We have been developing a biochemically functionalized electrospun fiber platform serving as an artificial stem cell niche that presents topographical and biochemical cues to impact the local regulation of stem cells.
Stem cell niche provides a complex array of biochemical and physical signals in a temporally and spatially defined fashion, engaging and instructing stem cells to proliferate, migrate and differentiate. Understanding its molecular and structural components of stem cell niche and their respective functions is critical for the development of effective methodologies for stem cell manipulation. Key niche factors include ECM-mediated adhesion and local regulation of “instructional” biochemical cues by manipulating the concentration and presentation pattern of signaling molecules.
We have been developing a biochemically functionalized electrospun fiber platform serving as an artificial stem cell niche that presents topographical and biochemical cues to impact the local regulation of stem cells. This series of studies will not only provide key insights into how niche factors regulate these important cell activities, but also apply engineering principles in designing functional scaffolds for stem cell manipulation for therapeutic applications.
1. Preparation of nanofiber platform with defined topographical features and surface-tethered bioactive cues
We have developed methods to electrospin a wide range of biocompatible polymeric fibers (biodegradable and non-biodegradable) with controlled diameters (~150 nm to 2 µm) and alignments (aligned and randomly oriented) (Fig. 1). In order to present the relevant biochemical cues from fiber surfaces, we have developed a surface grafting method to introduce various functional groups in a concentration range of 0.1 to 200 nmol/cm2 without compromising fiber integrity.
We are in the process of developing a versatile and robust scheme for surface conjugation of biochemical cues pertinent to stem cell adhesion, proliferation and differentiation, for example, key adhesion molecules (laminin and/or fibronectin) and growth factor (fibroblast growth factor-2, FGF-2). We are interested to investigate how surface presentation of signaling molecules influences the signaling activation and stem cell proliferation and differentiation.
2. Nanofiber-enhanced expansion of hematopoietic stem/progenitor cells
We have demonstrated that nanofiber topography and surface biochemical groups synergistically improve the self-renewal and proliferation of human umbilical cord blood-derived HSCs. A set of aminated nanofibers support the highest expansion of cryopreserved cord blood stem/progenitor cells, compared with similarly modified 2-D surface and other substrates. More interestingly, only on this set of nanofibers have we observed selective adhesion of CD34+ cells in unique pattern—abundant adherent colonies (Fig. 2), accounting for 27 to 43% of total expanded cells, whereas only very few cells adhered randomly on similarly modified 2-D surface. This result suggests a selective enrichment effect by these nanofibers during expansion. As HSCs are traditionally regarded as suspension cells, this evidence associates cell-substrate adhesion with HSC phenotype maintenance. We are currently investigating the efficiency of CD133+ cells on this nanofiber matrix, and collaborating with Prof. Vince Pompili and Prof. Das Hiranmoy at the Ohio State University to evaluating the therapeutic potential of these expanded cells.
3. Effect of nanofiber-guided cell alignment on the differentiation of neural stem cells
Figure 3. (a, b). Rat ANSCs differentiated on random and aligned LN-coated PCL nanofibers. The average diameter of the fibers is 250 nm. NSCs were cultured in the presence of 0.5% fetal bovine serum and 0.5 µM retinoic acid for 6 days. Differentiated cells formed extended long processes along fiber axis. Fibers provided guidance cue for process extension and outgrowth. Arrow in (b) indicates the axis of aligned fibers.
This study aims to integrate topographical and surface-tethered biochemical cues to potentiate the adhesion, contact guidance and growth factor signaling of neural stem cells (NSCs), thereby impacting the regulation of NSC proliferation and differentiation. Cell migration, neurite outgrowth and functional connection of the developing neurons are thought to be guided by complex, spatially and temporally ordered ECM-mediated contact guidance. Since distinct morphological characteristics often accompany stem cell differentiation, the question of whether one can conversely utilize topography-induced alterations in cell morphology to impact stem cell fate choices becomes scientifically interesting and relevant. We have demonstrated that nanofibers coated with laminin (LN), an important ECM component found in ventricular zone basal lamina, were much more efficient in mediating NSC adhesion and proliferation than collagen- and fibronectin (FN)-coated fibers. Most interestingly, nanofiber (~300 nm) alignment proves to be a strong cue in directing NSC differentiation under differentiation condition (0.5% FBS and 0.5 μM retinoic acid). NSCs adhere to aligned LN-coated nanofibers, extend processes along the fiber axis and drive more effective neuronal differentiation than LN-coated random fibers and 2-D membrane (Fig. 6). In contrast, random LN-coated fibers preferentially direct NSCs to differentiate towards oligodendrocyte lineage.
We will continue to systematically analyze effects of nanofiber-presented topographical cues and surface-tethered biochemical cues, independently or in combination, on the adhesion, proliferation and differentiation of human embryonic stem cells (hESCs) and hESC-derived neural progenitors and neural crest stem cells. These studies will provide an experimental framework for the development of a mechanistic model for the regulation of stem cell fate specification by niche cues.
4. Nanofiber nerve guide on nerve regeneration
Axonal regeneration is subject to various bioactive cues provided by ECM microenvironment, many of which secreted by glia cells in response to damage. Both guidance cues and neurotrophic cues are important for effective re-growth of the axons. Although numerous neurotrophic factors and cell adhesion cues that promote neural regeneration have been identified, the temporal and spatial delivery of these cues remains poorly defined. Taking advantage of nanofiber-provided adhesion guidance, we engineered a new nanofiber nerve guide with enhanced local delivery capability for neurotrophic factors and axonal guidance cues. In collaboration with Prof. Ahmet Hoke at Departments of Neurology and Neuroscience, we are investigating how these locally delivered cues influence axonal regeneration using a rat peripheral nerve repair model.