Dylan A. McCreedy

Combination therapies in the CNS: Engineering the environment

The inhibitory extracellular environment that develops in response to traumatic brain injury and spinal cord injury hinders axon growth thereby limiting restoration of function. Several strategies have been developed to engineer a more permissive central nervous system (CNS) environment to promote regeneration and functional recovery. The multi-faced inhibitory nature of the CNS lesion suggests that therapies used in combination may be more effective. In this mini-review we summarize the most recent attempts to engineer the CNS extracellular environment after injury using combinatorial strategies. The advantages and limits of various combination therapies utilizing neurotrophin delivery, cell transplantation, and biomaterial scaffolds are discussed. Treatments that reduce the inhibition by chondroitin sulfate proteoglycans, myelin-associated inhibitors, and other barriers to axon regeneration are also reviewed. Based on the current state of the field, future directions are suggested for research on combination therapies in the CNS.

Survival, differentiation, and migration of high-purity mouse embryonic stem cell-derived progenitor motor neurons in fibrin scaffolds after sub-acute spinal cord injury

Embryonic stem (ES) cells can be differentiated into many neural cell types that hold great potential as cell replacement therapies following spinal cord injury (SCI). Coupling stem cell transplantation with biomaterial scaffolds can produce a unified combination therapy with several potential advantages including enhanced cell survival, greater transplant retention, reduced scarring, and improved integration at the transplant/host interface. Undesired cell types, however, are commonly present in ES-cell derived cultures due to the limited efficiency of most ES cell induction protocols. Heterogeneous cell populations can confound the interaction between the biomaterial and specific neural populations leading to undesired outcomes. In particular, biomaterials scaffolds may enhance tumor formation by promoting survival and proliferation of undifferentiated ES cells that can persist after induction. Methods for purification of specific ES cell-derived neural populations are necessary to recognize the full potential of combination therapies involving biomaterials and ES cell-derived neural populations. We previously developed a method for enriching ES cell-derived progenitor motor neurons (pMNs) induced from mouse ES cells via antibiotic selection and showed that the enriched cell populations are depleted of pluripotent stem cells. In this study, we demonstrate the survival and differentiation of enriched pMNs within three dimensional (3D) fibrin scaffolds in vitro and when transplanted into a sub-acute dorsal hemisection model of SCI into neurons, oligodendrocytes and astrocytes.

Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels

Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins. Protein adsorption-resistant poly(ethylene glycol) (PEG) materials can be covalently modified to present precise concentrations of adhesion proteins or peptides without the unintended effects of non-specifically adsorbed proteins. Here, we describe an improved protocol that incorporates custom-engineered materials. We first reproduced the Efe et al. protocol on Matrigel-coated TCPS (the original material), reprogramming adult mouse tail-tip mouse fibroblasts (TTF) and mouse embryonic fibroblasts (MEF) to cardiomyocyte-like cells that demonstrated striated sarcomeric α-actinin staining, spontaneous calcium transients, and visible beating. We then designed poly(ethylene glycol) culture substrates to promote MEF adhesion via laminin and RGD-binding integrins. PEG hydrogels improved proliferation and reprogramming efficiency (evidenced by beating patch number and area, gene expression, and flow cytometry), yielding almost twice the number of sarcomeric α-actinin positive cardiomyocyte-like cells as the originally described substrate. These results illustrate that cellular reprogramming may be enhanced using custom-engineered materials.

Generation of v2a interneurons from mouse embryonic stem cells.

V2a interneurons of the ventral spinal cord and hindbrain play an important role in the central pattern generators (CPGs) involved in locomotion, skilled reaching, and respiration. However, sources of V2a interneurons for in vitro studies are limited. In this study, we developed a differentiation protocol for V2a interneurons from mouse embryonic stem cells (mESCs). Cells were induced in a 2(-)/4(+) induction protocol with varying concentrations of retinoic acid (RA) and the mild sonic hedgehog (Shh) agonist purmorphamine (Pur) in order to increase the expression of V2a interneuron transcription factors (eg, Chx10). Notch signaling, which influences the commitment of p2 progenitor cells to V2a or V2b interneurons, was inhibited in cell cultures to increase the percentage of V2a interneurons. At the end of the induction period, cell commitment was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, and flow cytometry to quantify expression of transcription factors specific to V2a interneurons and the adjacent ventral spinal cord regions. Low concentrations of RA and high concentrations of Pur led to greater expression of transcription factors specific for V2a interneurons. Notch inhibition favored V2a interneuron over V2b interneuron differentiation. The protocol established in this study can be used to further elucidate the pathways involved in V2a interneuron differentiation and help produce sources of V2a interneurons for developmental neurobiology, electrophysiology, and transplantation studies.

Transgenic enrichment of mouse embryonic stem cell-derived progenitor motor neurons.

Embryonic stem cells (ESCs) hold great potential for replacing neurons following injury or disease. The therapeutic and diagnostic potential of ESCs may be hindered by heterogeneity in ESC-derived populations. Drug selection has been used to purify ESC-derived cardiomyocytes and endothelial cells but has not been applied to specific neural lineages. In this study we investigated positive selection of progenitor motor neurons (pMNs) through transgenic expression of the puromycin resistance enzyme, puromycin N-acetyl-transferase (PAC), under the Olig2 promoter. The protein-coding region in one allele of Olig2 was replaced with PAC to generate the P-Olig2 cell line. This cell line provided specific puromycin resistance in cells that express Olig2, while Olig2(-) cells were killed by puromycin. Positive selection significantly enriched populations of Olig2(+) pMNs. Committed motoneurons (MNs) expressing Hb9, a common progeny of pMNs, were also enriched by the end of the selection period. Selected cells remained viable and differentiated into mature cholinergic MNs and oligodendrocyte precursor cells. Drug resistance may provide a scalable and inexpensive method for enriching desired neural cell types for use in research applications.

A new method for generating high purity motoneurons from mouse embryonic stem cells.

A common problem with using embryonic stem (ES) cells as a source for analysis of gene expression, drug toxicity, or functional characterization studies is the heterogeneity that results from many differentiation protocols. The ability to generate large numbers of high purity differentiated cells from pluripotent stem cells could greatly enhance their utility for in vitro characterization studies and transplantation in pre‐clinical injury models. Population heterogeneity is particularly troublesome for post‐mitotic neurons, including motoneurons, because they do not proliferate and are quickly diluted in culture by proliferative phenotypes, such as glia. Studies of motoneuron biology and disease, in particular amyotrophic lateral sclerosis, can benefit from high purity motoneuron cultures. In this study, we engineered a transgenic‐ES cell line where highly conserved enhancer elements for the motoneuron transcription factor Hb9 were used to drive puromycin N‐acetyltransferase expression in ES cell‐derived motoneurons. Antibiotic selection with puromycin was then used to obtain high purity motoneuron cultures following differentiation of mouse ES cells. Purity was maintained during maturation allowing the production of consistent, uniform populations of cholinergic ES cell‐derived motoneurons. Appropriate functional properties of purified motoneurons were verified by acetylcholinesterase activity and electrophysiology. Antibiotic selection, therefore, can provide an inexpensive alternative to current methods for isolating ES cell‐derived motoneurons at high purity that does not require specialized laboratory equipment and provides a unique platform for studies in motoneuron development and degeneration. Biotechnol. Bioeng. 2014;111: 2041–2055.

Differentiation of V2a interneurons from human pluripotent stem cells

Significance Spinal cord injury (SCI) significantly disrupts normal neural circuitry, leading to severe degradation of motor and sensory function. Excitatory interneurons that relay signals from the brain to neural networks throughout the spinal cord, including glutamatergic V2a interneurons that coordinate respiration and locomotion, are lost after SCI. Thus, transplantation of V2a interneurons after SCI could provide a novel therapy to restore functional connections between the brain and spared downstream neurons. This study describes the generation of V2a interneurons from human pluripotent stem cells, using developmentally relevant morphogenic signaling pathways. This work provides initial insight into the development of excitatory human interneurons and enables the examination of their therapeutic efficacy for SCI repair. The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10+ V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10+ cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10+ cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10+ cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI.

Reducing neuroinflammation by delivery of IL‐10 encoding lentivirus from multiple‐channel bridges

Abstract The spinal cord is unable to regenerate after injury largely due to growth‐inhibition by an inflammatory response to the injury that fails to resolve, resulting in secondary damage and cell death. An approach that prevents inhibition by attenuating the inflammatory response and promoting its resolution through the transition of macrophages to anti‐inflammatory phenotypes is essential for the creation of a growth permissive microenvironment. Viral gene delivery to induce the expression of anti‐inflammatory factors provides the potential to provide localized delivery to alter the host inflammatory response. Initially, we investigated the effect of the biomaterial and viral components of the delivery system to influence the extent of cell infiltration and the phenotype of these cells. Bridge implantation reduces antigen‐presenting cell infiltration at day 7, and lentivirus addition to the bridge induces a transient increase in neutrophils in the spinal cord at day 7 and macrophages at day 14. Delivery of a lentivirus encoding IL‐10, an anti‐inflammatory factor that inhibits immune cell activation and polarizes the macrophage population towards anti‐inflammatory phenotypes, reduced neutrophil infiltration at both day 7 and day 28. Though IL‐10 lentivirus did not affect macrophages number, it skewed the macrophage population toward an anti‐inflammatory M2 phenotype and altered macrophage morphology. Additionally, IL‐10 delivery resulted in improved motor function, suggesting reduced secondary damage and increased sparing. Taken together, these results indicate that localized expression of anti‐inflammatory factors, such as IL‐10, can modulate the inflammatory response following spinal cord injury, and may be a key component of a combinatorial approach that targets the multiple barriers to regeneration and functional recovery.

Three-Dimensional Imaging of the Developing Human Fetal Urogenital-Genital Tract: Indifferent Stage to Male and Female Differentiation

Recent studies in our lab have utilized three imaging techniques to visualize the developing human fetal urogenital tract in three dimensions: optical projection tomography, scanning electron microscopy and lightsheet fluorescence microscopy. We have applied these technologies to examine changes in morphology and differential gene expression in developing human external genital specimens from the ambisexual stage (<9 weeks fetal age) to well-differentiated male and female organs (>13 weeks fetal age). This work outlines the history and function of each of these three imaging modalities, our methods to prepare specimens for each and the novel findings we have produced thus far. We believe the images in this paper of human fetal urogenital organs produced using lightsheet fluorescence microscopy are the first published to date.

Semi-automated counting of axon regeneration in poly(lactide co-glycolide) spinal cord bridges

BACKGROUND Spinal cord injury (SCI) is a debilitating event with multiple mechanisms of degeneration leading to life-long paralysis. Biomaterial strategies, including bridges that span the injury and provide a pathway to reconnect severed regions of the spinal cord, can promote partial restoration of motor function following SCI. Axon growth through the bridge is essential to characterizing regeneration, as recovery can occur via other mechanisms such as plasticity. Quantitative analysis of axons by manual counting of histological sections can be slow, which can limit the number of bridge designs evaluated. In this study, we report a semi-automated process to resolve axon numbers in histological sections, which allows for efficient analysis of large data sets. NEW METHOD Axon numbers were estimated in SCI cross-sections from animals implanted with poly(lactide co-glycolide) (PLG) bridges with multiple channels for guiding axons. Immunofluorescence images of histological sections were filtered using a Hessian-based approach prior to threshold detection to improve the signal-to-noise ratio and filter out background staining associated with PLG polymer. RESULTS Semi-automated counting successfully recapitulated average axon densities and myelination in a blinded PLG bridge implantation study. COMPARISON WITH EXISTING METHODS Axon counts obtained with the semi-automated technique correlated well with manual axon counts from blinded independent observers across sections with a wide range of total axons. CONCLUSIONS This semi-automated detection of Hessian-filtered axons provides an accurate and significantly faster alternative to manual counting of axons for quantitative analysis of regeneration following SCI.