Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells
2011; 474 (7351): 399-?
RNAi in Budding Yeast
2009; 326 (5952): 544-550
The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.
View details for DOI 10.1038/nature10084
View details for Web of Science ID 000291647100050
View details for PubMedID 21602826
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes
2007; 25 (12): 1483-1487
RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' messenger RNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.
View details for DOI 10.1126/science.1176945
View details for Web of Science ID 000271019400033
View details for PubMedID 19745116
Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells
2006; 12 (2): 240-245
Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.
View details for DOI 10.1038/nbt1355
View details for Web of Science ID 000251457800039
View details for PubMedID 18059260
Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.
View details for DOI 10.1038/nm1342
View details for Web of Science ID 000235160600031
View details for PubMedID 16429146