Adam Filipczyk

Essential Roles of Sphingosine‐1‐Phosphate and Platelet‐Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum—sphingosine‐1‐phosphate (S1P), lysophosphatidic acid (LPA), and platelet‐derived growth factor (PDGF)—in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum‐free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri‐potentiality.

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.

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.

Biallelic Expression of Nanog Protein in Mouse Embryonic Stem Cells

Transcription factors (TFs) and their networks are central effectors controlling pluripotency (Young, 2011). Numerous involved TFs have been identified, but a subset of core pluripotency TFs regulates the majority of others. One such factor, Nanog, is expressed in pluripotent cells, is required for self-renewal of mouse embryonic stem cells (ESCs) in vitro, is able to force ESC self-renewal upon overexpression in the absence of LIF, and is necessary for the normal development of early mouse embryos (reviewed in Young, 2011).

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic–erythroid and granulocytic–monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic–erythroid versus granulocytic–monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Isolation, characterization, and differentiation of human embryonic stem cells

Publisher Summary The first derivation of human embryonic stem (ES) cells was reported in 1998, and although there have been a number of anecdotal reports of isolation of new ES cells since then, published data are based only on a few cell lines. Timely progress in this field depends upon the derivation of new ES cell lines, their proper characterization and comparison with existing isolates, and in-depth analysis of their differentiation under different conditions. Previous reviews have compared the properties of published human ES cells with those of pluripotent cell lines derived from nonhuman primates or with human embryonal carcinoma cell lines, and with the counterparts of both cell types in the mouse. These comparisons have given rise to some consensus regarding the canonical primate pluripotent cell phenotype; the data are limited, and the cultures are probably more heterogeneous than the published descriptions imply. The purpose of this chapter is to help provide some guidelines for human ES cell derivation, and a methodological basis for the characterization and comparison of stem cells and their differentiation in different laboratories. The chapter deals with the isolation, characterization, and differentiation of human embryonic stem (ES) cell lines from preimplantation blastocysts.

Network plasticity of pluripotency transcription factors in embryonic stem cells

Transcription factor (TF) networks are thought to regulate embryonic stem cell (ESC) pluripotency. However, TF expression dynamics and regulatory mechanisms are poorly understood. We use reporter mouse ESC lines allowing non-invasive quantification of Nanog or Oct4 protein levels and continuous long-term single-cell tracking and quantification over many generations to reveal diverse TF protein expression dynamics. For cells with low Nanog expression, we identified two distinct colony types: one re-expressed Nanog in a mosaic pattern, and the other did not re-express Nanog over many generations. Although both expressed pluripotency markers, they exhibited differences in their TF protein correlation networks and differentiation propensities. Sister cell analysis revealed that differences in Nanog levels are not necessarily accompanied by differences in the expression of other pluripotency factors. Thus, regulatory interactions of pluripotency TFs are less stringently implemented in individual self-renewing ESCs than assumed at present.

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.

Software tools for single-cell tracking and quantification of cellular and molecular properties

Software tools for single-cell tracking and quantification of cellular and molecular properties