Stefan Przyborski

Downregulation of NANOG Induces Differentiation of Human Embryonic Stem Cells to Extraembryonic Lineages

The homeobox transcription factor Nanog has been proposed to play a crucial role in the maintenance of the undifferentiated state of murine embryonic stem cells. A human counterpart, NANOG, has been identified, but its function and localization have not hitherto been described. We have used a combination of RNA interference and quantitative real‐time polymerase chain reaction to study NANOG in human embryonic stem and embryonic carcinoma cells. Transfection of NANOG‐specific small interfering RNAs reduced levels of NANOG transcript and protein and induced activation of the extraembryonic endoderm‐associated genes GATA4, GATA6, LAMININ B1, and AFP as well as upregulation of trophectoderm‐associated genes CDX2, GATA2, hCG‐alpha, and hCG‐beta. Immunostaining of preimplantation human embryos showed that NANOG was expressed in the inner cell mass of expanded blastocysts but not in earlier‐stage embryos, consistent with a role in the maintenance of pluripotency. Taken together, our findings suggest that NANOG acts as a gatekeeper of pluripotency in human embryonic stem and carcinoma cells by preventing their differentiation to extraembryonic endoderm and trophectoderm lineages.

An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder‐free culture systems. Unfortunately, the risks of cross‐transfer of pathogens from xenogeneic or allogeneic feeders or cell by‐products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses.

Human Induced Pluripotent Stem Cell Lines Show Stress Defense Mechanisms and Mitochondrial Regulation Similar to Those of Human Embryonic Stem Cells

The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient‐specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small number of mitochondria present in hESC; thus, it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of “bottleneck” situation after fertilization) and to show that hiPSC have antioxidant defenses similar to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterized two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whereas the other hiPSC clone is unable to silence the viral expression of OCT4 and c‐MYC, produce fully differentiated teratoma, and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show ROS stress defense mechanisms and mitochondrial biogenesis similar to hESC. Together our data suggest that, during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defense mechanisms against accumulation of ROS as the hESC. STEM CELLS 2010;28:661–673

Derivation of Human Embryonic Stem Cells from Day‐8 Blastocysts Recovered after Three‐Step In Vitro Culture

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5–7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three‐step culture conditions successfully support the development of day‐8 human blastocysts, which possess significantly (p <.01) more ICM cells than day‐6 blastocysts. Plating of ICMs isolated from day‐8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES‐NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM‐2, TG343, TRA1‐60, SSEA‐4, alkaline phosphatase, OCT‐4, NANOG, and REX‐1. Cytogenetic analysis of the hESCs revealed that hES‐NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.

Derivation of Human Embryonic Stem Cells from Developing and Arrested Embryos

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA‐1‐60, TRA‐1‐81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.

A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A

In this study, we show that NANOG, a master transcription factor, regulates S-phase entry in human embryonic stem cells (hESCs) via transcriptional regulation of cell cycle regulatory components. Chromatin immunoprecipitation combined with reporter-based transfection assays show that the C-terminal region of NANOG binds to the regulatory regions of CDK6 and CDC25A genes under normal physiological conditions. Decreased CDK6 and CDC25A expression in hESCs suggest that both CDK6 and CDC25A are involved in S-phase regulation. The effects of NANOG overexpression on S-phase regulation are mitigated by the down-regulation of CDK6 or CDC25A alone. Overexpression of CDK6 or CDC25A alone can rescue the impact of NANOG down-regulation on S-phase entry, suggesting that CDK6 and CDC25A are downstream cell cycle effectors of NANOG during the G1 to S transition.

Differentiation of Human Embryonic Stem Cells After Transplantation in Immune‐Deficient Mice

Our current knowledge of how human tissues grow and develop is limited. We need to increase our understanding of tissue formation if we are to fully realize the potential of stem cells as a source of material for research into health and disease and possible therapeutic applications. Transplanted pluripotent human embryonic stem cells (hESCs) provide a potential system to model and investigate cell differentiation in humans. hESCs transplanted into immune‐deficient mice form complex teratomas consisting of a range of differentiated somatic tissues, some of which appear highly organized and resemble structures normally identified in the embryo and adult. Analysis of such tumors may provide a unique opportunity to study organogenesis and lead to novel approaches in bioengineering and the growth of functioning structures composed of a range of alternative cell types. However, little has been done to characterize the developmental potential of hESCs after transplantation. This concise review presents evidence for the ability of hESCs to differentiate in vivo and highlights some of the prominent questions that need to be addressed if transplantation is to be used as a research tool to study hESC differentiation.

Lamin A/C is a risk biomarker in colorectal cancer

Background A-type lamins are type V intermediate filament proteins encoded by the gene LMNA. Mutations in LMNA give rise to diverse degenerative diseases related to premature ageing. A-type lamins also influence the activity of the Retinoblastoma protein (pRb) and oncogenes such a β-catenin. Consequently, it has been speculated that expression of A-type lamins may also influence tumour progression. Methodology/Principal Findings An archive of colorectal cancer (CRC) and normal colon tissue was screened for expression of A-type lamins. We used the Cox proportional hazard ratio (HR) method to investigate patient survival. Using CRC cell lines we investigated the effects of lamin A expression on other genes by RT-PCR; on cell growth by FACS analysis; and on invasiveness by cell migration assays and siRNA knockdown of targeted genes. We found that lamin A is expressed in colonic stem cells and that patients with A-type lamin-expressing tumours have significantly worse prognosis than patients with A-type lamin negative tumours (HR = 1.85, p = 0.005). To understand this finding, we established a model system based upon expression of GFP-lamin A in CRC cells. We found that expression of GFP-lamin A in these cells did not affect cell proliferation but did promote greatly increased cell motility and invasiveness. The reason for this increased invasiveness was that expression of lamin A promoted up-regulation of the actin bundling protein T-plastin, leading to down regulation of the cell adhesion molecule E-cadherin. Conclusions Expression of A-type lamins increases the risk of death from CRC because its presence gives rise to increased invasiveness and potentially a more stem cell-like phenotype. This report directly links A-type lamin expression to tumour progression and raises the profile of LMNA from one implicated in multiple but rare genetic conditions to a gene involved in one of the commonest diseases in the Western World.

The Netrin 1 Receptors Unc5h3 and Dcc Are Necessary at Multiple Choice Points for the Guidance of Corticospinal Tract Axons

Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and itsCaenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5 is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. We demonstrate that the Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, we show that corticospinal tract fibers respond differently to loss of UNC5H3. In addition, we observe corticospinal tract defects in mice homozygous for a spontaneous mutation that truncates the Dcctranscript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrateunc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates.

Human‐Serum Matrix Supports Undifferentiated Growth of Human Embryonic Stem Cells

One of the most frequently used matrices for feeder‐free growth of undifferentiated human embryonic stem cells (hESCs) is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of mouse embryonic fibroblast–conditioned medium. Unfortunately, application of Matrigel or medium conditioned by mouse embryonic feeder cells is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions. We demonstrate here that human serum as matrix and medium conditioned by differentiated hESCs reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal‐free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.