Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference-induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into cell types belonging to all three germ layers within the body, which makes them an interesting and intense field of research. Inefficient specific differentiation and contamination with unwanted cell types are the major issues in the use of ESCs in regenerative medicine. Lineage-specific progenitors generated from ESCs could be utilized to circumvent the issue. We demonstrate here that sustained activation of the Wnt pathway (using Wnt3A or an inhibitor of glycogen synthase kinase 3␤) in multiple mouse and human ESCs results in meso/endodermspecific differentiation. Using monolayer culture conditions, we have generated multipotential "mesendodermal progenitor clones" (MPC) from mouse ESCs by sustained Wnt pathway activation. MPCs express increased levels of meso/endodermal and mesendodermal markers and exhibit a stable phenotype in culture over a year. The MPCs have enhanced potential to differentiate along endothelial, cardiac, vascular smooth muscle, and skeletal lineages than undifferentiated ESCs. In conclusion, we demonstrate that the Wnt pathway activation can be utilized to generate lineage-specific progenitors from ESCs, which can be further differentiated into desired organ-specific cells.The unique property of pluripotent ESCs 2 to differentiate into three germ layers derivatives makes ESCs an ideal source of cells for regenerative therapy (1). One of the most pressing problems in developing ESC-based applications has been the inefficient differentiation of ESCs into the specific therapeutic cell type of choice and the presence of unwanted differentiated cells of other germ layers. A strategy to overcome this problem is to derive lineage-specific progenitor stem cells from ESCs.The advantages of lineage-specific progenitor cells over ESCs are that they differentiate into a limited number of cell types of a particular lineage and, therefore, the differentiation will be robust and more efficient. In addition, they can self-renew, and thus, can be maintained as a renewable source of cells.ESCs can aggregate to form embryoid bodies (EBs) (2, 3), which resemble an intact embryo, and thus, many protocols for in vitro differentiation of the ESCs utilize formation of EB as the first step. However, there can be a mixture of differentiating cells in using an EB approach, whereas differentiation using monolayer culture can yield more uniform and homogenous results. A successful strategy for the use of ESCs in regenerative medicine could involve formation of lineage-restricted progenitor cell using a monolayer culture system as a first step.Gene expression analysis and fate maps together indicate that endoderm and mesoderm are derived, at least in part, from bipotent mesendodermal cells that separate during gastrulation (4, 5) Active canonical Wnt signaling is detected in pregastrulating embryo, through primitive streak (PS) formation, and during gastrulation (6). The evolutionary conserved Wnt signaling pathway is absolutely essential for...

Angiogenesis is a highly regulated process that results from the sequential actions of naturally occurring stimulators and inhibitors. Here, we show that parathyroid hormone-related peptide, a peptide hormone derived from normal and tumor cells that regulates bone metabolism and vascular tone, is a naturally occurring angiogenesis inhibitor. Parathyroid hormone-related peptide or a ten-amino-acid peptide from its N terminus inhibits endothelial cell migration in vitro and angiogenesis in vivo by activating endothelial cell protein kinase A. Activation of protein kinase A inhibits cell migration and angiogenesis by inhibiting the small GTPase Rac. In contrast, inhibition of protein kinase A reverses the anti-migratory and anti-angiogenic properties of parathyroid hormone-related peptide. These studies show that parathyroid hormone-related peptide is a naturally occurring angiogenesis inhibitor that functions by activation of protein kinase A.

Receptors for the provisional ECM are important regulators of angiogenesis. One of these receptors, integrin α5β1, plays a critical role in tumor-and growth factor-induced angiogenesis, because antagonists of this integrin potently inhibit angiogenesis and tumor growth. Here we show that the integrin α5β1 promotes endothelial cell survival during angiogenesis in vivo by suppressing the activity of protein kinase A (PKA). Antagonists of integrin α5β1 activate PKA, which then leads to the activation of caspase-8 and induction of apoptosis. Direct activation of PKA by cAMP or by expression of the PKA catalytic subunit also induces endothelial cell apoptosis, resulting in angiogenesis inhibition in vivo. Our studies indicate that ligation of integrin α5β1 during angiogenesis suppresses an apoptotic program that is dependent on PKA. These studies also indicate that induction of endothelial cell apoptosis in vivo by genetic or pharmacological activation of PKA may be a useful strategy to inhibit angiogenesis.

Use of proteomic strategies to identify a risk classifier that estimates probability of distant recurrence in early-stage hormone receptor (HR)-positive breast cancer is relevant to physiological cellular function and therefore to intrinsic tumor biology. We used a 298-sample retrospective training set to develop an immunohistochemistry-based novel risk classifier called CanAssist-Breast (CAB) which combines 5 prognostically relevant biomarkers and 3 clinico-pathological parameters to arrive at probability of distant recurrence within 5 years from diagnosis. Five selected biomarkers, namely, CD44, ABCC4, ABCC11, N-cadherin, and pan-cadherin, were chosen based on their role in tumor metastasis. The chosen biomarkers represent the hallmarks of cancer and are distinct from other proliferation and gene expression–based prognostic signatures. The 3 clinico-pathological parameters integrated into the machine learning–based CAB algorithm are tumor size, tumor grade, and node status. These features are used to calculate a “CAB risk score” that classifies patients into low- or high-risk groups and predicts probability of distant recurrence in 5 years. Independent clinical validation of CAB in a retrospective study comprising 196 patients indicated that distant metastasis-free survival (DMFS) was significantly different in the 2 risk groups. The difference in DMFS between the low- and high-risk categories was 19% in the validation cohort (P = .0002). In multivariate analysis, CAB risk score was the most significant independent predictor of distant recurrence with a hazard ratio of 4.3 (P = .0003). CanAssist-Breast is a precise and unique machine learning–based proteomic risk-classifier that can assist in risk stratification of patients with early-stage HR+ breast cancer.