Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives

ABSTRACT The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus, it is a crucial parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine/disease modelling applications. However, the in vitro generation of posterior MNs corresponding to the thoracic/lumbosacral spinal cord has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well defined. Here, we determine the signals guiding the transition of human NMP-like cells toward thoracic ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal pre-/early neural state, whereas suppression of TGFβ-BMP signalling pathways promotes a ventral identity and neural commitment. Based on these results, we define an optimised protocol for the generation of thoracic MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the comparison of hPSC-derived spinal cord cells of distinct axial identities.

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Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives

10.1101/2020.06.24.168625 ◽

2020 ◽

Author(s):

Matthew Wind ◽

Antigoni Gogolou ◽

Ichcha Manipur ◽

Ilaria Granata ◽

Larissa Butler ◽

...

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Signalling Pathways ◽

Bona Fide ◽

Promising Solution ◽

Identity Based ◽

Bmp Signalling ◽

Ventral Spinal Cord

AbstractThe anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus it is a critical parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine and disease modelling applications. However, the in vitro generation of posterior spinal cord MNs has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the mammalian spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well-defined. Here we determine the signals guiding the transition of human NMP-like cells toward posterior ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal early neural state while suppression of TGFβ-BMP signalling pathways, combined with SHH stimulation, promotes a ventral identity. Based on these results, we define an optimised protocol for the generation of posterior MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the functional comparison of hPSC-derived spinal cord cells of distinct axial identities.

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Achyranthes bidentata polypeptide k enhances the survival, growth and axonal regeneration of spinal cord motor neurons in vitro

Neuroreport ◽

10.1097/wnr.0000000000001621 ◽

2021 ◽

Vol 32 (6) ◽

pp. 518-524

Author(s):

Ergai Cai ◽

Qiong Cheng ◽

Shu Yu ◽

Fei Ding

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Axonal Regeneration ◽

Achyranthes Bidentata

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Diminished ventral oligodendrocyte precursor generation results in the subsequent over-production of dorsal oligodendrocyte precursors of aberrant morphology and function

10.1101/2020.03.09.983908 ◽

2020 ◽

Author(s):

Lev Starikov ◽

Andreas H. Kottmann

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Ventricular Zone ◽

Normal Numbers ◽

Spinal Cord Development ◽

Oligodendrocyte Precursor ◽

And Function ◽

Sod1 Mouse ◽

Lateral Sclerosis ◽

Ventral Spinal Cord

AbstractOligodendrocyte precursor cells (OPCs) arise sequentially first from a ventral and then from a dorsal precursor domain at the end of neurogenesis during spinal cord development. Whether the sequential production of OPCs is of physiological significance has not been examined. Here we show that ablating Shh signaling from nascent ventricular zone derivatives and partially from the floor plate results in a severe diminishment of ventral derived OPCs but normal numbers of motor neurons in the postnatal spinal cord. In the absence of ventral vOPCs, dorsal dOPCs populate the entire spinal cord resulting in an increased OPC density in the ventral horns. These OPCs take on an altered morphology, do not participate in the removal of excitatory vGlut1 synapses from injured motor neurons, and exhibit morphological features similar to those found in the vicinity of motor neurons in the SOD1 mouse model of Amyotrophic Lateral Sclerosis (ALS). Our data indicates that vOPCs prevent dOPCs from invading ventral spinal cord laminae and suggests that vOPCs have a unique ability to communicate with injured motor neurons.

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Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition

Cells ◽

10.3390/cells8090951 ◽

2019 ◽

Vol 8 (9) ◽

pp. 951 ◽

Cited By ~ 2

Author(s):

Yong Dong ◽

Chengxiang Xia ◽

Qitong Weng ◽

Tongjie Wang ◽

Fangxiao Hu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Pluripotent Stem Cells ◽

Culture Condition ◽

Fusion Gene ◽

Nras Mutation ◽

Hematopoietic Stem ◽

Bona Fide

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

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Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients

Brain Sciences ◽

10.3390/brainsci10070407 ◽

2020 ◽

Vol 10 (7) ◽

pp. 407

Author(s):

Pierre-Antoine Faye ◽

Nicolas Vedrenne ◽

Federica Miressi ◽

Marion Rassat ◽

Sergii Romanenko ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Spinal Motor ◽

Electrophysiological Recordings ◽

Induced Pluripotent

Modelling rare neurogenetic diseases to develop new therapeutic strategies is highly challenging. The use of human-induced pluripotent stem cells (hiPSCs) is a powerful approach to obtain specialized cells from patients. For hereditary peripheral neuropathies, such as Charcot–Marie–Tooth disease (CMT) Type II, spinal motor neurons (MNs) are impaired but are very difficult to study. Although several protocols are available to differentiate hiPSCs into neurons, their efficiency is still poor for CMT patients. Thus, our goal was to develop a robust, easy, and reproducible protocol to obtain MNs from CMT patient hiPSCs. The presented protocol generates MNs within 20 days, with a success rate of 80%, using specifically chosen molecules, such as Sonic Hedgehog or retinoic acid. The timing and concentrations of the factors used to induce differentiation are crucial and are given hereby. We then assessed the MNs by optic microscopy, immunocytochemistry (Islet1/2, HB9, Tuj1, and PGP9.5), and electrophysiological recordings. This method of generating MNs from CMT patients in vitro shows promise for the further development of assays to understand the pathological mechanisms of CMT and for drug screening.

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Presynaptic Angiotensin II AT1 Receptors Enhance Inhibitory and Excitatory Synaptic Neurotransmission to Motoneurons and Other Ventral Horn Neurons in Neonatal Rat Spinal Cord

Journal of Neurophysiology ◽

10.1152/jn.00165.2005 ◽

2005 ◽

Vol 94 (2) ◽

pp. 1405-1412 ◽

Cited By ~ 14

Author(s):

Murat Oz ◽

Keun-Hang Yang ◽

Michael J. O'Donovan ◽

Leo P. Renaud

Keyword(s):

Spinal Cord ◽

Angiotensin Ii ◽

Ventral Horn ◽

Synaptic Activity ◽

Neonatal Rat ◽

Ang Ii ◽

Synaptic Neurotransmission ◽

Excitatory Neurotransmission ◽

Ventral Spinal Cord

In neonatal spinal cord, we previously reported that exogenous angiotensin II (ANG II) acts at postsynaptic AT1 receptors to depolarize neonatal rat spinal ventral horn neurons in vitro. This study evaluated an associated increase in synaptic activity. Patch clamp recordings revealed that 38/81 thoracolumbar (T7–L5) motoneurons responded to bath applied ANG II (0.3–1 μM; 30 s) with a prolonged (5–10 min) and reversible increase in spontaneous postsynaptic activity, selectively blockable with Losartan ( n = 5) but not PD123319 ( n = 5). ANG-II-induced events included both spontaneous inhibitory (IPSCs; n = 6) and excitatory postsynaptic currents (EPSCs; n = 5). While most ANG induced events were tetrodotoxin-sensitive, ANG induced a significant tetrodotoxin-resistant increase in frequency but not amplitude of miniature IPSCs ( n = 7/13 cells) and EPSCs ( n = 2/7 cells). In 35/77 unidentified neurons, ANG II also induced a tetrodotoxin-sensitive and prolonged increase in their spontaneous synaptic activity that featured both IPSCs ( n = 5) and EPSCs ( n = 4) when tested in the presence of selective amino acid receptor antagonists. When tested in the presence of tetrodotoxin, ANG II was noted to induce a significant increase in the frequency but not the amplitude of mIPSCs ( n = 9) and mEPSCs ( n = 8). ANG also increased spontaneous motor activity from isolated mouse lumbar ventral rootlets. Collectively, these observations support the existence of a wide pre- and postsynaptic distribution of ANG II AT1 receptors in neonatal ventral spinal cord that are capable of influencing both inhibitory and excitatory neurotransmission.

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Sulfatase 2 Modulates Fate Change from Motor Neurons to Oligodendrocyte Precursor Cells through Coordinated Regulation of Shh Signaling with Sulfatase 1

Developmental Neuroscience ◽

10.1159/000464284 ◽

2017 ◽

Vol 39 (5) ◽

pp. 361-374 ◽

Cited By ~ 9

Author(s):

Wen Jiang ◽

Yugo Ishino ◽

Hirokazu Hashimoto ◽

Kazuko Keino-Masu ◽

Masayuki Masu ◽

...

Keyword(s):

Spinal Cord ◽

Expression Pattern ◽

Motor Neurons ◽

Expression Patterns ◽

Precursor Cells ◽

Oligodendrocyte Precursor Cells ◽

Shh Signaling ◽

Sulfatase Activity ◽

Oligodendrocyte Precursor ◽

Ventral Spinal Cord

Sulfatases (Sulfs) are a group of endosulfatases consisting of Sulf1 and Sulf2, which specifically remove sulfate from heparan sulfate proteoglycans. Although several studies have shown that Sulf1 acts as a regulator of sonic hedgehog (Shh) signaling during embryonic ventral spinal cord development, the detailed expression pattern and function of Sulf2 in the spinal cord remains to be determined. In this study, we found that Sulf2 also modulates the cell fate change from motor neurons (MNs) to oligodendrocyte precursor cells (OPCs) by regulating Shh signaling in the mouse ventral spinal cord in coordination with Sulf1. In the mouse, Sulf mRNAs colocalize with Shh mRNA and gradually expand dorsally from embryonic day (E) 10.5 to E12.5, following strong Patched1 signals (a target gene of Shh signaling). This coordinated expression pattern led us to hypothesize that in the mouse, strong Shh signaling is induced when Shh is released by Sulf1/2, and this strong Shh signaling subsequently induces the dorsal expansion of Shh and Sulf1/2 expression. Consistent with this hypothesis, in the ventral spinal cord of Sulf1 knockout (KO) or Sulf2 KO mice, the expression patterns of Shh and Patched1 differed from that in wild-type mice. Moreover, the position of the pMN and p3 domains were shifted ventrally, MN generation was prolonged, and OPC generation was delayed at E12.5 in both Sulf1 KO and Sulf2 KO mice. These results demonstrated that in addition to Sulf1, Sulf2 also plays an important and overlapping role in the MN-to-OPC fate change by regulating Shh signaling in the ventral spinal cord. However, neither Sulf1 nor Sulf2 could compensate for the loss of the other in the developing mouse spinal cord. In vitro studies showed no evidence of an interaction between Sulf1 and Sulf2 that could increase sulfatase activity. Furthermore, Sulf1/2 double heterozygote and Sulf1/2 double KO mice exhibited phenotypes similar to the Sulf1 KO and Sulf2 KO mice. These results indicate that there is a threshold for sulfatase activity (which is likely reflected in the dose of Shh) required to induce the MN-to-OPC fate change, and Shh signaling requires the coordinated activity of Sulf1 and Sulf2 in order to reach that threshold in the mouse ventral spinal cord.

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HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

Stem Cells International ◽

10.1155/2016/9475981 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 20

Author(s):

Ji-Yon Kim ◽

So-Youn Woo ◽

Young Bin Hong ◽

Heesun Choi ◽

Jisoo Kim ◽

...

Keyword(s):

Stem Cells ◽

Peripheral Neuropathy ◽

Induced Pluripotent Stem Cells ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Motor Neuropathy ◽

Mitochondrial Movement ◽

Induced Pluripotent

The Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1) gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs) of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy.

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