Pluripotency, differentiation, and reprogramming: A gene expression dynamics model with epigenetic feedback regulation

Characterization of pluripotent states, in which cells can both self-renew and differentiate, and the irreversible loss of pluripotency are important research areas in developmental biology. In particular, an understanding of these processes is essential to the reprogramming of cells for biomedical applications, i.e., the experimental recovery of pluripotency in differentiated cells. Based on recent advances in dynamical-systems theory for gene expression, we propose a gene-regulatory-network model consisting of several pluripotent and differentiation genes. Our results show that cellular-state transition to differentiated cell types occurs as the number of cells increases, beginning with the pluripotent state and oscillatory expression of pluripotent genes. Cell-cell signaling mediates the differentiation process with robustness to noise, while epigenetic modifications affecting gene expression dynamics fix the cellular state. These modifications ensure the cellular state to be protected against external perturbation, but they also work as an epigenetic barrier to recovery of pluripotency. We show that overexpression of several genes leads to the reprogramming of cells, consistent with the methods for establishing induced pluripotent stem cells. Our model, which involves the inter-relationship between gene expression dynamics and epigenetic modifications, improves our basic understanding of cell differentiation and reprogramming

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation

Background: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments. Results: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heatkilled Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were dentified. Conclusion: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen

Total Hip Arthroplasty Using the S-ROM-A Prosthesis for Anatomically Difficult Asian Patients

Background. The S-ROM-A prosthesis has been designed for the Asian proximal femur with a small deformed shape and narrow canal. In this study, the clinical and radiological results using the S-ROM-A prosthesis for Japanese patients with severe deformity due to dysplasia and excessive posterior pelvic tilt were examined. Methods. 94 hips were followed up for a mean of 55 months, with a mean age at surgery of 61 years. The primary diagnoses were 94 coxarthritis cases, including 51 dysplasia and 37 primary OA, 1 avascular necrosis, 2 traumatic arthritis, and 3 Perthes disease. Thirty-one hips had been treated with osteotomy of the hip joints. Preoperative intramedullary canal shapes were stovepipe in 23 hips, normal in 51 hips, and champagne-flute in 5 hips. The maximum pelvic inclination angle was 56°. Results. The mean JOA score improved from 46 points preoperatively to 80 points at final follow-up. On radiological evaluation of the fixation of the implants according to the Engh classification, 92 (97%) hips were classified as "bone ingrown fixation." Conclusion. In primary THA, using the S-ROM-A prosthesis for Asian patients with proximal femoral deformity, even after osteotomy and with posterior pelvic tilt, provided good short- to midterm results

The gene regulatory network in the reprogramming simulation.

<p>We overexpressed the pluripotent genes <i>x</i>₁ and <i>x</i>₂, and added an external stimulus <i>ex</i>1 to activate gene <i>x</i>₄ in the four-gene network model (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004476#pcbi.1004476.g001" target="_blank">Fig 1C</a>). This induction triggered reprogramming, and cells started to oscillate once again. These reprogramming factors correspond with, for example, <i>x</i>₁ = <i>Oct4</i>, <i>x</i>₂ = <i>Sox2</i>, and <i>ex</i>1 = <i>Myc</i>.</p

Cell differentiation with the epigenetic variable.

<p>Time series of gene expression levels for <i>x</i>₁, <i>x</i>₂, <i>x</i>₃, <i>x</i>₄, and the epigenetic threshold variables <i>θ</i>₁₁(<i>t</i>) and <i>θ</i>₁₃(<i>t</i>). Expression levels of cells are plotted according to color, but most colors are overlaid and, therefore, difficult to discern. We set the parameters of the epigenetic variable as follows: <i>τ</i>_<i>epi</i> = 2.0 × 10³, <i>α</i> = 0.1, Θ_<i>ij</i> = 1.0. The initial value of the epigenetic variable <i>θ</i>_<i>ij</i>(0) was set as <i>K</i>_<i>ij</i> in the non-epigenetic model. The other parameters are the same as those used in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004476#pcbi.1004476.g004" target="_blank">Fig 4</a>. First, gene expression oscillated, and then the epigenetic variables in each cell changed gradually. <i>θ</i>₁₁(<i>t</i>) differentiated into two groups, and in one of these <i>x</i>₁ approached 0.</p

Time series of gene expression in reprogramming via overexpression of three genes and one external factor.

<p>Plotted here are time series of gene expression for <i>x</i>₁, <i>x</i>₂, <i>x</i>₃, <i>x</i>₄, <i>x</i>₅, and the epigenetic threshold variables <i>θ</i>₁₅. In this case, we used the following parameter set: <i>θ</i>₁₃(0) = <i>θ</i>₃₄(0) = <i>θ</i>₄₃(0) = 0.65, <i>θ</i>₁₅(0) = <i>θ</i>₃₁(0) = <i>θ</i>₂₁(0) = <i>θ</i>₅₁(0) = <i>θ</i>₄₂(0) = 1.0. In the differentiated state, we overexpressed genes <i>x</i>₁, <i>x</i>₂, and <i>x</i>₅ over a long period and added one external regulator. Induction of these factors changed the epigenetic threshold variables; gene expression then began to oscillate again and, later, differentiation occurred in a few cells.</p

Cellular state transition under noise.

<p>Time series of gene expression levels for <i>x</i>₁. Similar conditions to those described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004476#pcbi.1004476.g004" target="_blank">Fig 4</a> were adopted, except that a Gaussian noise term with the amplitude <i>σ</i> = 0.1 was included. Expression levels of cells are plotted according to color. Gene expression oscillation was irregular because of the noise. Irreversible transition from the oscillatory pluripotent to the differentiated state (<i>x</i>₁ ∼ 0) occurred for <i>σ</i> = 0.1.</p

Time series of single-cell dynamics.

<p>Time series of gene expression for <i>x</i>₁, <i>x</i>₂, <i>x</i>₃, and <i>x</i>₄. Each colored line represents expression levels of a single gene. Three different behaviors appeared in single-cell dynamics depending on the parameter <i>K</i>_<i>ij</i>. We set the parameter <i>K</i>₁₃ at (A) 0.98, (B) 0.58, and (C) 0.78. The other parameters were fixed at <i>K</i>₃₄ = 0.45, <i>K</i>₃₁ = 0.94, <i>K</i>₁₁ = 0.35, <i>K</i>₂₁ = 0.80, <i>K</i>₄₂ = 0.30, and <i>K</i>₄₃ = 0.45. A: The pluripotent genes <i>x</i>₁ and <i>x</i>₂ were highly expressed, and the differentiation genes <i>x</i>₃ and <i>x</i>₄ were suppressed. This state corresponds to FP. B: Pluripotent genes were suppressed, and differentiation genes were expressed. This state corresponds to FD. C: Oscillation of gene expression occurred, and this state corresponds to O.</p

Time series of gene expression with the occurrence of cell-cell interactions.

<p>Time series of gene expression levels for <i>x</i>₁, <i>x</i>₂, <i>x</i>₃, and <i>x</i>₄ for all cells, where cells divided per period = 25 until time = 125 to generate 32 cells. Expression levels of cells are plotted according to color, but most colors are overlaid and, therefore, difficult to discern. The diffusion coefficient <i>D</i> was set at <i>D</i> = 2, and the other parameter values are the same as those given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004476#pcbi.1004476.g002" target="_blank">Fig 2C</a>. The oscillatory state has pluripotency to allow for both self-renewal and differentiation. The oscillation of gene expression was initially desynchronized, and then a few cells switched to the differentiated state.</p

Time series of gene expression in the reprogramming simulation, with induction of pluripotent genes and external activation.

<p>Plotted here are the time series of gene expression levels for <i>x</i>₁, <i>x</i>₂, <i>x</i>₃, <i>x</i>₄, and the epigenetic threshold variables <i>θ</i>₁₁(<i>t</i>) and <i>θ</i>₁₃(<i>t</i>). Initially, all cells (e.g., 32 cells) were set at the differentiated state (<i>x</i>_1,2 = 0, <i>x</i>_3,4 = 0.8), with the epigenetic fixation threshold values set at 1.0 for the pluripotent genes Θ₃₁, Θ₂₁, and Θ₄₂, and at 0.78 for the differentiation regulators Θ₁₃, Θ₃₄, Θ₄₃. The auto-regulator Θ₁₁ was set at 0.50. We overexpressed genes <i>x</i>₁ and <i>x</i>₂ for a long period (<i>T</i>^<i>e</i> ∼ 100). The epigenetic variables in each cell changed gradually because of the overexpression of these genes. In addition, the gene <i>x</i>₄ was promoted by an external stimulus. After overexpression, gene expression began to oscillate again and a few cells showed differentiation. Thus, cells were reprogrammed.</p