Stem Cell Marker Expression in Early Stage Colorectal Cancer is Associated with Recurrent Intestinal Neoplasia.

BACKGROUND: Colorectal cancer (CRC) ranks second in cancer deaths worldwide and presents multiple management challenges, one of which is identifying high risk stage II disease that may benefit from adjuvant therapy. Molecular biomarkers, such as ones that identify stem cell activity, could better stratify high-risk cohorts for additional treatment. METHODS: To identify possible biomarkers of high-risk disease in early-stage CRC, a discovery set (n = 66) of advanced-stage tumors were immunostained with antibodies to stemness proteins (CD166, CD44, CD26, and LGR5) and then digitally analyzed. Using a second validation cohort (n = 54) of primary CRC tumors, we analyzed protein and gene expression of CD166 across disease stages, and extended our analyses to CD166-associated genes (LGR5, ASCL2, BMI1, POSTN, and VIM) by qRT-PCR. RESULTS: Stage III and metastatic CRC tumors highly expressed stem cell-associated proteins, CD166, CD44, and LGR5. When evaluated across stages, CD166 protein expression was elevated in advanced-stage compared to early-stage tumors. Notably, a small subset of stage I and II cancers harbored elevated CD166 protein expression, which correlated with development of recurrent cancer or adenomatous polyps. Gene expression analyses of CD166-associated molecules revealed elevated ASCL2 in primary tumors from patients who recurred. CONCLUSIONS: We identified a protein signature prognostic of aggressive disease in early stage CRC. Stem cell-associated protein and gene expression identified a subset of early-stage tumors associated with cancer recurrence and/or subsequent adenoma formation. Signatures for stemness offer promising fingerprints for stratifying early-stage patients at high risk of recurrence.

Expression of cancer stem cell markers in breast phyllodes tumor.

BACKGROUND: Phyllodes tumor (PT) is a rare tumor showing various malignant potential. The histological grade of PT is related to clinical outcome, but its relationship between gaining of malignant potential and underlying mechanism including cancer stem cell factor was not understood yet. OBJECTIVE: The main purpose of this study was to determine the expression pattern of cancer stem cell marker in PT and to understand its clinical and pathological implications. METHODS: CD44, CD166, ALDH1, and Ki-67 immunohistochemistry were performed on a tissue microarray from 185 cases of PT specimens (138 benign, 32 borderline, 15 malignant). The immunohistochemistry result and clinicopathological parameter of each cases were compared to analyze the implications of cancer stem cell markers on PT. RESULTS: Borderline/malignant PT showed higher CD44 expression of the stromal component than benign PT (p< 0.001). In lower histologic grade PT, CD166 showed increased expression in the epithelial component (p= 0.019), but decreased in the stromal component (p< 0.001). Stromal overgrowth was rarely observed as the number of positive cancer stem cell markers increased in the epithelial component (p< 0.001). In the stromal component, the number of positive cancer stem cell markers was related to higher histologic grade (p< 0.001), and increased stromal cellularity (p< 0.001), stromal atypia (p= 0.003), and stromal mitosis (p= 0.002). In benign PT, CD44 negativity (p= 0.013) and a decreased number of positive cancer stem cell markers (p= 0.012) in the epithelial component were related to poor prognosis. CONCLUSIONS: The cancer stem cell markers, CD44 and CD166, are expressed in both the epithelial and stromal components of phyllodes tumor. Besides, ALDH1 is only expressed in stromal component. In the stromal component, expression of cancer stem cell markers increases with higher PT histologic grade. In the epithelial component, the absence of cancer stem cell marker expression is related to poor clinical prognosis.

In silico Analysis, Molecular Docking, Molecular Dynamic, Cloning, Expression and Purification of Chimeric Protein in Colorectal Cancer Treatment.

INTRODUCTION: Colorectal cancer (CRC) is a type of cancer in humans that leads to high mortality and morbidity. CD166 and CD326 are immunoglobulins that are associated with cell migration. These molecules are included in tumorigenesis of CRC and serve a great marker of CRC stem cells. In the present study, we devised a novel chimeric protein including the V1-domain of the CD166 and two epitopes of CD326 to use in diagnostic or therapeutic applications. METHODS: In silico techniques were launched to characterize the properties and structure of the protein. We have predicted physicochemical properties, structures, stability, MHC class I binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. The sequence of chimeric gene was optimized for expression in prokaryotic host using online tools and cloned into pET-28a plasmid. The recombinant pET28a was transformed into the E. coli BL21DE3. Expression of recombinant protein was examined by SDS-PAGE and Western blotting. RESULTS: The designed chimeric protein retained high stability and the same immunogenicity as of the original proteins. Bioinformatics data indicated that the epitopes of the synthetic chimeric protein might induce B-cell- and T-cell-mediated immune responses. Furthermore, a gene was synthesized using the codon bias of a prokaryotic expression system. This synthetic gene expressed a bacterial expression system. The recombinant protein with molecular weights of 27kDa was expressed and confirmed by anti-his Western blot analysis. CONCLUSION: The designed recombinant protein may be useful as a CRC diagnostic tool and for developing a protective vaccine against CRC.

Brachyury: Strategies for Drugging an Intractable Cancer Therapeutic Target.

New approaches to drug discovery are unlocking enormous therapeutic potential residing in cancer-specific molecules. Brachyury is emerging as an exciting new drug target for the rare bone cancer chordoma. Here, recent advances targeting Brachyury in chordoma are discussed and how these might open doors to the targeting of other, more common cancer types.

Rapid aptasensor capable of simply detect tumor markers based on conjugated polyelectrolytes.

In this paper, a very simple, easily-operated and universal platform is proposed for tumor marker detection. In this strategy, tumor marker-specific aptamer, which can quench the fluorescence of polyfluorene-based cationic conjugated polyelectrolytes (PFN+), are used as recognizing probes. Upon addition of tumor marker, the aptamer can be assembled into the tumor marker-aptamer complex, resulting in fluorescence recovery of PFN+ and the detection of the targets. The most widely-used tumor markers, carcinoembryonic antigen (CEA) and fetoprotein (AFP) have been chosen as the model analytes for this work. The sensing method is capable of rapidly detect target protein within 5 min without complex handling procedure and expensive instruments. Compared with previous studies, the assay presented here is really simple and avoids either conjugated polyelectrolytes (CPEs) modification or oligonucleotide labeling. This method also shows a wide detection range of 3 orders of magnitude and the detection limit is 0.316 ng/mL for CEA and 1.76 ng/mL for AFP. Furthermore, the approach requires only a convenient"mix-and-detect" procedure and offers a universal platform for the sensitive detection of any target molecule of choice according to the selected aptamer.

Co-detection of ALDH1A1, ABCG2, ALCAM and CD133 in three A549 subpopulations at the single cell level by one-step digital RT-PCR.

Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression. In this study, one-step digital RT-PCR at the single cell level was developed to co-determine the expression of ALDH1A1, ABCG2, ALCAM and CD133 genes in A549 cancer stem cells that perform high ALDH activities (ALDH+ A549 cells), as well as in ALDH- A549 cells and A549 cells, with 36, 20 and 20 cell samples each. The results demonstrated that, when compared to single ALDH- or A549 cells, the majority of single ALDH+ A549 cells displayed a 1.5- and 2.0-fold increase in the gene expression of ALDH1A1 and ALCAM (P < 0.001), respectively. However, for ABCG2 and CD133, there was no significant difference (P > 0.05), which means that they are not appropriate as co-indicated markers to identify ALDH+ A549 cells. Conclusively, as a single cell level approach, one-step digital RT-PCR has potential in exploring efficient co-detection markers for the classification and identification of CSCs.

Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture.

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can provide sources for midbrain dopaminergic (mDA) neural progenitors (NPCs) for cell therapy to treat Parkinson's disease (PD) patients. However, the well-known line-to-cell line variability in the differentiation capacity of individual cell lines needs to be improved for the success of this therapy. To address this issue, we sought to identify mDA NPC specific cell surface markers for fluorescence activated cell sorting (FACS). Through RNA isolation after sorting for NPCs based on staining for cell-specific transcription factors followed by microarray, we identified two positive cell surface markers (CORIN and CD166) and one negative cell surface marker (CXCR4) for mDA NPC sorting. These three markers can enrich floor plate NPCs to 90% purity, and the sorted NPCs more efficiently differentiate to mature dopaminergic neurons compared to unsorted or CORIN+ alone mDA NPCs. This surface marker identification strategy can be used broadly to facilitate isolation of cell subtypes of interest from heterogeneous cultures.

Chordoma: Immunohistochemical Analysis of Brachury.

AIM: Chordomas are rare, slow growing but locally aggressive malignancies of the axial skeleton. Skull base chordomas, due to their intricate anatomical localization, pose significant challenges to managing physicians. In classical and chondroid chordomas, the disease course cannot be reliably determined using only morphological criteria. Brachyury (T Gene) was shown to play a central role in chordoma pathogenesis and several studies also showed that this gene also carries potential as a prognostic biomarker. This study aims to correlate Brachyury expression with the clinical course in surgically treated skull base chordomas. MATERIAL AND METHODS: Chordoma tumor samples from 14 patients with skull base chordomas, diagnosed using histopathological and immunohistochemistry criteria (epithelial membrane antigen (EMA), S100, pan cytokeratin (panCK)) were retrospectively analyzed for Brachyury expression using immunohistochemistry. Brachyury expression was graded using a 4 point semi-quantitative scoring system. Focal (grade II) and diffuse staining (grade III) were considered as overexpression. Patient recurrence-free survival and total survival were compared between Brachyury overexpressing and non-overexpressing groups using Kaplan-Meier survival analysis. RESULTS: Among the stained tumor samples, 85.7% were positive for brachyury expression. In both groups, there was one sample that was negative. We did not observe any significant difference among the groups for staining, grade and percentage of brachyury positive cells. CONCLUSION: Brachyury expression in tumor samples is not a sensitive indicator of prognosis in chordomas.

Parosteal extra-axial chordoma of the second metacarpal bone: a case report with literature review.

Extra-axial chordoma is a chordoma that occurs in non-axial locations. It is a very rare tumor, with 20 cases reported to date; 14 in bone and six in soft tissue. Of the 14 skeletal extra-axial chordomas, ten were intramedullary and four were intracortical. We report the first case of parosteal extra-axial chordoma arising in the second metacarpal bone, expressing brachyury on immunohistochemical analysis, and describe the pathologic and radiologic findings. We suggest that extra-axial chordoma can occur in parosteal bone lesions or the hand, without features of bone distribution or bone-specific sites.

Clinicopathologic features of four rare types of chordomas, confirmed by brachyury immunostaining.

BACKGROUND: A wide clinicopathologic spectrum of a chordoma exists. Brachyury constitutes as its most useful diagnostic immunohistochemical (IHC) marker. METHODS: During a 7-year-period, 4 unusual histopathologic types of chordomas were identified. Immunohistochemistry was performed by polymer technique. RESULTS: Clinicopathologic features of the 4 cases are as follows: Cases 1 and 2: Two tumors occurred in the sacrococcygeal and lumbosacral regions of a 42-year-old male and a 34-year-old female, respectively. Histopathologic examination showed areas of classical chordoma; juxtaposed to a high-grade, spindle cell sarcoma. By IHC, cytokeratin (CK), epithelial membrane antigen (EMA), S-100 protein, and brachyury were found to be distinctly positive in the differentiated chordomatous areas. Both these cases were diagnosed as dedifferentiated chordomas. The first patient, postresection and adjuvant radiation therapy (RT), died after 14 months of therapy. Case 3: A 58-year-old male presented with pain in his sacral region and urinary incontinence. Imaging disclosed a sacral mass. Histopathologic examination showed physaliphorous cells intimately admixed with, markedly pleomorphic cells, scattered mitotic figures, and focal tumor necrosis. By IHC, the tumor cells were positive for CK, AE1/AE3, S-100 protein, brachyury, and INI1/SMARCB1. The diagnosis of a poorly differentiated chordoma was offered. Despite surgical resection and adjuvant RT, the patient died within 18 months. Case 4: A 58-year-old male presented with a soft tissue lesion in his left leg. Histopathologic examination showed physaliphorous cells, embedded in a myxohyaline stroma. By IHC, the tumor cells were positive for EMA, S-100 protein, brachyury, and INI1. Diagnosis of an extra-axial, soft tissue chordoma was offered. CONCLUSIONS: These four unusual chordomas, confirmed by brachyury immunoexpression, constitute as one of the first such documentation from our country, revealing a wide clinicopathologic spectrum of chordomas. Dedifferentiated and poorly differentiated chordomas are associated with an aggressive clinical course. Further diagnostic implications are discussed herewith.

Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression.

Recent research has confirmed the presence of Mesenchymal stem cell (MSC)-like progenitors (MPC) in both normal and osteoarthritic cartilage. However, there is only limited information concerning how MPC markers are expressed with osteoarthritis (OA) progression. The purpose of this study was to compare the prevalence of various MPC markers in different OA grades. Human osteoarthritic tibial plateaus were obtained from ten patients undergoing total knee replacement. Each sample had been classified into a mild or severe group according to OARSI scoring. Tissue was taken from each specimen and mRNA expression levels of CD105, CD166, Notch 1, Sox9, Acan and Col II A1 were measured at day 0 and day 14 (2 weeks in vitro). Furthermore, MSC markers: Nucleostemin, CD90, CD73, CD166, CD105 and Notch 1 were studied by immunofluorescence. mRNA levels of MSC markers did not differ between mild and severe OA at day 0. At day 14, protein analysis showed that proliferated cells from both sources expressed all 6 MSC markers. Only cells from the mild OA subjects resulted in a significant increase of mRNA CD105 and CD166 after in vitro expansion. Moreover, cells from the mild OA subjects showed significantly higher levels of CD105, Sox9 and Acan compared with those from severe OA specimens. Results confirmed the presence of MSC markers in mild and severe OA tissue at both mRNA and protein levels. We found significant differences between cells obtained from mild compared to severe OA specimens suggests that mild OA derived cells may have a greater MSC potential.

Annotation of the Domestic Pig Genome by Quantitative Proteogenomics.

The pig is one of the earliest domesticated animals in the history of human civilization and represents one of the most important livestock animals. The recent sequencing of the Sus scrofa genome was a major step toward the comprehensive understanding of porcine biology, evolution, and its utility as a promising large animal model for biomedical and xenotransplantation research. However, the functional and structural annotation of the Sus scrofa genome is far from complete. Here, we present mass spectrometry-based quantitative proteomics data of nine juvenile organs and six embryonic stages between 18 and 39 days after gestation. We found that the data provide evidence for and improve the annotation of 8176 protein-coding genes including 588 novel and 321 refined gene models. The analysis of tissue-specific proteins and the temporal expression profiles of embryonic proteins provides an initial functional characterization of expressed protein interaction networks and modules including as yet uncharacterized proteins. Comparative transcript and protein expression analysis to human organs reveal a moderate conservation of protein translation across species. We anticipate that this resource will facilitate basic and applied research on Sus scrofa as well as its porcine relatives.

ALCAM a novel biomarker in patients with type 2 diabetes mellitus complicated with diabetic nephropathy.

BACKGROUND & AIM: Activated leukocyte cell adhesion molecule (ALCAM/CD166) functions analogue to the receptor of advanced glycation end products, which has been implicated in the development of diabetic nephropathy (DN). We investigated the expression of ALCAM and its ligand S100B in patients with DN. METHODS: A total of 34 non-diabetic patients, 29 patients with type 2 diabetes and normal albuminuria and 107 patients with type 2 diabetes complicated with DN were assessed for serum concentration of soluble ALCAM (sALCAM) by ELISA. Expression of ALCAM and S100B in kidney histology from patients with DN was determined by immunohistochemistry. Cell expression of ALCAM and S100B was analyzed through confocal immunofluorescence microscopy. RESULTS: Serum concentration of sALCAM was increased in diabetic patients with DN compared to non-diabetic (59.85±14.99ng/ml vs. 126.88±66.45ng/ml, P<0.0001). Moreover sALCAM correlated positively with HbA1c (R=0.31, P<0.0001), as well as with the stages of chronic kidney disease and negatively correlated with eGFR (R=-0.20, P<0.05). In diabetic patients with normal albuminuria sALCAM was increased compared to patients with DN (126.88±66.45ng/ml vs. 197.50±37.17ng/ml, P<0.0001). In diabetic patients, ALCAM expression was significantly upregulated in both the glomeruli and tubules (P<0.001). ALCAM expression in the glomeruli correlated with presence of sclerosis (R=0.25, P<0.001) and localized mainly in the podocytes supporting the hypothesis that membrane bound ALCAM drives diabetic nephropathy and thus explaining sALCAM decrease in diabetic patients with DN. The expression of S100B was increased significantly in the glomeruli of diabetic patients (P<0.001), but not in the tubules. S100B was as well localized in the podocytes. CONCLUSIONS: This study identifies for the first time ALCAM as a potential mediator in the late complications of diabetes in the kidney.

Effects of conventional CPB and mini-CPB on neutrophils CD162, CD166 and CD195 expression.

OBJECTIVE: Cardiac surgery is known to trigger a systemic inflammatory response. While the use of conventional cardiopulmonary bypass (CPB) results in profound inflammation, modified mini-CPB is considered less harmful. We evaluated the impact of cardiac surgery on the expression of CD162, CD166, CD195 molecules and their association with the type of CPB used. METHODS AND RESULTS: Twenty-four patients were enrolled in our study. Twelve of them were operated using conventional CPB while the other twelve patients underwent surgery with mini-CPB. Blood samples were analysed by flow cytometry. We observed a significant increase in median fluorescence intensity of CD162 and CD195 that peaked instantly after surgery and normalized to the baseline value on the 1st day post surgery, whereas CD166 was initially down-regulated and its median fluorescence intensity (MFI) value increased to the baseline in the next few days. CONCLUSION: We observed immediate changes in the expression of CD162, CD166, and CD195 molecules on the neutrophils after surgery in both study groups of patients. The intensity of the observed changes was significantly greater in the group of patients who underwent conventional CPB compared to patients who underwent mini-CPB cardiac surgery.

Comparative proteomics of umbilical vein blood plasma from normal and gestational diabetes mellitus patients reveals differentially expressed proteins associated with childhood obesity.

PURPOSE: Offspring obesity is one of long-term complications of gestational diabetes mellitus (GDM). The aim of this study is to identify proteins differentially expressed in the umbilical vein blood plasma, which could become markers for early diagnosis of childhood obesity. EXPERIMENTAL DESIGN: Umbilical vein plasma samples were collected from 30 control and 30 GDM patients in 2007-2008 whose offspring were suffering from obesity at 6-7 years old. Multiplexed isobaric tandem mass tag labeling combined with LC-MS/MS was used to identify differentially expressed proteins. Ingenuity pathway analysis was performed to identify canonical pathways, biological functions, and networks of interacting proteins. Western blotting was used to verify the expression of three selected proteins. RESULTS: A total of 318 proteins were identified, of which 12 proteins were upregulated in GDM group while 24 downregulated. Lipid metabolism was the top category identified by ingenuity pathway analysis. Three randomly chosen proteins were validated by Western blotting, which were consistent with LC-MS. CONCLUSION: There are significant differences of protein profile in the umbilical vein blood plasma between normal and GDM patients with obese offspring. The results indicate that a variety of proteins and biological mechanisms may contribute to childhood obesity.

Prognostic significance of ALCAM (CD166/MEMD) expression in cutaneous melanoma patients.

BACKGROUND: ALCAM (activated leukocyte cell adhesion molecule, CD166, MEMD) is a transmembrane protein of immunoglobulin superfamily (Ig-SF) and plays an important role in human malignant melanoma progression and formation of locoregional and distant metastases. The study using melanoma cell lines showed that overexpression of ALCAM is directly related with the increase of cytoaggregation and the ability to form cell nests. The aim of the study was to assess the expression and intracellular localization of ALCAM in primary skin melanomas and metastatic lesions from regional lymph nodes. Also, prognostic significance of ALCAM expression in primary tumor cells and metastatic lesion cells was evaluated in the context of 5-year observation. METHODS: Formalin-fixed paraffin-embedded tissue specimens from 104 primary cutaneous melanomas and 16 regional lymph nodes metastases were studied for the expression of ALCAM measured by immunohistochemistry. RESULTS: We demonstrate that high ALCAM expression in primary melanoma cells (IRS ≥8) is strongly correlated with unfavorable prognosis as compared with patients with lower ALCAM immunoreactivity in tumor compartment as regards cancer specific overall survival (CSOS) (P = 0.001) and disease free survival (DFS) (P < 0.001). Additionally lower ALCAM immunoreactivity in nodal metastatic foci was significantly statistically correlated with deeper melanoma invasion in the primary tumor according to Clark scale (P = 0.032). It was also found that decreased ALCAM expression (IRS <8) in nodal metastases shows a trend related with a correlation with shorter cancer specific overall survival (P = 0.083). Statistically significant correlations were also demonstrated between the presence of ulceration and decreased intensity of lymphocytic inflammatory infiltration and a high percentage of ALCAM-positive cells (P = 0.035, P = 0.01, respectively). CONCLUSIONS: High ALCAM expression in melanoma cells of the primary tumor can be used as a marker of negative outcome and may indicate a more invasive phenotype of cancer cells, which would require a more intensive therapeutic strategy. Low expression of ALCAM in regional lymph node metastases is a feature associated with unfavorable prognosis in patients with cutaneous melanoma. Our study is the first one to evaluate the effect of increased ALCAM expression on long-term survival in melanoma patients.

Nuclear Brachyury Expression Is Consistent in Chordoma, Common in Germ Cell Tumors and Small Cell Carcinomas, and Rare in Other Carcinomas and Sarcomas: An Immunohistochemical Study of 5229 Cases.

Brachyury is a transcription factor of the T-box family typically expressed in notochord and chordoma. Some studies report brachyury as highly specific for chordoma, whereas others have concluded that brachyury is expressed in many types of common carcinomas by reverse transcription polymerase chain reaction and immunohistochemistry and could be involved in the epithelial-mesenchymal transition and metastatic process. In this study, we immunohistochemically evaluated 5229 different tumors for nuclear brachyury expression using a new rabbit monoclonal antibody and automated immunostaining (Leica Bond Max). Only nuclear labeling was scored, and antibody dilution of 1:2000 was used. In normal tissues, only rare cells in seminiferous tubules were labeled; all other organs were negative. All chordomas (75/76), except a sarcomatous one, were positive, whereas chondrosarcomas were negative. Among epithelial tumors, positivity was often detected in embryonal carcinoma (74%) and seminoma (45%). Pulmonary small cell carcinoma was often positive (41%), whereas pulmonary and pancreatic adenocarcinomas only rarely showed nuclear brachyury positivity (3% to 4%). Common carcinomas such as ductal carcinomas of the breast or adenocarcinomas of the prostate only exceptionally showed nuclear positivity (<1%). No colorectal, hepatocellular, renal cell, squamous cell, thyroid or urothelial carcinoma, or mesothelioma showed nuclear brachyury positivity. Among mesenchymal and neuroectodermal tumors, only isolated cases of melanoma, malignant peripheral nerve sheath tumor, rhabdomyosarcoma, synovial sarcoma, and follicular lymphoma showed nuclear expression. However, as shown previously with lung carcinoma, experiments with lower antibody dilutions (1:200 to 1:500) showed weak cytoplasmic and nuclear labeling in breast cancers. In addition to chordoma, we show here for the first time that nuclear brachyury expression is prevalent in embryonal carcinoma, seminoma, and small cell carcinoma of the lung but very rare in common carcinomas, sarcomas, and melanoma. With these reservations, we have demonstrated the presence of nuclear brachyury immunoreactivity to be a sensitive and fairly specific marker for chordoma.

Activin A can induce definitive endoderm differentiation from human parthenogenetic embryonic stem cells.

OBJECTIVES: As activin/nodal signaling plays a key role in definitive endoderm (DE) differentiation, we have explored activin A-induced differentiation of DE from human parthenogenetic embryonic stem cells (hPESCs). RESULTS: Administration of 5 ng activin A/ml had no effect on the expression of markers of DE differentiation. However, higher concentrations of activin A (50 and 100 ng/ml) upregulated Sox17 and Cxcr4, as well upregulating the mesendodermal precursor marker, Brachyury. CONCLUSIONS: These findings demonstrate that low dose activin A can maintain the undifferentiated potency of hPESCs, whereas higher doses induce DE differentiation; 50 ng/ml is the optimal concentration for inducing DE from hPESCs.