Structurally distinct LewisX glycans distinguish subpopulations of neural stem/progenitor cells

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.     

Defining the heterogeneity of skeletal muscle‐derived side and main population cells isolated immediately ex vivo

Myoblast transfer therapy for Duchenne muscular dystrophy (DMD) largely fails due to cell death and inability of transplanted cells to engraft in diseased muscles. One method attempting to enrich for cell subpopulations is the Hoechst 33342 dye exclusion assay, yielding a side population (SP) thought to be progenitor enriched and a main population (MP). However, in vitro and transplant studies yielded inconsistent results relative to downstream progeny. Cell surface markers expressed by skeletal muscle‐derived MP and SP cells have not been fully characterized directly ex vivo. Using flow cytometry, MP and SP cells were characterized based on their expression of several well‐accepted progenitor cell antigens. Both the MP and SP populations are heterogeneous and overlapping in the cells they contain. The percentages of cells in each population vary with species and specific muscle examined. MP and SP populations contain both satellite and multipotent progenitor cells, based on expression of CD34, Sca‐1, Pax7, and M‐cadherin. Thus, isolation using this procedure cannot be used to predict downstream differentiation outcomes, and explains the conflicting literature on these cells. Hoechst dye also results in significant mortality of sorted cells. As defined subpopulations are easily obtained using flow cytometry, sorting immediately ex vivo based on accepted myogenic precursor cell markers will yield superior results in terms of cell homogeneity for transplantation therapy. J. Cell. Physiol. 222: 676–684, 2010. © 2009 Wiley‐Liss, Inc.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

Synchronized human diploid fibroblasts: progression capabilities of a subpopulation that fails to keep pace with the predominant, rapidly dividing cohort of cells

Highly synchronized cultures of HSF-55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA-specific stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S-phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early- and late-replicating species of human DNA.     

Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter

The disease diabetes mellitus arises as a consequence of a failure of the beta-cells in the islets of Langerhans of the pancreas to produce insulin in the amounts required to meet the needs of the body. Whole pancreas or islet transplants in patients with severe diabetes effectively restore insulin production. A lack of availability of donor pancreata requires the development of alternative sources of islets such as the ex vivo culture and differentiation of stem/progenitor cells. Earlier we discovered multipotential progenitor cells in islets isolated from adult human pancreata that express the neural stem cell marker nestin: nestin-positive islet-derived progenitor cells (NIPs). Recently it was shown that the exclusion of the Hoechst 33342 dye, which defines the pluripotential side population (SP) of hematopoietic stem cells, is mediated by the ATP-binding cassette transporter, ABCG2. Here we report that the human islet-derived NIPs contain a substantial subpopulation of SP cells that co-express ABCG2, MDR1, and nestin. Thus NIPs may be a potential source of adult pluripotential stem/progenitor cells useful for the production of islet tissue for transplantation into diabetic subjects.     

ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate

The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the “side population” (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145, and RWPE-1, but not in the BPH-1, LAPC-4, or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) human benign prostate tissue, and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.     

Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.

BACKGROUND: Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. METHODS: Hoechst 33342 staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. RESULTS: Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst 33342 staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst 33342 red fluorescence pattern. CONCLUSIONS: Hoechst 33342 staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis.     

FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen

Salmonella can cause a typhoid fever like disease in genetically susceptible mice. In contrast to in vitro cell culture models, most host cells in infected tissues contain only one or two Salmonella, but a small subpopulation of infected host cells contains many Salmonella. It has been proposed that these various subpopulations have differential relevance during infection but to test this, methods for isolating such Salmonella will be required. We developed a method to purify differentially infected host cells by flow cytometry using Salmonella expressing the green fluorescent protein (GFP). Critical parameters for this method were sufficient GFP expression to detect infected cells against strong host cell autofluorescence, and low variation in GFP content of individual Salmonella. We evaluated more than hundred different GFP-constructs but only one single-copy chromosomal P(sifB)-gfp fusion met these criteria and enabled differentiation of weakly and highly infected cells based on total GFP fluorescence. Confocal microscopy of sorted cells confirmed the successful separation of these subpopulations. In addition to isolation of infected cells, our method also enabled enumeration of the subpopulations and phenotypic characterization by staining with antibodies to surface markers. Surprisingly, a small subpopulation of highly infected host cells contained the majority of Salmonella but based on MHC II and ICAM I expression, this subpopulation was not more strongly activated than weakly infected cells. Our method will facilitate future characterization of the different subpopulations and the identification of bacterial and host factors that control Salmonella load and proliferation in vivo.     

Enrichment and characterization of mouse putative epidermal stem cells

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.     

Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies

Multivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol-fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine-conjugated wheat germ agglutinin (WGA), and a fluorescein-labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer-based multivariable pulse-height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S-phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.     

Identification of superficial zone articular chondrocyte stem/progenitor cells

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.     

Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.     

Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.     

Oxygen enhancement ratio in V79 spheroids

Chinese hamster V79 spheroids were stained with a nontoxic fluorescent stain, Hoechst 33342, which penetrates slowly into the spheroids. Single cells from these spheroids were then sorted by a fluorescence-activated cell sorter according to staining intensity (and therefore position in the spheroids). Flow cytometry characterization of the various cell subpopulations indicated that the innermost cells were more radiosensitive than expected on the basis of cell cycle position or cell thiol content. However, comparison of the radiosensitivities of cells sorted from equivalent depths from completely aerobic or anoxic V79 spheroids indicated that the oxygen enhancement ratio remained remarkably constant at 2.7 +/- 0.2 through the spheroid.     

Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.     

Netrin1 is required for neural and glial precursor migrations into the olfactory bulb

Netrin1 (NTN1) deficiency in mouse brain causes defects in axon guidance and cell migration during embryonic development. Here we show that NTN1 is required for olfactory bulb (OB) development at late embryogenesis and at early postnatal stages to facilitate the accumulation of proper numbers of granular and glomerular neuron subtypes and oligodendrocytes into the OB. In addition to the analysis of Ntn1−/− mice we made tissue and neurosphere cultures to clarify the role of NTN1 in the anterior forebrain. We propose that a subset of neural progenitors/precursors requires NTN1 to efficiently enter the rostral migratory stream to migrate into the OB. The analysis of postnatal Ntn1−/− OBs revealed a reduction of specific types of interneurons which have been shown to originate from particular subregions of the lateral ventricle walls. Based on Ntn1 expression in ventral parts of the ventricle walls, we observed a decrease in the mainly ventrally derived type II interneurons that express calcium-binding proteins calretinin and calbindin. Instead, no change in the numbers of dorsally derived tyrosine hydroxylase expressing interneurons was detected. In addition to the specific reduction of type II interneurons, our results indicate that NTN1 is required for oligodendroglial migration into the OB. Furthermore, we characterised the Ntn1 expressing subpopulation of neurosphere-forming cells from embryonic and adult brain as multipotent and self-renewing. However, NTN1 is dispensable for the proliferation of neurosphere forming progenitor cells and for their differentiation.     

Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells

Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.