Hematopoietic Differentiation And Targeted Gene Editing Of Wiskott-Aldrich Syndrome Specific Integration-free Induced Pluripotent Stem Cells

Progress in cell reprogramming revolutionizes the whole field of development biology, provides unprecedented opportunity for ex vivo study of human diseases and opens up innovative personalized cell therapy strategy to treat these diseases. Safety concern is one of the most important issues for the clinical application of those reprogrammed cells. Here we show that integration-free induced pluripotent stem cell (iPSCs) can be generated from the fibroblasts of a Wiskott-Aldrich Syndrome (WAS) patient. Those WAS-iPSCs give rise to hematopoietic cells when being differentiated in vitro, and show disease related phenotypes. Most importantly, we show the possibility that the WAS gene mutation in WAS-iPSCs can be corrected via an enhanced TALEN mediated homologous recombination strategy. Our work offers proof-of-principle that iPSCs can be generated and manipulated safely in vitro and serve as an alternative sauce for regenerative medicine. Chapter1Establishment and characterization of integration-free iPSCs from WAS patient fibroblastsWAS is an X-linked primary immunodeficiency disorder caused by loss-of-function mutations in the gene for the WAS protein. WAS protein is exclusively expressed in hematopoietic lineages and it modulates actin polymerization which is indispensable for the development of an organized immunological system for host defense and maintenance of tolerance. To establish an ex vivo model to study WAS, we reprogrammed the fibroblasts of a WAS patient with3episomal plasmids expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28and TP53shRNA. The established iPSC clones exhibited similar morphology and gene expression pattern as normal human embryonic stem cells (ESCs), and expressed pluripotency markers including Tra-1-60and Tra-1-81. PCR analysis of four WAS-iPSC clones failed to detect any residual episomal DNA, confirming the loss of the input plasmids once the iPSC clones were established. The WAS gene mutation in parental fibroblasts was inherited by all the iPSC clones we tested, indicating those WAS-iPSC clones could be used for in vitro disease modeling and gene manipulation.Chapter2Hematopoietic differentiation of WAS patient-specific iPSCs shows distinct disease related phenotypesIn vitro differentiation of iPSCs to specific tissues or cell types facilitates the study of normal development and disease progression. To determine whether the WAS disease phenotypes can be recapitulated in a dish, we differentiated the WAS-iPSCs into hematopoietic lineage ex vivo through a stromal cell co-culture system. In the presence of GM-CSF and M-CSF, we successfully expanded CD45+CD43+CD11c+progenitors which could give rise to CFU-G, CFU-M and CFU-GM in colony forming cell assay. Further differentiation with proper cytokines showed normal maturation of those progenitors into CD11c+CD14+CD163+macrophages, the vital cell population of innate and adaptive immunity. The WAS-iPSCs derived macrophages were functional in antigen uptake and processing, phagocytosis and chemotaxis. However, these macrophages failed to show the normal podosome formation, due to the defect in WAS gene. Thus these WAS-iPSC clones may provide an ideal ex vivo system to study the role of WAS protein in the development of a functional immune system, and provide a safer, unlimited source for stem cell gene therapy for WAS patients upon gene correction.Chapter3High efficiency TALEN-based WAS gene editing mediated by an integration defective lentiviral vectorSafely and efficiently correction of mutant genes in patient specific iPSCs is crucial for personalized stem cell gene therapy. Recent development in the design of Transcription Activator-Like Effector Nuclease (TALEN) provides a new strategy for targeted gene editing. However, the guideline to optimize TALEN design and the strategy for the delivery of TALEN and the gene editing template are not fully established, and the gene editing efficiency in mammalian cells especially in human iPSCs remains low. In the current study, we designed several TALEN pairs targeting a region in intron6of the WAS gene and showed that both the number of the TALEN repeats and the size of the spacer between the two TALEN-binding sites could modulate the genomic cutting efficiency. We also found that using an integration-defective lentiviral vector (IDLV) to deliver the gene-editing template significantly enhanced the gene editing efficiency. Our study not only showed a possible effective way to achieve WAS gene correction in the WAS-iPSC clones we established, but also facilitate the use of TALEN as a tool for potent genome editing in mammalian cells such as human iPSCs.