Pluripotent cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPS cells) have the potential to be the source of essentially unlimited numbers of differentiated cells for basic research, clinical, and preclinical applications. The goal is to produce a homogeneous population of differentiated cells in the cell preparation, without any remaining pluripotent cells. This is particularly important for cells that will be used for cell therapy, as there is a risk that pluripotent cells may form tumors. To ensure that cell preparations destined for clinical use are free of contaminating pluripotent cells, more sensitive and specific biomarkers for pluripotency need to be developed.
The aim of this project is to study the activity of every gene in the genome in a collection of human embryonic stem cells using massively parallel digital sequencing in order to identify biomarkers for pluripotence. We will then develop and validate a panel of these biomarkers, which can then be used to detect small numbers of residual pluripotent cells in hESC- and induced pluripotent stem-cell-derived differentiated cell preparations, and also to assess newly derived or reprogrammed cell lines for pluripotence.
Because our goal is to develop an assay that can be generalized to all pluripotent cell types, it is important to develop the assay using cell lines from a variety of sources. Of the over 400 hESC lines in existence, there are only 21 NIH-approved lines. Moreover, several of the NIH-approved hESC lines are not publicly available. The limited number of NIH-approved hESC lines represent a narrow range of derivation, culture, and passage techniques. In addition, because the NIH-approved lines were by definition derived prior to August 1, 2001, the publicly available cells are relatively high passage. It is therefore critical that we include non-NIH-approved cell lines in this study. In this application, we are proposing to exclusively study non-NIH-approved lines, as we have already been awarded funds from the NIH to study NIH-approved lines.
Since the hESC lines to be studied are non-NIH approved, this proposal is not eligible for federal funding.
Statement of Benefit to California:
Pluripotent cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPS cells) have the potential to be the source of essentially unlimited numbers of differentiated cells for basic research, clinical, and preclinical applications. The greatest current barrier to moving well-developed stem cell therapies into the clinic is the FDA’s concern about the safety of the cells once they have been transplanted. The FDA’s primary objective is to ensure that new therapies are safe and the agency’s approval is required before clinical trials can go forward. All cell therapy approaches call for the pluripotent stem cells to be differentiated into, for example, nerve cells, before they are transplanted. But there is a risk that if the cells are not completely differentiated, and a small number of pluripotent cells remains, the transplanted cells might develop into tumors. In order to ensure that cell preparations destined for clinical use are free of contaminating pluripotent cells, we require more sensitive and specific biomarkers for to detect pluripotent cells. We propose to develop new biomarkers that will identify small numbers of specific cell types within a large population of transplantable cells. We will do this by using massively parallel digital sequencing, also known as “next-generation sequencing,” to identify all of the amounts and types of RNAs found in pluripotent cells. This method allows us to detect “splice variants” that are alternative forms of messenger RNAs that are often very specifically limited to certain cell types. We will use these data to develop a panel of biomarkers that can detect tiny numbers of residual pluripotent cells in hESC- and induced pluripotent stem-cell-derived differentiated cell preparations. Californians will benefit from development of a technology that will be widely adopted for use by clinical investigators, and that may help win FDA approval for cell therapies.