Name: 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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Description: 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

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.

FCV were obtained from Manipal Hospital, Bengaluru, under Ethics Committee of Manipal Hospitals approval.
NOTE: See Table 1 for composition of all buffers and solutions.
1. Isolation of fibroblasts from fetal chorionic villi (FCV)
<ol>
	<li>Sample collection and tissue disintegration in collagenase
	<ol>
		<li>Collect FCV under sterile conditions in phosphate buffered saline (PBS) and transport (at room temperatur...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;, 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 &#945;/&#946;</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>&#160; Thermo Fisher Scientific,</td>
			<td>&#160;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 
			Storage: Room temperature. 
			PBST- 0.05% Tween 20 in 1X PBS. 
			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 &#160;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>&#946; 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 trans...

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 ...

Turner syndrome is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, cardiovascular disorders, and neurocognitive defects. In some cases, it causes fetal death.Although somatic cells of aneuploid fetuses are biologically valuable, they are short lived, thus limiting their use in research. Generation of iPSC is thus an effective method of cell preparation for perpetual conservation of the aneuploid traits. They are self-renewing and can differentiate into specialized cell types reminiscent of early embryonic development.Delivery of non-integrating episomal reprogramming plasmids through nuclear fiction is an efficient and reproducible method for generating completely reprogrammed and stable iPSC's. This method can also be used on cells which are difficult to program. In this protocol we describe regeneration of iPSCs from fetal material with aneupliod.Transfer the collected fetal chorionic villi to a sterile Petri dish. Wash it several times with PBS containing 1X antibiotic antimycotic solution. Remove the PBS completely by pipetting.Add 1 ml collagenase blend to the villi and incubate at 37 degrees Celsius for 10 minutes or till the tissue disintegrates. After incubation, neutralize the collagenase by adding media containing 10%fetal Bovine Serum. Transfer the contents of the Petri dish into a 15 ml tube.Centrifuge the digest to collect the disintegrated villi and released cells. Decant the supernatant carefully. Plate disintegrated villi along with released cells in a T25 culture flask and AmnioMAX media and grow till a confluent fibroblast culture is obtained.Expand fibroblasts and culture to prepare stocks for use in subsequent transfection and characterization experiments. Prepare cultures of alkali containing the reprogramming plasmids. Isolate the plasmids using Midi plasmid purification kit, as per manufacturer's instructions.Re-suspend each pallet in suitable volume of DNAse RNAse free water to obtain a final concentration of 1 microgram per milliliter. Verify the plasmids using ECoR1 restriction digestion kit, following manufacturer's instructions. Trypsinize the chorionic villi fibroblasts.Neutralize, and centrifuge to collect the cells. Decant the supernatant, and re-suspend the cells in 5 ml OPTI-MEM. Count the cells using a hemocytometer, and remove 1 million cells for nucleofection.Centrifuge to obtain cell pallet. Perform nucleofection using Amaxa NHDF Neucleofactor Kit. Prepare neucleofactor reagent by mixing 0.5 mL supplement and 2.25 mL nucleofactor solution provided in the kit.Remove 100 microliters of nucleofactor solution, and add 1 microgram of each plasmid to it. Gently re-suspend 1 million cells in this mix. Transfer the cell DNA suspension into cuvette, ensuring that the sample covers the bottom of the cuvette without any air bubbles.Cap the cuvette, and insert into the holder. Select the nucleofactor program D-23 for high efficiency, and apply. Remove cuvette out of the holder, and add 1 mL of AmnioMAX media.Transfer the contents gently into a tissue culture treated Petri dish, using the pipette provided in the kit. Incubate the cells in a humidified carbon dioxide incubator at 37 degrees Celsius. Maintain the cells in AmnioMAX media for 10 days, and then shift to pluripotency medium for 20 days.For iPSC expansion, manually dissect the embryonic stem cell-like colonies formed in the reprogramming dish using a pulled glass pipette. Propagate iPSCs appropriately, and massage every five to seven days. The iPCSs were positive for pluripotency marker alkaline phosphatase.Generate embryoid bodies by cutting the iPSC colonies into small pieces and plating them in low attachment Petri dishes in embryoid body medium. Ectoderm differentiation is induced by plating day four embryoid bodies in ectoderm differentiation medium. Similarly, mesoderm differentiation is induced by plating day eight embryoid bodies in mesoderm differentiation medium.For inducing endoderm differentiation, culture iPSCs in a monolayer in endoderm differentiation medium. Morphological changes in fibroblasts were monitored over the course of reprogramming. They express epithelial cell-like morphology and formed compact colonies.The cells also had a height nucleus to cytoplasmic ratio, typical of pluripotent cells in culture. The iPSCs possessed a 45 XO carrier type and were positive for pluripotency markers OCT4, NANOG, SOX 2, SSEA 4, TRA-1-81 and E-CADHERIN. The cells were stained for representative germ-layer markers.Turner Syndrome thus develops neurocognitive deficits. Neurons derived from Turner Syndrome iPSC can be an excellent model. This is modeled in a petri plate to understand the new line of normal disassociative Turner Syndrome.

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...