Aging alters bone-fat reciprocity by shifting in vivo mesenchymal precursor cell fate towards an adipogenic lineage

Abstract

Bone marrow derived mesenchymal progenitor cells (MPCs) play an important role in bone homeostasis. Age-related changes occur in bone resulting in a decrease in bone density and a relative increase in adipocity. Although in vitro studies suggest the existence of an age-related lineage switch between osteogenic and adipogenic fates, stem cell and microenvironmental contributions to this process have not been elucidated in vivo. In order to study the effects of MPC and microenvironmental aging on functional engraftment and lineage switching, transplantation studies were performed under non-myeloablative conditions in old recipients, with donor MPCs derived from young and old green fluorescent protein (GFP) transgenic mice. Robust engraftment by young MPCs or their progeny was observed in the marrow, bone-lining region and in the matrix of young recipients; however, significantly lower engraftment was seen at the same sites in old recipients transplanted with old MPCs. Differentiation of transplanted MPCs strongly favored adipogenesis over osteogenesis in old recipients irrespective of MPC donor age, suggesting that microenvironmental alterations that occur with in vivo aging are predominately responsible for MPC lineage switching. These data indicate that aging alters bone-fat reciprocity and differentiation of mesenchymal progenitors towards an adipogenic fate.

Keywords: Aging; Bone; Engraftment; Fat; Mesenchymal stem cells; Osteoporosis.

Figure 1

Donor MPCs from young and old animals display similar characteristics of proliferation and differentiation at early passage in vitro. (a) Growth curves through the fourth in vitro passage; (b) BrdU labeling index; (c) Sca-1⁺ CD45- MPCs by flow cytometry; (d) Differentiation of MPCs into adipocytes and osteoblasts. CPDL, cumulative population doubling level; AF, adipogenic factors; OF, osteogenic factors

Figure 2

Transplantation of young MPCs into old recipients increases osteoblast and adipocyte numbers. (A) Representative sections of the metaphyseal distal femur from Y|Y, Y|O, and O|O mice, stained with H&E, and showing osteoblasts (upper panels, arrows) or adipocytes (lower panels). (B) Bone volume (BV/TV, %). Higher magnification insets show the greater numbers of bone-lining osteoblasts in the Y|O transplantation group (compared to the O|O group) and more adipocytes in the Y|O and O|O groups compared to the Y|Y group. (C) Osteoblast number per bone surface (OB N/BS, /mm). (D,E) Adiposity increased in old femurs from recipients transplanted with young or old MSCs. (D) Adipocyte number (Adipocyte N/Total Area, /mm²). (E) Adipocyte Area (Adipocyte Area/Total Area, %). Data represent means + s.e.m. Statisical significance is *p<0.05, **p<0.01, and ****p<0.0001 compared to YIY group. Statisical significance is ^&p<0.05, compared to Y|O transplant group.

Figure 3

MPC osteogenic differentiation with aging is a cell autonomous process superimposed upon microenvironmental influences. Y, Young; O, Old; →, indicates the transplant pairings as donor cells → transplant recipient; Yellow arrowheads, bone-lining cells; Red arrowheads, osteocytes.

Figure 4

Engrafted MPCs differentiate into adipocytes only in old recipients and independent of donor MPC age. Higher magnification insets illustrate co-staining of both GFP (green) and FABP4 (red) in panels showing the merged images. Note that in the processing of tissue, sections were deparaffinized in xylene, which causes lipid extraction of adipocytes and limits immunostaining to the cellular periphery. Ad, adipocyte; b, bone