Galina Shapiro

Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation.

More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×10(5) microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation-induced gene therapies.

Human Induced Pluripotent Stem Cells Differentiate Into Functional Mesenchymal Stem Cells and Repair Bone Defects

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.

Human iPSCs Differentiate Into Functional MSCs and Repair Bone Defects

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.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs.

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. Bubbles and BMP-6 for bone repair Treatments for bone nonunions (fractures that fail to heal) include surgery and bone grafting. As an alternative to viral gene delivery, Bez et al. developed a two-step therapy. First, endogenous mesenchymal stem/progenitor cells were recruited to the bone nonunion by implanting a collagen sponge in the defect site. Two weeks later, bone morphogenetic protein-6 (BMP-6) plasmid DNA and microbubbles were injected into nonunions, and ultrasound was applied to oscillate the microbubbles, which helped the recruited progenitors take up the BMP-6. This therapy led to transient BMP-6 secretion, bone regeneration, and fracture healing over 6 weeks in critical-sized tibial nonunions in mini-pigs. More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

Recent Advances and Future of Gene Therapy for Bone Regeneration

Purpose of ReviewThe purpose of this review is to discuss the recent advances in gene therapy as a treatment for bone regeneration. While most fractures heal spontaneously, patients who present with fracture nonunion suffer from prolonged pain, disability, and often require additional operations to regain musculoskeletal function.Recent FindingsIn the last few years, BMP gene delivery by means of electroporation and sonoporation resulted in repair of nonunion bone defects in mice, rats, and minipigs. Ex vivo transfection of porcine mesenchymal stem cells (MSCs) resulted in bone regeneration following implantation in vertebral defects of minipigs. Sustained release of VEGF gene from a collagen-hydroxyapatite scaffold to the mandible of a human patient was shown to be safe and osteoinductive.SummaryIn conclusion, gene therapy methods for bone regeneration are systematically becoming more efficient and show proof-of-concept in clinically relevant animal models. Yet, on the pathway to clinical use, more investigation is needed to determine the safety aspects of the various techniques in terms of biodistribution, toxicity, and tumorigenicity.

Ultrasound-mediated transfection of endogenous stem cells for regenerative medicine

The oscillation of microbubbles has long been hypothesized to provide the opportunity to enhance gene delivery as a result of changes in membrane permeability; however, translationally-relevant therapeutic protocols have not yet been realized. We sought to develop and validate a protocol to transfect endogenous mesenchymal stem cells (MSCs) via the local injection of plasmids and microbubbles and the application of ultrasound. We apply this therapy in a pre-clinical model to solve an important clinical problem — that of healing segmental bone defects. More than two million bone-grafting procedures are performed each year using autografts or allografts and these standard of care therapies have substantial disadvantages.

Consideration of Biological Sex in Translating Regenerative Stem Cell Therapies

Regenerative cell therapies are emerging as the next major development in medicine. Stem cell therapies are defined as any treatment based on viable human stem cells including adult stem cells, embryonic stem cells, and induced pluripotent stem cells. Their ability to self-renew and potential to differentiate into multiple cell types has led to many clinical trials for tissue regeneration, immune modulation, and cancer therapy among other indications. The challenges surrounding commercial viability and clinical uptake of novel therapies are related to difficulties in establishing clinical utility and cost-effectiveness. Only recently has gender bias come to light as a potentially critical pitfall in translational research. Future stem cell strategies should be based on research that has considered sex as a biological variable throughout the biomedical development spectrum to maximize positive outcomes and avoid gender bias. In this review, we focus on the implications of biological sex on translational regenerative stem cell therapies, and consider the limitations of the present body of knowledge.

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.