Cite Harvard Ozurumba, B.
The invention further provides methods to determine the differentiation status of an embryonic cell by comparing its transcriptional patterns with those of ES cells at particular stages of differentiation.
The invention further provides a non-human primate embryonic germ EG cell which can also be used in several ways, including administering the differentiated EG cell line to a patient to treat a number of diseases.
The eventual clinical progression of embryonic stem cell ESCs derived tissues for therapeutic applications will require extensive studies of safety and efficacy in both small and large animal system. Because of the extensive evolutionary distance between rodents and humans, and already evident differences between mouse and primate ESCs, I there are limits to the use of mouse ESCs to demonstrate safety and efficacy of stem cell-based transplants.
Therefore, the present experiments rely heavily on non-human primate as the most relevant model for assessing the therapeutic potential of pluripotent stem cells. While a number of such species are germane options, several considerations point to Rhesus monkeys: The goals of the research described herein are: The present invention further provides a method of generating a chimeric primate embryo, which comprises reaggregating nhp ES cells with biopsied fertilized primate embryos.
The present invention provides a method of determining the differentiation status of an embryonic cell. In a specific embodiment, this method comprises the steps of determining the cell transcriptional pattern of the embyronic cell; comparing this embryonic cell transcriptional pattern to various prototype transcriptional patterns, where each of these prototype transcriptional patterns are derived from an embryonic cell of a specific embryonic cell differentiation status; determining which of these prototype transcriptional patterns most closely resembles the embryonic cell transcriptional pattern; and finally assigning the specific embryonic cell differentiation status corresponding to the prototype transcriptional pattern most closely matching the embryonic cell transcriptional pattern, to the embryonic cell.
In another specific embodiment of the present invention, the embryonic cell transcriptional pattern is determined by hybridizing labeled RNA, isolated from single colonies of cells, to microarray chips. In yet another specific embodiment of the present invention, these microarray chips display genomic DNA fragments originating from a species selected from the group consisting of mouse, human and Rhesus monkey.
In another specific embodiment, the specific embryonic cell differentiation status determined by the method of the present invention can be the inner cell mast, the epiblast, the mesoderm, endoderm, ectoderm, lateral plate mesoderm, gut and neuroectoderm, extraembryonic mesoderm, amniotic ectoderm and visceral endoderm.
In a specific embodiment of the present invention, a method is provided for deriving nhp EG cells, wherein non-human primates are mated to establish pregnancy; the pregnancy is terminated at between 28 and 45 days and an nhp fetus obtained; gonads are isolated from this fetus and placed on plates of feeder cells; the plates are cultured, then fixed and stained for TNAP1 a marker for PGCs; the PGCs are detected by TNAP staining; the PGCs placed into culture, supplemented with LIF, bFGF and forskolin; the culture is fed daily; EG cells are isolated by picking, then plated singly; and the EG colonies resulting are then tested for specific EG-expressed markers.
The present invention also provides for the composition that is a differentiated cell derived from an nhp EG cell. Further, the present invention provides for the composition that is the embryoid body derived from an nhp EG cell. Multidimensional scaling plot of relationships between pluripotent cell types of mouse and human origin.
All transcriptional profiling was done by the method of Tietjen et al. Each line represents a unique strand of DNA. Circles represent positions and methylation of individual CpGs as follows: The first and last CpG sites are numbered relative to the H19 transcriptional start site.
PO1 Primate Core B has now isolated of epiblasts from day 15 nhp embryos. These isolated samples can be placed into culture of frozen in OCT for laser capture experiments, directly dissected and processed for RNA analysis, or even cultured for epiblast-derived embryonic stem cells.
As such cell based transplantation therapies are likely to consist of allogeneic grafts and the most relevant endpoints for assessment will be comparable allogeneic grafts performed within a relevant species. Because of the extensive evolutionary distance between rodents and humans, and already evident differences between mouse and primate ESCs, there are limits to the use of mouse ESCs to demonstrate safety and efficacy of stem cell-based transplants.
Nevertheless, mice particularly immunodeficient strains can have great utility in demonstrating the pluripotency of primate ESCs through the development of teratomas induced by transplanting ESCs to ectopic sites.
However, it must be kept in mind that even such in vivo assays of pluripotency are surrogates for a more thorough assessment of differentiative capacity and long-term stability of the differentiated state of primate ESC derived cells, their pluripotency, epigenetic status and stability of their differentiated state.
This can only be ultimately achieved through even more fundamental assessment of the developmental capacity of pluripotent stem cells as a formal assessment of chimeric studies in embryos, in which contribution and function of ESC derived cells in all primary germ layers and their differentiated derivatives can be definitively assessed.
Based on the foregoing, the present research relies heavily on a non-human primate model as the most relevant model for assessing the therapeutic potential of pluripotent stem cells.
In studying a non-human primate model, a focus on a definitive assessment of pluripotency, capacity for functional differentiation, and epigenetic status in ESCs and EGCs will be undertaken. As used herein, pluripotency includes the characteristic of immortality and the plasticity to develop into any of the body's cell lineages perhaps excluding trophectoderm descendents like some extraembryonic and placental tissues.Activities: Developing proposals, Generation of Donor Reports,Partner Performance monitoring, Proposal review and recommendation, program advocacy, periodic program inventory check, preparation reports Team Building: Mentoring and monitoring a team of 50 members involved in the kaja-net.com://kaja-net.com 1%) who are not using family planning because of poor access and ineffective outreach It is the poorest Filipinos ( Where are we now?
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