Financial support for the above research was provided by Manipal Academy of Higher Education. Characterization of the line was conducted partially in the laboratory of M. M. Panicker at NCBS. We thank Anand Diagnostic Laboratory for assistance with karyotyping.

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<li>Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+Pluripotent+Stem+Cells+Generated+Without+Viral+Integration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced Pluripotent Stem Cells Generated Without Viral Integration.</a> Science. 322, 945-949 (2008).</li>
<li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus%2C+an+RNA+virus+that+does+not+integrate+into+the+host+genome%5BTitle%5D)+AND+%22Proceedings+of+the+Japan+Academy%2C+Series+B%22%5BJournal%5D)">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proceedings of the Japan Academy, Series B. 85, 348-362 (2009).</li>
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<li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human+induced+pluripotent+stem+cells+by+direct+delivery+of+reprogramming+proteins%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li>
<li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Induced+Pluripotent+Stem+Cells+Free+of+Vector+and+Transgene+Sequences%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences.</a> Science. 324, 797-801 (2009).</li>
<li>Okita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+more+efficient+method+to+generate+integration-free+human+iPS+cells%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8, 409-412 (2011).</li>
<li>Martí, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Characterization of pluripotent stem cells.</a> Nature Protocols. 8, 223-253 (2013).</li>
<li>Avior, Y., Sagi, I., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pluripotent+stem+cells+in+disease+modelling+and+drug+discovery%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Pluripotent stem cells in disease modelling and drug discovery.</a> Nature Reviews Molecular Cell Biology. 17, 170-182 (2016).</li>
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</ol>

The authors have nothing to disclose.

Generation of stable cellular models of cytogenetically abnormal fetal tissue is necessary for perpetuating defective phenotype. The iPSC route is the most effective method of cell preparation for perpetual conservation of defective properties20.
Pluripotent stem cells (PSC) display properties of self-renewal and differentiation into specialized cells reminiscent of early cleavage embryos21. Hence, PSCs can serve as excellent models to study early molecular, cellular and developmental defects in prematurely aborted fetuses.
In this article we have described human iPSC generation using nucleofection combined with the improved episomal vectors. The results show that this combination comprises a robust method for generating integration-free human iPSC lines as evidenced by the fact that single transfections were sufficient for successful reprogramming. We tracked the progressive conversion of FCV fibroblasts to pluripotent cells microscopically. 20 days post transfection we observed colonies of reprogrammed TSiPSCs surrounded with non-reprogrammed FCV fibroblasts. Morphologically, the derived human iPSCs resembled embryonic stem cells grown alongside in the lab. Typically, the cells aggregated as compact colonies with shiny borders. The cells of the colonies had large nuclei and tightly packed suggesting close membrane contact between the cells. The non-reprogrammed fibroblasts arched and surrounded these colonies. Upon transfer to iMEFs they continue proliferate in culture for over 30 transfers demonstrating the property of continued self-renewal.
As TSiPSCs were generated from 45XO fibroblasts we karyotyped the cells to check if they retained the chromosomal composition. The TSiPSCs maintained the 45XO karyotype in cell continuous culture suggesting a stable 45XO chromosome genetic makeup. To be useful as cellular resource representing 45XO aneuploidy the TSiPSCs should be free of exogenous DNA used in the reprogramming experiments. We checked to the presence of residual episomal plasmids by performing a genomic DNA PCR for episomal specific markers-OriP and EBNA. We found no trace of these markers in TSiPS cells after 15 passages suggesting that the TSiPSCs progressively lost episomal reprogramming vectors in prolonged culture.
The hallmark of a pluripotent cells is its potential to differentiate to cells of three germ lineages both in vitro and in vivo. To test this capability in the derived TSiPSCs we subjected them in vitro to embryoid body formation and differentiation assays directed by lineage specifying cytokines and growth factors. TSiPSCs formed embryoid bodies and differentiated into ectodermal cells expressing neuronal markers, mesodermal cells expressing cardiac markers and endodermal cells expressing SOX17 a biomarker of endoderm fate. We also tested the ability of TSiPSCs to differentiate into higher order 3D cerebral organoids using previously established protocols22. TSiPSCs progressively self-organise due to their own intrinsic developmental programs into mini tissues called organoids. TSiPSCs yielded cerebral organoids showing a cytoarchitecture similar to brain tissue with neuroepithelium surrounding a ventricle like cavity. However these organoids have to be further characterised extensively to reveal the exact cell types and compared with normal iPSCs to distinguish the intrinsic neural tissue patterning properties of TSiPSCs. These cerebral organoids and other types of brain organoids generated from TSiPSCs can be used to model developmental and functional inconsistencies that may contribute to the symptoms of neurological deficiencies of TS individuals.TSiPSCs exhibited biomarker characteristics of pluripotency as well as the hallmark trait of differentiation thus highlighting the success of reprogramming to induced pluripotency.
The above-described method has work efficiently in reprogramming dermal fibroblasts and mesenchymal cells derived from various sources in our lab (data of other lines not shown). In our experience, the following steps are critical for the success of the reprogramming experiment:
a) Quality of plasmid preparation: old preparations do not yield iPSCs. 
b) Quality of cells used for transfections: proliferating cells are essential for iPSC generation. 0.5 to 1 million cells per transfection yielded a reproducible reprogramming efficiency. 
c) Freshly reconstituted nucleofector reagents: reconstituted nucleofector reagents stored for over a month did not yield iPSCs. 
d) Maintenance of master cell bank by mechanical subculture of the iPSCs yielded stable lines. Enzymatic dissociation was used as per experiment requirement.
The future aim of the lab is to establish a panel of chromosomally abnormal iPSCs for downstream development, functional and disease modelling using this efficient method. Fetal aneuploidies cause pregnancy loss and organ malformations in live births. Aneuploid iPSCs derived from tissues of spontaneous abortuses are a valuable resource to model and study failed embryonic developmental events. In vitro 2D and 3D culture systems including embryoid bodies and tissue specific organoids22 will enable researchers to understand molecular and cellular irregularities such as aberrant cell proliferation and cell death in lineage specific cells that could manifest as developmental anomalies and pregnancy failures associated with aneuploidy syndromes.

Aneuploidies lead to birth defects/congenital malformations and pregnancy loss in humans. ~50%-70% of specimens from pregnancy losses show cytogenetic abnormalities. Aneuploid embryos lost early in pregnancy cannot be easily obtained for experimental analysis raising the need to develop other models closely representing human embryogenesis. Induced pluripotent stem cells (iPSCs) derived from cells diagnosed with genetic disorders have been used to model the representative genetic irregularities and their consequence on fetal development1,2,3,4. These iPSCs resemble epiblast cells of the developing embryo and can recapitulate the early events of embryo formation. They allow understanding and characterization of the developmental program of cell lineages and patterning in early mammalian embryos. iPSCs derived previously from skin fibroblasts and amniocytes from prenatal diagnostic tests of aneuploidy syndromes like monosomy X (Turner syndrome), trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome) and partial trisomy 11; 22 (Emanuel syndrome) have provided valuable insights regarding failed development4.
Turner syndrome (TS) is a rare condition characterized by female infertility, short stature, endocrine and metabolic disorders, an increased risk of autoimmune disease, and a predisposition to cardiovascular disease5. Though it is the only survivable monosomy syndrome it is also lethal to the developing embryo causing spontaneous abortions6. Surviving individuals with TS present with degrees of alteration of X-chromosomal material in their cells. Karyotypes range from complete loss of one X chromosome (45,XO) to mosaics like 45,XO/46,XX; 45,XO/47,XXX, the presence of ring chromosomes, the presence of Y-chromosomal material, etc5.
Diagnosis of the syndrome is generally done by karyotyping blood of symptomatic individuals and chorionic villi sampling (CVS) to detect early aneuploidy syndromes. Since aneuploidy syndromes account for ~30% of spontaneous abortions, it is routine to karyotype the product of conception (POC) upon a spontaneous abortion. These fetal cells including the chorionic villi possessing the cytogenetic abnormality and iPSCs derived from them provide a valuable source of biological material to study aneuploidy syndromes4,6. TS iPSCs have been previously established from amniocytes via retroviral reprogramming4, fibroblasts of chorionic villi (obtained through prenatal diagnosis)&#160;via retroviral reprogramming6, from blood mononuclear cells7 via Sendai virus reprogramming and from skin fibroblasts of TS individuals via lentiviral reprogramming4. Since the primary focus of our lab is to understand developmental failure, we have generated TS iPSCs from POC, specifically the chorionic villi component of a spontaneous abortion8. All the cells isolated from this fetal tissue had a 45XO karyotype and yielded iPSCs with the same karyotype. These iPSCs are unique as they are the first to be generated from an aborted fetus and provide a valuable resource to study aneuploidy related pregnancy failures. This article provides a detailed methodology of the generation of iPSCs from this unique cell source via episomal reprogramming.
The early methods of iPSC generation used viral transduction and transposons to deliver the reprogramming factors. Methods of inducing cells to pluripotency have evolved from using integrating retroviral vectors9, excisable lentiviral vectors10,11 and transposon-based methods12 to non-integrating adenoviral vectors13 and Sendai virus based vectors14. Retroviral and lentiviral based reprogramming, although efficient, involve integration of the reprogramming factors into the host chromosomes, causing insertion mutations which have unforeseen effects in the iPSCs. Furthermore, viral-based reprogramming prevents translational application of iPSCs. RNA-based systems15 and direct protein delivery16 have been explored to completely eliminate the potential risks associated with the use of viruses and DNA transfections. However, these methods have proven inefficient.
In 2011, Okita et al. reported improved efficiency of reprogramming by episomal plasmids augmented with TP53 suppression via shRNA. They also replaced cMYC with non-transforming LMYC (small cell lung carcinoma associated MYC) to enhance safety of the hiPSCs. These episomal plasmids express 5 reprogramming factors: OCT4, LIN28, SOX2, KLF4, LMYC and shRNA for TP5317,18. These vectors are maintained extra-chromosomally and lost from the reprogrammed cells upon continuous culture, thus making the lines transgene-free within 10-15 passages. Nucleofection is a specialized form of electroporation that delivers nucleic acids directly into the nucleus of host cells. It is an efficient method for delivery of the reprogramming plasmids into various cell types. Episomal plasmids are cost effective and compensate the high costs of nucleofection. This method is efficient and reproducible under optimized conditions yielding stable iPSCs from a variety of somatic cells. In this protocol, we describe the method for generation of iPSCs from fibroblasts isolated from fetal tissue by nucleofection of episomal reprogramming plasmids. Here are the detailed protocols for fibroblast isolation from fetal chorionic villi, plasmid purification, nucleofection, picking of colonies from the reprogramming plate and establishment of stable iPSCs.
It is essential to confirm the presence of pluripotency traits in the newly generated iPSCs. This includes demonstration of pluripotency related factors (e.g., alkaline phosphatase expression, NANOG, SSEA4, Tra 1-80, Tra 1-81, E-cadherin; usually shown with immunofluorescence or gene expression assays), identification of the three germ layers by in vitro differentiation assays to validate their differentiation potentials, karyotyping to determine chromosomal content, STR typing to establish identity with parent cells, verify loss of exogenous genes, and more stringent in vivo assays such as teratoma formation and tetraploid complementation. Here we describe characterization protocols of karyotyping, live cells alkaline phosphatase staining, detection of pluripotency related biomarkers by immunofluorescence, in vitro differentiation assays and method to demonstrate loss of exogenous genes19.

<table><tbody><tr><td>0.15% trypsin</td><td>Thermo Fisher Scientific</td><td>27250018</td><td>G Banding</td></tr><tr><td>2-mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>21985023</td><td>Pluripotency and Embryoid body medium</td></tr><tr><td>4&#039;, 6 diamidino-2-phenylindole</td><td>Sigma Aldrich</td><td>D8417</td><td>Immunocytochemistry</td></tr><tr><td>Activin A</td><td>Sigma Aldrich</td><td>SRP3003</td><td>Differentiation assays</td></tr><tr><td>Alkaline Phosphatase Live Stain</td><td>Thermo Fisher Scientific</td><td>A14353</td><td>AP staining</td></tr><tr><td>AMAXA Nucleofector II</td><td>Lonza</td><td>-</td><td>Nucleofection</td></tr><tr><td>AmnioMAX II complete media</td><td>Thermo Fisher Scientific, Gibco</td><td>11269016</td><td>Medium specific for foetal chorionic villi cell cultures</td></tr><tr><td>Ampicillin</td><td>HiMedia</td><td>TC021</td><td>Plasmid purification</td></tr><tr><td>Anti Mouse IgG (H+L) Alexa Fluor 488</td><td>Invitrogen</td><td>A11059</td><td>Immunocytochemistry</td></tr><tr><td>Anti Rabbit IgG (H+L) Alexa Fluor 488</td><td>Invitrogen</td><td>A11034</td><td>Immunocytochemistry</td></tr><tr><td>Anti Rabbit IgG (H+L) Alexa Fluor 546</td><td>Invitrogen</td><td>A11035</td><td>Immunocytochemistry</td></tr><tr><td>Antibiotic-Antimycotic</td><td>Thermo Fisher Scientific, Gibco</td><td>15240096</td><td>Contamination control</td></tr><tr><td>Anti-E-Cadherin</td><td>BD Biosciences</td><td>610181</td><td>Immunocytochemistry</td></tr><tr><td>Anti-Nanog</td><td>BD Biosciences</td><td>560109</td><td>Immunocytochemistry</td></tr><tr><td>Anti-OCT3/4</td><td>BD Biosciences</td><td>611202</td><td>Immunocytochemistry</td></tr><tr><td>Anti-SOX17</td><td>BD Biosciences</td><td>561590</td><td>Immunocytochemistry</td></tr><tr><td>Anti-SOX2</td><td>BD Biosciences</td><td>561469</td><td>Immunocytochemistry</td></tr><tr><td>Anti-SSEA4</td><td>BD Biosciences</td><td>560073</td><td>Immunocytochemistry</td></tr><tr><td>Anti-TRA 1-81</td><td>Millipore</td><td>MAB4381</td><td>Immunocytochemistry</td></tr><tr><td>basic Fibroblast Growth Factor[FGF2]</td><td>Sigma Aldrich</td><td>F0291</td><td>Pluripotency medium</td></tr><tr><td>Bone Morphogenetic Factor 4</td><td>Sigma Aldrich</td><td>SRP3016</td><td>Differentiation assays</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A3059</td><td>Blocking</td></tr><tr><td>Collagen Human Type IV</td><td>BD Biosciences</td><td>354245</td><td>Differentiation assays</td></tr><tr><td>Collagenase blend</td><td>Sigma Aldrich</td><td>C8051</td><td>Digestion of foetal chorionic villi</td></tr><tr><td>Dexamethasone</td><td>Sigma Aldrich</td><td>D4902</td><td>Differentiation assays</td></tr><tr><td>DMEM F12</td><td>Thermo Fisher Scientific</td><td>11320033</td><td>Differentiation assays</td></tr><tr><td>FastDigest EcoR1</td><td>Thermo Scientific</td><td>FD0274</td><td>Restriction digestion</td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F2518</td><td>Differentiation assays</td></tr><tr><td>Giemsa Stain</td><td>HiMedia</td><td>S011</td><td>G Banding</td></tr><tr><td>Glacial Acetic Acid</td><td>HiMedia</td><td>AS001</td><td>Fixative for karyotyping</td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>G7528</td><td>Differentiation assays</td></tr><tr><td>GlutaMAX</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Pluripotency and Embryoid body medium</td></tr><tr><td>Heparin sodium</td><td>Sigma Aldrich</td><td>H3149</td><td>Differentiation assays</td></tr><tr><td>Insulin solution human</td><td>Sigma Aldrich</td><td>I9278</td><td>Differentiation assays</td></tr><tr><td>Insulin Transferrin Selenite</td><td>Sigma Aldrich</td><td>I1884</td><td>Differentiation assays</td></tr><tr><td>KAPA HiFi PCR kit</td><td>Kapa Biosystems</td><td>KR0368</td><td>OriP, EBNA1 PCR</td></tr><tr><td>KaryoMAX Colcemid</td><td>Thermo Fisher Scientific</td><td>15210040</td><td>Mitotic arrest for karyotyping</td></tr><tr><td>KnockOut DMEM</td><td>Thermo Fisher Scientific</td><td>10829018</td><td>Pluripotency and Embryoid body medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828028</td><td>Pluripotency and Embryoid body medium</td></tr><tr><td>Luria Bertani agar</td><td>HiMedia</td><td>M1151F</td><td>Plasmid purification</td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>356234</td><td>Differentiation assays</td></tr><tr><td>MEM Non-essential amino acids</td><td>Thermo Fisher Scientific</td><td>11140035</td><td>Pluripotency and Embryoid body medium</td></tr><tr><td>Methanol</td><td>HiMedia</td><td>MB113</td><td>Fixative for karyotyping</td></tr><tr><td>Myosin ventricular heavy chain &amp;alpha;/&amp;beta;</td><td>Millipore</td><td>MAB1552</td><td>Immunocytochemistry</td></tr><tr><td>NHDF Nucleofector Kit</td><td>Lonza</td><td>VAPD-1001</td><td>Nucleofection</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>P6148</td><td>Fixing cells</td></tr><tr><td>pCXLE-hOCT3/ 4-shp53-F</td><td>Addgene</td><td>27077</td><td>Episomal reprogramming Plasmid</td></tr><tr><td>pCXLE-hSK</td><td>Addgene</td><td>27078</td><td>Episomal reprogramming Plasmid</td></tr><tr><td>pCXLE-hUL</td><td>Addgene</td><td>27080</td><td>Episomal reprogramming Plasmid</td></tr><tr><td>Penicillin Streptomycin</td><td>&amp;nbsp; Thermo Fisher Scientific,</td><td>&amp;nbsp;15070063</td><td>Pluripotency and Embryoid body medium</td></tr><tr><td>Phalloidin- Tetramethylrhodamine B isothiocyanate</td><td>Sigma Aldrich</td><td>P1951</td><td>Immunocytochemistry</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td>1 X PBS 1 tablet of PBS dissolved in 200mL of deionized water and sterilized by autoclaving&lt;br/&gt; Storage: Room temperature.&lt;br/&gt; PBST- 0.05% Tween 20 in 1X PBS.&lt;br/&gt; Storage: Room temperature.</td></tr><tr><td>Plasmid purification Kit- Midi prep</td><td>QIAGEN</td><td>12143</td><td>Plasmid purification</td></tr><tr><td>Potassium Chloride Solution</td><td>HiMedia</td><td>MB043</td><td>Hypotonic solution for karyotyping</td></tr><tr><td>QIAamp DNA Blood Kit</td><td>Qiagen</td><td>51104</td><td>Genomic DNA isolation</td></tr><tr><td>RPMI 1640</td><td>Thermo Fisher Scientific</td><td>11875093</td><td>Hepatocyte differentiation medium</td></tr><tr><td>Sodium Citrate</td><td>HiMedia</td><td>RM255</td><td>Hypotonic solution for karyotyping</td></tr><tr><td>Triton X-100</td><td>HiMedia</td><td>MB031</td><td>Permeabilisation</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Thermo Fisher Scientific, Gibco</td><td>25300054</td><td>Subculture of &amp;nbsp;foetal chorionic villi fibroblasts</td></tr><tr><td>Tween 20</td><td>HiMedia</td><td>MB067</td><td>Preparation of PBST</td></tr><tr><td>&amp;beta; III tubulin</td><td>Sigma Aldrich</td><td>T8578</td><td>Immunocytochemistry</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Sigma Aldrich</td><td>Y0503</td><td>Differentiation assays</td></tr></tbody></table>

generation of induced pluripotent stem cells from turner syndrome (45xo) fetal cells for downstream modelling of neurological deficits associated with the syndrome

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening is purely diagnostic and does not elucidate disease mechanism. Although cells from aneuploid fetuses are valuable biological material bearing the chromosomal aneuploidy, these cells are short lived, limiting their use for downstream research experiments. Generation of induced pluripotent stem cell (iPSC) models is an effective method of cell preparation for perpetual conservation of aneuploid traits. They are self-renewing and differentiate into specialized cells reminiscent of embryonic development. Thus, iPSCs serve as excellent tools to study early developmental events. Turner syndrome (TS) is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, endocrine, metabolic, autoimmune and cardiovascular disorders and neurocognitive defects. The following protocol describes isolation and culturing of fibroblasts from TS (45XO) fetal tissue, generation of integration free TSiPSCs through delivery of episomal reprogramming plasmids by nucleofection followed by characterization. The reprogramming TSiPSCs were initially screened by live cell alkaline phosphatase staining followed by extensive probing for pluripotency biomarkers. Selected colonies were mechanically dissected, passaged several times and stable self-renewing cells were used for further experiments. The cells expressed pluripotency transcription factors OCT4, NANOG, SOX2, cell surface markers SSEA 4 and TRA1-81 typical of pluripotent stem cells. The original 45XO karyotype was retained post reprogramming. The TSiPSCs were able to form embryoid bodies and differentiate into cells of endoderm, mesoderm and ectoderm expressing lineage specific biomarkers ((SRY BOX17), (MYOSIN VENTRICULAR HEAVY CHAIN&#945;/&#946;), (&#946;III TUBULIN)). The exogenous episomal plasmids were lost spontaneously and not detected after passage 15 in cells. These TSiPSCs are a valuable cellular resource for modelling defective molecular and cellular neurodevelopment causing neurocognitive deficits associated with Turner syndrome.

Generation of integration-free iPSCs from a spontaneously aborted fetus with 45XO karyotype 
We isolated fibroblasts from FCV with a Turner syndrome (TS) specific 45XO karyotype and nucleofected them with episomal reprogramming plasmids to generate TSiPSCs which can be used for downstream modelling of the syndrome, specifically the associated neurological deficits (Figure 1a&#38;b). We used nonintegrating episomal vectors and nucleofection for the transfection experiments (Figure 1 c&#38;d). We followed morphological changes of cells to monitor the success of reprogramming. The shift from the fibroblast to epithelial morphology, followed by a delineated compact colony formation was observed (Figure 2a). TSiPSCs acquired human embryonic stem cell like morphology with distinct edges and a high nucleus-to-cytoplasm ratio around day 20 post transfection (Figure 2b). In contrast, incompletely reprogrammed cells acquire epithelial morphologies but fail to form compact colonies. (Figure 2c).
Characterization of TSiPSCs 
Karyotyping of TSiPSCs revealed the 45XO karyotype associated with Turner Syndrome (Figure 3a). Immunofluorescence of TSiPSCs showed expression of pluripotency transcription factors OCT4, NANOG, SOX2, and cell surface markers SSEA4, E-Cadherin, and TRA-1-81. Human embryonic stem cells are the gold standard&#160;of pluripotent stem cells. We simultaneously performed immunofluorescence of HUES 1 which was used as positive control for comparison of pluripotency biomarker expression by TSiPSC (Figure 3b). Transgene free status of the TSiPSCs was demonstrated by a genomic DNA PCR for episomal plasmid markers OriP and EBNA. By passage 15, OriP and EBNA gene were lost in the TSiPSCs.The episomal genes OriP and EBNA were amplified and showed bands in passage 9 TSiPSCs indicating the presence of the episomal plasmids at this stage. However, these genes were not amplified in passage 15 TSiPSCs indicating a loss of the episomal plasmids and hence a transgene free state (Figure 3c).
In vitro differentiation assays 
The differentiation potential of TSiPSC lines was demonstrated in vitro. TSiPSCs upon aggregation in low attachment plates formed embryoid bodies (Figure 4a). Growth factor induced differentiation of TSiPSCs was used to generate cell types of the three germ layers. Immunofluorescence analysis using lineage specific biomarkers confirmed that TSiPSCs differentiated into representative derivatives of endoderm (SOX17), mesoderm (MYOSIN VENTRICULAR HEAVY CHAIN&#945;/&#946;) and ectoderm (&#946;III TUBULIN) (Figure 4b).
Cerebral organoid differentiation. 
TSiPSCs were differentiated as cerebral organoids in a stage wise manner. Single cell suspensions of TSiPSCs were aggregated into embryoid bodies to stimulate development of germ layers for initial 6 days followed by induction of neuroepithelial development for 5 days. The neuroepithelial aggregated were them embedded in Matrigel which provided the extracellular matrix and basement membrane components which support proper apicobasal orientation, outgrowth of neuroepithelial buds which expand and form lumens. Immunofluorescence with neuroepithelial marker NESTIN was performed to observe the overall morphology of the organoids (Figure 5b). The neuroepithelium surrounds a ventricle like cavity (Figure 5c - white line). The organoids morphologically display ventricular zones (VZ), sub ventricular zone (SVZ) and cortex like regions (Figure 5c - red, orange and yellow lines respectively)
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62240/62240fig01.jpg" /> 
Figure 1: Fibroblast isolation and reprogramming via nucleofection.&#160; (a) Microscopic image of fetal chorionic villi prior to collagenase treatment. (b) Fibroblasts isolated from fetal chorionic villi for reprogramming experiments. (c) Verification of reprogramming plasmids by EcoRI restriction digestion. (d) Schematic diagram of transfection protocol employed for iPSC generation from fetal chorionic villi fibroblasts using episomal reprogramming plasmids via nucleofection.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/62240fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62240/62240fig02.jpg" /> 
Figure 2: Establishment of Turner Syndrome induced pluripotent stem cells.&#160;(a) Cell morphology changes observed during the time course of reprogramming. (b) A fully reprogrammed TSiPSC colony. (c) A representative image of a colony with improperly reprogrammed cells.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/62240fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62240/62240fig03.jpg" /> 
Figure 3: Characterisation of TSiPSCs.&#160;&#160;(a) Karyotype of TSiPSCs. (b) Immunofluorescence analysis of pluripotency biomarkers OCT4, NANOG, SOX2, SSEA-4 and TRA-1-81 in TSiPSCs compared with embryonic stem cell HUES1.Nuclei are stained with 4&#39;, 6-diamidino-2-phenylindole. 3c. Demonstration of transgene free status of TSiPSCs. Lane 1- DNA ladder, Lane 2- OriP positive control with pCXLE-hSK, Lane 3- EBNA positive control with pCXLE-hSK, Lane 4-OriP with TSiPSCs, Lane 5-EBNA with TSiPSCs.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/62240fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62240/62240fig04.jpg" /> 
Figure 4: In vitro differentiation potential of TSiPSCs.&#160; (a) TSiPSC differentiated to Embryoid Bodies. (b) Immunofluorescence analyses of TSiPSCs for endodermal marker SOX17, mesodermal marker myosin ventricular heavy chain &#945;/&#946; and ectodermal markers &#946;III tubulin and SOX2. Nuclei are stained with 4&#39;, 6-diamidino-2-phenylindole.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/62240fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62240/62240fig05.jpg" /> 
Figure 5: Neuronal and cerebral organoid differentiation of TSiPSCs.&#160; (a) To understand cytoarchitecture of differentiated neurons, phalloidin staining of Actin was done. TSiPSC-derived neurons displayed pyramidal shaped neuronal soma (arrowhead) with dendrites and axons (arrows). Nuclei are stained with 4&#39;, 6-diamidino-2-phenylindole. Immunostaining. (a) Immunostaining for Nestin and actin to observe gross morphology of the organoids. (c) Staining for Nestin to visualize the apically and basally organized neuronal layers.Nuclei are stained with 4&#39;, 6-diamidino-2-phenylindole.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/62240fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of media, buffers, and solutions <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: PCR Reaction Mix <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/Table2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: PCR Cycling Program <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62240/Table3.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndr

Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

This protocol describes the generation of integration free iPSCs from fetal tissue fibroblasts through delivery of episomal plasmids by nucleofection followed by description of methods used for iPSC characterization and neuronal differentiation.

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening ...

generation-induced-pluripotent-stem-cells-from-turner-syndrome-45xo

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.1K Views.  Manipal Academy of Higher Education, Manipal, Karnataka, India-576104. Het syndroom van Turner is een zeldzame aandoening die gepaard gaat met een geheel of gedeeltelijk ontbrekend X-chromosoom. Het syndroom wordt gekenmerkt door onvruchtbaarheid, kleine gestalte, cardiovasculaire aandoeningen en neurocognitieve defecten. In sommige gevallen veroorzaakt het foetale sterfte.Hoewel somatische cellen van aneuploïde foetussen biologisch waardevol zijn, zijn ze van korte duur, waardoor hun gebruik in onderzoek wordt beperkt. Generatie van iPSC is dus een effe...

Video: Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neuroscience

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Biology

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Medicine

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

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