Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Intercellular network structure and regulatory motifs in the human hematopoietic system

The hematopoietic system is a distributed tissue that consists of functionally distinct cell types continuously produced through hematopoietic stem cell (HSC) differentiation. Combining genomic and phenotypic data with high‐content experiments, we have built a directional cell–cell communication network between 12 cell types isolated from human umbilical cord blood. Network structure analysis revealed that ligand production is cell type dependent, whereas ligand binding is promiscuous. Consequently, additional control strategies such as cell frequency modulation and compartmentalization were needed to achieve specificity in HSC fate regulation. Incorporating the in vitro effects (quiescence, self‐renewal, proliferation, or differentiation) of 27 HSC binding ligands into the topology of the cell–cell communication network allowed coding of cell type‐dependent feedback regulation of HSC fate. Pathway enrichment analysis identified intracellular regulatory motifs enriched in these cell type‐ and ligand‐coupled responses. This study uncovers cellular mechanisms of hematopoietic cell feedback in HSC fate regulation, provides insight into the design principles of the human hematopoietic system, and serves as a foundation for the analysis of intercellular regulation in multicellular systems.     

Theoretical and experimental analysis links isoform‐ specific ERK signalling to cell fate decisions

Cell fate decisions are regulated by the coordinated activation of signalling pathways such as the extracellular signal‐regulated kinase (ERK) cascade, but contributions of individual kinase isoforms are mostly unknown. By combining quantitative data from erythropoietin‐induced pathway activation in primary erythroid progenitor (colony‐forming unit erythroid stage, CFU‐E) cells with mathematical modelling, we predicted and experimentally confirmed a distributive ERK phosphorylation mechanism in CFU‐E cells. Model analysis showed bow‐tie‐shaped signal processing and inherently transient signalling for cytokine‐induced ERK signalling. Sensitivity analysis predicted that, through a feedback‐mediated process, increasing one ERK isoform reduces activation of the other isoform, which was verified by protein over‐expression. We calculated ERK activation for biochemically not addressable but physiologically relevant ligand concentrations showing that double‐phosphorylated ERK1 attenuates proliferation beyond a certain activation level, whereas activated ERK2 enhances proliferation with saturation kinetics. Thus, we provide a quantitative link between earlier unobservable signalling dynamics and cell fate decisions.     

Understanding cellular networks to improve hematopoietic stem cell expansion cultures

Efforts to develop culture technologies capable of eliciting robust human blood stem cell growth have met with limited success. Considering that adult stem cell cultures are complex systems, comprising multiple cell types with dynamically changing intracellular signalling environments and cellular compositions, this is not surprising. Typically treated as single-input single-output systems, adult stem cell cultures are better described as complex, non-linear, multiple-input multiple-output systems wherein the proliferation of subpopulations of cells leads to the formation of intercellular endogenously secreted protein interaction networks. Genomic and proteomic tools need to be applied to generate high-throughput (and ideally high-content) biological measurements of stem cell culture evolution. Datasets describing cellular interaction networks need to be integrated into predictive models of in vitro stem cell development. Ultimately, such models will serve as a starting point for the rational design of blood stem cell expansion bioprocesses utilizing dynamic system perturbations to achieve the preferential expansion of target cell populations.     

Notch signalling and haematopoietic stem cell formation during embryogenesis

The Notch signalling pathway is repeatedly employed during embryonic development and adult homeostasis of a variety of tissues. In particular, its frequent involvement in the regulation of stem and progenitor cell maintenance and proliferation, as well as its role in binary fate decisions in cells that are destined to differentiate, is remarkable. Here, we review its role in the development of haematopoietic stem cells during vertebrate embryogenesis and put it into the context of Notch's functions in arterial specification, angiogenic vessel sprouting and vessel maintenance. We further discuss interactions with other signalling cascades, and pinpoint open questions and some of the challenges that lie ahead. J. Cell. Physiol. 222:11–16, 2010. © 2009 Wiley‐Liss, Inc.     

Adventures in time and space: Nonlinearity and complexity of cytokine effects on stem cell fate decisions

Cytokines are central factors in the control of stem cell fate decisions and, as such, they are invaluable to those interested in the manipulation of stem and progenitor cells for clinical or research purposes. In their in vivo niches or in optimized cultures, stem cells are exposed to multiple cytokines, matrix proteins and other cell types that provide individual and combinatorial signals that influence their self‐renewal, proliferation and differentiation. Although the individual effects of cytokines are well‐characterized in terms of increases or decreases in stem cell expansion or in the production of specific cell lineages, their interactions are often overlooked. Factorial design experiments in association with multiple linear regression is a powerful multivariate approach to derive response‐surface models and to obtain a quantitative understanding of cytokine dose and interactions effects. On the other hand, cytokine interactions detected in stem cell processes can be difficult to interpret due to the fact that the cell populations examined are often heterogeneous, that cytokines can exhibit pleiotropy and redundancy and that they can also be endogenously produced. This perspective piece presents a list of possible biological mechanisms that can give rise to positive and negative two‐way factor interactions in the context of in vivo and in vitro stem cell‐based processes. These interpretations are based on insights provided by recent studies examining intra‐ and extra‐cellular signaling pathways in adult and embryonic stem cells. Cytokine interactions have been classified according to four main types of molecular and cellular mechanisms: (i) interactions due to co‐signaling; (ii) interactions due to sequential actions; (iii) interactions due to high‐dose saturation and inhibition; and (iv) interactions due to intercellular signaling networks. For each mechanism, possible patterns of regression coefficients corresponding to the cytokine main effects, quadratic effects and two‐way interactions effects are provided. Finally, directions for future mechanistic studies are presented. Biotechnol. Bioeng. 2010;106: 173–182. © 2010 Wiley Periodicals, Inc.     

Real‐time monitoring and control of soluble signaling factors enables enhanced progenitor cell outputs from human cord blood stem cell cultures

Monitoring and control of primary cell cultures is challenging as they are heterogenous and dynamically complex systems. Feedback signaling proteins produced from off‐target cell populations can accumulate, inhibiting the production of the desired cell populations. Although culture strategies have been developed to reduce feedback inhibition, they are typically optimized for a narrow range of process parameters and do not allow for a dynamically regulated response. Here we describe the development of a microbead‐based process control system for the monitoring and control of endogenously produced signaling factors. This system uses quantum dot barcoded microbeads to assay endogenously produced signaling proteins in the culture media, allowing for the dynamic manipulation of protein concentrations. This monitoring system was incorporated into a fed‐batch bioreactor to regulate the accumulation of TGF‐β1 in an umbilical cord blood cell expansion system. By maintaining the concentration of TGF‐β1 below an upper threshold throughout the culture, we demonstrate enhanced ex vivo expansion of hematopoietic progenitor cells at higher input cell densities and over longer culture periods. This study demonstrates the potential of a fully automated and integrated real‐time control strategy in stem cell culture systems, and provides a powerful strategy to achieve highly regulated and intensified in vitro cell manufacturing systems. Biotechnol. Bioeng. 2014;111: 1258–1264. © 2013 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Modelling the spatio-temporal cell dynamics reveals novel insights on cell differentiation and proliferation in the small intestinal crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days.     

BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions

The finding that morphogens, signalling molecules that specify cell identity, also act as axon guidance molecules has raised the possibility that the mechanisms that establish neural cell fate are also used to assemble neuronal circuits. It remains unresolved, however, how cells differentially transduce the cell fate specification and guidance activities of morphogens. To address this question, we have examined the mechanism by which the Bone morphogenetic proteins (BMPs) guide commissural axons in the developing spinal cord. In contrast to studies that have suggested that morphogens direct axon guidance decisions using non-canonical signal transduction factors, our results indicate that canonical components of the BMP signalling pathway, the type I BMP receptors (BMPRs), are both necessary and sufficient to specify the fate of commissural neurons and guide their axonal projections. However, whereas the induction of cell fate is a shared property of both type I BMPRs, axon guidance is chiefly mediated by only one of the type I BMPRs, BMPRIB. Taken together, these results indicate that the diverse activities of BMP morphogens can be accounted for by the differential use of distinct components of the canonical BMPR complex.     

A Dynamic Model for Stem Cell Homeostasis and Patterning in Arabidopsis Meristems

Plants maintain stem cells in their meristems as a source for new undifferentiated cells throughout their life. Meristems are small groups of cells that provide the microenvironment that allows stem cells to prosper. Homeostasis of a stem cell domain within a growing meristem is achieved by signalling between stem cells and surrounding cells. We have here simulated the origin and maintenance of a defined stem cell domain at the tip of Arabidopsis shoot meristems, based on the assumption that meristems are self-organizing systems. The model comprises two coupled feedback regulated genetic systems that control stem cell behaviour. Using a minimal set of spatial parameters, the mathematical model allows to predict the generation, shape and size of the stem cell domain, and the underlying organizing centre. We use the model to explore the parameter space that allows stem cell maintenance, and to simulate the consequences of mutations, gene misexpression and cell ablations.     

Microprinted feeder cells guide embryonic stem cell fate

We introduce a non‐contact approach to microprint multiple types of feeder cells in a microarray format using immiscible aqueous solutions of two biopolymers. Droplets of cell suspension in the denser aqueous phase are printed on a substrate residing within a bath of the immersion aqueous phase. Due to their affinity to the denser phase, cells remain localized within the drops and adhere to regions of the substrate underneath the drops. We show the utility of this technology for creating duplex heterocellular stem cell niches by printing two different support cell types on a gel surface and overlaying them with mouse embryonic stem cells (mESCs). As desired, the type of printed support cell spatially direct the fate of overlaid mESCs. Interestingly, we found that interspaced mESCs colonies on differentiation‐inducing feeder cells show enhanced neuronal differentiation and give rise to dense networks of neurons. This cell printing technology provides unprecedented capabilities to efficiently identify the role of various feeder cells in guiding the fate of stem cells. Biotechnol. Bioeng. 2011;108: 2509–2516. © 2011 Wiley Periodicals, Inc.