Regulation of Stem Cell Fate in Bioengineered Arrays of Hydrogel Microwells
Stem cell biology, since its inception 30 years ago, has been hindered by our limited ability to observe and direct the decisions of individual stem cells. In the case of adult tissue-specific stem cells, such as those from blood, muscle or pancreas, the numbers available for clinical use are extremely limited, as in tissue culture the cells either have limited viability, do not divide, or rapidly specialize and lose their stem cell properties and potential to contribute to tissue regeneration. To overcome this hurdle, over the past three years we have been developing and optimizing a novel technology that employs arrays of bioengineered hydrogel microwells to study the fate of single stem cells dynamically by timelapse microscopy. This technology has several distinct advantages: (1) The hydrogel material is hydrated and substantially softer than standard tissue culture plastic, which substantially increases stem cell viability; (2) The arrays consist of wells containing hundreds of microwells so that single stem cells can be monitored simultaneously, which is critical since the stem population is inherently diverse; (3) Finally, the hydrogels we developed can be chemically modified so that the stem cells are exposed to molecules found in the tissue – soluble or tethered. We have found that the viability of human fetal pancreatic progenitors is increased 1,000-fold when grown on our hydrogels, which could impact the development of a treatment for diabetes. We have also found that exposure of blood stem cells to specific proteins causes them to self-renew in culture, a step toward overcoming a major roadblock to their use in the treatment of hematologic malignancies. Finally, studies of human embryonic stem cells (hES) could benefit from this platform, as conditions improve. In the current grant, we propose to apply this technology to study factors that increase the function of young and old murine muscle stem cells. In addition, we propose to develop protocols for the isolation and characterization of human muscle stem cells, which will further help the translation of our findings to the clinic. A second novel technology with broad utility is presented here, non-invasive bioluminescence imaging, which we have developed in order to monitor muscle stem cell function in vivo. It entails a method for following stem cell numbers dynamically and quantitatively in mice using bioluminescence imaging. This method will allow the pattern of tissue formation to be monitored in real time without sacrificing the mice and will generate a more accurate picture of tissue regeneration following cell transplants. Together these technologies should advance the use of muscle stem cells for the treatment of age-related muscle wasting. In addition, these technologies should advance and benefit the entire stem cell field.
The state of California is the front-runner in stem cell research, having gathered not only private investments, as demonstrated by the numerous biotechnology companies that are developing innovative tools, but also extensive public funds via Prop 71, that allows the state, through CIRM to sponsor stem cell research in public and private institutions. In order to preserve the leadership position and encourage research on stem cells, the CIRM is calling for research proposals that could lead to significant breakthroughs or the development of technologies useful for studying stem cells in order to improve human health. We propose here to develop a platform that will establish a “gold standard” for monitoring and manipulating stem cells in culture. Adult stem cells are present in many tissues, but their regenerative potential is not currently fully realized. Although isolation of these cells is performed routinely, their expansion has been hindered by the lack of tools and knowledge of the factors capable of inducing their division without loss of their stem cell properties. Here we describe a highly innovative and powerful bioengineered platform capable of enhancing stem cell function in culture. We apply it here to muscle stem cells for the treatment of muscle wasting, which is a common problem in the aging population, detected by a progressive loss of muscle mass and decline in muscle function. A better understanding of muscle stem cell biology is greatly needed to effectively treat these disorders and our technological platform is focused on defining the conditions and factors required for expansion of muscle stem cells. This technology will contribute substantially to all types of stem cell research, including human embryonic stem cells and induced pluripotent stem cells.
Skeletal muscles provide necessary contractile forces to enable locomotion and breathing. Healthy skeletal muscle can recover from minor injuries, through a regeneration process involving skeletal muscle stem cells. However, there are several pathologic conditions (e.g. myopathies, aging-related sarcopenia) in which skeletal muscle is unable to sufficiently regenerate and ultimately atrophies. There is currently no cell-based therapy available to alleviate these disorders. Our objective is to develop innovative culture environments and investigate the mechanisms that could permit in vitro expansion of aged or diseased muscle stem cells.
In the first and second aims, we have developed a new stiffness-adjustable artificial support material that allows preservation of muscle stem cell viability in vitro (Aim 1) and, more importantly, permits maintenance of muscle stem cell potential as observed after in vivo transplantation of the cultured muscle stem cells (Aim 2). We have demonstrated that the rigidity of the support material on which the cells are expanded is critical for maintaining their transplantation potential. We observed that standard rigid culture surfaces negatively affect muscle stem cell viability and are unfavorable for preserving the muscle stem cell function. We noticed that a soft, very elastic culture material can dramatically enhance the survival of muscle stem cells. We have analyzed the genes expressed by the muscle stem cells isolated from young and aged mouse skeletal muscles as we reasoned that the poor stem cell potential of older stem cells could be resulting from the lack of key stem cell genes. Here we show that gene expression patterns differ between the aged and young muscle stem cells, and that identification of these differentially expressed genes could help us to investigate their potential for rejuvenating the aged cells. We observed that aged muscle stem cells seeded in culture and observed by time lapse videos have diminished behaviors in vitro. Our goal is to exploit this altered behavior to test our ten selected regulators for expanding and rejuvenating aged muscle stem cells.
In the third aim, we have developed a method to isolate muscle stem cells from human samples. Until recently, there has been a lack of information regarding the characterization of human stem muscle from skeletal tissue. By using flow cytometry, we have managed to identify a family of cell surface molecules that allows enrichment of the cells from a very heterogeneous population. These cells are highly myogenic, which means that in vitro they proliferate and generate muscle fibers, and we are now in the process of testing their behavior in culture on the artificial platform described above. In parallel, we are analyzing the genes expressed by these cells isolated from young or aged patients and we seek to compare these analyses with the mouse data that we have acquired.
We are pleased to report that we have been able to address all of the specific aims of the grant funded by CIRM. This progress goes a long way toward enabling therapies that will contribute to a substantially enhanced quality of life. The aging population is increasing in industrialized countries and aged-related muscular pathologies are becoming increasingly common leading to escalating medical costs and highly debilitating muscular weakness. It is well known that in young adults, skeletal muscle has a remarkable ability to heal itself and this capacity is solely due to the robust proliferative and differentiative potential of muscle stem cells. What is needed is a means of harnessing this stem cell potential by finding ways to increase stem cell numbers and function in cell culture for use in cell based therapies. This CIRM grant has enabled us to make progress that would otherwise not have been possible given the current NIH funding crisis. We are extremely grateful. Due to this grant we have succeeded in characterizing the functional differences between young and old muscle stem cells. Importantly, we have defined culture conditions that enable the maintenance, self-renewal, and expansion of muscle stem cells for the first time. (Aims 1 and 2). Finally, we have successfully applied our knowledge of adult mouse muscle stem cells to their human counterparts. We have identified markers for the prospective isolation of human muscle stem cells and growth conditions that allow their propagation in culture for cell based therapies (Aim3). Our findings have been well received with a seminal publication in Science this year and invitations to speak in symposia and at universities worldwide. Moreover our work was featured in the Wall Street Journal and on the front page of the New York Times in 2010. Another major advance in our laboratory, which was not part of this grant, was our development of the first mouse model that mimics the human genetic muscle wasting disease, Duchenne Muscular Dystrophy, which was published in Cell in 2010. Our future work will focus on testing muscle stem cell based therapies developed in the course of this grant in this muscle disease model. The findings will have relevance not only to DMD, but also more generally to sarcopenia and the profound muscle atrophy that accompanies aging.
- Nat Med (2014) Rejuvenation of the muscle stem cell population restores strength to injured aged muscles. (PubMed: 24531378)
- Integr Biol (Camb) (2012) A single cell bioengineering approach to elucidate mechanisms of adult stem cell self-renewal. (PubMed: 22327505)
- Adv Healthc Mater (2012) Protein-engineered biomaterials to generate human skeletal muscle mimics. (PubMed: 23184832)
- JAMA (2011) Re"evolutionary" regenerative medicine. (PubMed: 21177496)
- Cell (2011) DNA demethylation dynamics. (PubMed: 21925312)
- Adv Mater (2011) Nanogel star polymer architectures: a nanoparticle platform for modular programmable macromolecular self-assembly, intercellular transport, and dual-mode cargo delivery. (PubMed: 21901765)
- Stem Cell Res Ther (2011) Engineering a stem cell house into a home. (PubMed: 21345268)
- Nature (2010) Nuclear reprogramming to a pluripotent state by three approaches. (PubMed: 20535199)
- Proc Natl Acad Sci U S A (2010) skNAC, a Smyd1-interacting transcription factor, is involved in cardiac development and skeletal muscle growth and regeneration. (PubMed: 21071677)
- Cell Stem Cell (2010) Transient inactivation of Rb and ARF yields regenerative cells from postmitotic mammalian muscle. (PubMed: 20682446)
- Science (2010) Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. (PubMed: 20647425)
- Differentiation (2009) A home away from home: challenges and opportunities in engineering in vitro muscle satellite cell niches. (PubMed: 19751902)
- Nature (2009) Designing materials to direct stem-cell fate. (PubMed: 19940913)