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Bone marrow stromal or stem cells (BMSCs) remain a promising potential therapy for ischemic cardiomyopathy. The primary objective of this study was to evaluate the safety and feasibility of direct intramyocardial injection of autologous BMSCs in patients undergoing transmyocardial revascularization (TMR) or coronary artery bypass graft surgery (CABG). A phase I trial was conducted on adult patients who had ischemic heart disease with depressed left ventricular ejection fraction and who were scheduled to undergo TMR or CABG. Autologous BMSCs were expanded for 3 weeks before the scheduled surgery. After completion of surgical revascularization, BMSCs were directly injected into ischemic myocardium. Safety and feasibility of therapy were assessed. Cardiac functional status and changes in quality of life were evaluated at 1 year. A total of 14 patients underwent simultaneous BMSC and surgical revascularization therapy (TMR+BMSCs = 10; CABG+BMSCs = 4). BMSCs were successfully expanded, and no significant complications occurred as a result of the procedure. Regional contractility in the cell-treated areas demonstrated improvement at 12 months compared with baseline (TMR+BMSCs Δ strain: −4.6% ± 2.1%; P =.02; CABG+MSCs Δ strain: −4.2% ± 6.0%; P =.30). Quality of life was enhanced, with substantial reduction in angina scores at 1 year after treatment (TMR+BMSCs: 1.3 ± 1.2; CABG+MSCs: 1.0 ± 1.4). In this phase I trial, direct intramyocardial injection of autologous BMSCs in conjunction with TMR or CABG was technically feasible and could be performed safely. Preliminary results demonstrate improved cardiac function and quality of life in patients at 1 year after treatment. [ABSTRACT FROM AUTHOR]

In current orthopaedic practice, there is a need to increase the ability to reconstruct large segments of bone lost due to trauma, resection of tumors and skeletal deformities, or when normal regenerative processes have failed such as in non-unions and avascular necrosis. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells), when used in conjunction with appropriate carriers, represent a means by which to achieve bone regeneration in such cases. While much has been done at the bench and in pre-clinical studies, moving towards clinical application requires the generation of clinical grade cells. What is described herein is an FDA-approved cell manufacturing procedure for the ex vivo expansion of high quality, biologically active human BMSCs. This article is part of a Special Issue entitled Stem Cells and Bone. [ABSTRACT FROM AUTHOR]

Summary: Induced pluripotent stem cell (iPSC)-based cell therapies have great potential for regenerative medicine but are also potentially associated with tumorigenic risks. Current rodent models are not optimal predictors of efficiency and safety for clinical application. Therefore, we developed a clinically relevant nonhuman primate model to assess the tumorigenic potential and in vivo efficacy of both undifferentiated and differentiated iPSCs in autologous settings without immunosuppression. Undifferentiated autologous iPSCs indeed form mature teratomas in a dose-dependent manner. However, tumor formation is accompanied by an inflammatory reaction. On the other hand, iPSC-derived mesodermal stromal-like cells form new bone in vivo without any evidence of teratoma formation. We therefore show in a large animal model that closely resembles human physiology that undifferentiated autologous iPSCs form teratomas, and that iPSC-derived progenitor cells can give rise to a functional tissue in vivo. [Copyright &y& Elsevier]

Technologies on the development and differentiation of human induced pluripotent stem cells (hiPSCs) are rapidly improving, and have been applied to create cell types relevant to the bone field. Differentiation protocols to form bona fide bone-forming cells from iPSCs are available, and can be used to probe details of differentiation and function in depth. When applied to iPSCs bearing disease-causing mutations, the pathogenetic mechanisms of diseases of the skeleton can be elucidated, along with the development of novel therapeutics. These cells can also be used for development of cell therapies for cell and tissue replacement. • Creation of iPSCs marks a major advance in the cell biology. • Functional OCs and OBs can be differentiated from iPSCs. • Mutated iPSCs can model human skeletal diseases. [ABSTRACT FROM AUTHOR]

Postnatal skeletal stem cells are a unique class of progenitors with biological properties that extend well beyond the limits of stemness as commonly defined. Skeletal stem cells sustain skeletal tissue homeostasis, organize and maintain the complex architectural structure of the bone marrow microenvironment and provide a niche for hematopoietic progenitor cells. The identification of stem cells in the human post-natal skeleton has profoundly changed our approach to the physiology and pathology of this system. Skeletal diseases have been long interpreted essentially in terms of defective function of differentiated cells and/or abnormal turnover of the matrix that they produce. The notion of a skeletal stem cell has brought forth multiple, novel concepts in skeletal biology that provide potential alternative concepts. At the same time, the recognition of the complex functions played by skeletal progenitors, such as the structural and functional organization of the bone marrow, has provided an innovative, unifying perspective for understanding bone and bone marrow changes simultaneously occurring in many disorders. Finally, the possibility to isolate and highly enrich for skeletal progenitors, enables us to reproduce perfectly normal or pathological organ miniatures. These, in turn, provide suitable models to investigate and manipulate the pathogenetic mechanisms of many genetic and non-genetic skeletal diseases. This article is part of a Special Issue entitled Stem cells and Bone. [ABSTRACT FROM AUTHOR]

Abstract Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (μCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs. [ABSTRACT FROM AUTHOR]

Abstract The Signature Series Symposium "Cellular Therapies for Orthopaedics and Musculoskeletal Disease Proven and Unproven Therapies—Promise, Facts and Fantasy" was held as a pre-meeting of the 26th International Society for Cellular Therapy (ISCT) annual congress in Montreal, Canada, May 2, 2018. This was the first ISCT program that was entirely dedicated to the advancement of cell-based therapies for musculoskeletal diseases. Cellular therapies in musculoskeletal medicine are a source of great promise and opportunity. They are also the source of public controversy, confusion and misinformation. Patients, clinicians, scientists, industry and government share a commitment to clear communication and responsible development of the field. Therefore, this symposium convened thought leaders from around the world in a forum designed to catalyze communication and collaboration to bring the greatest possible innovation and value to patients with musculoskeletal conditions. [ABSTRACT FROM AUTHOR]

Use of human pluripotent stem cells (hPSCs) and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery (hPDD). Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. In this review, we dissect representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR , the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis. [ABSTRACT FROM AUTHOR]

Background aims. Ex vivo expansion and serial passage of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) is required to obtain sufficient quantities for clinical therapy. The BMSC confluence criteria used to determine passage and harvest timing vary widely, and the impact of confluence on BMSC properties remains controversial. The effects of confluence on BMSC properties were studied and confluence-associated markers were identified. Methods. BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony formation, immunosuppression, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed. Results. Confluence-dependent changes were detected in the expression of several cell surface markers: 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. The pigment epithelium-derived factor/vascular endothelial growth factor ratio increased with confluence, but 80% and 100% confluent BMSCs demonstrated a similar level of immunosuppression of mixed lymphocyte reactions. In addition, changes in lactate and glucose levels correlated with BMSC density. Conclusions. BMSC characteristics change as confluence increases. 100% confluent BMSCs may have compromised pro-angiogenesis properties but may retain their immunomodulatory properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry or microRNA expression can be used to confirm that the BMSCs have been harvested at the appropriate confluence. [ABSTRACT FROM AUTHOR]

WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1-/-) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1-/- mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1-/- mice had decreased trabecular bone volume/total volume and that both male and female Wisp1-/- mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1-/- mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1-/- mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1-/- mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1-/- bone marrow stromal cells had reduced expression of β-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1-/- mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones. [ABSTRACT FROM AUTHOR]

Abstract: Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (μCT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis. [Copyright &y& Elsevier]

Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues. [ABSTRACT FROM AUTHOR]

Abstract: Transplants of culture-expanded bone marrow stromal cells (BMSCs) combined with hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds successfully form cortico-cancellous bone to reconstruct the dog craniofacial skeleton. Yet, these transplants'' long-term stability in large animal models has not been evaluated. This study''s purpose was the evaluation of long-term BMSC transplant stability when used to augment the mandible. Here, autologous BMSC–HA/TCP transplants were introduced onto the unilateral dog mandible as onlay grafts, while contralateral control mandibles received HA/TCP onlays alone. Quantitative CT (qCT) scans were obtained both early and late after transplantation. Transplants were harvested up to 19 months later for histologic and mechanical analyses. In all dogs, BMSC transplants formed significantly greater amounts of bone over their control counterparts. The new bone formed an extensive union with the underlying mandible. BMSC transplants retained the majority of their initial volume, while control (HA/TCP only) transplants were nearly completely resorbed. By qCT, the extent of newly formed bone could be determined non-invasively. In summary, HA/TCP particles alone undergo a high degree of resorption, while autologous cultured BMSC–HA/TCP transplants provide long-term bony augmentation of the mandible. [Copyright &y& Elsevier]

Abstract: Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, which in turn are dependent on the bone''s mechanical environment. Male mice have a greater response to non-weight-bearing exercise than females, resulting in larger, stronger bones compared with control animals. The aim of this study was to test the hypothesis that short-term weight-bearing running during growth (21 days starting at 8 weeks of age; 30 min/day; 12 m/min; 5° incline; 7 days/week) would similarly have a greater impact on cross-sectional geometry and mechanical competence in the femora and tibiae of male mice versus females. Based on the orientation of the legs during running and the proximity of the tibia to the point of impact, this response was hypothesized to be greatest in the tibia. Exercise-related changes relative to controls were assayed by four-point bending tests, while volumetric bone mineral density and cross-sectional geometry were also assessed. The response to running was bone- and gender-specific, with male tibiae demonstrating the greatest effects. In male tibiae, periosteal perimeter, endocortical perimeter, cortical area, medial–lateral width and bending moment of inertia increased versus control mice suggesting that while growth is occurring in these mice between 8 and 11 weeks of age, exercise accelerated this growth resulting in a greater increase in bone tissue over the 3 weeks of the study. Exercise increased tissue-level strain-to-failure and structural post-yield deformation in the male tibiae, but these post-yield benefits came at the expense of decreased yield deformation, structural and tissue-level yield strength and tissue-level ultimate strength. These results suggest that exercise superimposed upon growth accelerated growth-related increases in tibial cross-sectional dimensions. Exercise also influenced the quality of this forming bone, significantly impacting structural and tissue-level mechanical properties. [Copyright &y& Elsevier]

Abstract: Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone''s geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local- and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone- and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo. [Copyright &y& Elsevier]

Abstract: Autologous grafts from axial and appendicular bones commonly used to repair orofacial bone defects often result in unfavorable outcome. This clinical observation, along with the fact that many bone abnormalities are limited to craniofacial bones, suggests that there are significant differences in bone metabolism in orofacial, axial and appendicular bones. It is plausible that these differences are dictated by site-specificity of embryological progenitor cells and osteogenic properties of resident multipotent human bone marrow stromal cells (hBMSCs). This study investigated skeletal site-specific phenotypic and functional differences between orofacial (maxilla and mandible) and axial (iliac crest) hBMSCs in vitro and in vivo. Primary cultures of maxilla, mandible and iliac crest hBMSCs were established with and without osteogenic inducers. Site-specific characterization included colony forming efficiency, cell proliferation, life span before senescence, relative presence of surface markers, adipogenesis, osteogenesis and transplantation in immunocompromised mice to compare bone regenerative capacity. Compared with iliac crest cells, orofacial hBMSCs (OF-MSCs) proliferated more rapidly with delayed senescence, expressed higher levels of alkaline phosphatase and demonstrated more calcium accumulation in vitro. Cells isolated from the three skeletal sites were variably positive for STRO 1, a marker of hBMSCs. OF-MSCs formed more bone in vivo, while iliac crest hBMSCs formed more compacted bone that included hematopoietic tissue and were more responsive in vitro and in vivo to osteogenic and adipogenic inductions. These data demonstrate that hBMSCs from the same individuals differ in vitro and in vivo in a skeletal site-specific fashion and identified orofacial marrow stromal cells as unique cell populations. Further understanding of site-specific properties of hBMSCs and their impact on site-specific bone diseases and regeneration are needed. [Copyright &y& Elsevier]

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-β (TGF-β)-binding proteoglycans, biglycan and decorin, prevents TGF-β from proper sequestration within the extracellular matrix. The excess TGF-β directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-β signaling in bgn/dcn-deficient bone marrow stromal cells is a ‘switch in fate’ from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass. [ABSTRACT FROM AUTHOR]