Right Column

RL1-00670-1: Derivation and comparative analysis of human pluripotent ESCs, iPSCs and SSCs: Convergence to an embryonic phenotype

Recommendation: Recommended for funding
Scientific Score: 82

First Year Funds Requested: $470,245
Total Funds Requested: $1,410,042

Public Abstract (provided by applicant)

This is an unprecedented time in stem cell biology and regenerative medicine. Today, we have cell lines and tools that did not exist just a few years ago. Indeed, human embryonic stem cells (hESCs) were derived from pre-implantation embryos just 10 years ago; more recently in the past year, cells with extensive similarities to ESCs have been derived via genetic reprogramming of ordinary fetal and adult skin cells in both mice and humans. These induced-pluripotent stem cells (IPSCs) have been shown to have many properties similar to hESCs. Also recently, and surprisingly in mice, a new source of cells that does not require genetic manipulation has been identified, namely mouse spermatogonial stem cells (mSSCs). These cells also demonstrate extensive similarity to mouse ESCs. However, human SSCs (hSSCs) have not yet been reported though our preliminary data presented here lends credence to their derivation. Our goal is to derive hIPSCs and hSSCs – two pluripotent cell types – from the same men and compare key characteristics to those of hESCs. We suspect human pluripotent cell types derived from these three different sources may differ in key characteristics including their ability to contribute to both the germ cell (egg and sperm) and somatic lineages (endoderm, mesoderm and ectoderm) and thus may provide an optimal or ideal resource for unique basic developmental genetic, pre-clinical and/or clinical applications. Specifically, our aims are to: 1) Derive additional hSSC and hIPSC lines. 2) Compare hSSCs, hESCs and IPSCs in terms of critical molecular, genetic and developmental characteristics. 3) Incorporate well-characterized first-generation hSSCs and IPSCs into a human pluripotent stem cell bank for broad distribution to the scientific community. Traditionally, development was considered to be a progression towards the irreversible reduction of potential to form diverse cell lineages (with the notable exception of germ cell development). However, in light of recent results, this view has been permanently altered. This proposal seeks to take advantage of unique resources and tools to derive novel cell lines, probe the breadth of potential of hIPSCs, hSSCs and hESCs and optimize use of appropriate pluripotent cell types for basic, pre-clinical and clinical applications. Note: It is necessary that we compare isogenic hIPSC and hSSC pairs with low-passage hESCs, grown under the same conditions; thus, this work must use “non-federal” hESCs and is not fundable by federal mechanisms.

Statement of Benefit to California (provided by applicant)

Human embryonic stem cells are classically derived from human embryos that are not suitable for, or are in excess of, the reproductive needs of infertile men and women who present to assisted reproductive clinics. Evidence suggests that human embryonic stem cells can differentiate to many different cell types in the body and in fact, perhaps all the different cell types present in the adult. Thus, much excitement surrounds the possibility that the potential of human embryonic stem cells might be used to develop novel cell-based therapeutics to ease the tremendous burden of common, chronic disease and injury to the citizens of California. Many diseases and injuries, from birth, to childhood and adulthood, have a cellular basis and indeed may arise in the germ cells, the egg and sperm, or early embryo. A particular cell type, or process within a group of cells that form a tissue, may be specifically defective in disorders that range from diabetes to cardiac and neurodegenerative disorders as well as prevalent cancers. Nonetheless, the hope of novel cell-based therapies must be balanced by the realization that immunological rejection after transplantation will be an obvious hurdle unless we can make pluripotent cell lines that are compatible to individual genetic makeup. In this application, we propose to derive isogenic pairs of human pluripotent stem cells (human induced pluripotent stem cells and human spermatogonial stem cells) from the same men and characterize the potential of different cell types to contribute to both germ line and somatic lineages, relative to human embryonic stem cells. This research will benefit those in California by using our team's extensive experience and tools to produce high quality, well-characterized lines for banking and distribution widely throughout the scientific community. These cell lines constitute a genetic match for potential cell-based therapies, and also provide a system for the study of human genetic disorders and/or pharmacological properties. Moreover, given the controversial nature of human embryonic stem cells, this research provides a systematic approach to explore our alternatives alongside human embryonic stem cells in order for the stem cell research community to best serve the citizens of California.

Review

The goal for this proposal is to derive human induced pluripotent stem cells (iPSC) and compare key characteristics to those of human embryonic stem cells (hESC). Recently, pluripotent cells have been derived from mouse spermatogonial stem cells (mSSCs), via a mechanism that may entail either reprogramming of endogenous spermatogonia in culture or selection of a pre-existing pluripotent population. Like ESCs and iPSCs, SSC-derived pluripotent cells express characteristic ESC markers and differentiate readily to all three germ layers. The applicant describes three specific aims to achieve his/her objectives. In the first specific aim, the applicant proposes to create isogenic human stem cell lines from spermatogonia (SSCs) and from dermal fibroblasts by reprogramming (iPSCs) using the combination of four factors that has been previously used for the derivation of human stem cell lines. The applicant proposes to produce a minimum of three isogenic pairs of stem cell lines. In the second aim, key properties of these isogenic stem cell lines will be compared with each other and with those of hESC cells in assays that examine gene expression patterns, epigenetic stability and differentiation potential into three germ layers. In the third aim, well characterized hSSCs and iPSCs will be incorporated into the hESC bank at the applicant’s institution for broad distribution to the scientific community.

There was a general agreement among the reviewers that this is one of the best applications considered. The strength of this proposal lies both in the well conceived experimental plan and in the strong consortium of collaborators that has been assembled by the principal investigator (PI). There is little doubt, given the exceptional research facilities available to the applicant at the host institution, coupled with the quality of the preliminary data that the applicant will deliver on the stated aims of this project.

The first aim, the proposal to generate pluripotent stem cell lines from spermatogonia, employs a well conceived strategy. Although details of the culture conditions remain to be optimized, reviewers felt that, given the track record and experience of the PI, the research team should be able to solve these problems. Reviewers agreed that the possibility of generating hESC from blastocysts obtained from couples from whom the male partner has donated biopsies (testis, skin) offers a very unique opportunity to conduct a comparison of the properties of isogenic human SSC and iPSC and related ESC. Success in this endeavor would underscore the value of this project. However, there was some concern about the likelihood of obtaining such matched cells. In the second specific aim, the applicant proposes to carry out comparisons of gene expression, genetic and epigenetic stability and differentiation potential of hSSCs, hESCs and human iPSCs. Reviewers agree that the PI and his/her team have extensive experience in performing these standard assays. Similarly, the capacity to conduct the transplant assays to analyze the contribution of the three stem cell populations are well within the applicant’s sphere of expertise, although some of the details of the in vivo transplantation assays were not provided in the application. To further assess the comparability of these cell lines in differentiation to the desired stem cell derivatives for clinical applications, the PI plans to study neuronal differentiation in collaboration with an another investigator at the PI’s institution. Reviewers agreed that the proposed collaborator has extensive experience in these assays. The applicant plans to incorporate the newly derived stem cell lines, following characterization, into the host institution’s hESC bank for distribution to the broad scientific community.

In conclusion, the panel agreed that the generation of pluripotent SSCs and iPS cells from the same individual will allow a comparison of the key stem cell features of these lines in a manner uncomplicated by differences in genetic background. By comparing these lines with the properties of hES cells, the proposed studies will provide data that represent a key step forward in defining objective criteria to evaluate the most appropriate stem cell type for a given therapeutic application. Because of these reasons, overall enthusiasm for this proposal was high.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None