Browsing by Subject "Myogenesis"

Technologies for engineering new functions into proteins are advancing biological research, biotechnology, and medicine at an astounding rate. Building on fundamental research of natural protein structure and function, scientists are identifying new protein domains with previously undescribed properties and engineering new proteins with expanded functionalities. Such tools are enabling the precise study of fundamental aspects of cellular behavior and the development of a new class of gene therapies that manipulate the expression of endogenous genes. The applications of these gene regulation technologies include but are not limited to controlling cell fate decisions, reprogramming cell lineage commitment, monitoring cellular states, and stimulating expression of therapeutic factors.

While the field has come a long way in the past 20 years, there are still many limitations. Historically, gene therapy and gene replacement therapies have relied on over-expression of natural transcription factors that activate specific endogenous gene networks. However, natural transcription factors are often inadequate for generating efficient, fast, and homogenous cellular responses. Furthermore, most natural transcription factors have complex structures and functions that are difficult to improve or alter by rational design. This thesis presents three novel and widely applicable methods for engineering transcription factors for programming cell fate decisions in primary human cells. MyoD is the master transcription factor defining the myogenic lineage. Expression of MyoD in certain non-myogenic lineages induces a coordinated change in differentiation state. We use MyoD as a model for developing our protein engineering techniques because myogenesis is a well-studied pathway that is characterized by an easily detected change in phenotype from mono-nucleated to multinucleated cells. Furthermore, efficient generation of myocytes in vitro presents an attractive patient-specific method by which to treat muscle-wasting diseases such as muscular dystrophy.

We first demonstrate that we can improve the ability of MyoD to convert human dermal fibroblasts and human adipose-derived stem cells into myocyte-like cells. By fusing potent modular activation domains to the MyoD protein, we increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. The engineered MyoD transcription factor induced myogenisis in a little as ten days, a process that takes three or more weeks with the natural MyoD protein.

While increasing the potency of transcriptional activation is one mechanism by which to improve transcription factor function, there are many other possible routes such as increasing DNA-binding affinity, increasing protein stability, altering interactions with co-factors, or inducing post-translational modifications. Endogenous regulatory pathways are complex, and it is difficult to predict specific amino acid changes that will produce the desired outcome. Therefore, we designed and implemented a high-throughput directed evolution system in mammalian cells that allowed us to enrich for MyoD variants that are successful at inducing expression of the myogenic gene network. Directed evolution presents a well-established and currently unexplored approach for uncovering amino acid substitutions that improve the intrinsic properties of transcription factors themselves without any prior knowledge. After ten rounds of selection, we identified amino acid substitutions in MyoD that increase expression of a subset of myogenic gene markers in primary human cells.

Rather than guide cell fate decisions by expressing an exogenous factor, it may be beneficial to activate expression of the endogenous gene locus. In comparison to delivering the transcription factor cDNA, expression from the endogenous locus may induce chromatin remodeling and activation of positive feedback loops to stimulate autologous expression more quickly. Recent discoveries of the principles of protein-DNA interactions in various species and systems has guided the development of methods for engineering designer enzymes that can be targeted to any DNA target site. We make use of the RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to induce expression of the endogenous MyoD gene in human induced pluripotent stem cells (iPSCs). Through complementary base pairing, chimeric guide RNAs (gRNAs) direct a Cas9 transcriptional activator to a target DNA sequence, leading to endogenous gene expression. A current limitation of CRISPR/Cas9-based gene regulation is the potency of transcriptional activation and delivery of the CRISPR/Cas9 components. To address these limitations, we first developed a platform to express Cas9 and up to four gRNAs from a single lentiviral vector. We then optimized the gRNAs and Cas9 transcriptional activator to induce endogenous MyoD expression and differentiate iPSCs into myocyte-like cells.

In summary, the objective of this work is to develop protein engineering techniques to improve both natural and synthetic transcription factor function for programming cell fate decisions in primary human cells. While we focus on myogenesis, each method can be easily adapted to other transcription factors and gene networks. Engineered transcription factors that induce fast and efficient remodeling of gene networks have widespread applications in the fields of biotechnology and regenerative medicine. Continuing to develop these tools for modulating gene expression will lead to an expanded number of disease models and eventually the efficient generation of patient-specific cellular therapies.

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.

The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro.

The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence.

Major results of the dissertation are as follows. Culture conditions of 100 μg/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.