Developing a novel 3D alginate platform for investigating the patterned differentiation of mouse embryonic stem cells

Standard 2D cell culture does not recreate the complex features of the in vivo environment, such as soluble factor gradients, cell migration into multiple planes, or cell-cell and cell-matrix interactions.1–3 3D cell culture addresses these limitations by using 3D biomaterial scaffolds, such as alginate hydrogels, to recreate the in vivo cell microenvironment in vitro. 4, 5 3D platforms can be used to create gradients of soluble factors, vary the biomaterial substrate stiffness, permit cell-matrix interactions or promote cell migration.6, 7 Currently available 3D platforms are prone to the burst release of soluble factors from the biomaterials, making it difficult to tightly control the soluble factor concentration.7 This limits the use of 3D platforms for investigating processes such as patterned neuronal differentiation, or cell fate specification in response to small changes in soluble factor concentration. This project proposes a novel 3D alginate platform for patterned differentiation. The first part of this thesis describes experiments to optimise alginate hydrogels for the encapsulation, aggregation and differentiation of embryonic stem cells (ESCs), and demonstrates that encapsulated ESCs form embryoid bodies containing cells from the three germ layers. Exogenous retinoic acid (RA) is used for in vitro neuronal differentiation protocols, but exogenous RA is not stable in cell culture and is easily degraded by light. The second part of the thesis outlines experiments to validate a cell-derived source of RA, which produces a stable concentration of RA in vitro and addresses the limitations of exogenous RA. The final section describes the novel 3D platform that combines the results from the previous sections using an adapted gradient maker protocol, to create 3D co-culture alginate tubes. The tubes support patterned differentiation of ESCs in response to the concentration gradient of cell-derived RA incorporated into the platform. The novel 3D platform produced in this project contributes a novel tool to the field of 3D cell culture. The 3D platform is a tool for investigating ESC differentiation in response to a 3D concentration gradient of a cell-derived source of retinoic acid. For experiments that require a gradient of RA, the ability to maintain a stable source of RA over several days is an advantage of using this 3D platform over the currently available alternatives. In addition, alginate hydrogels are highly tunable. Thus, the ability to tune the scaffold properties, change the cell types encapsulated, or introduce gradients of alternative soluble factors makes this a versatile tool for 3D culture

Localisation of oestrogen receptors in stem cells and in stem cell derived neurons of the mouse

Oestrogen receptors (ER) transduce the effects of the endogenous ligand, 17b-estradiol in cells to regulate a number of important processes such as reproduction, neuroprotection, learning and memory and anxiety. The ERa or ERb are classical intracellular nuclear hormone receptors while some of their variants or novel proteins such as the GPCR, GPER1/GPR30 are reported to localise in intracellular as well as plasma membrane locations. Though the brain is an important target for oestrogen with oestrogen receptors expressed differentially in various nuclei, subcellular organisation and crossttalk between these receptors is underexplored. Using an adapted protocol that is rapid, we first generated neurons from mouse embryonic stem cells. Our immunocytochemistry approach shows that the full length ERa (ERa-66) and for the first time, that an ERa variant, ERa-36, as well as GPER1 is present in embryonic stem cells. In addition, these receptors typically decrease their nuclear localisation as neuronal maturation proceeds. Finally, though these ERs are present in many subcellular compartments such as the nucleus and plasma membrane, we show that they are specifically not colocalised with each other, suggesting that they initiate distinct signalling pathways

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Neuralization of mouse embryonic stem cells in alginate hydrogels under retinoic acid and SAG treatment

This paper examines the differentiation of a mouse embryonic stem cell line (CGR8) into neurons, under retinoic acid (RA) and smoothened agonist (SAG) treatment. When stem cells underwent through an embryoid body (EB) formation stage, dissociation and seeding on glass coverslips, immunofluorescent labelling for neuronal markers (Nestin, b-Tubulin III, MAP2) revealed the presence of both immature neural progenitors and mature neurons. Undifferentiated CGR8 were also encapsulated in tubular, alginate-gelatin hydrogels and incubated in differentiation media containing retinoic acid (RA) and smoothened agonist (SAG). Cryo-sections of the hydrogel tubes were positive for Nestin, Pax6 and b-Tubulin III, verifying the presence of neurons and neural progenitors. Provided neural induction can be more precisely directed in the tubular hydrogels, these scaffolds will become a powerful model of neural tube development in embryos and will highlight potential strategies for spinal cord regeneration