The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step towards the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. While several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed in order to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here we report the use of a humanized version of a single lentiviral ‘stem cell cassette’ vector in order to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either 3 or 4 reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration, that upon removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, alpha-1 antitrypsin deficiency-related emphysema, scleroderma (SSc), and sickle cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

SUMMARY The use of human pluripotent stem cells for laboratory studies and cell-based therapies is hampered by their tumor-forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, we established endodermal progenitor (EP) cell lines from human embryonic and induced pluripotent stem cells. Optimized growth conditions were established that allow near unlimited (>1016) EP cell self-renewal in which they display a morphology and gene expression pattern characteristic of definitive endoderm. Upon manipulation of their culture conditions in vitro or transplantation into mice, clonally derived EP cells differentiate into numerous endodermal lineages, including monohormonal glucose-responsive pancreatic β-cells, hepatocytes, and intestinal epithelia. Importantly, EP cells are nontumorigenic in vivo. Thus, EP cells represent a powerful tool to study endoderm specification and offer a potentially safe source of endodermal-derived tissues for transplantation therapies.

Patients with Down syndrome (trisomy 21, T21) have hematologic abnormalities throughout life. Newborns frequently exhibit abnormal blood counts and a clonal preleukemia. Human T21 fetal livers contain expanded erythro-megakaryocytic precursors with enhanced proliferative capacity. The impact of T21 on the earliest stages of embryonic hematopoiesis is unknown and nearly impossible to examine in human subjects. We modeled T21 yolk sac hematopoiesis using human induced pluripotent stem cells (iPSCs). Blood progenitor populations generated from T21 iPSCs were present at normal frequency and proliferated normally. However, their developmental potential was altered with enhanced erythropoiesis and reduced myelopoiesis, but normal megakaryocyte production. These abnormalities overlap with those of T21 fetal livers, but also reflect important differences. Our studies show that T21 confers distinct developmental stage-and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development.

SummaryInduced pluripotent stem cells (iPSCs) provide an inexhaustible source of cells for modeling disease and testing drugs. Here we develop a bioinformatic approach to detect differences between the genomic programs of iPSCs derived from diseased versus normal human cohorts as they emerge during in vitro directed differentiation. Using iPSCs generated from a cohort carrying mutations (PiZZ) in the gene responsible for alpha-1 antitrypsin (AAT) deficiency, we find that the global transcriptomes of PiZZ iPSCs diverge from normal controls upon differentiation to hepatic cells. Expression of 135 genes distinguishes PiZZ iPSC-hepatic cells, providing potential clues to liver disease pathogenesis. The disease-specific cells display intracellular accumulation of mutant AAT protein, resulting in increased autophagic flux. Furthermore, we detect beneficial responses to the drug carbamazepine, which further augments autophagic flux, but adverse responses to known hepatotoxic drugs. Our findings support the utility of iPSCs as tools for drug development or prediction of toxicity.

Key Points• Ribosome biogenesis and hematopoiesis are impaired in iPSCs from DBA patients.• The abnormalities of DBA iPSCs are ameliorated by genetic restoration of the defective ribosomal protein genes.Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells. (Blood. 2013;122(6):912-921)