Investigate Molecular Mechanism of Reprogramming with Single-cell Analysis

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01417
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • We aim to develop new techniques to guide human stem cells to repair diseased or damaged tissues. In the past year, we have successfully developed new techniques and are able to guide human embryonic stem cells, ips cells and progenies to migrate and integrate with host tissues.
  • We aim to guide human stem cells to repair diseased or damaged tissues. In the past year, we have developed new techniques to guide neuronal stem cells to migrate in vitro, and a proprietorial technique to apply electrical cues in animal. This forms a strong basis that in the third year, we are ready to prepare to move to the next stage - to translate the results from this basic biology grant to a novel technique to possible clinical use.
  • The aim of this project is to develop technology to stimulate and guide human neural stem cells to repair and regenerate damaged tissues. The CIRM grant provided support that move both the science and technology forward and closer to clinical use.
  • Specifically, we have for the first time 1) demonstrated effectiveness of using electric stimulation to enhance migration of human stem cells to migrate directionally; 2) developed new techniques to stimulate cells in 3D and in vivo; 3) demonstrated synergistic effects of chemical guidance and electric stimulation on human stem cells.
  • We have successfully completed all the original proposed goals for year 3. Specific Aims that have been completed are: Aim 1B (Direct hNSCs in 2D), Aim 2A (Purinergic signaling), Aim 2B (Ca++ signaling). Aim 3A (Direct SCs in 3D), and Aim 3B (Transplant SCs in vivo and direct migration of SCs) are fully started as planned, with major part of 3A finished.
  • Our original hypothesis is that EFs are a powerful signal to direct migration of human embryonic stem cells (hESCs) and human nerve stem cells (hNSCs) through purinergic signaling. Three specific Aims with the following time scales were set in the proposal: Aim 1. To guide migration of hESCs and hNSCs. Aim 2. To elucidate purinergic receptor/Ca2+ signaling in EF-directed stem cell migration. Aim 3. To direct migration of hESCs and hNSCs in 3D. We have finished all proposed research and are writing up for publication.
  • This is report on a no cost extension of six month.
  • We have successfully conducted the experiments proposed and published 6 papers. We would like to request 1 year no-cost extension of the CIRM grant for the following reasons.
  • We have extend beyond the original proposal and carried out some in vivo experiments. We have
  • 1. Established stable human neural stem cell (hNSC) lines that stably expressing GFP for in vivo transplantation and tracking.
  • 2. Confirmed the electrotaxis response of those transduced cells;
  • 3. Developed proprietary techniques for stimulating and guiding transplanted cells in the brain;
  • 4. Manufactured prototype of stimulator for in vivo use;
  • 5. Successfully guided transplanted hNSCs in live rat brain.
  • We are currently analyzing those exciting data and are preparing manuscripts for high impact publication. We are preparing for early translational work. This will need to maintain active fund for use for analysis, publication purpose. We will be grateful if the CIRM grants this no-cost extension request. We have submitted three grant applications during this period of time and have published two important papers in high impact journals - one in EMBO Reports and one in Current Biology. Both papers have attracted media interview and commentary of experts.
  • The EMBO Reports paper proposes for the first time the existence of endogenous electric fields in the brain, which may guide migration of neural stem cells. This is a completely new mechanism no one proposed before and we have proved strong experimental evidences.
  • The Current Biology paper dissected the mechanisms of how cells sense and respond to a small electric fields.
  • We have submitted a patent application during this period of time.

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