Julie Glowacki

Background/Objectives It is known that failure to gain sufficient bone during skeletal growth and maturation phases predisposes to the development of senile osteoporosis as age-related bone loss ensues. There is limited knowledge about factors that are necessary for the pubertal growth spurt and achievement of peak bone mass. Diminution or disappearance of Juvenile Protective Factors (JPFs) after a given maturational stage could contribute to the onset of age-related declines in a variety of physiological functions, including bone physiology. Methods With available pediatric platelet-poor plasma (PPP) and mesenchymal/skeletal stem cells (MSCs), we tested whether proteomics and RNA-seq methodology have potential for the discovery of novel regulators of pubertal skeletal growth. Results Our data demonstrate that pediatric PPP rejuvenates age-related compromised MSC functions; that Mass Spectrometry (MS)-based proteomics identified known and novel circulating tissue growth/trophic factors in human PPP of pubertal, as compared with pre-pubertal, and post-pubertal subjects; and that the unbiased RNA-Seq approach revealed new genes and networks of genes that are dramatically elevated or diminished in pubertal MSCs. Conclusions The findings support the hypothesis that the characterization of pro-osteogenic JPFs could lead to the identification of novel therapeutic approaches to promote bone health in the elderly and of potential treatment regimens for senile osteoporosis.

A radiation combined injury is defined as an injury that occurs in the setting of irradiation, such as those expected after a nuclear accident, radiation dispersal device release (a ‘dirty bomb’), or a nuclear weapon detonation. There is much research on irradiation-associated burns and their healing, but there is less known about other injuries sustained in the context of irradiation. Animal models are limited in their correlations to clinical situations but can support research on specific questions about injuries and their healing. Mouse models of irradiation with skin or bone wounds are validated as highly reproducible and quantitative. They show dose-dependent impairment of wound healing, with later recovery. Irradiation-induced delay of bone wound healing was mitigated to different extents by single doses of gramicidin S-nitroxide JP4-039, a plasmid expressing manganese superoxide dismutase, amifostine/WR2721, or the bifunctional sulfoxide MMS-350. These models should be useful for research on mechanisms of radiation dermal and osseous damage and for further development of new radioprotectors. They also provide information of potential relevance to the effects of clinical radiation therapies.

Osteoblast differentiation of human mesenchymal stem cells (hMSCs) is stimulated by 1α,25‐dihydroxycholecalciferol [1α,25(OH) ₂ D ₃ ] and 25‐hydroxycholecalciferol [25(OH)D ₃ ]; the latter's effects require intracellular hydroxylation to 1α,25(OH) ₂ D ₃ . Thus, hMSCs are both a source of and target for 1α,25(OH) ₂ D ₃ . Megalin is a transmembrane receptor for serum d ‐binding protein (DBP) in kidney cells and is required for uptake of the 25(OH)D ₃ ‐DBP complex. We tested the hypothesis that megalin is required for D actions in hMSCs with cells from surgically discarded marrow for RT‐PCR, for effects of 25(OH)D ₃ and 1α,25(OH) ₂ D ₃ , for 1α,25(OH) ₂ D ₃ biosynthesis, for osteoblastogenesis, and for small interfering RNA for megalin (si‐Meg) and control (si‐Ctr). In hMSCs with high constitutive megalin expression, both 1α,25(OH) ₂ D ₃ and 25(OH)D ₃ stimulated osteoblastogenesis ( P < 0.05), but only 1α,25(OH) ₂ D ₃ did so in hMSCs with lower megalin (lo‐Meg, P < 0.001) or in si‐Meg cells ( P < 0.05). In addition, 1α,25(OH) ₂ D ₃ biosynthesis was significantly lower in lo‐Meg (46%, P = 0.034) and in si‐Meg (23%, P < 0.001) than each control. Leptin significantly stimulated megalin expression 2.1‐fold in lo‐Meg cells ( P < 0.01). These studies show that megalin is expressed in hMSCs and is required for the biosynthesis of 1α,25(OH) ₂ D ₃ and for the 25(OH)D ₃ /DBP complex to stimulate vitamin D receptor targets and osteoblastogenesis.—Gao, Y., Zhou, S., Luu, S., Glowacki, J. Megalin mediates 25‐hydroxyvitamin D ₃ actions in human mesenchymal stem cells. FASEB J. 33, 7684–7693 (2019). www.fasebj.org

Mesenchymal stem cells (MSCs) are a mixture of progenitors that are heterogeneous in their regenerative potential. Development of MSC therapies with consistent efficacy is hindered by the absence of an immunophenotype of MSC heterogeneity. This study evaluates decoy TRAIL receptor CD264 as potentially the first surface marker to detect cellular aging in heterogeneous MSC cultures. CD264 surface expression, regenerative potential, and metrics of cellular aging were assessed in vitro for marrow MSCs from 12 donors ages 20–60 years old. Male and female donors were age matched. Expression of CD264 was compared with that of p16, p21, and p53 during serial passage of MSCs. When CD264+ cell content was 20% to 35%, MSC cultures from young (ages 20–40 years) and older (ages 45–60 years) donors proliferated rapidly and differentiated extensively. Older donor MSCs containing < 35% CD264+ cells had a small size and negligible senescence despite the donor’s advanced chronological age. Above the 35% threshold, CD264 expression inversely correlated with proliferation and differentiation potential. When CD264+ cell content was 75%, MSCs were enlarged and mostly senescent with severely compromised regenerative potential. There was no correlation of the older donors’ chronological age to either CD264+ cell content or the regenerative potential of the donor MSCs. CD264 was upregulated after p53 and had a similar expression profile to that of p21 during serial passage of MSCs. No sex-linked differences were detected in this study. These results suggest that CD264 is a surface marker of cellular age for MSCs, not the chronological age of the MSC donor. CD264 is first upregulated in MSCs at an intermediate stage of cellular aging and remains upregulated as aging progresses towards senescence. The strong inverse correlation of CD264+ cell content to the regenerative potential of MSCs has possible application to assess the therapeutic potential of patient MSCs, standardize the composition and efficacy of MSC therapies, and facilitate aging research on MSCs.