Development-on-chip: in vitro neural tube patterning with a microfluidic device.

Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

Guidance of postural motoneurons requires MAPK/ERK signaling downstream of fibroblast growth factor receptor 1.

Identification of intracellular signaling pathways necessary for appropriate axon guidance is challenging because many CNS populations used to study these events contain multiple cell types. Here, we resolve this issue by using mouse embryonic stem (ES) cells that were directed to differentiate into a population of motoneurons that exclusively innervate epaxial muscles [medial median motor column (MMCm) motoneurons]. These ES cell-derived MMCm motoneurons, like their endogenous counterparts, express fibroblast growth factor receptor 1 (FGFR1) and selectively extend axons toward the epaxial trophin FGF8. Unlike wild-type MMCm motoneurons, FGFR1(-/-) MMCm motoneurons show guidance defects when transplanted into the neural tube of chick embryos. Furthermore, activation of FGFR1 selectively signals through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) for appropriate guidance in vitro, whereas overexpression of constitutively active MAPK/ERK in transplanted, or endogenous chick, MMCm cells causes guidance defects in vivo. These results indicate that MAPK/ERK activation downstream of FGFR1 is necessary for MMCm motor axon guidance and that ES cell-derived neurons provide an important tool for dissecting intracellular pathways required for axon guidance.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin.

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

Motoneurons derived from embryonic stem cells express transcription factors and develop phenotypes characteristic of medial motor column neurons.

Embryonic stem (ES) cells differentiate into functional motoneurons when treated with a sonic hedgehog (Shh) agonist and retinoic acid (RA). Whether ES cells can be directed to differentiate into specific subtypes of motoneurons is unknown. We treated embryoid bodies generated from HBG3 ES cells with a Shh agonist and RA for 5 d in culture to induce motoneuron differentiation. Enhanced green fluorescent protein (eGFP) expression was used to identify putative motoneurons, because eGFP is expressed under the control of the Hb9 promoter in HBG3 cells. We found that 96 +/- 0.7% of the differentiated eGFP+ motoneurons expressed Lhx3, a homeobox gene expressed by postmitotic motoneurons in the medial motor column (MMCm), when the treated cells were plated on a neurite-promoting substrate for 5 d. When the treated embryoid bodies were transplanted into stage 17 chick neural tubes, the eGFP+ motoneurons migrated to the MMCm, expressed Lhx3, projected axons to the appropriate target for MMCm motoneurons (i.e., epaxial muscles), and contained synaptic vesicles within intramuscular axonal branches. In ovo and in vitro studies indicated that chemotropic factors emanating from the epaxial muscle and/or surrounding mesenchyme likely guide Lhx3+ motoneurons to their correct target. Finally, whole-cell patch-clamp recordings of transplanted ES cell-derived motoneurons demonstrated that they received synaptic input, elicited repetitive trains of action potentials, and developed passive membrane properties that were similar to host MMCm motoneurons. These results indicate that ES cells can be directed to form subtypes of neurons with specific phenotypic properties.

Easy and rapid differentiation of embryonic stem cells into functional motoneurons using sonic hedgehog-producing cells.

Directing embryonic stem (ES) cells to differentiate into functional motoneurons has proven to be a strong technique for studying neuronal development as well as being a potential source of tissue for cell replacement therapies involving spinal cord disorders. Unfortunately, one of the mitogenic factors (i.e., sonic hedgehog agonist) used for directed differentiation is not readily available, and thus this technique has not been widely accessible. Here, we present a novel and simple method to derive motoneurons from ES cells using readily attainable reagents. ES cells were derived from a mouse in which enhanced green fluorescent protein (eGFP) was linked to a motoneuron specific promoter. The cells were plated onto a monolayer of 293 EcR-Shh cells that carry an integrated construct for the expression of sonic hedgehog (Shh) under ecdysone-inducible control. To initiate motoneuron differentiation, 293 EcR-Shh:ES cell cocultures were treated with ponasterone A (PA) and retinoic acid for 5 days. PA induces ecdysone, and thus drives Shh expression. To assess differentiation, putative ES cell-derived motoneurons were studied immunocytochemically and cultured on chick myotubes for functional analysis. We found that ES cells differentiated into eGFP+ cells that expressed transcription factors typical of motoneurons. Furthermore, ES cell-derived motoneurons were capable of forming functional connections with muscle fibers in vitro. Finally, when transplanted into the developing chick spinal cord, ES cell-derived motoneurons migrated to the ventral horn and projected axons to appropriate muscle targets. In summary, this simple treatment paradigm produces functional motoneurons that can be used for both developmental and preclinical studies. Disclosure of potential conflicts of interest is found at the end of this article.