Stephen Dalton

Highly efficient generation of human hepatocyte–like cells from induced pluripotent stem cells

There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient‐specific human hepatocytes from induced pluripotent stem (iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte‐like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010.)

LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.

Murine ES cells can be maintained as a pluripotent, self-renewing population by LIF/STAT3-dependent signaling. The downstream effectors of this pathway have not been previously defined. In this report, we identify a key target of the LIF self-renewal pathway by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3β dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, our results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency.

Architectural Protein Subclasses Shape 3D Organization of Genomes during Lineage Commitment

Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here, we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3D interactions that undergo marked reorganization at the submegabase scale during differentiation. Distinct combinations of CCCTC-binding factor (CTCF), Mediator, and cohesin show widespread enrichment in chromatin interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant subdomains. Conversely, Mediator/cohesin bridge short-range enhancer-promoter interactions within and between larger subdomains. Knockdown of Smc1 or Med12 in embryonic stem cells results in disruption of spatial architecture and downregulation of genes found in cohesin-mediated interactions. We conclude that cell-type-specific chromatin organization occurs at the submegabase scale and that architectural proteins shape the genome in hierarchical length scales.

Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3‐kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor β family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast‐conditioned medium under feeder‐free conditions, ∼70%–80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1‐II. Microarray and quantitative polymerase chain reaction‐based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin‐like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self‐renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC‐DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self‐renewal and early cell fate decisions.

Preserving the genetic integrity of human embryonic stem cells

transactions are the difficulty in accessing correct and complete information on potential partners, suppliers or market possibilities and the uncertainty of ensuring a partner’s commitment to formal contracts. The latter in particular, requires a judicial system that functions more efficiently and credibly. These conditions have nothing to do with TRIPS. Thus, the impact of TRIPS on either the commercial strategies of foreign companies or their strategic alliances with Indian companies is anyone’s guess, as it is only one parameter among many that will be used in making foreign investment decisions. From the perspective of Western firms, the implementation of TRIPS in India may encourage them to introduce new brand drugs because such products will now enjoy patent protection—a situation not possible since 1970. This will not mean, however, that high-priced, Western-manufactured products can be directly shoehorned into the Indian market. As K.S.N. Prasad, CEO of Shantha Biotechnics (Hyderabad, India), puts it: “Though TRIPS gives exclusive rights to Western companies to market their brand products in India—eliminating competition from local companies that copy inventions— these multinationals are unlikely to benefit from selling their products at high prices because Indian consumers simply cannot afford the high costs of drugs developed and manufactured abroad. Therefore, it will be necessary for Western and Indian companies to enter into strategic alliances so that novel drugs can be manufactured under license for local consumption. Such alliances will lead to a win-win situation for all, both biotech companies and the public.” To sum up, Indian biotech firms basically have three choices in the short-term as business innovation strategies2: first, they can focus on products that are either off-patent already or soon to be off-patent (essentially the generics market); second, they can collaborate with Western multinationals and biotech companies (two areas that are likely to witness an increase in collaborations are clinical trials and R&D outsourcing); or third, they can focus on innovations that the multinationals will not be interested in; that is, mainly ‘tropical’ or developing world diseases.

Functional analysis of a growth factor-responsive transcription factor complex

Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory factor, Elk-1. We constructed altered-binding specificity derivatives of SRF and Elk-1 that form a ternary complex at a mutated, inactive SRE; like Elk-1, the Elk-1 variant only binds its target as part of a ternary complex with SRF. Simultaneous expression of these SRF and Elk-1 derivatives restores serum-regulated activity to the mutated SRE in transfected cells. Efficient transcriptional activation is dependent on the regulated phosphorylation of Elk-1 C-terminal MAP kinase sites and requires the C-terminal sequences of SRF as well as SRF sequences that mediate ternary complex formation. These experiments provide direct evidence that SRF and Elk-1 functionally cooperate in the ternary complex at the SRE to regulate transcription.

Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities

Pluripotent cells of embryonic origin proliferate at unusually rapid rates and have a characteristic cell cycle structure with truncated gap phases. To define the molecular basis for this we have characterized the cell cycle control of murine embryonic stem cells and early primitive ectoderm-like cells. These cells display precocious Cdk2, cyclin A and cyclin E kinase activities that are conspicuously cell cycle independent. Suppression of Cdk2 activity significantly decreased cycling times of pluripotent cells, indicating it to be rate-limiting for rapid cell division, although this had no impact on cell cycle structure and the establishment of extended gap phases. Cdc2-cyclin B was the only Cdk activity that was identified to be cell cycle regulated in pluripotent cells. Cell cycle regulation of cyclin B levels and Y15 regulation of Cdc2 contribute to the temporal changes in Cdc2-cyclin B activity. E2F target genes are constitutively active throughout the cell cycle, reflecting the low activity of pocket proteins such as p107 and pRb and constitutive activity of pRb-kinases. These results show that rapid cell division cycles in primitive cells of embryonic origin are driven by extreme levels of Cdk activity that lack normal cell cycle periodicity.

Cell cycle control of embryonic stem cells

Embryonic stem cells have the capacity for unlimited proliferation while retaining their potential to differentiate into a wide variety of cell types. Murine, primate and human embryonic stem cells (ESCs) exhibit a very unusual cell cycle structure, characterized by a short G1 phase and a high proportion of cells in S-phase. In the case of mESCs, this is associated with a unique mechanism of cell cycle regulation, underpinned by the precocious activity of cyclin dependent protein kinase (Cdk) activities. As ES cells differentiate, their cell cycle structure changes dramatically so as to incorporate a significantly longer G1 phase and their mechanism of cell cycle regulation changes to that typically seen in other mammalian cells. The unique cell cycle structure and mechanism of cell cycle control indicates that the cell cycle machinery plays a role in establishment or maintenance of the stem cell state. This idea is supported by the frequent involvement of cell cycle regulatory molecules in cell immortalization.

The cell cycle and Myc intersect with mechanisms that regulate pluripotency and reprogramming.

Pluripotent stem cells have long-term proliferative capacity and an unusual mode of cell-cycle regulation and can divide independently of extrinsic mitogenic signals. The last few years has seen evidence emerge that links cell-cycle regulation to the maintenance and establishment of pluripotency. Myc transcription factors appear to be central to this regulation. This review addresses these links and discusses how cell-cycle controls and Myc impact on the maintenance and establishment of pluripotency.

Signaling Network Crosstalk in Human Pluripotent Cells: A Smad2/3-Regulated Switch that Controls the Balance between Self-Renewal and Differentiation

A general mechanism for how intracellular signaling pathways in human pluripotent cells are coordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining prodifferentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self-renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity after inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3β. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.