Marzena Zychowicz

Micro-stamped surfaces for the patterned growth of neural stem cells

We present a method for patterning neural stem cells based on pre-patterning polypeptides on a cell-repellent surface (poly(ethylene) oxide-like, PEO-like, plasma-deposited films). The method ensures cell attachment and stability for several weeks, as well as it allows cell migration and differentiation. Various patterns of approximately 1 nm thick cell adhesive poly-L-lysine (PLL) have been created on a cell-repellent PEO-like matrix by microcontact printing using different array configurations and printing conditions. The cell-repellent property of PEO-like film determined the confinement of the cells on the printed patterns. Optimization of the printing method showed that the most homogeneous patterns over large areas were obtained using PLL diluted in carbonate buffer (100mM) at pH 8.4. Neural stem cells cultured on the PLL patterns in low serum and in differentiating medium over 20 days exhibited a good confinement to the polypeptide domains. The number of cells attached increased linearly with the micro-stamped PLL area. The cells were able to extend random axon-like projections to the outside of the patterns and presented high amount of ramifications when cultured in differentiating medium. Migration and axon-like outgrowth have been successfully guided by means of an interconnected squares configuration. The surfaces are suitable for controlling the patterning of stem cells and provide a platform for the assessment of the way how different cell arrangements and culture conditions influence cell interactions and cell developmental processes.

Fabrication and characterization of protein arrays for stem cell patterning

Microarrays of fibronectin and other extracellular matrix (ECM) proteins were fabricated on plasma-deposited poly(ethyleneoxide) (PEO-like) film coated glass slides to study adhesion of stem cells. The arrays were generated by using a non-contact printing technology. The stability and the quality of the spots of fibronectin, used as protein model, were assessed by time of flight secondary ion mass spectrometry (ToF-SIMS), ellipsometry and atomic force microscopy (AFM). It was found that saturation with a mass density of 112 ± 4 ng/cm2 is reached when protein solutions at concentrations higher than 84 µg/ml are spotted. Fibronectin on the surface form a uniform sub-monolayer with a surface coverage that depends on the spotting solution concentration, as qualitatively demonstrated by AFM measurements. The active conformation of the spotted fibronectin was verified by performing an immunoassay with antibodies specific for the fibronectin RGD sequence by surface plasmon resonance (SPR) imaging. An immunorecognition efficiency of up to 22% was found for a spot with 3% coverage as estimated by ellipsometry. Human umbilical cord blood neural stem cells (HUCB-NSCs) were cultured on different ECM proteins (fibronectin, laminin, collagen I, collagen III and collagen V) arrays and showed protein concentration dependent adhesion on the micro-spots. The cell nuclei were stained for cell counting and preliminary specific cell staining was performed to evaluate the differentiation stage of HUCB-NSCs on such spots. The array platform developed in this study provides a promising approach to investigate in high throughput manner how surfaces patterned with extracellular matrix (ECM) proteins influence stem cell adhesion and development.

Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces.

Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1μM) concentration of MeHgCl. β-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to μm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.

Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer’s disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Functional properties of different collagen scaffolds to create a biomimetic niche for neurally committed human induced pluripotent stem cells (iPSC)

The biomimetic, standardized conditions for in vitro cultures of human neural progenitors derived from induced pluripotent stem cells (hiPSC-NPs) should meet the requirements to serve as the template and protective environment for therapeutically competent cell population. In this study, two different collagen scaffolds: bi-component consisting of collagen and chondroitin sulphate (Col-CS), and collagen modified by crosslinking agent 2,3-dialdehyde cellulose (Col-DAC) have been used for the first time to encapsulate hiPSC-NPs and compared for the ability to create permissive microenvironment enabling cell survival, growth and differentiation. In our previous report, physicochemical comparison of the scaffolds revealed different elasticity, and diverse size and distribution of the pores within the 3D structure. Binary systems of Col-CS and Col-DAC tested in the current study have the correct balance of properties to serve as a biomimetic niche: they accommodate hiPSC-NPs sustaining their ability to proliferate and differentiate into neural lineages. However, a dense, network structure and rounded in shape pores of the Col-DAC microenvironment resulted in differential cell distributions within the scaffolds, with a tendency for augmented formation of highly proliferating cell aggregates as compared to Col-CS scaffolds. In contrast, Col-CS, which exhibited formation of the network of ellipsoidal and inner interconnected parallel pore channels, promoted enhanced cell viability and neuronal differentiation.