SummaryInduced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into β-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient’s disease. GATA6 mutant β-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and β-like cell functionality in humans.

Highlights d HNF1A regulates endocrine cell development and suppresses an alpha cell fate d HNF1A plays an essential role in insulin secretion and metabolism d Human-specific lncRNA LINKA is an HNF1A target that regulates mitochondrial function

• When targeted to a single allele of the AAVS1 locus, the Gp1ba promoter drives a high level of expression specifically to megakaryocytes.• Transgene rescue in iPSCs provides a model for the return of surface aIIbb3 expression to near-normal levels in patients with type I GT.Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin aIIbb3. Hemizygous insertion of Gp1ba promoter-driven human aIIb complementary DNA into the AAVS1 locus of iPSCs led to high aIIb messenger RNA and protein expression and correction of surface aIIbb3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin aIIbb3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function. (Blood. 2014;123(5):753-757)