Longitudinal Analysis of Circulating Markers of Bone Turnover Across Multiple Decades in Osteoporotic Women.

PURPOSE: The objective of this study was to analyze changes in serum markers of bone turnover across multiple decades in osteoporotic women compared with nonosteoporotic controls, to determine their utility as potential predictors for osteoporosis. Early prediction of those at risk for osteoporosis can enable early intervention before the irreversible loss of critical bone mass. METHODS: Serum samples were obtained from 20 women given the diagnosis of osteoporosis after age 46 years and 20 age-matched women with normal bone mineral density from 4 time points in their life (ages 25-31, 32-38, 39-45, and 46-60 years). Serum levels of bone turnover markers (propeptide of type I collagen, parathyroid hormone, bone-specific alkaline phosphatase, osteocalcin, C-terminal telopeptide of type I collagen, sclerostin, osteoprotegerin, osteopontin, and 25-OH vitamin D) were measured using commercially available arrays and kits. We used logistic regression to assess these individual serum markers as potential predictors of osteoporosis, and mixed-effects modeling to assess the change in bone turnover markers between osteoporotic and control groups over time, then performed fivefold cross-validation to assess the classification ability of the models. RESULTS: Markers of bone turnover, bone-specific alkaline phosphatase, C-terminal telopeptide of type I collagen, sclerostin, and osteocalcin were all independent predictors at multiple time points; osteopontin was an independent predictor in the 39- to 45-year age group. Receiver operating characteristic analyses demonstrated moderately strong classification ability at all time points. Sclerostin levels among groups diverged over time and were higher in the control group than the osteoporotic group, with significant differences observed at time points 3 and 4. CONCLUSIONS: Serum markers of bone turnover may be used to estimate the likelihood of osteoporosis development in individuals over time. Although prospective validation is necessary before recommending widespread clinical use, this information may be used to identify patients at risk for developing low bone mineral density long before traditional screening would ostensibly take place. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic II.

Culture and characterization of various porcine integumentary-connective tissue-derived mesenchymal stromal cells to facilitate tissue adhesion to percutaneous metal implants.

BACKGROUND: Transdermal osseointegrated prosthesis have relatively high infection rates leading to implant revision or failure. A principle cause for this complication is the absence of a durable impervious biomechanical seal at the interface of the hard structure (implant) and adjacent soft tissues. This study explores the possibility of recapitulating an analogous cellular musculoskeletal-connective tissue interface, which is present at naturally occurring integumentary tissues where a hard structure exits the skin, such as the nail bed, hoof, and tooth. METHODS: Porcine mesenchymal stromal cells (pMSCs) were derived from nine different porcine integumentary and connective tissues: hoof-associated superficial flexor tendon, molar-associated periodontal ligament, Achilles tendon, adipose tissue and skin dermis from the hind limb and abdominal regions, bone marrow and muscle. For all nine pMSCs, the phenotype, multi-lineage differentiation potential and their adhesiveness to clinical grade titanium was characterized. Transcriptomic analysis of 11 common genes encoding cytoskeletal proteins VIM (Vimentin), cell-cell and cell-matrix adhesion genes (Vinculin, Integrin ß1, Integrin ß2, CD9, CD151), and for ECM genes (Collagen-1a1, Collagen-4a1, Fibronectin, Laminin-α5, Contactin-3) in early passaged cells was performed using qRT-PCR. RESULTS: All tissue-derived pMSCs were characterized as mesenchymal origin by adherence to plastic, expression of cell surface markers including CD29, CD44, CD90, and CD105, and lack of hematopoietic (CD11b) and endothelial (CD31) markers. All pMSCs differentiated into osteoblasts, adipocytes and chondrocytes, albeit at varying degrees, under specific culture conditions. Among the eleven adhesion genes evaluated, the cytoskeletal intermediate filament vimentin was found highly expressed in pMSC isolated from all tissues, followed by genes for the extracellular matrix proteins Fibronectin and Collagen-1a1. Expression of Vimentin was the highest in Achilles tendon, while Fibronectin and Col1agen-1a1 were highest in molar and hoof-associated superficial flexor tendon bone marrow, respectively. Achilles tendon ranked the highest in both multilineage differentiation and adhesion assessments to titanium metal. CONCLUSIONS: These findings support further preclinical research of these tissue specific-derived MSCs in vivo in a transdermal osseointegration implant model.

Alarming Cargo: The Role of Exosomes in Trauma-Induced Inflammation.

Severe polytraumatic injury initiates a robust immune response. Broad immune dysfunction in patients with such injuries has been well-documented; however, early biomarkers of immune dysfunction post-injury, which are critical for comprehensive intervention and can predict the clinical course of patients, have not been reported. Current circulating markers such as IL-6 and IL-10 are broad, non-specific, and lag behind the clinical course of patients. General blockade of the inflammatory response is detrimental to patients, as a certain degree of regulated inflammation is critical and necessary following trauma. Exosomes, small membrane-bound extracellular vesicles, found in a variety of biofluids, carry within them a complex functional cargo, comprised of coding and non-coding RNAs, proteins, and metabolites. Composition of circulating exosomal cargo is modulated by changes in the intra- and extracellular microenvironment, thereby serving as a homeostasis sensor. With its extensively documented involvement in immune regulation in multiple pathologies, study of exosomal cargo in polytrauma patients can provide critical insights on trauma-specific, temporal immune dysregulation, with tremendous potential to serve as unique biomarkers and therapeutic targets for timely and precise intervention.

Saracatinib is an efficacious clinical candidate for fibrodysplasia ossificans progressiva.

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-ß signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.

High Frequency Spectral Ultrasound Imaging Detects Early Heterotopic Ossification in Rodents.

Heterotopic ossification (HO) is a devastating condition in which ectopic bone forms inappropriately in soft tissues following traumatic injuries and orthopedic surgeries as a result of aberrant mesenchymal progenitor cell (MPC) differentiation. HO leads to chronic pain, decreased range of motion, and an overall decrease in quality of life. While several treatments have shown promise in animal models, all must be given during early stages of formation. Methods for early determination of whether and where endochondral ossification/soft tissue mineralization (HO anlagen) develop are lacking. At-risk patients are not identified sufficiently early in the process of MPC differentiation and soft tissue endochondral ossification for potential treatments to be effective. Hence, a critical need exists to develop technologies capable of detecting HO anlagen soon after trauma, when treatments are most effective. In this study, we investigate high frequency spectral ultrasound imaging (SUSI) as a noninvasive strategy to identify HO anlagen at early time points after injury. We show that by determining quantitative parameters based on tissue organization and structure, SUSI identifies HO anlagen as early as 1-week postinjury in a mouse model of burn/tenotomy and 3 days postinjury in a rat model of blast/amputation. We analyze single cell RNA sequencing profiles of the MPCs responsible for HO formation and show that the early tissue changes detected by SUSI match chondrogenic and osteogenic gene expression in this population. SUSI identifies sites of soft tissue endochondral ossification at early stages of HO formation so that effective intervention can be targeted when and where it is needed following trauma-induced injury. Furthermore, we characterize the chondrogenic to osteogenic transition that occurs in the MPCs during HO formation and correlate gene expression to SUSI detection of the HO anlagen.

BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma.

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma.

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-ß activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P < 0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P < 0.001) and 133.3-fold (P < 0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P < 0.001) and 2.7-fold reduction (P < 0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.

Orthopaedic osseointegration: Implantology and future directions.

Osseointegration (OI) is the direct anchorage of a metal implant into bone, allowing for the connection of an external prosthesis to the skeleton. Osseointegration was first discovered in the 1960s based on the microscopic analysis of titanium implant placed into host bone. New bone was observed to attach directly to the metal surface. Following clinical investigations into dentistry applications, OI was adapted to treat extremity amputations. These bone anchored implants, which penetrate the skin and soft tissues, eliminate many of the challenges of conventional prosthetic sockets, such as poor fit and suspension, skin breakdown, and pain. Osseointegrated implants show promise to improve prosthesis use, pain, and function for amputees. The successful process of transcutaneous metal integration into host bone requires three synergistic systems: the host bone, the metal implant, and the skin-implant interface. All three systems must be optimized for successful incorporation and longevity of the implant. Osseointegration begins during surgical implantation of the metal components through a complex interplay of cellular mechanisms. While implants can vary in design-including the original screw, press fit implants, and compressive osseointegration-they face common challenges to successful integration and maintenance of fixation within the host bone. Overcoming these challenges requires the understanding of the complex interactions between each element of OI. This review outlines (a) the basic components of OI, (b) the science behind both the bone-implant and the skin-implant interfaces, (c) the current challenges of OI, and (d) future opportunities within the field.

Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo.

Cancer cells and embryonic tissues share a number of cellular and molecular properties, suggesting that induced pluripotent stem cells (iPSCs) may be harnessed to elicit anti-tumor responses in cancer vaccines. RNA sequencing revealed that human and murine iPSCs express tumor-associated antigens, and we show here a proof of principle for using irradiated iPSCs in autologous anti-tumor vaccines. In a prophylactic setting, iPSC vaccines prevent tumor growth in syngeneic murine breast cancer, mesothelioma, and melanoma models. As an adjuvant, the iPSC vaccine inhibited melanoma recurrence at the resection site and reduced metastatic tumor load, which was associated with fewer Th17 cells and increased CD11b+GR1hi myeloid cells. Adoptive transfer of T cells isolated from vaccine-treated tumor-bearing mice inhibited tumor growth in unvaccinated recipients, indicating that the iPSC vaccine promotes an antigen-specific anti-tumor T cell response. Our data suggest an easy, generalizable strategy for multiple types of cancer that could prove highly valuable in clinical immunotherapy.

Palovarotene inhibits connective tissue progenitor cell proliferation in a rat model of combat-related heterotopic ossification.

Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1135-1144, 2018.

Inhibition of Mammalian Target of Rapamycin Signaling with Rapamycin Prevents Trauma-Induced Heterotopic Ossification.

A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism.

The traumatic bone: trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is a common occurrence after multiple forms of extensive trauma. These include arthroplasties, traumatic brain and spinal cord injuries, extensive burns in the civilian setting, and combat-related extremity injuries in the battlefield. Irrespective of the form of trauma, heterotopic bone is typically endochondral in structure and is laid down via a cartilaginous matrix. Once formed, the heterotopic bone typically needs to be excised surgically, which may result in wound healing complications, in addition to a risk of recurrence. Refinements of existing diagnostic modalities, like micro- and nano-CT are being adapted toward early intervention. Trauma-induced HO is a consequence of aberrant wound healing, systemic and local immune system activation, infections, extensive vascularization, and innervation. This intricate molecular crosstalk culminates in activation of stem cells that initiate heterotopic endochondral ossification. Development of animal models recapitulating the unique traumatic injuries has greatly facilitated the mechanistic understanding of trauma-induced HO. These same models also serve as powerful tools to test the efficacy of small molecules which specifically target the molecular pathways underlying ectopic ossification. This review summarizes the recent advances in the molecular understanding, diagnostic and treatment modalities in the field of trauma-induced HO.

Early local delivery of vancomycin suppresses ectopic bone formation in a rat model of trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is a debilitating sequela of high-energy injuries. It frequently requires surgical excision once symptomatic and there is no practical prophylaxis for combat-injured patients. In this study, we examined the effect of local vancomycin powder on HO formation in a small animal model of blast-related, post-traumatic HO. Male Sprague-Dawley rats were subjected to a polytraumatic extremity injury and amputation with or without methicillin-resistant Staphylococcus aureus infection. Animals were randomized to receive a single local application of vancomycin (20 mg/kg) at the time of injury (POD-0, n = 34) or on postoperative day-3 (POD-3, n = 11). Quantitative volumetric measurement of ectopic bone was calculated at 12-weeks post-injury by micro-CT. Bone marrow and muscle tissues were also collected to determine the bacterial burden. Blood for serum cytokine analysis was collected at baseline and post-injury. Vancomycin treatment on POD-0 suppressed HO formation by 86% and prevented bone marrow and soft tissue infections. We concurrently observed a marked reduction histologically in nonviable tissue, chronic inflammatory cell infiltrates, bone infection, fibrous tissue, and areas of bone necrosis within this same cohort. Delayed treatment was significantly less efficacious. Neither treatment had a marked effect on the production of pro-inflammatory cytokines. Our study demonstrates that local vancomycin treatment at the time of injury significantly reduces HO formation in both the presence and absence of infection, with decreased efficacy if not given early. These findings further support the concept that the therapeutic window for prophylaxis is narrow, highlighting the need to develop early treatment strategies for clinical management. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2397-2406, 2017.

Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic ossification.

Fibrodysplasia ossificans progressiva (FOP), a congenital heterotopic ossification (HO) syndrome caused by gain-of-function mutations of bone morphogenetic protein (BMP) type I receptor ACVR1, manifests with progressive ossification of skeletal muscles, tendons, ligaments, and joints. In this disease, HO can occur in discrete flares, often triggered by injury or inflammation, or may progress incrementally without identified triggers. Mice harboring an Acvr1R206H knock-in allele recapitulate the phenotypic spectrum of FOP, including injury-responsive intramuscular HO and spontaneous articular, tendon, and ligament ossification. The cells that drive HO in these diverse tissues can be compartmentalized into two lineages: an Scx+ tendon-derived progenitor that mediates endochondral HO of ligaments and joints without exogenous injury, and a muscle-resident interstitial Mx1+ population that mediates intramuscular, injury-dependent endochondral HO. Expression of Acvr1R206H in either lineage confers aberrant gain of BMP signaling and chondrogenic differentiation in response to activin A and gives rise to mutation-expressing hypertrophic chondrocytes in HO lesions. Compared to Acvr1R206H, expression of the man-made, ligand-independent ACVR1Q207D mutation accelerates and increases the penetrance of all observed phenotypes, but does not abrogate the need for antecedent injury in muscle HO, demonstrating the need for an injury factor in addition to enhanced BMP signaling. Both injury-dependent intramuscular and spontaneous ligament HO in Acvr1R206H knock-in mice were effectively controlled by the selective ACVR1 inhibitor LDN-212854. Thus, diverse phenotypes of HO found in FOP are rooted in cell-autonomous effects of dysregulated ACVR1 signaling in nonoverlapping tissue-resident progenitor pools that may be addressed by systemic therapy or by modulating injury-mediated factors involved in their local recruitment.

Sca-1+ cells from fetal heart with high aldehyde dehydrogenase activity exhibit enhanced gene expression for self-renewal, proliferation, and survival.

Stem/progenitor cells from multiple tissues have been isolated based on enhanced activity of cytosolic aldehyde dehydrogenase (ALDH) enzyme. ALDH activity has emerged as a reliable marker for stem/progenitor cells, such that ALDH(bright/high) cells from multiple tissues have been shown to possess enhanced stemness properties (self-renewal and multipotency). So far though, not much is known about ALDH activity in specific fetal organs. In this study, we sought to analyze the presence and activity of the ALDH enzyme in the stem cell antigen-1-positive (Sca-1+) cells of fetal human heart. Biochemical assays showed that a subpopulation of Sca-1+ cells (15%) possess significantly high ALDH1 activity. This subpopulation showed increased expression of self-renewal markers compared to the ALDH(low) fraction. The ALDH(high) fraction also exhibited significant increase in proliferation and pro-survival gene expression. In addition, only the ALDH(high) and not the ALDH(low) fraction could give rise to all the cell types of the original population, demonstrating multipotency. ALDH(high) cells showed increased resistance against aldehyde challenge compared to ALDH(low) cells. These results indicate that ALDH(high) subpopulation of the cultured human fetal cells has enhanced self-renewal, multipotency, high proliferation, and survival, indicating that this might represent a primitive stem cell population within the fetal human heart.

Contributions of muscle-resident progenitor cells to homeostasis and disease.

Adult skeletal muscle maintains a homeostatic state with modest levels of cellular turnover, unlike the skin or blood. However, the muscle is highly sensitive to tissue injury, which unleashes a cascade of regenerative and inflammatory processes. Muscle regeneration involves cross-talk between numerous cytokine signaling axes, and the coordinated activity of multiple muscle-resident and circulating progenitor populations. Satellite cells, closely associated with myofibers, are established as the canonical muscle stem cell, with self-renewal and myofiber-regenerating capacity. However, a heterogeneous group of mesenchymal progenitor cells residing within the muscle interstitium are also highly responsive to muscle injury and exhibit varying degrees of regenerative potential. These cells interact with satellite cells via direct and indirect mechanisms to regulate regeneration or repair. We describe the known phylogenetic and functional relationships of the multiple progenitor populations residing within skeletal muscle, their putative roles in the coordination of injury repair, and their possible contributions to health and disease.

Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer.

BACKGROUND: Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. METHODS AND RESULTS: After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. CONCLUSIONS: In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.

Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells.

INTRODUCTION: Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed 'mammospheres'. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression. METHODS: Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. RESULTS: In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser116 phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser116 phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116 residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser116 phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo. CONCLUSIONS: Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.

Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance.

The exact nature of the immune response elicited by autologous-induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic immune response characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B and perforin intragraft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.