Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons

Machado, Carolina Barcellos; Kanning, Kevin C.; Kreis, Patricia; Stevenson, D. S.; Crossley, Martin D.; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David W.; Blanc, Eric; Lieberam, Ivo

doi:10.1242/dev.108803

Cited by 18 publications

(33 citation statements)

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“…Among the ISL1 + /HB9 + cells that did not express FOXP1, 15±3% did not express LHX3 either, suggesting that this fraction of MNs had a molecular profile (FOXP1 -/LHX3 -) similar to that of MNs of the hypaxial motor column (HMC) containing MNs innervating respiratory muscles in vivo (data not shown). Consistent with this possibility, we observed that 10±2% of ISL1 + /HB9 + MNs expressed the transcription factor POU CLASS 3 HOMEOBOX 1/OCTAMER BINDING PROTEIN 6/SCIP (SCIP), known to be enriched in vivo in MNs of the phrenic motor column (PMC), a component of the HMC (Bermingham et al, 1996;Philipidou et al, 2014;Machado et al, 2014) (Figure 2C and D). Lastly, only 11±4% of the h-iPSC-derived MNs were FOXP1and expressed LHX3, thus exhibiting a profile (FOXP1 -/LHX3 + ) resembling that of MNs of the medial motor column (MMC) ( Figure 2C and D).…”

Section: Generation Of An Enriched Population Of Human Ipsc-derived Msupporting

confidence: 72%

Characterization of human-iPSCs derived spinal motor neurons by single-cell RNA sequencing

Thiry

Hamel

Pluchino

et al. 2019

Preprint

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AbstractHuman induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of brainstem and spinal cord MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, ∼19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of medial motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and diseases.

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Section: Generation Of An Enriched Population Of Human Ipsc-derived Msupporting

confidence: 72%

Characterization of human-iPSCs derived spinal motor neurons by single-cell RNA sequencing

Thiry

Hamel

Pluchino

et al. 2019

Preprint

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“…Although this hypothesis has not yet been proven experimentally, recent studies have provided supporting evidence of the differentiation of motor neuron identities (Machado et al . ; Jung & Dasen ).…”

Section: Introductionmentioning

confidence: 99%

“…In this scenario, addition and fixation of cervical vertebral counts in the lineage leading to mammals were evolutionarily linked to the individualization of the novelty, the diaphragm. Although this hypothesis has not yet been proven experimentally, recent studies have provided supporting evidence of the differentiation of motor neuron identities (Machado et al 2014;Jung & Dasen 2015).…”

Section: Introductionmentioning

confidence: 99%

Expansion of the neck reconstituted the shoulder–diaphragm in amniote evolution

Hirasawa¹,

Fujimoto²,

Kuratani³

2015

Dev Growth Differ

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The neck acquired flexibility through modifications of the head-trunk interface in vertebrate evolution. Although developmental programs for the neck musculoskeletal system have attracted the attention of evolutionary developmental biologists, how the heart, shoulder and surrounding tissues are modified during development has remained unclear. Here we show, through observation of the lateral plate mesoderm at cranial somite levels in chicken-quail chimeras, that the deep part of the lateral body wall is moved concomitant with the caudal transposition of the heart, resulting in the infolding of the expanded cervical lateral body wall into the thorax. Judging from the brachial plexus pattern, an equivalent infolding also appears to take place in mammalian and turtle embryos. In mammals, this infolding process is particularly important because it separates the diaphragm from the shoulder muscle mass. In turtles, the expansion of the cervical lateral body wall affects morphogenesis of the shoulder. Our findings highlight the cellular expansion in developing amniote necks that incidentally brought about the novel adaptive traits.

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“…To date, several studies using commercial rat serum in WEC by combining chemically defined medium have been reported [23][24][25] . For example, a mixture of 50% commercially available rat serum and 50% DMEM supports the normal growth of E8.5 mouse embryos for 36 hr 23 and the normal growth of E9.75 mouse embryos for 40 hr 24 .…”

Section: Discussionmentioning

confidence: 99%

“…For example, a mixture of 50% commercially available rat serum and 50% DMEM supports the normal growth of E8.5 mouse embryos for 36 hr 23 and the normal growth of E9.75 mouse embryos for 40 hr 24 . In another protocol, a mixture composed of 50% rat serum available from another company and 50% F12 medium supplemented with N-2 also supports the proper growth of E7.5 and E8.5 mouse embryos for 24 hr 25 .…”

Section: Discussionmentioning

confidence: 99%

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Takahashi

Makino

Kikkawa

et al. 2014

JoVE

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Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation. Video LinkThe video component of this article can be found at

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Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons

Cited by 18 publications

References 0 publications

Characterization of human-iPSCs derived spinal motor neurons by single-cell RNA sequencing

Characterization of human-iPSCs derived spinal motor neurons by single-cell RNA sequencing

Expansion of the neck reconstituted the shoulder–diaphragm in amniote evolution

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

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