Differentiation and characterization of neurons derived from rat iPSCs

Setien, Monica B.; Smith, Kylie R.; Howard, Kaleb; Williams, Kathleen; Suhr, Steve T.; Purcell, Erin K.

doi:10.1016/j.jneumeth.2020.108693

Cited by 9 publications

(7 citation statements)

References 55 publications

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“…Previous protocols have used gelatin-based coats; however, we opted for a dual coating approach using poly-lysine (PLL) and laminin (PLL/Lam). Research has demonstrated that dual coating (PLL/Lam) significantly enhances neuronal differentiation in rat iPSC models [ 57 ] and PC12 models [ 58 ]. This approach has shown superior results compared to single coatings such as gelatin and fibronectin.…”

Section: Discussionmentioning

confidence: 99%

“…This approach has shown superior results compared to single coatings such as gelatin and fibronectin. In fact, gelatin has been reported to be a poor substrate for neurite outgrowth or homogeneous differentiation in iPSC-based models [ 57 ]. On the other hand, Laminin has been shown to improve differentiation and neurite growth [ 59 , 60 ], particularly when used in conjunction with dual coatings [ 57 ].…”

Section: Discussionmentioning

confidence: 99%

“…In fact, gelatin has been reported to be a poor substrate for neurite outgrowth or homogeneous differentiation in iPSC-based models [ 57 ]. On the other hand, Laminin has been shown to improve differentiation and neurite growth [ 59 , 60 ], particularly when used in conjunction with dual coatings [ 57 ]. This rate is consistent between experiments ( Supplementary Table S1 ) and across lines, with the inter experiment conversion rate coefficient of variation being ~10% across all the lines studied.…”

Section: Discussionmentioning

confidence: 99%

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In Vitro Modeling as a Tool for Testing Therapeutics for Spinal Muscular Atrophy and IGHMBP2-Related Disorders

Sierra-Delgado,

Sinha-Ray,

Kaleem

et al. 2023

Biology

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Spinal Muscular Atrophy (SMA) is the leading genetic cause of infant mortality. The most common form of SMA is caused by mutations in the SMN1 gene, located on 5q (SMA). On the other hand, mutations in IGHMBP2 lead to a large disease spectrum with no clear genotype–phenotype correlation, which includes Spinal Muscular Atrophy with Muscular Distress type 1 (SMARD1), an extremely rare form of SMA, and Charcot–Marie–Tooth 2S (CMT2S). We optimized a patient-derived in vitro model system that allows us to expand research on disease pathogenesis and gene function, as well as test the response to the AAV gene therapies we have translated to the clinic. We generated and characterized induced neurons (iN) from SMA and SMARD1/CMT2S patient cell lines. After establishing the lines, we treated the generated neurons with AAV9-mediated gene therapy (AAV9.SMN (Zolgensma) for SMA and AAV9.IGHMBP2 for IGHMBP2 disorders (NCT05152823)) to evaluate the response to treatment. The iNs of both diseases show a characteristic short neurite length and defects in neuronal conversion, which have been reported in the literature before with iPSC modeling. SMA iNs respond to treatment with AAV9.SMN in vitro, showing a partial rescue of the morphology phenotype. For SMARD1/CMT2S iNs, we were able to observe an improvement in the neurite length of neurons after the restoration of IGHMBP2 in all disease cell lines, albeit to a variable extent, with some lines showing better responses to treatment than others. Moreover, this protocol allowed us to classify a variant of uncertain significance on IGHMBP2 on a suspected SMARD1/CMT2S patient. This study will further the understanding of SMA, and SMARD1/CMT2S disease in particular, in the context of variable patient mutations, and might further the development of new treatments, which are urgently needed.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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In Vitro Modeling as a Tool for Testing Therapeutics for Spinal Muscular Atrophy and IGHMBP2-Related Disorders

Sierra-Delgado,

Sinha-Ray,

Kaleem

et al. 2023

Biology

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“…Moreover, as it turned out, using the same method of differentiation into neurons with NGN2 overexpression meant that slightly different cultures were obtained from different protocols. The type and morphology of neurons depends on the specific cultivation conditions, the pluripotent stem cell line used, the presence of growth factors in the culture medium, and the type of substrate [6,[35][36][37]. Thus, the neuronal cultures obtained from rat iPSCs on different substrates (poly-d-lysine, Geltrex, gelatin and polylaminin) were different in morphology and maturity [35].…”

Section: Discussionmentioning

confidence: 99%

“…The type and morphology of neurons depends on the specific cultivation conditions, the pluripotent stem cell line used, the presence of growth factors in the culture medium, and the type of substrate [6,[35][36][37]. Thus, the neuronal cultures obtained from rat iPSCs on different substrates (poly-d-lysine, Geltrex, gelatin and polylaminin) were different in morphology and maturity [35]. NGN2-induced neurons that were cocultured on a Matrigelcoated substrate with mouse glial cells from day 2 of differentiation showed a fairly homogeneous culture, with features of excitatory neurons that expressed telencephal markers representative of 2/3 of the cortical layers.…”

Section: Discussionmentioning

confidence: 99%

IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals

et al. 2021

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The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON–NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

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