Search

Supplemental Figure 1. Consort Diagram 03-311. Supplemental Figure 2. Consort Diagram 211B. Supplemental Figure 3. Subtype-specific changes in Immune Index upon brief-exposure to nab-paclitaxel in the 211B trial. Supplemental Figure 4. Evaluation of the Immune Index upon brief-exposure to trastuzumab in the 03-311 and 211B trials according to ER/PR status. Supplemental Figure 5. Evaluation of the Immune Index upon brief-exposure to nab-paclitaxel in the 211B trial according to ER/PR status. Supplemental Figure 6. Correlation between Immune Index and tumor content. Supplemental Figure 7. Association of CD4+ Th1 and Treg signatures with response. Supplemental Table 1. Immune Index genes discriminative of response at the post-exposure timepoint in the discovery (03-311) and validation (211B) datasets. Supplemental Table 2. Immune cell subset signatures discriminative of response at baseline and post-exposure timepoints in the discovery (03-311) and validation (211B) datasets. Supplemental Table 3. CD8+ T-cell cytolytic activity genes discriminative of response at the post-exposure timepoint in the discovery (03-311) and validation (211B) datasets.

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.

Supplemental Figures. This file contains supplemental figures concerning analysis of megakaryocyte progenitor and erythroid progenitor analysis in the leukemia marrow, platelet and megakaryocyte progenitor analysis after DBZ treatment of leukemia mice, as well as CXCL12 promoter CHIP analysis

Supplemental Figure Legends

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. Cancer Res; 76(6); 1641–52. ©2016 AACR.

Supplemental Materials and Methods This file contains detailed methods of IHC, WB, CHIP assay, luciferase reporter assay, qRT-PCR and 2-photon imaging analysis and etc.

The endoplasmic reticulum unfolded protein response (UPR) is a conserved adaptive signaling in ER homeostasis and has emerged as critical in highly proliferating cells and potential treatment target for acute T-cell lymphoblastic leukemia (T-ALL).

Purpose [11C]methionine ([11C]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 11C decay limited further clinical development of [11C]Met. Synthetic amino acid analog anti-1-amino-3-[18F]fluoro-cyclobutyl-1-carboxylic acid ([18F]FCABC) was developed and FDA-approved for PET imaging of recurrent prostate cancer. This study investigated “repurposed” [18F]FACBC for PET imaging of primary liver cancer such as hepatocellular carcinoma (HCC) in comparison with [11C]Met. Methods [11C]Met was synthesized in the lab, and [18F]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 [11C]Met and [18F]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. [11C]Met and [18F]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 [18F]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 [18F]FACBC and [11C]Met showed uptake in HCC through the use of a clinically relevant animal model of woodchuck HCC. The uptake and retention of [18F]FACBC and [11C]Met depend on their metabolism and also rely on the distribution of their principal amino acid transporters.

Malignant polyps are polypoid lesions that appear benign endoscopically but harbor invasive adenocarcinoma microscopically. Patient with diagnosis of malignant polyp can be managed by surgical resection or endoscopic surveillance. Current literature on long term recurrence is sparse. A total of 76 patients with malignant polyp and follow-up period of over one year are included. Of these, 28 patients underwent endoscopic polypectomy followed by surveillance (group 1). Forty-eight patients underwent segmental colectomy (group 2). In group 1, three patients developed local recurrent pT3 adenocarcinoma (5.9 to 9.7 years) and one patient developed liver metastasis (7.3 years). One patient presented with malignant polyp in another segment of colon (4.0 years). Two of the malignant polyps with local recurrence do not have commonly reported high-risk features, including tumor ≤ 1 mm from resection margin, presence of lymphovascular invasion and high grade tumor, they had invasion depth of >4 mm and harbored a TP53 missense mutation. In group 2, during the follow-up period (1.0-21.8 years, median 9.3 years), none of the patients developed local recurrence. In this study, surveillance group had a local late recurrence rate of 10.7% versus no local recurrence in surgical resection group (0%). Our study shows that depth of invasion of over 4 mm in malignant polyp is a risk factor for late local recurrence if managed by endoscopic surveillance. Further study is needed to explore whether certain molecular alterations, such as TP53 mutation, is a risk factor for late recurrence.

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 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.

AimDevelop an objective assay to detect c-MYC protein expression using multiparametric flow cytometry (FCM) as an alternative to immunohistochemistry (IHC).Methods57 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 ac-MYCgene rearrangement in aggressive and high-grade lymphomas was also assessed.Resultsc-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.c-MYCgene was rearranged by fluorescence in situ hybridisation in 6/9 samples with high c-MYC expression (>40%) by FCM and 6/14 by IHC.ConclusionsWe 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.

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.

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 18F-labeled clofarabine ([18F]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 [18F]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 [18F]CFA injection to evaluate the reduction in liver background uptake. Application of forodesine before hot [18F]CFA injection did not reduce the background uptake. Instead, it increased the background by 11.6&ndash, 36.3%. Application of probenecid also increased the liver background uptake by 16.6&ndash, 32.1%. Cold CFA application did reduce the liver background uptake of [18F]CFA, comparing to the baseline scan. Combining cold CFA with [18F]CFA for PET imaging of liver cancers is a promising strategy, worthy of further clinical evaluation.

Introduction. 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. Methods. 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. Results. 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. Conclusions. 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.

Purpose [11C]methionine ([11C]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 11C decay limited further clinical development of [11C]Met. Synthetic amino acid analog anti-1-amino-3-[18F]fluoro-cyclobutyl-1-carboxylic acid ([18F]FCABC) was developed and FDA-approved for PET imaging of recurrent prostate cancer. This study investigated “repurposed” [18F]FACBC for PET imaging of primary liver cancer such as hepatocellular carcinoma (HCC) in comparison with [11C]Met. Methods [11C]Met was synthesized in the lab, and [18F]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 [11C]Met and [18F]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. [11C]Met and [18F]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 [18F]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 [18F]FACBC and [11C]Met showed uptake in HCC through the use of a clinically relevant animal model of woodchuck HCC. The uptake and retention of [18F]FACBC and [11C]Met depend on their metabolism and also rely on the distribution of their principal amino acid transporters.

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.

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.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.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.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.

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. Cancer Res; 76(6); 1641–52. ©2016 AACR.

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.

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.

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.

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.

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.

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.

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.

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.

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 withatriple-fusionreporter,fluc-mrfp-ttk(encodingfireflyluciferase, 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 forthe expression ofeachfunctionalcomponentofthetriple-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 atonespot(cube).Cubeswereremovedfromsomeanimals,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.

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-CAd). 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-CAd 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 (r2 > .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.

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 hyperplastic polyp (HP), and determine whether Hes1 immunostaining can help differentiate between these two entities. Serrated polyps with cytological dysplasia (sessile serrated adenoma with cytological 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 to the normal expression of Hes1 in HP (35/35,100%). In SSA/p with cytological 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.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor beta and 10-7 M dexamethasone in 15-ml polypropylene tubes ("aggregate cultures"). Adipogenic potential was quantitated by flow cytometry of Nile Red-stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase-positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10-7 M vitamin D3 and 150 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow-derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.

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.

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.

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 in vitro, 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-NH2-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.

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.

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.

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.

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.

This study tested the tissue engineering hypothesis that construction of an osteochondral composite graft could be accomplished using multipotent progenitor cells and phenotype-specific biomaterials. Rat bone marrow-derived mesenchymal stem cells (MSCs) were culture-expanded and separately stimulated with transforming growth factor-beta1 (TGF-beta1) for chondrogenic differentiation or with an osteogenic supplement (OS). MSCs exposed to TGF-beta1 were loaded into a sponge composed of a hyaluronan derivative (HYAF-11) for the construction of the cartilage component of the composite graft, and MSCs exposed to OS were loaded into a porous calcium phosphate ceramic component for bone formation. Cell-loaded HYAFF-11 sponge and ceramic were joined together with fibrin sealant, Tisseel, to form a composite osteochondral graft, which was then implanted into a subcutaneous pocket in syngeneic rats. Specimens were harvested at 3 and 6 weeks after implantation, examined with histology for morphologic features, and stained immunohistochemically for type I, II, and X collagen. The two-component composite graft remained as an integrated unit after in vivo implantation and histologic processing. Fibrocartilage was observed in the sponge, and bone was detected in the ceramic component. Observations with polarized light indicated continuity of collagen fibers between the ceramic and HYAFF-11 components in the 6-week specimens. Type I collagen was identified in the neo-tissue in both sponge and ceramic, and type II collagen in the fibrocartilage, especially the pericellular matrix of cells in the sponge. These data suggest that the construction of a tissue-engineered composite osteochondral graft is possible with MSCs and different biomaterials and bioactive factors that support either chondrogenic or osteogenic differentiation.