Due to the lack of reliable antibodies the
Due to the lack of reliable antibodies, the homozygous knockout was verified by reverse transcription and qPCR analyses of different KLF15 locus regions. This revealed the anticipated absence of transcripts encoding for KLF15 (n=8 different passages). GAPDH was equally expressed and used for normalization. RT-PCR confirmed the truncation of the KLF15 transcript (Fig. 1C).
Genomic integrity was demonstrated by standard G-banding karyotype analysis of the KO line (Fig. 1D). Pluripotency was assessed by immunofluorescence using the stemness markers OCT4, TRA1-60, SSEA-4, and by flow cytometry showing 99.3% OCT4 and 88.1% TRA1-60 positive retinoic acid receptor (Fig. 1E). Spontaneous differentiation capacity into all three germ layers was tested by formation of embryoid bodies (EBs) in the KLF15-KO and -WT line. This was demonstrated by marker expression on mRNA- and protein level of α-Feto-Protein (AFP, endodermal); cardiac TroponinT and α-Smooth Muscle Actin (TNNT2, α-SMA, mesodermal); and Microtubule-Associated Protein 2 (MAP2) as well as βIII-Tubulin (ectodermal) at different stages (Fig. 1F and G).
Finally, we investigated the suitability of the KLF15-KO line for studies in cardiomyocytes. We performed directed 2D differentiation followed by lactate selection and obtained spontaneously beating cardiomyocytes (videos provided as supplemental data). The KLF15-KO and -WT cells showed comparable expression of the cardiac markers α-Sarcomeric-Actinin, gap junction protein Connexin-43 (CX43), cell adhesion component N-Cadherin (NCAD), and transcription factor TBX5 (Fig. 1H).
In summary, we generated a homozygous KLF15-KO hESC line with normal karyotype and full pluripotency. KLF15-KO cells have the capacity to differentiate into cardiomyocytes, indicating that KLF15 is not essential for their development as shown previously in mice. Moreover, we showed that the KLF15-KO line has the potential to generate all three germ layers. Since KLF15 is important for the homeostasis of several organs, the KLF15-KO line represents a valuable tool for in vitro translational studies and disease modelling.
Materials and methods
Acknowledgements We thank Daria Reher, Iris Quentin, and Sandra Georgi for superb technical support. We thank Dr. Boris Greber for generously providing material and protocols. This work was supported by the DFG grant ZE300-3 to LCZ; SFB1002 C04and S1 to WHZ and C07 to LCZ; and the DZHK (German Center for Cardiovascular Disease).
Resource table Resource utility
Resource details Multiple Endocrine Neoplasia type 2 (MEN2) are autosomal dominant hereditary diseases, related to mutations of the RET (REarranged during Transfection) proto-oncogene (Donis-Keller et al., 1993). MEN2B is a rare syndrome which associates medullary thyroid cancer (MTC), pheochromocytoma, and phenotypical features such as a typical facies, ophthalmologic abnormalities (thickened and everted eyelids, inability to cry tears, and prominent corneal nerves), skeletal malformations (marfanoid body habitus, pes cavus, pectus excavatum, high-arched palate, scoliosis, and slipped capital femoral epiphyses), and a generalized ganglioneuromatosis throughout the aerodigestive tract. Most patients have abdominal symptoms characterized by bloating, intermittent constipation, and diarrhea (Wells et al., 2013). It affects therefore organs originating from endodermal and ectodermal layers. Mouse models have been unsuccessful in recapitulating the full clinical spectrum of MEN2B (Acton et al., 2000; Smith-Hicks et al., 2000). The most common mutation, accounting for over 95% of all mutations associated with the classic MEN2B, affects codon 918 (Wells et al., 2013). Mutations at codon 918 are classified as highest risk, according to the American Thyroid Association (ATA) guidelines (Wells et al., 2015). A 23year-old woman was diagnosed with left thyroid nodule with adenomegaly and elevated calcitonin of 4610pg/ml in 1989. She had a high blood pressure, flush and phenotypical features: triangular face, thicken eyelid, prominent corneal nerves, muscular hypotrophy, marfanoid habitus but she did not have ganglioneuromatosis on the lips, tongue and eye lids. There was no familial history of medullary thyroid cancer or pheochromocytoma. Urinary metanephrines were elevated and scintigraphy revealed a bilateral adrenal MIBG uptake. First, the patient underwent bilateral adrenalectomy which revealed a bilateral pheochromocytoma with no capsular nor vascular invasion. Afterwards, a total thyroidectomy with bilateral lateral and central neck dissection was performed and revealed a multifocal medullary thyroid cancer with extra thyroid extension and bilateral and central lymph nodes metastasis. The tumor was classified pT3(m)N1bMx. Post-surgery calcitonin was 228pg/ml with no evidence of structural disease. Eighteen months later, patient relapsed with liver, lung, lymph nodes and bone involvement and chemotherapy had no effect; the patient experienced progressive disease (calcitonin 19,500pg/ml) and died 4years after initial diagnosis.