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Protocol

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Materials

References

Developmental Biology

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

Published: December 4th, 2021

DOI:

10.3791/62240

1Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104, 2Department of Medical Genetics, Manipal Hospitals
* These authors contributed equally

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 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 CHAINα/β), (βIII 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.

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

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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)

  1. Sample collection and tissue disintegration in collagenase
    1. Collect FCV under sterile conditions in phosphate buffered saline (PBS) and transport (at room temperatur.......

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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&b). We used nonintegrating episomal vectors and nucleofection for the trans.......

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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.......

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

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