Edward W. Scott

PU.1 Functions in a Cell-Autonomous Manner to Control the Differentiation of Multipotential Lymphoid–Myeloid Progenitors

Transcription factor PU.1 is required for the development of lymphoid and myeloid progenitors during fetal hematopoiesis. By generating chimeric animals using PU.1-/- ES cells or PU.1(-/-) hematopoietic progenitors, we demonstrate that PU.1 functions in an exclusively cell-autonomous manner to regulate the development of the lymphoid-myeloid system. Multipotential lymphoid-myeloid progenitors (AA4.1+, Lin-) are significantly reduced in PU.1(-/-) embryos and fail to differentiate into B lymphoid or myeloid cells in vitro. These results suggest that the lymphoid and myeloid lineages develop in the fetal liver from a common hematopoietic progenitor not shared with erythrocytes and megakaryocytes. Finally, the Ikaros gene is expressed in PU.1 mutant embryos, suggesting that PU.1 and Ikaros are independently required for specification of embryonic lymphoid cell fates.

PU.1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation

We have previously shown using gene targeting that PU.1 is essential for the development of lymphoid and myeloid lineages during fetal liver hematopoiesis. We now show that PU.1 is required for the maturation of yolk sac-derived myeloid progenitors and for the differentiation of ES cells into macrophages. The role of PU.1 in regulating target genes, thought to be critical in the development of monocytes and granulocytes, has been analyzed. Early genes such as GM-CSFR, G-CSFR, and myeloperoxidase are expressed in PU.1-/- embryos and differentiated PU.1-/- ES cells. However, the expression of genes associated with terminal myeloid differentiation (CD11b, CD64, and M-CSFR) is eliminated in differentiated PU.1-/- ES cells. Development of macrophages is restored with the introduction of a PU.1 cDNA regulated by its own promoter. The PU.1-/- ES cells represent an important model for analyzing myeloid cell development.

Abstract 107: p53 mutation at codon 273 in colorectal cancer and colitis-derived ALDHhigh cells promotes sphere formation in vitro

Introduction: p53 protein is a cell cycle regulator that functions as a tumor suppressor. Mutations in the p53 gene results in uncontrolled cell division, leading to diverse types of human cancer. In the case of colorectal cancer, p53 is the last gene to be mutated in the adenoma-to-carcinoma sequence. However, in colitis, early mutations in p53 occur in the course of dysplasia-to-carcinoma transition. Analysis of these mutations in colitis and colorectal cancer can provide clues regarding the pathogenesis of colitis to cancer.Recently, we have isolated and propagated ALDHhigh colon cancer stem cells (CCSCs) from colorectal cancer patients and precursor-CCSCs (pCCSCs) from colitic patients, which may be propagated as spheres. These CCSCs and pCCSCs may be used as a model system to study the transition from colitis to cancer. To determine if any of the p53 mutations correlate with sphere formation we analyzed p53 mutations in the existing sphere isolates. Methods: We performed sequence analysis of p53 mRNA and genomic DNA on 6 ALDHhigh sphere isolates derived from human colon cancer and 2 from colitic colon. Results: 5 out of 6 (83%) colorectal cancer derived ALDHhigh sphere isolates showed mutation at 273rd codon on the 8th exon (Arg→ His), which is one of the hot spots of p53 mutations in colorectal cancer. The same mutation was also observed in the ALDHhigh sphere isolates derived from colitic patients. This mutation corresponds to the amino acid residue that is in direct contact with DNA (contact mutants), but this mutation does not result in alterations in the spatial structure of p53. However, the defect in DNA binding ability of p53 allows the cell cycle to proceed unchecked and thus this mutation correlates with sphere forming ability. Conclusions: Mutations of p53 at codon 273 on exon 8 correlates with the sphere forming ability of the ALDHhigh cells derived from human colon cancer and colitic colon. Mutational knock-in and wild-type p53 overexpression studies would be vital to further confirm the role of this mutation in sphere formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 107. doi:1538-7445.AM2012-107

Detailed Analysis of Bone Marrow From Patients With Ischemic Heart Disease and Left Ventricular DysfunctionNovelty and Significance

Rationale: Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI). Objective: To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction. Methods and Results: BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34+ cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%–2.7% in other cohorts; P<0.05). BM-derived endothelial colonies were significantly decreased (P<0.05). Increased BM CD11b+ cells associated with worse LV ejection fraction (LVEF) after AMI (P<0.05). Increased BM CD34+ percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P=0.03, for patients with AMI and +6.6% versus −0.02%; P=0.021 for patients with chronic IHD). In addition, decreased BM CD34+ percentage in patients with chronic IHD correlated with decrement in LVEF (−2.9% versus +0.7%; P=0.0355). Conclusions: In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment. Clinical Trial Registrations: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00684021, NCT00684060, and NCT00824005Rationale: Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI).nnObjective: To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction.nnMethods and Results: BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34+ cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%–2.7% in other cohorts; P <0.05). BM-derived endothelial colonies were significantly decreased ( P <0.05). Increased BM CD11b+ cells associated with worse LV ejection fraction (LVEF) after AMI ( P <0.05). Increased BM CD34+ percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P =0.03, for patients with AMI and +6.6% versus −0.02%; P =0.021 for patients with chronic IHD). In addition, decreased BM CD34+ percentage in patients with chronic IHD correlated with decrement in LVEF (−2.9% versus +0.7%; P =0.0355).nnConclusions: In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment.nnClinical Trial Registrations: URL: . Unique identifiers: [NCT00684021][1], [NCT00684060][2], and [NCT00824005][3]nn# Novelty and Significance {#article-title-31}nn [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00684021&atom=%2Fcircresaha%2F115%2F10%2F867.atomn [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00684060&atom=%2Fcircresaha%2F115%2F10%2F867.atomn [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00824005&atom=%2Fcircresaha%2F115%2F10%2F867.atom

Detailed Analysis of Bone Marrow From Patients With Ischemic Heart Disease and Left Ventricular DysfunctionNovelty and Significance: BM CD34, CD11b, and Clonogenic Capacity as Biomarkers for Clinical Outcomes

Rationale: Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI). Objective: To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction. Methods and Results: BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34+ cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%–2.7% in other cohorts; P<0.05). BM-derived endothelial colonies were significantly decreased (P<0.05). Increased BM CD11b+ cells associated with worse LV ejection fraction (LVEF) after AMI (P<0.05). Increased BM CD34+ percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P=0.03, for patients with AMI and +6.6% versus −0.02%; P=0.021 for patients with chronic IHD). In addition, decreased BM CD34+ percentage in patients with chronic IHD correlated with decrement in LVEF (−2.9% versus +0.7%; P=0.0355). Conclusions: In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment. Clinical Trial Registrations: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00684021, NCT00684060, and NCT00824005Rationale: Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI).nnObjective: To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction.nnMethods and Results: BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34+ cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%–2.7% in other cohorts; P <0.05). BM-derived endothelial colonies were significantly decreased ( P <0.05). Increased BM CD11b+ cells associated with worse LV ejection fraction (LVEF) after AMI ( P <0.05). Increased BM CD34+ percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P =0.03, for patients with AMI and +6.6% versus −0.02%; P =0.021 for patients with chronic IHD). In addition, decreased BM CD34+ percentage in patients with chronic IHD correlated with decrement in LVEF (−2.9% versus +0.7%; P =0.0355).nnConclusions: In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment.nnClinical Trial Registrations: URL: . Unique identifiers: [NCT00684021][1], [NCT00684060][2], and [NCT00824005][3]nn# Novelty and Significance {#article-title-31}nn [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00684021&atom=%2Fcircresaha%2F115%2F10%2F867.atomn [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00684060&atom=%2Fcircresaha%2F115%2F10%2F867.atomn [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00824005&atom=%2Fcircresaha%2F115%2F10%2F867.atom

Abstract 5193: Wnt signaling in colitis derived colon cancer initiating cells promotes the tumorigenicity in colitis to cancer transition

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnIntroduction: Wnt (Wingless) signaling in colon stem cells is involved in the development and maintenance of crypt architecture. In addition, activating mutations in the Wnt signaling pathway initiate and perpetuate colon cancer initiating cells (CCIC). Recently, we have isolated and propagated CCICs from colitis. Here, we hypothesize that Wnt signaling in the colitis derived CCICs potentiates colitis-associated tumor initiation.nnMethods: In order to test the above hypothesis, we have designed a dual fusion Wnt reporter construct (TTLG). The construct comprises of luminescent firefly luciferase and enhanced green fluorescent protein (eGFP), which are preceded by the TCF/LEF binding site. TTLG was transduced into colitis sphere isolates that were derived from Aldehyde dehydrogenase (ALDH) high cells. Immunohistochemistry validated the staining for activated β-Catenin in TTLG GFP high vs eGFP low colitis sphere isolates Tumorigenicity was evaluated by injecting eGFP high and eGFP low cells (1000 each) in the flanks of NOD-SCID mice. Secondary tumors were generated from 10% eGFP bright vs dimmest cells (1000, 100 and 10 each) obtained from eGFP high xenograft derived cells. In parallel, in vitro limiting dilution assays (LDA) were performed.nnResults: The eGFP high sphere cell population had nuclear localized Active Beta Catenin staining, indicating active Wnt signaling (95%). Conversely, little to no (<5%) Active Beta Catenin (ABC) staining was observed in the nucleus of eGFP low sphere cells. In vitro studies demonstrated that the clonogenic potential of eGFP high colitis sphere cells was significantly higher than that of eGFP low cells. This was further confirmed by in vivo studies, where the tumors generated from eGFP high cells grew more rapidly and larger when compared to eGFP low cells. The ability of the eGFP low cells to induce the tumor relates to the fact that eGFP low cells are a subset of the ALDH high population. Furthermore, secondary tumors that were generated from the 10% eGFP bright cells derived from eGFP high xenograft were initiated within few days after injection. At the time of harvest, the tumors formed from 10% eGFP dimmest cells were less than ¼ the size of the 10% eGFP bright cells derived tumor. The clonogenic potential of these 10% eGFP bright cells was about 50-100 fold higher than the 10% eGFP dimmest cells.nnConclusions: The Wnt high ALDH high cells have higher clonogenic potential when compared to Wnt low ALDH high cells. Also, the tumors generated from Wnt high ALDH high colitis cells demonstrated decreased tumor latency and increased tumorigenicity compared to Wnt low ALDH high colitis cells. Collectively, our observations indicate that Wnt high CCICs from colitis promote colitis-associated tumorigenicity.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5193. doi:10.1158/1538-7445.AM2011-5193

Abstract 5189: An IL-8 dependent autocrine circuit promotes colorectal cancer tumorigenicity

Introduction: It is well established that chronic inflammation of the colon increases the incidence of colorectal cancer. One plausible mechanism involves inflammation-associated signals targeting the cancer stem cell (CSC). The CSC hypothesis posits that solid tumors originate from a rare cell population with stem cell-like properties, including self-renewal, and the ability to initiate tumor growth. We have previously demonstrated that CSC-like cells can be enriched from sporadic colorectal tumors (CRC) based on high level aldehyde dehydrogenase (ALDH) enzyme activity and expression of the epithelial cell adhesion molecule (EpCAM). The ALDH high /EpCAM + population was shown to initiate tumors in NOD-SCID mice, and termed the colon cancer initiating cell (CCIC). CRC-derived CCICs were able to be propagated in vitro under serum-free conditions as non-adherent spheres. IL-8, an inflammatory chemokine with known proliferative and angiogenic properties, is detected in the serum of patients with CRC. We have shown that antibodies targeting IL-8 inhibited the growth of CRC tumor xenografts. We hypothesize that an IL-8-dependent autocrine circuit promotes colorectal cancer tumorigenicity. Methods: Immunochemical analysis was used to detect expression of the IL8 receptors, CXCR1 and CXCR2, by CRC-derived spheres and tumor xenografts. IL-8 secretion by CRC-derived spheres was quantified by an IL-8 ELISA. To assess effects of IL-8 on tumor growth, continuous release pellets containing IL-8 (2 ng/ml) or a placebo were implanted into the flanks of NOD-SCID mice prior to injection of CRC-derived CCICs. Tumor volume was measured biweekly. Tumor vessel density was quantified by immunochemical detection of MECA-32 + blood vessels. To assess effects of IL-8 on CRC-derived CCIC sphere formation and growth, cells were plated in the presence or absence of exogenous IL-8 (0 to 20 ng/ml). Sphere number and size were enumerated on days 2 and 7. Results: Immunochemical analysis detected selective expression of the IL-8 receptor, CXCR1, by both CRC-derived spheres and xenografts. Significant amounts of IL-8 were secreted by CRC-derived spheres (3000 cells; 1513 pg/ml). The tumor growth rate of mice receiving an exogenous source of IL-8 was significantly higher than the placebo group (p=0.008). The resulting tumors exhibited an increased level of angiogenesis. The addition of increasing concentrations of exogenous IL-8 to CRC-derived CCIC cultures increased the sphere size at day 2 (10 and 20 ng/ml; p Conclusion: Collectively, these data suggest that colorectal cancer tumor initiation and growth can be modulated by the inflammation-associated chemokine IL-8 via an autocrine circuit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5189. doi:10.1158/1538-7445.AM2011-5189