Upregulation of Adhesion Molecules Sustains Matrix-Free Growth of Human Embryonic Stem Cells

Canonical WNT signals are transduced through Frizzled (FZD) family receptor and LRP5/LRP6 co-receptor to upregulate FGF20, JAG1, DKK1, WISP1, CCND1 and MYC genes for cell-fate determination, while non-canonical WNT signals are transduced through FZD family receptor and ROR2/PTK7/RYK co-receptor to activate RHOA/RHOU/RAC/CDC42, JNK, PKC, NLK and NFAT signaling cascades for the regulation of tissue polarity, cell movement, and adhesion. We previously reported molecular cloning and characterization of human FZD5, which showed six amino-acid substitutions with human Hfz5. FZD5, functioning as WNT5A receptor, is the key molecule in the fields of oncology, regenerative medicine, cardiology, rheumatology, diabetology, and gastroenterology. Here, comparative integromics analyses on FZD5 orthologs were performed by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee FZD5 and cow Fzd5 genes were identified within NW_104292.1 and AC166656.2 genome sequences, respectively. FZD5 orthologs were seven-transmembrane proteins with extracellular Frizzled domain, leucine zipper motif around the 5th transmembrane domain, and cytoplasmic DVL- and PDZ-binding motifs. Ser523 and Ser529 around the DVL-binding motif of FZD5 orthologs were putative aPKC phosphorylation sites. POU5F1 (OCT4)-binding site linked to SP1-binding site within the 5'-promoter region of human FZD5 gene was evolutionarily conserved among mammalian FZD5 orthologs. POU5F1 was more related to POU2F and POU3F subfamily members. POU5F1 was preferentially expressed in undifferentiated human embryonic stem (ES) cells, pancreatic islet, and diffuse-type gastric cancer. POU2F1 (OCT1) was expressed in ES cells, fetal liver/spleen, adult colon, POU2F2 in ES cells, fetal liver/spleen, and POU2F3 in diffuse-type gastric cancer. Multiple SP1/KLF family members, other than KLF2 or KLF4, were expressed in undifferentiated human ES cells. Together, these facts indicate that POU5F1 and POU2F subfamily members play a pivotal role for the FZD5 expression in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer.

Little is known about the cell-surface molecules that are related to the undifferentiated and pluripotent state of human embryonic stem cells (hESCs). Here, we generated a panel of murine monoclonal antibodies (MAb) against undifferentiated hESCs by a modification of a previously described decoy immunization strategy. H9 hESCs were differentiated in the presence of retinoic acid and used as a decoy immunogen. Twelve Balb/c mice were immunized in the right hind footpads with differentiated H9 cells and in the left hind footpads with undifferentiated H9 cells. After immunization, the left popliteal lymph node cells were collected and were fused with mouse myeloma cells. The fusion resulted in 79 hybridomas secreting MAbs that bound to the undifferentiated H9 cells as shown by flow cytometric analysis. Of these, 70 MAbs bound to the undifferentiated H9 cells, but only weakly or not at all to the differentiated H9 cells. We characterized 37 MAbs (32 IgGs, 5 IgMs) recognizing surface molecules that were down-regulated during embryoid body cell formation. One of the MAbs, L125-C2, was confirmed to immunoprecipitate CD9, previously known as a surface molecule on the undifferentiated hESCs. To investigate the relationship between the MAbs and hESC-specific antibodies, two representative MAbs, viz., L125-C2 and 291-D4, were selected and studied by multi-color flow cytometric analysis. This showed that more than 60% of L125-C2- and 291-D4-positive cells were also positive for the expression of hESC-specific surface molecules such as SSEA3, SSEA4, TRA-1-60, and TRA-1-81, indicating the close relationship between the two MAbs and the hESC-specific surface molecules. Our results suggest that the decoy immunization strategy is an efficient method for isolating a panel of MAbs against undifferentiated hESCs, and that the generated MAbs should be useful for studying the surface molecules on hESCs in the pluripotent and undifferentiated state.

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA-4, TRA-1-60 and TRA-1-81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5-fluorouracil, indomethacin and non-embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down-regulated in a dose-dependent manner after treatment with embryotoxic chemicals. After treatment with 5-fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up-regulation of development, cell cycle and apoptosis-related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development-, cell cycle- and apoptosis-related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage.

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

Kynurenine, which is generated from tryptophan by indoleamine 2,3-dioxygenase 1 (IDO1), binds to the aryl hydrocarbon receptor (AhR). Here, we report that kynurenine was produced by undifferentiated human embryonic stem cells (hESCs) and by induced pluripotent stem cells (iPSCs). In undifferentiated hESCs, kynurenine stimulated the AhR to promote the expression of self-renewal genes. The kynurenine-AhR complex also stimulated the expression of IDO1 and AHR, activating a positive feedback loop. Inhibition of IDO1 activity reduced the proliferation of undifferentiated ESCs but did not stimulate their differentiation. Substantial amounts of free kynurenine were present in the culture medium, providing a paracrine signal for maintenance of the undifferentiated state. Kynurenine was not present in the medium of differentiated ESCs or iPSCs. When ESCs were induced to undergo ectodermal differentiation, the abundance of kynurenine in the medium was reduced through activation of the main kynurenine catabolic pathway mediated by kynurenine aminotransferase 2 (KAT2, also known as AADAT), resulting in the secretion of 2-aminoadipic acid (2-AAA) into the culture medium. Inhibition of KAT2 activity blocked ectodermal differentiation. Thus, kynurenine metabolism plays an important role in the maintenance of the undifferentiated state and in ectodermal differentiation. Furthermore, kynurenine in the culture medium is a biomarker for the undifferentiated state, whereas the presence of 2-AAA in the culture medium is a biomarker of ESCs and iPSCs that have committed to differentiate along the ectoderm lineage.

Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the alpha1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.

Purpose: Neuroblastoma (NB) is one of the most common childhood tumors, accounting for ~15% of all pediatric cancer deaths. Improved neuroblastoma disease modeling is needed to improve our understanding and ultimately lead to the development of more effective therapies. NB is an embryonal tumor of the sympathetic nervous system believed to arise from sympathoadrenal (SA) precursor cells belonging to the primitive neural crest. Because the neural crest is a transient embryologic structure, there have been few studies comparing NB tumors to normal human neural crest stem cells (NCSC). However, recent advances have enabled the in vitro differentiation of neural crest cells from pluripotent stem cells. We compared gene expression profiles of pluripotent stem cells, NCSC, and NB tumors to characterize the gene expression profile of the putative cell of origin of NB as the first step in the development of a human NCSC based model of NB. Methods: Human embryonic and induced pluripotent stem (iPS) cells were differentiated to early NCSC and SA precursors in vitro using modifications of previously published protocols. Specifically, undifferentiated human embryonic stem cell GFP reporter and iPS cell lines were induced to differentiate into SA cells in a sequential, stepwise manner over the course of ~4 weeks. After differentiation, SA precursors were identified by expression of the lineage-specific transcription factors ASCL1 and PHOX2B, as well as other markers including tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). We subsequently compared the gene expression profiles of these cells to early, multi-potent neural crest stem cells, autonomic and enteric neurons, 12 NB cell lines and 39 non-MYCN amplified primary NB tumors (3 stage 1, 5 stage 2, 3 stage 3, 24 stage 4, and 4 stage 4S) using Illumina HT-12 bead arrays. These profiles were also compared to the gene expression profiles of laser-capture microdissected fetal adrenal neuroblasts previously reported by De Preter et. al. (Genome Biology 2006, 7:R84). Results were normalized using quantile normalization and analyzed with principle components analysis (PCA); differential expression analysis was performed using the Linear Models for Microarray Data (Limma) method. Pathway enrichment analysis was performed using DAVID annotation tools and cross-platform normalization was performed using the CONOR package in the R software program. All analyses were corrected for multiple comparisons and results considered significant when False Discovery Rate (FDR) was <0.05. Results: PCA showed that undifferentiated iPS cells and early NCSC cluster relatively closely together and away from primary tumors and fetal adrenal neuroblasts. The NB cell lines and primary tumors do not cluster closely together suggesting there are significant differences between them. Gene expression profiles of in vitro differentiated ASCL1 and PHOX2B(+) SA precursor cells were similar to fetal adrenal neuroblasts and more closely resembled NB tumors than did earlier multi-potent NCSC. Comparison between SA precursor cells, NCSC, and primary tumors identified numerous differentially expressed genes (FDR<0.05) the majority of which are regulators of embryologic and/or neural development (SOX3, DPPA4, HES5, LIN28, IRX2, SOX21, PAX6, FABP7, and OTX2 among others). Most were overexpressed in normal neural crest derivatives relative to NB cell lines and tumors. Conclusions: This study, the first of its kind for this disease, demonstrates the feasibility and potential utility of using human pluripotent stem cells and in vitro derived sympathoadrenal precursors cells to simulate early NB pathogenesis. Gene expression profiling of NB identified down regulation of multiple developmentally important genes of normal neural crest differentiation. Further refinement of this in vitro neuroblastoma tumor model as well as investigations of its disrupted gene pathways is underway. Note: This abstract was not presented at the conference. Citation Format: Stephen S. Roberts, Yudelca Ogando, Irina Ostrovnaya, Faranak Fattahi, Irene Cheung, Nai-Kong V. Cheung, Lorenz Studer, Mark Tomishima. Using directed differentiation of human pluripotent stem cells and gene expression profiling to characterize the cell of origin of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A08.

There is evidence demonstrating the regulation of microRNAs in a myriad of vascular biology events such as cardiogenesis, but the role of miRNA in controlling human embryonic stem cell fate...

Nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions

A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1-60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column ('flow-through' fraction) and those retained ('labelled' fraction') were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 +/- 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the 'flow-through' fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.

We have recently shown that Skp2 levels are high in undifferentiated human embryonic stem cells, but decline rapidly following induction of differentiation, thereby leading to accumulation of p27. Changes in Skp2 levels were found to be caused mainly by its rate of degradation. Here we show that the activity of APC/CCdh1, the ubiquitin ligase that targets Skp2 for degradation, increases markedly during the differentiation process of human embryonic stem cells. APC/CCdh1 is present but inactive in undifferentiated embryonic stem cells and becomes active in the differentiated state. The rise in APC/CCdh1 activity with differentiation appears to be due, at least in part, to a dramatic decline in the levels of its inhibitor Emi1. In addition, protein kinase activity also appears to contribute to the suppression of APC/CCdh1 activity in undifferentiated stem cells, possibly by inhibitory phosphorylation of Cdh1.

Human embryonic stem cells will remain undifferentiated or undergo differentiation when grown in conditioned or non-conditioned medium, respectively. The factors and signaling events that control the maintenance of the undifferentiated state are not well characterized and their identification is of major importance. Based on the data from global expression analyses, we set out to identify genes and the signaling pathways controlling them that are regulated in the early phase of the differentiation process. This study shows that nodal and the inhibitors of Nodal signaling, lefty-A and lefty-B, are down-regulated very early upon differentiation. High expression of these genes in undifferentiated cells is maintained by activation of the transcription factor Smad2/3, downstream of the activin-linked kinases (ALK) 4/5/7. Treatment of differentiating cells with Activin A leads to activation of Smad2/3 and expression of nodal, lefty-A and lefty-B, while inhibition of ALK4/5/7 by the kinase inhibitor SB-431542 blocks activation of Smad2/3 and expression of these genes in the undifferentiated state. In addition, when cells are maintained undifferentiated by treatment with the GSK3-inhibitor, BIO, high expression of nodal, lefty-A, and lefty-B also requires activation of ALK4/5/7. Conversely, BMP signaling leading to Smad1/5/8 activation via ALK2/3/6 is blocked in undifferentiated cells and becomes activated upon differentiation. Taken together, these observations establish that Smad2/3 is activated in undifferentiated hESCs and required for the expression of genes controlling Nodal signaling. Moreover, there appears to be cross-talk between inhibition of GSK3, a hallmark of Wnt signaling and the Activin/Nodal pathway.

Like other cell populations, undifferentiated human embryonic stem cells (hESCs) express a characteristic set of proteins and mRNA that is unique to the cells regardless of culture conditions, number of passages, and methods of propagation. We sought to identify a small set of markers that would serve as a reliable indicator of the balance of undifferentiated and differentiated cells in hESC populations. Markers of undifferentiated cells should be rapidly downregulated as the cells differentiate to form embryoid bodies (EBs), whereas markers that are absent or low during the undifferentiated state but that are induced as hESCs differentiate could be used to assess the presence of differentiated cells in the cultures. In this paper, we describe a list of markers that reliably distinguish undifferentiated and differentiated cells. An initial list of approximately 150 genes was generated by scanning published massively parallel signature sequencing, expressed sequence tag scan, and microarray datasets. From this list, a subset of 109 genes was selected that included 55 candidate markers of undifferentiated cells, 46 markers of hESC derivatives, four germ cell markers, and four trophoblast markers. Expression of these candidate marker genes was analyzed in undifferentiated hESCs and differentiating EB populations in four different lines by immunocytochemistry, reverse transcription-polymer-ase chain reaction (RT-PCR), microarray analysis, and quantitative RT-PCR (qPCR). We show that qPCR, with as few as 12 selected genes, can reliably distinguish differentiated cells from undifferentiated hESC populations.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs. The antigen recognized by MAb2 is expressed on the cell surface of undifferentiated hESCs; three diffused bands with molecular mass between 30 and 60 kDa in the lysates of hESCs were diminished during hESC differentiation into neural cells. The expression of MAb2 antigen was also observed on the plasma membrane of lung cancer cells, and MAb2 detected 55, 50, and 35 kDa protein bands in the cell lysates. Immunoprecipitation followed by proteomics analyses identified CD147/basigin as a MAb2 antigen. Finally, the positive expression of CD147/basigin protein in undifferentiated hESCs was confirmed. These results suggested that CD147/basigin could be another undifferentiated hESC marker.

High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells