Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentally-restricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI.

Role of EZH2 in Uterine Gland Development.

Enhancer of zeste homolog 2 (EZH2) is a core component of polycomb repressive complex 2 that plays a vital role in transcriptional repression of gene expression. Conditional ablation of EZH2 using progesterone receptor (Pgr)-Cre in the mouse uterus has uncovered its roles in regulating uterine epithelial cell growth and stratification, suppressing decidual myofibroblast activation, and maintaining normal female fertility. However, it is unclear whether EZH2 plays a role in the development of uterine glands, which are required for pregnancy success. Herein, we created mice with conditional deletion of Ezh2 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase that is expressed in mesenchyme-derived cells of the female reproductive tract. Strikingly, these mice showed marked defects in uterine adenogenesis. Unlike Ezh2 Pgr-Cre conditional knockout mice, deletion of Ezh2 using Amhr2-Cre did not lead to the differentiation of basal-like cells in the uterus. The deficient uterine adenogenesis was accompanied by impaired uterine function and pregnancy loss. Transcriptomic profiling using next generation sequencing revealed dysregulation of genes associated with signaling pathways that play fundamental roles in development and disease. In summary, this study has identified an unrecognized role of EZH2 in uterine gland development, a postnatal event critical for pregnancy success and female fertility.

Circulating neutrophil activation in dogs with naturally occurring spinal cord injury secondary to intervertebral disk herniation.

OBJECTIVE: To investigate the time course of circulating neutrophil priming and activity in dogs with spinal cord injury secondary to intervertebral disk herniation that undergo decompressive surgery. ANIMALS: 9 dogs with spinal cord injury and 9 healthy dogs (controls). PROCEDURES: For dogs with spinal cord injury, blood samples were collected on the day of hospital admission and 3, 7, 30, and 90 days after injury and decompressive surgery. A single blood sample was collected from the control dogs. Flow cytometry analysis was performed on isolated neutrophils incubated with antibody against CD11b and nonfluorescent dihydrorhodamine 123, which was converted to fluorescent rhodamine 123 to measure oxidative burst activity. RESULTS: Expression of CD11b was increased in dogs with spinal cord injury 3 days after injury and decompressive surgery, relative to day 7 expression. Neutrophils expressed high oxidative burst activity both 3 and 7 days after injury and decompressive surgery, compared with activity in healthy dogs. CLINICAL RELEVANCE: For dogs with spinal cord injury, high CD11b expression 3 days after injury and decompressive surgery was consistent with findings for rodents with experimentally induced spinal cord injury. However, the high oxidative burst activity 3 and 7 days after injury and decompressive surgery was not consistent with data from other species, and additional studies on inflammatory events in dogs with naturally occurring spinal cord injury are needed.

Differentiation of V2a interneurons from human pluripotent stem cells.

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.

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.

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.

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.

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.

Tissue-engineered fibrin scaffolds containing neural progenitors enhance functional recovery in a subacute model of SCI.

Two recurring problems with stem/neural progenitor cell (NPC) transplantation therapies for spinal cord injury (SCI) are poor cell survival and uncontrolled cell differentiation. The current study evaluated the viability and differentiation of embryonic stem cell-derived neural progenitor cells (ESNPCs) transplanted within fibrin scaffolds containing growth factors (GFs) and a heparin-binding delivery system (HBDS) to enhance cell survival and direct differentiation into neurons. Mouse ESNPCs were generated from mouse embryonic stem cells (ESCs) using a 4-/4+ retinoic acid (RA) induction protocol that resulted in a population of cells that was 70% nestin positive NPCs. The ESNPCs were transplanted directly into a rat subacute dorsal hemisection lesion SCI model. ESNPCs were either encapsulated in a fibrin scaffold; encapsulated in fibrin containing the HBDS, neurotrophin-3 (NT-3) and platelet derived growth factor (PDGF-AA); or encapsulated in fibrin scaffolds with NT-3 and PDGF-AA without the HBDS. We report that the combination of GFs and fibrin scaffold (without HBDS) enhanced the total number of ESNPCs present in the treated spinal cords and increased the number of ESNPC-derived NeuN positive neurons 8 weeks after transplantation. All experimental groups treated with ESNPCs exhibited an increase in behavioral function 4 weeks after transplantation. In a subset of animals, the ESNPCs over-proliferated as evidenced by SSEA-1 positive/Ki67 positive ESCs found at 4 and 8 weeks. These results demonstrate the potential of tissue-engineered fibrin scaffolds to enhance the survival of NPCs and highlight the need to purify cell populations used in therapies for SCI.