Novel Method For Isolation Research Articles

Optimization of novel method for isolation of high purity food grade α-linolenic acid from Linum usitatissimum seeds

Omega-3 (ω-3) fatty acids are derived from marine sources and offer disadvantages like heavy metal toxicity, unfit for vegetarians, sinking sources of fish, fishy smell, and side effects. Long-term fish oil consumption increases oxidative stress, inflammation, and risk of chronic diseases. Polyunsaturated fatty acids (PUFAs) are abundantly available in plant seeds like Perilla frutescence, Linum usitatissimum, etc. Plant-based PUFAs are either not available in the market or available as complete extracts.The present study was undertaken to standardize the method for extraction and isolation of α-linolenic acid (C18:3, ω-3) from Linum usitatissimum. The gas chromatograph-FID was used to characterise lipids. A complete mixture of lipids was extracted through cold maceration and saponified to separate saturated lipids. The unsaturated lipids, thus obtained were subjected to urea crystallization. Following the segregation of monounsaturated fatty acids, the lipids were esterified by a chemical method to segregate ω-3 PUFAs. The ω-3 PUFAs were subjected to column chromatography to isolate α-linolenic acid (C18:3, ω-3).

An enzyme-based system for extraction of small extracellular vesicles from plants

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.

ThTP8.6 A novel method for stoma isolation in a complex abdominal wound treated with vacuum assisted closure

Abstract Introduction Vacuum assisted closure (VAC) is a well established method in the treatment of abdominal wounds where the fascia is intact. The presence of one or multiple intestinal stomas or fistulae may present challenges in successful application of VAC therapy. Key concerns with the presence of intestinal stomas alongside open abdominal wounds is the risk of contamination with stoma effluent or the potential for the negative pressure to create enterocutaneous fistulae. Here we describe a novel method for isolating a small bowel stoma existing within a large abdominal wound, allowing successful resolution having failed conventional management. Method No device currently exists to allow a functioning stoma to be isolated within an abdominal wound requiring VAC therapy. This method relies on modification of a standard silicone fistula crown, this is then integrated into the standard VAC dressing, using stoma seals to provide successful vacuum. Stoma output is then successfully isolated, protecting surrounding tissue and supporting wound granulation without concern of wound effluent contamination. Discussion Of key importance in this case is the proximity of clean wounds with skin grafts done by plastic surgeons also being managed with VAC therapy and the importance of preventing contamination to allow successful granulation without further debridement of an already large wound. Conclusion Although this situation is rare with the increasing use of vacuum assisted closure devices for the management of wounds across many specialities this scenario is certainly not unprecedented. The technique described allowed successful treatment of a complex wound with significant existing comorbidities allowing resolution of a very challenging situation.

A Novel Method for Human Adipose-Derived Stem Cell Isolation and Cryopreservation.

Adipose-derived stem cells (ADSCs) are isolated from abundant adipose tissue and have the capacity to differentiate into multiple cell lineages. ADSCs have raised big interest in therapeutic applications in regenerative medicine and demonstrated to fulfill the criteria for a successful cell therapy. There are several methods for isolation of ADSCs from adipose tissue and cryopreservation of ADSCs. Here, novel methods for the isolation and cryopreservation of ADSCs are presented and focused. Microscopic pieces of adipose tissue were placed on transwell inserts, and the ADSCs were induced to migrate to the lower wells for 1 week. We compared the properties of our ADSCs with those isolated by enzymatic digestion and enzyme-free method of culture plate, and our ADSCs were found to be more stable and healthier. In addition, we proposed a novel cryoprotectant solution (FNCP) containing pectin and L-alanine, which was compared with standard cryoprotectant solution. Overall, our methods proved more useful for ADSCs isolation than other methods and did not require consideration of "minimal manipulation" by the U.S. Food and Drug Administration (FDA). Furthermore, our FNCP did not contain dimethyl sulfoxide and fetal bovine serum, therefore stable storage is possible in xeno-free and animal-free cryopreservation solutions.

A novel method for isolation of functional stallion spermatozoa for artificial insemination

In vitro semen purification techniques have been developed that seek to mimic the in vivo selection process, in order to generate the highest possible chance of oocyte fertilization following artificial insemination (Morrell and Rodriguez-Martinez. Vet Med Int. 2011:894767). Since this practice began, there have been many methods developed in an attempt to isolate functional spermatozoa for AI, yet only one method is routinely used – density gradient centrifugation using commercial preparations such as EquiPure. Here, we have developed a novel method of isolating functional spermatozoa and compared sperm quality to spermatozoa isolated with EquiPure. The AI-Port system (Memphasys, Sydney, Australia), comprises a disposable cartridge consisting of a sample chamber – where extended semen is deposited, and a harvest chamber – where isolated spermatozoa is extracted. These chambers are separated by a 5 µm nylon woven membrane, through which highly motile spermatozoa are able to swim from the sample chamber into the harvest chamber over 20 min, leaving behind seminal plasma and other cell types such as leukocytes. Spermatozoa were collected from miniature pony stallions, and immediately extended with EquiPlus semen extender. The ejaculate was split, and spermatozoa were isolated into BWW media with EquiPure (as per manufacturer's instructions; Nidacon, International), or the AI-Port system. Comparisons were conducted between spermatozoa isolated with the AI-Port and EquiPure, which revealed that the yield of AI-Port samples was significantly lower (25.5±5.9%) incomparison to EquiPure samples (44.9±10.2%; Kruskal-Wallis), potentially solvable with prototype improvement. Viability (Eosin-nigrosin) of AI-Port samples was significantly higher than EquiPure samples (94.4±1.1 vs 81.5±2.1%; P≤0.01; ANOVA; respectively). In regard to motility, total motility for AI-Port samples was significantly higher than EquiPure samples (93±1.3 vs 89.5±2.9%; P≤0.05; ANOVA; respectively). Whilst there was no significant difference for progressive motility between AI-Port and EquiPure samples (45.7±3.2 vs 45.6±3.4%; ANOVA; respectively). Both samples were higher than the progressive motility of the initial non-purified ejaculate (30±4.1%; P≤0.05). Mitochondrial superoxide was measured with flow cytometry and MitoSOX red. Likewise, there was no significant difference in superoxide levels between AI-Port and EquiPure samples (10.2±3.1 vs 5.6±0.8%; ANOVA; respectively). However, both samples had a reduced level of superoxide generation compared to the initial non-purified ejaculate (32.5±20.2%; P≤0.05). The DNA integrity was measured with the Halo test, which confirmed AI-Port samples had less DNA damage than EquiPure™ samples (2.0±0.5 vs 10.2±2.1%; ANOVA; P≤0.01; respectively). In summary, the AI-Port effectively isolates high-quality spermatozoa at a comparable or superior level to EquiPure. Its cost-effectiveness and user-friendliness make it a viable option for improving artificial insemination success rates in breeding programs.

A novel method for isolation and flow cytometry analysis of intraepithelial lymphocytes from colon biopsies

Investigating the immune responses of the intestine in response to different insults is predominantly limited to indirect methods such as circulating markers of intestinal health or gene expression from dissections. We describe here a validated protocol for the isolation and subsequent flow cytometry analysis of intestinal intraepithelial lymphocytes (IEL) from colonic biopsy samples. Colon biopsy samples were collected with endoscopy forceps from Holstein dairy bull calves at d 2, 28, and 42 of life. The biopsies were put into an isolation solution of Hanks' balanced salt solution, and fetal bovine serum followed by digestion solution. The solution was filtered and the flow-through, containing IEL, was stained with fluorescent antibodies for flow cytometry analysis. Density gradient separation of the isolate yielded higher viability and cleaner samples for flow cytometry analysis. Anti-bovine γ chain of the T cell receptor was used to identify populations of gamma delta (γδ) T cells via flow cytometry. In addition, γδ T cell subsets were identified using an anti-bovine antibody against the coreceptor workshop cluster 1. This method allowed for the precise identification of lymphocyte populations and evaluation of the proportion of different subsets of γδ T cells from intestinal IEL over time. The technique described here will allow the research community to characterize intestinal immune function over time and improve our understanding of how different management and nutritional strategies affect intestinal health.

423 Novel method for isolation of small airway epithelial cells by bulk enzymatic digestion

423 Novel method for isolation of small airway epithelial cells by bulk enzymatic digestion

Novel Method for Isolation of Porcine Epidemic Diarrhea Virus with the Use of Suspension Vero Cells and Immunogenicity Analysis.

In February and December of 2019, two large-scale outbreaks of diarrhea were observed in the same swine farm with 3,000 sows in Shanghai, China. We successfully isolated two porcine epidemic diarrhea virus (PEDV) isolates (strains shxx1902 and shxx1912 in February and December, respectively) from clinical samples in this farm using suspension Vero cells. A third PEDV strain (SH1302) tested positive in another farm of Shanghai, China, in 2013 and was also isolated using suspension Vero cells. The three isolates were better adapted to growth in adherent Vero cells through serial passages in the suspension Vero cells. The three isolated strains were detected positive by an immunofluorescence assay (IFA) and observed through electron microscopy. Phylogenetic analysis of the complete genomic sequence demonstrated that shxx1902 (the 5th passage) and shxx1912 (the 5th passage) clustered with a new GII subgroup (GII-c), which consisted of SINDEL strains from America (e.g., OH851), and their S gene belonged to GII-a. Both strains(the 35th passage) have incurred dramatic changes in their genomes compared with the 5th passage. The 5th and 35th passages of SH1302 belonged to the GI-b genotype. The anti-N protein antibody titer of the strain shxx1902 was elevated to the same level as the vaccine strain (CV777) in mice. The use of the suspension Vero cells to isolate and propagate PEDV provides an effective approach for studies of the epidemiological characteristics and vaccine development of this virus.

A novel method for isolation and culture of primary swine gastric epithelial cells

BackgroundCulturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.ResultsMechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.ConclusionsA new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

Routine and novel methods for isolation of extracellular vesicles

Extracellular vesicles (EV) play an important role in many physiological and pathological processes. Three main classes of EV are recognized, based on their biogenesis: exosomes, microvesicles and apoptotic bodies. Exosomes are extracellular-vesicles of 30 to 150 nm found in many bodily fluids (blood, urine, milk, cerebrospinal fluid, etc.). Due to their cellular origin and role in physiological and pathological processes, exosomes present in body fluids are considered a unique source of non-invasive and clinically relevant biomarkers. Analysis of exosomes can provide insight into the state of the parent-cell from which they originated. However, there is great heterogeneity in the methodologies used for exosome purification affecting the results of downstream analysis. The most commonly used methods for purification are based on ultracentrifugation (UC), ultrafiltration (UF) and precipitation. However, these are hard to standardize, leading to confounding and misleading results during downstream analyses, especially when highly-sensitive techniques such as mass spectrometry are used. Furthermore, loss of certain fractions or damage of EVs can lead to loss in obtained protein and RNA profile. Consequently, there is an emerging need to obtain consensus protocols for exosome isolation and identification of specific sub-populations. This manuscript will critically review the most commonly used techniques for EV purification such as UC, UF, size-exclusion, precipitation and immunoaffinity (IA) methods. We will also review the use of nano-antibodies for the development of novel IA protocols and identification of new EV biomarkers.

Pt-induced crosslinks promote target enrichment and protection from serum nucleases

Identifying the interactions of small molecules with biomolecules in complex cellular environments is a significant challenge. As one important example, despite being widely used for decades, much is still not understood regarding the cellular targets of Pt(II)-based anticancer drugs. In this study we introduce a novel method for isolation of Pt(II)-bound biomolecules using a DNA hybridization pull-down approach. Using a modified Pt reagent, click-ligation of a DNA oligonucleotide to both a Pt(II)-bound DNA hairpin and bovine serum albumin (BSA) are demonstrated. Subsequent hybridization to a biotin-labeled oligonucleotide allows for efficient isolation of Pt(II)-bound species by streptavidin pulldown. We also find that platinated bovine serum albumin readily crosslinks to DNA in the absence of click ligation, and that a fraction of BSA-bound Pt(II) can transfer to DNA over time. Interestingly, in in vitro studies, fragmented mammalian DNA that is crosslinked to BSA through Pt(II) exhibits significantly increased protection from degradation by serum nucleases.

A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood.

Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours

BackgroundDysregulation in post-translational modifications of histones and their modifiers are now well-recognized as a hallmark of cancer and can be used as biomarkers and potential therapeutic targets for disease progression and prognosis. In most solid tumours, a biopsy is challenging, costly, painful or potentially risky for the patient. Therefore, non-invasive methods like ‘liquid biopsy’ for analysis of histone modifications and their modifiers if possible will be helpful in the better clinical management of cancer patients.MethodsHere, we have developed a cost-effective and time-efficient protocol for isolation of circulating histones from serum of solid tumor, HCC, called Dual Acid Extraction (DAE) protocol and have confirmed by mass spectrometry. Also, we measured the activity of HDACs and HATs in serum samples.ResultsThe serum purified histones were profiled for changes in histone PTMs and have shown a comparable pattern of modifications like acetylation (H4K16Ac), methylation (H4K20Me3, H3K27Me3, H3K9Me3) and phosphorylation (γ-H2AX and H3S10P) to paired cancer tissues. Profiling for the histone PTM changes in various other organs of normal and tumor bearing animal suggests that the changes in the histone PTMs observed in the tumor serum is indeed due to changes in the tumor tissue only. Further, we demonstrate that the observed hypo-acetylation of histone H4 in tissue and serum samples of tumor bearing animals corroborated with the elevated HDAC activity in both samples compared to normal. Interestingly, human normal and tumor serum samples also showed elevated HDAC activity with no significant changes in HAT activity.ConclusionsOur study provides the first evidence in the context of histone PTMs and modifiers that liquid biopsy is a valuable predictive tool for monitoring disease progression. Importantly, with the advent of drugs that target specific enzymes involved in the epigenetic regulation of gene expression, liquid biopsy-based ‘real time’ monitoring will be useful for subgrouping of the patients for epi-drug treatment, predicting response to therapy, early relapse and prognosis.

Matrix metalloproteinases as reagents for cell isolation.

Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase™. Samples isolated with MMPs and Liberase™ had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase™. However, ADSCs isolated by Liberase™ displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to<1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.

A novel method for isolation and recovery of ceramic nanoparticles and metal wear debris from serum lubricants at ultra-low wear rates

Ceramics have been used to deliver significant improvements in the wear properties of orthopaedic bearing materials, which has made it challenging to isolate wear debris from simulator lubricants. Ceramics such as silicon nitride, as well as ceramic-like surface coatings on metal substrates have been explored as potential alternatives to conventional implant materials. Current isolation methods were designed for isolating conventional metal, UHMWPE and ceramic wear debris. In this paper, we describe a methodology for isolation and recovery of ceramic or ceramic-like coating particles and metal wear particles from serum lubricants under ultra-low and low wear performance. Enzymatic digestion was used to digest the serum proteins and sodium polytungstate was used as a novel density gradient medium to isolate particles from proteins and other contaminants by ultracentrifugation. This method demonstrated over 80% recovery of particles and did not alter the size or morphology of ceramic and metal particles during the isolation process. Statement of SignificanceImprovements in resistance to wear and mechanical damage of the articulating surfaces have a large influence on longevity and reliability of joint replacement devices. Modern ceramics have demonstrated ultra-low wear rates for hard-on-hard total hip replacements. Generation of very low concentrations of wear debris in simulator lubricants has made it challenging to isolate the particles for characterisation and further analysis. We have introduced a novel method to isolate ceramic and metal particles from serum-based lubricants using enzymatic digestion and novel sodium polytungstate gradients. This is the first study to demonstrate the recovery of ceramic and metal particles from serum lubricants at lowest detectable in vitro wear rates reported in literature.

Abstract 1597: A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients

Abstract Multiple Myeloma is bone marrow tumor that frequently results in the aberrant release of neoplastic plasma cells into circulation. The CELLSEARCH® system (Janssen Diagnostics, Raritan, NJ) has been used to capture, enumerate, and perform FISH analysis on circulating multiple myeloma cells (CMMC) from patients with active multiple myeloma, smoldering myeloma, and MGUS (Gross, et.al. Poster #1825, ASH 2011). This method utilizes a blood fixative and subsequently permeabilizes the cells, which confounds the ability to do mRNA analysis on these samples. Here we present a novel method to isolate pure circulating myeloma cells that is compatible with downstream genetic analysis. Blood was obtained through a commercial vendor (Conversant Bio) from multiple myeloma patients. Samples were processed in parallel using the existing method for enumeration and a novel method for genetic profiling. For CMMC enumeration, blood was collected in CellSave tubes (Janssen Diagnostics) and CMMC were enriched using paramagnetic ferrofluid recognizing CD138 and CS1 antigens. Cells were permeabilized and stained on the CELLSEARCH® AUTOPREP with CD38 (PE), CD45 (APC), CD19 (APC) and DAPI. CMMC were enumerated using the CELLTRACKS ANALYZER II® scanning platform. CMMC were defined as DAPI +, CD138+, CD38+, CD45-, CD19-. For genetic profiling, 4 ml EDTA blood was enriched using CD138 and CS1 ferrofluids on the AUTOPREP with no subsequent permeabilization. CMMC were stained offline using CD38 (Texas Red), CD45 (FITC), CD19 (FITC), CD3 (FITC) and NucBlue. Samples were transferred to glass a bottomed petri dish and observed through an inverted microscope using a Texas Red/FITC dual filter, individual FITC and Texas red filters, and a DAPI filter. CMMC were defined as NucBlue+, CD138+, CD38+, CD45-, CD19-, and CD3-. Pools of individual CMMC were picked from the sample using an Eppendorf Transferman micromanipulator. For comparison, contaminating leukocytes were also collected for analysis. RNA from picked cells was amplified using the SMARTer Ultra Low Input Kit (Clontech) and qPCR was performed for myeloma and leukocyte specific markers. Detection of CMMC using the novel method for genetic profiling was found to be concordant with the existing method for enumeration. We found this approach to provide a deep and unbiased characterization of gene pathways activated in CMMC by RT qPCR and RNASeq. This method will have applications in longitudinal studies of active myeloma patients as a way to gather information about relapse and minimal residual disease between bone marrow draws. Citation Format: Greg Brittingham, Chandra Rao, Peter Vulfson, Vipul Bhargava, Denis Smirnov, Brad Foulk. A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2015-1597

Upregulation of Nanog and Sox-2 genes following ectopic expression of Oct-4 in amniotic fluid mesenchymal stem cells.

Octamer-binding transcription factor 4 (Oct-4), an important gene regulating stem cell pluripotency, is well-known for its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. The aim of this study was to assess the effect of ectopic expression of Oct human amniotic fluid stem cells. We developed a novel method for isolation of putative human amniotic fluid-derived multipotent stem cells. These cells showing mesenchymal stem cell phenotypes (human amniotic fluid-derived mesenchymal stem cells, hAFMSCs) were transfected with a plasmid carrying genes for Oct-4 and the green fluorescent protein (GFP). The stably transfected cells, hAFMSCs-Oct4/GFP, were selected by using G418 and found to express the GFP reporter gene under the control of Oct-4 promoter. We found that hAFMSCs developed by our method possess very high self-renewal ability (about 78 cumulative population doublings) and multilineage differentiation potency. Significantly, the hAFMSCs-Oct4/GFP cells showed enhanced expression of the three major pluripotency genes Oct-4, Nanog, and Sox-2, and increased colony-forming ability and growth rate compared with the parental hAFMSCs. We demonstrated that the ectopic expression of Oct-4 gene in hAFMSCs with high self-renewal ability could upregulate Nanog and Sox-2 gene expression and enhance cell growth rate and colony-forming efficiency. Therefore, the ectopic expression of Oct-4 could be a strategy to develop pluripotency in hAFMSCs for clinical applications.

ALDH expression characterizes G1-phase proliferating beta cells during pregnancy.

High levels of aldehyde dehydrogenase (ALDH) activity have been detected in various progenitor and stem cells. Thus, Aldefluor fluorescence, which represents precisely the ALDH activity, has been widely used for the identification, evaluation, and isolation of stem and progenitor cells. Recently, ALDH activity was detected in embryonic and adult mouse pancreas, specifically in adult centroacinar and terminal duct cells supposed to harbor endocrine and exocrine progenitor cells in the adult pancreas. Nevertheless, ALDH activity and aldeflour fluorescence have not been examined in beta cells. Here, we report a dynamic increase in the number of aldeflour+ beta cells during pregnancy. Interestingly, nearly all these aldeflour+ beta cells are positive for Ki-67, suggesting that they are in an active cell cycle (G1, S and M phases). To determine precisely at which phase beta cells activate ALDH activity and thus become aldeflour+, we co-stained insulin with additional proliferation markers, phosphohistone3 (PHH3, a marker for M-phase proliferating cells) and Bromodeoxyuridine (BrdU, a marker for S-phase proliferating cells). Our data show little aldeflour+ beta cells that were positive for either PHH3, or BrdU, suggesting that beta cells activate ALDH and become Aldefluor+ when they enter G1-phase of active cell cycle, but may downregulate ALDH when they leave G1-phase and enter S phase. Our data thus reveal a potential change in ALDH activity of proliferating beta cells during pregnancy, which provides a novel method for isolation and analysis of proliferating beta cells. Moreover, our data also suggest that caution needs to be taken on interpretation of Aldefluor lineage-tracing data in pancreas.

Development of a novel method for isolation of four developmentally distinct myeloid derived dendritic cells pre-cursors: phenotypic and functional analyses (HEM3P.283)

Abstract Myeloid cell progenitors have the potential to give rise to dendritic cells which have a high capacity to stimulate adaptive immune responses as well as to cells with potent immunosuppressive activity. However, the stage in their development in which these functions are gained is poorly defined. Here, we developed a method for isolating progenitor populations at specific stages of development. Bone marrow cells were lineage depleted and cultured in GM-CSF to drive their differentiation toward a myeloid lineage for three days. Cells were then sorted based on Ly6C and CSF-1R (CD115) expression, two markers whose expression is known to change throughout myeloid ontogeny. We then monitored the developmental progression of each population based on the expression of these markers as well as other hallmarks of dendritic cell or myeloid suppressor cell phenotypes. We also measured the function of each population relative to antigen uptake and degradation as well as their response to stimulation by TLR agonists. Collectively, our results indicate that myeloid progenitors progress along this pathway: Ly6C-CD115- -&amp;gt; Ly6C+CD115- -&amp;gt; Ly6C+CD115+ -&amp;gt; Ly6C-CD115+ -&amp;gt; Ly6C-CD115+. Phenotypically, these populations represent CMP, MDSC, monocytes, immature DC, and more mature DC, respectively. These findings not only enhance our understanding of myeloid cell development and differentiation driven by GM-CSF, but also inform the design of GM-CSF-derived DC based vaccine strategies.

A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering

The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.