Dedifferentiation rescues senescence of progeria cells but only while pluripotent

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease in which children develop pathologies associated with old age. HGPS is caused by a mutation in the LMNA gene, resulting in the formation of a dominant negative form of the intermediate filament, nuclear structural protein lamin A, termed progerin. Expression of progerin alters the nuclear architecture and heterochromatin, affecting cell cycle progression and genomic stability. Two groups recently reported the successful generation and characterization of induced pluripotent stem cells (iPSCs) from HGPS fibroblasts. Remarkably, progerin expression and senescence phenotypes are lost in iPSCs but not in differentiated progeny. These new HGPS iPSCs are valuable for characterizing the role of progerin in driving HGPS and aging and for screening therapeutic strategies to prevent or delay cell senescence.

splice donor site, resulting in synthesis of a dominant negative, incompletely processed form of lamin A, termed progerin [2]. Th e expression of progerin alters nuclear structure and heterochromatin, aff ecting cell cycle progression, gene expression, and genomic stability. Progerin is hypothesized to promote sequestration of DNA repair and replication proteins, resulting in a more frequent stalling of replication forks and thereby replication-dependent double-strand breaks [3]. Progerin is farnesylated on its C-terminus, leading to concentration of the truncated protein at the nuclear periphery and leading to rigidity of the normally dynamic nuclear lamina [4], the structure meshwork lining the nuclear membrane. Currently, inhibitors of farnesylation are the only available strategy for treating HGPS but are incompletely eff ective because they are nonspecifi c.
Interestingly, the cryptic splice site in LMNA is sporadically used in cells from normal individuals, leading to a low-level expression of progerin [5]. Furthermore, fi broblasts from individuals who are more than 80 years old show nuclear abnormalities and changes in heterochromatin markers typical of cells from adolescent patients with HGPS. Th us, progerin may contribute to both accelerated and normal aging. However, establishing a direct causal role for progerin (or anything else) in aging is particularly challenging.
Progerin also leads to activation of NOTCH, a crucial regulator of stem cell diff erentiation [6]. Th us, consti tutive expression of progerin in human mesenchymal stem cells (MSCs) induces aberrant expression of diff erentiation markers [6]. Th is supports the notion that HGPS is caused by dysfunction of adult stem cell populations (in particular mesenchymal progenitors), resulting in stem cell exhaustion. But this raises the question of whether patients with HGPS will ever be amenable to autologous stem cell therapy to delay their degenerative symptoms.
Th e recent development of technology to reprogram somatic diff erentiated cells into induced pluripotent stem cells (iPSCs) allows the generation of stem cells from virtually anyone, including patients with rare genetic diseases that lead to premature stem cell exhaustion [7]. iPSCs, which pro lifer ate extensively in culture, can be Abstract Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease in which children develop pathologies associated with old age. HGPS is caused by a mutation in the LMNA gene, resulting in the formation of a dominant negative form of the intermediate fi lament, nuclear structural protein lamin A, termed progerin. Expression of progerin alters the nuclear architecture and heterochromatin, aff ecting cell cycle progression and genomic stability. Two groups recently reported the successful generation and characterization of induced pluripotent stem cells (iPSCs) from HGPS fi broblasts. Remarkably, progerin expression and senescence phenotypes are lost in iPSCs but not in diff erentiated progeny. These new HGPS iPSCs are valuable for characterizing the role of progerin in driving HGPS and aging and for screening therapeutic strategies to prevent or delay cell senescence.
used to examine the pathogenesis of disease by diff erentiating cells into specifi c lineages. For example, motor neurons diff erentiated from iPSCs produced from patients with spinal muscular atrophy are smaller, have impaired pre-synaptic maturation, and degenerate more rapidly than motor neurons produced from normal iPSCs [8]. Disease-specifi c iPSCs are ideal for screening drugs that improve production of fully functional diff erentiated cells. In addition, the iPSCs can be forcibly diff erentiated into specifi c types of progenitor cells, such as MSCs, of therapeutic value.
To study the biology of HGPS, two groups [9,10] recently generated iPSCs from fi broblasts of patients with HGPS by transduction with retroviral vectors encoding OCT4, SOX2, KLF4, and c-MYC. HGPS fi broblasts express progerin and have altered nuclear morphology, reduced proliferation, and loss of heterochromatin markers, relative to their normal counterparts. Interestingly, iPSCs could be generated from early-passage, but not latepassage, HGPS fi broblasts [9] and were generated with less effi ciency from HGPS fi broblasts compared with normal fi broblasts [10], suggesting that progerin-positive fi bro blasts from normal, aged individuals may pose a challenge to dediff erentiation. Th e HGPS iPSCs lost expression of progerin and had morphology, proliferation, and hetero chromatic markers similar to those of . iPSCs can be expanded while maintaining their pluripotent state (passage 50×) or can be induced to diff erentiate into fi ve diff erent lineages, including smooth muscle cells (SMCs). Fibroblasts from HGPS patients (in orange) express increased levels of progerin and have numerous senescence phenotypes (decreased proliferation, abnormal nuclear morphology, and altered expression of heterochromatin markers) in comparison with normal fi broblasts. Conversion of these cells to iPSCs attenuates expression of progerin and senescence phenotypes so that HGPS iPSCs can be serially passaged and remain pluripotent. However, when induced to diff erentiate into SMCs, these iPSCs re-express progerin and undergo premature senescence. If progerin expression is knocked down in the HGPS iPSC-derived SMCs, senescence is again attenuated. Conversely, ectopic expression of progerin in SMCs derived from normal iPSCs induces senescence. This establishes that progerin drives senescence of diff erentiated mesenchymal cells. shRNA, short hairpin RNA.  (Figure 1). Th us, the defects associated with HGPS are lost in pluripotent iPSCs. However, upon diff erentiation of the HGPS iPSCs toward embryoid bodies, progerin was again upregulated. Diff erentiation of the HGPS iPSCs toward smooth muscle cells (SMCs) resulted in altered nuclear morphology, loss of certain heterochromatic markers, and premature senes cence. Knockdown of progerin in HGPS iPSCs by using short hairpin RNA (shRNA) attenuated replicative senescence. In contrast, expression of progerin in primary human SMCs induced nuclear abnormalities while attenuating proliferation. Th ese data strongly support the conclusion that progerin is directly respon sible for the cellular senescence phenotypes associated with HGPS. Furthermore, this demonstrates the utility of iPSCs for discriminating between factors that drive a specifi c phenotype (in this case, cell senescence and accelerated aging) as compared with being a passive biomarker or consequence of change.

fibroblasts iPSCs SMCs
Zhang and colleagues [10] examined the diff erentiation capacity of HGPS iPSCs in detail, confi rming the multipotency of these cells. MSCs and vascular smooth muscle cells (VSMCs) derived from HGPS iPSCs are sensitive to stress such as substratum deprivation, serum starvation, electrical stimulation, and hypoxia. Moreover, in contrast to normal MSCs, MSCs derived from the HGPS iPSCs are unable to repair ischemic muscle damage caused by ligation of the femoral artery. Interestingly, neuralderived progenitor cells derived from HGPS iPSCs expressed a lower level of progerin in comparison with VSMCs and MSCs. Th is is consistent with the lack of neurodegeneration in patients with HGPS and the fact that most symptoms in HGPS are related to defects in tissues of mesenchymal origin.
Th ese bodies of work illustrate the utility of iPSCs for identifying mechanisms of pathology caused by inherited mutations because of the potentially reversible expression of mutant protein in diff erentiated and dediff erentiated cells. In this case, the authors demonstrate that the mutant form of lamin A, progerin, drives senescence of diff erentiated, but not pluripotent, cells in HGPS. In addition, HGPS iPSCs off er a unique reagent for characterization of the eff ects of progerin on cellular diff erentiation, nuclear morphology, epigenetic regulation of gene expression, genomic instability, stem cell function, and cellular senescence. Th is is applicable not only to patients with HGPS but also to normal, older individuals.
Indeed, the HGPS iPSCs have already been used to provide insight into diff erences in expression of progerin between cell lineages and response of cells to stress. Th e HGPS iPSCs can also be used to identify therapies that aff ect the expression, splicing, farnesylation, and function of progerin or to correct nuclear lamina fl uidity by screening for drugs that correct diff erentiation defects.
Correction of the HGPS mutation by homologous recombination or knockdown of the dominant progerin by using shRNA could give rise to progenitor cell populations able to treat some of the pathologies associated with HGPS. Importantly, given the possible role of progerin in natural aging, the development of strategies to reduce the level or activity of progerin could be applied to treating degenera tion associated with aging in the general population.