Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using an Hb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

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Transcription factor directed differentiation of embryonic stem cells in vitro

Fertility and Sterility ◽

10.1016/j.fertnstert.2009.07.1334 ◽

2009 ◽

Vol 92 (3) ◽

pp. S172

Author(s):

R.M. Anchan ◽

S.A. Lachke ◽

B. Gerami-Naini ◽

K.E. Naber ◽

J. Eaton ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Directed Differentiation

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

Development ◽

10.1242/dev.194415 ◽

2021 ◽

Vol 148 (6) ◽

Author(s):

Matthew Wind ◽

Antigoni Gogolou ◽

Ichcha Manipur ◽

Ilaria Granata ◽

Larissa Butler ◽

...

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Lumbosacral Spinal Cord ◽

Bona Fide ◽

Promising Solution ◽

Bmp Signalling ◽

Crucial Parameter ◽

Ventral Spinal Cord

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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Modeling motor neuron resilience in ALS using stem cells

10.1101/399659 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ilary Allodi ◽

Jik Nijssen ◽

Julio Aguila Benitez ◽

Christoph Schweingruber ◽

Andrea Fuchs ◽

...

Keyword(s):

Stem Cells ◽

Motor Neuron ◽

Motor Neurons ◽

Embryonic Stem ◽

Spinal Motor Neurons ◽

Neuronal Progenitors ◽

Oculomotor Neurons ◽

Bona Fide

SUMMARYOculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

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Small Molecule Inhibitors of LcrF, a Yersinia pseudotuberculosis Transcription Factor, Attenuate Virulence and Limit Infection in a Murine Pneumonia Model

Infection and Immunity ◽

10.1128/iai.01305-09 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4683-4690 ◽

Cited By ~ 50

Author(s):

Lynne K. Garrity-Ryan ◽

Oak K. Kim ◽

Joan-Miquel Balada-Llasat ◽

Victoria J. Bartlett ◽

Atul K. Verma ◽

...

Keyword(s):

Transcription Factor ◽

Small Molecule ◽

Type Iii Secretion ◽

Mammalian Cells ◽

Yersinia Pseudotuberculosis ◽

Secretion System ◽

Bacterial Burden ◽

Small Molecule Library ◽

Null Mutants

ABSTRACT LcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersinia type III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersinia infection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitro assay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosis toward macrophages in vitro. In a murine model of Y. pseudotuberculosis pneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

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307 REPROGRAMMING OF PIG SOMATIC CELLS TO PLURIPOTENCY WITH SLEEPING BEAUTY TRANSPOSON VECTORS CONTAINING THE PORCINE TRANSCRIPTION FACTOR SEQUENCES

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab307 ◽

2013 ◽

Vol 25 (1) ◽

pp. 300

Author(s):

S. Petkov ◽

M. Nowak-Imialek ◽

P. Hyttel ◽

H. Niemann

Keyword(s):

Transcription Factor ◽

Retroviral Vectors ◽

Culture Conditions ◽

Sleeping Beauty ◽

Patient Specific ◽

Differentiation Potential ◽

Serum Replacement ◽

Gfp Reporter ◽

Fetal Fibroblasts

Induced pluripotent stem cells (iPSC), developed by Yamanaka and co-workers (Takahashi et al., 2006), hold significant potential for the development of regenerative therapies due to the possibilities of deriving patient-specific pluripotent cells. In this aspect, the pig is an important animal model for testing iPSC-based applications for the human medicine. However, even though significant progress has been made, the derivation of porcine iPSC lines fully equivalent to those from mouse and human has been elusive. To date, most of the reported putative pig iPSC lines have been derived with the use of lentiviral or retroviral vectors harboring the mouse or human transcription factor sequences. Here, we report the construction of Sleeping Beauty (SB) transposon vectors with porcine cDNA sequences coding for OCT4, SOX2, NANOG, C-MYC, and KLF4, in addition to the human LIN28. By using standard cloning techniques, we produced 2 polycistronic SB-CAG-pOSMK-ires-Tomato and SB-Ef1a-pNANOG-ires-hLIN28 transposon vectors and we transfected them together with the SB100X transposase into pig fetal fibroblasts (pFF) harboring a mouse OCT4-GFP reporter construct (Nowak-Imialek et al., 2010). Both the basic transposon and transposase vectors were generously provided by Dr. Zoltan Ivics from Paul Ehrlich Institute, Langen, Germany. In each experiment, 2 × 106 pFF were electroporated with 3 µg of each transposon together with 0.5 µg of SB100X. Two days after transfection, the cells were transferred to mouse embryonic fibroblast (MEF) feeders and cultured with iPSC medium [DMEM with antibiotics, nonessential amino acids, 20% Knockout serum replacement, 5 ng mL–1 human recombinant basic fibroblast growth factor (bFGF), and 1000 U mL–1 ESGRO]. Two weeks post-transfection, multiple compact colonies were apparent (mean = 2195; SEM = 166; n = 3), which were 95% alkaline phosphatase-positive and ~80% expressed the OCT4-GFP reporter. Reverse transcription-PCR showed that these colonies expressed high levels of endogenous OCT4, SOX2, NANOG, REX1, UTF1, CDH1, and TDH. The cultures were passaged by trypsin disaggregation, followed by seeding on fresh feeders at density 10 × 103 cells cm–2. The established cell lines proliferated as compact, mouse iPSC-like colonies that retained their OCT4 reporter expression as well as the expression of the endogenous pluripotency genes for at least 30 passages. The expression of the transgenes was persistent and showed that no silencing had occurred, even in long-term culture. When subjected to in vitro differentiation protocols, the putative iPSC formed mainly large trophectodermal (TE) vesicles (positive for TE markers CDX2, PAG, and HAND1), fibroblast-like, and neuronal-like cells. These cells still expressed the transgenes as well as most endogenous pluripotency markers, demonstrating limited differentiation capacity. Because the stable transgene expression and the suboptimal culture conditions are the most likely causes of this limited differentiation potential, we are currently working on generating transgene-free iPSC lines under improved cell culture conditions.

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A Curcumin Derivative Activates TFEB and Protects Against Parkinsonian Neurotoxicity in Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21041515 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1515 ◽

Cited By ~ 2

Author(s):

Ziying Wang ◽

Chuanbin Yang ◽

Jia Liu ◽

Benjamin Chun-Kit Tong ◽

Zhou Zhu ◽

...

Keyword(s):

Transcription Factor ◽

Animal Models ◽

Small Molecule ◽

Therapeutic Target ◽

Cell Models ◽

Neuroprotective Effects ◽

Lysosome Biogenesis ◽

Transcription Factor Eb ◽

Curcumin Derivative

TFEB (transcription factor EB), which is a master regulator of autophagy and lysosome biogenesis, is considered to be a new therapeutic target for Parkinson’s disease (PD). However, only several small-molecule TFEB activators have been discovered and their neuroprotective effects in PD are unclear. In this study, a curcumin derivative, named E4, was identified as a potent TFEB activator. Compound E4 promoted the translocation of TFEB from cytoplasm into nucleus, accompanied by enhanced autophagy and lysosomal biogenesis. Moreover, TFEB knockdown effectively attenuated E4-induced autophagy and lysosomal biogenesis. Mechanistically, E4-induced TFEB activation is mainly through AKT-MTORC1 inhibition. In the PD cell models, E4 promoted the degradation of α-synuclein and protected against the cytotoxicity of MPP+ (1-methyl-4-phenylpyridinium ion) in neuronal cells. Overall, the TFEB activator E4 deserves further study in animal models of neurodegenerative diseases, including PD.

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Disruption of IRElα through its Kinase Domain Attenuates Multiple Myeloma

10.1101/495242 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jonathan M Harnoss ◽

Adrien Le Thomas ◽

Scot A Marsters ◽

David A Lawrence ◽

Min Lu ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

Small Molecule ◽

Therapeutic Target ◽

Plasma Cells ◽

Critical Role ◽

Kinase Domain ◽

Kinase Inhibition

AbstractMultiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138− cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1α inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1α with kinase-based small-molecule inhibitors in MM.Significance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1α mitigates ER stress by expanding the ER’s protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1α elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1α as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1α-XBP1s pathway plays a critical role in MM growth. We further show that IRE1α’s kinase domain is an effective and safe potential small-molecule target for MM therapy.

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Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification

Chemical Communications ◽

10.1039/c8cc01764f ◽

2018 ◽

Vol 54 (38) ◽

pp. 4774-4777 ◽

Cited By ~ 13

Author(s):

Yongpeng Yao ◽

Shanshan Li ◽

Jiaqian Cao ◽

Weiwei Liu ◽

Keqiang Fan ◽

...

Keyword(s):

Transcription Factor ◽

Small Molecule ◽

Strand Displacement ◽

Strand Displacement Amplification

We demonstrate a novel small molecule biosensing strategy by coupling the recognition of aTF with SDA reaction in vitro.

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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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R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma

eLife ◽

10.7554/elife.43026 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 8

Author(s):

Jeroen Overman ◽

Frank Fontaine ◽

Jill Wylie-Sears ◽

Mehdi Moustaqil ◽

Lan Huang ◽

...

Keyword(s):

Transcription Factor ◽

Small Molecule ◽

Therapeutic Potential ◽

Metastatic Cancer ◽

Vascular Diseases ◽

Infantile Hemangioma ◽

Small Molecule Inhibitor ◽

Clinical Model ◽

Molecule Inhibitor

Propranolol is an approved non-selective β-adrenergic blocker that is first line therapy for infantile hemangioma. Despite the clinical benefit of propranolol therapy in hemangioma, the mechanistic understanding of what drives this outcome is limited. Here, we report successful treatment of pericardial edema with propranolol in a patient with Hypotrichosis-Lymphedema-Telangiectasia and Renal (HLTRS) syndrome, caused by a mutation in SOX18. Using a mouse pre-clinical model of HLTRS, we show that propranolol treatment rescues its corneal neo-vascularisation phenotype. Dissection of the molecular mechanism identified the R(+)-propranolol enantiomer as a small molecule inhibitor of the SOX18 transcription factor, independent of any anti-adrenergic effect. Lastly, in a patient-derived in vitro model of infantile hemangioma and pre-clinical model of HLTRS we demonstrate the therapeutic potential of the R(+) enantiomer. Our work emphasizes the importance of SOX18 etiological role in vascular neoplasms, and suggests R(+)-propranolol repurposing to numerous indications ranging from vascular diseases to metastatic cancer.

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