Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming.

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

Differentiation is coupled to changes in the cell cycle regulatory apparatus of human embryonic stem cells.

Mouse embryonic stem cells (mESC) exhibit cell cycle properties entirely distinct from those of somatic cells. Here we investigated the cell cycle characteristics of human embryonic stem cells (hESC). HESC could be sorted into populations based on the expression level of the cell surface stem cell marker GCTM-2. Compared to mESC, a significantly higher proportion of hESC (GCTM-2(+) Oct-4(+) cells) resided in G(1) and retained G(1)-phase-specific hypophosphorylated retinoblastoma protein (pRb). We showed that suppression of traverse through G(1) is sufficient to promote hESC differentiation. Like mESC, hESC expressed cyclin E constitutively, were negative for D-type cyclins, and did not respond to CDK-4 inhibition. By contrast, cyclin A expression was periodic in hESC and coincided with S and G(2)/M phase progression. FGF-2 acted solely to sustain hESC pluripotency rather than to promote cell cycle progression or inhibit apoptosis. Differentiation increased G(1)-phase content, reinstated cyclin D activity, and restored the proliferative response to FGF-2. Treatment with CDK-2 inhibitor delayed hESC in G(1) and S phase, resulting in accumulation of cells with hypophosphorylated pRb, GCTM-2, and Oct-4 and, interestingly, a second pRb(+) GCTM-2(+) subpopulation lacking Oct-4. We discuss evidence for a G(1)-specific, pRb-dependent restriction checkpoint in hESC closely associated with the regulation of pluripotency.

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 culture of human embryonic stem cells.

Human embryonic stem (ES) cells are cultured cell lines derived from the inner cell mass of the blastocyst that can be grown indefinitely in their undifferentiated state, yet also are capable of differentiating into all cells of the adult body as well as extraembryonic tissue. Detailed investigation of the properties of embryonal carcinoma cells of both the mouse and human as well as mouse and primate ES cells led to the initial isolation and subsequent culture of human ES cells. The methodologies that were developed to culture and characterize these cell lines have provided a template for the development of human ES cells. The existing data illustrate a number of important differences and similarities between human ES cells and the other cell lines. This review aims to provide a brief historic account of the development of the mammalian pluripotent stem cell field; describe how this led to the isolation, culture, and characterization of human ES cells; and discuss the potential implications of recent advances.