Sarah C. Heilshorn

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1+ ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1+ ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5+ ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

LKB1/STRAD promotes axon initiation during neuronal polarization.

Axon/dendrite differentiation is a critical step in neuronal development. In cultured hippocampal neurons, the accumulation of LKB1 and STRAD, two interacting proteins critical for establishing epithelial polarity, in an undifferentiated neurite correlates with its subsequent axon differentiation. Downregulation of either LKB1 or STRAD by siRNAs prevented axon differentiation, and overexpression of these proteins led to multiple axon formation. Furthermore, interaction of STRAD with LKB1 promoted LKB1 phosphorylation at a PKA site S431 and elevated the LKB1 level, and overexpressing LKB1 with a serine-to-alanine mutation at S431 (LKB1(S431A)) prevented axon differentiation. In developing cortical neurons in vivo, downregulation of LKB1 or overexpression of LKB1(S431A) also abolished axon formation. Finally, local exposure of the undifferentiated neurite to brain-derived neurotrophic factor or dibutyryl-cAMP promoted axon differentiation in a manner that depended on PKA-dependent LKB1 phosphorylation. Thus local LKB1/STRAD accumulation and PKA-dependent LKB1 phosphorylation represents an early signal for axon initiation.

Biomaterial Design Strategies for the Treatment of Spinal Cord Injuries

The highly debilitating nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Many experts agree that the greatest hope for treatment of spinal cord injuries will involve a combinatorial approach that integrates biomaterial scaffolds, cell transplantation, and molecule delivery. This manuscript presents a comprehensive review of biomaterial-scaffold design strategies currently being applied to the development of nerve guidance channels and hydrogels that more effectively stimulate spinal cord tissue regeneration. To enhance the regenerative capacity of these two scaffold types, researchers are focusing on optimizing the mechanical properties, cell-adhesivity, biodegradability, electrical activity, and topography of synthetic and natural materials, and are developing mechanisms to use these scaffolds to deliver cells and biomolecules. Developing scaffolds that address several of these key design parameters will lead to more successful therapies for the regeneration of spinal cord tissue.

Two-component protein-engineered physical hydrogels for cell encapsulation

Current protocols to encapsulate cells within physical hydrogels require substantial changes in environmental conditions (pH, temperature, or ionic strength) to initiate gelation. These conditions can be detrimental to cells and are often difficult to reproduce, therefore complicating their use in clinical settings. We report the development of a two-component, molecular-recognition gelation strategy that enables cell encapsulation without environmental triggers. Instead, the two components, which contain multiple repeats of WW and proline-rich peptide domains, undergo a sol–gel phase transition upon simple mixing and hetero-assembly of the peptide domains. We term these materials mixing-induced, two-component hydrogels. Our results demonstrate use of the WW and proline-rich domains in protein-engineered materials and expand the library of peptides successfully designed into engineered proteins. Because both of these association domains are normally found intracellularly, their molecular recognition is not disrupted by the presence of additional biomolecules in the extracellular milieu, thereby enabling reproducible encapsulation of multiple cell types, including PC-12 neuronal-like cells, human umbilical vein endothelial cells, and murine adult neural stem cells. Precise variations in the molecular-level design of the two components including (i) the frequency of repeated association domains per chain and (ii) the association energy between domains enable tailoring of the hydrogel viscoelasticity to achieve plateau shear moduli ranging from ≈9 to 50 Pa. Because of the transient physical crosslinks that form between association domains, these hydrogels are shear-thinning, injectable, and self-healing. Neural stem cells encapsulated in the hydrogels form stable three-dimensional cultures that continue to self-renew, differentiate, and sprout extended neurites.

Local and Long-Range Reciprocal Regulation of cAMP and cGMP in Axon/Dendrite Formation

Promoting Axon Formation How do neurons initiate one axon and lots of dendrites? Using an in vitro assay involving stripes of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), Shelly et al. (p. 547) show that an increase in cAMP initiates axon formation while an increase in cGMP initiates dendrites. Moreover, cAMP and cGMP reciprocally inhibit each other via the activation of specific phosphodiesterases, as well as protein kinase A and protein kinase G. Finally, long-range self-inhibition of cAMP can explain why only one axon, yet multiple dendrites, is initiated in single hippocampal neurons in culture. Locally increasing cAMP in one neurite causes long-range cAMP reduction in the rest of the neurites, accompanied by corresponding reciprocal changes in cGMP. Cyclic adenosine monophosphate and cyclic guanosine monophosphate regulate axon formation and exert opposite actions on dendrite formation. Cytosolic cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) often mediate antagonistic cellular actions of extracellular factors, from the regulation of ion channels to cell volume control and axon guidance. We found that localized cAMP and cGMP activities in undifferentiated neurites of cultured hippocampal neurons promote and suppress axon formation, respectively, and exert opposite effects on dendrite formation. Fluorescence resonance energy transfer imaging showed that alterations of the amount of cAMP resulted in opposite changes in the amount of cGMP, and vice versa, through the activation of specific phosphodiesterases and protein kinases. Local elevation of cAMP in one neurite resulted in cAMP reduction in all other neurites of the same neuron. Thus, local and long-range reciprocal regulation of cAMP and cGMP together ensures coordinated development of one axon and multiple dendrites.

Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124

Plumbing in the Brain Superficial similarities of vasculature in different parts of the body may mask organ-specific developmental nuances. The vasculature of the brain uniquely has to insulate the organ from insults that the rest of the body must tolerate. Kuhnert et al. (p. 985) analyzed the developmental uniqueness of the brains vasculature through study of a G protein–coupled receptor, GPR124, initially identified by its actions in the vasculature of colon cancer. GPR124 is also involved in normal development of the brains vasculature. Mice expressing low levels of GPR124 did not develop adequate vasculature in the brain and died from hemorrhages. Mice with too much GPR124 developed a tangled, thin-walled, excessive vasculature in the brain. Although the overexpressing mice survived, they were prone to neurological symptoms such as ataxia. GPR124 seems to control the normal development of the endothelial cells, particularly in the forebrain and ventral neural tube. A factor is identified that determines the amount of vasculature in the brain, and, in doing so, affects brain function. The orphan G protein–coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor–independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.

Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.

This study examines the spreading and adhesion of human umbilical vein endothelial cells (HUVEC) on artificial extracellular matrix (aECM) proteins containing sequences derived from elastin and fibronectin. Three aECM variants were studied: aECM 1 contains lysine residues periodically spaced within the protein sequence and three repeats of the CS5 domain of fibronectin, aECM 2 contains periodically spaced lysines and three repeats of a scrambled CS5 sequence, and aECM 3 contains lysines at the protein termini and five CS5 repeats. Comparative cell binding and peptide inhibition assays confirm that the tetrapeptide sequence REDV is responsible for HUVEC adhesion to aECM proteins that contain the CS5 domain. Furthermore, more than 60% of adherent HUVEC were retained on aECM 1 after exposure to physiologically relevant shear stresses (</=100dynes/cm(2)). Finally, the levels of thrombogenic markers (tissue plasminogen activator and plasminogen activator inhibitor-1) secreted by HUVEC monolayers on aECM 1 were found to be similar to those secreted by HUVEC monolayers cultured on fibronectin. These characteristics, along with the physical strength and elasticity of crosslinked films prepared from these materials, make aECM proteins promising candidates for application in small-diameter vascular grafts.

Adaptable hydrogel networks with reversible linkages for tissue engineering.

Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-formation of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, an overview of adaptable-hydrogel design considerations and linkage selections is presented, with a focus on various cell-compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering.

High Speed Water Sterilization Using One-Dimensional Nanostructures

The removal of bacteria and other organisms from water is an extremely important process, not only for drinking and sanitation but also industrially as biofouling is a commonplace and serious problem. We here present a textile based multiscale device for the high speed electrical sterilization of water using silver nanowires, carbon nanotubes, and cotton. This approach, which combines several materials spanning three very different length scales with simple dying based fabrication, makes a gravity fed device operating at 100000 L/(h m(2)) which can inactivate >98% of bacteria with only several seconds of total incubation time. This excellent performance is enabled by the use of an electrical mechanism rather than size exclusion, while the very high surface area of the device coupled with large electric field concentrations near the silver nanowire tips allows for effective bacterial inactivation.

Multifunctional coatings to simultaneously promote osseointegration and prevent infection of orthopaedic implants.

The two leading causes of failure for joint arthroplasty prostheses are aseptic loosening and periprosthetic joint infection. With the number of primary and revision joint replacement surgeries on the rise, strategies to mitigate these failure modes have become increasingly important. Much of the recent work in this field has focused on the design of coatings either to prevent infection while ignoring bone mineralization or vice versa, to promote osseointegration while ignoring microbial susceptibility. However, both coating functions are required to achieve long-term success of the implant; therefore, these two modalities must be evaluated in parallel during the development of new orthopaedic coating strategies. In this review, we discuss recent progress and future directions for the design of multifunctional orthopaedic coatings that can inhibit microbial cells while still promoting osseointegration.