Tools and Technologies II
Stem cells hold great potential to treat many devastating injuries and diseases. However, stem cell research has been stymied by the difficulty in obtaining stem cells from sources other than human embryos. Recently, generation of stem cells by reprogramming normal human cells has been achieved. These human induced pluripotent stem cells (hiPSCs) still faces challenges because they use viral vectors to introduce the four reprogramming genes into adult cells. The use of virus vectors raises safety concerns because unwanted and potentially dangerous genes could be introduced into the hiPSCs. A safer approach for generating hiPSCs has recently been demonstrated by introducing reprogramming proteins themselves into human somatic cells rather than the genes. Nanoparticles (NPs) are promising vectors for effective and safe delivery of biomolecules into specific types of cells or tissues. Our joint team has developed a unique approach leveraging the power of supramolecular chemistry and digital microreactors to screen NP-based vectors for optimal protein-delivery performance. The goal of this proposal is to develop a new class of NP-based vectors, capable of encapsulating and delivering reprogramming proteins in order to achieve highly efficient generation of hiPSCs providing the benefits to stem cell researchers without the risks to human health. As the proposed project unfolds, we anticipate making contributions in the following key areas: First, we envision that the successful demonstration of proposed research will establish safer and more efficient protein delivery reagents for generation of iPSCs. Our new approach will be able to effectively eliminate potential risk of modifying the target cell genome by viruses and/or foreign genetic materials. Second, our new protein delivery method will provide a robust and widely applicable approach for the manipulation cellular behavior and could enable broader and more economical application of reprogramming methodology. For example, generation of human stem cell based disease models for drug screening would now be feasible and cost-effective. Third, microfluidic image cytometry (MIC) technology, previously developed by an ongoing CIRM Tools and Technologies grant, will be employed to monitor characteristics including (i) growth rates, (ii) pluripotency/differentiation status (OCT4, NANOG, SSEA4, TRA-1-60, TRA-1-80 and SSEA1), and (iii) a phenotype assay for parallel detection of DAPI (cell cycle), pHistone H3 (M-Phase), EdU (S-Phase) and Caspase-3/7 (apoptosis) of the reprogramming protein-treated human somatic cells during the reprogramming processes. Since the reprogramming of hiPSCs is not well understood, the resulting phenotypic/signaling signatures can help to elucidate the mechanisms dominating the reprogramming process.
Statement of Benefit to California:
The proposed project will benefit the state of California and its citizens as follows: 1. Safer and more efficient approach for cell-based treatment. The proposed research can establish protein delivery reagents for generation of human induced pluripotent stem cells (hiPSCs). hiPSCs hold great potential for regenerative medicine to treat many devastating injuries and diseases such as Alzheimer’s disease, Parkinson’s disease, diabetes, cancer, rheumatoid arthritis and spinal cord injuries. 2. New technology for cell reprogramming. Our new protein delivery method provides a robust and widely applicable approach for manipulation cellular behaviors, enabling broader and more economical applications of reprogramming methodology, for example, generation of human stem cell based disease models for drug screening. 3. New intellectual property. The proposed research activities are based upon three innovative technologies, including (i) self-assembled supramolecular nanoparticles for highly efficient delivery of biomolecules, (ii) digital microreactors for large-scale screening, and (iii) microfluidic image cytometry (MIC) technology for monitoring phenotypical characteristics of single cells. We note that there are five patent applications and provisional patents generated at [REDACTED] to cover the intellectual properties (IPs) associated with these inventions. Two of these have recently been licensed to two separate California companies. We foresee the proposed research activities will continue to generate crucial IP that could create business opportunities in California. 4. New business opportunities. The joint research team has established strong partnership with three California companies, [REDACTED]. [REDACTED] has licensed the supramolecular nanoparticle-related IPs from [REDACTED] and is committed to help the joint research team on exploring the use of SNPs as protein delivery reagents for generation of hiPSCs. [REDACTED] has licensed MIC technology-related IPs from [REDACTED] is willing to provide the most advanced MIC platform for phenotypical characterization of nanoparticles-treated cells. [REDACTED] will ensure high quality and sufficient supply of the four reprogramming transcription factors (TFs, i.e., OCT4, SOX2, KLF4 and c-MYC) over the three-year research period. 5. Education and jobs for next-generation California scientists. Our team is composed of three research groups at [REDACTED] with expertise covering synthetic chemistry, nanoparticles, microfluidics, stem cell biology and reprogramming. In addition to creating approximately ten highly skilled jobs, the proposed research activities will create an interdisciplinary education environment for training the next generation of California citizens at all levels, including high school, undergraduate, graduate students, as well as postdoctoral fellows.
This proposal is focused on the development of novel nanoparticle (NP) vectors to deliver reprogramming factors for induced pluripotent stem cell (iPSC) generation. The applicant identifies the lack of safe, efficient, non-integrating methods for iPSC generation as a translational bottleneck. There are three Specific Aims: (1) to synthesize a variety of building blocks for self-assembling NP-based delivery vectors; (2) to prepare a combinatorial library of NPs carrying reprogramming factor proteins and screen for the complexes and combinations that result in optimal reprogramming of human embryonic stem cell (hESC)-derived fibroblasts; and (3) to validate the optimized NP vectors by reprogramming adult patient fibroblasts into iPSCs. The reviewers agreed that this proposal addresses a significant bottleneck in the translation of iPSC-based therapies. Viral integration is a serious safety concern, and while protein-based reprogramming has been successful, published approaches are inefficient and slow. Reviewers also praised the applicant’s innovative and clever approach, using NP complexes to provide flexibility and microfluidic technology to create combinatorial libraries of these vectors. However, they did note that the significance and potential impact of the proposal are reduced by the recent publication of an efficient, non-viral, synthetic RNA reprogramming method. Reviewers commented that the applicant does not adequately justify the use of protein-based reprogramming over other non-integrating DNA- and RNA-based approaches. They also cautioned that because proteins are expensive to produce and aren’t amplified by cells, they might not be the best access point for reprogramming strategies. Reviewers found some aspects of the research plan to be well-designed and feasible, but others to be inadequately justified and challenging. They appreciated the use of the microfluidic reactor approach to generate a diversity of NP vectors and described the multi-well plate screen as a balance between stringency and ease-of-screening. They also noted that Aim 3 would provide strong validation of the approach. However, reviewers felt that the experimental design was weakened significantly by the use of microfluidics to assay the reprogrammed state. They noted that this approach is not well justified over multi-well plate imaging and the small culture area of each chamber in the microfluidic chip will make it difficult to achieve adequate cell numbers to compare the reprogramming efficiency of different NP complexes. Reviewers cautioned that culturing cells for 21 days or longer in a microfluidic format is challenging and has not been demonstrated by the applicant. Finally, while reviewers appreciated the detailed timeline and milestones they noted that only one potential pitfall is acknowledged. The reviewers described the Principal Investigator (PI) as well suited to oversee the chemical aspects of the proposal and appreciated his/her significant experience with microfluidics and NPs. They praised the strong interdisciplinary team assembled, including Co-Investigators with extensive stem cell expertise. In general, the reviewers found the research team to be well qualified to carry out the proposed research. Overall, while reviewers appreciated that this proposal utilizes innovative approaches to address an important translational bottleneck, they were not convinced that it would offer significant advantages over existing reprogramming techniques. Reviewers were impressed by some aspects of the research plan but raised serious concerns about others and noted that proposed complex approaches were not well justified over simpler alternatives.