Genetic studies have placed the Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/β-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.

FGF Receptor-1 (FGFR1), a membrane-targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal-like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA-induced differentiation while full-length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co-associate with 5'-Fluorouridine-labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf-2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter-gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation.

The regenerating (Reg) protein family comprises C-type lectin-like proteins discovered independently during pancreatitis and pancreatic islet regeneration. However, an increasing number of studies provide evidence of participation of Reg proteins in the proliferation and differentiation of diverse cell types. Moreover, Reg family members are associated with various pathologies, including diabetes and forms of gastrointestinal cancer. These findings have led to the emergence of key roles for Reg proteins as anti-inflammatory, antiapoptotic and mitogenic agents in multiple physiologic and disease contexts. Yet, there are significant gaps in our knowledge regarding the regulation of expression of different Reg genes. In addition, the pathways relaying Reg-triggered signals, their targets and potential cross-talk with other cascades are still largely unknown. In this review, the expression patterns of different Reg members in the pancreas and extrapancreatic tissues are described. Moreover, factors known to modulate Reg levels in different cell types are discussed. Several signaling pathways, which have been implicated in conferring the effects of Reg ligands to date, are also delineated. Further efforts are necessary for elucidating the biological processes underlying the action of Reg proteins and their involvement in various maladies. Better understanding of the function of Reg genes and proteins will be beneficial in the design and development of therapies utilizing or targeting this protein group.

Heart diseases are major causes of morbidity and mortality linked to extensive loss of cardiac cells. Embryonic stem cells (ESCs) give rise to cardiomyocyte-like cells, which may be used in heart cell replacement therapies. Most cardiogenic differentiation protocols involve the culture of ESCs as embryoid bodies (EBs). Stirred-suspension bioreactor cultures of ESC aggregates may be employed for scaling up the production of cardiomyocyte progeny but the wide range of EB sizes and the unknown effects of the hydrodynamic environment on differentiating EBs are some of the major challenges in tightly controlling the differentiation outcome. Here, we explored the cardiogenic potential of mouse ESCs (mESCs) and human ESCs (hESCs) encapsulated in poly-L-lysine (pLL)-coated alginate capsules. Liquefaction of the capsule core led to the formation of single ESC aggregates within each bead and their average size depended on the concentration of seeded ESCs. Encapsulated mESCs were directed along cardiomyogenic lineages in dishes under serum-free conditions with the addition of bone morphogenetic protein 4 (BMP4). Human ESCs in pLL-layered liquid core (LC) alginate beads were also differentiated towards heart cells in serum-containing media. Besides the robust cell proliferation, higher fractions of cells expressing cardiac markers were detected in ESCs encapsulated in LC than in solid beads. Furthermore, we demonstrated for the first time that ESCs encapsulated in pLL-layered LC alginate beads can be coaxed towards heart cells in stirred-suspension bioreactors. Encapsulated ESCs yielded higher fractions of Nkx2.5- and GATA4-positive cells in the biore-actor compared to dish cultures. Differentiated cells formed beating foci that responded to chronotropic agents in an organotypic manner. Our findings warrant further development and implementation of microencapsulation technologies in conjunction with bioreactor cultivation to enable the production of stem cell-derived cardiac cells appropriate for clinical therapies and applications.

Embryonic stem cells (ESCs) with their unlimited capacity for self-renewal and ability to differentiate along multiple cell lineages are a superb starting material for biotechnology applications and cellular therapies. However, realization of the potential of ESCs requires the development of scalable systems for their production in large quantities and in a regulated manner. Here, we describe a methodology for the expansion of mouse ESCs (mESCs) as pluripotent aggregates in a stirred suspension bioreactor and in medium without serum. Initially, the culture of feeder cell-independent mESCs in dishes was adapted to serum-free conditions. Also, we explored whether spinner flasks equipped with a triangle-shaped impeller and baffles support the culture of mESC aggregates. Serum-free culture in these vessels resulted in an almost 20-fold increase in the live mESC concentration over 4 days without significant loss of cell viability. Even after consecutive passages, mESCs retained high expression of pluripotency markers Oct3/4, Rex1 and SSEA-1. More importantly, when differentiation was induced these cells adopted fates of all three germ layers namely neuroectoderm, cardiac mesoderm and definitive endoderm. These findings demonstrate that stem cells can be propagated under serum-free conditions in a scalable stirred-suspension culture without loss of their pluripotency.