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Transcriptome of the developing Ataxia-Telangiectasia cerebellum: RNA Sequencing of human iPSC-derived cerebellar progenitors

Authors:
Sam Nayler at University of Oxford
Sam Nayler
Darya Vanichkina at The University of Sydney
Darya Vanichkina
Refik Kanjhan at The University of Queensland
Refik Kanjhan
Jian Sun
Jian Sun
Jian Sun
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Abstract

We report the use of patient-derived induced pluripotent stem cells (iPSCs) to generate neuronal-like cells similar to those affected in the neurological disorder Ataxia-Telangiectasia (A–T), namely Purkinje and granule cells of the cerebellum. Generation of cerebellar-like cells from murine ESCs and human embryonic stem cells has previously been reported, however isolation and manipulation of discrete sub-populations of cerebellar neuronal cells has proven challenging. Building on these studies, we successfully induce widescale expression of mid–hindbrain markers EN1 and GBX2, and the transcription factors MATH1 and PTF1, which demarcate rhombic lip (granule cell) and ventricular zone (Purkinje, Golgi, and Stellate cell) progenitors, respectively, We then expand these progenitors to produce cells that are morphologically similar to granule cells, which also express markers characteristic of this cell type. To gain insight into the early events that occur during the formation of the cerebellum and how these may be affected in the absence of ATM, RNA sequencing of neuronal progenitors was performed. After 34 days of differentiation, both control and AT samples downregulated pluripotency genes and upregulated neural commitment and anterior/posterior patterning gene programs, including EN1, ISL1, MEIS1, SHH, REELIN, LHX9, WNTLESS, NFIX and members of the HOX gene family. Previously characterized pluripotency-regulating and neurogenesis-associated long non-coding RNAs and small RNA precursors were identified as differentially expressed during development, and novel transcripts with no previous reports of neurological function were observed. Comparative pathway analysis between control and AT neurons revealed gene expression patterns indicative of neurological disease, in particular progressive motor neuropathy and cerebellar ataxia, major hallmarks of A–T. This exemplifies the concept that iPS cells can be used for disease modeling purposes and presents a unique view on a window of human brain development, which has not been previously investigated. We were able to detect gene expression evidence that is congruous with a number of prominent theories based on other cellular systems and animal models regarding the nature of the neurodegeneration in A–T including dysregulation of genes involved with ROS, DNA repair and cell cycle regulation, and neuronal long term potentiation. Overall, we report validation of a methodology to generate a mixed population of cerebellar-like cells, and provide a deep transcriptomic characterization of the pluripotent cells and neurons differentiated from them. These data are a valuable resource for researchers to formulate and test hypotheses regarding the early developmental events that occur in the absence of ATM. It does however represent only a snapshot in time and ideally, further timepoints, clones and patients/ATM mutants should be sequenced in order to garner more statistical confidence.
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7 Transcriptome of the developing Ataxia-Telangiectasia
cerebellum: RNA Sequencing of human iPSC-derived
cerebellar progenitors
Sam Nayler1,2, Darya Vanichkina3*, Refik Kanjhan4, Jian Sun1,
Othmar Korn1, Ryan Taft3,5,6, Martin Lavin2, Ernst Wolvetang1*
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane,
QLD 4072, Australia
We repor t th e us e of patient- derived indu ced plu ripotent stem cell s (iPSCs) to g enera te neurona l-like cells
similar to those affected in the neurological d isorder Ataxia-Telang iectas ia (A–T), namely Purkinje and granul e
cells of the cerebellum. Generation of cerebellar-like cells from murine ESCs and human embryonic stem cells
has previously been repor ted, however isolation and m anipulation of discrete su b-pop ulatio ns of cereb ellar
neuronal cells has proven challenging. Building on these studies, we successfully induce widescale expression of
mid–hindbrain markers EN1 and GBX2, and the transcription factors MATH1 and PTF1, which demarcate rhombic
lip (granu le cell) and vent ricul ar zo ne ( Purkinje, Golgi, and Stella te cell) p rogen itors , respe ctive ly, We then
expand these proge nitors to produce cells t hat are morphologically similar to granule cells, whic h also e xpress
markers characteristic of this cell type. To gain i nsight into the ea rly events that occur dur ing the fo rmation of
the cereb ellum and how these may be affe cted in the a bsence of ATM, RNA sequencing of neuronal p rogenito rs
was perfor med. After 3 4 days of diff erentiati on, bot h con trol and A -T sampl es downregulated pluripo tency
gen es and u pregul ated n eural commitment and an terior/posterior pat terning g ene p rogram s, in cludin g EN1,
ISL1 , MEIS 1, SHH, REELIN, LHX9, WNTLESS , NFIX a nd me mbers of the H OX gen e family. Previously characterized
pluripoten cy-reg ulating a nd ne urogen esis-as sociated long non-coding RNAs and s mall RNA precu rsors were
identified a s dif ferent ially expres sed d uring develo pment , and novel tr anscripts w ith no p revious r eport s of
neurological function were observed.
Comparative pathway analysis between control and A-T neurons revealed gene expression patterns indicative of
neurological dis ease, in par ticular progres sive motor neuropathy and cerebellar ataxia, majo r hallma rks of A–T.
This exemp lifies the concept that iPS cells can b e used for disease modeling purp oses and presents a unique
view on a window of human br ain deve lopmen t, whic h has not been previously i nvestigated . We were able to
detec t gene ex pression evidence that is congruo us with a n umber of p rominent theories bas ed on other cellular
systems and a nimal mod els reg arding the na ture of the n eurode genera tion in A–T includ ing dysregula tion of
genes involved with ROS , DNA rep air and cell c ycle regulation, and neuronal lon g term potentiation. Overall, we
repo rt validat ion of a methodology to genera te a mixe d popul ation of cerebellar-l ike cells , and p rovide a deep
transcriptomic characterization of the pluripotent cells and neurons differentiated from them.
Th ese data a re a valua ble reso urce for research ers to for mula te a nd t est hypo theses regard ing the early
deve lopmental events t hat occur in the absence of ATM. It d oes however repr esent only a snap shot in ti me
and idea lly, further timepoints , clone s and patients/ATM mutants sho uld be sequenced in order to garner mo re
statistical confidence.
... On day 7, the cells were switched to GN induction medium supplemented with Shh and Jag1 and cultured for an additional 5 days, with 50% media changes performed every other day. Importantly, Western blots of whole cell extracts prepared from day-12 cultures revealed the expression of Math1 (also known as Atoh1), a transcription factor required for GN differentiation (Fig. 2, BGN precursors^) [34,41,[61][62][63][64][65] (note, however, that Math1 is not GN-specific). Moreover, day-12 cultures contained large numbers of cells exhibiting a typical GN morphology and expressing NeuN, a marker expressed by cerebellar GNs [66][67][68][69] (Fig. S1) (note, however, that NeuN is not GN-specific). ...
... We referred to other cells in our final mESC-derived cultures as GN-like rather than GNs because we have not shown categorically that they are bona fide GNs. That said, our GN precursor cultures express the transcription factor Math1 (Fig. 2), which, while not GNspecific, is required for GN differentiation [34,41,[60][61][62][63][64][65]. Moreover, cells in our final co-cultures that exhibit the morphology of GNs stain with an antibody to the alpha6 subunit of GABRA6, which is often used as a GN marker [96][97][98] (Fig. 5). ...
An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons
Article
Full-text available
  • Jun 2019
  • CEREBELLUM
While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure–function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCβ4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.
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