Juehua Yu

Polymer Nanofiber‐Embedded Microchips for Detection, Isolation, and Molecular Analysis of Single Circulating Melanoma Cells

Circulating tumor cells (CTCs)[1] are cancer cells shed from either the primary tumors or metastatic sites. The presence and number of CTCs in peripheral blood can provide clinically significant data on prognosis and therapeutic response patterns, respectively[2]. Thus, as with traditional invasive tumor biopsies that enable gold-standard pathological analysis, CTCs can be regarded as “liquid biopsies” of the tumor, which enable repeated and relatively non-invasive characterization of tumor evolution, especially important during therapeutic interventions. Currently, FDA-cleared CellSearch™ Assay is costly and inefficient in capturing CTCs, and the enriched CTCs are typically contaminated with a large number of white blood cells (WBCs). As a result, the diagnostic value of CTCs has been underutilized. Over the past decade, a diversity of CTC detection technologies[2d, 3] have been developed to overcome the challenges encountered by the immunomagnetic separation-based CellSearch™ Assay.

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

High‐Purity Prostate Circulating Tumor Cell Isolation by a Polymer Nanofiber‐Embedded Microchip for Whole Exome Sequencing

Handpick single cancer cells: a modified NanoVelcro Chip is coupled with ArcturusXT laser capture microdissection (LCM) technology to enable the detection and isolation of single circulating tumor cells (CTCs) from patients with prostate cancer (PC). This new approach paves the way for conducting next-generation sequencing (NGS) on single CTCs.

Dnmt3a Regulates Both Proliferation and Differentiation of Mouse Neural Stem Cells

DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early‐passage neural stem cells (NSCs) derived from Dnmt3a‐deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild‐type counterparts. However, successive passaging to passage 6 (P6), when wild‐type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a−/− NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1−/− NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a−/− NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a−/− NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC‐derived‐NSCs.

Dnmt1-dependent DNA methylation is essential for photoreceptor terminal differentiation and retinal neuron survival.

Epigenetic regulation of the genome is critical for the emergence of diverse cell lineages during development. To understand the role of DNA methylation during retinal network formation, we generated a mouse retinal-specific Dnmt1 deletion mutation from the onset of neurogenesis. In the hypomethylated Dnmt1-mutant retina, neural progenitor cells continue to proliferate, however, the cell cycle progression is altered, as revealed by an increased proportion of G1 phase cells. Despite production of all major retinal neuronal cell types in the Dnmt1-mutant retina, various postmitotic neurons show defective differentiation, including ectopic cell soma and aberrant dendritic morphologies. Specifically, the commitment of Dmnt1-deficient progenitors towards the photoreceptor fate is not affected by DNA hypomethylation, yet the initiation of photoreceptor differentiation is severely hindered, resulting in reduction and mislocalization of rhodopsin-expressing cells. In addition to compromised neuronal differentiation, Dnmt1 deficiency also leads to rapid cell death of photoreceptors and other types of neurons in the postnatal retina. These results indicate that Dnmt1-dependent DNA methylation is critical for expansion of the retinal progenitor pool, as well as for maturation and survival of postmitotic neurons.

Identification of miRNA Signatures during the Differentiation of hESCs into Retinal Pigment Epithelial Cells

Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.

Selective demethylation and altered gene expression are associated with ICF syndrome in human-induced pluripotent stem cells and mesenchymal stem cells

Immunodeficiency, centromeric instability and facial anomalies type I (ICF1) syndrome is a rare genetic disease caused by mutations in DNA methyltransferase (DNMT) 3B, a de novo DNA methyltransferase. However, the molecular basis of how DNMT3B deficiency leads to ICF1 pathogenesis is unclear. Induced pluripotent stem cell (iPSC) technology facilitates the study of early human developmental diseases via facile in vitro paradigms. Here, we generate iPSCs from ICF Type 1 syndrome patient fibroblasts followed by directed differentiation of ICF1-iPSCs to mesenchymal stem cells (MSCs). By performing genome-scale bisulfite sequencing, we find that DNMT3B-deficient iPSCs exhibit global loss of non-CG methylation and select CG hypomethylation at gene promoters and enhancers. Further unbiased scanning of ICF1-iPSC methylomes also identifies large megabase regions of CG hypomethylation typically localized in centromeric and subtelomeric regions. RNA sequencing of ICF1 and control iPSCs reveals abnormal gene expression in ICF1-iPSCs relevant to ICF syndrome phenotypes, some directly associated with promoter or enhancer hypomethylation. Upon differentiation of ICF1 iPSCs to MSCs, we find virtually all CG hypomethylated regions remained hypomethylated when compared with either wild-type iPSC-derived MSCs or primary bone-marrow MSCs. Collectively, our results show specific methylome and transcriptome defects in both ICF1-iPSCs and differentiated somatic cell lineages, providing a valuable stem cell system for further in vitro study of the molecular pathogenesis of ICF1 syndrome. GEO accession number: GSE46030.

Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin downregulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

Down-regulation of pancreatic and duodenal homeobox-1 by somatostatin receptor subtype 5: a novel mechanism for inhibition of cellular proliferation and insulin secretion by somatostatin

Somatostatin (SST) is a regulatory peptide and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells. SST’s actions are mediated by a family of seven transmembrane domain G protein-coupled receptors that comprise five distinct subtypes (SSTR1-5). SSTR5 is one of the major SSTRs in the islets of Langerhans. Homeodomain-containing transcription factor pancreatic and duodenal homeobox-1 (PDX-1) is essential for pancreatic development, β cell differentiation, maintenance of normal β cell functions in adults and tumorigenesis. Recent studies show that SSTR5 acts as a negative regulator for PDX-1 expression and that SSTR5 mediates somatostatin’s inhibitory effect on cell proliferation and insulin expression/excretion through down-regulating PDX-1 expression. SSTR5 exerts its inhibitory effect on PDX-1 expression at both the transcriptional level by down-regulating PDX-1 mRNA and the post-translational level by enhancing PDX-1 ubiquitination. Identification of PDX-1 as a transcriptional target for SSTR5 may help in guiding the choice of therapeutic cancer treatments.

PDX1 associated therapy in translational medicine.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely poor prognosis and a low median survival due to lack of the early and reliable detection and effective therapeutic options, despite improvements observed for many other cancers in last decade. Pancreatic and duodenal homeobox 1 (PDX1), which is a homeodomain-containing transcription factor and a key regulator for insulin gene expression, β cell maturation and proper β cell function maintenance in the pancreas. Our previous studies revealed that PDX1 promotes tumorigenesis and it is a promising therapeutic target for PDAC. For translational purposes, we developed three therapeutic platforms utilizing RNA interference (RNAi), gene therapy and small inhibitory drug targeting PDX1, and further validated them in PDAC preclinical models both in vitro and in vivo. These PDX1 targeted therapies significantly inhibited PDX1 expression in PDAC cells, ablated PDX1-expressing human PDAC xenograft tumor growth, and prolonged survival in the PDAC mouse models. The data from these preclinical studies proved the translational potentials of PDX1 targeted therapies in PDAC and suggest that the strategy of developing PDX1 targeted therapies would permit a rapid bench-to-bedside translation of other relevant gene therapies, which would eventually benefit the patients suffering from this deadly disease.