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Abstract

Insulin resistance (IR) is a precursor and accelerating factor for Type 2 diabetes (T2D), the greatest health challenge facing Qatar and the world today. Psoriasis, is an immune-mediated, chronic skin disorder that can aggregate in families, because of its strong genetic predisposition. It has been found that patients with psoriasis for more than two years, regardless the severity of the disease, are at a very high risk of developing insulin resistance and diabetes. Although several studies highlighted the link between IR and skin disorders, no reports studied the relationship between IR, T2D, and epidermal dysfunction using induced pluripotent stem cells (iPSCs). Therefore, our aim in this study was to generate patient-specific hiPSCs from IR Qatari patients (associated with psoriasis or T2D) and differentiate them into insulin target cells. Blood samples were collected from Qatari individuals with a family history of IR associated with T2D or psoriasis as well as from healthy individuals. The Ficoll-Paque density gradient method was used to separate the peripheral blood mononuclear cells (PBMCs). The isolated PBMCs were cultured in vitro for 5 days in StemPro-34 culture medium before transduction. PBMCs were reporgammed using Sendai viral vectors encoding four pluripotency factors including OCT4, SOX2, C-MYC, and KLF4. After 20-30 days of the transduction, several undifferentiated colonies of high morphological quality (defined border and high nuclear to cytoplasmic ratio) were manually picked up and transferred to new matrigel-coated plates to establish different clones. Several hiPSC clones were established from each individual (sample) and only three clones were maintained after extensive characterization of all clones. The generated hiPSC clones were characterized using different techniques including immunostaining, RT-PCR, Western blotting, alkaline phosphatase assay, embroid body (EB) formation, karyotyping, and hPSC ScoreCard assay. H1-human embryonic stem cell (H1-hiESC) line was used as a positive control in all experiments. Similar to hESCs, hiPSC clones expressed pluripotency markers, such as OCT4, SOX2, NANOG, KLF4, C-MYC, SSEA4, TRA-60, TRA-81, REX-1, DPPA4, and TERT at mRNA and protein levels. All hiPSC lines showed standard hESC morphology, normal karyotype and stained positive for alkalaine phosphatase. Only the hiPSC lines that showed similar characteristics as those of hESCs were maintained and expanded in culture. To confirm the pluripotent ability of the generated hiPSCs, we used EB technique to differentiate patient-specific hiPSCs into three germ layers in vitro. Immunostaining and RT-PCR analyses showed that these hiPSCs can differentiate into endodermal (SOX17+/FOXA2+), mesodermal (BRACHYURY+) and ecodermal (NESTIN+) lineages. To further validate the multilineage differentiation potential of the generated hiPSCs, we used the hPSC ScoreCard assay in vitro. These findings indicate that hiPSCs generated in this study are pluripotent, fully reprogrammed stem cells having the ability to differentiate into any cell type of the body. Thus, we will differentiate patient-specific hiPSCs into insulin target cells carrying the genetic background of the patients to identify signaling mechanism involved in the inherited form of IR and to understand the genetic link between IR, T2D, and psoriasis. To our knowledge, this is the first study to generate hiPSCs from diabetic and psoriatic patients in the MENA region.

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/content/papers/10.5339/qfarc.2018.HBPD138
2018-03-15
2019-11-16
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http://instance.metastore.ingenta.com/content/papers/10.5339/qfarc.2018.HBPD138
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