The goal of this project is to generate important new insights concerning reprogramming of primate somatic cells to the pluripotent state employing somatic cell nuclear transfer (SCNT) and direct reprogramming approaches and to conduct comparative pluripotency assessments using expression profiling, genetic and epigenetic analysis and in vitro and in vivo differentiation assays. Our main hypothesis is that primate pluripotent cells experimentally derived using these two alternative approaches are equivalent to each other and to embryonic stem cells (ESCs) isolated from fertilized embryos. Another goal of this application is to evaluate, for the first time, the potential of monkey ESCs derived from fertilized or SCNT embryos and induced pluripotent (iPS) cells to generate chimeras upon injection into developing embryos. To achieve these goals we propose the following specific aims: 1). to create monkey pluripotent cells by epigenetic and genetic reprogramming of somatic cells. In Experiment 1, we will derive ESCs by SCNT from adult monkey skin cells and test our working hypothesis that experimental upregulation of critical pluripotent factors - OCT4, SOX2, NANOG and CARM1- in cytoplasts will enhance reprogramming and increase the current efficiency of SCNT embryo development and ESC isolation. In Experiment 2, we will generate monkey iPS cells from the same monkey skin cells by lentiviral transduction of genes encoding critical reprogramming factors OCT4, SOX2, KLF4, C-MYC, NANOG and LIN28 under control of doxycyclin-inducible promoters. 2). Examine pluripotency of novel pluripotent cells. Our working hypothesis here is that both SCNT and direct reprogramming can support complete reprogramming of somatic cells to the pluripotent state. To test this assumption, in Experiment 1 we will interrogate genetic, cytogenetic, epigenetic and transcriptional profiles of novel cell lines. In Experiment 2, cell lines will be subjected to in vivo differentiation in teratomas in SCID mice and to in vitro directed differentiation into mesoderm (cardiomyocytes), ectoderm (neuronal phenotypes) and endoderm (pancreatic beta-cells).3). Determine the potential of monkey pluripotent cells to contribute to chimeras. We hypothesize that similar to their mouse counterparts, primate ESCs and iPS cells have the potential to integrate and participate in development of chimeric offspring. To test this hypothesis, we propose to inject GFP-expressing monkey ESCs and iPS cells into monkey preimplantation embryos and transfer the resultant chimeric embryos into recipients to establish pregnancies. Chimeric fetuses and full-term offspring will subsequently be studied for tissue distribution and germ line colonization.
In this application we will study the potential of autologous (patient-matched) stem cells derived by two alternative approaches in the nonhuman primate model. The outcomes of these experiments, in turn, will allow the production of useful preclinical monkey models for testing therapeutic applications involving autologous stem cells for the treatment of wide range of degenerative diseases.
|Folmes, Clifford Dl; Ma, Hong; Mitalipov, Shoukhrat et al. (2016) Mitochondria in pluripotent stem cells: stemness regulators and disease targets. Curr Opin Genet Dev 38:1-7|
|Ma, Hong; Marti Gutierrez, Nuria; Morey, Robert et al. (2016) Incompatibility between Nuclear and Mitochondrial Genomes Contributes to an Interspecies Reproductive Barrier. Cell Metab 24:283-94|
|Izpisua Belmonte, Juan Carlos; Callaway, Edward M; Caddick, Sarah J et al. (2015) Brains, genes, and primates. Neuron 86:617-31|
|Wolf, Don P; Mitalipov, Nargiz; Mitalipov, Shoukhrat (2015) Mitochondrial replacement therapy in reproductive medicine. Trends Mol Med 21:68-76|
|Mitalipov, Shoukhrat; Amato, Paula; Parry, Samuel et al. (2014) Limitations of preimplantation genetic diagnosis for mitochondrial DNA diseases. Cell Rep 7:935-7|
|Kang, Eunju; Wu, Guangming; Ma, Hong et al. (2014) Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. Nature 509:101-4|
|Wolf, Don P; Mitalipov, Shoukhrat (2014) Mitochondrial replacement therapies can circumvent mtDNA-based disease transmission. Cell Metab 20:6-8|
|Mitalipov, Shoukhrat; Wolf, Don P (2014) Clinical and ethical implications of mitochondrial gene transfer. Trends Endocrinol Metab 25:5-7|
|Daughtry, Brittany; Mitalipov, Shoukhrat (2014) Concise review: parthenote stem cells for regenerative medicine: genetic, epigenetic, and developmental features. Stem Cells Transl Med 3:290-8|
|Janovick, Jo Ann; Stewart, M David; Jacob, Darla et al. (2013) Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy. Proc Natl Acad Sci U S A 110:21030-5|
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