Tools and Technologies II
Elucidating how genetic variation contributes to disease susceptibility and drug response requires human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc. Our project combines technology development and scaling methods to increase throughput and reduce cost of hiPSC generation at least 10-fold, enabling the demonstration, and criterion for success, that we can generate 300 useful hiPSC lines (6 independent lines each for 50 individuals) by the end of this project. Thus, we propose to develop an efficient, cost effective, and minimally labor-intensive pipeline of methods for hIPSC identification and characterization that will enable routine generation of tens to hundreds of independent hIPSC lines from human patients. We will achieve this goal by adapting two simple and high throughput methods to enable analysis of many candidate hIPSC lines in large pools. These methods are already working in our labs and are called "fluorescence cell barcoding" (FCB) and expression cell barcoding (ECB). To reach a goal of generating 6 high quality hIPSC lines from one patient, as many as 60 candidate hIPSC colonies must be expanded and evaluated individually using labor and cost intensive methods. By improving culturing protocols, and implementing suitable pooled analysis strategies, we propose to increase throughput at least 10-fold with a substantial drop in cost. In outline, the pipeline we propose to develop will begin with the generation of 60 candidate hIPSC lines per patient directly in 96 well plates. All 60 will be analyzed for diagnostic hIPSC markers by FCB in 1 pooled sample. The 10 best candidates per patient will then be picked for expression and multilineage differentiation analyses with the goal of finding the best 6 from each patient for digital karyotype analyses. Success at 10-fold scaleup as proposed here may be the first step towards further scaleup once these methods are fully developed. Aim 1: To develop a cost-effective and minimally labor-intensive set of methods/pipeline for the generation and characterization high quality hIPSC lines from large numbers of human patients. We will test suitability/develop a set of methods that allow inexpensive and rapid characterization of 60 candidate hIPSC lines per patient at a time. Aim 2: To demonstrate/test/evaluate the success and cost-effectiveness of our pipeline by generating 6 high quality hIPSC lines from each of 50 human patients from [REDACTED]. We will obtain skin biopsies and expand fibroblasts from 50 patients, and generate and analyze a total of 6 independent hIPSC lines from each using the methods developed in Aim 1.
Statement of Benefit to California:
Many Californians suffer from diseases whose origin is poorly understood, and which are not treatable in an effective or economically advantageous manner. Part of solving this problem relies upon elucidating how genetic variation contributes to disease susceptibility and drug response and better understanding disease mechanism. Achieving these goals can be accelerated through the use of human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc. If successful, our project will lead to breakthroughs in understanding of disease, development of better therapies, and economic development in California as businesses that use our methods are launched. In addition, new therapies will bring cost-savings in healthcare to Californians, stimulate employment since Californians will be employed in businesses that develop and sell these therapies, and relieve much suffering from the burdens of chronic disease.
This proposal is focused on the development of tools to increase the throughput and lower the cost of generating induced pluripotent stem cells (iPSCs) from patient fibroblasts. The specific goals are a ten-fold reduction in both the cost and time of iPSC generation, and the establishment of six independent iPSC lines from each of 50 human Alzheimer’s disease (AD) patients. Many diseases, including AD, are thought to have a heterogeneous genetic basis. Identifying how this genetic variation contributes to disease susceptibility and drug response will require iPSC lines from many patients. The applicant identifies the high cost and labor intensiveness of iPSC generation as bottlenecks to the translation of novel therapies. Two specific aims are proposed: (1) to develop methods to allow inexpensive and rapid generation and characterization of 60 candidate iPSC lines per patient; and (2) to evaluate and validate the methods developed in Aim 1 by generating six high quality iPSC lines from each of 50 different AD patients. The reviewers agreed that this proposal addresses a significant translational bottleneck. For many diseases, genetic heterogeneity will require the generation of many iPSC lines in order to encompass the genetic variation of the patient population. The low efficiency of current methods and the high cost of analyzing each of the candidate iPSC lines make it cost prohibitive to produce hundreds of lines. The reviewers found the applicant’s approach to this problem to be logical and highly innovative. They noted that the fluorescence cell barcoding (FCB) and expression cell barcoding (ECB) technologies have existed for a few years but have not been applied in this context. Reviewers did raise a minor concern that, due to the complexity of these technologies, it may be difficult to transfer them to other laboratories, potentially limiting the impact of the proposal. In addition, it was noted that while the generation of many AD patient iPSC lines will be an important advance, a more significant bottleneck may be the lack of specific and efficient differentiation protocols for phenotyping and drug screening. Reviewers praised the carefully designed research plan and the extensive preliminary data supporting its feasibility. Importantly, the applicant demonstrates that the FCB approach can distinguish 60 cell lines from one another and presents promising data from a pilot screen on primary iPSC lines. Reviewers did note that certain aspects of the proposal are technologically demanding and not guaranteed of success, including Aim 1, Step 1, but they appreciated that the applicant identifies key risks and proposes alternative approaches. One reviewer highlighted some possible technical issues that should be carefully considered in the course of carrying out the proposed studies. This reviewer noted that the use of feeder-free conditions might affect the genomic stability of cells, which will have to be carefully assessed using the proposed approach. In addition, the reviewer felt that iPSCs may not only grow better on feeders but also may culture better when mechanically passaged. This reviewer cautioned that there will inevitably be a high dropout level of clones and the applicant will have to pay careful attention to cloning efficiency and karyotype. Another minor concern was that performing FCB on both surface markers and intracellular transcription factors may be complicated by the permeabilization procedure and may require substantial optimization. One reviewer suggestion was for the applicant to consider the use of aggrewells for the embryoid body (EB) differentiation protocol, as they produce a uniform EB size distribution which could reduce heterogeneity. The reviewers described the Principal Investigator (PI) as outstanding and well-positioned to take advantage of the patient cell lines that should result from this proposal. They also praised the Co-Investigator, who is an expert in bioinformatics and will anchor much of the FCB and ECB technology. In general, the reviewers found the research team to be a major strength. Overall, the reviewers were extremely impressed with this proposal. They raised a few minor technical concerns, but the strong research team and extensive preliminary data convinced them of the research plan’s feasibility. Reviewers were optimistic that this proposal could be a breakthrough in the development of high-throughput, low-cost methods for patient-specific iPSC line generation.