Mai X. Luong

Pluripotency: Toward a gold standard for human ES and iPS cells

With the advent of technologies for the derivation of embryonic stem cells and reprogrammed stem cells, use of the term “pluripotent” has become widespread. Despite its increased scientific and political importance, there are ambiguities with this designation and a common standard for experimental approaches that precisely define this state in human cells remains elusive. Recent studies have revealed that reprogramming may occur via many pathways which do not always lead to pluripotency. In addition, the pluripotent state itself appears to be highly dynamic, leading to significant variability in the results of molecular studies. Establishment of a stringent set of criteria for defining pluripotency will be vital for biological studies and potential clinical applications in this rapidly evolving field. In this review, we explore the various definitions of pluripotency, examine the current status of pluripotency testing in the field and provide an analysis of how these assays have been used to establish pluripotency in the scientific literature. J. Cell. Physiol. 220: 21–29, 2009.

Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility.

ABSTRACT Murine CDP/Cux, a homologue of the Drosophila Cut homeoprotein, modulates the promoter activity of cell cycle-related and cell-type-specific genes. CDP/Cux interacts with histone gene promoters as the DNA binding subunit of a large nuclear complex (HiNF-D). CDP/Cux is a ubiquitous protein containing four conserved DNA binding domains: three Cut repeats and a homeodomain. In this study, we analyzed genetically targeted mice (Cutl1tm2Ejn, referred to as ΔC) that express a mutant CDP/Cux protein with a deletion of the C terminus, including the homeodomain. In comparison to the wild-type protein, indirect immunofluorescence showed that the mutant protein exhibited significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (ΔC−/−) mice, indicating the functional loss of CDP/Cux protein in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wild-type and ΔC−/− MEFs in culture. However, specific histone genes (H4.1 and H1) containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice have stunted growth (20 to 50% weight reduction), a high postnatal death rate of 60 to 70%, sparse abnormal coat hair, and severely reduced fertility. The deregulated hair cycle and severely diminished fertility in Cutl1tm2Ejn/tm2Ejn mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions.

A Call for Standardized Naming and Reporting of Human ESC and iPSC Lines

Human embryonic and induced pluripotent stem cell lines are being generated at a rapid pace and now number in the thousands. We propose a standard nomenclature and suggest the use of a centralized database for all cell line names and a minimum set of information for reporting new derivations.

The Cell Cycle Control Element of Histone H4 Gene Transcription Is Maximally Responsive to Interferon Regulatory Factor Pairs IRF-1/IRF-3 and IRF-1/IRF-7

Interferon regulatory factors (IRFs) are transcriptional mediators of interferon-responsive signaling pathways that are involved in antiviral defense, immune response, and cell growth regulation. To investigate the role of IRF proteins in the regulation of histone H4 gene transcription, we compared the transcriptional contributions of IRF-1, IRF-2, IRF-3, and IRF-7 using transient transfection assays with H4 promoter/luciferase (Luc) reporter genes. These IRF proteins up-regulate reporter gene expression but IRF-1, IRF-3, and IRF-7 are more potent activators of the H4 promoter than IRF-2. Forced expression of different IRF combinations reveals that IRF-2 reduces IRF-1 or IRF-3 dependent activation, but does not affect IRF-7 function. Thus, IRF-2 may have a dual function in histone H4 gene transcription by acting as a weak activator at low dosage and a competitive inhibitor of other strongly activating IRFs at high levels. IRF-1/IRF-3 and IRF-1/IRF-7 pairs each mediate the highest levels of site II-dependent promoter activity and can up-regulate transcription by 120–150-fold. We also find that interferon γ up-regulates IRF-1 and site II-dependent promoter activity. This up-regulation is not observed when the IRF site is mutated or if cells are preloaded with IRF-1. Our results indicate that IRF-1, IRF-2, IRF-3, and IRF-7 can all regulate histone H4 gene expression. The pairwise utilization of distinct IRF factors provides a flexible transcriptional mechanism for integration of diverse growth-related signaling pathways.

Tumor suppressor pRB functions as a co-repressor of the CCAAT displacement protein (CDP/cut) to regulate cell cycle controlled histone H4 transcription

The CCAAT displacement protein (CDP‐cut/CUTL1/cux) performs a key proliferation‐related function as the DNA binding subunit of the cell cycle controlled HiNF‐D complex. HiNF‐D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell‐cycle dependent histone genes, which are transcriptionally and coordinately activated at the G1/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF‐D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, we show that CDP represses the H4 gene promoter and that pRB functions with CDP as a co‐repressor. Direct physical interaction between CDP and pRB was observed in glutathione‐S‐transferase (GST) pull‐down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two‐hybrid experiments and co‐immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co‐localized in situ. Using a series of pRB mutants, we find that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters. J. Cell. Physiol. 196: 541–556, 2003.

International stem cell registries

Rapid advances in stem cell research have led to the derivation of hundreds of human embryonic stem (hES) cell lines in centers throughout the world, as well as the development of new technologies for producing pluripotent stem cells. These cell lines have unique characteristics and were derived using a variety of ethical guidelines. Stem cell registries have been developed in order to collect, organize, and disseminate cell line-specific information. In this review, we describe the current state of the field by providing an overview of the unique qualities and mandates of the three major stem cell registries: the European hES Cell Registry, the Registry of hES Cell Line Provenance developed by the International Society for Stem Cell Research, and the International Stem Cell Registry of hES and induced pluripotent stem cell lines established at the University of Massachusetts Medical School. While each registry has its own unique mandate and features, there is some overlap in the goals and information provided. This review discusses the challenges and prospects for an integrated approach in which all three registries effectively collaborate to minimize duplication and facilitate information exchange within the stem cell community.

Human embryonic stem cell registries: Value, challenges and opportunities†

The accelerating pace of human embryonic stem cell (hESC) research has created an urgent need for the development of hESC registries, information repositories intended to gather, organize and disseminate hESC information. Although of enormous value to this evolving field, registries face significant challenges to their development. These challenges include addressing the legal and ethical issues surrounding hESC derivation as well as complex intellectual property concerns. In addition to these issues, registries must develop tools to efficiently gather, validate and present many different types of hESC information from a variety of sources. Given the pace and regulatory complexities of this field, it is important that registries develop cooperative mechanisms to avoid duplication and more efficiently support hESC research. J. Cell. Biochem. 105: 625–632, 2008.

Biobanks for Pluripotent Stem Cells

To date, over 700 hESC lines have been used in over 1450 published studies (International Stem Cell Registry (ISCR)), signifying remarkable progress in a rapidly growing field. However, the findings in a number of these reports have been controversial, with some difficult to reproduce. This situation may have resulted from many factors, including varying competencies for cell line handling, differing culture conditions and usage of cells that have undergone deleterious changes. This chapter provides an overview of the fundamental principles of hPSC banking. A detailed section on current practices by stem cell providers and a discussion about the future of banking are also presented