Somatic cell reprogramming refers to conversion of differentiated somatic cells to pluripotent stem cells. This reprogramming event demonstrates that adult cells can be turn into stem cells. It was speculated that the four reprogram factors: Oct-4, Sox-2, c-Myc and Klf-4 change cells into stem cells. The molecular mechanism involved in reprogramming is currently unknown. Studying the intermediate stages of reprogramming is critical for understanding its mechanism. However, identification and isolation of sufficient cells in various reprogramming intermediate stages for molecular analysis is very challenging with conventional techniques. To overcome this hurdle, we have successfully developed new tools for monitoring, isolating and analyzing single-cells from the continuous spectrum of somatic cell reprogramming. We will generated human cells lines for drug-inducible reprogramming, and obtain single-cell gene expression profiles at various intermediate stages with our novel microfluidic system. Because each individual cell is at a specific reprogramming stage, profiling entire population of cells will cover all reprogram intermediate stages. With bioinformatics analysis, the stepwise changes of pluripotency will be identified for uncovering the mechanism of reprogramming. Our approach circumvents the challenging requirement of isolating cells from specific reprogramming intermediate stages. Our proposal will provide new tools and knowledge to the scientific community. It will facilitate the development of more efficient and safer reprogramming method in regenerative medicine. The knowledge made manifest by our study will also deepen our understanding of stem cell, and provide fundamental knowledge for stem cell therapy.
Statement of Benefit to California:
This grant proposes to study the molecular mechanism of somatic cell reprogramming by single-cell analysis. Somatic cell reprogramming is a method to turn adult cells into stem cells. With this ability, stem cells can be generated from patient’s own skin or blood in large quantity for repairing damaged tissues and studying diseases. Because these stem cells are generated from the patient’s own cells, immune rejection will be avoid in transplantation and stem cell therapies. Stem cell made with reprogramming is the most promising source of stem cells for clinical application. However, the efficiency of current reprogramming technology is very low (less than 1%) and the molecular mechanism of reprogramming is unknown. Before reprogramming can be applied to clinical practices, its molecular mechanism must be studied and understood. This proposal specifically proposes a single-cell analysis platform to uncover the molecular mechanism of reprogramming. This proposal will create and provide new cell lines and new nanotechnology tools to the scientific community. The novel methods and the knowledge made manifest by our study will stimulate and facilitate the commercial development of safer and efficiency methods to produce stem cells for clinical applications. Therefore, this proposal has a significant health and financial impacts for the state of California.
The goal of this proposal is to investigate the molecular mechanisms of cellular reprogramming by analyzing how the four reprogramming factors (RF’s), Oct4, Sox2, cMyc, and Klf4, alter the transcriptional networks in human cells and lead to a stepwise change in cell pluripotency. In Aim 1, the applicant plans to generate stable human cell lines harboring inducible vectors encoding RFs in order to allow for drug-inducible reprogramming. In Aim 2, single-cell gene expression profiles will be obtained at various intermediate stages in the reprogramming process using a novel microfluidic system. Finally, in Aim 3, the applicant will apply a bioinformatics analysis to identify the stepwise changes in gene expression leading to pluripotency.
Reviewers found that the proposal addresses important questions about the generation of induced pluripotent cells and could have substantial impact in understanding cellular reprogramming. Moreover, reviewers found the approaches developed by the applicant to be appropriately powerful, innovative, and unique. Although the proposal was viewed as extremely ambitious, reviewers felt that, if successful, the experiments would provide a reference database of information that researchers could use to study the dynamics and heterogeneity of reprogramming.
Reviewers’ enthusiasm was substantially dampened by their assessment of the proposal’s experimental design and feasibility. Reviewers judged the proposal as overly ambitious with some aspect poorly planned or lacking adequate detail. For example, reviewers had serious concerns about the applicant’s choice of reporter gene used as a marker of pluripotency. Other major problems included the enormous volume of PCR analysis required by the proposed experiments (which may make the approach unfeasible), and inadequate consideration of the timing and frequency of sampling during reprogramming experiments. There were additional concerns about cellular heterogeneity and whether adequate criteria to identify and confirm the achievement of pluripotency were developed. Reviewers also expressed serious reservations about the number of genes to be analyzed in Aim 2, which was judged as too low. Finally, description of experiments planned to validate gene regulatory relationships identified in Aim 3 were largely inadequate.
The applicant is a relatively young principle investigator who is well trained and has a strong publication record relevant to the proposed research. Reviewers agreed that this applicant has the necessary experience with both technology and stem cell biology to perform the proposed research. Additionally, the research environment appears excellent.
In summary, this proposal utilizes novel techniques and tools to examine the role of individual reprogramming factors in the generation of iPS cells. However, critical deficiencies in experimental design raised serious concerns about the project’s feasibility.