Building the cell: design principles of cellular architecture

The astounding structural complexity of a cell arises from the action of a relatively small number of genes, raising the question of how this complexity is achieved. Self-organizing processes combined with simple physical constraints seem to have key roles in controlling organelle size, number, shape and position, and these factors then combine to produce the overall cell architecture. By examining how these parameters are controlled in specific cell biological examples we can identify a handful of simple design principles that seem to underlie cellular architecture and assembly.     

Full factorial screening of human embryonic stem cell maintenance with multiplexed microbioreactor arrays

Use of human pluripotent stem cells (hPSCs) in regenerative medicine applications relies on control of cell fate decisions by exogenous factors. This control can be hindered by the use of undefined culture components, poorly understood autocrine/paracrine effects, spatiotemporal variations in microenvironmental composition inherent to static culture formats, and signal cross-talk between multiple factors. We recently described microbioreactor arrays that provide a full factorial spectrum of exogenous factors, and allow gradual accumulation of paracrine factors through serial culture chambers. We combined these with defined biochemical conditions, and in situ reporter gene- and immunofluorescence-based readouts to create an hPSC screening platform with enhanced data throughput and microenvironmental control. HES3-EOS-C(3+)-EiP reporter hESCs were screened against FGF-2, TGF-β1, and retinoic acid in a modified mTeSR-1 medium background. Differential pluripotency marker expression reflected mTeSR-1's maintenance capacity, and differentiation in response to removal of maintenance factors or addition of retinoic acid. Interestingly, pluripotency marker expression was downregulated progressively through serial chambers. Since downstream chambers are exposed to greater levels of paracrine factors under continuous flow, this effect is thought to result from secreted factors that negatively influence pluripotency. The microbioreactor array platform decodes factor interplay, and has a broad application in deciphering microenvironmental control of cell fate.     

A proposed design for the cryopreservation of intact and adherent human embryonic stem cell colonies

Summary Recently, it was demonstrated that the application of slow-cooling cryopreservation protocols to adherent human embryonic stem (hES) cell colonies, cultured on matrigel or murine embryonic fibroblast feeder layers, resulted in marked improvement in postthaw viability and reduction in cell differentiation. However, the use of commercially available culture plates for this purpose presents several limitations. Most obviously, these plates are not designed for cryopreservation or to withstand the low temperatures encountered during liquid nitrogen cryopreservation, or both. The physical storage of cryopreserved plates is another consideration, in addition to difficulty in maintaining sterile conditions in liquid nitrogen storage and during the thaw phase in a water bath. Hence, a redesign of the cell culture plate for the cryopreservation of adherent hES cell colonies is proposed. In this model, a culture plate made of synthetic materials resistant to storage at −196° C of liquid nitrogen is designed, with readily attachable screw-cap culture wells that function as a replacement for cryovial storage. The detachable wells facilitate storage and after thawing can easily be reattached to a specially designed holding plate. Currently, there are no commercially available cell culture plates using this design concept. The proposed design is envisioned to facilitate the cryopreservation of intact adherent hES cell colonies that could assist the development of automated systems for handling bulk quantities of cells.     

Challenges to human embryonic stem cell patents

The patenting of human embryonic stem (hES) cells has produced one of the most unusual and fraught situations in the history of science, ethics, and law. This Commentary examines legal and moral challenges to three foundational patents held by the Wisconsin Alumni Research Foundation (WARF). We conclude that, in the United States, technical challenges may, paradoxically, produce a stronger patent position for WARF. In the European Union, moral challenges mean confusion for member states. We demonstrate that hES cell intellectual property will be guided and bound by a welter of moral, technical, and legal inputs, with discrete national and jurisdictional dimensions.     

microRNAs regulate human embryonic stem cell division

RNA interference-mediated suppression of DICER and DROSHA in human embryonic stem cells (hESCs) attenuates cell proliferation, supporting a role for an intact microRNA (miRNA) pathway in the control of hESC cell division. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the cycB/CDK complex and CDKN1A, which encodes p21, a cycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.

Supplemental information can be found here.     

Neurotrophins mediate human embryonic stem cell survival

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.     

Derivation of human embryonic stem cell lines

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.     

Population estimation of human embryonic stem cell cultures

Traditionally, the population of human embryonic stem cell (hESC) culture is estimated through haemacytometer counts, which include harvesting the cells and manually analyzing a fraction of an entire population. Obviously, through this highly invasive method, it is not possible to preserve any spatial information on the cell population. The goal of this study is to identify a fast and consistent method for in situ automated hESC population estimation to quantitatively estimate the cell growth. Therefore, cell cultures were fixed, stained, and their nuclei imaged through high‐resolution microscopy, and the images were processed with different image analysis techniques. The proposed method first identifies signal and background by computing an image specific threshold for image segmentation. By applying a morphological operator (watershed), we split most physically overlapping nuclei, leading to a pixel area distribution of isolated signal areas on the image. On the basis of this distribution, we derive a nucleus area model, describing the distribution of the area of cell debris, single nuclei, and small groups of connected nuclei. Through the model, we can give a quantitative estimation of the population. The focus of this study is on low‐density human embryonic stem cell populations; hence cultures were measured at days 2–3 after seeding. Compared with manual cell counts, the automatic method achieved higher accuracy with <6% error. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Albumin-associated lipids regulate human embryonic stem cell self-renewal

Background

Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal.

Methodology/Principal Findings

In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect.

Conclusions/Significance

Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems.     

Culture development for human embryonic stem cell propagation: molecular aspects and challenges

Basic fibroblast growth factor and members of the transforming growth factor-beta superfamily are important regulators of human embryonic stem cell (hESC) self-renewal. Extensive cross-talk between the intracellular signaling pathways activated by these factors contributes to maintenance of the undifferentiated hESC state. Understanding the molecular regulation of hESC self-renewal will facilitate the design of improved systems for hESC propagation and provide a foundation for strategies to direct the differentiation of hESCs to clinically relevant cell types.     

Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.     

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.