Recent reports indicate that skin fibroblasts can be reprogrammed, to express an induced pluripotent stem (iPS) cell-like phenotype comparable to that of embryonic stem cells (ESC). If reduced to practice this would make every patient their own donor for tissue replacement therapy and eliminate ethical concerns regarding ESC. However even if iPS cells can be differentiated into clinically relevant tissues, several obstacles remain that will limit the translation of iPS cell derived therapies to the clinic. Two are addressed in this application. 1) The reprogramming protocol currently uses retroviral transduction to generate iPS cells which raises the unacceptable concern of insertional mutagenesis. 2) A large animal pre-clinical model is needed to test the long term safety and efficacy of transplanting iPS cell derived tissues. The first obstacle will be addressed by testing the hypothesis that a cocktail of small molecules can be identified that induce reprogramming. The second issue is addressed by establishing a canine iPS model.
Three specific aims are proposed:
In aim 1, we will first generate canine iPS cells using retroviral mediated expression of different combinations of transcription factors as reported. The resulting iPS cells will be compared to the canine ESC that we recently described.
In aim 2, we will develop reporter constructs for the transcription factors shown to be required for reprogramming in aim 1. These reporters will be used to generate cell lines for high throughput screening (HTS) to identify small molecules that will induce expression of the relevant transcription factors and eliminate the need for retroviral vectors. The HTS will be performed in collaboration with a Seattle based biotechnology company, Fate Therapeutics.
In aim 3, we will use combinations of small molecules identified through HTS to reprogram canine fibroblasts and generate iPS. The experiments proposed in the application are designed to provide the Principal Investigator, Aravind Ramakrishnan, with an intense training experience focused on the cell and molecular biology of ESC. The training program involves scientific investigation and didactic study under the mentorship of Dr. Beverly Torok-Storb. The applicant will also benefit from the guidance of Drs. Randall Moon, C. Anthony Blau, and Carol Ware who will form his advisory committee. If the studies proposed here are successful, Dr Ramakrishnan will progress to inducing differentiation of the iPS cells into transplantable cell types that can be evaluated for safety and efficacy in the dog. Given that the transcription factors are highly conserved, it is likely that the same molecules will also reprogram human cells. Therefore once safety and efficacy has been established in the canine model, protocols can be translated to clinical trials.
There has been considerable interest in using stem cells to develop cell replacement therapies for degenerative diseases. It has clearly been demonstrated that ESC are capable of giving rise to every tissue in the body. Thus, there has been great excitement generated in the scientific community regarding the promise of ESC for treating disease and understanding early development. However, there are many obstacles to overcome prior to the transition of ESC based cell replacement therapies to the clinic. The proposed studies are to address some of the existing problems.
|Du, Jianhai; Yanagida, Aya; Knight, Kaitlen et al. (2016) Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium. Proc Natl Acad Sci U S A 113:14710-14715|
|Kim, Eunhee; Ilagan, Janine O; Liang, Yang et al. (2015) SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition. Cancer Cell 27:617-30|
|Ilagan, Janine O; Ramakrishnan, Aravind; Hayes, Brian et al. (2015) U2AF1 mutations alter splice site recognition in hematological malignancies. Genome Res 25:14-26|
|Hubbard, Jesse J; Sullivan, Spencer K; Mills, Jason A et al. (2014) Efficient iPS cell generation from blood using episomes and HDAC inhibitors. J Vis Exp :e52009|
|Balakrishnan, Ilango; Yang, Xiaodong; Brown, Joseph et al. (2014) Genome-wide analysis of miRNA-mRNA interactions in marrow stromal cells. Stem Cells 32:662-73|
|Ramakrishnan, Aravind; Torok-Storb, Beverly; Pillai, Manoj M (2013) Primary marrow-derived stromal cells: isolation and manipulation. Methods Mol Biol 1035:75-101|
|Ramakrishnan, Aravind; Sandmaier, Brenda M (2010) Optimizing reduced-intensity conditioning regimens for myeloproliferative neoplasms. Expert Rev Hematol 3:23-33|
|Ramakrishnan, Aravind; Torok-Storb, Beverly J (2010) The role of the marrow microenvironment in hematopoietic stem cell transplantation. Cell Ther Transplant 2:7-12|