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Truncated vitronectin with E-cadherin enables the xeno-free derivation of human embryonic stem cells – Scientific Reports

  • Time Stamp: September 11, 2023
  • Source Node: 26155

Donor testing

Embryo donors were tested for the presence of HIV1/2, hepatitis B, hepatitis C, and syphilis with negative results as previously described19. Testing was performed according to procedures of the CAR University Hospital Brno embryological laboratory (Brno, Czech Republic).

Embryo preparation and transport

Embryos were thawed using Warm Cleave or Warm Blast media (Vitrolife, Västra Frölunda, Sweden) at the CAR University Hospital Brno embryological laboratory (Brno, Czech Republic), a day or two days (according to frozen stages) before the transport as previously described19. For lower embryo stages, the cultivation to the blastocyst stage in Blastocyst Medium (cat. no. G20722, Cook Medical, Bloomington, USA) was performed. After reaching the blastocyst stage, the disruption of the zona pellucida was executed using a laser (OCTAX NaviLase). Prepared embryos in the hatched blastocyst stage were transferred to the Sydney IVF gamete buffer medium (cat. no. G48258, Cook Medical, Bloomington, USA) and transported using a temperature-controlled transport incubator at 37 °C (portable incubator, Minitube).

Derivation

The embryo derivation procedure was initiated immediately upon receipt of the embryos, following established protocols19. Each individual embryo was carefully introduced into a well of a 4-well dish (Thermofisher Scientific, San Jose, CA, USA) filled with pre-warmed Sydney IVF gamete buffer medium, situated on a stereomicroscope plate maintained at 37 °C. Subsequently, the embryo was delicately transferred into a small droplet of Sydney IVF gamete buffer medium (cat. no. G48258, Cook Medical, Bloomington, USA), overlaid with Sydney IVF culture oil (cat. no. G44990, Cook Medical, Bloomington, USA), using a denuding micropipette (cat. 005-300-A, Microtech IVF, Czech Republic). Throughout the process, each embryo was handled separately, employing biopsy micropipettes (cat. 004-35-30A, Microtech IVF, Czech Republic) and holding micropipettes (cat. 001-120-30H, Microtech IVF, Czech Republic). The inner cell mass (ICM) was aspirated using a biopsy micropipette and carefully positioned in parallel with a holding micropipette, slightly overlapping to enable efficient mechanical biopsy using a rapid swinging motion (previously published video: https://www.mdpi.com/1422-0067/23/20/12500#B41-ijms-23-12500). Subsequently, the denuding micropipette (cat. 005-150-C, Microtech IVF, Czech Republic) was employed to manipulate the ICM, which was then transferred into an individual well of a 4-well dish pre-coated with 10.0 µg/mL (1.6 µg/cm2) of recombinant VTN-N (cat. no. A14700, ThermoFisher Scientific), and 2.2 µg/mL (0.34 µg/cm2) of E-cadherin (R&D Systems) diluted in phosphate-buffered saline (PBS). Coating of 4-well dishes with VTN-N/E-cadherin was performed for 1 h at room temperature. Throughout the entire derivation process, the microscope plates were maintained at a constant temperature of 37 °C. E-cadherin was added only for the derivation until the first passage. The derivation medium consisted of NutriStem® hPSC XF Medium (Biological Industries, Beit-Haemek, Israel), supplemented with 20 mg/mL of human serum albumin (Vitrolife), and 10 µM of the ROCK inhibitor (Y27632, GMP, Bio-techne). The derivation medium was used only for the first three days of the derivation, then the NutriStem® hPSC XF Medium was changed daily.

Culture conditions

The derived hESCs were cultured under hypoxic culture conditions (5% O2, 5% CO2, 37 °C) for the first three passages, then under normoxic conditions (5% CO2, 37 °C) in a NutriStem® hPSC XF Medium (Biological Industries) with a daily medium change as previously described19. The cells were passaged mechanically by an insulin syringe (B.Braun) and cultured on 5.0 µg/mL (0.6 µg/cm2) recombinant VTN-N (cat. no. A14700, ThermoFisher Scientific) for the first three passages17. From passage four, non-enzymatic subculturing utilizing 0.5 mM EDTA was conducted once the cell population achieved 70% confluence. To promote single cell survival and facilitate the passaging process, a ROCK inhibitor (Y27632, GMP, Bio-techne) at a concentration of 10 µM was applied, both 1 h before and immediately after the passage for the following 24 h. Following the one-hour treatment with the ROCK inhibitor, the cells were gently washed with phosphate-buffered saline (PBS, Gibco), dissociated into clumps using 0.5 mM EDTA (Invitrogen), and subsequently transferred onto culture surfaces coated with 5.0 µg/mL (0.6 µg/cm2) recombinant VTN-N (cat. no. A14700, ThermoFisher Scientific).

Determination of growth curve and population doubling time

Cells were cultured in 24-well plates at a seeding density of 2 × 104 cells per well and harvested between days 1 and 6 post-seeding. The viability of cells was assessed using the Countess III Automated Cell Counter (Thermo Fisher Scientific) with trypan blue exclusion. A growth curve was constructed to depict the cell population dynamics over time. The proliferation rate, expressed as Population Doubling Time (PDT), was determined at the inflection point of the growth curve. The PDT was calculated using the formula PDT = T ln(2)/ln(A/A0), where T represents the cultivation time in hours, A is the final cell number, A0 corresponds to the initial cell number and ln is natural logarithm20.

Flow cytometry

Cells were washed in phosphate-buffered saline (PBS, Gibco), resuspended in a PBS/EDTA (Invitrogen)/bovine serum albumin (BSA, Pan Biotech) solution, and incubated with antibodies for 10 min at 4 °C, after which the cells were rinsed with PBS, spun for 4 min/300 g, resuspended in PBS, and analyzed by a BD FACS Canto II device (BD Biosciences) using FACSDiva (BD Biosciences) and Flowing Software (The University of Turku) as previously described19. The antibodies used were: human anti-TRA-1-60-PE, 1:75 (cat: 130-122-921, Miltenyi Biotec), human anti-SSEA-4-PE, 1:150 (cat: 130-122-914, Miltenyi Biotec), and human anti-TRA-1-81-APC, 1:90 (cat: 17-8883-42, Thermo Fisher Scientific, San Jose, CA, USA).

EB formation and cell differentiation

Differentiation to the three germ layers was supported by the medium: DMEM/F12 (Gibco), 15% knockout-serum replacement (Gibco), 1% non-essential amino acids (Sigma), 1% Glutamax (Gibco), 1% ZellShield (Minerva Biolabs), and 0.2% 2-Mercaptoethanol (Gibco) as previously described19. The cells were passed into a low attachment 96-well dish (20 × 103 cells/well) and cultured for 1 week until embryoid bodies were formed. The embryoid bodies were then transferred to 4-well dishes coated with 5.0 µg/mL (0.6 µg/cm2) recombinant VTN-N (cat. no. A14700, ThermoFisher Scientific), where they were allowed to attach and culture for another 14 days.

Immunocytochemistry

The cells were fixed using cold 4% paraformaldehyde (Sigma) for a duration of 20 min, followed by permeabilization using 0.2% Triton × (Sigma) for 30 min. Subsequently, a blocking step was performed for 1 h in a solution of 2.5% Bovine Serum Albumin (BSA, Pan Biotech) in phosphate-buffered saline (PBS, Gibco), supplemented with 0.1% Tween 20 (Sigma), as previously described19. For immunostaining, the fixed cells were exposed to primary antibodies overnight at 4 °C. The primary antibodies used were as follows: mouse anti-OCT3/4 at 1:200 dilution (cat: sc-5279, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-NANOG at 1:200 dilution (cat: 4903, Cell Signaling Technology), mouse anti-SOX2 at 1:100 dilution (cat: MAB2018, R&D Systems), mouse anti-α-actin at 1:200 dilution (cat: sc-130616, Santa Cruz Biotechnology), goat anti-FOXA2 at 1:200 dilution (cat: AF2400, R&D Systems), mouse anti-β3 Tubulin at 1:200 dilution (cat: sc-850005, Santa Cruz Biotechnology), goat anti-PDX1 at 1:17 dilution (cat: AF2419, R&D Systems), rabbit anti-brachyury at 1:200 dilution (cat: sc-20109, Santa Cruz Biotechnology), and mouse anti-OTX2 at 1:200 dilution (cat: sc-514195, Santa Cruz Biotechnology). Subsequently, secondary antibodies were applied for 1 h, using the following dilutions: anti-mouse Alexa 555 at 1:500 (cat: 4409, Cell Signaling Technology), anti-rabbit Alexa 488 at 1:500 (cat: 4412, Cell Signaling Technology, Danvers, MA, USA), and donkey anti-goat NL557 at 1:500 (cat: 4412, Cell Signaling). To visualize cell nuclei, the stained cells were treated with 1 µg/mL of DAPI (4′,6-diamidino-2-phenylindole) and subsequently observed under a fluorescence microscope. Image acquisition was performed using Acquiarium software (v2012-06-12, Faculty of Informatics, Masaryk University).

Karyotyping

Cells were mitotically arrested by adding 0.4 μg/mL KaryoMAX™ Colcemid™ Solution (Thermofisher) and subsequently incubated for 2 h under standard culture conditions (5% CO2, 37 °C) as previously described19. Following the detachment of cells with TrypLe express and a 25 min treatment with the hypotonic solution (DMEM/F12 with demineralized water in ratio 1:3), the cells were fixed with 4 °C methanol and acetic acid (3:1). A karyotype analysis was performed by the Cytogenetic Laboratory Brno (Brno, Czech Republic) with Giemsa-banding and microscopic examination. At least 40 metaphase spreads/samples were analyzed at a resolution of 450–500 bands/haploid set.

Ethics approval and consent to participate

The study was approved by the Ethics Board of the Faculty of Medicine of Masaryk University (name of the project: Clinical grade human embryonic stem cells—derivation and characterization, approval number: 16/2017, date of approval: 26/06/2017). All research was performed in accordance with relevant regulations. Informed consent was obtained from all embryo donors involved in the project.

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