New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.

The hypoxia-inducible factor renders cancer cells more sensitive to vitamin C-induced toxicity.

Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells.

Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology.

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells.

Vitamin C (ascorbate) is a widely used medium supplement in embryonic stem cell culture. Here, we show that ascorbate causes widespread, consistent, and remarkably specific DNA demethylation of 1,847 genes in human embryonic stem cells (hESCs), including important stem cell genes, with a clear bias toward demethylation at CpG island boundaries. We show that a subset of these DNA demethylated genes displays concomitant gene expression changes and that the position of the demethylated CpGs relative to the transcription start site is correlated to such changes. We further show that the ascorbate-demethylated gene set not only overlaps with gene sets that have bivalent marks, but also with the gene sets that are demethylated during differentiation of hESCs and during reprogramming of fibroblasts to induced pluritotent stem cells (iPSCs). Our data thus identify a novel link between ascorbate-mediated signaling and specific epigenetic changes in hESCs that might impact on pluripotency and reprogramming pathways.

Ascorbate promotes epigenetic activation of CD30 in human embryonic stem cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to adapt to various culture conditions. Phenotypic and epigenetic changes brought about by the culture conditions can, however, have significant impacts on their use in research and in clinical applications. Here, we show that diploid hESCs start to express CD30, a biomarker for malignant cells in Hodgkin's disease and embryonal carcinoma cells, when cultured in knockout serum replacement (KOSR)-based medium, but not in fetal calf serum containing medium. We identify the commonly used medium additive, ascorbate, as the sole medium component in KOSR responsible for CD30 induction. Our data show that this epigenetic activation of CD30 expression in hESCs by ascorbate occurs through a dramatic loss of DNA methylation of a CpG island in the CD30 promoter. Analysis of the phenotype and transcriptome of hESCs that overexpress the CD30 signaling domain reveals that CD30 signaling leads to inhibition of apoptosis, enhanced single-cell growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. Collectively, our data show that hESC culture media that contain ascorbate trigger CD30 expression through an epigenetic mechanism and that this provides a survival advantage and transcriptome changes that may help adapt hESCs to in vitro culture conditions.

CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells.

The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.

Characterization and gene expression profiling of five new human embryonic stem cell lines derived in Taiwan.

Many human embryonic stem cell (hESC) lines have been reported, but only a few of them have been fully characterized. In this report, five new hESC lines were derived from 32 discarded blastocysts in Taiwan, and these lines were continuously cultured on mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer in the hESC medium for more than 44 passages and underwent freezing/thawing processes. All five hESC lines expressed characteristic undifferentiated hESC markers, such as SSEA-4, TRA-1-81, alkaline phosphatase, TERT, and the transcription factors POU5F1 (OCT4) and NANOG. hESC lines T1 and T3 possess normal female karyotypes, whereas lines T4 and T5 are normal male, but line T2 is male trisomy 12 (47XY,+12). hESC lines T1, T2, T3, and T5 were able to produce teratomas in severe combined immunodeficient (SCID) mice, and line T4 could only form embryoid bodies (EBs) in vitro. Global gene expression profiles of these five newly derived hESC lines were analyzed using the Affymetrix human genome U133 plus 2.0 GeneChip. The results showed that 4,145 transcripts, including 19% of unknown functions, were detected in all five hESC lines. Comparison of the 4,145 genes commonly expressed in the five hESC lines with those genes expressed in teratomas produced by the hESC line T1 and placenta revealed 40 genes exclusively expressed in all five hESC lines. These 40 genes include the previously reported stemness genes, such as POU5F1 (OCT4), NANOG, TDGF1 (CRIPTO), SALL4, LECT1, and BUB1 responsible for self-renewal and pluripotent differentiation. The global gene expression analysis also indicated that the transforming growth factor-beta (TGF-beta)/activin branch components inhibin BC, ACVR2A, ACVR1 (ALK2), TGFBR1 (ALK5), and SMAD2 were found to be highly expressed in undifferentiated states of these five hESC lines and decreased upon differentiation. In short, the hESC nature of these five hESC lines is supported by the undifferentiated state, extensive renewal capacity, and pluripotency, including the ability to form teratomas and/or EBs. These cell lines will be useful for human embryonic stem cell biology and drug development.

Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides.

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.