Regulated expression of transgene production and function is of great importance for gene therapy. Such regulation can potentially be used to monitor and control complex biological processes. We report here a regulated stem cell-based system for controlling bone regeneration, utilizing genetically engineered mesenchymal stem cells (MSCs) harboring a tetracycline-regulated expression vector encoding the osteogenic growth factor human BMP-2. We show that doxycycline (a tetracycline analogue) is able to control hBMP-2 expression and thus control MSC osteogenic differentiation both in vitro and in vivo. Following in vivo transplantation of genetically engineered MSCs, doxycycline administration controlled both bone formation and bone regeneration. Moreover, our findings showed increased angiogenesis accompanied by bone formation whenever genetically engineered MSCs were induced to express hBMP-2 in vivo. Thus, our results demonstrate that regulated gene expression in mesenchymal stem cells can be used as a means to control bone healing.

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Intervertebral disc degeneration (IVDD) occurs as a result of an imbalance of the anabolic and catabolic processes in the intervertebral disc, leading to an alteration in the composition of the extracellular matrix (ECM), loss of nucleus pulposus (NP) cells, excessive oxidative stress and inflammation. Degeneration of the IVD occurs naturally with age, but mechanical trauma, lifestyle factors and certain genetic abnormalities can increase the likelihood of symptomatic disease progression. IVDD, often referred to as degenerative disc disease (DDD), poses an increasingly substantial financial burden due to the aging population and increasing incidence of obesity in the United States. Current treatments for IVDD include pharmacological and surgical interventions, but these lack the ability to stop the progression of disease and restore the functionality of the IVD. Biological therapies have been evaluated but show varying degrees of efficacy in reversing disc degeneration long-term. Stem cell-based therapies have shown promising results in the regeneration of the IVD, but face both biological and ethical limitations. Exosomes play an important role in intercellular communication, and stem cell-derived exosomes have been shown to maintain the therapeutic benefit of their origin cells without the associated risks. This review highlights the current state of research on the use of stem-cell derived exosomes in the treatment of IVDD.

Microbubble-enhanced, ultrasound-mediated BMP-6 gene delivery to endogenous progenitor cells induces rapid and efficient repair of critical-sized, nonunion bone fractures in mini-pigs.

Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self-renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short-term exposure of embryoid bodies to transforming growth factor-β was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC-derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow-derived MSCs (BM-MSCs). Ectopic injections of BMP6-overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6-overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self-renewal without tumorigenic ability. Compared with BM-MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture. Significance: Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration of various skeletal conditions; however, availability of autologous MSCs is very limited. This study demonstrates a new method to differentiate human fibroblast-derived induced pluripotent stem cells (iPSCs) to cells with MSC properties, which we comprehensively characterized including differentiation potential and transcriptomic analysis. We showed that these iPS-derived MSCs are able to regenerate nonunion bone defects in mice more efficiently than bone marrow-derived human MSCs when overexpressing BMP6 using a nonviral transfection method.

Many clinical conditions require regeneration or implantation of bone. This is one focus shared by neurosurgery and orthopedics. Current therapeutic options (bone grafting and protein-based therapy) do not provide satisfying solutions to the problem of massive bone defects. In the past few years, gene- and stem cell–based therapy has been extensively studied to achieve a viable alternative to current solutions offered by modern medicine for bone-loss repair. The use of adult stem cells for bone regeneration has gained much focus. This unique population of multipotential cells has been isolated from various sources, including bone marrow, adipose, and muscle tissues. Genetic engineering of adult stem cells with potent osteogenic genes has led to fracture repair and rapid bone formation in vivo. It is hypothesized that these genetically modified cells exert both an autocrine and a paracrine effects on host stem cells, leading to an enhanced osteogenic effect. The use of direct gene delivery has also shown much promise for in vivo bone repair. Several viral and nonviral methods have been used to achieve substantial bone tissue formation in various sites in animal models. To advance these platforms to the clinical setting, it will be mandatory to overcome specific hurdles, such as control over transgene expression, viral vector toxicity, and prolonged culture periods of therapeutic stem cells. This review covers a prospect of cell and gene therapy for bone repair as well as some very recent advancements in stem cell isolation, genetic engineering, and exogenous control of transgene expression.