MicroRNA-200 Loaded Lipid Nanoparticles Promote Intestinal Epithelium Regeneration in Canonical MicroRNA-Deficient Mice.

Intestinal epithelium undergoes regeneration after injuries, and the disruption of this process can lead to inflammatory bowel disease and tumorigenesis. Intestinal stem cells (ISCs) residing in the crypts are crucial for maintaining the intestinal epithelium's homeostasis and promoting regeneration upon injury. However, the precise role of DGCR8, a critical component in microRNA (miRNA) biogenesis, in intestinal regeneration remains poorly understood. In this study, we provide compelling evidence demonstrating the indispensable role of epithelial miRNAs in the regeneration of the intestine in mice subjected to 5-FU or irradiation-induced injury. Through a comprehensive pooled screen of miRNA function in Dgcr8-deficient organoids, we observe that the loss of the miR-200 family leads to the hyperactivation of the p53 pathway, thereby reducing ISCs and impairing epithelial regeneration. Notably, downregulation of the miR-200 family and hyperactivation of the p53 pathway are verified in colonic tissues from patients with active ulcerative colitis (UC). Most importantly, the transient supply of miR-200 through the oral delivery of lipid nanoparticles (LNPs) carrying miR-200 restores ISCs and promotes intestinal regeneration in mice following acute injury. Our study implies the miR-200/p53 pathway as a promising therapeutic target for active UC patients with diminished levels of the miR-200 family. Furthermore, our findings suggest that the clinical application of LNP-miRNAs could enhance the efficacy, safety, and acceptability of existing therapeutic modalities for intestinal diseases.

Transforming Growth Factor Beta Promotes Inflammation and Tumorigenesis in Smad4-Deficient Intestinal Epithelium in a YAP-Dependent Manner.

Transforming growth factor beta (TGF-ß), a multifunctional cytokine, plays critical roles in immune responses. However, the precise role of TGF-ß in colitis and colitis-associated cancer remains poorly defined. Here, it is demonstrated that TGF-ß promotes the colonic inflammation and related tumorigenesis in the absence of Smad family member 4 (Smad4). Smad4 loss in intestinal epithelium aggravates colitis and colitis-associated neoplasia induced by dextran sulfate sodium (DSS) and azoxymethane/dextran sulfate sodium (AOM/DSS), leading to over-activated immune responses and increased TGF-ß1 levels. In Smad4-deficient organoids, TGF-ß1 stimulates spheroid formation and impairs intestinal stem cell proliferation and lineage specification. YAP, whose expression is directly upregulated by TGF-ß1 after Smad4 deletion, mediates the effect of TGF-ß1 by interacting with Smad2/3. Attenuation of YAP/TAZ prevents TGF-ß1-induced spheroid formation in Smad4-/- organoids and alleviates colitis and colitis-associated cancer in Smad4-deficient mice. Collectively, these results highlight an integral role of the TGF-ß/Smad4 axis in restraining intestinal inflammation and tumorigenesis and suggest TGF-ß or YAP signaling as therapeutic targets for these gastrointestinal diseases intervention.

Discovery and construction of prognostic model for clear cell renal cell carcinoma based on single-cell and bulk transcriptome analysis.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common malignant kidney tumor in adults. Single-cell transcriptome sequencing can provide accurate gene expression data of individual cells. Integrated single-cell and bulk transcriptome data from ccRCC samples provide comprehensive information, which allows the discovery of new understandings of ccRCC and the construction of a novel prognostic model for ccRCC patients. METHODS: Single-cell transcriptome sequencing data was preprocessed by using the Seurat package in R software. Principal component analysis (PCA) and the t-distributed stochastic neighbor embedding (t-SNE) algorithm were used to perform cluster classification. Two subtypes of cancer cells were identified, pseudotime trajectory analysis and gene ontology (GO) analysis were conducted with the monocle and clusterProfiler packages. Two novel cancer cell biomarkers were identified according to the single-cell sequencing and were confirmed by The Cancer Genome Atlas (TCGA) data. T cell-related marker genes according to single-cell sequencing were screened by a combination of Kaplan-Meier (KM) analysis, univariate Cox analysis, least absolute shrinkage and selection operator (Lasso) regression and multivariate Cox analysis of TCGA data. Four survival predicting genes were screened out to develop a risk score model. A nomogram consisting of the risk score and clinical information was constructed to predict the prognosis for ccRCC patients. RESULTS: A total of 5,933 cells were included in the study after quality control. Fifteen cell clusters were classified by PCA and t-SNE algorithm. Two clusters of cancer cells with distinct differentiation status were identified. Besides, GO analysis revealed that biological processes were different between the two subgroups. Egl-9 family hypoxia-inducible factor 3 (EGLN3) and nucleolar protein 3 (NOL3) were specifically expressed in cancer cell clusters, bulk RNA sequencing data from TCGA confirmed their high expression in ccRCC tissues. GTSE1, CENPF, SMC2 and H2AFV were screened out and applied to the construction of risk score model. A nomogram was generated to predict prognosis of ccRCC by combing the risk score and clinical parameters. CONCLUSIONS: We integrated single-cell and bulk transcriptome data from ccRCC in this study. Two subtypes of ccRCC cells with different biological characteristics and two potential biomarkers of ccRCC were discovered. A novel prognostic model was constructed for clinical application.

Convolutional neural network with group theory and random selection particle swarm optimizer for enhancing cancer image classification.

As an epitome of deep learning, convolutional neural network (CNN) has shown its advantages in solving many real-world problems. Successful CNN applications on medical prognosis and diagnosis have been achieved in recent years. Their common goal is to recognize the insights from the subtle details from medical images by building a suitable CNN model with maximum accuracy and minimum error. The CNN performance is extremely sensitive to the parameter tuning for any given network structure. To approach this concern, a novel self-tuning CNN model is proposed with a significant characteristic of having a metaheuristic-based optimizer. The most optimal set of parameters is often found via our proposed method, namely group theory and random selection-based particle swarm optimization (GTRS-PSO). The insights of symmetric essentials of model structure and parameter correlation are extracted, followed by the hierarchical partitioning of parameter space, and four operators on those partitions are designed for moving neighborhoods and formulating the swarm topology accordingly. The parameters are updated by a random selection strategy at each interval of partitions during the search process. Preliminary experiments over two radiology image datasets: breast cancer and lung cancer, are conducted for a comprehensive comparison of GTRS-PSO versus other optimization algorithms. The results show that CNN with GTRS-PSO optimizer can achieve the best performance for cancer image classifications, especially when there are symmetric components inside the data properties and model structures.

Overexpression of MAX dimerization protein 3 (MXD3) predicts poor prognosis in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of malignant kidney tumor. The molecular mechanism of ccRCC is complicated, and few effective prognostic predictors have been applied to clinical practice. MAX dimerization protein 3 (MXD3) is generally considered a transcription factor of the MYC/MAX/MAD transcriptional network. This study aimed to investigate the impact of MXD3 in ccRCC. METHODS: Gene expression profiles and clinical data of ccRCC were downloaded from The Cancer Genome Atlas (TCGA) database. MXD3 expression levels between tumors and adjacent normal tissues were compared. The influence of MXD3 on overall survival (OS) was evaluated using the Kaplan-Meier method. Associations between MXD3 expression and clinical features were assessed with the Kruskal test and Wilcoxon test. Univariate and multivariate Cox analyses were performed to observe the impact of MXD3 expression and clinical features on prognosis. The correlation between MXD3 and ccRCC immune infiltration was estimated with TIMER. The DNA methylation levels of the MXD3 promoter were obtained from UALCAN. Gene set enrichment analysis (GSEA) was conducted to explore the biological signaling pathways. RESULTS: MXD3 was overexpressed in ccRCC tumor tissues compared with adjacent normal kidney tissues. High expression of MXD3 was significantly correlated with poor prognosis. MXD3 expression levels were associated with tumor grade, tumor stage, tumor (T) classification and metastasis (M) classification. Univariate and multivariate Cox analyses showed that high expression of MXD3 was an independent risk factor for OS in ccRCC. MXD3 expression was positively correlated with the infiltrating levels of B cells and myeloid dendritic cells, and negatively correlated with macrophages. The MXD3 promoter region tended to be hypomethylated in ccRCC compared with normal tissues. GSEA identified homologous recombination, base excision repair, and glycerophospholipid metabolism as differentially enriched in ccRCC with high MXD3 expression. CONCLUSIONS: This study suggests that high expression of MXD3 is an independent risk factor for poor prognosis in ccRCC. MXD3 expression potentially contributes to regulation of immune infiltration and cell proliferation in ccRCC, and the aberrant expression of MXD3 in tumor tissues could be caused by hypomethylation of gene promoter. MXD3 could be an effective prognostic biomarker and potential therapeutic target for ccRCC.

White learning methodology: A case study of cancer-related disease factors analysis in real-time PACS environment.

BACKGROUND AND OBJECTIVE: Bayesian network is a probabilistic model of which the prediction accuracy may not be one of the highest in the machine learning family. Deep learning (DL) on the other hand possess of higher predictive power than many other models. How reliable the result is, how it is deduced, how interpretable the prediction by DL mean to users, remain obscure. DL functions like a black box. As a result, many medical practitioners are reductant to use deep learning as the only tool for critical machine learning application, such as aiding tool for cancer diagnosis. METHODS: In this paper, a framework of white learning is being proposed which takes advantages of both black box learning and white box learning. Usually, black box learning will give a high standard of accuracy and white box learning will provide an explainable direct acyclic graph. According to our design, there are 3 stages of White Learning, loosely coupled WL, semi coupled WL and tightly coupled WL based on degree of fusion of the white box learning and black box learning. In our design, a case of loosely coupled WL is tested on breast cancer dataset. This approach uses deep learning and an incremental version of Naïve Bayes network. White learning is largely defied as a systemic fusion of machine learning models which result in an explainable Bayes network which could find out the hidden relations between features and class and deep learning which would give a higher accuracy of prediction than other algorithms. We designed a series of experiments for this loosely coupled WL model. RESULTS: The simulation results show that using WL compared to standard black-box deep learning, the levels of accuracy and kappa statistics could be enhanced up to 50%. The performance of WL seems more stable too in extreme conditions such as noise and high dimensional data. The relations by Bayesian network of WL are more concise and stronger in affinity too. CONCLUSION: The experiments results deliver positive signals that WL is possible to output both high classification accuracy and explainable relations graph between features and class.

Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion.

Targeting immune checkpoints, such as PD-L1 and its receptor PD-1, has opened a new avenue for treating cancers. Understanding the regulatory mechanism of PD-L1 and PD-1 will improve the clinical response rate and efficacy of PD-1/PD-L1 blockade in cancer patients and the development of combinatorial strategies. VGLL4 inhibits YAP-induced cell proliferation and tumorigenesis through competition with YAP for binding to TEADs. However, whether VGLL4 has a role in anti-tumor immunity is largely unknown. Here, we found that disruption of Vgll4 results in potent T cell-mediated tumor regression in murine syngeneic models. VGLL4 deficiency reduces PD-L1 expression in tumor cells. VGLL4 interacts with IRF2BP2 and promotes its protein stability through inhibiting proteasome-mediated protein degradation. Loss of IRF2BP2 results in persistent binding of IRF2, a transcriptional repressor, to PD-L1 promoter. In addition, YAP inhibits IFNγ-inducible PD-L1 expression partially through suppressing the expression of VGLL4 and IRF1 by YAP target gene miR-130a. Our study identifies VGLL4 as an important regulator of PD-L1 expression and highlights a central role of VGLL4 and YAP in the regulation of tumor immunity.

HBx gene transfection affects the cycle of primary renal tubular epithelial cells through regulating cyclin expression.

Hepatitis B virus X protein (HBx) has been previously demonstrated to be associated with the regulation of cell proliferation; however, the exact mechanisms underlying this effect remain unclear. The present study aimed to investigate the regulatory mechanism of HBx on the cycle progression of primary renal tubular epithelial cells. Primary renal tubular epithelial cells of Sprague Dawley (SD) rats were separated and cultured. The morphology of cultured cells was characterized by immunohistochemical analysis and the results demonstrated that primary renal tubular epithelial cells with the expected morphology and distribution were successfully separated and cultured from SD rats. HBx gene pcDNA3.1/myc vector and empty vector were constructed and transfected into cells as HBx and empty groups, respectively. Following transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were determined by reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. The results demonstrated that following HBx gene transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were significantly upregulated, compared with the empty control group (P<0.05). Furthermore, cell apoptosis and the cell cycle were evaluated by Annexin V­fluorescein isothiocyanate/propidium iodide staining and flow cytometry. HBx gene transfection significantly inhibited the cell apoptosis (P<0.05), promoted cell cycle progression from the G1 to S phase and arrested the cell cycle in the S phase. Therefore, the results of the present study indicated that HBx gene transfection may regulate the apoptosis and cell cycle of primary renal tubular epithelial cells by affecting the expression of cyclins. The results of the present study may improve the understanding of pathogenesis associated with HBV­associated glomerulonephritis, and may also provide insight and theoretical support for the future design and development of drugs for the treatment of hepatitis B virus.

MEKK2 and MEKK3 suppress Hedgehog pathway-dependent medulloblastoma by inhibiting GLI1 function.

Hedgehog (Hh) pathway plays a pivotal role in diverse aspects of development and postnatal physiology. Perturbation of Hh signaling and activation of GLI1 (glioma-associated oncogene 1), a dedicated transcription factor for Hh pathway, are highly associated with several cancers, such as medulloblastoma and basal cell carcinoma. Dynamic and precise control of GLI1 activity is thus important to ensure proper homeostasis and tumorigenesis. Here we show that MEKK2 (MAP3K2) and MEKK3 (MAP3K3) inhibit GLI1 transcriptional activity and oncogenic function through phosphorylation on multiple Ser/Thr sites of GLI1, which reduces GLI1 protein stability, DNA-binding ability, and increases the association of GLI1 with SUFU. Interestingly, MEKK2 and MEKK3 are responsible for FGF2-mediated inhibition on Hh signaling. Moreover, expression of MEKK2 and MEKK3 inhibits medulloblastoma cell proliferation and negatively correlates with Hh pathway activity in medulloblastoma clinical samples. Together, these findings reveal a novel noncanonical GLI1 regulation and provide a potential therapeutic target for the treatment of cancers with aberrant Hh pathway activation, such as medulloblastoma.

Hepatic loss of Lissencephaly 1 (Lis1) induces fatty liver and accelerates liver tumorigenesis in mice.

The liver is a major organ in lipid metabolism, and its malfunction leads to various diseases. Nonalcoholic fatty liver disease, the most common chronic liver disorder in developed countries, is characterized by the abnormal retention of excess lipid within hepatocytes and predisposes individuals to liver cancer. We previously reported that the levels of Lissencephaly 1 (LIS1, also known as PAFAH1B1) are down-regulated in human hepatocellular carcinoma. Following up on this observation, we found that genetic deletion of Lis1 in the mouse liver increases lipid accumulation and inflammation in this organ. Further analysis revealed that loss of Lis1 triggers endoplasmic reticulum (ER) stress and reduces triglyceride secretion. Attenuation of ER stress by addition of tauroursodeoxycholic acid (TUDCA) diminished lipid accumulation in the Lis1-deficient hepatocytes. Moreover, the Golgi stacks were disorganized in Lis1-deficient liver cells. Of note, the Lis1 liver-knockout mice exhibited increased hepatocyte ploidy and accelerated development of liver cancer after exposure to the liver carcinogen diethylnitrosamine (DEN). Taken together, these findings suggest that reduced Lis1 levels can spur the development of liver diseases from steatosis to liver cancer and provide a useful model for delineating the molecular pathways that lead to these diseases.

Imaging Macrophage Accumulation in a Murine Model of Chronic Pancreatitis with 125I-Iodo-DPA-713 SPECT/CT.

Pancreatitis remains a diagnostic challenge in patients with mild to moderate disease, with current imaging modalities being inadequate. Given the prominent macrophage infiltration in chronic pancreatitis, we hypothesized that 125I-iodo-DPA-713, a small-molecule radiotracer that specifically targets macrophages, could be used with SPECT/CT to image pancreatic inflammation in a relevant experimental model. Methods: Chronic pancreatitis was induced with cerulein in C57BL/6 mice, which were contrasted with saline-injected control mice. The animals were imaged at 7 wk after induction using N,N-diethyl-2-(2-(3-125I-iodo-4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (125I-iodo-DPA-713) SPECT/CT or 18F-FDG PET/CT. The biodistribution of 125I-iodo-DPA-713 was determined under the same conditions, and a pair of mice was imaged using a fluorescent analog of 125I-iodo-DPA-713, DPA-713-IRDye800CW, for correlative histology. Results: Pancreatic 125I-iodo-DPA-713 uptake was significantly higher in treated mice than control mice (5.17% ± 1.18% vs. 2.41% ± 0.34% injected dose/g, P = 0.02), as corroborated by imaging. Mice imaged with 18F-FDG PET/CT showed cerulein-enhanced pancreatic uptake in addition to a moderate signal from healthy pancreas. Near-infrared fluorescence imaging with DPA-713-IRDye800CW showed strong pancreatic uptake, focal liver uptake, and gastrointestinal uptake in the treated mice, whereas the control mice showed only urinary excretion. Ex vivo fluorescence microscopy revealed a large influx of macrophages in the pancreas colocalizing with the retained fluorescent probe in the treated but not the control mice. Conclusion: These data support the application of both 125I-iodo-DPA-713 SPECT/CT and DPA-713-IRDye800CW near-infrared fluorescence to delineate pancreatic, liver, or intestinal inflammation in living mice.

Adult enteric nervous system in health is maintained by a dynamic balance between neuronal apoptosis and neurogenesis.

According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Metal-Organic Framework/Graphene Quantum Dot Nanoparticles Used for Synergistic Chemo- and Photothermal Therapy.

In this study, a simple one-pot method was used to prepare a multifunctional platform for synergistic chemo- and photothermal therapy,, which is composed of zeolitic imidazolate framework-8 (ZIF-8) as drug nanocarriers and the embedded graphene quantum dots (GQDs) as local photothermal seeds. The structure, drug release behavior, photothermal effect, and synergistic therapeutic efficiency of the ZIF-8/GQD nanoparticles were systematically investigated. Using doxorubicin (DOX) as a model anticancer drug, the results showed that monodisperse ZIF-8/GQD nanoparticles with a particle size of 50-100 nm could encapsulate DOX during the synthesis procedure and trigger DOX release under acidic conditions. The DOX-loaded ZIF-8/GQD nanoparticles could efficiently convert near-infrared (NIR) irradiation into heat and thereby increase the temperature. More importantly, with breast cancer 4T1 cells as a model cellular system, the results indicated that the combined chemo- and photothermal therapy with DOX-ZIF-8/GQD nanoparticles exhibited a significant synergistic effect, resulting in a higher efficacy to kill cancer cells compared with chemotherapy and photothermal therapy alone. Hence, ZIF-8/GQD nanoparticles would be promising as versatile nanocarriers for synergistic cancer therapy.

Intranasal versus intraperitoneal delivery of human umbilical cord tissue-derived cultured mesenchymal stromal cells in a murine model of neonatal lung injury.

Clinical trials investigating mesenchymal stromal cell (MSC) therapy for bronchopulmonary dysplasia have been initiated; however, the optimal delivery route and functional effects of MSC therapy in newborns remain incompletely established. We studied the morphologic and functional effects of intranasal versus i.p. MSC administration in a rodent model of neonatal lung injury. Cultured human cord tissue MSCs (0.1, 0.5, or 1 × 10(6) cell per pup) were given intranasally or i.p. to newborn severe combined immunodeficiency-beige mice exposed to 90% O2 from birth; sham controls received an equal volume of phosphate-buffered saline. Lung mechanics, engraftment, lung growth, and alveolarization were evaluated 8 weeks after transplantation. High-dose i.p. MSC administration to newborn mice exposed to 90% O2 resulted in the restoration of normal lung compliance, elastance, and pressure-volume loops (tissue recoil). Histologically, high-dose i.p. MSC administration was associated with alveolar septal widening, suggestive of interstitial matrix modification. Intranasal MSC or lower-dose i.p. administration had no significant effects on lung function or alveolar remodeling. Pulmonary engraftment was rare in all the groups. These findings suggest that high-dose systemic administration of human cultured MSCs can restore normal compliance in neonatally injured lungs, possibly by paracrine modulation of the interstitial matrix. Intranasal delivery had no obvious pulmonary effects.

Homing and long-term engraftment of long- and short-term renewal hematopoietic stem cells.

Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.

Transgastric versus laparoendoscopic single-site peritoneoscopy in a rat model: effects on motility, inflammation, and nociception.

BACKGROUND: Natural orifice translumenal endoscopic surgery (NOTES) and laparoendoscopic single-port surgery (LESS) are emerging approaches to abdominal surgery that have been advocated as potentially causing fewer physiologic derangements and less pain. This study aimed to compare these procedures in a novel rat model by assessing peritoneal inflammation, gastric motility, and nociception in response to peritoneoscopy performed via NOTES and LESS. METHODS: Adult male rats underwent peritoneoscopy via either transgastric NOTES or LESS using the same type of endoscope and were allowed to recover for 2 to 4 h. Liquid gastric emptying was assessed using phenol red, and cytokine levels were analyzed in peritoneal washings. Thoracic spinal cord segments were stained for Finkel-Biskins-Jinkins osteosarcoma gene (FOS) to assess activation of nociceptive pathways. RESULTS: The NOTES procedure significantly delayed both postsurgical recovery time compared with LESS (115 ± 25 vs. 82 ± 20 min, respectively; P = 0.04) and liquid gastric emptying (26.7 ± 11.1% vs. 57 ± 10.5%; P = 0.004). Several cytokines such as interleukin-1ß (IL-1ß), IL-6, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1ß) were significantly elevated in the NOTES group compared with the LESS group. However, the two groups did not differ significantly in spinal FOS activation. CONCLUSIONS: The NOTES approach is feasible in an experimental rat model, facilitating a scientific approach to hypothesis testing through specific methods and instruments. The transgastric NOTES approach in rats is associated with a worse physiologic outcome in terms of gastric motility and peritoneal inflammation but does not differ significantly from LESS in activation of pain pathways.

Heterogeneity of non-cycling and cycling synchronized murine hematopoietic stem/progenitor cells.

Purified long-term multilineage repopulating marrow stem cells have been considered to be homogenous, but functionally these cells are heterogeneous. Many investigators urge clonal studies to define stem cells but, if stem cells are truly heterogeneous, clonal studies can only define heterogeneity. We have determined the colony growth and differentiation of individual lineage negative, rhodamine low, Hoechst low (LRH) stem cells at various times in cytokine culture, corresponding to specific cell cycle stages. These highly purified and cycle synchronized (98% in S phase at 40 h of culture) stem cells were exposed to two cytokine cocktails for 0, 18, 32, or 40 h and clonal differentiation assessed 14 days later. Total heterogeneity as to gross colony morphology and differentiation stage was demonstrated. This heterogeneity showed patterns of differentiation at different cycle times. These data hearken to previous suggestions that stem cells might be similar to radioactive isotopes; decay rate of a population of radioisotopes being highly predictable, while the decay of individual nuclei is heterogeneous and unpredictable (Till et al., 1964). Marrow stem cells may be most adequately defined on a population basis; stem cells existing in a continuum of reversible change rather than a hierarchy.

Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

Methotrexate (MTX) is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma (PCNSL). Important pharmacological properties that directly bear on the use of MTX in PCNSL, such as mechanisms that govern its uptake into brain tumors, are poorly defined, but are amenable to investigation in mouse models. In order to pursue such preclinical pharmacological studies, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of MTX and its metabolite, 7-hydroxymethotrexate (7-OH MTX) in plasma and microdialysate samples from brain tumors and cerebrospinal fluid (CSF) is needed. The plasma assay was based on 10 microl samples and following a protein precipitation procedure enabled direct injection onto a LC/MS/MS system using positive electrospray ionization. A column switching technique was employed for desalting and the clean-up of microdialysate samples from brain tissues. The methods were validated for MTX and 7-OH MTX in both plasma and microdialysate samples from brain tumor and CSF, and produced lower limits of quantification (LLOQ) in plasma of 3.7 ng/ml for MTX and 7.4 ng/ml for 7-OH MTX, and in microdialysate samples of 0.7 ng/ml for both MTX and 7-OH MTX. The utility of the method was demonstrated by estimation of pharmacokinetic (PK) and brain distribution properties of MTX and 7-OH MTX in conscious mice. The method has the advantages of low sample volume, rapid clean-up, and the simultaneous measurement of MTX and 7-OH MTX in plasma and brain tissues allowing detailed PK studies to be completed in individual mice.