A critical bottleneck to translate the promise of regenerative medicine to the clinic is the ability to efficiently harvest, expand, and deliver sufficient numbers of viable stem cells. While relatively large numbers of patient-specific, multipotent human adipocyte stem cells (hASC) can be harvested from adults, these cells must be re-delivered to the patient (either with or without intervening culture steps) in sufficient quantity for functional regeneration. We propose development of a clinically translatable biomaterial that is used both to improve the efficiency of stem cell expansion and to enhance the effectiveness of stem cell delivery. Current in vitro stem cell expansion protocols are time-, space-, energy-, and cost-intensive and often result in the spontaneous loss of self-renewal in addition to a heterogeneous population of differentiated cells. Furthermore, the most non-invasive method of stem cell delivery to the patient, direct cell injection, commonly results in less than 5% cell viability. Our specific aims demonstrate the flexibility of a single biomaterial to address these bottlenecks in three different clinical paradigms: 1) direct re-injection of hASC immediately following cell isolation from the patient, 2) ex vivo expansion and differentiation of hASC prior to transplantation, and 3) in vitro reprogramming of hASC into induced pluripotent stem cells (iPSC). Due to the urgency of translational outcomes and the complementary, non-overlapping experimental design, the following aims will be pursued in parallel:
Aim 1. Utilize a novel, protein-based, self-assembling biomaterial to achieve greater than 95% viability of transplanted hASC by direct injection. Injection protocols will be optimized in vitro and validated in vivo using a subcutaneous mouse model with non-invasive bioluminescence imaging. We hypothesize that cell delivery within a biomaterial will significantly improve viability by providing flow-protection during injection, localization at the target site, and scaffolding to promote cell adhesion.
Aim 2. Improve efficiency of hASC expansion and differentiation using a three-dimensional (3D) niche mimic for bone tissue regeneration. Biomaterial delivery of bone morphogenetic protein 2 (BMP2) and hydroxy apatite (HA) nanoparticles will be optimized ex vivo to enhance osteogenic differentiation and validated in vivo using a mouse cranial critical defect model. We hypothesize that customization of the biomaterial for optimal mechanics, BMP2 delivery, and HA content will enhance 3D bone tissue formation.
Aim 3. Optimize materials and methods for reprogramming of hASC into iPSC using a 3D in vitro culture environment and nonviral minicircle DNA. Recently, hASC have demonstrated enhanced iPSC reprogramming efficiency compared to other cell types. We hypothesize our 3D cultures will greatly reduce reagent, space, and cost requirements and improve efficiency of iPSC preparation compared to traditional 2D culture methods.
A critical bottleneck in translating the promise of regenerative medicine to the clinic is (i) the efficient preparation and (ii) the successful delivery of sufficient numbers of stem cells. While relatively large numbers of patient-specific, human adipocyte (i.e., fat-derived) stem cells (hASC) can be harvested from adults, these cells must be re-delivered to the patient in sufficient quantity for functional regeneration. We propose development of a clinical biomaterial that can be used both to improve the efficiency of stem cell expansion and to enhance the effectiveness of stem cell delivery. Current stem cell expansion protocols are time-, space-, energy-, and cost-intensive. Furthermore, the most non-invasive method of stem cell delivery to the patient, direct cell injection, commonly results in death for more than 95% of the transplanted cells. We hypothesize that an optimized biomaterial scaffold will greatly reduce the time-, space-, energy-, and cost-requirements for stem cell culture, resulting in a great cost-savings for California. We further hypothesize that these biomaterials will improve the efficiency of stem cell transplantation, enabling the transplantation of more than 95% living, functional cells, resulting in greatly improved clinical outcomes for California patients.
The goal of this proposal is to develop novel biomaterial formulations for improving survival, expandability and delivery of therapeutic stem cell populations. By targeting these areas, the applicant hopes to overcome three bottlenecks in the translation of stem cell-based therapies to the clinic. First, a self-assembling, protein-based hydrogel will be optimized for reducing cell damage that occurs during injection of human adipose-derived stem cells (hASC) for regenerative applications. A series of in vitro and in vivo studies will be employed to enhance cell viability, integrity and localization to the intended site. For the second Aim, the proposed biomaterials will be engineered to provide three-dimensional (3D) cues and mechanical context for improved ex vivo expansion and osteogenic differentiation of hASC. The efficacy of these protocols will be assessed using an in vivo model of bone repair. For the third aim, the applicant proposes to develop a hydrogel-based gene delivery platform for viral-free reprogramming of hASCs to pluripotency. By combining 3D culture environments with minicircle DNA technology, the applicant hopes to reduce costs of cellular reprogramming while improving the overall safety and efficiency of this process.
Reviewers had mixed impressions of the overall significance and impact of this proposal. In general, they found the biomaterial aspect to be unique and highly innovative, citing several major advantages over alternative hydrogel formulations including an ability to be mechanically tuned, chemically defined, and experimentally manipulated under normal physiological conditions. They also agreed that a lack of proven and reproducible strategies for assuring cell viability after transplant represents a significant barrier to the development of cell therapies. Consequently, the rationale of Aim 1 for developing hydrogels to protect cells and promote their survival during experimental manipulation and delivery was strongly supported. Reviewers believed that even modest success in this aim could rapidly translate to important advances in regenerative medicine. In contrast, reviewers did not consider the remaining goals of the proposal to be compelling. They acknowledged efficient osteogenic differentiation and bone formation would advance the field. However, the group expressed considerable concern that the approaches of Aim 2 would merely repeat the successes and failures of similar scaffold and factor release studies in generating nonfunctional bone. Reviewers also expressed a general lack of enthusiasm for the reprogramming studies in Aim 3, which they found poorly justified scientifically and of limited translational significance.
Reviewers described the research plan as straightforward and well written, with clearly defined aims and efficient use of time and resources. The preliminary data were compelling and supportive of proposed approaches as well as the capabilities of the applicant team. While many potential pitfalls were acknowledged, reviewers nonetheless identified a number of weaknesses in the experimental design. For example, several worried that the tissues to be derived in Aim 2 would not possess the appropriate structural or mechanical properties of native bone. While the plans for optimization were elegant, some reviewers believed the proposed characterization of the regenerated bone inadequate to assess the mechanical properties critical to success. Others felt the proposal lacked important details, such as discussion of statistical analyses to be employed, or a more comprehensive description of the myriad controls and experimental variables that would need to be managed. Some reviewers questioned whether cell dosage and subsequent survival would be sufficient for detection using the imaging approaches proposed in Aim 1. It was also unclear to what extent the hydrogel might interact with vectors or their intracellular processing in Aim 3. Finally, while they appreciated many of the individual elements, reviewers felt the overall strategy lacked cohesiveness and would have preferred a more unified focus of investigation.
The principal investigator (PI) was described as an accomplished young scientist and established leader in protein and peptide-based biomaterials. Collaborators were described as outstanding, recognized pioneers in the use of hASC and cellular reprogramming approaches. While the reviewers were convinced that the PI could ably conduct the materials synthesis and characterization, they nonetheless viewed the expertise of the collaborators as essential to the success of the project. While they would have preferred a specific commitment of effort, their concerns were somewhat assuaged by strong letters of support from the individuals in question.
Overall, while reviewers acknowledged the strength of the investigators and the innovation of the proposed technology, only the first aim was considered significant and compelling in its potential for translational impact. After a discussion of programmatic considerations, reviewers recommended Aim 1 for funding.
- A motion was made to move Specific Aim 1 of this application into Tier 1, Recommended for Funding. Reviewers discussed the broader impacts of the proposed hydrogels for regenerative applications, including potential use for treating facial wounds and the repercussions of wasting disorders such as HIV/AIDS. The proposed materials may offer some advantages over competing materials in the current marketplace for soft tissue reconstruction. Reviewers discussed the emergence of clinical data for utility of hASC and agreed that there might be potential for nearer term translation of formulations as proposed in Aim 1. Motion to fund Specific Aim 1 carried.