Preparation and Delivery of Clinically Relevant Numbers of Stem Cells Using 3D Hydrogels
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.
A key hurdle preventing translation of stem cell therapies to the clinic is the lack of efficient strategies to transplant and retain viable cells. Cell transplantation by direct injection is favored for its minimal invasiveness but commonly results in poor cell viability. Use of thixotropic hydrogels as injectable cell-delivery vehicles is one potential strategy to overcome this limitation. We report a protein-engineered hydrogel designed to meet four criteria for use in stem cell injection protocols: (i) gentle cell encapsulation at constant physiological conditions without the need for chemical crosslinkers, (ii) shear-thinning under reasonable hand-injection force through a syringe needle, (iii) rapid gel recovery to localize cells at the injection site, and (iv) cell-adhesive ligands and mechanical properties conducive to three-dimensional (3D) cell culture. This Mixing-Induced Two-Component Hydrogel (MITCH) is synthesized using protein-engineering technology to yield monodisperse block-copolymers that hetero-assemble upon simple mixing. Human and mouse adipose-derived stem cells (ASCs) remain viable and display a well-spread 3D morphology within MITCH. Use of MITCH to deliver ASCs to the subcutaneous dorsa of athymic mice resulted in significantly greater viable cell retention at the implant site compared to Type I collagen or buffer alone up to two weeks post-transplantation.
Although stem cells have tremendous potential to regenerate damaged or diseased tissue, scientists must develop efficient methods to deliver the stem cells to the sites in the body where they are needed. Injection of stem cells through a syringe needle directly into the tissue site is a simple procedure to perform, but it exposes the stem cells to damaging mechanical forces that can injure the cells. In year 1 of this project, we developed a gel that encapsulates stem cells and protects them from these damaging mechanical forces. This gel was able to maintain excellent viability of transplanted stem cells for up to one week. In year 2 of the project, we further developed this new gel material with the goal of extending the lifetime of transplanted stem cells. In particular, we synthesized two new gel formulations. The first gel is able to co-encapsulate the stem cells together with pro-survival factors. The presence of these pro-survival factors was found to increase the retention of transplanted stem cells. The second gel is able to stiffen after it is injected into the body; thereby enabling it to last for longer times. Our long-term goal is to increase the percentage of stem cells that can survive the transplantation process and participate in the regeneration of damaged tissue.
Since the previous report, efforts have focused on expanding the stem cell-protective hydrogel technology to other biomaterial formulations to confirm the broad applicability of this technology and to optimize the technology for specific disease and injury therapies.
During the past year, three manuscripts were accepted for publication, and an additional three manuscripts are in preparation. The technology has been protected by the Stanford Office of Technology Licensing with a patent disclosure to the US Patent Office.