Quantifying anthropogenic emission of iron in marine aerosol in the Northwest Pacific with shipborne online measurements.

Anthropogenic emissions are recognized as significant contributors to atmospheric soluble iron (Fe) in recent years, which may affect marine primary productivity, especially in Fe-limited areas. However, the contribution of different emission sources to Fe in marine aerosol has been primarily estimated by modeling approaches. Quantifying anthropogenic Fe based on field measurements remains a great challenge. In this study, online multi-element measurements and Positive Matrix Factorization (PMF) were combined for the first time to quantify sources of atmospheric Fe and soluble Fe in the Northwest Pacific during a cruise in spring 2015. Fe concentration in 624 atmospheric PM2.5 samples measured online was 74.58 ± 90.87 ng/m3. The PMF results showed anthropogenic activities, including industrial coal combustion, biomass burning, and maritime transport, were important in this region, contributing 31.4 % of atmospheric Fe on average. In addition, anthropogenic Fe concentration resolved by PMF was comparable to the simulation results of the CMAQ (Community Multiscale Air Quality) and GEOS-Chem (Goddard Earth Observing System-Chemical transport) models, with better correlation to CMAQ (r = 0.76) than GEOS-Chem (r = 0.26). This study developed a new method to estimate atmospheric soluble Fe, which integrates Fe source apportionment results and Fe solubility from different sources. Soluble Fe concentration was estimated as 3.93 ± 5.14 ng/m3, of which 87.0 % was attributed to anthropogenic emissions. Notably, ship emission alone contributed 27.5 % of soluble Fe, though its contribution to total Fe was only 2.2 %. Finally, the total deposition fluxes of atmospheric Fe (37.11 ± 38.43 µg/m2/day) and soluble Fe (1.85 ± 2.13 µg/m2/day) were estimated. This study developed a new methodology for quantifying contribution of anthropogenic emissions to Fe in marine aerosol, which could greatly help the assessment of impacts of human activities on marine environment.

Estimation of sea surface nitrate from space: Current status and future potential.

Sea surface nitrate (SSN) plays an important role in assessing phytoplankton growth and new production in the ocean. Field sampling of SSN data is important, but limited by data quantity both spatially and temporally. Satellite remote sensing can contribute through providing spatial and temporal data to such assessments. During the past 30 years many studies have been published focusing on SSN retrievals from satellites to a greater or less extent. In this study, we reviewed the progresses of SSN estimation from satellites in both open ocean and coastal waters. Because of the lack of electromagnetic properties of SSN, satellite retrievals of SSN were most realized by developing relationships between SSN and related environmental variables (e.g., sea surface temperature, chlorophyll-a concentration, sea surface salinity), using traditional empirical regressions and novel machine learning techniques. We synthesized most of the peer-reviewed studies for both open and coastal oceans, in terms of study areas, model inputs, regression formulas, and model uncertainties. In general, regional SSN algorithms were most developed in coastal oceans with upwelling or river discharges. The published SSN algorithms had varying uncertainties with a wide range of 0.83-6.87 µmol/L, and the uncertainties were significantly reduced in recent studies, with more field measurements available and better understanding of the physical and biogeochemical processes in driving nitrate dynamics.

Manufacturing of human corneal endothelial grafts.

PURPOSE: Human corneal endothelial cells (HCECs) play a significant role in maintaining visual function. However, these cells are notorious for their limited proliferative capacity in vivo. Current treatment of corneal endothelial dysfunction resorts to corneal transplantation. Herein we describe an ex vivo engineering method to manufacture HCEC grafts suitable for transplantation through reprogramming into neural crest progenitors. METHODS: HCECs were isolated by collagenase A from stripped Descemet membrane of cadaveric corneoscleral rims, and induced reprogramming via knockdown with p120 and Kaiso siRNAs on collagen IV-coated atelocollagen. Engineered HCEC grafts were released after assessing their identity, potency, viability, purity and sterility. Phase contrast was used for monitoring cell shape, graft size, and cell density. Immunostaining was used to determine the normal HCEC phenotype with expression of N-cadherin, ZO-1, ATPase, acetyl-α-tubulin, γ-tubulin, p75NTR, α-catenin, ß-catenin, and F-actin. Stability of manufactured HCEC graft was evaluated after transit and storage for up to 3 weeks. The pump function of HCEC grafts was measured by lactate efflux. RESULTS: One HCEC graft suitable for corneal transplantation was generated from 1/8th of the donor corneoscleral rim with normal hexagonal cell shape, density, and phenotype. The manufactured grafts were stable for up to 3 weeks at 37 °C or up to 1 week at 22 °C in MESCM medium and after transcontinental shipping at room temperature by retaining normal morphology (hexagonal, >2000 cells/mm2, >8 mm diameter), phenotype, and pump function. CONCLUSIONS: This regenerative strategy through knockdown with p120 and Kaiso siRNAs can be used to manufacture HCEC grafts with normal phenotype, morphology and pump function following prolonged storage and shipping.

VEGF mimetic peptide-conjugated nanoparticles for magnetic resonance imaging and therapy of myocardial infarction.

To reduce the mortality of myocardial infarction (MI), accurate detection of the infarct and appropriate prevention against ischemia/reperfusion (I/R) induced cardiac dysfunction are highly desired. Considering that vascular endothelial growth factor (VEGF) receptors are overexpressed in the infarcted heart and VEGF mimetic peptide QK binds specifically to VEGF receptors and activates vascularization, the PEG-QK-modified, gadolinium-doped carbon dots (GCD-PEG-QK) were formulated. This research aims to investigate the magnetic resonance imaging (MRI) capability of GCD-PEG-QK on myocardial infarct and their therapeutic effect on I/R-induced myocardial injury. These multifunctional nanoparticles exhibited good colloidal stability, excellent fluorescent and magnetic property, and satisfactory biocompatibility. Intravenous injection of GCD-PEG-QK nanoparticles post myocardial I/R displayed accurate MRI of the infarct, enhanced efficacy of QK peptide on pro-angiogenesis, and amelioration of cardiac fibrosis, remodeling and dysfunction, probably via the improvement on QK's in vivo stability and MI-targeting. Collectively, the data suggested that this theranostic nanomedicine can realize precise MRI and effective therapy for acute MI in a non-invasive manner.

Poly-α, ß-d, l-Aspartyl-Arg-Gly-Asp-Ser-Based Urokinase Nanoparticles for Thrombolysis Therapy.

The most concerning adverse effects of thrombolytic agents are major bleeding and intracranial hemorrhage due to their short half-life, low fibrin specificity, and high dosage. To alleviate bleeding side effects during thrombolytic therapy which would bring about the risk of aggravation, we try to find a novel biodegradable delivery nanosystem to carry drugs to target the thrombus, reduce the dosage of the drug, and system side effects. A novel urokinase/poly-α, ß-d, l-aspartyl-Arg-Gly-Asp-Ser complex (UK/PD-RGDS) was synthesized and simply prepared. Its thrombolytic potency was assayed by the bubble-rising method and in vitro thrombolytic activity by the thrombus clot lysis assay separately. The in vivo thrombolytic activity and bleeding complication were evaluated by a rat model of carotid arteriovenous bypass thrombolysis. The thrombolytic potency (1288.19 ± 155.20 U/mg) of the UK/PD-RGDS complex nano-globule (18-130 nm) was 1.3 times that of commercial UK (966.77 ± 148.08 U/mg). In vivo, the UK/PD-RGDS complex (2000 IU/kg) could reduce the dose of UK by 90% while achieving the equivalent thrombolysis effect as the free UK (20,000 IU/kg). Additionally, the UK/PD-RGDS complex decreased the tail bleeding time compared with UK. The organ distribution of the FITC-UK/PD-RGDS complex was explored in the rat model. The UK/PD-RGDS complex could provide a promising platform to enhance thrombolytic efficacy significantly and reduce the major bleeding degree.

Environmental impacts of three Asian dust events in the northern China and the northwestern Pacific in spring 2021.

In March 2021, China experienced three dust events (Dust-1, 2, 3), especially the first of which was reported as the strongest one in recent ten years. Their environmental impacts have received great attention, demanding comprehensive study to assess such impacts quantitatively. Multiple advanced measurement methods, including satellite, ground-based lidar, online aerosol speciation instrument, and biogeochemical Argo float, were applied to examine and compare the transport paths, optical and chemical properties, and impacts of these three dust events on urban air quality and marine ecosystem. The results showed that Dust-1 exhibited the largest impacts on urban area, increasing PM10 concentration in Beijing, Shuozhou, and Shijiazhuang up to 7525, 3819, and 2992 µg m-3, respectively. However, due to fast movement of the Mongolian low-pressure cyclone, the duration of northwest wind over the land was quite short (e.g., only 10 h in Beijing), which prevented the transport of dust plume to the northwestern Pacific, resulting in limited impact on the ocean. Dust-2 and Dust-3, though weaker in intensity, were transported directly to the sea, and led to a substantial increase in chlorophyll-a concentration (up to near 3 times) in the northwestern Pacific, comparing to climatological value. This indicates that the impacts of dust events on ocean was not necessarily and positively correlated to their impacts on land. Based on the analyses of land-ocean-space integrated observational data and synoptic systems, this study examined how marine ecosystem responded to three significant Asian dust events in spring 2021 and quantitatively assessed the overall impacts of mega dust storms both on land and ocean, which could also provide an interdisciplinary research methodology for future research on strong aerosol emission events such as wildfire and volcanic eruption.

Study on the application value of BACs-on-Beads technology combined with chromosome karyotype analysis in prenatal diagnosis.

Background: Bacterial artificial chromosome (BAC) marker-microsphere identification/separation technique [BACs-on-Beads (BoBs)] not only has a high detection rate for major chromosomal changes, but also for the other 9 microdeletion syndromes. In this study, the application value of BoBs combined with karyotype detection in prenatal diagnosis was evaluated. Methods: The amniotic fluid samples of 132 pregnant women with prenatal diagnosis indications in Harbin Red Cross Central Hospital from June 2018 to June 2019 were collected and subjected to the detection of BoBs and routine karyotyping. Results: Among the 132 pregnant women's amniotic fluid samples, 30 cases were abnormal in BoBs detection, with a detection rate of 22.73%, and 29 cases were abnormal in chromosome karyotype analysis, with a detection rate of 21.97%. Among them, 1 case of DiGeorge Type I microdeletion syndrome BoBs was successfully detected. The karyotype analysis failed to detect the same syndrome; the total coincidence rate of two methods was 99.24%, the positive coincidence rate was 100.00%, and the negative coincidence rate was 99.03%; the sensitivity, specificity and positive predictive value (PPV), and negative predictive value (NPV) of the chromosome karyotype analysis was 96.67%, 100%, and 99.03%, respectively; the accuracy, specificity, and PPV/NPV of BoBs detection were 100%. Conclusions: When BoBs technology is combined with chromosome karyotype analysis, it can increase the detection rate of fetal chromosomal abnormalities, which could provide a basis for clinical prevention and follow-up diagnosis and treatment.

Southern Ocean carbon export efficiency in relation to temperature and primary productivity.

Satellite remote sensing and numerical models are widely used to estimate large-scale variations in ocean carbon export, but the relationship between export efficiency (e-ratio) of sinking organic carbon out of the surface ocean and its drivers remains poorly understood, especially in the Southern Ocean. Here, we assess the effects of temperature and primary productivity on e-ratio by combining particulate organic carbon export flux from in situ measurements during 1997-2013, environmental parameters from satellite products, and outputs from ocean biogeochemical models in the Southern Ocean. Results show that "High Productivity Low E-ratio" (HPLE) is a common phenomenon in the Subantarctic Zone and the Polar Frontal Zone, but not the Antarctic Zone. The e-ratio shows little dependence on temperature below 6 °C. Our results support the hypothesis that the HPLE phenomenon is due to the large contribution of non-sinking organic carbon. Both temperature and ballast minerals play less important roles in controlling e-ratio than ecosystem structure at low temperatures. These findings suggest that non-sinking organic carbon, ecosystem structure, and region-specific parameterizations of e-ratio are key factors to quantify the carbon export in the Southern Ocean.

Advances in culture, expansion and mechanistic studies of corneal endothelial cells: a systematic review.

Human corneal endothelial cells are notorious for their restricted proliferative ability in vivo and in vitro. Hence, injury or dysfunction of these cells may easily result in blindness. Currently, the only treatment is to transplant a donor cornea that contains a healthy corneal endothelium. However there is a severe global shortage of donor corneas and there remains an unmet clinical need to engineer human corneal grafts with healthy corneal endothelium. In this review, we present current advances in the culture, expansion, and molecular understandings of corneal endothelial cells in vitro in order to help establish methods of engineering human corneal endothelial grafts.

Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model.

In this article, human limbal niche cells (LNC) or bone marrow derived mesenchymal stem cells (BMMSC) were used to prevent limbal stem cell deficiency (LSCD) in an alkali burn rabbit model and their results were compared. The epithelial cell defect area, corneal neovascularization, and the print cell cytometry were quantified to grade the severity of LSCD. Three months after the alkali burn, a partial LSCD was observed in the control group (no treatment) indicated by chronic corneal epithelial defects, positive corneal fluorescein staining, neovascularization and goblet cell migration. In contrast, the severity of LSCD in both the LNC and BMMSC transplantation groups was dramatically reduced as shown by smaller epithelial cell defects, decreased fluorescein sodium staining, decreased neovascularization and decreased goblet cell density. Interestingly, the LNC group was shown to more effectively prevent LSCD than the BMMSC group. Further analysis indicated subconjunctivally transplanted LNCs were more powerful than BMMSCs to prevent LSCD, at least partially, due to increased activation of SCF-c-Kit signal. We conclude that LNCs are a more powerful resource than BMMSCs to prevent LSCD in an alkali burn rabbit model, at least partially due to increased activation of SCF signaling.

Limbal niche cells are a potent resource of adult mesenchymal progenitors.

Limbal niche cells located in the limbal Palisades of Vogt are mesenchymal stem cells that reside next to limbal basal epithelial cells. Limbal niche cells are progenitors that express embryonic stem cell markers such as Nanog, Nestin, Oct4, Rex1, Sox2 and SSEA4, mesenchymal cell markers such as CD73, CD90 and CD105, and angiogenesis markers such as Flk-1, CD31, CD34, VWF, PDGFRß and α-SMA, but negative for CD45. In addition, the stemness of limbal niche cells can be maintained during their cell culture in a three-dimension environment. Furthermore, expanded limbal niche cells have the capability to undergo adipogenesis, chondrogenesis, osteogenesis and endogenesis in vitro, indicating that they are in fact a valuable resource of adult progenitors. Furthermore studies on how the limbal niche cells regulate the aforementioned stemness and corneal fate decision are warranted, as those investigations will shed new light on how mesenchymal progenitors reverse limbal stem cell deficiency and lead to new methods for limbal niche cell treatment.

Characterization and Prospective of Human Corneal Endothelial Progenitors.

Corneal endothelial cells play a critical role in maintaining corneal transparency and dysfunction of these cells caused by aging, diseases (such as Fuch's dystrophy), injury or surgical trauma, which can lead to corneal edema and blindness. Due to their limited proliferative capacity in vivo, the only treatment method is via transplantation of a cadaver donor cornea. However, there is a severe global shortage of donor corneas. To circumvent such issues, tissue engineering of corneal tissue is a viable option thanks to the recent discoveries in this field. In this review, we summarize the recent advances in reprogramming adult human corneal endothelial cells into their progenitor status, the expansion methods and characteristics of human corneal endothelial progenitors, and their potential clinical applications as corneal endothelial cell grafts.

Human Corneal Endothelial Cells Expanded In Vitro Are a Powerful Resource for Tissue Engineering.

Human corneal endothelial cells have two major functions: barrier function mediated by proteins such as ZO-1 and pump function mediated by Na-K-ATPase which help to maintain visual function. However, human corneal endothelial cells are notorious for their limited proliferative capability in vivo and are therefore prone to corneal endothelial dysfunction that eventually may lead to blindness. At present, the only method to cure corneal endothelial dysfunction is by transplantation of a cadaver donor cornea with normal corneal endothelial cells. Due to the global shortage of donor corneas, it is vital to engineer corneal tissue in vitro that could potentially be transplanted clinically. In this review, we summarize the advances in understanding the behavior of human corneal endothelial cells, their current engineering strategy in vitro and their potential applications.

The Role of Cyclooxygenase-2 in Colorectal Carcinogenesis.

Colorectal cancer is a major worldwide health care problem that accounts for 1 million new cases each year. The risk factors for this disease include hereditary factors, environmental agents, and inflammatory stimuli that affect the gastrointestinal tract. Among these risk factors, cyclooxygenase-2 (COX-2) is one of the major players in the progression of colorectal cancer; however, the detailed mechanism of its role in causing colorectal cancer is still not well understood. In addition, the role of COX-2 signaling through the interaction in the epithelial and stromal compartments on colorectal carcinogenesis has not been fully illustrated. In the present review, we provide published evidence to demonstrate that (1) COX-2 signaling plays a major role in the progression of colorectal cancer, (2) activation of COX-2 in the stromal compartment also contributes to colorectal carcinogenesis, and (3) inhibition of COX-2 signaling by COX-2 inhibitors might be an effective method to control colorectal cancer. We have also summarized recent advances and insights from mechanistic studies of colorectal cancer to help prevent and control this deadly disease and provide our opinion regarding the importance of risk reduction and disease prevention for colorectal cancer.

The Limbal Epithelial Progenitors in the Limbal Niche Environment.

Limbal epithelial progenitors are stem cells located in limbal palisades of vogt. In this review, we present the audience with recent evidence that limbal epithelial progenitors may be a powerful stem cell resource for the cure of human corneal stem cell deficiency. Further understanding of their mechanism may shed lights to the future successful application of stem cell therapy not only to the eye tissue, but also to the other tissues in the human body.

Senescence Mediated by p16INK4a Impedes Reprogramming of Human Corneal Endothelial Cells into Neural Crest Progenitors.

Human corneal endothelial cells (HCECs) have limited proliferative capacity due to "contact-inhibition" at G1 phase. Such contact-inhibition can be delayed from Day 21 to Day 42 by switching EGF-containing SHEM to LIF/bFGF-containing MESCM through transient activation of LIF-JAK1-STAT3 signaling that delays eventual nuclear translocation of p16INK4a. Using the latter system, we have reported a novel tissue engineering technique by implementing 5 weekly knockdowns with p120 catenin (p120) and Kaiso siRNAs since Day 7 to achieve effective expansion of HCEC monolayers to a transplantable size with a normal HCEC density, through reprogramming of HCECs into neural crest progenitors by activating p120-Kaiso-RhoA-ROCK-canonical BMP signaling. Herein, we noted that a single knockdown with p120-Kaiso siRNAs at Day 42 failed to achieve such reprogramming when contact inhibition transitioned to senescence with nuclear translocation of p16INK4a. In contrast, 5 weekly knockdowns with p120-Kaiso siRNAs since Day 7 precluded senescence mediated by p16INK4a by inducing nuclear translocation of Bmi1 because of sustained activation of JAK2-STAT3 signaling downstream of p120-Kaiso-RhoA-ROCK signaling. STAT3 or Bmi1 siRNA impeded nuclear exclusion of p16INK4a and suppressed the reprogramming induced by p120-Kaiso siRNAs, suggesting that another important engineering strategy of HCEC lies in prevention of senescence mediated by nuclear translocation of p16INK4a.

Engineering of Human Corneal Endothelial Grafts.

Human corneal endothelial cells (HCEC) play a pivotal role in maintaining corneal transparency. Unlike in other species, HCEC are notorious for their limited proliferative capacity in vivo after diseases, injury, aging, and surgery. Persistent HCEC dysfunction leads to sight-threatening bullous keratopathy with either an insufficient cell density or retrocorneal membrane due to endothelial-mesenchymal transition (EMT). Presently, the only solution to restore vision in eyes inflicted with bullous keratopathy or retrocorneal membrane relies upon transplantation of a cadaver human donor cornea containing a healthy corneal endothelium. Due to a severe global shortage of donor corneas, in conjunction with an increasing trend toward endothelial keratoplasty, it is opportune to develop a tissue engineering strategy to produce HCEC grafts. Prior attempts of producing these grafts by unlocking the contact inhibition-mediated mitotic block using trypsin-EDTA and culturing of single HCEC in a bFGF-containing medium run the risk of losing the normal phenotype to EMT by activating canonical Wnt signaling and TGF-ß signaling. Herein, we summarize our novel approach in engineering HCEC grafts based on selective activation of p120-Kaiso signaling that is coordinated with activation of Rho-ROCK-canonical BMP signaling to reprogram HCEC into neural crest progenitors. Successful commercialization of this engineering technology will not only fulfill the global unmet need but also encourage the scientific community to re-think how cell-cell junctions can be safely perturbed to uncover novel therapeutic potentials in other model systems.

Stabilization of source-separated human urine by chemical oxidation.

The inhibitory effect of ozone and hydrogen peroxide (HP) on urea hydrolysis in stored urine was investigated and compared. Ozone showed less effect on urea hydrolysis due to the complicated composition of urine (including a large amount of urease-producing bacteria) and bacteria regeneration. Ozone concentration and total heterotrophic bacteria analysis demonstrated that residual ozone concentration decreased by 43% within 15 hr from 13.50 to 7.72 mg/L in the one-time ozonation urine test, and finally completely decomposed within 4 days. In addition, bacteria regenerated quickly after ozone completely decomposed. However, HP showed a significant effect on inhibiting urea hydrolysis not only in stored urine but also in fecal-contaminated urine. The suitable doses of applied HP to inhibit urea hydrolysis in stored urine, concentrations of 0.5 and 1.0 g feces per liter of fecal-contaminated urine, were 0.03, 0.16 and 0.23 mol/L, respectively. The urea concentrations after 2 months stored were 7,145, 7,109 and 7,234 mg/L, respectively.

Quantification of Mesenchymal Stem Cells (MSCs) at sites of human prostate cancer.

Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious and inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes and chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs and BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.

Human prostate cancer initiating cells isolated directly from localized cancer do not form prostaspheres in primary culture.

BACKGROUND: Recent experimental studies suggest that hierarchical expansion from a minor population of cancer cells with an unlimited self-renewal capacity, termed cancer initiating cells (CICs), drives both lethality and heterogeneity of prostate cancer. Human prostate CICs have been established from only two primary prostate cancer patients, with the remaining established CIC lines being derived from metastatic sites from <10 patients. This suggests that the established CIC lines are significant "outliers" and may not be representative of the prostate CICs seen clinically. Thus, there is an urgent need to develop new approaches to achieve the "routine" establishment of CIC containing lines, particularly derived from primary prostate cancers. METHODS: In the present studies, we confirmed that in serum free, high Ca(2+) (i.e., DMEN: F12) growth factor defined (GFD) media plus androgen, a large (n = 10) series of established human prostate cancer cell lines derived from both localized and metastatic sites characteristically self-associate in suspension and grow as unattached spheroids, termed prostaspheres which contain CICs based upon their self-renewal in vitro and tumorigenicity in vivo. RESULTS: Unfortunately, however, while dissociated single cells from human primary prostate cancer tissues are viable, contain CICs as documented by their ability to take and proliferate as xenografts, and produce prostaspheres when plated with serum free, high Ca(2+) /GFD-media plus androgen onto standard tissue culture flask, these prostasphere do not contain CICs. CONCLUSION: The development of reproducibly methods to culture CICs isolated directly from localized cancers is still an urgent unmeet need of the prostate cancer research community.