Generation and cardiac differentiation of a human induced pluripotent stem cell line UALGi002-A from a female patient with Left-Ventricular Noncompaction Cardiomyopathy

Left Ventricular Noncompaction Cardiomyopathy (LVNC) is characterized by abnormal number and prominence of trabeculations of the left ventricle of the heart. Although LVNC has been associated with mutations in several genes encoding for transcriptional regulators, ion channels, sarcomeric and mitochondrial proteins, approximately 60% of LVNC patients do not present these genetic alterations. Here, we describe an induced pluripotent stem cell (hiPSC) line (UALGi002-A) originated from a LVNC female patient (LVNC-hiPSC) who does not present any previously known mutations associated to LVNC. The LVNC-hiPSC exhibited full pluripotency and differentiation potential and retained a normal karyotype after reprogramming. Moreover, the LVNC-hiPSC differentiated into contracting cardiomyocytes. This cellular model will be useful


Resource utility
Left Ventricular Noncompaction Cardiomyopathy (LVNC) is a heart disorder characterized by endomyocardial noncompaction.The UALGi002-A cell line, derived from a female patient with LVNC offers a useful tool for molecular, cellular and physiological analysis to study mechanisms involved in this cardiomyopathy.

Resource details
Mononucleated cells were collected from 4 mL of peripheral blood sample from a 51-year-old female.The patient has a history of palpitations and was clinically diagnosed with left ventricular trabeculations located in the middle and apical segments, with a diastolic ratio of >2.3, compatible with Left Ventricular Noncompaction Cardiomyopathy (LVNC).A genetic study of this patient showed no clinically relevant mutations in LDB3, TAZ, LMNA/C, MYH7, MYBPC3, TNNT2, ACTC1, TPM1, CSRP3, TCAP, SGCD, and PLN genes that have been previously associated with LVNC.LVNC is characterized by the presence of vast trabeculations likely due to endomyocardial morphogenesis arrest during embryogenesis.Clinical manifestations and their onset are variable, and may include heart failure, thromboembolism, ventricular arrhythmias, and ultimately sudden cardiac death (Finsterer et al., 2017).The human induced pluripotent stem cell (hiPSC) line UALGi002-A was generated using the CytoTune® iPSC-Reprogramming kit (Thermo Fisher Scientific, Invitrogen), encoding for the reprogramming factors hOCT3/4, hc-MYC, hKLF4, and hSOX2 (Takahashi and Yamanaka, 2006), according to manufacturer's instructions.Clonal hiPSC lines were established and further characterized (Table 1), formed colonies with a standard stem-like morphology visible by phase contrast (Fig. 1A) and were positive for Alkaline Phosphatase (AP) activity (Fig. 1B).The expression of pluripotency markers was demonstrated by immunofluorescence staining of NANOG, SOX2, OCT4 and SSEA4 proteins (Fig. 1C), as well as by detection of NANOG expressing hiPSCs by flow cytometry (Fig. 1D), and by detection of NANOG, OCT4, SOX2 and REX1 transcripts expression by RT-PCR (Supplementary Fig. S1A).Viral clearance was confirmed at passage 11 (Fig. 1E).
Genomic integrity was assessed by karyotype analysis, showing that UALGi002-A, at passage 22, presented a normal diploid (46, XX) chromosomal content (Fig. 1F).The capacity of hiPSCs to differentiate into three germ layers was confirmed by embryoid body (EB) differentiation assay, and by the detection of ectoderm (TUJ1), mesoderm (α-SMA) and endoderm (AFP) markers expression (Fig. 1G).In addition, hiPSC were differentiated into contracting cardiomyocytes as previously described (Lian et al., 2013), and expressed the sarcomeric protein α-actinin (Figure H).DNA fingerprinting was used to prove the genetic identity between hIPSCs and parental mononucleated blood cells (archived with journal).Mycoplasma was regularly tested negative throughout cell culture indicating that UALGi002-A line is mycoplasma-free (Supplementary Fig. S1B).

Immunocytochemistry
Cells were grown in glass coverslips coated with Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix and washed in ice-cold PBS before fixation in 4% PFA, for 15 min.Fixed cells were washed twice with PBS and placed in blocking solution (2% bovine serum albumin in 0.2% Triton-X100/PBS) for 1 h, at room temperature.Next, cells were incubated for 1 h at room temperature with the primary antibody (Table 2).After incubation, samples were washed 3 times with 0.2% Triton X100/PBS, and incubated with the secondary antibodies for 1 h, at room temperature (Table 2).After 3 washes, sections were mounted with Fluoromount G mounting medium (Thermo Fisher Scientific) containing 4,6-diamidino-2-phenylindole (DAPI) and analyzed on a Axioimager Z2 fluorescence microscope (Carl Zeiss).

In vitro differentiation assay
Three germ layers differentiation in vitro was performed by EB formation.hiPSC colonies were lifted manually and cultured in nonadherent conditions in mTeSR1 medium, containing 0.4% of polyvinyl alcohol, for 48 h.Next, EBs were seeded on glass coverslips coated with Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix and cultured for 3 weeks in differentiation medium (DMEM, 10% FBS, 1% Pen/Strep, 1% GlutaMAX, 1% MEM-NEAA).Directed cardiomyocyte differentiation was performed following the GiWi protocol, as described by Lian and co-workers (Lian et al., 2013).The coverslips were fixed   2) on Axioimager Z2/Apotome fluorescence microscope (Carl Zeiss).

Flow cytometry
hiPSCs were dissociated using TrypLE Select (Gibco) for 3 min at RT, centrifuged at 300 g for 5 min and 100,000 cells resuspended in 200 μl of ice-cold 0.5% PFA in PBS, for 20 min.Fixed cells were washed twice in PBS/0.5% BSA/0.1% Triton X100 and incubated with the primary antibody for 1 h at 4 • C (Table 2).After incubation, samples were washed 3 times with PBS/0.5% BSA/0.1% Triton X100, and incubated with the secondary antibodies for 1 h, at 4 • C (Table 2), protected from light.The cells were analyzed using a FACScalibur cell analyzer (BD Biosciences) and data was analyzed by CytExpert 2.0 software.

Alkaline phosphatase activity
Alkaline phosphatase staining was carried out using Alkaline Phosphatase Staining Kit II (Stemgent, MA).

RT-PCR for detection of viral clearance
Total RNA was isolated from cultured hiPSC with RNeasy Mini Kit (Qiagen).1 μg of total RNA was used as template to obtain cDNA, using NZY First-Strand cDNA Synthesis Kit (nzytech).Viral clearance was analyzed using the primers described in Table 2. RT-PCR reaction was performed using DreamTaq DNA Polymerase (Thermo Scientific) and PCR products were visualized on a 2% agarose gel.

Karyotype analyses
Genome integrity of the hiPSC was evaluated by G-banding at 400-550 band resolution, with a minimum of 20 metaphase spreads analyzed (Genomed, Lisbon, Portugal).

Fingerprinting
Genomic DNA from PBMC and hiPSC was extracted using QIAamp DNA Blood mini kit (Qiagen).Fingerprinting analyses was performed using Promega's PowerPlex 16 kit and analyzed on ABI PRISM 3100 using GeneMapper (Thermo Fisher) by STABVida, Lisbon, Portugal.

Mycoplasma detection
The presence of mycoplasma was tested regularly by PCR (Uphoff and Drexler, 2001) using the Primers listed in Table 2.

Fig. 1 .
Fig. 1.Characterisation of the induced pluripotent stem cell line (UALGi002-A) from a patient with LVNC.(A) Phase contrast micrograph of UALGi002-A colony cultured in feeder-free conditions.(B) Alkaline phosphatase positive staining.(C) Immunofluorescence for pluripotency markers NANOG, SOX2, OCT4 and SSEA4.Nuclei were counterstained with DAPI (blue).(D) Flow cytometry of nuclear NANOG pluripotency marker.(E) RT-PCR analysis of Sendai virus and reprogramming transgenes at day 7 after transduction (SeV C+) and at passage 11 in UALGi002-A line (V8 P11).(F) Representative metaphase showing normal diploid 46, XY karyotype.(G) Immunocytochemistry for ectodermal (TUJ1), mesodermal (α-SMA) and endodermal (AFP) markers.Nuclei were counterstained with DAPI (blue).(H) Detection by immunofluorescence of the sarcomeric protein α-actinin (green) and staining of the nuclei by DAPI (blue).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1
Characterization and validation.

Table 2
Reagents details.CGC CTG AGT AGT ACG TTC GC CGC CTG AGT AGT ACG TAC GC TGC CTG AGT AGT ACA TTC GC CGC CTG GGT AGT ACA TTC GC CGC CTG AGT AGT ATG CTC GC TGC CTG GGT AGT ACA TTC GC Reverse primers: GCG GTG TGT ACA AGA CCC GA GCG GTG TGT ACA AAA CCC GA GCG GTG TGT ACA AAC CCC GA S.M. Calado et al.