Klaus A. Becker

Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase

Competency for self‐renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH‐designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15–16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT‐2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220NPAT containing Cajal bodies revealed that the durations of the S (∼8 h), G2 (∼4 h), and M phases (∼1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti‐tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5–3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT‐PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin‐dependent kinases (CDKs), we observed high mRNA levels for the G1‐related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication. J. Cell. Physiol. 209: 883–893, 2006.

X‐inactivation reveals epigenetic anomalies in most hESC but identifies sublines that initiate as expected

The clinical and research value of human embryonic stem cells (hESC) depends upon maintaining their epigenetically naïve, fully undifferentiated state. Inactivation of one X chromosome in each cell of mammalian female embryos is a paradigm for one of the earliest steps in cell specialization through formation of facultative heterochromatin. Mouse ES cells are derived from the inner cell mass (ICM) of blastocyst stage embryos prior to X‐inactivation, and cultured murine ES cells initiate this process only upon differentiation. Less is known about human X‐inactivation during early development. To identify a human ES cell model for X‐inactivation and study differences in the epigenetic state of hESC lines, we investigated X‐inactivation in all growth competent, karyotypically normal, NIH approved, female hESC lines and several sublines. In the vast majority of undifferentiated cultures of nine lines examined, essentially all cells exhibit hallmarks of X‐inactivation. However, subcultures of any hESC line can vary in X‐inactivation status, comprising distinct sublines. Importantly, we identified rare sublines that have not yet inactivated Xi and retain competence to undergo X‐inactivation upon differentiation. Other sublines exhibit defects in counting or maintenance of XIST expression on Xi. The few hESC sublines identified that have not yet inactivated Xi may reflect the earlier epigenetic state of the human ICM and represent the most promising source of NIH hESC for study of human X‐inactivation. The many epigenetic anomalies seen indicate that maintenance of fully unspecialized cells, which have not formed Xi facultative heterochromatin, is a delicate epigenetic balance difficult to maintain in culture. J. Cell. Physiol. 216: 445–452, 2008.

Establishment of histone gene regulation and cell cycle checkpoint control in human embryonic stem cells

Rapid self‐renewal of human embryonic stem (ES) cells (NIH designation WA01 and WA09) is accommodated by an abbreviated cell cycle due to a reduction in the G1 phase. Thus, molecular mechanisms operative in ES cells may expedite the cellular commitment to progress into S phase to initiate replication of DNA and biosynthesis of histone proteins to form new chromatin. Here we show that the selective cell cycle regulated expression of individual histone H4 gene copies, which is typical for somatic cell types, is already firmly established in human ES cells. This early establishment of H4 gene regulation, which is E2F independent, is consistent with co‐expression of the cognate transcriptional regulators HiNF‐P and p220NPAT. Human ES cells differ from somatic cells in the expression of members of the E2F family and RB‐related pocket proteins (p105RB1, p107RBL1, and p130RBL2/RB2) that control expression of genes encoding enzymes for nucleotide metabolism and DNA synthesis. Human ES cells rapidly and robustly (>200‐fold) induce the cyclin dependent kinase (CDK) inhibitor p21WAF1/CIP1 upon gamma‐irradiation. This DNA damage response promptly reduces histone gene expression as well as mRNA levels for HiNF‐P and p220NPAT and causes accumulation of unprocessed histone H4 precursor RNAs. Furthermore, while E2F4, E2F5 and p130RBL2/RB2 are the major E2F and pocket protein mRNAs in actively proliferating ES cells, expression levels of E2F5, E2F6, and p105RB1 are most strongly elevated during cell cycle arrest in cells responding to DNA damage. Our data suggest that the brief G1 phase of ES cells is supported by a potent p21WAF1/CIP1 related DNA damage response that functions through several mechanisms to rapidly inhibit cell cycle progression. This response may alter the E2F/pocket protein combinations that control E2F dependent genes and block H4 gene expression by inhibiting histone‐specific transcription factors and processing of histone gene transcripts, as well as by destabilizing histone mRNAs. J. Cell. Physiol. 210: 517–526, 2007.

The abbreviated pluripotent cell cycle.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self‐renewal of tissue‐specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory, and structural. The primary temporal context that the pluripotent self‐renewal cell cycle of hESCs is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the embryonic stem cell (ESC) cell cycle. This supports the requirements of pluripotent cells to self‐propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated ESC cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell‐based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. J. Cell. Physiol. 228: 9–20, 2013.

Staged assembly of histone gene expression machinery at subnuclear foci in the abbreviated cell cycle of human embryonic stem cells

Human embryonic stem (hES) cells have an abbreviated G1 phase of the cell cycle. How cells expedite G1 events that are required for the initiation of S phase has not been resolved. One key regulatory pathway that controls G1/S-phase transition is the cyclin E/CDK2-dependent activation of the coactivator protein nuclear protein, ataxia–telangiectasia locus/histone nuclear factor-P (p220NPAT/HiNF-P) complex that induces histone gene transcription. In this study, we use the subnuclear organization of factors controlling histone gene expression to define mechanistic differences in the G1 phase of hES and somatic cells using in situ immunofluorescence microscopy and fluorescence in situ hybridization (FISH). We show that histone gene expression is supported by the staged assembly and modification of a unique subnuclear structure that coordinates initiation and processing of transcripts originating from histone gene loci. Our results demonstrate that regulatory complexes that mediate transcriptional initiation (e.g., p220NPAT) and 3′-end processing (e.g., Lsm10, Lsm11, and SLBP) of histone gene transcripts colocalize at histone gene loci in dedicated subnuclear foci (histone locus bodies) that are distinct from Cajal bodies. Although appearance of CDK2-phosphorylated p220NPAT in these domains occurs at the time of S-phase entry, histone locus bodies are formed ≈1 to 2 h before S phase in embryonic cells but 6 h before S phase in somatic cells. These temporal differences in the formation of histone locus bodies suggest that the G1 phase of the cell cycle in hES cells is abbreviated in part by contraction of late G1.

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells

Human embryonic stem (ES) cells have an expedited cell cycle (∼15 h) due to an abbreviated G1 phase (∼2.5 h) relative to somatic cells. One principal regulatory event during cell cycle progression is the G1/S phase induction of histone biosynthesis to package newly replicated DNA. In somatic cells, histone H4 gene expression is controlled by CDK2 phosphorylation of p220NPAT and localization of HiNF‐P/p220NPAT complexes with histone genes at Cajal body related subnuclear foci. Here we show that this ‘S point’ pathway is operative in situ in human ES cells (H9 cells; NIH‐designated WA09). Immunofluorescence microscopy shows an increase in p220NPAT foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220NPAT foci is evident in S phase, the increase in the number of p220NPAT foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220NPAT at CDK dependent epitopes is most pronounced in S phase when cells exhibit elevated levels of cyclins E and A. Our data indicate that subnuclear organization of the HiNF‐P/p220NPAT pathway is rapidly established as ES cells emerge from mitosis and that p220NPAT is subsequently phosphorylated in situ. Our findings establish that the HiNF‐P/p220NPAT gene regulatory pathway operates in a cell cycle dependent microenvironment that supports expression of DNA replication‐linked histone genes and chromatin assembly to accommodate human stem cell self‐renewal. J. Cell. Physiol. 213: 9–17, 2007.

Human embryonic stem cells are pre-mitotically committed to self-renewal and acquire a lengthened G1 phase upon lineage programming

Self‐renewal of human embryonic stem (hES) cells proceeds by a unique abbreviated cell cycle with a shortened G1 phase and distinctions in molecular cell cycle regulatory parameters. In this study, we show that early lineage‐commitment of pluripotent hES cells modifies cell cycle kinetics. Human ES cells acquire a lengthened G1 within 72 h after lineage‐programming is initiated, as reflected by loss of the pluripotency factor Oct4 and alterations in nuclear morphology. In hES cells that maintain the pristine pluripotent state, we find that autocrine mechanisms contribute to sustaining the abbreviated cell cycle. Our data show that naïve and mitotically synchronized pluripotent hES cells are competent to initiate two consecutive S phases in the absence of external growth factors. We conclude that short‐term self‐renewal of pluripotent hES cells occurs autonomously, in part due to secreted factors, and that pluripotency is functionally linked to the abbreviated hES cell cycle. J. Cell. Physiol. 222:103–110, 2010.

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 p220NPAT/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 p220NPAT causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220NPAT, decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220NPAT 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 p220NPAT/HiNF‐P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. J. Cell. Physiol. 222: 456–464, 2010.

The Architectural Organization of Human Stem Cell Cycle Regulatory Machinery

Two striking features of human embryonic stem cells that support biological activity are an abbreviated cell cycle and reduced complexity to nuclear organization. The potential implications for rapid proliferation of human embryonic stem cells within the context of sustaining pluripotency, suppressing phenotypic gene expression and linkage to simplicity in the architectural compartmentalization of regulatory machinery in nuclear microenvironments is explored. Characterization of the molecular and architectural commitment steps that license human embryonic stem cells to initiate histone gene expression is providing understanding of the principal regulatory mechanisms that control the G1/S phase transition in primitive pluripotent cells. From both fundamental regulatory and clinical perspectives, further understanding of the pluripotent cell cycle in relation to compartmentalization of regulatory machinery in nuclear microenvironments is relevant to applications of stem cells for regenerative medicine and new dimensions to therapy where traditional drug discovery strategies have been minimally effective.

The Ultrastructural Signature of Human Embryonic Stem Cells

The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure—gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764–774, 2017.