Unsorted Control Research Articles

Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

IgA-Biome Profiles Correlate with Clinical Parkinson's Disease Subtypes.

Parkinson's disease is a heterogeneous neurodegenerative disorder with distinctive gut microbiome patterns suggesting that interventions targeting the gut microbiota may prevent, slow, or reverse disease progression and severity. Because secretory IgA (SIgA) plays a key role in shaping the gut microbiota, characterization of the IgA-Biome of individuals classified into either the akinetic rigid (AR) or tremor dominant (TD) Parkinson's disease clinical subtypes was used to further define taxa unique to these distinct clinical phenotypes. Flow cytometry was used to separate IgA-coated and -uncoated bacteria from stool samples obtained from AR and TD patients followed by amplification and sequencing of the V4 region of the 16 S rDNA gene on the MiSeq platform (Illumina). IgA-Biome analyses identified significant alpha and beta diversity differences between the Parkinson's disease phenotypes and the Firmicutes/Bacteroides ratio was significantly higher in those with TD compared to those with AR. In addition, discriminant taxa analyses identified a more pro-inflammatory bacterial profile in the IgA+ fraction of those with the AR clinical subclass compared to IgA-Biome analyses of those with the TD subclass and with the taxa identified in the unsorted control samples. IgA-Biome analyses underscores the importance of the host immune response in shaping the gut microbiome potentially affecting disease progression and presentation. In the present study, IgA-Biome analysis identified unique proinflammatory microbial signature in the IgA+ fraction of those with AR that would have otherwise been undetected using conventional microbiome analysis approaches.

Chondrogenic differentiation of mouse CD105+ stem/progenitor cells on amino-group-functionalized biosilica-hydrogel scaffolds

Mesenchymal stem/progenitor cells (MSPCs) are critical for tissue regeneration. Moreover, the CD105 antigen identifies early MSPCs with increased chondrogenic differentiation ability. We hypothesized that amine-(NH2)-functionalized biosilica incorporating hydrogel scaffolds, seeded with mCoSPCs105+ would contribute to creating tissue-engineered scaffolds, capable of de novo cartilage synthesis. Scaffolds were characterized by water uptake, lysozyme degradation, axial compression, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. Differentiation stimulus of scaffold functionalization was evaluated using Alcian blue staining. Cartilage-forming abilities of mCoSPCs105+ were evaluated using Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Biosilica particle incorporation into scaffolds resulted in increased water uptake capacity and compression force withstanding abilities. Amine-(NH2)-group functionalization of biosilica led to significantly increased stem cell differentiation potential, by Alcian blue staining, in the first 3 weeks. Scaffold attachment and viable cell proliferation were observed for 6 weeks under chondrogenic differentiation. Downregulation of Runx2, an increase of Col10a1, Ihh, and maintenance of Sox9, was seen under these culture conditions. mCoSPCs105+ gene expression pattern was defined by the significant upregulation of Col1a1, Col2a1, Prg4, and Agc-1 over 6 weeks of incubation compared to the unsorted control. Immunostaining of cell-seeded scaffolds revealed significantly higher secretion of proteins relevant to cartilage extracellular matrix. The preselecting of CD105+ phenotype in MSPCs may enhance tissue regeneration of fibrocartilage and biosilica nanoparticles may be a beneficial additive in tissue engineering of scaffolds.

Abstract 5324: Isolation of small number of tumor cells for proliferation studies using DEPArray technology

Abstract Cancer is universally recognized as a heterogeneous disease. The different cell clones present in a tumor can display distinct molecular and phenotypic profiles, which may influence proliferation capabilities, metastatic potential and response to therapy. To better understand cancer progression and possibly design effective therapeutic strategies, it is fundamental to design cell-sorting technologies able to isolate rare, viable tumor cell clones from tissue or liquid biopsies. Fluorescence-activated cell sorting (FACS) is the most widely used technique to isolate cell populations from heterogeneous samples. However, FACS analysis presents technical limitations when applied to rare cell populations and/or samples with low cellularity and limitations associated with the low total number of target cells in the sample. DEParray™ PLUS (DA) is a highly automated image-based cell sorting technology capable of isolating pure, single and rare cells from heterogeneous samples. Here we evaluate the capabilities of the DA system to isolate a small number of viable tumor cells and assess the proliferation capacity of DA-sorted cells in comparison with FACS-sorted cells. Human Jurkat T lymphoblast cell line were cultured in RPMI medium and a double staining procedure was optimized to label cells with CellTraceFarRed (CTFR), division tracking dye and Live/Dead (LD) Fixable marker. CTFR/LD staining was performed for each experiment and, at days 0 and 7 of culture, CTFR and LD fluorescences were analyzed by means of BD CANTO II Flow cytometry. At day 0, FACS analysis discriminate dead from live cells, while at day 7 evaluate cell proliferation and viability after 7 days in culture. Comparison tests were carried out following two different recovery schemes: 1 recovery of 100 CTFR+/LD- cells pool and 10 recoveries of 10 CTFR+/LD- cells pool using DA Plus and BD FACSAria Fusion. For each experiment, the results were compared to cells seeded manually, which represent our unsorted control. When comparing the results obtained with a 100-cells pool, no significant difference in cell viability and proliferation were observed between DA-sorted and FACSAria-sorted cells after 7 days in culture. On the other hand, the 10 cells pool sorted with DA PLUS showed a significantly higher viability and proliferation rate compared to those sorted with FACSAria at day 7. Moreover, cells sorted by DA-technology showed less variation between experiments, highlighting a more robust performance when sorting a small number of cells. Overall, the results confirm the unique capability of the DA system to sort a small number of cells, which maintain a higher cell viability and proliferation capacity compared to those sorted with FACS. This opens up the possibility to isolate rare tumor cell clones for functional studies that can help to better understand tumor progression and response to therapy. Citation Format: Maximilian Sergio, Chiara Rossi, Valeria Stivani, Ilaria Maffei, Marco Rossi, Gianpiero Parisi, Gianni Medoro. Isolation of small number of tumor cells for proliferation studies using DEPArray technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5324.

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

Sorting cells alters their redox state and cellular metabolome

A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP+, and NAD+ to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.

Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a control. Sperm binding to oviduct cells was reduced by more than half in the three sorted samples when compared to the control sperm that were not sorted. The percentage of sperm that were motile 24 h after sorting was also decreased significantly in each of the sorted sample groups when compared to the unsorted control. In contrast, sorting did not decrease the percentage of sperm that bound purified soluble glycans or the location on sperm to which they bound. There was also no difference in sperm acrosome status among the four groups. In summary, sorting reduced sperm binding to the complex matrix around oviductal cell aggregates but sperm binding to purified soluble oviduct glycans was not affected. The requirement for higher affinity and motility to bind glycans immobilized on oviduct cells may explain this difference. The reduction in sperm fertility observed following sex-sorting may be explained partially by a reduced or altered ability to bind to the oviduct epithelium.

Label-Free, High-Throughput Purification of Satellite Cells Using Microfluidic Inertial Separation.

Skeletal muscle satellite cells have tremendous therapeutic potential in cell therapy or skeletal muscle tissue engineering. Obtaining a sufficiently pure satellite cell population, however, presents a significant challenge. We hypothesized that size differences between satellite cells and fibroblasts, two primary cell types obtained from skeletal muscle dissociation, would allow for label-free, inertial separation in a microfluidic device, termed a "Labyrinth," and that these purified satellite cells could be used to engineer skeletal muscle. Throughout tissue fabrication, Labyrinth-purified cells were compared with unsorted controls to assess the efficiency of this novel sorting process and to examine potential improvements in myogenic proliferation, differentiation, and tissue function. Immediately after dissociation and Labyrinth sorting, cells were immunostained to identify myogenic cells and fibroblast progenitors. Remaining cells were cultured for 14 days to form a confluent monolayer that was induced to delaminate and was captured as a 3D skeletal muscle construct. During monolayer development, myogenic proliferation (BrdU assay on Day 4), differentiation and myotube fusion index (α-actinin on Day 11), and myotube structural development (light microscopy on Day 14) were assessed. Isometric tetanic force production was measured in 3D constructs on Day 16. Immediately following sorting, unsorted cells exhibited a myogenic purity of 39.9% ± 3.99%, and this purity was enriched approximately two-fold to 75.5% ± 1.59% by microfluidic separation. The BrdU assay on Day 4 similarly showed significantly enhanced myogenic proliferation: in unsorted controls 47.0% ± 2.77% of proliferating cells were myogenic, in comparison to 61.7% ± 2.55% following purification. Myogenic differentiation and fusion, assessed by fusion index quantification, showed improvement from 82.7% ± 3.74% in control to 92.3% ± 2.04% in the purified cell population. Myotube density in unsorted controls, 18.6 ± 3.26 myotubes/mm2, was significantly enriched in the purified cell population to 33.9 ± 3.74 myotubes/mm2. Constructs fabricated from Labyrinth-purified cells also produced significantly greater tetanic forces (143.6 ± 16.9 μN) than unsorted controls (70.7 ± 8.03 μN). These results demonstrate the promise of microfluidic sorting in purifying isolated satellite cells. This unique technology could assist researchers in translating the regenerative potential of satellite cells to cell therapies and engineered tissues.

A new method for gene expression analysis of pure lymphocytes recovered from heterogeneous fixed mouse tissue.

e23089 Background: Tumor infiltrating lymphocytes (TILs) are biomarkers that play a critical role in cancer diseases, including differential diagnosis, determination of prognosis, prediction of response to treatment, and evaluation of disease progression. Gene expression analysis in TILs derived from fresh tissue may not accurately depict the gene profile of the tissue microenvironment as it can change aggressively during lymphocyte isolation and RNA extraction. In addition, tissue sample size can limit the isolation of TILs with current technologies. In this study, we demonstrate the use of the DEPArray™platform to isolate pure populations of lymphocytes from a fixed mouse tissue for RNA analysi. Methods: Mouse splenocytes were activated in vitro with anti-CD3 and -CD28 for 72hs. Cells were harvested, fixed with 2% paraformaldehyde (PFA) for 20 min at RT, and stained for either CD4 or CD8 expression. Gene expression analysis of CD45, ADORA2A, GLS and GAPDH was performed in CD4+ and CD8+ DEPArray™sorted cells using the TaqMan PreAmp Cells-to-Ct kit. Results: The table below summarizes the Ct values for CD45, ADORA2A, GLS and GAPDH expression in 300 fixed unsorted control and DEPArray™sorted lymphocytes. Conclusions: We have demonstrated the feasibility of gene expression analysis on pure populations of CD4+ and CD8+ cells isolated from a fixed tissue using the DEPArray™ platform. The advantage of this approach is the DEPArray’s ability to identify and isolate subpopulations of cells from complex heterogeneous samples and/or specimens that are limited by size or content. This methodology will be applied for isolation of TILs in syngeneic and xenograft models of cancers for downstream RNA applications. [Table: see text]

Birth of puppies of predetermined sex after artificial insemination with a low number of sex-sorted, frozen-thawed spermatozoa in field conditions.

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen-thawed spermatozoa of 100×106 unsorted (a dose in practice) and 4×106 sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P>0.05), but lower than controls (11/20; 55.0%) (P<0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P<0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm-sexing technology potentially applicable for elite breeding units.

Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions

To study the ability of human spermatogonial stem cells (hSSCs) to proliferate invitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create invitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA-)/epithelial cell surface antigen (EPCAM+) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells invitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA-/EPCAM+ resulted in an enrichment of 27% VASA+/UTF1+ hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm2 after 2weeks invitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4weeks invitro. We found that hSSCs show limited proliferation invitro under mSSC culture conditions. Coculture of HLA-/EPCAM+ sorted cells with testicular feeders improved the germ cell/somatic cell ratio.

Abstract 2494: Adhesive signature technology for tumor initiating cell purification in cancer research

Abstract In spite of major therapeutic advances, cancer relapse and low rates of patient response persist. This failure is due in part to a small subpopulation of tumor initiating cells (TICs) with stem cell like properties that are responsible for the growth of the tumor and the progression of metastasis. Currently, no efficient and reliable methods to isolate TICs for study exist. Our lab has developed a methodology to isolate cells based on their unique adhesion binding strength to a matrix. The novel technology (micro-Stem cell High- Efficiency Adhesion based Recovery [μSHEAR]) consists of a microfluidic device that applies varying degrees of detachment shear forces on cells. Using this device, human pluripotent stem cells and their progeny have been isolated with high reproducibility, yield (&amp;gt;97%), purity (95-99%), and survival (&amp;gt;95%) rates (Singh et al, Nature Methods 2013). The process is fast (10 min), label free, and scalable. The objective of this research is to isolate the rare TICs from the general cancer cell population by exploiting differences in adhesion strength. To study these differences, we measured the adhesion strength (adhesive signature) of a panel of breast cancer cell lines to fibronectin using a hydrodynamic assay. Based on this screen, we selected the MDA-MB- 231, MDA-MB-453, and MCF7 cell lines for purification of TICs using the uSHEAR technology. Briefly, microfluidic channels were sterilized and coated with fibronectin. MDA-MB- 231, MDA-MB-453 or MCF7 breast cancer cells were enzymatically disassociated, pipetted into the inlet reservoir, and cultured in the device for 24h before detachment experiments. Cells were exposed to different shear forces to selectively detach cell sub-populations. Recovered cell/colonies were counted, seeded into a mammosphere formation assay (MFA), and analyzed after 10 days The ability to form mammospheres is characteristic of TICs. After uSHEAR mediated separation of breast cancer cells into three fractions, the strongest adhering fraction consistently produced larger and more mammospheres. Furthermore, as the selection adhesive force for the adhered fraction was increased, greater increases in mammosphere number and size were observed. After 10 days, the mammospheres were disassociated and the number of cells quantified. A 5-15 fold increase in the final number of cells was observed in the strongly adherent fractions of cells, compared to 0.8-1.7 fold increase in the unsorted controls. Our results show that cancer cells with a higher mammosphere formation potential, a hallmark of characteristic TICs, have a higher adhesion strength. Furthermore, these cells can be enriched via adhesion based separation giving rise to more and larger mammospheres than their less adherent counterparts. Our results suggest that TICs could be separated based on adhesive forces. Future studies are aimed at characterizing the isolated cells as TICs and testing the isolation strategy in primary tumors. Citation Format: Efrain A. Cermeno, Austin P. Veith, Susan N. Thomas, Andres J. Garcia. Adhesive signature technology for tumor initiating cell purification in cancer research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2494.

Association Between Candidate Performance-related Gene Polymorphisms And Strength In Iranian Elite Athletes

One of the most important elements gives us the potential to individualize as an elite endurance runner or world champion weight lifter is DNA sequence variation. Investigations has discussed that about 66 percent of the variability in the athletic position is under the influence of genetics. There are many sport-related traits which can be explained by common. One of the traits whose heritability may depend on SNPs is maximal muscle strength. Unlike the vast number of studies in genetic profile of endurance performance, the genetic influence on elite strength performance has received less attention. PURPOSE: The purpose of this study was to investigate the relation between the polymorphisms of candidate genes (ACTN3, PGC-1α, ACE, CKMM, and PPARγ) with strength trait in Iranian elite athletes. METHODS: The subjects of this study included 100 Iranian elite athletes from different disciplines, as well as 100 sedentary healthy controls. Body mass index (BMI), waist-hip ratio (WHR), body fat percentage (%BF) has been measured for physical characteristic of subjects. Grip strength test has been conducted to measure maximal strength. Genomic DNA was extracted from saliva. Genetic polymorphism evaluation performed by RFLP and double check with sequencing methods. One-way ANOVA was used to assess differences between genotype groups for investigated phenotypes. RESULTS: Measuring Mean differences showed that %BF (athlete: 12.78±4.48, non-athlete: 17.9±5.84), WHR (athlete: 0.83±0.5, non-athlete: 0.80±0.06) and grip strength (athlete: 49.54±0.5, non-athlete: 40.16±0.8) were significantly different in two groups (P≤0.05). We have shown that ACTN3 (P=0.027), ACE (P=0.001), and CKMM (P=0.041) polymorphisms were significantly associated with strength phenotype in elite athletes. While the ACTN3 (P=0.040) polymorphism was the only one that had a significant association in unsorted control population. CONCLUSIONS: Considering Iranian population as Caucasians, these results may lead us to clarify the polygenic profiles of athletes in this ethnicity, although we have to out spread this study to different disciplines of athletic populations. Further investigations are warranted to study the mechanisms by which these polymorphisms may affect skeletal muscle to produce force.

Performance and carcass characteristics when sorting feedlot cattle on the basis of phenotype, and leptin genotype along with differential use of β-adrenergic agonists

Kononoff, P. J., Defoor, P. J., Engler, M. J., Swingle, R. S., James, S. Y., Deobald, H. M., Deobald, R. L., Woronuk, G. N. and Marquess, F. L. S. 2015. Performance and carcass characteristics when sorting feedlot cattle on the basis of phenotype, and leptin genotype along with differential use of β-adrenergic agonists. Can. J. Anim. Sci. 95: 455–463. Crossbred steers were used in a randomized complete block design to evaluate a feedlot cattle sorting system. The sorting system combined information on live body weight, ultrasound fat thickness, coat color, and genotype on the leptin R25C genotype. Using described sorting criteria, a total of four groups (Groups 1–4) of cattle were created along with a randomly selected unsorted control (Group 5), with Groups 1 through 5 representing experimental treatments, and placed in one of five pens within each block. Block was replicated 10 times thus n equaled 50. All cattle were administered zilpaterol hydrochloride except Group 3, which we hypothesized would have a greater proportion of cattle reaching a higher value Quality Grade, and as a result administered ractopamine hydrochloride. The initial body weight of cattle was similar (P=0.426) between the control Group and Groups 1–4. Initial fat, as measured by ultrasound, was also observed to be similar (P=0.256) between these two groups. Overall, when cattle included in Groups 1–4 were compared with Group 5, hot carcass weight was greater (P=0.040), while the proportion of excessively heavy weight carcasses was less (P=0.049). Additionally, hot carcass weight gain tended (P=0.096) to be higher for Groups 1–4 compared with Group 5. No differences (P≥0.129) were observed for calculated yield grade. On average, cattle included in the Sorting Groups 1–4 returned $23 more per head than those in Group 5.

Can Arthroscopically Harvested Synovial Stem Cells Be Preferentially Sorted Using Stage-Specific Embryonic Antigen 4 Antibody for Cartilage, Bone, and Adipose Regeneration?

The aim of this study was to investigate the relation between stage-specific embryonic antigen 4 (SSEA4) expression and synovium-derived stem cell (SDSC) lineage differentiation. Human SDSCs were collected during arthroscopic surgery from 4 young patients with anterior cruciate ligament injuries. Passage 2 SDSCs were sorted by fluorescence-activated cell sorting using phycoerythrin-conjugated monoclonal antibody against SSEA4 into 3 groups: SSEA4(+) cells, SSEA4(-) cells, and unsorted control cells. After 1 more passage, expanded cells from each group were evaluated for SSEA4 expression by use of flow cytometry as well as multilineage differentiation capacities, including chondrogenesis, adipogenesis, and osteogenesis, using biochemical analysis, histologic analysis, immunostaining, and real-time polymerase chain reaction. After cell sorting, 1 more passage expansion decreased SSEA4(+) cells from 99.8% to 79.2% and increased SSEA4(-) cells from 4.4% to 53.3% compared with 70.3% in the unsorted cell population. SSEA4(-) SDSCs with a lower cell proliferation exhibited higher chondrogenic potential (in terms of the ratio of glycosaminoglycan to DNA [P < .001] and COL2A1 [type II collagen] messenger RNA [mRNA] [P < .001]) and adipogenic potential (in terms of oil red O staining and quantitative assay [P= .007], LPL [lipoprotein lipase] mRNA [P= .005], and CEBP [CCAAT/enhancer-binding protein alpha] mRNA [P= .010]). In contrast, SSEA4(+) SDSCs retained cell expansion and enhanced osteogenic capacity, as evidenced by intense calcium deposition stained by alizarin red S and a significantly elevated expression of OPN (osteopontin) mRNA (P= .007). In this study, for the first time, we showed the benefit of using the surface marker SSEA4 in SDSCs to preferentially sort a mixed population of cells. SSEA4(+) SDSCs indicated a strong potential for osteogenesis rather than chondrogenesis and adipogenesis. SDSC-based mesenchymal tissue regeneration can be easily achieved by arthroscopic harvesting followed by quick cell sorting.

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

It's not just leukocytes in cytometry

most common objects of investigation for cytometricanalysis, may it be image or flow cytometry, are leukocytesand cells or cell constituents isolated from cells or tissues.However, there are more than just leukocytes to be looked atin the world of cytometry.Erythrocytes, as the transport vehicles for oxygen, are ofcentral relevance in several red blood cell-related diseases. Thisincludes anemia resulting from various acquired or congenitaldiseases, erythrocytosis, and erythrocyte deformation in sicklecell anemia or spherocytosis, among others. These diseasesmay lead to altered erythrocyte count, reduced hemoglobincontent, shape alterations, or alterations of the cells’ elasticity.All of these changes can be analyzed quantitatively on the sin-gle erythrocyte level using the cytometry approach. Possibly,the most advanced and challenging methods are those thattake advantage of novel optical techniques and perform cyto-metry in vivo (1). By innovative in vivo flow cytometry tech-nology, many of the abovementioned erythrocyte characteris-tics can be obtained (2) but it is still a glimpse into the futurefor most researchers. The image cytometry-based technologydeveloped by Tomaiuolo and colleagues from Napoli, Italy(this issue, page 1040) is presently more accessible. Theauthors compared two flow-based image cytometry methodsto measure the morphology of a large number of erythrocytesflowing through a large (50 lm, unbound) or small (4.7–10lm, confined) capillary and images being captured by ahigh-speed camera. The authors report that their techniqueprovides results comparable to standard technologies such asCoulter Counter with regard to erythrocyte volume, area andvolume distribution. In addition, it renders additionalinformation not available by standard methods such as defor-mation in small capillaries or sphericity index.Philipp and colleagues from Kiel, Germany (this issue,page 1048) addressed the question of how to isolate viableMalaria parasite (Plasmodium falciparum) infected erythro-cytes by fluorescence activated cell sorting. In the past, manyresearchers developed flow cytometry methods to analyze dif-ferent stages of development of Plasmodium spec. within theerythrocyte. The majority of these methods rely on stainingthe DNA of the parasite by a DNA-specific fluorochrome andas an example, the dye YOYO-1 can clearly distinguish differ-ent stages of development (3). However, not all of these dyesare suitable for life sorting because labeling, among others,may need erythrocyte permeability. Philipp and colleaguestested three different DNA dyes and reported that VybrantDyeCycle Violet, a fluorochrome specific to the violet spectralrange, provides best results with regard to sensitive detectionand viable sorting of erythrocytes harboring immature para-sites. Test results for in vitro maturation and reinfection rate ofthese sorted early developmenta l stage parasites is identical tothat of unsorted controls. This new improved method providesan important tool for the investigation of P. falciparum matura-tion and for therapeutic drug development to fight malaria.In bacteriology, a small but scientifically strong and activecytometry community is pushing the limits to improve theanalysis and functional characterizationof mixed bacterial com-munities (seeasexample: Ref. 4). In clinical reality, mixedinfec-tions by different bacterial strains are the usual situation. R€ugerand colleagues from Magdeburg and K€othen, Germany (thisissue, page 1055) raised the questionregarding how to discrimi-

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM+ enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM+ enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.

Rad6B acts downstream of Wnt signaling to stabilize β-catenin: Implications for a novel Wnt/β-catenin target

BackgroundAberrant Wnt/β-catenin signaling is associated with breast cancer even though genetic mutations in Wnt signaling components are rare. We have previously demonstrated that Rad6B, an ubiquitin conjugating enzyme, stabilizes β-catenin via polyubiqutin modifications that render β-catenin insensitive to proteasomal degradation. Rad6B is a transcriptional target of β-catenin, creating a positive feedback loop between Rad6B expression and β-catenin activation.MethodsTo isolate subpopulations expressing high or low Rad6B levels, we transfected MDA-MB-231 or WS-15 human breast cancer cells with ZsGreen fluorescent reporter vector in which the expression of ZsGreen was placed under the control of Rad6B promoter. ZsGreenhigh and ZsGreenlow subpopulations, reflective of high and low Rad6B promoter activity, respectively, were isolated by FACS. To determine the relevance of Wnt signaling in Rad6B-mediated β-catenin stabilization/activation, the ZsGreenhigh cells were transfected with signaling-defective Wnt coreceptor LRP6Δ173. Rad6B expression and promoter activity were determined by RT-PCR, Western blot and Rad6B promoter-mediated luciferase assays. β-catenin levels and transcriptional activity were determined by Western blot and TOP/FOP Flash reporter assays. Tumor formation and morphologies of ZsGreenlow, ZsGreenhigh, and ZsGreenhigh/LRP6Δ173 cells compared to unsorted vector controls were evaluated in nude mice. Expression of Wnt signaling related genes was profiled using the Wnt signaling pathway RT2 Profiler PCR arrays.ResultsZsGreenhigh subpopulations showed high Rad6B expression and Rad6B promoter activity as compared to ZsGreenlow cells. ZsGreenhigh (high Rad6B expressors) also showed elevated β-catenin levels and TOP/Flash activity. Inhibiting Wnt signaling in the high Rad6B expressors decreased ZsGreen fluorescence, Rad6B gene expression, β-catenin levels and TOP/Flash activity. Tumors derived from high Rad6B expressors were predominantly composed of cells with epithelial mesenchymal transition (EMT) phenotype as compared to control tumors that were composed of both cuboidal and EMT-type cells. Tumors derived from low Rad6B expressors lacked EMT phenotype. Inhibition of LRP6 function in the high Rad6B expressors abrogated the EMT phenotype. Gene expression profiling showed upregulation of several Wnt signaling pathway regulators in high Rad6B expressors that were downregulated by interference of Wnt signaling with mutant LRP6 or by Rad6B silencing.ConclusionsThese data reveal a functional link between the canonical Wnt pathway and Rad6B in β-catenin activation and breast cancer progression.

Breast cancer stem-like cells show dominant homologous recombination due to a larger S-G2 fraction

The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation resistant when sorted for the two stem cell markers CD24 and ESA. In this study, we examined a possible mechanism that might underlie this phenotype by looking at cell cycle profile and the effect this has on DNA repair pathways. The cell cycle profile showed that there were more CD24- ESA+ sorted MDA-MB231 cells in the S- and G2-phases compared with the unsorted cells, 60 and 38% respectively. Cyclin D and E protein levels supported the cell cycle profile and highlighted the possible involvement of homologous recombination (HR) repair in the radioresistant phenotype. To further support this, CD24- ESA+ sorted MDA-MB231 cells demonstrated statistically significant more RAD51 and less γ-H2AX foci 2 h post 4Gy ionising radiation, compared with the unsorted population. Inhibition of the HR pathway effectively sterilised the CD24- ESA+ sorted MDA-MB231 cells but had no effect on the unsorted cells or MDA468 control breast cancer cell line. Although the changes we saw were specific to MDA-MB231, these results merit further investigation and can be crucial in identifying a mechanism responsible for cancer stem cells treatment resistance in primary tumors.