Circulating tumor cell counts are prognostic of overall survival in SWOG S0421: a phase III trial of docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer.

PURPOSE: Circulating tumor cell (CTC) enumeration has not been prospectively validated in standard first-line docetaxel treatment for metastatic castration-resistant prostate cancer. We assessed the prognostic value of CTCs for overall survival (OS) and disease response in S0421, a phase III trial of docetaxel plus prednisone with or without atrasentan. PATIENTS AND METHODS: CTCs were enumerated at baseline (day 0) and before cycle two (day 21) using CellSearch. Baseline counts and changes in counts from day 0 to 21 were evaluated for association with OS, prostate-specific antigen (PSA), and RECIST response using Cox regression as well as receiver operator characteristic (ROC) curves, integrated discrimination improvement (IDI) analysis, and regression trees. RESULTS: Median day-0 CTC count was five cells per 7.5 mL, and CTCs < versus ≥ five per 7.5 mL were significantly associated with baseline PSA, bone pain, liver disease, hemoglobin, alkaline phosphatase, and subsequent PSA and RECIST response. Median OS was 26 months for < five versus 13 months for ≥ five CTCs per 7.5 mL at day 0 (hazard ratio [HR], 2.74 [adjusting for covariates]). ROC curves had higher areas under the curve for day-0 CTCs than for PSA, and IDI analysis showed that adding day-0 CTCs to baseline PSA and other covariates increased predictive accuracy for survival by 8% to 10%. Regression trees yielded new prognostic subgroups, and rising CTC count from day 0 to 21 was associated with shorter OS (HR, 2.55). CONCLUSION: These data validate the prognostic utility of CTC enumeration in a large docetaxel-based prospective cohort. Baseline CTC counts were prognostic, and rising CTCs at 3 weeks heralded significantly worse OS, potentially serving as an early metric to help redirect and optimize therapy in this clinical setting.

Inhibition of protease-activated receptor 1 ameliorates intestinal radiation mucositis in a preclinical rat model.

PURPOSE: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. METHODS AND MATERIALS: Rats underwent fractionated X-irradiation (5 Gy×9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. RESULTS: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. CONCLUSION: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

Pharmacological targeting of the thrombomodulin-activated protein C pathway mitigates radiation toxicity.

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

Procalcitonin as a predictive biomarker for total body irradiation-induced bacterial load and lethality in mice.

Sepsis is the leading cause of mortality in intensive care units. Early detection and intervention are critical to prevent death. The acute radiation syndrome is characterized by damage of the gastrointestinal and hematopoietic systems. Translocation of intestinal microflora combined with immune system compromise may lead to septicemia and death. This work examined the utility of procalcitonin, a clinical sepsis biomarker, in a mouse model of radiation toxicity. C57/BL6 mice were exposed to total body irradiation (TBI). Intestinal mucosal permeability was measured in vivo, and liver bacterial load and plasma levels of procalcitonin (PCT), lipopolysaccharide (LPS), and LPS-binding protein were measured at baseline and at 3.5, 7, and 10 days after TBI. The value of early PCT in predicting subsequent lethality was determined by receiver operating characteristic analysis. Four days after TBI, a dose-dependent increase in permeability of the intestinal mucosa was observed, whereas bacterial translocation was present from day 7 onward. There was a high positive correlation between bacterial translocation and all sepsis biomarkers, with PCT exhibiting the strongest correlation. Moreover, plasma PCT levels were elevated already from day 3.5 onward, whereas LPS was elevated from day 7 and LPS-binding protein only 10 days after TBI. Receiver operating characteristic analysis revealed that PCT levels measured 3.5 days after TBI predicted lethality at 10 days. These data demonstrate the value of PCT as an early biomarker in radiation-induced bacteremia for mouse studies and suggest that clinical results from other septic conditions may apply to postradiation septicemia in humans.

Isolation, characterization, and in vitro propagation of infantile hemangioma stem cells and an in vivo mouse model.

BACKGROUND: Infantile hemangiomas (IH) are the most common benign tumors of infancy. The typical clinical course consists of rapid growth during the first year of life, followed by natural and gradual involution over a multi-year time span through unknown cellular mechanisms. Some tumors respond to medical treatment with corticosteroids or beta-blockers, however, when this therapy fails or is incomplete, surgical extirpation may be necessary. Noninvasive therapies to debulk or eliminate these tumors would be an important advance. The development of an in vitro cell culture system and an animal model would allow new insights into the biological processes involved in the development and pathogenesis of IH. RESULTS: We observed that proliferative stage IH specimens contain significantly more SALL4+ and CD133+ cells than involuting tumors, suggesting a possible stem cell origin. A tumor sphere formation assay was adapted to culture IH cells in vitro. Cells in IH tumor spheres express GLUT1, indicative of an IH cell of origin, elevated levels of VEGF, and various stem/progenitor cell markers such as SALL4, KDR, Oct4, Nanog and CD133. These cells were able to self-renew and differentiate to endothelial lineages, both hallmarks of tumor stem cells. Treatment with Rapamycin, a potent mTOR/VEGF inhibitor, dramatically suppressed IH cell growth in vitro. Subcutaneous injection of cells from IH tumor spheres into immunodeficient NOD-SCID mice produced GLUT1 and CD31 positive tumors with the same cellular proliferation, differentiation and involution patterns as human hemangiomas. CONCLUSIONS: The ability to propagate large numbers of IH stem cells in vitro and the generation of an in vivo mouse model provides novel avenues for testing IH therapeutic agents in the future.

Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4.

BACKGROUND: Sall4 is a key factor for the maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). Our previous studies have shown that Sall4 is a robust stimulator for human hematopoietic stem and progenitor cell (HSC/HPC) expansion. The purpose of the current study is to further evaluate how Sall4 may affect HSC/HPC activities in a murine system. METHODS: Lentiviral vectors expressing Sall4A or Sall4B isoform were used to transduce mouse bone marrow Lin-/Sca1+/c-Kit+ (LSK) cells and HSC/HPC self-renewal and differentiation were evaluated. RESULTS: Forced expression of Sall4 isoforms led to sustained ex vivo proliferation of LSK cells. In addition, Sall4 expanded HSC/HPCs exhibited increased in vivo repopulating abilities after bone marrow transplantation. These activities were associated with dramatic upregulation of multiple HSC/HPC regulatory genes including HoxB4, Notch1, Bmi1, Runx1, Meis1 and Nf-ya. Consistently, downregulation of endogenous Sall4 expression led to reduced LSK cell proliferation and accelerated cell differentiation. Moreover, in myeloid progenitor cells (32D), overexpression of Sall4 isoforms inhibited granulocytic differentiation and permitted expansion of undifferentiated cells with defined cytokines, consistent with the known functions of Sall4 in the ES cell system. CONCLUSION: Sall4 is a potent regulator for HSC/HPC self-renewal, likely by increasing self-renewal activity and inhibiting differentiation. Our work provides further support that Sall4 manipulation may be a new model for expanding clinically transplantable stem cells.

Comparison of circulating MicroRNA 141 to circulating tumor cells, lactate dehydrogenase, and prostate-specific antigen for determining treatment response in patients with metastatic prostate cancer.

Our aim was to determine the utility of circulating micro RNA miR-141 as a potential biomarker of therapeutic response in prostate cancer (CaP) patients. We compared the values of miR-141 in plasma of 21 CaP patients to the levels of prostate specific antigen (PSA), circulating tumor cells (CTC) and lactate dehydrogenase (LDH). Data suggest a strong correlation of miR-141 values and clinical course. For prostate cancer (CaP), the measurement of prostate-specific antigen (PSA) and radiographic studies do not adequately predict response to therapy and survival, and, therefore, new relevant biomarkers are needed. We and other researchers have shown that longitudinal measurements of PSA, circulating tumor cells (CTC), and lactate dehydrogenase (LDH) may aid in predicting response to therapy. Results of recent studies have determined that circulating microRNA (miRNA) miR-141 is detected in plasma of patients with CaP. We, therefore, compared the temporal changes of miR-141 with the levels of CTC, LDH, and PSA in 21 patients with CaP, and longitudinally examined these markers alone or in combinations to determine the utility of miR-141 in the predicting a patient's clinical course and response to therapy. Levels of miR-141 in plasma of 21 patients with CaP were measured by using quantitative reverse transcription-polymerase chain reaction. A total of 35 intervals were assessed. Directional changes (increasing or decreasing) in PSA, CTC, and miR-141 had sensitivity in predicting clinical outcome (progression vs. nonprogressing) of 78.9%. Logistic regression modeling of the probability of clinical progression demonstrates that miR-141 levels predicted clinical outcomes with an odds ratio of at least 8.3. miR-141 also had the highest correlation with temporal changes of PSA with a correlation of R = 0.77 (P < .001). In this retrospective study, miR-141 demonstrated a similar ability to predict clinical progression when compared with other clinically validated biomarkers. Furthermore, miR-141 demonstrated high correlation with changes of the other biomarkers.

Circulating tumor cells as a predictive biomarker in patients with hormone-sensitive prostate cancer.

UNLABELLED: Little information exists regarding the utility circulating tumor cell (CTC) enumeration in hormone sensitive prostate cancer. We enumerated CTC in 33 consecutive patients undergoing androgren deprivation therapy (ADT) at our institution. Multivariate analysis revealed baseline CTC as the only independent predictor of progression to CRPC. These data suggest that baseline CTC may identify those unlikely to benefit from ADT. INTRODUCTION: Circulating tumor cell (CTC) enumeration by using the Cellsearch platform has established prognostic and predictive value in patients with metastatic castration-resistant prostate cancer (mCRPC). Limited information exists regarding the clinical utility of CTC enumeration in metastatic hormone-sensitive prostate cancer (mHSPC). The goal of this study was to prospectively determine the relative clinical utility of CTCs in mHSPC. PATIENTS AND METHODS: We analyzed serial CTC in conjunction with other classic biomarkers in 33 consecutive patients treated at the Nevada Cancer Institute with HSPC initiating androgen deprivation therapy and correlated these patients with prognostic prostate-specific antigen (PSA) endpoints and onset of CRPC. RESULTS: Initial CTC correlated positively with lactate dehydrogenase and alkaline phosphatase, and were unrelated to PSA and testosterone. In univariate analysis, baseline CTC, alkaline phosphatase, lactate dehydrogenase, testosterone, and follow-up CTC were individual predictors of progression to CRPC. In a multivariate Cox regression, only baseline CTC retained independent predictive value. Threshold analysis revealed the cutpoint that optimized specificity and sensitivity of the test to be 3 cells per 7.5 mL whole blood. Baseline CTC also correlated well with PSA nadir benchmarks. CONCLUSIONS: Initial CTC values predict the duration and magnitude of response to hormonal therapy. CTC enumeration may identify patients at risk of progression to CRPC before initiation of androgen deprivation therapy.

Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells.

Induced pluripotent stem (iPS) cells are derived from reprogrammed somatic cells and are similar to embryonic stem (ES) cells in morphology, gene/protein expression, and pluripotency. In this study, we explored the potential of iPS cells to differentiate into alveolar Type II (ATII)-like epithelial cells. Analysis using quantitative real time polymerase chain reaction and immunofluorescence staining showed that pulmonary surfactant proteins commonly expressed by ATII cells such as surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC) were upregulated in the differentiated cells. Microphilopodia characteristics and lamellar bodies were observed by transmission electron microscopy and lipid deposits were verified by Nile Red and Periodic Acid Schiff staining. C3 complement protein, a specific feature of ATII cells, was present at high levels in culture supernatants demonstrating functionality of these cells in culture. These data show that the differentiated cells generated from iPS cells using a culture method developed previously (Rippon et al., 2006) are ATII-like cells. To further characterize these ATII-like cells, we tested whether they could undergo epithelial to mesenchymal transition (EMT) by exposure to drugs that induce lung fibrosis in mice, such as bleomycin, and the combination of transforming growth factor beta1 (TGF(b1)) and epidermal growth factor (EGF). When the ATII-like cells were exposed to either bleomycin or a TGF(b1)-EGF cocktail, they underwent phenotypic changes including acquisition of a mesenchymal/fibroblastic morphology, upregulation of mesenchymal markers (Col1, Vim, a-Sma, and S100A4), and downregulation of surfactant proteins and E-cadherin. We have shown that ATII-like cells can be derived from skin fibroblasts and that they respond to fibrotic stimuli. These cells provide a valuable tool for screening of agents that can potentially ameliorate or prevent diseases involving lung fibrosis.

Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties.

BACKGROUND: The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. METHODOLOGY/PRINCIPAL FINDING: The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44(+) cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44(+) cells consisted of mixed CD44(+) and CD44(-) cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44(+) and CD44(+) cells derived from CD44(+)-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44(-) cells. Furthermore, freshly sorted CD44(+) cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44(-) cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. CONCLUSION/SIGNIFICANCE: Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment.

Increases in quantitative D-dimer levels correlate with progressive disease better than circulating tumor cell counts in patients with refractory prostate cancer.

Changes in quantitative D-dimer levels, circulating tumor cell (CTC) counts, and prostate-specific antigen (PSA) levels were measured in 28 patients with refractory castration-resistant prostate cancer to assess their concordance during the course of therapy and their relationship with risk of progressive disease. A significant correlation was identified between changes in PSA and both CTC counts and D-dimer levels (r = 0.67 and 0.58, respectively; P < .001). In addition, there was a significant correlation between changes in CTC count and D-dimer level (r = 0.62; P < .001). A significantly stronger concordance between these biomarkers was noted for increasing values (sensitivity, 72%-77.8%) compared with decreasing values (specificity, 43.8%-71.4%). Notably, increases in PSA and D-dimer levels, not CTC counts, were associated with increased risks for progressive disease (P < .024). Increases in quantitative D-dimer levels correlate with progressive disease better than CTC counts in patients with refractory prostate cancer.

Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells.

Diabetes mellitus is characterized by either the inability to produce insulin (type 1 diabetes) or as insensitivity to insulin secreted by the body (type 2 diabetes). In either case, the body is unable to move blood glucose efficiently across cell membranes to be used. This leads to a variety of local and systemic detrimental effects. Current treatments for diabetes focus on exogenous insulin administration and dietary control. Here, we describe a potential cure for diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity. Using induced pluripotent stem (iPS) cells, we were able to derive beta-like cells similar to the endogenous insulin-secreting cells in mice. These beta-like cells secreted insulin in response to glucose and corrected a hyperglycemic phenotype in two mouse models of type 1 and 2 diabetes via an iPS cell transplant. Long-term correction of hyperglycemia was achieved, as determined by blood glucose and hemoglobin A1c levels. These data provide an initial proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2.

Generation of murine hepatic lineage cells from induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be generated from somatic cells of individuals by retrodifferentiation using defined transcription factors. Similar to embryonic stem (ES) cells, iPS cells can be differentiated into a variety of specific cell types. However, to date, no detailed hepatic differentiation of mouse iPS cells has been reported. In this study, we successfully developed a stepwise protocol to induce hepatic differentiation of iPS cells reprogrammed from mouse tail tip fibroblasts. At day 25 of differentiation, the iPS cell-derived hepatocytes morphologically resemble mouse primary hepatocytes with a distinct polygonal shape. Immunostaining and reverse transcription-polymerase chain reaction analysis revealed expression of specific hepatic markers including alpha-fetoprotein, albumin and alpha-1-anti-trypsin. In addition, these iPS cell-derived hepatocytes successfully demonstrated mature liver cell functions in vitro. Furthermore, in vivo assays revealed that the mouse iPS cell-derived hepatocytes successfully engrafted into the recipient livers with typical hepatic morphology. Thus, iPS cell-derived hepatocytes may hold great promise as a unique system for basic liver research and liver regeneration in the near future.

Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts.

We have successfully developed both spontaneous and inductive cardiomyocyte differentiation of iPS cells reprogrammed from human foreskin fibroblasts. The reprogrammed iPS cells morphologically resemble human cardiomyocytes which can beat. RT-PCR and immunostaining show that cardiac markers are expressed that are comparable to the differentiation pattern of authentic human embryonic stem cells, indicating the existence of both immature and mature differentiated cardiomyocytes. 5-Azacytidine greatly enhanced the efficiency of cardiomyocyte differentiation, whereas dimethylsulfoxide had no effect. Low serum and bone morphogenetic protein-2 marginally improved differentiation efficiency. iPS cell-derived cardiomyocytes changed their beat frequency in response to cardiac drugs, which included ion channel blockers and alpha/beta adrenergic stimulators. Derived cardiomyocytes look promising as an in vitro system for potential drug screen and/or toxicity, making this system closer to practical use in the near future.

Thrombotic events in patients with cancer receiving antiangiogenesis agents.

Tumor-associated neoangiogenesis has recently become a suitable target for antineoplastic drug development. In this overview, we discuss specific drug-associated hemostatic complications, the already known pathogenetic mechanisms involved, and the effect of varying antithrombotic strategies. Multiple agents with angiogenic inhibitory capacity (thalidomide, lenalidomide, bevacizumab, sunitinib, sorafenib, and sirolimus) have obtained US Food and Drug Administration approval, and many others have entered clinical trials. Arterial and venous thromboembolism and hemorrhage have emerged as significant toxicities associated with the use of angiogenesis inhibitors. We present a detailed analysis of the literature on thrombotic complication of antiangiogenic drugs. Close attention to hemostatic complications during antiangiogenic treatment is warranted. Further studies are required to better understand the pathophysiologic mechanisms involved and to define a safe prophylactic strategy.

Circulating tumor cells in patients with castration-resistant prostate cancer baseline values and correlation with prognostic factors.

PURPOSE: Circulating tumor cells (CTC) have been recently accepted by the Food and Drug Administration of the United States as a prognostic tool in advanced prostate cancer. However, a number of questions remain about the use of the test. The optimal clinical cut-off has never been determined. Also, the predictive value of CTCs in the setting of low-burden advanced prostate cancer has not been evaluated. Herein we describe our experience with the CellSearch method of CTC enumeration. EXPERIMENTAL DESIGN: CTCs enumerated from 100 patients with castration-resistant prostate cancer were correlated with clinicopathologic characteristics and conventional biomarkers, such as prostate-specific antigen and lactate dehydrogenase. Patients received ongoing medical oncologic follow-up for up to 26 months, and overall survival status was documented. RESULTS: Forty-nine of the patients (49%) were alive at the end of the study. CTC counts correlate well with overall survival (P < 0.001) but are also tightly interrelated to other biomarkers. Threshold analysis identified 4 CTC/7.5 cc (compared with the approved value of 5) as an optimal cut-off value with respect to correlation with survival outcomes as well as predictive of metastatic disease. Univariate analysis confirmed a tight interrelationship between cut-off CTC values and biomarkers. Multivariate analysis with bootstrap sampling validation identified lactate dehydrogenase (P = 0.002) and CTCs (P = 0.001) as independently prognostically significant. CONCLUSIONS: Baseline CTC values provide important prognostic information and specific prediction of metastatic disease. Their presence correlates with classic biomarkers.

Hypercoagulable states in patients with multiple myeloma can affect the thalidomide-associated venous thromboembolism.

The therapeutic use of thalidomide in patients with multiple myeloma is often complicated by the development of venous thromboembolism. The objective of the present study was to identify hypercoagulable states associated with development of venous thromboembolism in thalidomide-treated multiple myeloma patients. We screened 49 consecutive multiple myeloma patients treated with thalidomide at baseline for hypercoagulability. With a median follow-up of 11 months, 10 of 49 multiple myeloma patients developed a thrombotic episode. Laboratory assays revealed an underlying abnormality in nine of the 10 patients; hypercoagulable screenings were normal in 36 of the 39 patients who did not develop venous thromboembolism (P < 0.0001). Our retrospective study results suggest that the multiple myeloma patients with thromboembolic complications during treatment with thalidomide have a frequent concomitant underlying thrombophilic state.

Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.

Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.

Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells.

Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls.