Cyclin D2 and the CDK substrate p220NPAT are required for self‐renewal of human embryonic stem cells

Self‐renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB‐dependent growth control. We investigated whether self‐renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220^NPAT/HiNF‐P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220^NPAT causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220^NPAT, decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220^NPAT are principal cell cycle regulators that determine competency for self‐renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220^NPAT/HiNF‐P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. J. Cell. Physiol. 222: 456–464, 2010. © 2009 Wiley‐Liss, Inc.     

Reprogramming the pluripotent cell cycle: restoration of an abbreviated G1 phase in human induced pluripotent stem (iPS) cells

Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are reprogrammed to possess stem cell like properties. However, the extent to which iPS cells exhibit unique properties of the human embryonic stem (hES) cell cycle remains to be established. hES cells are characterized by an abbreviated G1 phase (～ 2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e., histone locus bodies (HLBs)]. We therefore examined cell cycle parameters of iPS cells in comparison to hES cells. Analysis of DNA synthesis [5-bromo-2'-deoxy-uridine (BrdU) incorporation], cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immunofluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase (～ 2.5 h) and an abbreviated cell cycle (16-18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within 1.5-2 h). Thus, reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency.     

Differentiation of human embryonic stem cells induces condensation of chromosome territories and formation of heterochromatin protein 1 foci

Abstract Human embryonic stem cells (hES) are unique in their pluripotency and capacity for self-renewal. Therefore, we have studied the differences in the level of chromatin condensation in pluripotent and all-trans retinoic acid-differentiated hES cells. Nuclear patterns of the Oct4 (6p21.33) gene, responsible for hES cell pluripotency, the C-myc (8q24.21) gene, which controls cell cycle progression, and HP1 protein (heterochromatin protein 1) were investigated in these cells. Unlike differentiated hES cells, pluripotent hES cell populations were characterized by a high level of decondensation for the territories of both chromosomes 6 (HSA6) and 8 (HSA8). The Oct4 genes were located on greatly extended chromatin loops in pluripotent hES cell nuclei, outside their respective chromosome territories. However, this phenomenon was not observed for the Oct4 gene in differentiated hES cells, for the C-myc gene in the cell types studied. The high level of chromatin decondensation in hES cells also influenced the nuclear distribution of all the variants of HP1 protein, particularly HP1α, which did not form distinct foci, as usually observed in most other cell types. Our experiments showed that unlike C-myc , the Oct4 gene and HP1 proteins undergo a high level of decondensation in hES cells. Therefore, these structures seem to be primarily responsible for hES cell pluripotency due to their accessibility to regulatory molecules. Differentiated hES cells were characterized by a significantly different nuclear arrangement of the structures studied.     

Mesenchymal differentiation propensity of a human embryonic stem cell line

Objectives: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL‐002. Materials and methods: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real‐time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. Results: Undifferentiated KCL‐002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox‐2, Oct‐4 and TRA 1‐60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR‐2, α‐actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. Conclusions: The data presented suggest that the KCL‐002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Brd4‐Brd2 isoform switching coordinates pluripotent exit and Smad2‐dependent lineage specification

Pluripotent stem cells (PSCs) hold great clinical potential, as they possess the capacity to differentiate into fully specialised tissues such as pancreas, liver, neurons and cardiac muscle. However, the molecular mechanisms that coordinate pluripotent exit with lineage specification remain poorly understood. To address this question, we perform a small molecule screen to systematically identify novel regulators of the Smad2 signalling network, a key determinant of PSC fate. We reveal an essential function for BET family bromodomain proteins in Smad2 activation, distinct from the role of Brd4 in pluripotency maintenance. Mechanistically, BET proteins specifically engage Nodal gene regulatory elements (NREs) to promote Nodal signalling and Smad2 developmental responses. In pluripotent cells, Brd2‐Brd4 occupy NREs, but only Brd4 is required for pluripotency gene expression. Brd4 downregulation facilitates pluripotent exit and drives enhanced Brd2 NRE occupancy, thereby unveiling a specific function for Brd2 in differentiative Nodal‐Smad2 signalling. Therefore, distinct BET functionalities and Brd4‐Brd2 isoform switching at NREs coordinate pluripotent exit with lineage specification.     

Identification of microRNAs expressed highly in pancreatic islet‐like cell clusters differentiated from human embryonic stem cells

Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self‐renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES‐T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet‐like cell clusters derived from T3 cells), which expressed pancreatic islet cell‐specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES‐T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein‐coding mRNAs. The T3pi showed very high expression of microRNAs, miR‐186, miR‐199a and miR‐339, which down‐regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics.     

Identification of microRNAs regulated by activin A in human embryonic stem cells

Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self‐renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES‐T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder‐free Matrigel in MEF‐conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self‐renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES‐T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell‐specific microRNAs miR‐372, miR‐302d, miR‐367, and miR‐200c, as well as three other microRNAs miR‐199a, miR‐19a, and miR‐217, were found to be up‐regulated, whereas five miRNAs miR‐19b, miR‐221, miR‐222, let‐7b, and let‐7c were down‐regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24, and COX6A1 genes, targeted by seven over‐expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4, and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under‐expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self‐renewal and pluripotency of hES cells. J. Cell. Biochem. 109: 93–102, 2010. © 2009 Wiley‐Liss, Inc.     

Geminin escapes degradation in G1 of mouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog

Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast. Here we show that geminin is present in G1 phase of mouse pluripotent cells in contrast to somatic cells, where anaphase-promoting complex/cyclosome (APC/C)-mediated proteasomal destruction removes geminin in G1. Silencing geminin directly or by depleting the APC/C inhibitor Emi1 causes loss of stem cell identity and trophoblast differentiation of mouse embryonal carcinoma and embryonic stem cells. Depletion of cyclins A2 or B1 does not induce this effect, even though both of these APC/C substrates are also present during G1 of pluripotent cells. Crucially, geminin antagonizes the chromatin-remodeling protein Brg1 to maintain expression of Oct4, Sox2, and Nanog. Our results define a pluripotency pathway by which suppressed APC/C activity protects geminin from degradation in G1, allowing sustained expression of core pluripotency factors. Collectively, these findings link the cell cycle to the pluripotent state but also raise an unexplained paradox: How is cell-cycle progression possible in pluripotent cells when oscillations of key regulatory proteins are lost?     

Identification and expansion of human osteosarcoma‐cancer‐stem cells by long‐term 3‐aminobenzamide treatment

A novel cancer stem‐like cell line (3AB‐OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG‐63 cells by long‐term treatment (100 days) with 3‐aminobenzamide (3AB). 3AB‐OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle‐shaped, polygonal‐shaped, and rounded‐shaped. With respect to MG‐63 cells, 3AB‐OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self‐renewal ability and much higher levels of cell cycle markers which account for G1‐S/G2‐M phases progression. Differently from MG‐63 cells, 3AB‐OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP‐binding cassette transporter ABCG2‐dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG‐63 cells. 3AB‐OS cells are much less committed to osteogenic and adipogenic differentiation than MG‐63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF‐1α, FLIP‐L, Bcl‐2, XIAP, IAPs, and survivin). 3AB‐OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting. J. Cell. Physiol. 219: 301–313, 2009. © 2009 Wiley‐Liss, Inc.