High efficiency and high fidelity somatic cell nuclear reprogramming

Funding Type: 
SEED Grant
Grant Number: 
RS1-00174
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Embryonic stem (ES) cells possess a remarkable property of pluripotency to give rise to all cells of the organism; therefore holding great promise for regenerative medicine. ES cells are envisioned as potential sources for use in cell replacement therapies. However, as with any allogeneic material, ES cells generated from fertilized embryos, and derivatives of such cells, inevitably face risks of immunorejection when transplanted into a host. A solution to the problem of rejection is to derive ES cells from embryos cloned from a host patient’s own cells, because any replacement cells would be genetically identical to the host. ES-like cell lines have been generated from somatic cells via somatic cell nuclear transfer (SCNT) technology. By this technique the developmental fate of a somatic cell nucleus can be reprogrammed following transfer into an enucleated oocyte, resulting in a cloned embryo that can be used to derive novel ES cells. The nuclear transferred ES cell lines have been shown to possess the same characteristics for self-renewal and unlimited differentiation capacity as those of conventional ES cell lines derived from normal embryos produced by fertilization. Although these cloning experiments are direct evidence that a terminally differentiated cell can be fully reset to a pluripotent ES cell state, the fidelity of reprogrammed nuclei is very inefficient as evidenced by the extremely low frequency (<5%) of producing healthy offspring in animals. Thus, the current available SCNT technique makes ES cell related therapies only an academic possibility at this time. Alternative approaches in deriving stem cells are based on genome-wide analysis on genes that are involved in the molecular control of pluripotency. Takahashi and Yamanaka (2006) have recently reported the induction of pluripotent stem cells from mouse embryonic and adult fibroblast cells by introducing four factors (Oct3/4, Sox2, c-Myc and Klf4) under ES cell culture conditions. All four factors play a crucial role in maintaining the undifferentiated state and proliferation of ES cells in culture. The resultant induced pluripotent stem (iPS) cells are similar but not identical to bona fide ES. The iPS cells are not fully reprogrammed and have a limited capacity to stably integrate into normal tissues in vivo. Thus, this approach is unlikely to sufficiently reprogram somatic cells to a state that will be optimal for human therapeutic cloning. The proposed research is to develop a new high-efficiency nuclear transfer (HENT) approach to obtain high fidelity ES cells. The approach is based on the current existing SCNT method in combination with transcription factors that are known to be required for ES cell renewal and pluripotency. The long-term goal of the proposed research is to apply HENT to derive high fidelity human ES cells and subsequently for cell replacement therapies.
Statement of Benefit to California: 
Efficient derivation of host patient-specific embryonic stem (ES) cells is a fundamental step, currently a bottleneck, for cell replacement therapies and regenerative medicine. The proposed research aims to increase the efficiency of ES cell derivation from somatic cells through a modified nuclear transplantation technique. The result of such work will potentially benefit the state of California and its citizens in many aspects including health, life, costs, and leadership in biomedicine. ∑ About half of California families have a child or adult who suffers or will suffer from devastating medical conditions that could potentially be treated or cured with stem cell therapies. ∑ Development of ES cell therapies that treat diseases and injuries with the ultimate goal to cure them will improve the California health care system and reduce the long-term health care cost burden on California. ∑ Intellectual property resulted from the research will provide an opportunity for the state to benefit from royalties, patents, and licensing fees. ∑ The research will contribute to the advancement of the biomedical research in California to world leadership.
Progress Report: 
  • Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
  • One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
  • To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
  • It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
  • Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).

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