Tools and Technologies II
Stem Cell Use:
Cell Line Generation:
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.
An important problem in stem cell and regenerative medicine research has been the ability to quickly and cheaply generate and characterize reprogrammed stem cells from defined human patients. The primary goal of our project is to address this need by developing new technologies that allow stem cell lines to be characterized in large mixed pools as opposed to one by one. Our new methods use flow cytometry and highly sensitive methods for detecting the activity of genes in the cell lines. We made excellent progress in the first year and reduced flow cytometry methods to practice taking advantage of a method called fluorescence cell barcoding. Methods for analyzing activity of genes and chromosome number are in progress and being tested. Our ultimate goal is to reduce cost tenfold and increase speed by about tenfold and our methods development is on track to accomplish this aim.
A key bottleneck in reprogramming technology to make induced pluripotent stem (IPS) cell lines is the ability to make large numbers of lines from large numbers of patients in a way that is cost effective and minimizes labor. Our project has focused primarily on dropping the cost of characterization of candidate lines. We have made a number of discoveries about the behavior of candidate reprogrammed lines that allow us to drop cost and labor needed for candidate reprogrammed line characterization. We measured the frequency of candidate lines that were well-behaved in a large retroviral reprogramming experiment, which allows us to rigorously estimate how many candidate lines must be picked and analyzed if 4-6 high-quality lines are to be generated for every patient fibroblast sample subjected to typical retroviral reprogramming technology. We then continued our work on developing a combination of different array and microfluidic chip technologies to measure the chromosome number in each candidate line and the ability of each line to be pluripotent, i.e., to be able to generate many different type of cells similar to embryonic stem cells. We are optimistic that our work will simplify and drop the cost of the characterization process so that it costs far less than before our work was initiated.
Reprogrammed stem cell lines, i.e., induced pluripotent stem cell lines, have the potential to revolutionize research into causes of disease and genetic contributions to the causes of disease. One key limitation, however, is the ability to generate large numbers of different stem cell lines from different people to sample the range of genetic variation in the human population as it relates to disease development. A key bottleneck is the speed and cost with which reprogrammed stem cell lines can be generated and validated for usefulness. We have succeeded in developing a streamlined workflow for characterization of reprogrammed stem cell lines that drops the cost for characterization from several thousand dollars to a few hundred dollars and increases the speed and number of lines that can be handled substantially. We take advantage of novel genetic characterization methods to analyze genetic stability and the pattern of gene expression as it reveals the capabilities of the stem cell lines. We are finishing up the loose ends on this project now and should have a high quality publication prepared for submission shortly that describes this simple and inexpensive workflow that we have developed with modern gene characterization methods.