Early Translational I
The successful development of human embryonic stem cell (hESC) based therapies to treat human disease will hinge upon the preparation of high quality cells for implantation, as heterogeneous cell preparations can pose a safety risk to the patient and even compromise efficacy. In particular, implantation of differentiated cell populations with heterogeneous composition into numerous tissues, including muscle and brain, can lead to teratoma formation due to undifferentiated cell contaminants in the graft. Furthermore, inappropriately differentiated cells can lead to adverse side effects. Therefore, cell graft quality remains a major bottleneck for human embryonic stem cell based regenerative medicine efforts. We have assembled a strong team – with highly complementary biological, engineering, and clinical expertise – to address this critical bottleneck by applying advanced technologies for cell differentiation and purification. First, we have developed a novel microfluidic cell separations system in which surfaces are functionalized with antibodies against key cell surface markers, and the subsequent microfluidic flow of a cell suspension over these surfaces results in spatial separation of cells based on expression of these markers. This scalable system will be applied towards the purification of high quality differentiated cell preparations. Second, stem cells arguably differentiate heterogeneously in culture in part because, in contrast to native microenvironments that present them with regulated signals to control their function, typical culture systems expose them to heterogeneous and contradictory conditions that imprecisely control cell behavior. Synthetic, bioactive materials presenting biological signals will be utilized to provide chemically defined and homogeneous conditions for uniform cell differentiation. Graft quality is a bottleneck for most disease targets, and we propose to test these technologies in two critical tissues: skeletal muscle and brain. Specifically, human embryonic stem cells will be differentiated into skeletal muscle cells and dopaminergic neurons – using both established differentiation conditions and bioactive materials engineered for this application. Cells will in some cases be purified prior to implantation into animal models of skeletal muscle injury and Parkinson’s Disease. We propose that parallel progress in cell differentiation and purification technologies will enhance both the safety of the graft, by reducing the incidence of teratoma formation, and potentially its therapeutic efficacy in these important animal models of human disease and injury.
Statement of Benefit to California:
This proposal will address a critical bottleneck that impedes the translation of pluripotent stem cells to the clinic. By overcoming this bottleneck, this project will strongly enhance the biomedical, technological, and economic development of stem cell therapeutics in California. The most important net benefit will be for the treatment of human diseases. Specifically, the tumor-forming properties of human embryonic stem cells (hESCs) are part and parcel with their advantageous properties of continuous self-renewal and pluripotent differentiation; however, they also pose considerable challenges for their safe use in humans. In particular, any serious adverse events in a clinical trial involving hESC-derived cells will severely damage the field. Therefore, the ability to deplete tumor-forming cells from populations of cells differentiated from hESCs will greatly enhance their clinical safety, as well as their utility as models of human disease and development. The coupled technology platforms to be used in this proposed work – microdevices for cell separations, and more homogeneous cell culture and differentiation systems based on bioactive materials – have broad applications not only for stem cells but also many other scientific and biomedical efforts, such that the results of this project will enable many fields. Furthermore, this proposal directly addresses several central objectives of this RFA – pre-transplant manipulations of cells to prevent teratoma formation, as well as development of cell differentiation and purification methods that result in more consistent yield of cells of the desired phenotype, and that are scalable and more cost-effective – indicating that CIRM believes that the proposed capabilities are a priority for California’s stem cell effort. While the potential applications of the proposed technology are broad, we will first apply it to two specific and urgent biomedical problems: the depletion of teratoma-forming cells from hESC-derived populations of myocytes and dopaminergic neurons. By assessing the performance of this technology in two models of human disease, the preclinical impact of the work will be directly established. Furthermore, in addition to enhancing the safety of tranplanted cells, this work has the short and longer term potential to greatly increase the uniformity and yield of therapeutically important cells derived from hESCs, thereby potentially impacting therapeutic efficacy. The co-investigators have a strong record of translating basic science, engineering, and medicine into practice, both through medical schools and interactions with biotech companies in California. In addition, this collaborative project will focus diverse research groups with many students on an important interdisciplinary project at the interface of science and engineering, thereby training a future workforce and contributing to the technological and economic leadership of California.
This proposal addresses a major bottleneck in the development of stem cell therapies: the potential for contaminating undifferentiated cells to form teratomas following transplantation. The applicant proposes to develop advanced technologies for cell differentiation and purification with the goal of producing homogenous cell populations for transplantation. In Aim 1 the applicant will use novel microfluidic cell separation technologies to purify differentiated cells derived from human embryonic stem cells (hESCs). Markers of teratoma-forming cells will be identified and antibodies to these markers will be used in microfluidic devices for cell separation. Resulting purified populations of dopaminergic (DA) neurons and myocytes will be tested for teratoma-formation in nude mice and then for functional efficacy in a rat model of Parkinson’s disease (PD), and mouse models of traumatic muscle injury and muscular dystrophy. In Aim 2 the applicant proposes to develop biomimetic interfaces and media conditions to improve the efficiency of hESC differentiation into desired cell types. The applicant has developed bioactive polymeric surfaces functionalized with peptides that interact specifically with stem cells to promote differentiation. These surfaces are called interpenetrating polymer networks (IPNs) and will be modified and optimized for uniform hESC differentiation into DA neurons and myocytes. The resulting cells will be subjected to the sorting technologies developed in Aim 1 and tested for function in the same rat and mouse models of disease. Reviewers agreed that the development of technologies to address the bottlenecks identified in this proposal could have significant impact if successful. The potential for teratoma formation following transplantation of heterogeneous cell populations is a critical bottleneck in the translation of stem cell therapies. The use of microfluidic cell sorting strategies to purify differentiated cells has several advantages over fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). FACS can be slow, damaging, and require the separation of antibody from cells while MACS can also be damaging and only allows for single-stage selection (one antibody). On the other hand, microfluidics can be multi-stage, gentle, inexpensive and moderately scalable. Reviewers noted that the research plan is well-written and clearly articulated, but raised questions about its feasibility. They were particularly concerned that the antibodies selected for testing in Aim 1 might not be effective. One panelist noted that the success of the proposed microfluidic technology relies on antibody-to-antigen affinity and cell capture and with similar column-like approaches has been inefficient. For this reason, although one reviewer appreciated that the applicant would benchmark the novel microfluidic approach against MACS sorting, several panelists thought it likely that MACS would outperform the microfluidics technology. Reviewers also commented on the limited discussion of pitfalls for Aim 1, and suggested that the proposed alternative approaches seem contradictory to the aim’s objectives. Reviewers had similar concerns about Aim 2, noting that there is little evidence IPNs will work as anticipated and that many different bioactive peptides may have to be tried before the right ones are identified. They also felt that the rationale for testing variable modulus is debatable. One reviewer placed biomechanical properties third in a hierarchy of factors important in cell differentiation, behind biochemical signals and matrix adhesive components. Another reviewer noted that pitfalls and alternative approaches are barely discussed for Aim 2. Concerns about scale-up were also raised. One reviewer cautioned that sorting 105 cells/minute in a single microfluidic channel will subject cells to shear. Another panelist noted that 2D culture methods are inherently less scalable than 3D methods. A reviewer commented that the NOD/SCID mouse, or a derivative lacking the IL-2 receptor (NOG), would be a better test bed for the tumorigenic potential of cells than the nude mouse. A recent paper from Sean Morrison’s lab showed that NOG mice may be more permissive to tumor formation (and therefore a better readout) when used as hosts, compared to the NOD/SCID mice. Another reviewer noted that the rationale for selecting PD and muscle injury/disease as models for testing cells was not provided, nor was justification for the number of animals to be tested. This reviewer was also concerned whether the tight timeline was realistic for the extensive amount of work proposed. Reviewers praised the applicant’s productive track record in applying engineering approaches to problems in stem cell biology. They described the assembled research team, with varied expertise in biology, engineering and medicine, as “a strength of the proposal”. Reviewers did notice that the applicant has a number of other grants and raised the possibility of overcommitment. The budget was judged to be reasonable, well-balanced and justified, with the exception of travel expenditures, which seemed high. Reviewers described the research environment and resources as outstanding. Overall, while reviewers praised the potential impact of this proposal and its novel approaches to a critical bottleneck, they raised doubts about its feasibility and its readiness for translation to the clinic.