Name: Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome (Video) | JoVE Journal
Uploaded: 2021-12-04T13:30:00.000+00:00
Duration: 9 min 39 s
Description: Learn to generate integration-free iPSCs from Turner syndrome fetal fibroblasts using episomal plasmid nucleofection. This protocol enables neuronal differentiation for modeling neurocognitive deficits.

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5 minutes

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.

<ol><li>Verlinsky, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+embryonic+stem+cell+lines+with+genetic+disorders%5BTitle%5D)+AND+%22Reproductive+BioMedicine+Online%22%5BJournal%5D)">Human embryonic stem cell lines with genetic disorders.</a> Reproductive BioMedicine Online. 10, 105-110 (2005).</li>
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<li>Gravholt, C. H., Viuff, M. H., Brun, S., Stochholm, K., Andersen, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Turner+syndrome%3A+mechanisms+and+management%5BTitle%5D)+AND+%22Nature+Reviews+Endocrinology%22%5BJournal%5D)">Turner syndrome: mechanisms and management.</a> Nature Reviews Endocrinology. 15, 601-614 (2019).</li>
<li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uniparental+disomy+of+the+entire+X+chromosome+in+Turner+syndrome+patient-specific+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Discovery%22%5BJournal%5D)">Uniparental disomy of the entire X chromosome in Turner syndrome patient-specific induced pluripotent stem cells.</a> Cell Discovery. 1, 15022(2015).</li>
<li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+an+induced+pluripotent+stem+cell+line+from+an+adult+male+with+45%2CX%2F46%2CXY+mosaicism%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">Generation of an induced pluripotent stem cell line from an adult male with 45,X/46,XY mosaicism.</a> Stem Cell Research. 27, 42-45 (2018).</li>
<li>Parveen, S., Panicker, M. M., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+an+induced+pluripotent+stem+cell+line+from+chorionic+villi+of+a+Turner+syndrome+spontaneous+abortion%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">Generation of an induced pluripotent stem cell line from chorionic villi of a Turner syndrome spontaneous abortion.</a> Stem Cell Research. 19, (2017).</li>
<li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+Pluripotent+Stem+Cells+from+Adult+Human+Fibroblasts+by+Defined+Factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors.</a> Cell. 131, 861-872 (2007).</li>
<li>Soldner, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parkinson%27s+Disease+Patient-Derived+Induced+Pluripotent+Stem+Cells+Free+of+Viral+Reprogramming+Factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors.</a> Cell. 136, 964-977 (2009).</li>
<li>Somers, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+Transgene-Free+Lung+Disease-Specific+Human+Induced+Pluripotent+Stem+Cells+Using+a+Single+Excisable+Lentiviral+Stem+Cell+Cassette%5BTitle%5D)+AND+%22STEM+CELLS%22%5BJournal%5D)">Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette.</a> STEM CELLS. 28, 1728-1740 (2010).</li>
<li>Woltjen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((piggyBac+transposition+reprograms+fibroblasts+to+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.</a> Nature. 458, 766-770 (2009).</li>
<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>
<li>Warren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+Efficient+Reprogramming+to+Pluripotency+and+Directed+Differentiation+of+Human+Cells+with+Synthetic+Modified+mRNA%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA.</a> Cell Stem Cell. 7, 618-630 (2010).</li>
<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>
<li>Ambartsumyan, G., Clark, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+and+early+human+embryo+development%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">Aneuploidy and early human embryo development.</a> Human Molecular Genetics. 17, 10-15 (2008).</li>
<li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+cerebral+organoids+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Generation of cerebral organoids from human pluripotent stem cells.</a> Nature Protocols. 9, 2329-2340 (2014).</li>
</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 earl...

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

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&amp;b). We used nonintegrating episomal vectors and nucleofection for the transfe...

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.

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

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This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

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.

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

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.

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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

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

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.

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

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

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.

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

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

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.

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

This protocol outlines a methodology for recapitulating Down syndrome (DS) impaired neurogenesis using DS human iPSCs. The protocol identified biphasic cell cycle defect as the cause of impaired neurogenesis in Down syndrome. It provides a robust platform for understanding cellular and molecular mechanisms underlying the abnormal neurogenesis associated with DS.

In Vitro Modeling of Down Syndrome Neurogenesis Using Human-Induced Pluripotent Stem Cells

Here we describe a protocol that facilitates the differentiation of human induced pluripotent stem cells into functional forebrain-specific astrocytes. This enables investigations into the role of glial cells in the pathogenesis of neurodevelopmental disorders, such as Fragile X Syndrome, and modeling of other brain disorders.

Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein

Learn to generate integration-free iPSCs from Turner syndrome fetal fibroblasts using episomal plasmid nucleofection. This protocol enables neuronal differentiation for modeling neurocognitive deficits.

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

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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