Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.

Promoting teaching quality by portfolio assessment in Cell Engineering classroom / 生物工程学报

We introduce the portfolio assessment into the classroom teaching reform in the curriculum of Cell Engineering, a specialty course in bioengineering & biotechnology. We established a complete classroom evaluation system that was divided the classroom assessment system of portfolio into four stages including the preparation stage, training stage, implementation stage and exhibition stage. We also discuss the feasibility and necessity of implementing the portfolio evaluation method in the course of cell engineering, the construction of evaluation system, and the key points and matters needing attention in the implementation process. The classroom reform is very productive, not only the classroom atmosphere has been activated, students' learning initiative and autonomy has been enhanced, but also the students' ability to analyze and solve professional problems related to cell engineering technology has been improved. The implementation of classroom teaching reform of this course can provide reference for other similar professional courses in colleges and universities.

In vitro generation of mature midbrain-type dopamine neurons by adjusting exogenous Nurr1 and Foxa2 expressions to their physiologic patterns

Developmental information aids stem cell biologists in producing tissue-specific cells. Recapitulation of the developmental profile of a specific cell type in an in vitro stem cell system provides a strategy for manipulating cell-fate choice during the differentiation process. Nurr1 and Foxa2 are potential candidates for genetic engineering to generate midbrain-type dopamine (DA) neurons for experimental and therapeutic applications in Parkinson's disease (PD), as forced expression of these genes in neural stem/precursor cells (NPCs) yields cells with a complete battery of midbrain DA neuron-specific genes. However, simple overexpression without considering their expression pattern in the developing midbrain tends to generate DA cells without adequate neuronal maturation and long-term maintenance of their phenotype in vitro and in vivo after transplantation. We here show that the physiological levels and timing of Nurr1 and Foxa2 expression can be replicated in NPCs by choosing the right vectors and promoters. Controlled expression combined with a strategy for transgene expression maintenance induced generation of fully mature midbrain-type DA neurons. These findings demonstrate the feasibility of cellular engineering for artificial cell-fate specification.

Avances en bioingeniería dental y su aplicación en ortodoncia y ortopedia dentofacial: Una revisión de literatura / Advances in dental bioengineering and their application in orthodontics and dentofacial orthopedics: A literature review

conocer la importancia de la terapia génica, celular y tisular como enfoque novedoso de la medicina regenerativa para la biomecánica ortodóntica y ortopedia dentofacial, ampliando su campo de acción, corrigiendo defectos estructurales y ofreciendo alternativas biológicas de alta calidad para la consulta especialista. Los avances en tecnología, acceso a información y exigencias actuales crean una necesidad de innovación estratégica por el clínico; el objetivo de esta revisión es describir los enfoques actuales en bioingeniería dental y terapia génica con sus aplicaciones clínicas enfocadas directamente en la consulta de ortodoncia y ortopedia dentofacial. Esto va a permitir ampliar la visión del especialista, crear necesidad de actualización constante y operar bajo evidencia científica. Se realizó una búsqueda en las bases de datos: Scopus, Medline y ScienceDirect, con las palabras clave: Bioengineering, Stem Cells, Gen Therapy. Finalmente se pudo concluir que el enfoque de la consulta odontológica va dirigido a la medicina regenerativa como terapia convencional, innovadora y de alta calidad; ofreciendo resultados con gran cercanía a la biología humana natural, eficacia a largo plazo y menor porcentaje de efectos adversos, ampliando la visión de todas aquellas fronteras en el manejo clínico del paciente

The advances in dental bioengineering disclose the importance of genetic, cellular and tissue therapies as the latest approach to the regenerative medicine for biomechanical orthodontic and dentofacial orthopedics; extending its scope, correcting structural flaws and offering biological alternatives with high quality technology for specialist practices. In this review, some recent findings related to genetic advances in dental bioengineering are provided and applied to the induction of the potential cell forming in the craniofacial complex and its clinical applications. A special research was made using some databases such as: Scopus, Medline and Science Direct, with the following keywords: Bioengineering, Stem cells, and Gen therapy. Finally, we can conclude that the focus of dental practice is led to regenerative medicine as a conventional, innovative and high quality therapy, giving closely results to natural human biology, long term effectivity and less adverse effects. Then we can scope out the vision of all clinic procedures limits with all patients

Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis

Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-beta1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis.

Advances in improving mammalian cells metabolism for recombinant protein production

Background: The production of recombinant proteins for therapeutic use represents a great impact on the biotechnology industry. In this context, established mammalian cell lines, especially CHO cells, have become a standard system for the production of such proteins. Their ability to properly configure and excrete proteins in functional form is an enormous advantage which should be contrasted with their inherent technological limitations. These cell systems exhibit a metabolic behaviour associated with elevated cell proliferation which involves a high consumption of glucose and glutamine, resulting in the rapid depletion of these nutrients in the medium and the accumulation of ammonium and lactate. Both phenomena contribute to the limitation of cell growth, the triggering of apoptotic processes and the loss of quality of the recombinant protein. Results: In this review, the use of alternative substrates and genetic modifications (host cell engineering) are analyzed as tools to overcome those limitations. In general, the results obtained are promising. However, metabolic and physiological phenomena involved in CHO cells are still barely understood. Thus, most of publications are focused on specific modifications rather than giving a systemic perspective. Conclusions: A deeper insight in the integrated understanding of metabolism and cell mechanisms is required in order to define complementary strategies at these two levels, so providing effective means to control nutrients consumption, reduce by-products and increase process productivity.

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

Medicina regenerativa: posibles aplicaciones de la medicina regenerativa en las afecciones del aparato locomotor. protocolo de investigación clínica. Informe preliminar / Regenerative medicine: possible applications of regenerative medicine in locomotor system diseases. Clinical research protocol. Preliminary report

El deterioro funcional de un órgano o tejido es un problema médico grave y de alto costo, ya que la sustitución de estas estructuras involucra, entre otros, múltiples factores de orden científico, económico y ético. La medicina busca en la actualidad convertir en realidad la regeneración de los tejidos mediante el desarrollo de terapias para restaurar, en el cuerpo humano, las células y los tejidos envejecidos o dañados. En esta comunicación preliminar presentamos una serie de pacientes donde se utilizaron células mesenquimales, mononucleares, autólogas, obtenidas de la médula ósea de la cresta ilíaca del propio enfermo, para tratar distintas afecciones. Desde noviembre de 2005 hasta mayo 2011, tratamos 23 pacientes, con un promedio de edad de 55,39 años. Once pseudoartrosis; 2 retardos de consolidación; 3 alargamientos óseos; 1 defecto osteo-tegumentario de tibia; 3 pacientes con necrosis óseas asépticas; 1 quiste óseo de cuello femoral; 4 artrosis incipientes de rodilla; 1 lesión del manguito rotador del hombro y 1 entesitis crónica aquiliana. El procedimiento se realizó en todos los casos en forma ambulatoria. Todos los procedimientos se realizaron en quirófano bajo anestesia general breve y con rigurosa asepsia. Utilizamos métodos reglados para cosechar células madre mesenquimáticas mononucleares de médula ósea del propio enfermo. Igualmente de manera reglada se cosecha sangre periférica del paciente para obtener, una vez procesada, plasma enriquecido con plaquetas y lisado de plaquetas. Logramos consolidación clínica y radiológica en 10 de los 11 casos de peudoartrosis (90,90 por ciento), en los 2 casos de retardo de consolidación, en los 3 alargamientos óseos y en el defecto osteo-tegumentario de tibia...

The functional impairment of an organ or tissue is a serious and high cost medical problem. The replacement of these structures involves multiple scientific, economical and ethical issues. All medical physicians and researchers are working nowadays to make possible tissue regeneration by developing therapies to restore damaged, aged or dead cells and tissues of the human body. At this preliminary report we present a series of patients, with various pathologies, in which we used autologous mononuclear mesenchymal cells, harvested from the bone marrow of the patient's iliac crest. Since November 2005 until May 2011, 23 patients, with an average age of 55,39 years, were treated: 11 non unions; 2 delayed unions; 3 bone lengthening; 1 tibia severe lesion of soft tissue and loss of bone; 3 aseptic osteo necrosis; 1 femoral neck bone cyst, 4 mild knee osteoarthritis, 1 rotator cuff injury and 1 chronic Achilles enthesitis. All were ambulatory patients. Performed in an operating room, with strict aseptic conditions, under short general anesthesia. The autologous bone marrow with mesenchymal mononuclear stem cells was harvested under predetermined protocols. Also, pre determined protocols were used to collect the patient's peripheral blood to be sent to the laboratory to obtain platelet rich plasma (PRP) and concentrated lyses platelets in plasma. We achieved clinical and radiological osseous union in: 10 of 11 non unions (90, 90 percent), in 2 delayed unions, in 3 bone lengthening and also in the tibia's severe soft tissue and bone defect. Based on this experience we can conclude that the stimulation of bone consolidation with a concentrated autologous mesenchymal stem cells, obtained from the patient's iliac crest, resulted to be a safe, reliable, minimally invasive procedure with a high rate of success...