Thomas Ambrosi

All Publications

• Delayed Union of a Diaphyseal Forearm Fracture Associated With Impaired Osteogenic Differentiation of Prospectively Isolated Human Skeletal Stem Cells. JBMR plus Goodnough, L. H., Ambrosi, T. H., Steininger, H., DeBaun, M. R., Abrams, G. D., McAdams, T. R., Gardner, M. J., Chan, C. K., Bishop, J. A. 2020; 4 (10): e10398

  Abstract

  Delayed union or nonunion are relatively rare complications after fracture surgery, but when they do occur, they can result in substantial morbidity for the patient. In many cases, the etiology of impaired fracture healing is uncertain and attempts to determine the molecular basis for delayed union and nonunion formation have been limited. Prospectively isolating skeletal stem cells (SSCs) from fracture tissue samples at the time of surgical intervention represent a feasible methodology to determine a patient's biologic risk for compromised fracture healing. This report details a case in which functional in vitro readouts of SSCs derived from human fracture tissue at time of injury predicted a poor fracture healing outcome. This case suggests that it may be feasible to stratify a patient's fracture healing capacity and predict compromised fracture healing by prospectively isolating and analyzing SSCs during the index fracture surgery. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

  View details for DOI 10.1002/jbm4.10398

  View details for PubMedID 33103027

• Human skeletal stem cell aging AGING-US Ambrosi, T. H., Goodnough, L., Chan, C. F. 2020; 12 (17): 16669–71

  View details for Web of Science ID 000572850000003

• Articular cartilage regeneration by activated skeletal stem cells. Nature medicine Murphy, M. P., Koepke, L. S., Lopez, M. T., Tong, X., Ambrosi, T. H., Gulati, G. S., Marecic, O., Wang, Y., Ransom, R. C., Hoover, M. Y., Steininger, H., Zhao, L., Walkiewicz, M. P., Quarto, N., Levi, B., Wan, D. C., Weissman, I. L., Goodman, S. B., Yang, F., Longaker, M. T., Chan, C. K. 2020

  Abstract

  Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

  View details for DOI 10.1038/s41591-020-1013-2

  View details for PubMedID 32807933

• Geriatric fragility fractures are associated with a human skeletal stem cell defect. Aging cell Ambrosi, T. H., Goodnough, L. H., Steininger, H. M., Hoover, M. Y., Kim, E., Koepke, L. S., Marecic, O., Zhao, L., Seita, J., Bishop, J. A., Gardner, M. J., Chan, C. K. 2020: e13164

  Abstract

  Fragility fractures have a limited capacity to regenerate, and impaired fracture healing is a leading cause of morbidity in the elderly. The recent identification of a highly purified bona fide human skeletal stem cell (hSSC) and its committed downstream progenitor cell populations provides an opportunity for understanding the mechanism of age-related compromised fracture healing from the stem cell perspective. In this study, we tested whether hSSCs isolated from geriatric fractures demonstrate intrinsic functional defects that drive impaired healing. Using flow cytometry, we analyzed and isolated hSSCs from callus tissue of five different skeletal sites (n=61) of patients ranging from 13 to 94years of age for functional and molecular studies. We observed that fracture-activated amplification of hSSC populations was comparable at all ages. However, functional analysis of isolated stem cells revealed that advanced age significantly correlated with reduced osteochondrogenic potential but was not associated with decreased in vitro clonogenicity. hSSCs derived from women displayed an exacerbated functional decline with age relative to those of aged men. Transcriptomic comparisons revealed downregulation of skeletogenic pathways such as WNT and upregulation of senescence-related pathways in young versus older hSSCs. Strikingly, loss of Sirtuin1 expression played a major role in hSSC dysfunction but re-activation by trans-resveratrol or a small molecule compound restored in vitro differentiation potential. These are the first findings that characterize age-related defects in purified hSSCs from geriatric fractures. Our results provide a foundation for future investigations into the mechanism and reversibility of skeletal stem cell aging in humans.

  View details for DOI 10.1111/acel.13164

  View details for PubMedID 32537886

• FGF21, not GCN2, influences bone morphology due to dietary protein restrictions. Bone reports McNulty, M. A., Goupil, B. A., Albarado, D. C., Castano-Martinez, T., Ambrosi, T. H., Puh, S., Schulz, T. J., Schurmann, A., Morrison, C. D., Laeger, T. 2020; 12: 100241

  Abstract

  Background: Dietary protein restriction is emerging as an alternative approach to treat obesity and glucose intolerance because it markedly increases plasma fibroblast growth factor 21 (FGF21) concentrations. Similarly, dietary restriction of methionine is known to mimic metabolic effects of energy and protein restriction with FGF21 as a required mechanism. However, dietary protein has been shown to be required for normal bone growth, though there is conflicting evidence as to the influence of dietary protein restriction on bone remodeling. The purpose of the current study was to evaluate the effect of dietary protein and methionine restriction on bone in lean and obese mice, and clarify whether FGF21 and general control nonderepressible 2 (GCN2) kinase, that are part of a novel endocrine pathway implicated in the detection of protein restriction, influence the effect of dietary protein restriction on bone.Methods: Adult wild-type (WT) or Fgf21 KO mice were fed a normal protein (18kcal%; CON) or low protein (4kcal%; LP) diet for 2 or 27weeks. In addition, adult WT or Gcn2 KO mice were fed a CON or LP diet for 27weeks. Young New Zealand obese (NZO) mice were placed on high-fat diets that provided protein at control (16kcal%; CON), low levels (4kcal%) in a high-carbohydrate (LP/HC) or high-fat (LP/HF) regimen, or on high-fat diets (protein, 16kcal%) that provided methionine at control (0.86%; CON-MR) or low levels (0.17%; MR) for up to 9weeks. Long bones from the hind limbs of these mice were collected and evaluated with micro-computed tomography (muCT) for changes in trabecular and cortical architecture and mass.Results: In WT mice the 27-week LP diet significantly reduced cortical bone, and this effect was enhanced by deletion of Fgf21 but not Gcn2. This decrease in bone did not appear after 2weeks on the LP diet. In addition, Fgf21 KO mice had significantly less bone than their WT counterparts. In obese NZO mice dietary protein and methionine restriction altered bone architecture. The changes were mediated by FGF21 due to methionine restriction in the presence of cystine, which did not increase plasma FGF21 levels and did not affect bone architecture.Conclusions: This study provides direct evidence of a reduction in bone following long-term dietary protein restriction in a mouse model, effects that appear to be mediated by FGF21.

  View details for DOI 10.1016/j.bonr.2019.100241

  View details for PubMedID 31921941

• A Revised Perspective of Skeletal Stem Cell Biology FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Ambrosi, T. H., Longaker, M. T., Chan, C. F. 2019; 7

  View details for DOI 10.3389/fcell.2019.00189

  View details for Web of Science ID 000485330300001

• Identification of the Human Skeletal Stem Cell. Cell Chan, C. K., Gulati, G. S., Sinha, R., Tompkins, J. V., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E. Y., Leavitt, T., Lu, W., Nguyen, A., Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43

  Abstract

  Stem cell regulation and hierarchical organization ofhuman skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

  View details for PubMedID 30241615

• Loss of the Hematopoietic Stem Cell Factor GATA2 in the Osteogenic Lineage Impairs Trabecularization and Mechanical Strength of Bone MOLECULAR AND CELLULAR BIOLOGY Tolkachov, A., Fischer, C., Ambrosi, T. H., Bothe, M., Han, C., Muenzner, M., Mathia, S., Salminen, M., Seifert, G., Thiele, M., Duda, G. N., Meijsing, S. H., Sauer, S., Schulz, T. J., Schupp, M. 2018; 38 (12)

  Abstract

  The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs) GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and co-localizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.

  View details for DOI 10.1128/MCB.00599-17

  View details for Web of Science ID 000433419600008

  View details for PubMedID 29581184

  View details for PubMedCentralID PMC5974429

• Loss of periostin occurs in aging adipose tissue of mice and its genetic ablation impairs adipose tissue lipid metabolism. Aging cell Graja, A., Garcia-Carrizo, F., Jank, A. M., Gohlke, S., Ambrosi, T. H., Jonas, W., Ussar, S., Kern, M., Schürmann, A., Aleksandrova, K., Blüher, M., Schulz, T. J. 2018; 17 (5): e12810

  Abstract

  Remodeling of the extracellular matrix is a key component of the metabolic adaptations of adipose tissue in response to dietary and physiological challenges. Disruption of its integrity is a well-known aspect of adipose tissue dysfunction, for instance, during aging and obesity. Adipocyte regeneration from a tissue-resident pool of mesenchymal stem cells is part of normal tissue homeostasis. Among the pathophysiological consequences of adipogenic stem cell aging, characteristic changes in the secretory phenotype, which includes matrix-modifying proteins, have been described. Here, we show that the expression of the matricellular protein periostin, a component of the extracellular matrix produced and secreted by adipose tissue-resident interstitial cells, is markedly decreased in aged brown and white adipose tissue depots. Using a mouse model, we demonstrate that the adaptation of adipose tissue to adrenergic stimulation and high-fat diet feeding is impaired in animals with systemic ablation of the gene encoding for periostin. Our data suggest that loss of periostin attenuates lipid metabolism in adipose tissue, thus recapitulating one aspect of age-related metabolic dysfunction. In human white adipose tissue, periostin expression showed an unexpected positive correlation with age of study participants. This correlation, however, was no longer evident after adjusting for BMI or plasma lipid and liver function biomarkers. These findings taken together suggest that age-related alterations of the adipose tissue extracellular matrix may contribute to the development of metabolic disease by negatively affecting nutrient homeostasis.

  View details for DOI 10.1111/acel.12810

  View details for PubMedID 30088333

  View details for PubMedCentralID PMC6156450

• The emerging role of bone marrow adipose tissue in bone health and dysfunction JOURNAL OF MOLECULAR MEDICINE-JMM Ambrosi, T. H., Schulz, T. J. 2017; 95 (12): 1291–1301

  Abstract

  Replacement of red hematopoietic bone marrow with yellow adipocyte-rich marrow is a conserved physiological process among mammals. The extent of this conversion is influenced by a wide array of pathological and non-pathological conditions. Of particular interest is the observation that some marrow adipocyte-inducing factors seem to oppose each other, for instance obesity and caloric restriction. Intriguingly, several important molecular characteristics of bone marrow adipose tissue (BMAT) are distinct from the classical depots of white and brown fat tissue. This depot of fat has recently emerged as an active part of the bone marrow niche that exerts paracrine and endocrine functions thereby controlling osteogenesis and hematopoiesis. While some functions of BMAT may be beneficial for metabolic adaptation and bone homeostasis, respectively, most findings assign bone fat a detrimental role during regenerative processes, such as hematopoiesis and osteogenesis. Thus, an improved understanding of the biological mechanisms leading to formation of BMAT, its molecular characteristics, and its physiological role in the bone marrow niche is warranted. Here we review the current understanding of BMAT biology and its potential implications for health and the development of pathological conditions.

  View details for DOI 10.1007/s00109-017-1604-7

  View details for Web of Science ID 000415312100005

  View details for PubMedID 29101431

• Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration CELL STEM CELL Ambrosi, T. H., Scialdone, A., Graja, A., Gohlke, S., Jank, A., Bocian, C., Woelk, L., Fan, H., Logan, D. W., Schuermann, A., Saraiva, L. R., Schulz, T. J. 2017; 20 (6): 771-+

  Abstract

  Aging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.

  View details for DOI 10.1016/j.stem.2017.02.009

  View details for Web of Science ID 000402716800017

  View details for PubMedID 28330582

  View details for PubMedCentralID PMC5459794

• Muscle mitochondrial stress adaptation operates independently of endogenous FGF21 action MOLECULAR METABOLISM Ost, M., Coleman, V., Voigt, A., van Schothorst, E. M., Keipert, S., van der Stelt, I., Ringel, S., Graja, A., Ambrosi, T., Kipp, A. P., Jastroch, M., Schulz, T. J., Keijer, J., Klaus, S. 2016; 5 (2): 79–90

  Abstract

  Fibroblast growth factor 21 (FGF21) was recently discovered as stress-induced myokine during mitochondrial disease and proposed as key metabolic mediator of the integrated stress response (ISR) presumably causing systemic metabolic improvements. Curiously, the precise cell-non-autonomous and cell-autonomous relevance of endogenous FGF21 action remained poorly understood.We made use of the established UCP1 transgenic (TG) mouse, a model of metabolic perturbations made by a specific decrease in muscle mitochondrial efficiency through increased respiratory uncoupling and robust metabolic adaptation and muscle ISR-driven FGF21 induction. In a cross of TG with Fgf21-knockout (FGF21(-/-)) mice, we determined the functional role of FGF21 as a muscle stress-induced myokine under low and high fat feeding conditions.Here we uncovered that FGF21 signaling is dispensable for metabolic improvements evoked by compromised mitochondrial function in skeletal muscle. Strikingly, genetic ablation of FGF21 fully counteracted the cell-non-autonomous metabolic remodeling and browning of subcutaneous white adipose tissue (WAT), together with the reduction of circulating triglycerides and cholesterol. Brown adipose tissue activity was similar in all groups. Remarkably, we found that FGF21 played a negligible role in muscle mitochondrial stress-related improved obesity resistance, glycemic control and hepatic lipid homeostasis. Furthermore, the protective cell-autonomous muscle mitohormesis and metabolic stress adaptation, including an increased muscle proteostasis via mitochondrial unfolded protein response (UPR(mt)) and amino acid biosynthetic pathways did not require the presence of FGF21.Here we demonstrate that although FGF21 drives WAT remodeling, the adaptive pseudo-starvation response under elevated muscle mitochondrial stress conditions operates independently of both WAT browning and FGF21 action. Thus, our findings challenge FGF21 as key metabolic mediator of the mitochondrial stress adaptation and powerful therapeutic target during muscle mitochondrial disease.

  View details for DOI 10.1016/j.molmet.2015.11.002

  View details for Web of Science ID 000368693500002

  View details for PubMedID 26909316

  View details for PubMedCentralID PMC4735627

• A Focused Low-Intensity Pulsed Ultrasound (FLIPUS) System for Cell Stimulation: Physical and Biological Proof of Principle IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Puts, R., Ruschke, K., Ambrosi, T. H., Kadow-Romacker, A., Knaus, P., Jenderka, K., Raum, K. 2016; 63 (1): 91–100

  Abstract

  Quantitative ultrasound (QUS) is a promising technique for bone tissue evaluation. Highly focused transducers used for QUS also have the capability to be applied for tissue-regenerative purposes and can provide spatially limited deposition of acoustic energy. We describe a focused low-intensity pulsed ultrasound (FLIPUS) system, which has been developed for the stimulation of cell monolayers in the defocused far field of the transducer through the bottom of the well plate. Tissue culture well plates, carrying the cells, were incubated in a special chamber, immersed in a temperature-controlled water tank. A stimulation frequency of 3.6 MHz provided an optimal sound transmission through the polystyrene well plate. The ultrasound was pulsed for 20 min daily at 100-Hz repetition frequency with 27.8% duty cycle. The calibrated output intensity corresponded to I(SATA) = 44.5 ± 7.1 mW/cm2, which is comparable to the most frequently reported nominal output levels in LIPUS studies. No temperature change by the ultrasound exposure was observed in the well plate. The system was used to stimulate rat mesenchymal stem cells (rMSCs). The applied intensity had no apoptotic effect and enhanced the expression of osteogenic markers, i.e., osteopontin (OPN), collagen 1 (Col-1), the osteoblast-specific transcription factor-Runx-2 and E11 protein, an early osteocyte marker, in stimulated cells on day 5. The proposed FLIPUS setup opens new perspectives for the evaluation of the mechanistic effects of LIPUS.

  View details for DOI 10.1109/TUFFC.2015.2498042

  View details for Web of Science ID 000367616200008

  View details for PubMedID 26552085