Zuzana Holubcová

MicroRNAs Regulate p21Waf1/Cip1 Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells

Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53‐p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC‐specific miRNAs such as those of the miR‐302 family, miR‐371‐372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC‐enriched miRNA family miR‐302 (miR‐302a, miR‐302b, miR‐302c, and miR‐302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR‐302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. STEM CELLS2012;30:1362–1372

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

Embryonic stem cells progress very rapidly through the cell cycle, allowing limited time for cell cycle regulatory circuits that typically function in somatic cells. Mechanisms that inhibit cell cycle progression upon DNA damage are of particular importance, as their malfunction may contribute to the genetic instability observed in human embryonic stem cells (hESCs). In this study, we exposed undifferentiated hESCs to DNA‐damaging ultraviolet radiation‐C range (UVC) light and examined their progression through the G1/S transition. We show that hESCs irradiated in G1 phase undergo cell cycle arrest before DNA synthesis and exhibit decreased cyclin‐dependent kinase two (CDK2) activity. We also show that the phosphatase Cdc25A, which directly activates CDK2, is downregulated in irradiated hESCs through the action of the checkpoint kinases Chk1 and/or Chk2. Importantly, the classical effector of the p53‐mediated pathway, protein p21, is not a regulator of G1/S progression in hESCs. Taken together, our data demonstrate that cultured undifferentiated hESCs are capable of preventing entry into S‐phase by activating the G1/S checkpoint upon damage to their genetic complement. STEM CELLS 2010;28:1143–1152

Human Embryonic Stem Cells Suffer from Centrosomal Amplification

Propagation of human embryonic stem cells (hESCs) in culture tends to alter karyotype, potentially limiting the prospective use of these cells in patients. The chromosomal instability of some malignancies is considered to be driven, at least in part, by centrosomal overamplification, perturbing balanced chromosome segregation. Here, we report, for the first time, that very high percentage of cultured hESCs has supernumerary centrosomes during mitosis. Supernumerary centrosomes were strictly associated with an undifferentiated hESC state and progressively disappeared on prolonged propagation in culture. Improved attachment to culture substratum and inhibition of CDK2 and Aurora A (key regulators of centrosomal metabolism) diminished the frequency of multicentrosomal mitoses. Thus, both attenuated cell attachment and deregulation of machinery controlling centrosome number contribute to centrosomal overamplification in hESCs. Linking the excessive number of centrosomes in mitoses to the ploidy indicated that both overduplication within a single cell cycle and mitotic failure contributed to generation of numerical centrosomal abnormalities in hESCs. Collectively, our data indicate that supernumerary centrosomes are a significant risk factor for chromosome instability in cultured hESCs and should be evaluated when new culture conditions are being implemented. STEM CELLS 2011;29:46–56

Lineage specific composition of cyclin D–CDK4/CDK6–p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin

Abstract.  Objectives: This article is to study the role of G1/S regulators in differentiation of pluripotent embryonic cells. Materials and methods: We established a P19 embryonal carcinoma cell‐based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G1/S regulators were analysed with respect to growth and differentiation parameters of the cells. Results and Conclusions: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E–CDK2 but not of cyclin D–CDK4/6–p27 complexes. In an exponentially growing P19 cell population, the cyclin D1–CDK4 complex is detected, which is replaced by cyclin D2/3–CDK4/6–p27 complex following density arrest. During endodermal differentiation kinase‐inactive cyclin D2/D3–CDK4–p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2–CDK4–p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D–CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G1/S transition‐regulating machinery in early embryonic cells.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Soluble Cripto-1 Induces Accumulation of Supernumerary Centrosomes and Formation of Aberrant Mitoses in Human Embryonic Stem Cells

Chromosomal instability evoked by abnormalities in centrosome numbers has been traditionally considered as a hallmark of aberrant, typically cancerous or senescent cells. We have reported previously that pristine human embryonic stem cells (hESC) suffer from high frequency of supernumerary centrosomes and hence may be prone to undergo abnormal mitotic divisions. We have also unraveled that this phenomenon of multicentrosomal mitoses vanishes with prolonged time in culture and with initiation of differentiation, and it is strongly affected by the culture substratum. In this study, we report for the first time that Cripto-1 protein (teratocarcinoma-derived growth factor 1, epidermal growth factor-Cripto/FRL-1/Cryptic) produced by hESC represents a factor capable of inducing formation of supernumerary centrosomes in cultured hESC. Elimination of Cripto-1 signaling on the other hand restores the normal number of centrosomes in hESC. Linking the secretory phenotype of hESC to the centrosomal metabolism may help to develop better strategies for propagation of stable and safe bioindustrial and clinical grade cultures of hESC. From a broader point of view, it may lead to unravelling Cripto-1 as a micro-environmental factor contributing to adverse cell behaviors in vivo.