You are hereNovember 18, 2013 | Pluripotent Stem Cells
Stem Cell Defects Uncovered in DS
Down's syndrome (DS) is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21 and is the most common chromosome abnormality in humans, associated with physical growth delays and a severe degree of intellectual disability. It is also associated with early aging and related disorders and, as recent research has linked aging to impaired or exhausted stem cells (Liu and Rando), suggests that DS may also be associated with adult stem cell defects. In a recent report in Nature, researchers from the laboratory of Michael F. Clarke at Stanford University School of Medicine, California, USA have begun to unravel the molecular underpinnings of DS through the examination of one of the genes known to be triplicated in human DS: the deubiquitinase Usp16 gene. The group's exciting findings suggest that overexpression of Usp16 leads to dysfunction in multiple stem cells populations through epigenetic mechanisms which may give rise to the DS related phenotype (Adorno et al).
The studies involved the analysis of tissue-specific stem cells in two Down's Syndrome mouse models (Ts65Dn and Ts1Cje mice) which display gene triplication and neurological impairment similar to that in humans (Reeves et al, Sago et al (1998) and Sago et al (2000)). Importantly, Usp16 is uniquely triplicated in the Ts65Dn model and so Ts1Cje can be used as a control for normal Usp16 expression. Interestingly, the overexpression of Usp16 mRNA in Ts65Dn mice in each of the stem cell populations examined was associated with the loss of stem cell-like characteristics. Ts65Dn HSCs demonstrated lower haematopoietic chimaerism in recipient mice due in part to fewer KLS (c-Kit+ Lineage− Sca+) cells in the Ts65Dn mice, with no multi-lineage engraftment of Ts65Dn bone marrow cells observed in secondary transplants. The mechanisms behind HSC dysfunction were then analysed concentrating on Usp16 and its ability to counteract the ubiquitination function of the Polycomb repressive complex 1 (PRC1) (Joo et al) which contains Bmi1, known to be essential for self-renewal of tissue specific stem cells, including those investigated in this study (Molofsky et al, Park et al, and Pietersen et al). In line with higher Usp16 levels, Ts65Dn HSCs had a two-fold reduction of ubiquitinated chromatin foci while a reduction of Usp16 mRNA to control levels led to a 100% increase in the colony forming ability of Ts65Dn HSCs, allowing them to engraft in recipient mice and perform multipotent differentiation upon serial transplantation.
Neural stem cells analysis concentrated on the subventricular zone (SVZ), a key area of adult neurogenesis in the brain, through dissection and enrichment of Lin− neural progenitor cells (NPCs). Unlike wild type cells, progenitors from Ts65Dn did not form neurospheres at high efficiency and lost Sox2 expression, but mutation of one of the normal alleles of Usp16 recovered the loss of neurosphere formation ability and Sox2 expression.
Analysis of stem-like cells in breast tissue found a reduction of Lin- cells with signs of abnormal development in Ts65Dn mice. Furthermore 3D in vitro colony assays found a reduced number of colonies in Ts65Dn CD49f+ CD24med Lin- cells known to have regenerative potential, while in vivo mammary transplantation assays also demonstrated a functional defect in Ts65Dn mice. As before, these defects could all be rescued by a reduction in Usp16 mRNA.
In the final part of the study, the researchers concentrated on uncovering an additional mechanism, concentrating on Cdkn2a, the locus for p16Ink4a and p19Arf, a well-known Bmi1/PRC1 target loci (Jacobs et al), and also studied the role of USP16 in human Down's syndrome. p16Ink4a/p19Arf expression increases with aging and is associated with senescence-associated loss of proliferation and analysis of fibroblasts from Ts65Dn mice showed decreased proliferation, increased senescence and increased p16Ink4a/p19Arf expression compared to controls, with downregulation of Usp16 leading to a fall in p16Ink4a/p19Arf expression. Overall H2AK119 and Ink4a/Arf specific H2AK119 ubiquitination was also decreased in Ts65Dn mice linking Usp16 overexpression to the dysregulation of gene expression.
Following this, USP16 overexpression was studied in human foreskin fibroblasts and human NPCs, finding that this slowed proliferation in both cell types and reduced neurosphere formation in NPCs, while downregulation of USP16 or BMI1 led to increased proliferation of human DS fibroblasts.
Overall, this data shows that Usp16 over-expression in DS mice leads to stem cell dysfunction, and the mechanism behind this may be shared between mouse and human. Their exciting study suggests a key role for epigenetic modifications through dysregulation of the normal balance of enzymatic activities between Usp16 and the Bmi1-containg Prc1 polycomb complex which may underlie many of the disease related changes observed in DS, providing an excellent platform for further in depth epigenetic analyses. Additionally, the data also suggests that the USP16 pathway may be targeted to aid some of the associated pathologies of Down's Syndrome allowing for better quality of life, and perhaps even life extension, in these patients.
Jacobs, J. J. et al.
The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus.
Nature 397, 164–168 (1999)
Joo, H.-Y. et al.
Regulation of cell cycle progression and gene expression by H2A deubiquitination.
Nature 449,1068–1072 (2007)
Liu, L. & Rando, T. A.
Manifestations and mechanisms of stem cell aging.
J. Cell Biol. 193,257–266 (2011)
Molofsky, A. V. et al.
Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation.
Nature 425,962–967 (2003)
Park, I.-K. et al.
Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells.
Nature 423,302–305 (2003)
Pietersen, A. M. et al.
Bmi1 regulates stem cells and proliferation and differentiation of committed cells in mammary epithelium.
Curr. Biol.18,1094–1099 (2008)
Reeves, R. H. et al.
A mouse model for Down syndrome exhibits learning and behaviour deficits.
Nature Genet.11,177–184 (1995)
Sago, H. et al.
Genetic dissection of region associated with behavioural abnormalities in mouse models for Down syndrome.
Pediatr. Res.48,606–613 (2000)
Sago, H. et al.
Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities.
Proc. Natl Acad. Sci. USA 95,6256–6261 (1998)
Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.