Identification of the Proliferation/Differentiation Switch in the Cellular Network of Multicellular Organisms

The protein–protein interaction networks, or interactome networks, have been shown to have dynamic modular structures, yet the functional connections between and among the modules are less well understood. Here, using a new pipeline to integrate the interactome and the transcriptome, we identified a pair of transcriptionally anticorrelated modules, each consisting of hundreds of genes in multicellular interactome networks across different individuals and populations. The two modules are associated with cellular proliferation and differentiation, respectively. The proliferation module is conserved among eukaryotic organisms, whereas the differentiation module is specific to multicellular organisms. Upon differentiation of various tissues and cell lines from different organisms, the expression of the proliferation module is more uniformly suppressed, while the differentiation module is upregulated in a tissue- and species-specific manner. Our results indicate that even at the tissue and organism levels, proliferation and differentiation modules may correspond to two alternative states of the molecular network and may reflect a universal symbiotic relationship in a multicellular organism. Our analyses further predict that the proteins mediating the interactions between these modules may serve as modulators at the proliferation/differentiation switch

A Novel Correlation Networks Approach for the Identification of Gene Targets

Correlation networks are emerging as a powerful tool for modeling temporal mechanisms within the cell. Particularly useful in examining coexpression within microarray data, studies have determined that correlation networks follow a power law degree distribution and thus manifest properties such as the existence of “hub” nodes and semicliques that potentially correspond to critical cellular structures. Difficulty lies in filtering coincidental relationships from causative structures in these large, noise-heavy networks. As such, computational expenses and algorithm availability limit accurate comparison, making it difficult to identify changes between networks. In this vein, we present our work identifying temporal relationships from microarray data obtained from mice in three stages of life. We examine the characteristics of mouse networks, including correlation and node degree distributions. Further, we identify high degree nodes (“hubs”) within networks and define their essentiality. Finally, we associate Gene Ontology annotations to network structures to deduce relationships between structure and cellular functions

Role of AMP-activated protein kinase in regulating hypoxic survival and proliferation of mesenchymal stem cells

Aims Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. Methods and results MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. Conclusion MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulatio

Transcriptome and Network Changes in Climbers at Extreme Altitudes

Extreme altitude can induce a range of cellular and systemic responses. Although it is known that hypoxia underlies the major changes and that the physiological responses include hemodynamic changes and erythropoiesis, the molecular mechanisms and signaling pathways mediating such changes are largely unknown. To obtain a more complete picture of the transcriptional regulatory landscape and networks involved in extreme altitude response, we followed four climbers on an expedition up Mount Xixiabangma (8,012 m), and collected blood samples at four stages during the climb for mRNA and miRNA expression assays. By analyzing dynamic changes of gene networks in response to extreme altitudes, we uncovered a highly modular network with 7 modules of various functions that changed in response to extreme altitudes. The erythrocyte differentiation module is the most prominently up-regulated, reflecting increased erythrocyte differentiation from hematopoietic stem cells, probably at the expense of differentiation into other cell lineages. These changes are accompanied by coordinated down-regulation of general translation. Network topology and flow analyses also uncovered regulators known to modulate hypoxia responses and erythrocyte development, as well as unknown regulators, such as the OCT4 gene, an important regulator in stem cells and assumed to only function in stem cells. We predicted computationally and validated experimentally that increased OCT4 expression at extreme altitude can directly elevate the expression of hemoglobin genes. Our approach established a new framework for analyzing the transcriptional regulatory network from a very limited number of samples

Is My Network Module Preserved and Reproducible?

In many applications, one is interested in determining which of the properties of a network module change across conditions. For example, to validate the existence of a module, it is desirable to show that it is reproducible (or preserved) in an independent test network. Here we study several types of network preservation statistics that do not require a module assignment in the test network. We distinguish network preservation statistics by the type of the underlying network. Some preservation statistics are defined for a general network (defined by an adjacency matrix) while others are only defined for a correlation network (constructed on the basis of pairwise correlations between numeric variables). Our applications show that the correlation structure facilitates the definition of particularly powerful module preservation statistics. We illustrate that evaluating module preservation is in general different from evaluating cluster preservation. We find that it is advantageous to aggregate multiple preservation statistics into summary preservation statistics. We illustrate the use of these methods in six gene co-expression network applications including 1) preservation of cholesterol biosynthesis pathway in mouse tissues, 2) comparison of human and chimpanzee brain networks, 3) preservation of selected KEGG pathways between human and chimpanzee brain networks, 4) sex differences in human cortical networks, 5) sex differences in mouse liver networks. While we find no evidence for sex specific modules in human cortical networks, we find that several human cortical modules are less preserved in chimpanzees. In particular, apoptosis genes are differentially co-expressed between humans and chimpanzees. Our simulation studies and applications show that module preservation statistics are useful for studying differences between the modular structure of networks. Data, R software and accompanying tutorials can be downloaded from the following webpage: http://www.genetics.ucla.edu/labs/horvath/CoexpressionNetwork/ModulePreservation

Computational Modeling and Reverse Engineering to Reveal Dominant Regulatory Interactions Controlling Osteochondral Differentiation: Potential for Regenerative Medicine

The specialization of cartilage cells, or chondrogenic differentiation, is an intricate and meticulously regulated process that plays a vital role in both bone formation and cartilage regeneration. Understanding the molecular regulation of this process might help to identify key regulatory factors that can serve as potential therapeutic targets, or that might improve the development of qualitative and robust skeletal tissue engineering approaches. However, each gene involved in this process is influenced by a myriad of feedback mechanisms that keep its expression in a desirable range, making the prediction of what will happen if one of these genes defaults or is targeted with drugs, challenging. Computer modeling provides a tool to simulate this intricate interplay from a network perspective. This paper aims to give an overview of the current methodologies employed to analyze cell differentiation in the context of skeletal tissue engineering in general and osteochondral differentiation in particular. In network modeling, a network can either be derived from mechanisms and pathways that have been reported in the literature (knowledge-based approach) or it can be inferred directly from the data (data-driven approach). Combinatory approaches allow further optimization of the network. Once a network is established, several modeling technologies are available to interpret dynamically the relationships that have been put forward in the network graph (implication of the activation or inhibition of certain pathways on the evolution of the system over time) and to simulate the possible outcomes of the established network such as a given cell state. This review provides for each of the aforementioned steps (building, optimizing, and modeling the network) a brief theoretical perspective, followed by a concise overview of published works, focusing solely on applications related to cell fate decisions, cartilage differentiation and growth plate biology. Particular attention is paid to an in-house developed example of gene regulatory network modeling of growth plate chondrocyte differentiation as all the aforementioned steps can be illustrated. In summary, this paper discusses and explores a series of tools that form a first step toward a rigorous and systems-level modeling of osteochondral differentiation in the context of regenerative medicine

Investigation of histone lysine methylation in stem cell differentiation using inhibitor peptide

In an effort to expand the histone code, we examine a novel site of methylation on lysine 43 of histone H2B. In mouse embryonic stem cells (ESCs), KDM5b acts as the lysine demethylase for H2BK43me2, diminishing this histone mark as cells differentiate. We utilize a synthetic peptide mimetic corresponding to amino acids 37-49 of histone H2B in order to sterically inactivate KDM5b enzyme. The addition of inhibitor peptide into culture enhanced stem cell differentiation, upregulating cell cycle and neural-specific markers while downregulating the expression of pluripotency genes. Global gene analysis patterns of peptide-treated ESCs were representative of differentiated cell populations. Applying a novel inhibition method, we reveal an accelerated rate of stem cell differentiation through the upregulation of KDM5b targets. Our investigation serves to elucidate the role of histone H2BK43 methylation in an epigenetically regulated model of development

Microenvironmental control of epithelial cell fate

Cancer is a devastating condition, yet its prevalence is not surprising when one considers the possibility that growth and motility define the default state of epithelial cells. What is more surprising is that epithelial cells can be induced to a “fragile quiescent state” in multicellular organisms through constant inhibitory influences from extracellular sources. In other words, the immotile and growth-constrained behavior we associate with epithelia (quiescence) is not the default state cells in a multicellular organism, but rather must be exogenously induced by tissue-specific (and systemic) factors. Quiescence therefore, is a fragile existence for any cell, as the removal of differentiating signals should cause the cell to revert to a migratory, stem-like default state. It will be argued that cancer is better understood as a disease of tissues rather than individual cells, and the complexities of tissues cannot be inferred from the study of cells in isolation. In-vitro studies which have been used to explain carcinogenesis will be critically reviewed and their relevance to in-vivo conditions will be questioned. Complex signaling mechanisms define the relationship between the epithelium and other cells in a metazoan tissue. These signals originate both from the stromal/mesenchymal compartments and between epithelial cells. Studies have shown that carcinogenic insults which affect the stroma alone can turn an otherwise normal epithelium cancerous, while transplanting “cancerous” epithelial cells into an otherwise normal stroma does not result in neoplasm formation. Experimental evidence has confirmed that the differentiation fate of any epithelial cell is malleable depending on its environmental context. Further, it was previously thought that epithelium had to dramatically change identity in order to be capable of migration. It will be shown that collective motility is an endogenous capability of epithelial cells, and multiple non-mammalian and mammalian in-vivo studies of collective epithelial motility will be reviewed to better understand epithelial motility in cancer. In fact, it will be shown that the creation of adhered epithelial sheets requires the same morphological changes necessary for motility. Lastly, evidence that heterogeneous cell populations contribute to epithelial cell migration in development and metastasis will be presented

A Study to Improve the Differentiation of Human Embryonic Stem Cells to Functional Hepatocytes

Human embryonic stem cells (hESCs) possess 2 unique properties (1) pluripotency and (2) self-renewal, and therefore, hold great promise for biomedical application and regenerative medicine. In vitro differentiation of hESCs is a vital tool to generate unlimited human hepatocytes. To date, several multi-step protocols have been established to generate hepatocyte-like cells from undifferentiated hESCs via definitive endoderm (DE) formation. However, hESC derived-hepatocytes in these systems exhibit some immature characteristics, thus it remains a challenge on how to further improve hepatic differentiation. In addition, the molecular mechanisms regulating the differentiation are still ambiguous, making in vitro differentiation a difficult task. The ultimate aim of this project is to improve the differentiation of hESCs to functional hepatocytes. In this thesis, the work includes two main parts: (1) modulating signalling pathways to explore molecular mechanisms controlling DE differentiation; (2) developing a 3-dimensional (3D) culture system to improve the functionality of hESC-derived hepatocytes. DE formation is a critical step for the production of hepatocytes. In the first part of this thesis, I showed that suppression of PI3K signalling using the LY 294002 inhibitor (LY) during hESC differentiation significantly improves Activin A (AA)-induced DE generation, which subsequently augments hepatocyte production. Further mechanistic interrogation of this phenomenon has revealed that dual treatment of hESCs with AALY enhances the Activin downstream signalling, Smad2/3 phosphorylation and their nuclear translocation with Smad4. Furthermore, dual treatment with AALY also affects the disruption of β-catenin/E-cadherin complexes, which cooperatively contribute to distinctive morphological changes that may signify the occurrence of EMT and hence improved specification of DE. These findings suggest that suppression of PI3K/Akt modulates both Nodal/Activin and β-catenin pathways, the two most important signalling involved in mesendoderm and DE cell fate specification, therefore improved DE differentiation of hESCs. Liver development in vivo is regulated by cell–cell contacts in a 3D environment and the absences of this may account for, at least partly, some of the immature features of hESC-derived hepatocytes. In the second part of my thesis, based on initial encouraging results obtained from HepG2 cells, I applied alginate based 3D culture system to hESCs after they are differentiated into DE cells and optimised culture conditions. The results confirmed that 3D culture microenvironment enhanced hepatic differentiation and functionality of hESC-derived hepatocytes in compared to the monolayer format. Collectively, this study has demonstrated a significant cornerstone in the strategies to improve hepatic differentiation of hESCs by addressing the molecular signalling and micro-niche cues that govern hepatocyte lineage commitment. Hence, this will pave the way for the use of these hepatocytes in future regenerative therapies and biomedical applications