Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies

Rosati, Jessica; Ferrari, Daniela; Altieri, Filomena; Tardivo, Silvia; Ricciolini, Claudia; Fusilli, Caterina; Zalfa, Cristina; Profico, Daniela Celeste; Pinos, Francesca; Bernardini, Laura; Torres, Bárbara; Manni, Isabella; Piaggio, Giulia; Binda, Elena; Copetti, Massimiliano; Lamorte, Giuseppe; Mazza, Tommaso; Carella, Massimo; Gelati, Maurizio; Valente, Enza Maria; Simeone, Antonio; Vescovi, Angelo L.

doi:10.1038/s41419-018-0990-2

Cited by 42 publications

(38 citation statements)

References 63 publications

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“…S1B). The quantitative RT-PCR assay further confirmed that the proportion of OTX2 expression was nearly ten-fold higher than that of NANOG and ESRRB in the piPSC-dox line that was generated in our lab and reported previously [29] (Fig. 1B).…”

Section: Conjoint Analysis Of Pipsc Rna and Mirna Transcriptomessupporting

confidence: 86%

“…The piPSC-dox cells generated in this laboratory were cultured in LF2i medium [29], which included DMEM (Hyclone, USA) supplemented with 15% FBS (SeraPro, Germany), 0.1 mM nonessential amino acids (NEAA, Gibico, USA), 1 mM L-glutaMAX (Gibico, USA), 10 ng/mL LIF (Merck Millipore, USA), 10 ng/mL bFGF (PeproTech, USA), 0.1 mM β-mercaptoethanol (β-met, Sigma Aldrich, USA), 3 µM CHIR99021 (StemRD, USA), 2 µM SB431542 (StemRD. USA), 1 μg/mL doxycycline (Dox, Sigma Aldrich, USA).…”

Section: Cell Culturementioning

confidence: 99%

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Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression

et al. 2018

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2019) Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression, RNA Biology, 16:1, 82-92, ABSTRACTPorcine OTX2 was found to be highly activated in porcine iPS cells (piPSCs) that were reported by different laboratories worldwide. To reveal the regulatory function of OTX2 in porcine reprogrammed cells, we screened porcine miRNA-seq databases and found two miRNAs, miR-1343 and miR-545, that could specifically bind to 3ʹUTR of OTX2 and suppress endogenous OTX2 expression in piPSCs. Knockdown of OTX2 by miR-1343 and miR-545 could significantly increase the expression of SOX2 and ESRRB, but did not alter the expressions of OCT4 and KLF4, and improve the pluripotency of piPSCs. The promoter-based assays showed that OTX2 potentially bound to the promoter region of SOX2 and ESRRB and suppressed their expression. On the other hand, SOX2 could interact with OTX2 promoter. Ectopic expression of SOX2 could significantly decrease OTX2 promoter activity, showing that there is a negative feedback loop between SOX2 and OTX2. Additionally, SOX2 and ESRRB significantly stimulated miR-1343 expression in piPSCs, but OTX2 down regulated the expression of miR-1343 in either direct or indirect manners. In summary, this study demonstrates that there is a regulatory network mediated by miR-1343, in which downregulation of OTX2 by miR-1343 can elevate the expression of pluripotent genes that were then sustain the pluripotency of piPSCs. ARTICLE HISTORY

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Section: Conjoint Analysis Of Pipsc Rna and Mirna Transcriptomessupporting

confidence: 86%

Section: Cell Culturementioning

confidence: 99%

Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression

et al. 2018

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“…Further studies are needed to explore this possibility, as it could have implications for future applications. Indeed, we cannot rule out that this contributed to the low in vivo survival in nude mice in our experiment compared with published rates of 40% for iPS‐NSCs or 20% for fetal NSCs 38 …”

Section: Discussionmentioning

confidence: 65%

“…Three animals were sacrificed at 3 weeks to assess survival and proliferation and seven animals were sacrificed at 6 months. Transplantation experiments and analyses were performed as previously described for fetal NSCs 29,38 . For immunohistochemical analysis, brains were perfused‐fixed with ice‐cold 4% paraformaldehyde (Sigma #158127), postfixed overnight, cryoprotected and sectioned in a cryostat.…”

Section: Methodsmentioning

confidence: 99%

Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage

Fernández‐Muñoz

Rosell‐Valle²,

Ferrari

et al. 2020

Stem Cells Translational Medicine

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Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology.

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“…Due to their ability to self-renew and differentiate into the nervous tissue, they offer signi cant therapeutic potential in the treatment of neurological diseases such as spinal cord injury (SCI) [2], Alzheimer's disease (AD) [3], and Multiple sclerosis (MS) [4]. However, many currently available cell lines present us with severe obstacles relating to donor tissue acquisition, heterogeneity, availability, and related technical or ethical issues [5]. Besides,it is essential that cells must be transported from one place to another around the world for research and treatment.…”

Section: Introductionmentioning

confidence: 99%

Generation of UCiPSCs-Derived Neurospheres for Cell Therapy and its Application in Cell Shipment at Ambient Temperature

Zhao

Han

et al. 2020

Preprint

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BackgroundNeural stem cells（NSCs）therapy remains one of the most potential approaches for neurological disorders treatment. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized our technique to cell therapy. Meanwhile, it is often required that NSCs are stored and transported long distances for research or treatment. Although high survival rates could be maintained, conventional methods of cell transport (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore，the establishment of a safe, affordable, and frequent obtained hiPSCs and hiNSCs, with characteristics that match fetal hNSCs and a simple, low-cost way to store and transport, are incredibly urgent. MethodsWe reprogrammed human urinary cells to iPSCs using a virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis and in vitro and in vivo differentiation capabilities, etc).ResultsHere, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique and their differentiation into neural networks. Moreover，hiNSCs survived longer as neurospheres at ambient temperature than those cultured in a monolayer. Approximately 7 days, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to ambient temperature that were placed under standard culture conditions (37 ℃, 5% CO2) recovered their typical morphology, and retained their ability to proliferate and differentiate. ConclusionsIn this study, we provided a simple method for the storage of NSCs as neurospheres at ambient temperature as an alternative to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at ambient temperature and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

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Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies

Cited by 42 publications

References 63 publications

Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression

Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression

Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage

Generation of UCiPSCs-Derived Neurospheres for Cell Therapy and its Application in Cell Shipment at Ambient Temperature

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