Derivation of Specific Neural Populations From Pluripotent Cells for Understanding and Treatment of Spinal Cord Injury

White, Nicholas; Sakiyama‐Elbert, Shelly E.

doi:10.1002/dvdy.24680

Cited by 16 publications

(10 citation statements)

References 93 publications

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“…1) 21,22 . Additional protocols for specific neural subtypes including glia, motor neurons, as well as ventral and dorsal interneurons have been comprehensively reviewed by White et al 24 . This protocol for V2a interneurons describes how to induce committed V2a interneurons from mouse PSCs in 6 days and from human PSCs in 17 days.…”

Section: Comparison With Other Methods To Differentiate Neural Cellsmentioning

confidence: 99%

V2a interneuron differentiation from mouse and human pluripotent stem cells

et al. 2019

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V2a interneurons are located in the hindbrain and spinal cord where they provide rhythmic input to major motor control centers. Many of the phenotypic properties and functions of excitatory V2a interneurons have yet to be fully defined, which could lead to novel regenerative therapies for traumatic injuries and drug targets for chronic degenerative diseases. Here, we describe how to produce V2a interneurons from mouse and human pluripotent stem cells (PSCs), as well as strategies to characterize and mature the cells for further analysis. The described protocols are based on a sequence of small molecule treatments to induce differentiation into V2a interneurons. We also include a detailed description of how to phenotypically characterize, mature, and freeze the cells. The mouse and human protocols are similar in the sequence of small molecules used, but

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Section: Comparison With Other Methods To Differentiate Neural Cellsmentioning

confidence: 99%

V2a interneuron differentiation from mouse and human pluripotent stem cells

et al. 2019

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“…Several groups have investigated the potential of induced pluripotent stem cell (iPSC) derived propriospinal neurons as a potential therapy to restore respiratory motor function. For example, iPSC derived V2a and V3 neurons have been generated from mouse or human cells (Brown et al, 2014;Xu et al, 2015;Iyer et al, 2016;Butts et al, 2017Butts et al, , 2019White and Sakiyama-Elbert, 2019). iPSC derived V2a neurons have been transplanted into the spinal cord where they survive and appear to form synapses with host neurons (Butts et al, 2017;Zholudeva et al, 2018a).…”

Section: Propriospinal Neurons Modulate Respiratory Motor Output Follmentioning

confidence: 99%

Role of Propriospinal Neurons in Control of Respiratory Muscles and Recovery of Breathing Following Injury

Jensen

Alilain

Crone

2020

Front. Syst. Neurosci.

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Respiratory motor failure is the leading cause of death in spinal cord injury (SCI). Cervical injuries disrupt connections between brainstem neurons that are the primary source of excitatory drive to respiratory motor neurons in the spinal cord and their targets. In addition to direct connections from bulbospinal neurons, respiratory motor neurons also receive excitatory and inhibitory inputs from propriospinal neurons, yet their role in the control of breathing is often overlooked. In this review, we will present evidence that propriospinal neurons play important roles in patterning muscle activity for breathing. These roles likely include shaping the pattern of respiratory motor output, processing and transmitting sensory afferent information, coordinating ventilation with motor activity, and regulating accessory and respiratory muscle activity. In addition, we discuss recent studies that have highlighted the importance of propriospinal neurons for recovery of respiratory muscle function following SCI. We propose that molecular genetic approaches to target specific developmental neuron classes in the spinal cord would help investigators resolve the many roles of propriospinal neurons in the control of breathing. A better understanding of how spinal circuits pattern breathing could lead to new treatments to improve breathing following injury or disease.

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“…To reconnect the lost circuits, cell transplantation is a potential method of introducing cells in and around the injury site to support growth of spared neurons through release of neurotrophic factors and for cell replacement. For cell transplantation, several cell lineages have been investigated to determine their contribution to repair (see this review for more information: [ 1 ]), but the propriospinal, interneuron (IN) populations have been shown to create local connections post-injury [ 2 – 4 ]. INs are classified as dorsal or ventral, which contribute largely to sensory or motor function, respectively [ 5 ], and several studies have worked toward deconstructing the role of the different IN subtypes and the interplay between them, which drives normal function [ 6 ], as well as their potential as therapeutic populations [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

A transgenic mouse embryonic stem cell line for puromycin selection of V0V interneurons from heterogenous induced cultures

2022

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Background Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0V) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0V INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0V INs. This study generated a transgenic mESC line to enrich V0V INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies. Methods The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0V IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0V INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs). Results V0V IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting. Conclusions Evx1-PAC mESCs can be used to purify V0V IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0V INs.

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Derivation of Specific Neural Populations From Pluripotent Cells for Understanding and Treatment of Spinal Cord Injury

Cited by 16 publications

References 93 publications

V2a interneuron differentiation from mouse and human pluripotent stem cells

V2a interneuron differentiation from mouse and human pluripotent stem cells

Role of Propriospinal Neurons in Control of Respiratory Muscles and Recovery of Breathing Following Injury

A transgenic mouse embryonic stem cell line for puromycin selection of V0V interneurons from heterogenous induced cultures

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