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The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However,the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end,we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog. View Publication

The tyrosine kinase Fyn plays a key role in oligodendrocyte differentiation and myelination in the central nervous system,but the molecules responsible for regulating Fyn activation in these processes remain poorly defined. Here we show that receptor-like protein-tyrosine phosphatase alpha (PTPalpha) is an important positive regulator of Fyn activation and signaling that is required for the differentiation of oligodendrocyte progenitor cells (OPCs). PTPalpha is expressed in OPCs and is up-regulated during differentiation. We used two model systems to investigate the role of PTPalpha in OPC differentiation: the rat CG4 cell line where PTPalpha expression was silenced by small interfering RNA,and oligosphere-derived primary OPCs isolated from wild-type and PTPalpha-null mouse embryos. In both cell systems,the ablation of PTPalpha inhibited differentiation and morphological changes that accompany this process. Although Fyn was activated upon induction of differentiation,the level of activation was severely reduced in cells lacking PTPalpha,as was the activation of Fyn effector molecules focal adhesion kinase,Rac1,and Cdc42,and inactivation of Rho. Interestingly,another downstream effector of Fyn,p190RhoGAP,which is responsible for Rho inactivation during differentiation,was not affected by PTPalpha ablation. In vivo studies revealed defective myelination in the PTPalpha(-/-) mouse brain. Together,our findings demonstrate that PTPalpha is a critical regulator of Fyn activation and of specific Fyn signaling events during differentiation,and is essential for promoting OPC differentiation and central nervous system myelination. View Publication

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived,patient-specific pluripotent cells for use in cell-based regenerative therapies. However,current methods of cell culture are tedious and expensive,and the mechanisms underlying cell proliferation are not understood. In this study,we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion,survival,and proliferation. We show that iPSC lines generated using Oct-3/4,Sox-2,Nanog,and Lin-28 express a repertoire of integrins similar to that of hESCs,with prominent expression of subunits alpha5,alpha6,alphav,beta1,and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin,in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel,as shown by immunological blockade experiments. On vitronectin,the integrin alphavbeta5 was required for initial attachment,but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore,iPSCs cultured on vitronectin for 9 passages retained normal karyotype,pluripotency marker expression,and capacity to differentiate in vitro. These studies suggest that vitronectin,or derivatives thereof,might substitute for Matrigel in a more defined system for iPSC culture. View Publication

Patients with chronic ulcerative colitis are at increased risk of developing colorectal cancer. Although current hypotheses suggest that sporadic colorectal cancer is due to inability to control cancer stem cells,the cancer stem cell hypothesis has not yet been validated in colitis-associated cancer. Furthermore,the identification of the colitis to cancer transition is challenging. We recently showed that epithelial cells with the increased expression of aldehyde dehydrogenase in sporadic colon cancer correlate closely with tumor-initiating ability. We sought to determine whether ALDH can be used as a marker to isolate tumor-initiating populations from patients with chronic ulcerative colitis. We used fluorescence-activated cell sorting to identify precursor colon cancer stem cells from colitis patients and report both their transition to cancerous stem cells in xenografting studies as well as their ability to generate spheres in vitro. Similar to sporadic colon cancer,these colitis-derived tumors were capable of propagation as sphere cultures. However,unlike the origins of sporadic colon cancer,the primary colitic tissues did not express any histologic evidence of dysplasia. To elucidate a potential mechanism for our findings,we compared the stroma of these different environments and determined that at least one paracrine factor is up-regulated in the inflammatory and malignant stroma compared with resting,normal stroma. These data link colitis and cancer identifying potential tumor-initiating cells from colitic patients,suggesting that sphere and/or xenograft formation will be useful to survey colitic patients at risk of developing cancer. View Publication

The cancer stem cell (CSC) hypothesis implicates the development of new therapeutic approaches to target the CSC population. Characterization of the pathways that regulate CSCs activity will facilitate the development of targeted therapies. We recently reported that the enzymatic activity of ALDH1,as measured by the ALDELFUOR assay,can be utilized to isolate normal and malignant breast stem cells in both primary tumors and cell lines. In this study,utilizing a tumorsphere assay,we have demonstrated the role of retinoid signaling in the regulation of breast CSCs self-renewal and differentiation. Utilizing the gene set enrichment analysis (GSEA) algorithm we identified gene sets and pathways associated with retinoid signaling. These pathways regulate breast CSCs biology and their inhibition may provide novel therapeutic approaches to target breast CSCs. View Publication

The lack of natural killer (NK) cell-specific markers,as well as the overlap among several common surface antigens and functional properties,has obscured the delineation between NK cells and dendritic cells. Here,novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells,which lack CD7. In contrast to CD7+CD56+ NK cells,CD7(neg)CD56+ cells lack expression of NK cell-associated markers,but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7,we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells,indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally,only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore,using CD7 to separate CD56+ NK cells and CD56+ myeloid cells,we demonstrate that unlike resting CD7+CD56+ NK cells,the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells,thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo. View Publication

Uterine leiomyomas (also known as uterine fibroids) are the most common benign tumors of female reproductive tract and are the single most common indication for hysterectomies. Despite their high prevalence,the exact pathogenesis of these benign tumors is still unknown. One possible mechanism for leiomyoma formation is dysregulation of mesenchymal stem cell activity. Mesenchymal stem cells have been identified in both human and murine uteri and cancer stem cells have been identified in female reproductive malignancies. We compared stem/progenitor cell characteristics in both normal myometrium and the corresponding leiomyoma of patient's undergoing hysterectomies. We found that leiomyoma cells form fewer mesenchymal stem cell colonies and exhibit less Hoechst dye-excluding side population (SP) activity,which is a function associated with progenitor cells in other tissues,than cells isolated from normal myometrium. Whereas in normal myometrium,we observed heterogeneous expression of CD90,a cell surface marker associated the with differentiation potential of uterine fibroblasts,in leiomyomas,we observed homogenous expression of CD90,suggesting leiomyoma cells are more terminally differentiated. Furthermore,we found that while leiomyoma cells could only produce CD90 expressing cells,both CD90+ and CD90- myometrial cells could reestablish their original heterogeneous CD90 profile when expanded in vitro. These results suggest that normal myometrium contains cells with stem/progenitor cell activities that are absent in leiomyomas. View Publication

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies,yet the molecular effectors of this process are relatively uncharacterized. For extravasation,studies based predominantly on static transwell assays support a general model,whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However,this model of chemotaxis cannot readily reconcile how chemokines influence intravasation,as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus,to analyze how T cells integrate perivascular chemokine signals under physiologic flow,we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment,transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However,when exposedto abluminal CXCL12,transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent,augmented by apical flow,blocked by antibodies to integrins,and reduced by AMD3100 in a dose-dependent manner. Moreover,CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream. View Publication

The presentation of proteins on surfaces is fundamental to numerous cell culture and tissue engineering applications. While a number of physisorption and cross-linking methods exist to facilitate this process,few avoid denaturation of proteins or allow control over protein orientation,both of which are critical to the functionality of many signal proteins and ligands. Often recombinant protein sequences include a poly-histidine tag to facilitate purification. We utilize this sequence to anchor proteins to biosurfaces via a peptide bonded to the surface which conjugates with the poly-histidine tag in the presence of zinc rather than nickel,which is more traditionally used to conjugate poly-histidine tags to surfaces. We demonstrate that this strategy enables the display of proteins on 2D and 3D surfaces without compromising protein function through direct cross-linking or physisorption. View Publication

Experimentation with the progenitor/stem cells in adult prostate epithelium can be inconvenient due to a tight time line from tissue acquisition to cell isolation and to downstream experiments. To circumvent this inconvenience,we developed a simple technical procedure for culturing epithelial cells derived from human prostate tissue. In this study,benign prostate tissue was enzymatically digested and fractionated into epithelium and stroma,which were then cultured in the medium designed for prostate epithelial and stromal cells,respectively. The cultured cells were analyzed by immunocytochemical staining and flow cytometry. Prostate tissue-regenerating capacity of cultured cells in vitro was determined by co-culturing epithelial and stromal cells in dihydrotestosterone-containing RPMI. Cell lineages in formed acini-like structures were determined by immunohistochemistry. The culture of epithelial cells mainly consisted of basal cells. A minor population was negative for known lineage markers and positive for CD133. The culture also contained cells with high activity of aldehyde dehydrogenase. After co-culturing with stromal cells,the epithelial cells were able to form acini-like structures containing multiple cell lineages. Thus,the established culture of prostate epithelial cells provides an alternative source for studying progenitor/stem cells of prostate epithelium. View Publication

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses,the function of some,like virion protein 11/12 (VP11/12),have not been clearly defined. Previously,we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis,M. J. Wagner,R. C. Doepker,J. M. Maciejko,C. M. Crider,K. R. Jerome,and J. R. Smiley,J. Virol. 82:6098-6108,2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here,we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394),a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However,we were unable to detect an interaction between ICP0 and Lck in T lymphocytes,and we show that ICP0,US3,and UL13 are not strictly required for Lck activation. In contrast,VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus,VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. View Publication

Mtb influences DC activity and T cell-mediated immune responses. We show that the treatment of immature monocyte-derived DC with Mtb elicited the formation of mature DC,producing TNF-alpha,IL-1beta,IL-6,and IL-23 and instructing CD4(+) cells to secrete IFN-gamma and IL-17. Mtb-induced cytokine release by DC depended on dectin-1 receptor engagement,whereas MR or DC-SIGN stimulation inhibited this process. A selective dectin-1 binding by the receptor agonist glucan was sufficient to enable DC to generate Th1/Th17 lymphocytes,showing features comparable with those induced by Mtb-treated DC. Interestingly,DC-SIGN or MR engagement inhibited Th17 and increased Th1 generation by glucan- or Mtb-treated DC. Our results indicate that Mtb modulates the lymphocyte response by affecting DC maturation and cytokine release. Dectin-1 engagement by Mtb enables DC to promote a Th1/Th17 response,whereas DC-SIGN and MR costimulation limits dectin-1-dependent Th17 generation and favors a Th1 response,probably by interfering with release of cytokines. View Publication