Human stem cells hold great promise to treat a variety of devastating diseases, but there are many barriers to successful clinical trials. One major challenge in embryonic stem (ES) cell research is to maintain cultures of human ES cells. There are variations in culture conditions that affect the quality of human ES cells, as well as cellular differentiation and genomic alterations. To advance ES cell research, it is essential to establish optimal culture conditions for ES cells. A second major challenge is to direct ES cells to differentiate into specific lineages. Since the development of mouse ES cells in 1980, there has not been a single reported case in which differentiation of an ES cell was guided to a specific cell type. A better understand is needed of the intrinsic and extrinsic factors that governing ES cell differentiation.
Over the past two decades, several laboratories including ours have shown that a mouse model implanted with intact human tissues provides the best physiologically-relevant model to study human stem cell biology. Based on our experience, we hypothesized that the engrafted human tissues in SCID mice could provide a physiologically relevant microenvironment to direct human ES cells to differentiate into mature cells of corresponding tissues. To test this, we used two NIH-approved ES cell lines (H1 and HSF-6) and three types of human fetal tissues (thymus, lung and pancreas). Human ES cells were injected directly into each tissue type engrafted in SCID mice. In most engrafted human tissues, we observed an aggressively growing tumor, which displayed characteristics of primitive, undifferentiated tumors rather than teratomas.
A better understanding the basis for this interesting phenomenon observed in our experimental system could have significant impacts in two important areas related to human ES cell research: tumorigenicity and expansion of human ES cells. In Aim 1, we will characterize these tumor-like cells as being ES cell-like or EC cell-like. If the cells resemble ES cells, it would suggest that we have identified a physiologically-relevant model system in which human ES cells could be expanded while maintaining their ES cell activity. In Aim 2, we will identify human tissue-derived factors responsible for ES cell expansion, which could mean the feasibility to develop a MEF-free system for robust human ES cell expansion. In addition, the identification of human tissue-derived factors could also provide an important tool to investigate intrinsic and extrinsic factors that govern self-renewal of human ES cells. However, if these tumor-like cells turn out to resemble EC cells, then it would suggest that EC cells are possible outcomes and could raise serious safety concerns for clinical use of ES cells. The identification of human tissue-derived factors (Aim 2), may lead to investigations of intrinsic and extrinsic factors important in EC development and of ways to prevent ES to EC transitions.
Statement of Benefit to California:
Over the past two decades, several laboratories, including ours, have shown that the SCID-hu mouse, an animal model that has intact human tissues implanted, provides the best physiologically-relevant model to study human stem cell biology. Based on our extensive experience with SCID-hu mice, we hypothesized that this model might also provide a physiologically-relevant microenvironment to direct human ES cells to differentiate into mature cells of corresponding tissues. Using two NIH-certified cell lines and three different human tissues in SCID mice, we observed aggressive tumor growth in most engrafted human tissues. These tumors displayed histological characteristics of primitive, undifferentiated tumors rather than teratomas, and molecular studies suggest that these tumor-like cells may be ES cells. A better understanding of the basis for this interesting phenomenon could have significant impacts in two important areas related to human ES cell research: tumorigenicity and expansion of human ES cells.
Benefit to California: Currently, culturing and expanding human ES cells in vitro is a major challenge. In order to advance human ES cell research, it is essential to establish optimal human ES cell culture conditions. One of the possible outcome of our proposal is the identification of human ES cell proliferation factor(s). By working with the Geron Corporation (Menlo Park, CA), we may be able to develop an optimal ex vivo culture system to expand high quality human ES cells for potential clinical application and to distribute to researchers in California who are interested in doing human ES cell research. Another possible outcome of our proposal is to alleviate the risks and concerns of using human ES cell-derived products in clinical application. Again, we will work with the Geron Corporation and medical centers in California, including the City of Hope, to investigate this issue. The outcome of this proposal could help to establish California as a premier state in human ES cell research.
SYNOPSIS: In previous experiments the applicants have engrafted SCID mice with human fetal tissue, including pancreas, lung and thymus. The goal was to determine whether these tissues might provide a physiologically relevant microenvironment that would support the engraftment and differentiation of human embryonic stem cells (hESCs) in vivo. Instead they observed that the ESCs grew into primitive, undifferentiated tumor-like structures. In Aim 1 the applicants propose to determine whether the cells within these tumor-like structures retain ESC-like characteristics, or if they have become more embryonal carcinoma (EC)-like. This will be done by examining their patterns of gene expression, as well as their growth characteristics in vitro and in vivo. In Aim 2, it is proposed to biochemically fractionate the fetal tissues to identify factors that promote the growth of ESCs in vitro, or that promote their conversion to EC-like cells.
SIGNIFICANCE AND INNOVATION: It is proposed that if it can be shown that the tumor-like cells growing in vivo retain ESC characteristics, this will represent a new method for ESC expansion. Furthermore, the biochemical fractionation of fetal tissue extracts would then provide a new tool for the in vitro expansion of ESCs and possibly provide insight into factors that control their self-renewal. Conversely, if these tumors result from a transition to an EC-like phenotype, then the experiments might provide insight into the factors that control this transition and thus possibly a means to prevent this transition from occurring.
The reviewers felt that this proposal has minimal innovation and only modest significance. Derivation of tumors from hESCs has been demonstrated in other models. This proposal seems to describe tumors that are distinct from the typical teratomas. However, these more homogeneous tumors derived from hESCs are of unclear etiology and significance. The overall rationale of why this is an important model for understanding hESCs or tumor biology remains of relatively limited general interest.
STRENGTHS: Strengths of this proposal rests on the fact that this is a novel idea for which the applicants have been solely responsible for generating the preliminary data. The applicants have prior experience in studying neural progenitor cells. The Principal Investigator (PI) obviously has experience with growth of hESCs and already has strong preliminary data that co-injection of undifferentiated hESCs along with engrafted human fetal tissue in a SCID-HU mouse model leads to the development of tumors that are felt to be distinct from teratomas, and may give some insight into hESC biology. The aims proposed are rational and coherent and in logical order, and of appropriate scope for this funding mechanism. Analyses of specific genes derived from these tumors in comparison to ESCs and EC cells is appropriate.
WEAKNESSES: The reviewers noted serious weaknesses with this proposal. Aim 2 is very poorly conceived. First of all, it is not yet established that the fetal tissue-induced ESC proliferation observed in vivo can be reconstituted in vitro with crude lysates. Even if this could be shown, there is no consideration at all of the many problems involved in purifying growth factors from living tissue. Are these factors proteins? Are they labile? Are they secreted? Are combinations of factors required? Are they membrane-bound or matrix associated? Neither the PI nor anyone else associated with this proposal has expertise in biochemical fractionation and protein purification. Dr. Yazaki, who will perform these experiments, has primarily been involved in live animal imaging. There appears to be direct overlap with the two NCI grants that the applicant currently has: the clinical oncology research career development program and the Developmental potential of human embryonic stem cells in vivo.
Again, the overall approach and aims, while interesting, are not highly significant or important at this stage of hESC research. Many of the aims such as those to attempt to grow ESCs or other cells from the tumor either in vitro or in vivo do not provide a clear mechanism to identify etiology or pathology behind these tumors. Studies proposed in the second aim use extracts from human fetal tissue to mediate differentiation of the hESCs in this type of tumor model seems very unusual; this reviewer is not persuaded that there is any compelling evidence such a strategy will lead to lineage-specific differentiation of the hESCs. To the reviewer's knowledge, there is no specific previous evidence in either mouse or human ESC models that co-culture of ESCs with particular tissue will lead to lineage-specific development.
One reviewer pointed out that a clearer significance and experimental design beyond development and characterization of tumors or tissues derived from hESCs in the SCID-HU model is needed, and that a clearer plan to translate these studies into more efficient lineage-specific differentiation of hESCs is necessary.
DISCUSSION: There was no discussion following the reviewers' comments.