell genotyping and (2) parasite DNA purification for subsequent whole genome sequencing using shotgun technologies.MethodsThe method described allows isolation of Plasmodium falciparum trophozoites, genotyping and whole genome sequencing from the blood of infected patients. For trophozoite isolation, parasite and host nuclei are stained using propidium iodide (PI) followed by flow cytometry and cell sorting to separate trophozoites from host cells. Before genotyping or sequencing, whole genome amplification is used to increase the amount of DNA within sorted samples. The method has been specifically designed to deal with frozen blood samples.Results and conclusionThe results demonstrate that single trophozoite genotyping is possible and that cell sorting can be successfully applied to reduce the contaminating host DNA for subsequent whole genome sequencing of parasites extracted from infected blood samples.

The Age of Olfactory Bulb Neurons in Humans

Continuous turnover of neurons in the olfactory bulb is implicated in several key aspects of olfaction. There is a dramatic decline postnatally in the number of migratory neuroblasts en route to the olfactory bulb in humans, and it has been unclear to what extent the small number of neuroblasts at later stages contributes new neurons to the olfactory bulb. We have assessed the age of olfactory bulb neurons in humans by measuring the levels of nuclear bomb test-derived (14)C in genomic DNA. We report that (14)C concentrations correspond to the atmospheric levels at the time of birth of the individuals, establishing that there is very limited, if any, postnatal neurogenesis in the human olfactory bulb. This identifies a fundamental difference in the plasticity of the human brain compared to other mammals.

Adoptive transfer of Regulatory T cells from burn-injured rats depressed T cell responses in the recipient sham rats (179.1)

Abstract Immunosuppression occurs after burn injury with or without septic complications. We hypothesize that CD4+CD25+ T cells (Treg) are responsible for transmitting inhibitory signals to the IL-2-producing T cells in burn related inflammatory/infectious conditions. Enriched Treg were obtained from day-3 burn rats by flow cytometry and magnetic microbead cell sorting and given intravenously to sham rats. The rats were then sacrificed day 1 and day 3 post-adoptive transfer and T cell proliferation responses were assessed by thymidine assay. The results indicated that when burn Treg were adoptively transferred to sham rats, T cell proliferation (CD4+, CD4+CD25+ and CD4+CD25-) of sham rats was significantly depressed. Furthermore, the results showed that adoptive transfer of Treg did not influence the phenotypic expression of CD4+ T cells of the recipient rats. The percentage expressions of CD25, CD11a (LFA), and CD62L of the recipient rats remained same as before adoptive transfer. Experiments are currently underway to expand these findings to burn plus septic rats. These preliminary findings however allow us to hypothesize that while burn injury alone can produce attenuated CD4+ T cell mediated responses as a result of actions of Treg; burn injury with septic complications may further cause an irreversible loss of T cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with burn plus a critical infection.

Abstract 5359: Gene expression characterization of different cancer stem cell isolation methods in head and neck cancer

Abstract Background: Head and neck squamous cell cancer (HNSCC) is a heterogeneous cancer that continues to pose major clinical problems. There is growing evidence that cancer stem cells (CSCs) may be an important factor in disease progression and resistance to current treatments. Establishing that stem cells are present in HNSCC and characterizing their global gene expression patterns provides the rationale for this study. Methods: A series of 5 HNSCC cell lines that have been maintained at low passage number such that they maintain phenotypic and morphological characteristics similar to the primary tumor were used in this study. Three different strategies were used to isolate stem cells by flow cytometry sorting: 1) Hoechst 33342 “side” population, 2) Aldefluor, and 3) CD44 bright cells. Global gene expression for each isolated CSC-enriched population and its associated CSC-depleted population was analyzed using Affymetrix Human Exon 1.0ST arrays. Gene set enrichment analysis was performed using Ariadne Pathway Studio. Results: The different methods of identifying CSCs produced widely different populations. In side population cells, there were 77 differentially expressed genes (p<0.05 and 1.5-fold cutoff) while only 59 and 17 genes were differentially expressed in the CD44 bright and Aldefluor bright populations, respectively. Not a single gene was commonly altered between any two of the isolation methods. Gene set enrichment analysis, however, was able to identify categories that differed between the CSC-enriched and CSC-depleted populations. Biological processes of cell cycle, cell division, mitosis, translational elongation, and spindle assembly were highly regulated. Microtubule binding/microtubule plus-end binding and structural constituent of ribosome were commonly altered molecular functions. Conclusions: This study suggests that current methods for isolating putative stem cells in HNSCC result in diverse populations with differing expression of specific genes. However, when examined for global expression patterns, several important categories of genes are commonly altered between the CSC-enriched and CSC-depleted populations. Some of the known characteristics of stem cells such as asymmetric division and unlimited growth potential are suggested by the highly regulated categories of cell division and spindle assembly. From these studies we hope to gain greater insight into the importance of stem cells in HNSCC as well as improve the capability of identifying these potentially important new targets for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5359. doi:1538-7445.AM2012-5359

Abstract 3298: Arsenic induces human bronchial epithelial cell transformation featured with tumor-initiating cells

Abstract Arsenic is a well-known human carcinogen. Chronic exposure to arsenic is associated with cancers in various tissues, including skin, urinary bladder, lung, liver, kidney and prostate. However, the mechanism underlying arsenic-induced carcinogenesis remains elusive. Here, we demonstrate that long-term (six month) exposure to low levels of sodium arsenite (0.25 µ M) in human bronchial epithelial cell line (BEAS-2B) results in altered morphology, increased cell proliferation and anchorage-independent colony formation in soft agar, indicative of malignant transformation of these cells. Moreover, tumorigenesis analysis revealed that s.c. inoculation of the arsenic-transformed cells can induce tumors in nude mice. The tumors can be observed after 2-3 weeks even only 500 arsenic-transformed cells were injected, suggesting that these transformed cells induced by long-term and low-dose arsenic treatment are highly tumorigenic. Additional studies indicated that some of these arsenic-transformed cells exhibit characteristics of tunor-initiating cells (TICs) or cancer stem cells (CSCs), such as overexpression of Oct3/4, Nanog, Sox2, Klf4, and C-myc. Tumor sphere formation assay, a general procedure in defining TICs or CSCs, suggested that about 5% of these arsenic-transformed cells can form tumor spheres in non-adherent plates. The tumor sphere forming cells were enriched through serial passage of the cells collected from tumor spheres. Flow cytometry sorting showed that 90% of these sphere cells are CD49f positive, whereas CD133, CD44, EpCAM, and c-kit were not detected. Overexpression of CD49f was confirmed by immunoblotting, which indicated an increased CD49f level in the arsenic-transformed cells relative to the non-transformed BEAS-2B cells. Finally, the ability of sphere formation and colony formation in soft agar was compared between the cells showed the strongest and the weakest CD49f expression and suggested that the cells with the strongest CD49f expression are more capable of forming tumor spheres and colonies. In conclusion, our data suggested that the transformed cells induced by arsenic have features of TICs or CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3298. doi:1538-7445.AM2012-3298

Isolation, proliferation and differentiation of thyroid stem cell from human thyroid nodule tissue

To explore the presence of a characteristic stem cell population (side population, SP) in human thyroid gland and perform sphere culture method for the isolation and proliferation of thyroid stem cell. Flow cytometry and cell sorting were performed to identify and isolate the ABCG2-positive SP cells from primary thyroid cells. The comparison of gene profiles and morphology between SP and main population (MP) were performed. Primary thyroid cells were also cultured in neurosphere-like growth condition for sphere formation. Gene profile of developed spheres was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and thyroid lineage commitment was then induced in a differentiating condition. In stem cell-derived thyrocytes, embedded in collagen to form follicles, TSH-dependent (125)iodide uptake was measured. The SP was identified as a population enriched in stem cells with typical morphology, and characteristics of self-renewal and differentiation. Nonadherent clonal spheres developed in thyroid cell cultures, displaying an expression pattern resembling that of SP cells and in response to TSH and serum these sphere cells differentiated into thyrocytes expressing PAX8, thyroglobulin, sodium iodide symporter, thyroid-stimulating hormone receptor and thyroperoxidase mRNA. And there was TSH-dependent (125)iodide uptake. It is shown first time that human thyroid contains an undescribed population of cells with SP phenotype and clonal expansion capacity. Moreover sphere culture method is developed for the isolation and proliferation of thyroid stem cell.

Paroxysmal nocturnal hemoglobinuria and concurrent JAK2V617F mutation

Paroxysmal nocturnal hemoglobinuria and concurrent JAK2V617F mutation

Flow-cytometry and cell sorting: An efficient approach to investigate productivity and cell physiology in mammalian cell factories

The performance of cell lines used for the production of biotherapeutic proteins typically depends on the number of cells in culture, their specific growth rate, their viability and the cell specific productivity (qP). Therefore both cell line development and process development are trying to (a) improve cell proliferation to reduce lag-phase and achieve high number of cells; (b) delay cell death to prolong the production phase and improve culture longevity; (c) and finally, increase qP.All of these factors, when combined in an optimised process, concur to increase the final titre and yield of the recombinant protein. As cellular performance is at the centre of any improvement, analysis methods that enable the characterisation of individual cells in their entirety can help in identifying cell types and culture conditions that perform exceptionally well. This observation of cells and their complexity is reflected by the term “cytomics” and flow cytometry is one of the methods used for this purpose. With its ability to analyse the distribution of physiological properties within a population and to isolate rare outliers with exceptional properties, flow cytometry ideally complements other methods used for optimisation, including media design and cell engineering. In the present review we describe approaches that could be used, directly or indirectly, to analyse and sort cellular phenotypes characterised by improved growth behaviour, reduced cell death or high qP and outline their potential use for cell line and process optimisation.

Interleukin-1 accounts for intrarenal Th17 cell activation during ureteral obstruction

Interleukin 17A-secreting T-helper 17 (Th17) cells are pathogenic in inflammatory kidney diseases, but their intrarenal regulation is poorly understood. In order to better define Th17 cell dynamics during interstitial inflammation, we utilized the mouse unilateral ureteral obstruction model to analyze inflammatory cell subtypes by multicolor flow cytometry and cell sorting and by effects on in vitro-generated Th17 cells. Interleukin 17A expression localized to CCR6(+)CCR4(+/-)CD4(+) T-cells and progressively increased in obstructed kidneys. The number of CCR6(+)CD4(+) T-cells increased over 10-fold by 72 h, were enriched for interleukin 17A production, and were highly proliferative based on in vivo bromodeoxyuridine incorporation. Secreted products of leukocytes isolated from obstructed kidneys enhanced the interleukin 17A production of in vitro-generated Th17 cells. This Th17-enhancing activity was identified as interleukin-1 produced by renal dendritic cells and monocytes. The in vivo validity of these findings was confirmed in mice lacking the interleulin-1 receptor and in mice treated with a recombinant interleukin-1 receptor antagonist, each of which exhibited reduced intrarenal Th17 activity compared with control mice. Thus, the inflamed kidney accumulates CCR6(+) Th17 cells that undergo activation and proliferation. Production of interleukin 1 family cytokines by resident dendritic cells and infiltrating monocytes enhances intrarenal Th17 activation in acute kidney injury.

Focus on bioinstrumentation and biotechnologies

Focus on bioinstrumentation and biotechnologies

Phosphatidyl-Serine-Positive Cells with High Sensitivity to the Alzheimer's Disease Aβ Peptides Display Distinctive Mitochondrial Characteristics

The Alzheimer's Aβ peptides exhibit cell-selective toxic effects in cultured cells and in neurons of Alzheimer's disease (AD) brains. As we previously showed ex vivo neuronal cells and cell lines consisted of subpopulations of cells characterized by differential levels of a common surface membrane target for the phosphatidyl-serine (PtdSer) specific binder annexin V and for the Alzheimer's Aβ peptidesbinding. These findings enable us to efficiently separate by flow cytometry sorting PtdSer positive cells on a variety of neuronal cell lines and ex vivo neurons and analyze their Aβ-binding affinity. We collected compelling data confirming the involvement of PtdSer as one of the surface membrane signal molecules for Aβ. This investigation used the persistent presence of PtdSer on the outer leaflet of the plasma membrane of PC12 cells to identify, sort and further culture the subpopulation of cells that are PtdSer positive (PtdSer+) and have high affinity to bind Aβ, and consequently are more likely to be harmed by Aβ. Various viability tests showed that the group of cells sorted as PtdSer+ was the most sensitive to Aβ toxic effects. The caspase3/7 activation level of the PtdSer+ subpopulation of cells was similar to one of the PtdSer negative (PtdSer-) subpopulation and the control cells. Therefore we assume, PtdSer+ cells are not undergoing apoptosis. Additionally, PtdSer+ cells have persistent higher levels of PtdSer on the outer leaflet of the plasma membrane, significantly lower levels of cytosolic ATP, lower mitochondrial mass and mitochondrial membrane potential, and slightly higher production of reactive oxidative species compared to PtdSer- and control cells. We hypothesize that all of these distinctive cellular and mitochondrial conditions are the basis for the high sensitivity to Aβ displayed by some selective cell lines and neurons.

A novel Ly6C/Ly6G‐based strategy to analyze the mouse splenic myeloid compartment

Currently, there is no standardized panel for immunophenotyping myeloid cells in mouse spleen using flow cytometry. Markers such as CD11b, CD11c, F4/80, Gr-1, Ly6C, and Ly6G have long been used to identify various splenic cell myeloid populations. Flow cytometry and fluorescence-activated cell sorting (FACS) analysis demonstrated that Ly6G/Ly6C markers are superior to Gr-1 for identifying splenic neutrophils, eosinophils, and subsets of monocytes/macrophages. Moreover, these experiments showed that F4/80 is not required for identifying these myeloid subsets and that many of the commercially available preparations of anti-F4/80 antibodies stain poorly for this antigen in spleen. Taken together, we have now developed an informative flow cytometry panel that can be combined with other cell markers to further delineate subpopulations of mouse splenic myeloid cells. This panel will be highly useful to investigators in the flow cytometry field, as there is a critical need to standardize the analysis of myeloid cell subsets.

Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

Over the last several decades, harmful algal blooms (HABs) have become a serious environmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species (Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.), were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%–91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%–12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.

Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata.

This study evaluates the potential of flow cytometry for chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes were characterized and content of chromosome peaks was determined. Peaks of chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the chromosome could be sorted with purities exceeding 95%. The remaining chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops chromosomes using sorted chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.

CD10: A tool to crack the role of stem cells in breast cancer

Keller et al. (1) highlighted the use of the CD10 and epithelial cell adhesion molecule (EpCAM) markers to discriminate basal from luminal breast epithelial subpopulations. Based on the same two markers, we have previously developed a simple, robust, and reliable flow cytometry sorting technique to separate luminal populations cleanly from the rest of precursor cells (2). The various functional assays we used have established that EpCAM+ separates luminal progenitors from other epithelial populations present in the CD10+ fraction (stem cells, early and bipotent progenitors, and later myoepithelial lineage cells). In addition, we have identified CD10-enzyme as an easy-to-use cell surface tool to isolate sphere-forming cells from a variety of other normal human adult tissues. Despite the similar results on the characterization of the two epithelial subpopulations, there is an apparent discrepancy between our findings, which suggest that this sphere-forming ability is linked to CD10+ cells (2), and the paper of Keller et al. (1), which related this property to EpCAM+ cells. This difference is likely attributable to the sorting strategy, the culture media, and the criteria used for mammosphere scoring. As noted by Keller et al. (figure S2C and corresponding legend of ref. 1), they defined mammosphere based on physical criteria that are influenced by the processing protocol applied. In our case, we have considered as mammospheres a group of cells (over 100) growing as floating, dense, and compact aggregates and surrounded by a membrane-like structure that makes them look very much like embryonic bodies. Regardless of these differences, both our results and those of Keller et al. (1) suggest a potential value of CD10 expression in mammary stem cells. We have also demonstrated the key role of CD10 not only as a cell surface marker but as a full functional regulator of normal mammary progenitor cells. Indeed, our work also indicates that a gradient of biological peptides generated by CD10-enzyme cleavage is involved, with β1-integrins, in maintaining stem cells/early progenitors and regulating their conversion to luminal progenitors. This illustrates the dual role of CD10 in this system as already pointed out by Kenny et al. (3) years ago. In addition, different data in the literature suggest that CD10 and its enzymatic functions might act as a stem cell regulator in a number of cellular compartments (reviewed in 4).