Publications by authors named "Amad Awadallah"

32 Publications

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Cancer Immunol Res 2021 Mar 13;9(3):348-361. Epub 2021 Jan 13.

Department of Pathology, Case Western Reserve University, Cleveland, Ohio.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8 T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that -mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925430PMC
March 2021

Peritumoral/vascular expression of PSMA as a diagnostic marker in hepatic lesions.

Diagn Pathol 2020 Jul 23;15(1):92. Epub 2020 Jul 23.

Department of Pathology, University Hospital Cleveland Medical Center, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio, 44106, USA.

Background: The differential diagnosis between primary cholangiocarcinoma and metastatic pancreatobiliary adenocarcinoma is histologically challenging due to lack of distinct morphological features and reliable molecular markers. Prostate-specific membrane antigen (PSMA) is expressed in prostate epithelium and upregulated on the surface of prostatic adenocarcinoma cells. Studies have shown PSMA enzymatic activity is involved in malignancy-driven neoangiogenesis in the endothelium of tumor-associated neovasculature in breast, lung, thyroid, hepatocellular carcinoma (HCC) and urothelial cancer. Recently, PSMA-targeted imaging technology (PSMA PET-CT) detected the presence of PSMA in primary cholangiocarcinoma. However histological correlation with PSMA expression other mass lesions in the liver has not yet been studied.

Methods: 72 cases of liver mass resection were collected at a tertiary hospital from 2011 to 2019. Immunohistochemical stains for PSMA and CD34 were performed. The expression of PSMA in tumor cells and associated neovascular endothelium were analyzed separately and the locations of vascular structures were confirmed by CD34 expression.

Results: Among 72 cases, 28 cases (22/72, 38.9%) showed PSMA peritumoral/vascular expression only, 3 cases (3/72, 4.2%) showed tumor cell expression only, and 2 cases (2/72, 2.8%) showed both tumor cell and peritumoral/vascular expression. The remainder (39/72, 54.2%) showed no expression. Particularly, most of primary cholangiocarcinoma showed PSMA vascular expression (13/15, 86.7%), while none of the 18 cases of metastatic pancreatobiliary adenocarcinoma were positive for PSMA (0/18, 0%) (p < 0.01). Outside of pancreatobiliary adenocarcinoma, none of the metastatic tumors, including colon and lung cancers, expressed PSMA. In 8 cases of metastatic prostate carcinoma, 3 showed PSMA expressions in tumor cells only (3/8, 37.5%) and 2 expressed PMSA in both tumor cells and neovasculature (2/8, 25.0%). Out of 22 HCC cases, 15 (15/22, 68.2%) were positive for PSMA in tumor vasculature. None of the 5 hepatic adenoma expressed PSMA (0/5, 0%).

Conclusion: Significantly enhanced tumor-associated neovascular PSMA expression was identified in primary cholangiocarcinoma, compared to metastatic pancreatobiliary adenocarcinoma. Our findings potentially provide a sensitive marker in differential diagnosis between otherwise morphologically indistinguishable cases.
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http://dx.doi.org/10.1186/s13000-020-00982-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376868PMC
July 2020

[F] Clofarabine for PET Imaging of Hepatocellular Carcinoma.

Cancers (Basel) 2019 Nov 7;11(11). Epub 2019 Nov 7.

Radiology, Case Western Reserve University, Cleveland, OH 44106, USA.

Clinical diagnosis of hepatocellular carcinoma (HCC) relies heavily on radiological imaging. However, information pertaining to liver cancer treatment such as the proliferation status is lacking. Imaging tumor proliferation can be valuable in patient management. This study investigated F-labeled clofarabine ([F]CFA) targeting deoxycytidine kinase (dCK) for PET imaging of dCK-dependent proliferation in HCC. Since clinical PET scans showed a high liver background uptake of [F]CFA, the aim of this study was to reduce this liver background uptake. A clinically relevant animal model of spontaneously developed HCC in the woodchucks was used for imaging experiments. Several modifiers were tested and compared with the baseline PET scan: Forodesine, probenecid, and cold clofarabine, all applied before the hot [F]CFA injection to evaluate the reduction in liver background uptake. Application of forodesine before hot [F]CFA injection did not reduce the background uptake. Instead, it increased the background by 11.6-36.3%. Application of probenecid also increased the liver background uptake by 16.6-32.1%. Cold CFA application did reduce the liver background uptake of [F]CFA, comparing to the baseline scan. Combining cold CFA with [F]CFA for PET imaging of liver cancers is a promising strategy, worthy of further clinical evaluation.
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http://dx.doi.org/10.3390/cancers11111748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896045PMC
November 2019

Uniform and Robust Nuclear Expression of HES1 in Neuroendocrine Neoplasms.

Int J Surg Pathol 2019 Dec 24;27(8):844-851. Epub 2019 Jun 24.

University Hospitals Cleveland Medical Center, Cleveland, OH, USA.

. Neuroendocrine neoplasms (NENs) are neoplasms that most commonly arise from gastrointestinal tract, pancreas, and lung. HES1 is a downstream target of Notch signaling pathway. The current literature about HES1 expression in NENs is sparse and inconsistent. . In this study, we evaluated HES1 expression by immunohistochemistry in a total of 32 cases of NENs, including 13 well-differentiated neuroendocrine tumors from gastrointestinal tract, 10 cases of well-differentiated neuroendocrine tumors of pancreas, 9 cases from lung, including 4 cases of typical carcinoid, 1 case of atypical carcinoid, and 4 cases of neuroendocrine carcinoma. The intensity of the stain was scored from - to +++, and the distribution of the staining of HES1 was evaluated. . HES1 demonstrates uniform robust (+++) nuclear staining pattern in the tumor cells of all the NENs (32/32), regardless of the origin of the system and the grade of the tumor. . HES1 is uniformly expressed in NENs with robust nuclear expression pattern. Our finding suggests that NOTCH1 or HES1 inhibitor is a potential therapeutic choice for neuroendocrine neoplasms.
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http://dx.doi.org/10.1177/1066896919854166DOI Listing
December 2019

PET imaging of hepatocellular carcinoma with anti-1-amino-3-[F]fluorocyclobutanecarboxylic acid in comparison with L-[S-methyl-C]methionine.

EJNMMI Res 2019 May 22;9(1):47. Epub 2019 May 22.

Radiology, Case Western Reserve University, Cleveland, OH, USA.

Purpose: [C]methionine ([C]Met) was used for cancer imaging based on upregulated amino acid transport and protein synthesis in different tumor types. However, the short half-life of C decay limited further clinical development of [C]Met. Synthetic amino acid analog anti-1-amino-3-[F]fluoro-cyclobutyl-1-carboxylic acid ([F]FCABC) was developed and FDA-approved for PET imaging of recurrent prostate cancer. This study investigated "repurposed" [F]FACBC for PET imaging of primary liver cancer such as hepatocellular carcinoma (HCC) in comparison with [C]Met.

Methods: [C]Met was synthesized in the lab, and [F]FACBC was purchased from a commercial outlet. A clinically relevant animal model of spontaneously developed HCC in the woodchucks was used for PET imaging. Bioinformatics analysis was performed for the expression of amino acid transporters responsible for radiotracer uptake and validated by PCR. Dynamic PET scans of [C]Met and [F]FACBC were acquired within 1 week. Standardized uptake value (SUV) was calculated for regions of interest (ROIs) defined over HCC and a liver background region. H&E staining and immunohistochemical (IHC) staining were performed with harvested tissues post-imaging.

Results: Higher expression of ACST2 and LAT1 was found in HCC than in the surrounding liver tissues. PCR validated this differential expression. [C]Met and [F]FACBC displayed some differences in their uptake and retention in HCC. Both peaked in HCC with an SUV of 3.5 after 10 min post-injection. Met maintained a plateaued contrast uptake in HCC to that in the liver while [F]FCABC declined in HCC and liver after peak uptake. The pathological assessment revealed the liver tumor as moderately differentiated similar to the human HCC and proliferative.

Conclusion: Both [F]FACBC and [C]Met showed uptake in HCC through the use of a clinically relevant animal model of woodchuck HCC. The uptake and retention of [F]FACBC and [C]Met depend on their metabolism and also rely on the distribution of their principal amino acid transporters.
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http://dx.doi.org/10.1186/s13550-019-0519-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531569PMC
May 2019

Flow Cytometry Identifies a Spectrum of Maturation in Myeloid Neoplasms Having Plasmacytoid Dendritic Cell Differentiation.

Cytometry B Clin Cytom 2020 01 5;98(1):43-51. Epub 2019 Jan 5.

Department of Pathology, University Hospitals of Cleveland Medical Center/Case Western Reserve University School of Medicine, Cleveland, Ohio.

Background: Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN.

Methods: We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5-26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN.

Results: Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation.

Conclusions: These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized. © 2019 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21761DOI Listing
January 2020

A multicolour flow cytometric assay for c-MYC protein in B-cell lymphoma.

J Clin Pathol 2018 Oct 16;71(10):906-915. Epub 2018 May 16.

Department of Pathology, University Hospitals Cleveland Medical Center and Seidman Comprehensive Cancer Center, Cleveland, Ohio, USA.

Aim: Develop an objective assay to detect c-MYC protein expression using multiparametric flow cytometry (FCM) as an alternative to immunohistochemistry (IHC).

Methods: 57 patient samples and 11 cell line samples were evaluated. Cell suspensions were obtained and c-MYC staining was performed in combination with CD45 and CD19 and, in some samples, CD10. The percentage of c-MYC+ cells by FCM was correlated with the percentage determined by IHC. The relationship between c-MYC protein expression and the presence of a gene rearrangement in aggressive and high-grade lymphomas was also assessed.

Results: c-MYC expression by FCM and IHC demonstrated a high degree of correlation in a training set of 33 patient cases, r=0.92, 11 cell line samples, r=0.81 and in a validation set of 24 aggressive and high-grade B-cell lymphomas, r=0.85. gene was rearranged by fluorescence in situ hybridisation in 6/9 samples with high c-MYC expression (>40%) by FCM and 6/14 by IHC.

Conclusions: We have developed a reliable multicolour FCM assay to detect c-MYC expression suitable for clinical laboratories that should be helpful to accurately quantify c-MYC expression in B-cell lymphomas.
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http://dx.doi.org/10.1136/jclinpath-2018-205075DOI Listing
October 2018

Endochondral Ossification in Critical-Sized Bone Defects via Readily Implantable Scaffold-Free Stem Cell Constructs.

Stem Cells Transl Med 2017 07 8;6(7):1644-1659. Epub 2017 Jun 8.

Biomedical Engineering.

The growing socioeconomic burden of musculoskeletal injuries and limitations of current therapies have motivated tissue engineering approaches to generate functional tissues to aid in defect healing. A readily implantable scaffold-free system comprised of human bone marrow-derived mesenchymal stem cells embedded with bioactive microparticles capable of controlled delivery of transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) was engineered to guide endochondral bone formation. The microparticles were formulated to release TGF-β1 early to induce cartilage formation and BMP-2 in a more sustained manner to promote remodeling into bone. Cell constructs containing microparticles, empty or loaded with one or both growth factors, were implanted into rat critical-sized calvarial defects. Micro-computed tomography and histological analyses after 4 weeks showed that microparticle-incorporated constructs with or without growth factor promoted greater bone formation compared to sham controls, with the greatest degree of healing with bony bridging resulting from constructs loaded with BMP-2 and TGF-β1. Importantly, bone volume fraction increased significantly from 4 to 8 weeks in defects treated with both growth factors. Immunohistochemistry revealed the presence of types I, II, and X collagen, suggesting defect healing via endochondral ossification in all experimental groups. The presence of vascularized red bone marrow provided strong evidence for the ability of these constructs to stimulate angiogenesis. This system has great translational potential as a readily implantable combination therapy that can initiate and accelerate endochondral ossification in vivo. Importantly, construct implantation does not require prior lengthy in vitro culture for chondrogenic cell priming with growth factors that is necessary for current scaffold-free combination therapies. Stem Cells Translational Medicine 2017;6:1644-1659.
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http://dx.doi.org/10.1002/sctm.16-0222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689752PMC
July 2017

Juvenile myelomonocytic leukemia with prominent CD141+ myeloid dendritic cell differentiation.

Hum Pathol 2017 10 13;68:147-153. Epub 2017 Apr 13.

Department of Pediatrics, University Hospitals Cleveland Medical Center, Cleveland, OH 44106.

Myeloid malignancies showing CD141+ myeloid dendritic cell (MDC) differentiation have not been documented. Here, we describe a patient with juvenile myelomonocytic leukemia in which a prominent CD141+ cell population was identified most consistent with CD141+ MDCs based on phenotypic similarity with normal CD141+ MDCs. Molecular studies demonstrated a KRAS mutation. The findings from the spleen and bone marrow are described. This is the first well-documented demonstration of CD141+ MDC differentiation of a hematopoietic neoplasm.
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http://dx.doi.org/10.1016/j.humpath.2017.03.025DOI Listing
October 2017

Expression of miR-145-5p During Chondrogenesis of Mesenchymal Stem Cells.

J Stem Cell Res (Overl Park) 2017 3;1(3):1-10. Epub 2017 Oct 3.

Radiology, Case Western Reserve University, Ohio, US.

Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926818PMC
October 2017

Disparate response of articular- and auricular-derived chondrocytes to oxygen tension.

Connect Tissue Res 2016 07 29;57(4):319-33. Epub 2016 Apr 29.

a Matrix Biology Program , Benaroya Research Institute at Virginia Mason , Seattle , WA , USA.

Purpose/aim: To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1-7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes.

Materials And Methods: Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression.

Results: Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension.

Conclusions: This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.
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http://dx.doi.org/10.1080/03008207.2016.1182996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984267PMC
July 2016

Immune Signatures Following Single Dose Trastuzumab Predict Pathologic Response to PreoperativeTrastuzumab and Chemotherapy in HER2-Positive Early Breast Cancer.

Clin Cancer Res 2016 07 3;22(13):3249-59. Epub 2016 Feb 3.

Case Western Reserve University School of Medicine, Cleveland, Ohio.

Purpose: Recent data suggest that intrinsic subtype and immune cell infiltration may predict response to trastuzumab-based therapy. We studied the interaction between these factors, changes in immune signatures following brief exposure to trastuzumab, and achievement of pathologic complete response (pCR) to subsequent preoperative trastuzumab and chemotherapy in HER2-positive breast cancer.

Experimental Design: In patients enrolled on two multicenter trials (03-311 and 211B), tumor core biopsies were obtained at baseline and after brief exposure to single-agent trastuzumab or nab-paclitaxel. Gene expression profiles were assessed to assign PAM50 subtypes, measure immune cell activation, and were correlated with response.

Results: The pCR rate was significantly higher in HER2-enriched tumors in the Discovery, 03-311 (36%, P = 0.043) dataset, as compared with other subtypes, which validated in 211B (50%, P = 0.048). Significant increases in a signature of immune cell admixture (Immune Index) were observed only following brief exposure to trastuzumab in HER2-enriched tumors (Discovery/03-311, P = 0.05; Validation/211B, P = 0.02). Increased Immune Index was predictive of response after brief exposure (03-311, P = 0.03; 211B, P = 0.04), but not at baseline, in addition to increased expression of a CD4(+) follicular helper T-cell signature (03-311, P = 0.05; 211B, P = 0.04). Brief exposure to trastuzumab significantly increased gene expression of the T-cell marker PD-1 in HER2-enriched tumors (Discovery/03-311, P = 0.045) and PD-1 positivity by IHC (Validation/211B, P = 0.035).

Conclusions: Correlations between pCR rates, increases in Immune Index and markers of T-cell activity following brief exposure to trastuzumab in HER2-enriched tumors provide novel insights into the interaction between tumor biology, antitumor immunity, and response to treatment, and suggest potential clinically useful biomarkers in HER2(+) breast cancers. Clin Cancer Res; 22(13); 3249-59. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439498PMC
July 2016

Aberrant Notch Signaling in the Bone Marrow Microenvironment of Acute Lymphoid Leukemia Suppresses Osteoblast-Mediated Support of Hematopoietic Niche Function.

Cancer Res 2016 Mar 22;76(6):1641-52. Epub 2016 Jan 22.

Department of Pathology, Case Western Reserve University, Cleveland, Ohio. Department of Pathology, University Hospitals Case Medical Center, Cleveland, Ohio.

More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794354PMC
March 2016

CD1c(+) myeloid dendritic cells in myeloid neoplasia.

Cytometry B Clin Cytom 2016 07 22;90(4):337-48. Epub 2015 Dec 22.

Department of Pathology, University Hospitals Case Medical Center and Seidman Cancer Center Case Western Reserve University, Cleveland, Ohio, 44106.

We determined the normal level and phenotype of CD1c(+) myeloid dendritic cells (MDCs) in blood and bone marrow and evaluated the level of CD1c(+) MDCs in 295 myeloid neoplasms. CD1c(+) MDCs were increased above the mean level of non-neoplastic hospital controls in 18.0% (53/295) of myeloid malignancies, increased three standard deviations above the control mean in 14.2% (42/295) with a 10-fold or more increase compared to mean in 6.8% (20/295). Increased CD1c(+) MDCs were associated with chronic myelomonocytic leukemia (CMML) (12/24, 50%) and acute myeloid leukemia (AML) (31/140, 22%) with a strong association with AML with the inv(16) cytogenetic abnormality. The cells were not increased in chronic myelogenous leukemia (CML) and rarely increased in non-CML myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). Immunohistochemical staining of cases with increased CD1c(+) MDCs did not reveal clustering of the cells unlike that observed with myeloid neoplasms associated with increased plasmacytoid dendritic cells. Our findings indicate CD1c(+) MDC elevations are not uncommon in myeloid leukemias and are associated with CMML and AML, particularly AML with inv(16). © 2015 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21332DOI Listing
July 2016

Loss of Hes1 Differentiates Sessile Serrated Adenoma/Polyp From Hyperplastic Polyp.

Am J Surg Pathol 2016 Jan;40(1):113-9

*Department of Pathology, University Hospitals Case Medical Center †Department of Pathology, Case Western Reserve University, Cleveland, OH.

Sessile serrated adenoma/polyp (SSA/p) is a precancerous lesion, and its differential diagnosis from hyperplastic polyp (HP) could be challenging in certain circumstances based on morphology alone. Hes1 is a downstream target of Notch-signaling pathway and plays an important role in intestinal development by regulating differentiation of enterocytes. In this study, we evaluated the expression patterns of Hes1 in SSA/p and HP, and determine whether Hes1 immunostaining can help differentiate between these 2 entities. Serrated polyps with cytologic dysplasia (SSA with cytologic dysplasia, tubular adenoma, and traditional serrated adenoma) were also studied. Hes1 is ubiquitously expressed in the nuclei of normal colon epithelial cells. The complete loss or a very weak expression of Hes1 is observed in the majority of the SSA/p in the study (58/63, 92%) compared with the normal expression of Hes1 in HP (35/35,100%). In SSA/p with cytologic dysplasia, dysplastic area demonstrated cytoplasmic and/or nuclear staining for Hes1. Tubular adenoma and traditional serrated adenoma showed variability of Hes1 staining within the polyp with a mixed positive and negative staining pattern. Our study suggests that loss of Hes1 could be used as a sensitive and specific marker to differentiate SSA/p from HP, which helps the diagnosis in morphologically challenging cases.
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http://dx.doi.org/10.1097/PAS.0000000000000531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008857PMC
January 2016

CRABP-II is a highly sensitive and specific diagnostic molecular marker for pancreatic ductal adenocarcinoma in distinguishing from benign pancreatic conditions.

Hum Pathol 2014 Jun 31;45(6):1177-83. Epub 2014 Jan 31.

Department of Pathology, University Hospitals Case Medical Center, Cleveland, OH 44106; Case Western Reserve University, Cleveland, OH 44106. Electronic address:

CRABP-II, a retinoic acid binding protein, shuffles retinoic acid from cytoplasm into nucleus and forms a complex with nuclear retinoic acid receptor to facilitate transcriptional activities of retinoic acid. In this study, we studied the expression patterns of CRABP-II in pancreatic ductal adenocarcinoma (PDAC) compared with those in normal pancreas, chronic pancreatitis, and precancerous lesions. We showed no detectable expressions of CRABP-II in normal pancreatic parenchyma, normal ductal epithelium, and chronic pancreatitis. In contrast, the expression of CRABP-II was readily detected in all PDACs including metastatic PDACs. CRABP-II staining was also observed and progressively increased from pancreatic intraepithelial neoplasia 1 to 3. In addition, when fine needle aspiration specimens were evaluated from patients with PDAC, CRABP-II was positive in 55.6% cases if cytology diagnosis was "atypia," and in 87.5% cases, if "malignancy." Our study suggests that CRABP-II is highly and specifically expressed in PDAC and is more commonly expressed in high-grade precursor cancerous lesions than in low-grade lesions. Therefore, overexpression of CRABP-II is a late event of pancreatic carcinogenesis, and it could be used as a diagnostic marker to distinguish PDAC from other benign pancreatic conditions in both resection and cytology specimens.
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http://dx.doi.org/10.1016/j.humpath.2014.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621761PMC
June 2014

Utilization of CDX2 expression in diagnosing pancreatic ductal adenocarcinoma and predicting prognosis.

PLoS One 2014 29;9(1):e86853. Epub 2014 Jan 29.

Department of Pathology, University Hospitals Case Medical Center, Cleveland, Ohio, United States of America ; Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States of America.

CDX2, a master transcriptional regulator of intestinal cell differentiation and survival, has been used as a marker to indicate colorectal lineage in adenocarcinomas of unknown origin. Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes for adenocarcinomas of unknown origin, but CDX2 expression in pancreatic disease remains unclear. In this study, we systemically and extensively investigated the expression and role of CDX2 in PDAC. We reported that CDX2 expression is weak and heterogeneous is all normal pancreas and chronic pancreatitis. It is largely expressed in epithelial-lining cells of pancreatic ducts including main ducts, inter-lobular ducts, intra-lobular ducts, intercalated ducts and centroacinar cells, but not in acinar cells or islet cells. CDX2 expression is down regulated during the transformation process from PanIN to PDAC. Only one third of PDACs retain some degree of CDX2 expression, and this group of PDACs have reduced median survival time compared to that of CDX2 negative group (308 days vs. 586 days, p = 0.0065). Metastatic PDACs remain similar expression pattern to that of the primary sites. Our study clearly demonstrates CDX2 expression in pancreatic diseases including PDAC, which is practically important when CDX2 is used to establish the primary sites of adenocarcinomas of unknown origin. In addition, our study also provides CDX2 as a prognostic marker for PDAC and implicates an important role of CDX2 in the development of normal pancreas and PDAC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086853PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906088PMC
September 2014

Serial transplantation and long-term engraftment of intra-arterially delivered clonally derived mesenchymal stem cells to injured bone marrow.

Mol Ther 2014 Jan 25;22(1):160-8. Epub 2013 Sep 25.

Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, Ohio, USA.

It has been hypothesized that mesenchymal stem cells (MSCs) home to sites of injury. Nevertheless, efficient delivery of MSCs to target organs and description of their ultimate fate remain major challenges. We provide evidence that intra-arterially (IA) injected MSCs selectively engraft from the circulation as perivascular cells in the bone marrow (BM) after a localized radiation injury. Luciferase-expressing MSCs, derived from a conditionally immortalized clone (BMC-9) representing a pure population of cells, were arterially delivered into mice irradiated in one leg. Cell distribution was measured by bioluminescent imaging and final destination assessed by luciferase immunolocalization. IA injections resulted in engraftment only in the irradiated leg where cells localize and proliferate abluminal to the BM vasculature, a phenomenon not replicated with intravenous injections or with IA injections of kidney cells harvested from the same donor used for MSCs. Furthermore, MSCs harvested from the engrafted marrow and serially transplanted retain the ability to selectively engraft at sites of injury. This study demonstrates that MSCs can serially engraft at sites of injury from the circulation, that they reside in the perivascular space, and that arterial delivery is more efficient than venous delivery for cell engraftment.
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http://dx.doi.org/10.1038/mt.2013.221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980027PMC
January 2014

Ossified choroid plexus papilloma of the fourth ventricle: elucidation of the mechanism of osteogenesis in benign brain tumors.

J Neurosurg Pediatr 2013 Jul 3;12(1):13-20. Epub 2013 May 3.

Division of Pediatric Neurosurgery, Rainbow Babies and Children's Hospital, Cleveland, Ohio, USA.

True ossification within benign brain tumors is rare, and the molecular mechanism for this process is poorly understood. The authors report a case of ossified choroid plexus papilloma (CPP) and analyze it to help elucidate the underlying molecular basis of osteogenesis in benign brain tumors. A 21-year-old man presented with headache and depression that progressed over years. Computed tomography, MRI, and angiography demonstrated a large heavily calcified fourth ventricular tumor with a vascular blush and no hydrocephalus. The tumor was resected and was found to be an ossified CPP. Immunohistochemical staining for VEGF, Sox2, BMP-2, osterix, osteopontin, and osteocalcin was performed in an attempt to elucidate the mechanism of bone formation. The tumor was extensively ossified with mature bone trabeculae. Immunostaining for VEGF was positive. Additional staining showed the presence of osteocalcin in this ossified tumor but not in samples of nonossified CPPs collected from other patients. Staining for osterix and osteopontin was equivocally positive in the ossified CPP but also in the nonossified CPPs examined. The presence of osteocalcin in the ossified CPP demonstrates that there is true bone formation rather than simple calcification. Its appearance within cells around the trabeculae suggests the presence of osteoblasts. The presence of osterix suggests that a pluripotent cell, or one that is already partially differentiated, may be differentiated into an osteoblast through this pathway. This represents the first systematic immunohistochemical analysis of osteogenesis within choroid plexus tumors.
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http://dx.doi.org/10.3171/2013.3.PEDS12400DOI Listing
July 2013

Methods for producing scaffold-free engineered cartilage sheets from auricular and articular chondrocyte cell sources and attachment to porous tantalum.

Biores Open Access 2012 Aug;1(4):157-65

Department of Biomedical Engineering, Case Western Reserve University , Cleveland, Ohio. ; Department of Orthopaedics, Case Western Reserve University , Cleveland, Ohio. ; Hope Heart Matrix Biology Program, Benaroya Research Institute , Seattle, Washington.

Scaffold-free cartilage engineering techniques may provide a simple alternative to traditional methods employing scaffolds. We previously reported auricular chondrocyte-derived constructs for use in an engineered trachea model; however, the construct generation methods were not reported in detail. In this study, methods for cartilage construct generation from auricular and articular cell sources are described in detail, and the resulting constructs are compared for use in a joint resurfacing model. Attachment of cartilage sheets to porous tantalum is also investigated as a potential vehicle for future attachment to subchondral bone. Large scaffold-free cartilage constructs were produced from culture-expanded chondrocytes from skeletally mature rabbits, and redifferentiated in a chemically-defined culture medium. Auricular constructs contained more glycosaminoglycan (39.6±12.7 vs. 9.7±1.9 μg/mg wet weight, mean and standard deviation) and collagen (2.7±0.45 vs. 1.1±0.2 μg/mg wet weight, mean and standard deviation) than articular constructs. Aggregate modulus was also higher for auricular constructs vs. articular constructs (0.23±0.07 vs. 0.12±0.03 MPa, mean and standard deviation). Attachment of constructs to porous tantalum was achieved by neocartilage ingrowth into tantalum pores. These results demonstrate that large scaffold-free neocartilage constructs can be produced from mature culture-expanded chondrocytes in a chemically-defined medium, and that these constructs can be attached to porous tantalum.
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http://dx.doi.org/10.1089/biores.2012.0231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559237PMC
August 2012

Monosodium Urate and Tumor Necrosis Factor-α Increase Apoptosis in Human Chondrocyte Cultures.

Rheumatology (Sunnyvale) 2012 Dec;2:113

Department of Orthopaedics, Case Western Reserve University School of Medicine, University Hospitals Case Medical Center, Cleveland, Ohio, USA 44106 ; Benaroya Research Institute at Virginia Mason, Seattle, WA, USA 98101-2795.

Monosodium urate and tumor necrosis factor-α, are two potent mediators of separate inflammatory response pathways in arthritic joints where inflammation may be accompanied by the loss of chondrocyte vitality via apoptosis. To address this possibility , chondrocyte cultures were employed to determine the extent to which monosodium urate and recombinant TNF-α altered the frequency of apoptotic chondrocytes. Apoptosis as a function of the activation of p38 kinase, C-Jun-terminal kinase, signal transducer and activator of transcription-3 and/or the activity of xanthine oxidase was also studied. Using normal human chondrocytes, monosodium urate or recombinant tumor necrosis factor-α increased the frequency of apoptosis and activity of xanthine oxidase. However, the xanthine oxidase-specific inhibitor, febuxostat, failed to blunt this response. Monosodium urate, tumor necrosis factor-α or the Janus kinase inhibitor, AG-490, increased the frequency of apoptotic nuclei in macroaggregate pellet cultures initiated from juvenile human chondrocytes, but not in pellet cultures derived from mesenchymal stem cells. In OA chondrocytes, activation of p38, C-Jun-NH-kinase and signal transducer and activator of transcription-3 preceded apoptosis. Activation of signal transducer and activator of transcription-3 also was seen in pellet cultures initiated from juvenile chondrocytes and MSCs incubated with MSU, recombinant tumor necrosis factor-α or febuxostat, but apoptosis was increased only in the pellet cultures derived from juvenile chondrocytes. Although AG-490 or the combination of AG-490 and febuxostat inhibited signal transducer and activator of transcription-3 activation, apoptosis was unaffected. These results showed that recombinant tumor necrosis factor-α, monosodium urate and AG-490 increased apoptosis in normal human chondrocytes, OA chondrocytes and human juvenile chondrocyte pellet cultures, but not in chondrocyte pellet cultures initiated from MSCs. The increased frequency of apoptotic chondrocytes in response to recombinant tumor necrosis factor-α or monosodium urate was not dependent on either activation of STAT3 or the activity of XO.
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http://dx.doi.org/10.4172/2161-1149.1000113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959644PMC
December 2012

Significance of Notch1-signaling pathway in human pancreatic development and carcinogenesis.

Appl Immunohistochem Mol Morphol 2013 May;21(3):242-7

Department of Pathology, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

In animal studies, Notch1-signaling pathway plays an important role in the pancreatic embryogenesis by promoting pancreatic progenitor cells self-renewal and exocrine linage development. The persistent activation of Notch pathway could arrest the organ development and keep cells at an undifferentiated stage. Studies have shown that Notch1-signaling pathway is upregulated in invasive pancreatic ductal adenocarcinoma (PDAC). Here we examined the expression pattern of Notch1 and Hes1 in human fetal pancreatic tissues to elucidate the role of Notch1 in human pancreatic embryonic development. We also compared Notch1 expression in tissues from PDAC, chronic pancreatitis and pancreatic intraepithelial neoplasm. Our data show that Notch1/Hes1-signaling pathway is activated during early pancreatic embryogenesis and reaches the highest at birth. After pancreas is fully developed, Notch1/Hes1 pathway is inactivated even though Notch1 protein cell-surface expression is upregulated. We also showed that the expression of both Notch1 and Hes1 are present in 50% (33/66) of PDACs, but not in pancreatic intraepithelial neoplasms. These findings indicate that Notch1 activation is only apparent in late stage of pancreatic carcinogenesis, suggesting that treatment with Notch-signaling inhibitors including γ-secretase should be selectively used for PDACs with confirmed Notch1-signaling activation.
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http://dx.doi.org/10.1097/PAI.0b013e3182655ab7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621772PMC
May 2013

Loss of ARID1A/BAF250a expression in ovarian endometriosis and clear cell carcinoma.

Int J Clin Exp Pathol 2012 5;5(7):642-50. Epub 2012 Sep 5.

Department of Pathology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Ovarian endometriosis has been associated with increased risk for ovarian clear cell carcinoma (CCC). Atypical endometriosis shares common molecular alterations with CCC and therefore, has been proposed as a precursor lesion of CCC, although it is unclear if benign endometriosis is pre-neoplastic. In this study, we examined some molecular alterations in ovarian benign endometriosis, atypical endometriosis, and CCC in comparison to papillary serous carcinoma (PSC). These included BAF250a (encoded by ARID1A), a recently identified major tumor suppressor in ovarian CCC, as well as hepatocyte nuclear factor (HNF)-1b, estrogen receptor (ER), progesterone receptor (PR), and P53. We confirmed that CCC but not PSC had loss of BAF250a expression, HNF-1b up-regulation, loss of ER expression and P53 expression. We further showed that both atypical endometriosis and adjacent CCC had loss of BAF250a expression (38.5% vs. 57.7%), HNF-1b up-regulation (53.8% vs. 92.3%), and loss of ER (84.6% vs. 92.3%) and PR (76.9% vs. 84.6%) expression. Importantly, about 20% of benign ovarian endometriosis had loss of BAF250a expression, 33% with HNF-1b up-regulation, 23% loss of ER expression and 50% loss of PR expression, respectively. The concurrent rate of loss of BAF250a expression, HNF-1b up-regulation, and loss of ER expression was not observed in any benign endometriosis, and was increased to 23.1% in atypical endometriosis, and was further increased to 42.3% in CCC. Therefore, the molecular alterations accumulate in a stepwise manner along the transformation process from benign endometriosis through atypical endometriosis to CCC. These data suggest that a portion of benign ovarian endometriosis has already undergone genetic alterations that lead to aberrant protein expression, possibly conferring a higher risk for malignant transformation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438773PMC
February 2013

Follicular center helper T-cell (TFH) marker positive mycosis fungoides/Sezary syndrome.

Mod Pathol 2013 Jan 24;26(1):32-43. Epub 2012 Aug 24.

Department of Pathology, Seidman Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA.

We identified 11 patients with CD10(+) cutaneous T-cell lymphoma by flow cytometry. All cases were CD4(+) and CD8(-). Three patients had extensive lymphadenopathy, systemic symptoms and an aggressive clinical course consistent with angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma. However, 8 of the 11 patients had a prolonged disease course with gross morphology, histology and tumor cell phenotype indistinguishable from mycosis fungoides or Sezary syndrome. Immunohistochemical studies confirmed CD10 expression in seven of the eight cases and revealed the lymphoma cells were Bcl-6(+), PD-1(+), and EBV(-). Two had significant expression of CXCL-13(+). The findings indicate that lymphoma cells from mycosis fungoides or Sezary syndrome may express follicular center helper T-cell markers CD10, Bcl-6, and PD-1 and occasionally CXCL-13. The expression of these markers in some cases of mycosis fungoides/Sezary syndrome suggests follicular center helper T-cell differentiation and may lead to confusion in distinguishing mycosis fungoides/Sezary syndrome from other follicular center helper T-cell marker positive T-cell lymphomas with cutaneous manifestations.
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http://dx.doi.org/10.1038/modpathol.2012.124DOI Listing
January 2013

Targeting improves MSC treatment of inflammatory bowel disease.

Mol Ther 2010 Jul 13;18(7):1365-72. Epub 2010 Apr 13.

Department of Orthopedics, Case Western Reserve University, Cleveland, Ohio, USA.

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is an inflammatory autoimmune disease characterized by T-cell infiltration to the colon. Mesenchymal stem cells (MSCs) have the potential to rescue IBD owing to their immunosuppressive capabilities and clinical studies have shown positive influence on intestinal graft versus host disease. We demonstrate here a new method to coat MSCs with antibodies against addressins to enhance their delivery to the colon and thereby increase the therapeutic effectiveness. Bioluminescence imaging (BLI) demonstrated that vascular cell adhesion molecule antibody (Ab)-coated MSCs (Ab(VCAM-1)- MSCs) had the highest delivery efficiency to inflamed mesenteric lymph node (MLN) and colon compared to untreated MSCs, Ab(isotype)-MSCs, and Ab(MAdCAM)-MSCs. Therapeutically, when mice with IBD were injected with addressin Ab-coated MSCs, they showed dramatically improved survival rates, higher IBD therapeutic scores, and significantly improved body weight gain compared to mice injected with MSCs only, isotype Ab, free Ab plus MSCs, or vehicle-only controls. These data demonstrate that anti-addressin Ab coating on MSC increased cell delivery to inflamed colon and increased the efficacy of MSC treatment of IBD. This is the first study showing an increased therapeutic efficacy when stem cells are first coated with antibodies specifically target them to inflamed sites.
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http://dx.doi.org/10.1038/mt.2010.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911249PMC
July 2010

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging.

Physiol Genomics 2009 Mar 30;37(1):23-34. Epub 2008 Dec 30.

Department of Biomedical Engineering, University Hospitals, Case Medical Center, Case Western Reserve University, Cleveland, Ohio, USA.

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.
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http://dx.doi.org/10.1152/physiolgenomics.00300.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661103PMC
March 2009

Fabrication of a neotrachea using engineered cartilage.

Laryngoscope 2008 Apr;118(4):593-8

Department of Orthopaedics/Otolaryngology-Head and Neck Surgery, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Objectives: Surgical management of long-segment tracheal stenosis is an ongoing problem. Many types of tracheal prostheses have been tried but with limited success because of immune rejection, graft ischemia, or restenosis. Tissue engineered cartilage may offer a solution to this problem, although scaffolds, which are currently often used for support, can lead to biocompatibility problems. This study investigated the feasibility of scaffold-free cartilage to tissue engineer a vascularized neotrachea in rabbits.

Study Design: Animal study.

Methods: Autologous neotracheal constructs were implanted in the abdomen of six New Zealand white rabbits. Auricular chondrocytes were used to engineer scaffold-free cartilage sheets. A muscle flap raised from the external abdominal oblique muscle and the engineered cartilage were wrapped around a silicone stent to fabricate a vascularized neotrachea in vivo. In two of the six rabbits, a full thickness skin graft was used to create an epithelial lining. The constructs were harvested after either 6 or 10 weeks.

Results: All neotracheal constructs were healthy with well-vascularized and integrated layers. The implanted engineered cartilage underwent a remodeling process, forming a solid tracheal framework. Constructs harvested after 10 weeks proved to have significantly better mechanical properties than after 6 weeks and were comparable with the rabbit's native trachea.

Conclusion: Scaffold-free engineered cartilage can successfully fabricate a well-vascularized, autologous neotrachea with excellent mechanical properties. The results suggest that this approach can be used to reconstruct tracheal defects in rabbits.
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http://dx.doi.org/10.1097/MLG.0b013e318161f9f8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504720PMC
April 2008

Imaging of mesenchymal stem cell transplant by bioluminescence and PET.

J Nucl Med 2007 Dec 15;48(12):2011-20. Epub 2007 Nov 15.

Department of Nuclear Medicine/Radiology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Unlabelled: Dynamic measurements of infused stem cells generally require animal euthanasia for single-time-point determinations of engraftment. In this study, we used a triple-fusion reporter system for multimodal imaging to monitor human mesenchymal stem cell (hMSC) transplants.

Methods: hMSCs were transduced with a triple-fusion reporter, fluc-mrfp-ttk (encoding firefly luciferase, monomeric red fluorescent protein, and truncated herpes simplex virus type 1 sr39 thymidine kinase) by use of a lentiviral vector. Transduced cells were assayed in vitro for the expression of each functional component of the triple-fusion reporter. Transduced and control hMSCs were compared for their potential to differentiate into bone, cartilage, and fat. hMSCs expressing the reporter were then loaded into porous, fibronectin-coated ceramic cubes and subcutaneously implanted into NOD-SCID mice along with cubes that were loaded with wild-type hMSCs and empty cubes. Mice were imaged repeatedly over 3 mo by bioluminescence imaging (BLI), and selected animals underwent CT and PET imaging.

Results: Osteogenic, adipogenic, and chondrogenic potential assays revealed retained differentiation potentials between transduced and wild-type hMSCs. Signals from the cubes loaded with reporter-transduced hMSCs were visible by BLI over 3 mo. There was no signal from the empty or wild-type hMSC-loaded control cubes. PET data provided confirmation of the quantitative estimation of the number of cells at one spot (cube). Cubes were removed from some animals, and histologic evaluations showed bone formation in cubes loaded with either reporter-transduced or wild-type hMSCs, whereas empty controls were negative for bone formation.

Conclusion: The triple-fusion reporter approach resulted in a reliable method of labeling stem cells for investigation in small-animal models by use of both BLI and small-animal PET imaging. It has the potential for translation into future human studies with clinical PET.
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http://dx.doi.org/10.2967/jnumed.107.043166DOI Listing
December 2007

Hyaluronan-based scaffolds to tissue-engineer cartilage implants for laryngotracheal reconstruction.

Laryngoscope 2007 Oct;117(10):1745-9

Department of Orthopaedics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Objectives: Donor site morbidity, including pneumothorax, can be a considerable problem when harvesting cartilage grafts for laryngotracheal reconstruction (LTR). Tissue-engineered cartilage may offer a solution to this problem. This study investigated the feasibility of using Hyalograft C combined with autologous chondrocytes to tissue engineer cartilage grafts for LTR in rabbits.

Study Design: Animal study.

Methods: Eighteen New Zealand white rabbits underwent LTR: 12 rabbits received autologous tissue-engineered cartilage grafts and 6 animals, serving as a positive control group, native auricular cartilage. To determine any differences in response to the site of implantation and any potential immune response to the scaffold, a second piece of engineered neocartilage and a non-cell-loaded scaffold were inserted paralaryngeally into a subset of the rabbits. The rabbits were sacrificed 3, 6, 8, 10, and 12 weeks after the LTR and their larynx examined.

Results: None of the 18 rabbits showed signs of respiratory distress. A smooth, noninflammatory scar was visible intraluminally. Histologically, the native auricular cartilage implants showed excellent integration without any signs of inflammation or cartilage degradation. In contrast, all tissue-engineered grafts and empty scaffolds revealed marked signs of an unspecific foreign body reaction, leading to a complete degradation of the neocartilage, whether implanted para- or intralaryngeally.

Conclusion: In contrast to the success with which Hyalograft C has been applied in articular defect repair, our results indicate that, in rabbits, Hyalograft C initiates a foreign body reaction if implanted intra- or paralaryngeally, leading to cartilage degradation and possible graft failure. These findings suggest limitations on the environment in which Hyalograft C can be applied.
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http://dx.doi.org/10.1097/MLG.0b013e31811434aeDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504717PMC
October 2007

Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration.

Stem Cells 2007 Oct 21;25(10):2575-82. Epub 2007 Jun 21.

Department of Orthopaedics, Case Western Reserve University, University Hospitals of Cleveland, 6 Floor Hanna Building, 11100 Cedar Avenue, Cleveland, Ohio 44106, USA.

Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical-scale ex vivo production of bone marrow-derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC-C); and (c) an adherent subset of TRC-C (TRC-C(Ad)). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC-C(Ad) and TRC-C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r(2) > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.
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http://dx.doi.org/10.1634/stemcells.2007-0204DOI Listing
October 2007