Naïve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo

Abstract

Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by segmental accelerated aging and early death from coronary artery disease or stroke. Nearly 90% of HGPS sufferers carry a G608G mutation within exon 11 of LMNA, producing a truncated form of prelamin A, referred to as "progerin". Here, we report the isolation of naïve multipotent skin-derived precursor (SKP) cells from dermal fibroblast cultures from HGPS donors. These cells form spheres and express the neural crest marker, nestin, in addition to the multipotent markers, OCT4, Sox2, Nanog and TG30; these cells can self-renew and differentiate into smooth muscle cells (SMCs) and fibroblasts. The SMCs derived from the HGPS-SKPs accumulate nuclear progerin with increasing passages. A subset of the HGPS-naïve SKPs express progerin in vitro and in situ in HGPS skin sections. This is the first in vivo evidence that progerin is produced in adult stem cells, and implies that this protein could induce stem cells exhaustion as a mechanism contributing to aging. Our study provides a basis on which to explore therapeutic applications for HGPS stem cells and opens avenues for investigating the pathogenesis of other genetic diseases.

Keywords: Adult stem cells; HGPS; Lamin A; Progeria; Progerin.

Fig. 1.. Generation of SKP spheres from human foreskin biopsy and from respective foreskin primary fibroblast culture.

(A) The method for SKP sphere cultures is outlined starting from a typical skin biopsy (skin image), followed by the dermal cell suspension which was either used directly for SKP cultures or for the establishment of primary fibroblast cultures as described in Materials and Methods. Typical 3D SKP spheres at day 10, 18 and 21 in culture are shown. SKP denotes skin-derived precursor and Fib-SKPs denotes SKPs derived from primary fibroblast cultures. Experiments repeated four times. (B) Immunofluorescence analysis performed on SKPs derived from foreskin biopsies (left) and SKPs derived from foreskin fibroblast cultures (right) taken between day 16 to day 18 in culture and stained with the indicated antibodies (n = 3). Nuclei were counterstained with a DNA stain dapi (blue). Scale bar: 20 µm.

Fig. 2.. Typical SKP spheres were derived from HGPS primary dermal fibroblast cultures.

(A) Immunofluorescence detection of lamin B1, lamin A and progerin in HGPS and normal fibroblast cultures from PPD 20 to 35 are shown. The percentage of cells showing dysmorphic nuclei is indicated (n = 3). Scale bar: 20 µm. (B) Western Blot analysis of total HGPS and normal fibroblast lysates were probed as indicated (n = 3). (C) In vitro formation of SKP spheres derived from HGPS-003 (HGADFN003) from day 0 to 17 in culture. Typical 3D spheres derived from four HGPS and four normal fibroblast strains and a human mesenchymal stem cell line (BMOI.55) from day 19 to 21 in culture are shown. At least five SKP cultures have been established for each fibroblast strains used in this study. Scale bar: 100 µm.

Fig. 3.. SKP spheres derived from HGPS fibroblasts express stem cell markers.

(A) SKP-spheres derived from normal fibroblasts (left) and HGPS fibroblasts (right) from day 18 to 21 in culture were immunostained for indicated stem cell proteins (n = 3; similar stainings were obtained in SKP-spheres derived from the other fibroblast strains). (B) Real time PCR analysis of stem cell markers (Nestin, Oct4, Sox2 and Nanog) on HGPS and normal SKP sphere cultures from day 21 to 24 relative to the parental fibroblast cultures. All values are presented as mean ± S.D. (P<0.05; n = 4). (C) HGPS-SKPs were capable of self-renewal and proliferation. Limiting dilution assays showed that a single cell derived from an HGPS003-SKP sphere taken at day 18 re-formed a sphere denoted SKP2-HGPS003 (phase contrast microscopy). A typical SKP2 sphere appeared after approximately 30 days. SKP2 spheres from HGPS and normal cells at day 25 to 30 were double stained with anti-Ki67 and anti-Nestin antibodies. Nuclei are stained blue with DAPI. Scale bars: 20 µm.

Fig. 4.. Characterization of smooth muscle cells-derived from HGPS-SKPs.

(A) SKP-spheres derived from HGPS and normal fibroblasts were directed to differentiate into smooth muscle cells (SMCs). Four SMCs directed differentiation experiments were performed using two normal (GMO3348E, GMO8398A) and three HGPS-SKP strains (HGADFN03, HGADFN164, HGADFN178). Phase contrast imaging recapitulating the different culture steps from the fibroblast culture to the SKP sphere formation and to the SMCs differentiation (upper panel). SMCs from passage 2 (P2) and 5 (P5) were immunostained for indicated SMC markers and progerin. At passage 2 the percent of αSMA-positive and progerin-positive cells are indicated (n = 3). Scale bar: 20 µm. (B) Real-time PCR analysis of the SMC markers, myosin heavy chain (MHC), α-smooth muscle actin (αSMA) and smoothelin as indicated relative to the parental HGPS SKP-sphere cultures. All values are presented as mean ± S.D. (P<0.05; n = 3).

Fig. 5.. Progerin is expressed in naïve HGPS-SKPs in vitro.

(A) HGPS-SKP and Normal-SKP spheres immunostained with anti-progerin and anti-nestin antibodies or anti-lamin A (LMNA) and anti-lamin A/C (LMNA/C) (lower panels), and counterstained with a DNA stain (dapi). Lower panel, immunofluorescence analysis of dissociated SKP spheres derived from normal (GMO3348E) and HGPS (HGPS164) cultures at day 18 were stained with anti-progerin and nestin antibodies as indicated (n = 4). Arrows in Merge image indicate progerin-positive cells. Scale bar: 20 µm. (B) Real-time PCR analysis of lamin A/C in HGPS-SKP spheres and normal fibroblast-SKP spheres collected at day 21 in relation to respective parental fibroblast cultures. All values are presented as mean ± S.D. (P<0.05; n = 4). (C) Semi-quantitative PCR analysis of lamin A and progerin transcripts in fibroblasts and corresponding SKP sphere cultures as indicated (n = 3).

Fig. 6.. Progerin is expressed in nestin-positive cells in vivo in HGPS skin sections.

In situ localization of progerin and nestin on normal human foreskin sections and on skin sections derived from a subject with HGPS (HGADFN143). DNA was stained with dapi. Morphologic entities are indicated: epidermis (ep), dermis (de), Arrows indicate progerin- and nestin-positive cells on Merge images. Scale bar: 50 µm. Zoom in by a factor of 4.