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Department of Biomedical Sciences

 

 

 
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梅雅俊(Yaa-Jyuhn Meir)

Yaa-Jyuhu Meir

Position

Assistant Professor

Education

Ph.D. in Cell Biology, Vanderbilt University, USA

E-mail

jmeir@mail.cgu.edu.tw

Office Tel

+886-3-211-8800 ext.3641

Fax

+886-3-211-8700

Laboratory

Stem Cell & Molecular Genetics Laboratory

Spciality

Stem Cell Biology, Gene therapy, and Molecular Genetics

Lab & Research Interest

Multicellular organisms contain a special group of cells, named stem cells, which possess the capacity for both self-renewal and differentiation; in other words, stem cells serve as a reservoir of new cells to replace aged or dying cells.  With such remarkable capabilities, stem cells have long been considered as the “Fountain of Youth” for their ability to repair and/or replace lesions or aged tissues, and have been highlighted as the future direction of clinical sciences as well as an important trend in global bio-industry.  This idea has been further boosted by the seminal finding of nuclear reprogramming which reverses the developmental timeline to restore stem cell characteristics to highly differentiated cells.  The resulting cells, which possess many characteristics of embryonic stem cells (ESCs), are called induced pluripotent stem cell (iPSCs).  This pivotal finding not only provides a source of stem cells for cell replacement therapy, but also creates unprecedented opportunities for studying the development of organisms and the etiology of diseases.  Yet, unraveling the mechanisms by which ESCs maintain pluripotency and iPSCs acquire stem cell characteristics relies heavily on a highly efficient genome manipulation platform for probing the dynamic molecular networks of pluripotency.

Transposons are one of the most popular genome manipulation tools that have been applied to the study of gene function in plants and invertebrates.  Although 40% of the vertebrate genome is comprised of transposon-like elements, the mobilization of DNA transposons has never been detected in mammals.  Therefore, these transposons have been considered as relics or junk elements acquired during genome evolution.  The loss of mobilization ability of DNA transposons during the evolution of mammalian genome may serve the purpose of maintaining the integrity of genomic DNA, allowing the genome to reach physiological homeostasis for adaptation.   Work from our laboratory and others found that the piggyBac transposon, derived from cabbage looper moth, is able to jump efficiently in different mammalian genomes even when carrying exogenous genes (Wu, et al., 2006).  Since then, the piggyBac transposon, a nonviral vector system, has gained momentum for genome manipulation.  It not only has high transposition efficiency in various mammalian cells, but it also has the unique property of being tracelessly removable after genome manipulation.  Thus, the piggyBac transposon-based vector system has been considered as one of the best therapeutic vehicles for gene therapy, since it will not cause “genome pollution” or an immune response as seen with viral vectors.  Currently, my laboratory has advanced the piggyBac transposon-based genome manipulation system to allow efficient probing of the structure and function of the mammalian genome.  By adopting piggyBac as a model transposable element, one can potentially study the role of mobilizing elements during genome evolution.  Meanwhile, this well-established genome manipulation platform can be applied to unravel genomic plasticity by detecting the dynamic process of chromatin remodeling during differentiation, nuclear reprogramming, and regeneration.

To study the intriguing questions of how cell fates are determined, how cells maintain their differentiated status, and which signals can alter cell identity to promote regeneration, my laboratory uses our well-established piggyBac-mediated iPSC generation platform.  Accordingly, the research goals of my laboratory are as follows:

Establishing a highly efficient genome manipulation platform

The piggyBac-based genome manipulation system comprises two major elements, a donor plasmid and a helper plasmid.  The donor plasmid carries a gene of interest bracketed by a short stretch of repetitive nucleotide sequences, termed the Terminal Repeat Domains (TRDs).  The helper plasmid contains the coding region of the piggyBac transposase driven by a constitutively active or inducible promoter.  Once the transposase is expressed, it recognizes the TRDs on the donor plasmid and excises the TRD-bracketed element to form a looped protein-DNA complex.  After reaching a suitable TTAA-tetranucleotide sequence in the host chromosome, the transposase catalyzes insertion of the excised element into the target site.  Thus, it is conceivable that the transposition efficiency largely depends on the catalytic activity of piggyBac transposase and the composition of the TRD nucleotide sequences.

To improve the transposition activity of piggyBac, we first trimmed down the TRDs from their original length of 245/313 (left/ right) nucleotides to 67/40 nucleotides.  Shortening the TRDs not only diminished the cryptic enhancer effect, but also increased the transposition activity 3~4-fold (Meir, et al., BMC Biotechnology 11(1): 28 (2011)).  On the other hand, we engineered the piggyBac transposase by protein fusion and generated two recombinant transposases, TPLGMH and ThyPLGMH, which can catalyze efficient hopping-in and hopping-out to ameliorate genome lesions in the host chromosome during transposition.  Additionally, we are currently constructing a “two-component system” in which the helper plasmid is replaced with a purified ThyPLGMH protein to avoid constitutive expression of transposase that may cause instability of the targeted genome.

Generating iPSCs using the piggyBac-based genome manipulation system

The superiority of the piggyBac-based genome manipulation system is that it is able to remove the transgene after achieving the goal of transgenesis without leaving “footprints” in the host genome.  Due to this unique property, the piggyBac-based vector system emerged as the most celebrated therapeutic vehicle for current gene therapy.  My laboratory advanced this technology to generate iPSCs from mouse embryonic fibroblasts (MEFs).  After activating the endogenous circuitry of pluripotency, the transgene is no longer needed and can be induced to hop out in order to preserve the integrity of the host genome, and to induce differentiation of cell types of interest.  Figure 1 displays the dynamic process of iPSC clone formation within a three-week time frame after initiating the expression of Yamanaka factors carried by the piggyBac-based vector.  In addition to expressing several canonical pluripotency markers including OCT4, Sox2, Nanog, mpppA4, and SSEA1, the resulting iPSCs also generate teratomas in NOD SCID mice.  Currently, we are generating iPSC-based disease models for Sanfillippo syndrome in mouse and for Taiwanese Sialidosis type I in human by collaborating with Chang Gung Memorial Hospital to evaluate the efficacy of regeneration therapy and to develop personalized medicine as well. 

Studying the dynamic epigenetic processes during iPSC formation

When a highly differentiated cell acquires stem cell characteristics, the process of cell differentiation is reversed, allowing us to study the plasticity of the genome during nuclear reprogramming.  Yet, the current technology for creating iPSCs by exogenously introducing Yamanaka factors results in a heterogeneous population which obscures the genetic pathways governing the dynamic process of nuclear reprogramming.  To overcome this difficulty, an inducible triple transgenic “All-iPS mouse” was created with the following genomic modifications: (1) constitutive rtTA expression from the ROSA26 locus; (2) a pluripotent marker Oct4-ires-GFP knocked-in to the endogenous Oct4 locus; and (3) tetO-regulated iPSC quartet factors knocked-in to the Col1a locus.  The exact genetic pathway that allows each individual cell type derived from this animal to regain pluripotency may directly relate to the origin of the cell as well as its differentiation status.  By creating individual clones, each representing an independent reprogramming event, we can identify factors that directly cause a specific phenotype (e.g. accelerating or preventing iPSC formation), which might otherwise be obscured by the heterogeneous cell population.  To this end, we will use a piggyBac-mediated dominant gene entrapment system in conjunction with the triple transgenic “All-iPS mouse” to unravel the potential master regulators at each step during iPSC formation. 

Cellular reprogramming is a multifactorial and multistep procedure during which several fundamental cellular processes are coordinated in a sequential manner until the pluripotent state is reached.  Although iPSCs and nuclear transplantation are artificial ways of acquiring stem cell characteristics, there is increasing evidence from model animal studies that somatic reprogramming also occurs in vivo to replenish aged and damaged cells to maintain tissue homeostasis.  Perhaps some cells inappropriately acquire stem cell characteristics and consequently become tumor stem cells through a similar dedifferentiation process as seen in iPSC formation.  Therefore, genetically dissecting the reprogramming process will provide a deeper insight into how programming factors reverse normal development and whether the mechanisms that normally prevent reprogramming in vivo are also involved in cancer.  The identification of the different players involved in regaining pluripotency in distinct cell types will allow us to elucidate the principles of mammalian development and the mechanisms that normally prevent cells from undergoing malignant transformation.  Ultimately, these studies may allow us to generate a desired cell state from existing cell types for cell replacement therapy, as well as promoting drug development in an individual setting.

 

Bioengineering an iPSC niche

As stem cells have been thought as the “Fountain of Youth”, researchers have mainly explored the intrinsic capacity of stem cells for tissue replacement and regeneration by unraveling the mechanisms underlying self-renewal as well as maintenance of pluripotency.   A growing body of evidence in different model organisms indicates that the behavior of stem cells is critically influenced by their neighboring cells/tissues.  In that vein, the dynamic status of stem cell fate is tightly controlled through the concerted action of stem cell-intrinsic factors as well as signals derived from the milieu or niche in which the stem cell resides.  The stem cell niche serves as a functional unit for development and regeneration in many tissues, and it also nourishes resident stem cells and provides local or systemic instructions for immediate response to the body’s needs.  Thus, decisions about the fate of a stem cell depend on the neighboring tissues, extrinsic signals, and its own intrinsic properties.  This context-dependent combinatorial complexity of the stem cell niche poses an enormous challenge to studying the underlying mechanisms of stem cell biology.  To unravel the interplay of signals derived from intrinsic and extrinsic factors, there is a need to reconstruct the stem cell niche in an in vitro system that recapitulates the dynamic features of the interactions between stem cells and their microenvironment.

In spite of the fact that the expression of extrinsic Yamanaka factors alone is sufficient to allow a differentiated cell to acquire stem cell properties, the efficiency of iPSC formation reported in the original studies was quite low (0.001–0.05%).  Besides that, the process of nuclear reprogramming may generate harmful mutations, or antigens that are unexpectedly attacked by the immune system of the donor.  These limitations, which impede the use of iPSCs in the clinical setting, may be due to lack of an appropriate microenvironment to guide and coordinate the sequential reprogramming events.  Currently, we are bioengineering the iPSC culture system to improve the quality of iPSC production by identifying novel players that are involved in the nuclear reprogramming pathway. This will in turn allow us to establish a more efficient culture system.

Small molecule drug screening

The discovery of the phenomenon of nuclear reprogramming not only provides new opportunities to understand the plasticity of the genome, but also creates novel platforms for bio-industrial drug screening that recapitulate the potential drug response in patients.  Since it has been shown that iPSCs can model diseases in a dish, there is an unprecedented opportunity to perform high-throughput drug screening to validate compounds targeting specific disease-related pathways.  My laboratory has currently adopted the “All-iPSC mouse” system to screen the compounds in Chinese herbs and to look for the molecules that, individually or in combination, promote iPSC formation.

The unique features of stem cells create tremendous potential for cell replacement therapy in regeneration medicine.  Performing cell replacement therapy, however, needs understanding of the biology of stem cells and the nature of their surrounding microenvironments to avoid depletion of transplanted stem cells.  Additionally, unraveling how the niche forms, which signals are needed for stem cells in different tissues, and how the interplay between intrinsic and extrinsic signals determines the action of stem cells, should ameliorate age-dependent stem cell depletion and increase the homing efficiency of transplanted stem cells for future stem cell therapy.  The goal of my research is to adopt iPSCs as a paradigm to understand developmental biology, and ultimately to apply the knowledge gained about organogenesis through the combination of advanced genome manipulation platforms and nuclear reprogramming technology.  Undoubtedly, the phenomenon of nuclear reprogramming and the mechanism of iPSC formation will open novel avenues for the medical sciences and provide new insights into biology as well.

Publications

Publications:

  1. J. Reproduction and Fertility 94, 431-436 (1992). Jiann-Ping Wang, Wann-Yee Her, Yaa-Jyuhn James Meir, Ts’an-Shiuu Lir, Hsiu-Luan Chang, Fore-Lien Haung. Seasonal variation in cell cycle during early development of the mouse embryo. 
  2. Development 124, 1699-1709 (1997). SCI 7.60 Amy Pflugrad*, Yaa-Jyuhn James Meir*, Tom M. Barnes, and David M. Miller, III. The Groucho-like transcription factor UNC-37 functions with the neural specificity gene unc-4 to govern motor neuron identity in C. elegans. (*These authors contributed equally to this work) 
  3. Genes and Development 13(21), 2774-2786 (1999). SCI 15.61 Angela R. Winnier*, Yaa-Jyuhn James Meir*, Jennifer M. Ross, Nektarios Tavernerakis, Monica Driscoll, Takeshi Ishihara, Isao Katsura, and David M. Miller, III. UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans. (*These authors contributed equally to this work) 
  4. Molecular Microbiology 60(2), 331-348 (2006).  SCI 6.20 Bently Lim, Yaa-Jyuhn James Meir, and Fitnat Yildiz. Cyclic-di-GMP signal transduction system in Vibrio cholerae: Modulation of Rugosity and Biofilm formation. 
  5. Proc Natl Acad Sci USA 103(41), 15008-13 (2006). SCI 10.23 Wu SC*, Yaa-Jyuhn James Meir*, Coates CJ, Handler AM, Moisyadi S, Pelczar P, Kaminski JM (2006). piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and Mos1 in mammalian cells. (* These authors contributed equally to this work.)  
  6. Current Biology 17(7), 592-8 (2007). SCI 11.73 Sakaguchi-Nakashima A., Yaa-Jyuhn James Meir, Yishi J., Matsumoto K., & Hisamoto N. LRK-1, a C. elegans PARK8-Related Kinase, Regulates Axonal-Dendritic Polarity of SV Proteins.

Patents:

  1. Methods and Compositions for Drug-Free Selection in Genetic Engineering. (US patent no. 12/588,708; 10/26/2009) 
  2. A Transposon-Mediated Genetic Engineering System with a Self-Activating Reporter for a Rapid Indication of Transposition. (US patent no. 61-131-298; June 2008)