Development of a novel explant culture method for the isolation of mesenchymal stem cells from human breast tumor

Background: Mesenchymal stem cells (MSCs) were isolated from various sources, including various types of tumors. However choosing an appropriate isolation method is an important step in obtaining cells with optimal quality and yield in companion with economical considerations. The purpose of this study was to isolate more pure MSCs from human breast tumor tissue by a modified explant culture method.

Methods and Materials: The tumor tissues (n = 8) were cut into 1 to 3-mm cube-like pieces (explant). Each explant was placed in a well of 24-well format plates, cultured in Dulbecco’s Modified Eagle’s medium (DMEM), and maintained at 37°C with 5% humidified incubator. Morphological phenotypes of the cells were surveyed by an inverted microscope and wells with rather homogenous fibroblast-like morphology cell were considered as positive and selected for more expansion and characterization.

Results: A total of 185 wells, 63.7% of wells were positive that were chosen for expansion. Flowcytometry analysis demonstrated that isolated cells were positive for CD73, CD44, CD29, CD105, and CD90 but negative for CD11b, CD45, CD34, and HLA?DR. In addition, cells possessed the capability of multipotential differentiation into osteoblasts and adipocytes.     

一种新型肠上皮干细胞分离方法的建立

目的 探讨密度梯度离心法作为一种新型肠上皮干细胞分离方法的可行性.方法 通过腹腔注射大剂量5-氟尿嘧啶(5-FU)制备肠黏膜严重损伤小鼠模型,取出损伤的肠黏膜消化成单细胞悬液,利用Percoll密度梯度离心法分离细胞体积大小不同的细胞群.HE染色观察各群细胞形态特征,免疫细胞化学及RT-PCR检测各群细胞musashi-1(msi-1)的表达.结果 分离的大部分细胞位于50%和70%密度梯度的Percoll分离液体层,50%密度梯度分离液体层中的细胞符合干细胞的形态特征,免疫细胞化学检测其msi-1表达阳性率约为93%,RT-PCR显示该群细胞msi-1 mRNA表达较强.结论 建立了一种简便、有效的分离具有活性的肠上皮干细胞的方法.     

Varicella-zoster virus: isolation and propagation in human melanoma cells at 36 and 32 degrees C.

Cell lines derived from human malignant melanoma tumors are susceptible to infection with varicella-zoster virus (VZV). Within 5 days after inoculation of vesicular fluid, cytopathic changes appeared in melanoma cell monolayer cultures that were incubated at either 36 or 32 degrees C. The VZV isolates at the two temperatures were serially propagated by passage of trypsin-dispersed infected cells. A plaque assay was developed utilizing melanoma cell monolayers overlaid with nutrient medium containing carboxymethylcellulose. By this assay method, the growth cycle of a VZV isolate propagated at 36 degrees C was studied and compared with that of another VZV isolate grown at 32 degrees C. With equivalent infected-cell inocula at a ratio on one inoculum cell to eight uninfected cells, the yield of cell-free virus at an incubation temperature of 32 degrees C was slightly higher than at 36 degrees C, although the peak occurred 60 h, rather than 36 h, postinfection. It was also found that the titer of low-passage VZV propagated at 36 degrees C was 0.5 to 1 log higher when assayed at 32 degrees C rather than at 36 degrees C.     

Herpes simplex virus DNA isolation from infected cells with a novel procedure.

Herpes Simplex Virus type 1-infected cells were extracted in the presence of 0.25% Triton X-100-0.2 M NaCl. Viral DNA associated with proteins was found in the supernatant after low-speed centrifugation. Only viral DNA was recovered by this procedure, as shown by CsCl density analysis after deproteinization. Full-length viral DNA molecules were observed in the electron microscope.     

The isolation and differentiation of human adipose-derived stem cells using membrane filtration

Human adipose-derived stem cells (hADSCs) were purified from a suspension of human adipose tissue cells (stromal vascular fraction) by the conventional culture method and by membrane filtration through polyurethane (PU) foam membranes. hADSCs can be obtained from a suspension of human adipose tissue cells using the membrane filtration method in less than 30?min, whereas the conventional culture method requires 5-12 days. hADSCs that express the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the recovery solution from the PU membranes; no hADSCs were isolated in the permeate. After filtration, the cells expressing the mesenchymal stem cell markers were 3-4.5 times more concentrated than in the initial suspension of human adipose tissue cells, with the level of concentration depending on the surface modification of the PU membrane. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the recovery solutions, whereas CD34(+) cells could not be purified by the conventional culture method. The hADSCs in the recovery solution demonstrated a superior capacity for osteogenic differentiation than did the cells in the suspension of human adipose tissue cells. These results suggested that the hADSCs with the capability for osteogenic differentiation adhered to the PU membranes.     

Autologous graft-versus-host disease: a novel approach for antitumor immunotherapy.

Autologous bone marrow transplantation (BMT) is a therapeutic option for the treatment of lymphohematopoietic malignancies and solid tumors. Despite the intensive cytoreductive therapy, however, the rates of tumor recurrence after autologous BMT remain unacceptably high. Current studies suggest that the administration of cyclosporine (CsA) disrupts the reconstitution of self-tolerance following autologous BMT leading to the induction of an autoimmune graft-versus-host disease (GVHD). Studies in a rat tumor model and preliminary clinical trials suggest that this autoimmune or autologous GVHD provides a significant antitumor effect. Moreover, the antitumor effect of autologous GVHD can be enhanced by administration of gamma-interferon, which upregulates the antigen recognized by the autoreactive effector cells of autologous GVHD. These studies indicate that the induction of an autoimmune GVHD after autologous BMT may be a promising immunotherapeutic approach for treatment of certain neoplastic diseases.     

用糖基化磷脂酰肌醇B7-1锚定肿瘤细胞膜进行免疫治疗

目的 研制抗肿瘤免疫治疗的新疫苗.方法 用蛋白转化法将B7-1锚定在肿瘤细胞膜上,免疫C57BL-6小鼠后,一组小鼠取脾细胞进行T细胞扩增和细胞毒T淋巴细胞(CTL)功能检测,另一组小鼠进行肿瘤细胞接种试验.结果 用GPI-B7-1修饰的肿瘤细胞膜免疫小鼠后诱发了肿瘤特异性T细胞扩增和CTL.且该疫苗有一定的保护小鼠免受肿瘤细胞侵袭的作用.结论 GPI蛋白转化法为人类抗肿瘤免疫治疗提供了新的、有效的修饰肿瘤细胞膜的方法.     

Electrophoretic isolation of extrachromosomal DNA from tumor cells

Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.     

Focal degeneration of basal cells and the resultant auto-immunoreactions: a novel mechanism for prostate tumor progression and invasion

The development of human prostate cancer is believed to be a multistep process, progressing sequentially from normal, to hyperplasia, to prostatic intraepithelial neoplasia (PIN), and to invasive and metastatic lesions. High grade PIN has been generally considered as the direct precursor of invasive lesions, and the progression of PIN is believed to be triggered primarily, if not solely, by the overproduction of proteolytic enzymes predominately by cancer cells, which result in the degradation of the basement membrane. These theories, however, are hard to reconcile with two main facts: (1) only about 30% untreated PIN progress to invasive stage, while none of the current approaches could accurately identify the specific PIN or individuals at greater risk for progression, and (2) results from recent world-wide clinical trials with a wide variety of proteolytic enzyme inhibitors have been very disappointing, casting doubt on the validity of the proteolytic enzyme theory. Since over 90% of prostate cancer-related deaths result from invasion-related illness and the incidence of PIN could be up to 16.5-25% in routine or ultrasound guided prostate biopsy, there is an urgent need to uncover the intrinsic mechanism of prostate tumor invasion. Promoted by the facts that the basal cell population is the source of several tumor suppressors and the absence of the basal cell layer is the most distinct feature of invasive lesions, our recent studies have intended to identify the early alterations of basal cell layers and their impact on tumor invasion using multidisciplinary approaches. Our studies revealed that a subset of pre-invasive tumors contained focal disruptions (the absence of basal cells resulting in a gap greater than the combined size of at least three epithelial cells) in surrounding basal cell layers. Compared to their non-disrupted counterparts, focally disrupted basal cell layers had several unique features: (1) significantly lower proliferation; (2) significantly lower p63 expression; (3) significantly higher apoptosis; and (4) significantly higher leukocyte infiltration and stromal reactions. Compared to their counterparts distant from focal disruptions or overlying non-disrupted basal cell layers, epithelial cells overlying focal basal cell layer disruptions showed the following unique features: (1) significantly higher proliferation; (2) significantly higher expression of cell cycle control-, cell growth-, and stem cell-related genes; and (3) physical continuity with adjacent invasive lesions. Together, these findings suggest that focal basal cell layer disruptions could substantially impact the molecular profile and biological presentations of the overlying epithelial cells. Based on these and other findings, we have proposed that prostate tumor invasion is triggered by a localized degeneration of aged or injured basal cells and the resultant auto-immunoreactions. Our hypothesized steps for prostate tumor invasion include the following: (1) due to inherited or environmental factors, some patients contained cell cycle control- and renewal-related defects in the basal cell population that cause elevated basal cell degenerations; (2) the degradation products of degenerated basal cells or diffusible molecules of the overlying epithelial cells attract leukocyte infiltration; (3) leukocytes discharge their digestive enzymes upon the direct physical contact, resulting in a focal disruption in the basal cell layer, which leads to several focal alterations: (a) a focal loss of tumor suppressors and paracrine inhibitory function; (b) a focal increase of the permeability for growth-required nutrients and oxygen; (c) a focal increase of growth factors; (d) direct physical contact between epithelial and stromal cells; and (e) the exposure of the overlying epithelial cells directly to the stromal tissue fluid. These alterations individually or collectively stimulate or favor a clonal proliferation and stromal invasion of tumor progenitor or stem cells. Our hypothesis differs from the traditional theories in several aspects, including the triggering factor for the initiation of tumor invasion, the stage of tumor invasion, the cellular origin of invasive lesions, the significance of immunoreactive and stromal cells, and the potential approaches for early detection, treatment, and prevention of invasion. Our hypothesis represents a novel in vivo model as to the cellular mechanism leading to prostate tumor invasion. If confirmed, it could lead to a new direction to search for more effective approaches to combat prostate cancer. It could also have an immediate impact on patient care through improved pathologic evaluation of prostate tumor biopsies. More importantly, our hypothesis might be applicable, and significantly impact the detection, treatment, and prevention of other epithelium-derived tumors.     

A novel mechanism for regulating clonal propagation of mouse ES cells

Self-renewal and differentiation of embryonic stem (ES) cells are controlled by the combinatorial action of extracellular signals and regulation of gene expression. For characterizing the entire molecular mechanism governing these events, we first established a feeder- and serum-free culture system in which mouse ES cells could propagate in clonal density in keeping with proper pluripotency. Supplementation of peptide hormones such as adrenocorticotropic hormone (ACTH) is required to remove serum, and the key event in this phenomenon may be the inhibition of the adenylyl cyclase (AC) activity, as it replaces the effect of these peptides. Because ES cells themselves produce the same activity, the finding suggests a novel mechanism in which activation of AC restricts clonal propagation of pluripotent stem cells.     

The use of a combined enrichment–filtration technique for the isolation of Campylobacter spp. from clinical samples

A combined non-selective enrichment-filtration technique was investigated for the isolation of Campylobacter spp. from clinical samples. In total, 479 samples were tested by direct culture, enrichment subculture and enrichment-filtration. The enrichment-filtration technique was used with both selective and non-selective media. Direct culture and enrichment subculture yielded 13 and seven isolates, respectively, while enrichment-filtration yielded 18 and 14 isolates on selective and non-selective agar, respectively. Thus, the combination of enrichment-filtration with selective agar produced a 38.5% increase in the number of isolates (p <0.05). All isolates were identified as Campylobacter jejuni.     

A novel approach for immunization,screening and characterization of selected scFv libraries using membrane fractions of tumor cells

Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.     

Thymic dendritic cells and B cells: isolation and function.

The thymus is the primary organ in which T cells undergo rearrangement of T cell receptor alpha and beta genes, positive selection for affinity to self MHC products, and elimination (negative selection) of reactivity to self antigens. These events require an interaction of the developing T cell with other cell types in the thymus. The latter include epithelial cells, macrophages, dendritic cells, and the recently described thymic B cells the majority of which are CD5+. Here we review the identification and isolation of thymic dendritic cells and CD5+ B cells. We consider phenotype, ontogeny, and function, including possible contributions to the induction of self tolerance. Thymic dendritic cells are similar to spleen dendritic cells, but are larger and exhibit a few differences in phenotype. Dendritic cells from both organs are equally potent accessory cells for the MLR and lectin-induced, T cell proliferation. Thymic dendritic cells have higher levels of Fc receptors and support anti-CD3 dependent mitogenesis. Thymic CD5+ B cells share phenotypic features with peritoneal CD5+ B cells. However thymic B cells neither proliferate nor form antibody producing cells in response to the stimulation with LPS or anti-IgM plus IL-4, but do respond to stimulation with MHC class II-restricted helper T cells. Thymic dendritic cells and CD5+ B cells both appear at a similar time in ontogeny, about 14 d of gestation, which is the time T cell differentiation begins to take place. Dendritic cells from spleen, which are potent activators for peripheral T cells, are also potent inactivators for thymic-derived cytotoxic T cells. A correlation between reactivity to MIs products and the expression of TCR-V beta genes is well documented, and B cells are the primary APC for this antigen. Therefore, thymic CD5+ B cells may be a good tool for the investigation of tolerance to M1s products.     

A novel approach for reliable microarray analysis of microdissected tumor cells from formalin-fixed and paraffin-embedded colorectal cancer resection specimens

We present a novel approach for microarray analysis of RNA derived from microdissected cells of routinely formalin-fixed and paraffin-embedded (FFPE) cancer resection specimens. Subsequent to RNA sample preparation and hybridization to standard GeneChips (Affymetrix), RNA samples yielded 36.43 ± 9.60% (FFPE), 49.90 ± 4.43% (fresh-frozen), and 53.9% (cell line) present calls. Quality control parameters and Q-RT-PCR validation demonstrated reliability of results. Microarray datasets of FFPE samples were informative and comparable to those of fresh-frozen samples. A systematic measurement difference of differentially processed tissues was eliminated by a correction step for comparative unsupervised data analysis of fresh-frozen and FFPE samples. Within FFPE samples, unsupervised clustering analyses clearly distinguished between normal and malignant tissues as well as to further separate tumor samples according to histological World Health Organization (WHO) subtypes. In summary, our approach represents a major step towards integration of microarrays into retrospective studies and enables further investigation of the relevance of microarray analysis for clinico-pathological diagnostics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls

Detection of circulating tumor cells (CTCs) morphologically may be a promising new approach in clinical oncology. We tested the reliability of a cytomorphologic approach to identify CTCs: 808 blood samples from patients with benign and malignant diseases and healthy volunteers were examined using the isolation by size of epithelial tumor cell (ISET) method. Cells having nonhematologic features (so-called circulating nonhematologic cells [CNHCs]) were classified into 3 categories: CNHCs with malignant features, CNHCs with uncertain malignant features, and CNHCs with benign features. CNHCs were found in 11.1% and 48.9% of patients with nonmalignant and malignant pathologies, respectively (P < .001). CNHCs with malignant features were observed in 5.3% and in 43.1% of patients with nonmalignant and malignant pathologies, respectively. Cytopathologic identification of CTCs using the ISET method represents a promising field for cytopathologists. The possibility of false-positive diagnosis stresses the need for using ancillary methods to improve this approach.     

Modified selective medium for isolation of Campylobacter spp. from feces: comparison with Preston medium, a blood-free medium, and a filtration system.

Our previously described (H. Goossens, M. De Boeck, and J. P. Butzler, Eur. J. Clin. Microbiol. 2:389-393, 1983) selective medium, consisting of cefoperazone (15 mg/liter), rifampin (10 mg/liter), colistin (10,000 IU/liter), and amphotericin B (2 mg/liter) (medium M1), for the isolation of Campylobacter jejuni and Campylobacter coli from stool specimens was modified as follows: cefoperazone (30 mg/liter), rifampin (10 mg/liter), and amphotericin B (2 mg/liter) (medium M2). A comparative study of the isolation of Campylobacter spp. from stool specimens was carried out with medium M1; medium M2; a selective blood-free medium consisting (per liter) of charcoal (4 g), ferrous sulfate (0.25 g), sodium pyruvate (0.25 g), casein hydrolysate (3 g), sodium deoxycholate (1 g), nutrient broth no. 2 (25 g), agar (12 g), and cefoperazone (32 mg) (medium M3); and Preston medium containing (per liter) trimethoprim (10 mg), rifampin (10 mg), polymyxin B (5,000 IU), and cycloheximide (100 mg) (medium M4). We also included a filtration system in which membrane filters were applied directly to the surface of the nonselective blood-free medium distributed in small petri dishes. A total of 5,276 stool specimens were tested: 2,788 stool specimens were tested on M1 and M3 in study 1; 2,488 stool specimens were inoculated on the four selective media in study 2, and the last 986 specimens of the 2,488 were tested in parallel with the filtration system. In study 2, 128 Campylobacter strains were isolated from 126 different patients; 85.0, 88.3, 82.5, and 66.6% of these strains were isolated on M1, M2, M3, and M4, respectively. No contaminating fecal flora was found on 65.4, 70.7, 62.4, and 40.3% of the M1, M2, M3, and M4 plates, respectively. Furthermore, C. coli was found to be more susceptible to antibiotics present in the selective media, particularly colistin and polymyxin B, than was C. jejuni. We therefore recommend M2 for the isolation of Campylobacter spp. Finally, the filtration method was found to be easy and cheap; although the sensitivity was low, this method allowed the isolation of new Campylobacter spp. which seem to be associated with diarrhea.     

PhenoChipping of psychotic disorders: a novel approach for deconstructing and quantitating psychiatric phenotypes.

Psychiatric phenotypes as currently defined are primarily the result of clinical consensus criteria rather than empirical research. We propose, and present initial proof of principle for, a novel approach to characterizing psychiatric phenotypes. We have termed our approach PhenoChipping, by analogy with, and borrowing paradigms and tools from, gene expression microarray studies (GeneChipping). A massive parallel profiling of cognitive and affective state is done with a PhenoChip composed of a battery of existing and new quantitative psychiatric rating scales, as well as hand neuromotor measures. We present preliminary data from 104 subjects, 72 with psychotic disorders (bipolar disorder-41, schizophrenia-17, schizoaffective disorder-14), and 32 normal controls. Microarray data analysis software and visualization tools were used to investigate: 1. relationships between phenotypic items ("phenes"), including with objective motor measures, and 2. relationships between subjects. Our analyses revealed phenotypic overlap among, as well as phenotypic heterogeneity within, the three major psychotic disorders studied. This approach may be useful in helping us move beyond current diagnostic classifications, and suggests a combinatorial building-block (Lego-like) structure underlies psychiatric syndromes. The adaptation of microarray informatic tools for phenotypic analysis readily facilitates direct integration with gene expression profiling of lymphocytes in the same individuals, a strategy for molecular biomarker identification. Empirically derived clusterings of (endo)phenotypes and of patients will better serve genetic, pharmacological, and imaging research, as well as clinical practice.     

Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.     

High frequency pressure propagation in viscoelastic tubes: a new experimental approach.

A new method for estimating the high-frequency characteristics of wave propagation (phase velocity and attenuation per wavelength) in hydraulic lines is presented in this work. It consists of measuring the ratio of pressure amplitudes at two distinct sections of an occluded tube at different frequencies, and minimizing the difference between the experimental pattern and the theoretical one predicted on the basis of the transmission line theory. In this work the method is used for estimating the high frequency propagation characteristics of two different latex tubes. The values obtained are then compared with those provided by the more traditional three-point pressure method. The results of our trials demonstrate that the new method furnishes reliable estimations of the asymptotic values of phase velocity and attenuation per wavelength, provided the frequencies used during the experiment are sufficiently high. Moreover, the method turns out quite robust as to the influence of noise and possible measurement errors. For this reason it seems particularly suitable for studying wave propagation under difficult experimental conditions, such as those met with when measurements are performed on blood vessels in vivo. Finally, some discrepancies between our experimental results and the predictions of the transmission line theory are pointed out, and their possible origin examined.