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Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors,the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors,however,may be needed to guide cell growth and differentiation. To provide access to such new materials,we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait,approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested,and seven cell-binding peptides were identified. Of these,six sequences possess EC cell-binding ability. Specifically,when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold,they mediate cell adhesion. The corresponding soluble peptides block this adhesion,indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR),they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells. View Publication

PURPOSE: To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage. EXPERIMENTAL DESIGN: We performed immunohistochemical analyses of 12 proteins [CD44,CD24,ALDH1,vimentin,osteonectin,EPCR,caveolin 1,connexin 43,cytokeratin 18 (CK18),MUC1,claudin 7,and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only,135 cases of IDC with ductal carcinoma in situ (DCIS),35 cases of DCIS with microinvasion,and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry. RESULTS: CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors,especially in luminal A subtype. The expression of vimentin,osteonectin,connexin 43,ALDH1,CK18,GATA3,and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells. CONCLUSIONS: Our findings suggest that in breast cancer,the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage. View Publication

Memantine is classified as an NMDA receptor antagonist. We recently reported that memantine promoted the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus. However,the molecular mechanism responsible for the memantine-induced promotion of cellular proliferation remains unknown. In this study we searched for a factor that mediates memantine-induced cellular proliferation,and found that pigment epithelium-derived factor (PEDF),a broad-acting neurotrophic factor,is up-regulated in the dentate gyrus of adult mice after the injection of memantine. PEDF mRNA expression increased significantly by 3.5-fold at 1 day after the injection of memantine. In addition,the expression level of PEDF protein also increased by 1.8-fold at 2 days after the injection of memantine. Immunohistochemical study using anti-PEDF antibody showed that the majority of the PEDF-expressing cells were protoplasmic and perivascular astrocytes. Using a neurosphere assay,we confirmed that PEDF enhanced cellular proliferation under the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) but was not involved in the multilineage potency of hippocampal progenitor cells. Over expression of PEDF by adeno-associated virus,however,did not stimulate cellular proliferation,suggesting PEDF per se does not promote cellular proliferation in vivo. These findings suggest that the memantine induced PEDF up-regulation is involved in increased proliferation of hippocampal progenitor cells. View Publication

Human embryonic stem (hES) cell production of heparan sulfate influences cell fate and pluripotency. Human ES cells remain pluripotent in vitro through the action of growth factors signaling,and the activity of these factors depends on interaction with specific receptors and also with heparan sulfate. Here,we tested the hypothesis that matrix-associated heparan sulfate is enough to maintain hES cells under low fibroblast growth factor-2 concentration in the absence of live feeder cells. To pursue this goal,we compared hES cells cultured either on coated plates containing live murine embryonic fibroblasts (MEFs) or on a matrix derived from ethanol-fixed MEFs. hES cells were analyzed for the expression of pluripotency markers and the ability to form embryoid bodies. hES cells cultured either on live mouse fibroblasts or onto a matrix derived from fixed fibroblasts expressed similar levels of Oct-4,SOX-2,Nanog,TRA-1-60 and SSEA-4,and they were also able to form cavitated embryoid bodies. Heparan sulfate-depleted matrix lost the ability to support the adherence and growth of hES cells,confirming that this glycosaminoglycan,bound to the extracellular matrix,is enough for the growth and attachment of hES cells. Finally,we observed that the ethanol-fixed matrix decreases by 30% the levels of Neu5Gc in hES cells,indicating that this procedure reduces xeno-contamination. Our data suggest that matrix-bound heparan sulfate is required for the growth and pluripotency of hES cells and that ethanol-fixed MEFs may be used as a live cell"-free substrate for stem cells." View Publication

The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover,these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology,positive staining for alkaline phosphatase,normal karyotype,and expression of hESC-like markers including Nanog,Rex1,Oct4,TRA-1-60,TRA-1-80,SSEA-3,and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers,and could as well be readily differentiated into functional motor neurons. Among other things,our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming,differentiation ability,stability of the resulting lineages,and the risk of associated abnormalities. View Publication

We report here that butyrate,a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent,greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment,the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (textgreater100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines,including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors,show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation,we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming,butyrate treatment enhanced histone H3 acetylation,promoter DNA demethylation,and the expression of endogenous pluripotency-associated genes,including DPPA2,whose overexpression partially substitutes for butyrate stimulation. Thus,butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover,butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells,including cells from patients that are more refractory to reprogramming. View Publication

A subset of cells,tentatively called cancer stem cells (CSCs),in breast cancer have been associated with tumor initiation,drug resistance,and tumor persistence or aggressiveness. They are characterized by CD44 positivity,CD24 negativity,and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed,flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue,with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years,we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1,CD44,and cytokeratin to measure the coexpression of these proteins,putative CSCs appear in variable sized clusters and in 27 cases (of 490),which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size,histological grade,nodal status,ER-,PR,- and HER2-status. In this cohort,ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer. View Publication

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines,of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice,we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH(br) cells) from the OS99-1 xenografts were much less frequent,averaging 3% of the entire tumor population,compared to those isolated directly from the OS99-1 cell line. ALDH(br) cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH(lo) cells),generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH(br) cells illustrated the stem cell characteristics of self-renewal,the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A,Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma. View Publication

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only ∼ 44% of the seeded cells were rescued,but an optimized heat shock treatment combined with Ri significantly increased cell survival to ∼ 60%. Mechanistically,our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2,hES3) that were used at higher passages (textgreater 86). In contrast,rapid down regulation of Oct4,Tra-1-60,and SSEA4 was observed for ESI049,a clinically compliant line,used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless,our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. textcopyright 2010 Elsevier B.V. All rights reserved. View Publication

BACKGROUND: We recently reported that gastrointestinal (GI) cancer cells can be reprogrammed to a pluripotent state by the ectopic expression of defined embryonic stem (ES)-like transcriptional factors. The induced pluripotent cancer (iPC) cells from GI cancer were sensitized to chemotherapeutic agents and differentiation-inducing treatment during a short-term culture,although a phenotype induced by long-term culture needs to be studied. METHODS: A long-term cultured (Lc)-iPC cells were produced in GI cancer cell lines by virus-mediated introduction of four ES-like genes-c-MYC,SOX2,OCT3/4,and KLF4-followed by a culture more than three months after iPC cells induction. An acquired state was studied by expression of immature-related surface antigens,Tra-1-60,Tra-1-81,Tra-2-49,and Ssea-4; and epigenetic trimethyl modification at lysine 4 of histone H3. Sensitivity to chemotherapeutic agents and tumorigenicity were studied in Lc-iPC cells. RESULTS: Whereas the introduction of defined factors of iPC cells once induced an immature state and sensitized cells to therapeutic reagents,the endogenous expression of the ES-like genes except for activated endogenous c-MYC was down-regulated in a long-term culture,suggesting a high magnitude of the reprogramming induction by defined factors and the requirement of therapeutic maintenance in Lc-iPC cells from cholangiocellular carcinoma HuCC-T1 cells,which harbor TP53(R175H) and KRAS(G12D). The Lc-iPC cells showed resistance to 5-fluorouracil in culture,and high tumorigenic ability with activated endogenous c-MYC in immunodeficient mice. CONCLUSION: The Lc-iPC cells from HuCC-T1 might be prone to an undesirable therapeutic response because of an association with the activated endogenous c-MYC. To consider the possible therapeutic approach in GI cancer,it would be necessary to develop a predictive method for evaluating the improper reprogramming-associated aggressive phenotype of iPC cells. View Publication

Both innate and adaptive immune systems are considered important for cancer prevention,immunosurveillance,and control of cancer progression. It is known that,although both systems initially eliminate emerging tumor cells efficiently,tumors eventually escape immune attack by a variety of mechanisms,including differentiation and recruitment of immunosuppressive CD11b(+)Gr-1(+) myeloid suppressor cells into the tumor microenvironment. However,we show that CD11b(+)Gr-1(+) cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover,like dendritic cells,immunostimulatory CD11b(+)Gr-1(+) cells can strongly cross-prime,augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b(+)Gr-1(+) cells from ascites of ovarian cancer-bearing mice results in the significant regression of s.c. tumors even without being pulsed with exogenous tumor Ag prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b(+)Gr-1(+)) cells,thereby mediating highly effective antitumor immunity in an adoptive transfer model of immunity. View Publication

Compared with adults,pediatric mastocytosis has a relatively favorable prognosis. Interestingly,a difference was also observed in the status of c-kit mutations according to the age of onset. Although most adult patients have a D(816)V mutation in phosphotransferase domain (PTD),we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus KIT-PTD mutants were introduced into rodent Ba/F3,EML,Rat2,and human TF1 cells to investigate their biologic effect. Both ECD and PTD mutations induced constitutive receptor autophosphorylation and ligand-independent proliferation of the 3 hematopoietic cells. Unlike ECD mutants,PTD mutants enhanced cluster formation and up-regulated several mast cell-related antigens in Ba/F3 cells. PTD mutants failed to support colony formation and erythropoietin-mediated erythroid differentiation. ECD and PTD mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT,whereas AKT was only activated by ECD mutants. Consistently,AKT inhibitor suppressed ECD mutant-dependent proliferation,clonogenicity,and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EML-PTD cells,suggesting the differential role of AKT in those mutants. Overall,our study implied different pathogenesis of pediatric versus adult mastocytosis,which might explain their diverse phenotypes. View Publication