Gustavo Tiscornia

Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes

The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Production and purification of lentiviral vectors

Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (106 viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (109 viral particles/ml) purified preparation requires 2 more days.

A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes in vitro and in vivo. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F1 progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating “knockdown” mice will aid in functional genomics.

Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector

Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types of both mice and humans by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. The delivery of these transcription factors has mostly entailed the use of integrating viral vectors (retroviruses or lentiviruses), carrying the risk of both insertional mutagenesis and oncogenesis due to misexpression of these exogenous factors. Therefore, obtaining iPS cells that do not carry integrated transgene sequences is an important prerequisite for their eventual therapeutic use. Here we report the generation of iPS cell lines from mouse embryonic fibroblasts with no evidence of integration of the reprogramming vector in their genome, achieved by nucleofection of a polycistronic construct coexpressing Oct4, Sox2, Klf4, and c-Myc.

Diseases in a dish: modeling human genetic disorders using induced pluripotent cells

The derivation of induced pluripotent cells (iPSCs) from individuals suffering from genetic syndromes offers new opportunities for basic research into these diseases and the development of therapeutic compounds. iPSCs can self renew and can be differentiated to many cell types, offering a potentially unlimited source of material for study. In this review we discuss the conceptual and practical issues to consider when attempting to model genetic diseases using iPSCs.

MicroRNAs in embryonic stem cell function and fate

Since their discovery in the early 1990s, microRNAs (miRs) have gone from initially being considered an oddity to being recognized as a level of gene expression regulation that is integral to the normal function of cells and organisms. They are implicated in many if not all biological processes in animals, from apoptosis and cell signaling to organogenesis and development. Our understanding of cell regulatory states, as determined primarily by transcription factor (TF) profiles, is incomplete without consideration of the corresponding miR profile. The miR complement of a cell provides robust and redundant control over the output of hundreds of possible targets for each miR. miRs are common components of regulatory pathways, and in some cases can constitute on-off switches that regulate crucial fate decisions. In this review, we summarize our current knowledge about the biogenesis and regulation of miRs and describe their involvement in the pathways that regulate cell division, pluripotency, and reprogramming to the pluripotent state.

Design and cloning of lentiviral vectors expressing small interfering RNAs

RNA interference (RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs (shRNAs), combined with the versatility and robustness of lentiviral vector–mediated gene delivery to a wide range of cell types offers the possibility of long-term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines (or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.

Neuronopathic Gaucher's disease: induced pluripotent stem cells for disease modelling and testing chaperone activity of small compounds

Gauchers disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-β-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-β-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-β-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.

pH-Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases

A general approach is reported for the design of small-molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self-inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH-responsive glycomimetics targeting human glucocerebrosidase or α-galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed.