Tools and Technologies I
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.
The proposal focuses on the identification of new biomarkers for pluripotency in human embryonic stem cells (hESCs). To achieve this goal, the applicant proposes to perform massive parallel sequencing of the entire transcriptome of various hESC lines. These data will be used to identify hESC-associated biomarkers and develop an assay panel for assessment of pluripotency by quantitative PCR. This tool would allow for detection of small numbers of residual pluripotent cells in differentiated cell populations, or assessment of pluripotency in newly derived hESC lines and in induced pluripotent stem cell (iPSC) lines. Reviewers agreed that the primary objective of this proposal is an important one. Identification of new biomarkers for pluripotency would enable detection of undifferentiated cells in differentiated cell preparations derived from hESCs and iPSCs, an important prerequisite for these cells’ use in therapies. In addition, these biomarker molecules could themselves be of interest and play roles in the maintenance of pluripotency. However, all reviewers strongly criticized the presentation, specifically the applicant did not clearly state how biomarkers specific to the pluripotent state will be identified and validated. Based on this lack of clarity in the research design, they felt that the proposal would not achieve its objectives. Reviewers disagreed about whether the focus on non-NIH-approved hESC lines is scientifically justified. One noted that the applicant is already doing similar work on NIH-approved lines and commented that it would seem easier to take the markers identified in NIH lines and test them in non-NIH lines rather than doing another large study in parallel. But another reviewer found the focus on non-NIH-approved hESC lines to be justified because of the limited number and availability of the NIH-approved lines and their availability usually only at high passage number. Furthermore, one reviewer raised concerns about the quality of the sequencing data presented in the preliminary data. Reviewers questioned the applicant’s expertise in the fields of genomics and stem cell biology but appreciated the experience and strong credentials of the senior consultant. They worried about the applicant’s lack of team-leading experience, but still felt that the research group was qualified to carry out the work. Reviewers also raised concerns about the budget, including a lack of detail regarding the consultant/subcontract budget and a discrepancy between the requested supply budget and its justification. Overall, while this proposal addresses a major roadblock in stem cell research, the omission of critical validation experiments seriously diminishes its potential to achieve the proposed objectives. Reviewers considered this to be a fatal flaw.