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.
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.
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.
This project is directed toward the ultimate objective of providing optimal human stem cell candidates for application to the study of human developmental genetics and as the potential basis for the development via translational/preclinical studies of novel cellular therapies for regenerative medicine applications. In the first of three specific aims, the applicant proposes to create isogenic human stem cell lines from spermatogonia (SSCs) and from dermal fibroblasts by reprogramming (IPSCs) using the combination of 4 factors identified by the Thomson lab. 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 the germ cell lineage and into endoderm, mesoderm and ectoderm. In the third aim, well characterized hSSCs and IPSCs will be incorporated into the Stanford hESC bank for distribution for broad distribution to the scientific community.
Reviewer One Comments
This is a project to derive and compare isogenic human iPS, SSC, and also ES. It will surely be important to better understand the similarity and difference of various stem cell populations. The ability to compare isogenic cells is appealing. The ultimate goal of this particular proposal was not very well stated. The PI, Dr. Reijo Pera is an expert in hES derivation and germ cell differentation, so the rationale is to consider which cell sources will provide the optimal source for germ cell development. Overall this is a clearly important goal.
The first Aim is to generate human SSC and iPS lines. Male donors will be recruited and asked to donate skin biopsies. Since the PI has good access to donors, presumably such willing men will be identified. Efforts are currently underway to optimize the SSC culture. The PI has a proven track record at generating hES lines, so there is confidence that additional lines will be derived. It was not clear why the PI pursues SSC rather than to derive ES-like colonies from SSC, as has been achieved by different groups in mouse. This lack of explanation made the proposal difficult to follow.
Experiments in Aim 2 will compare the SSC, ES, and iPS lines. While the ES lines will not be isogenic, the PI will try to recruit couples from whom the men have donated biopsies to provide embryos, although this would seem to be problematic. 3 lines each will be evaluated in a variety of assays including epigenetic status of key reprogramming genes, imprinted loci, ability to form germ cells in response to BMPs, in vitro differentiation and ability to from hematopoietic or neural cell types. Some of these comparisons seem rather arbitrary, but are presumably driven by the research interests of the PI and her colleagues. There is substantial expertise available for analysis and the comparisons are likely to generate interesting information.
The third Aim is to bank and distribute the lines.
Responsiveness to RFA:
The proposal will result in the generation of new hES lines and so is responsive. It is not yet clear if SSC lines will be established nor if they will generate pluripotent resources. The PI is an expert in ES derivation and is already generating lines. She is PI already on funded CIRM grants for facilities, training, and research projects (app. 10 million total costs), 2 R01 grants, and others, in addition to serving as Director of the Stanford Center for ES Research. Overall, this is a feasible project by an outstanding investigator, although the rationale and significance could have been more clearly stated particularly in terms of priorities and focus.
Reviewer Two Comments
This is an important study which will provide information that is both unique and valuable. The generation of pluripotent SSC and iPS cells from the same individual will allow for the first time 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 toward the end of defining objective criteria to allow informed choice as to the most appropriate stem cell type for a given therapeutic application.
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 PI Dr Reijo Pera. In addition to the PI who is a well established, highly respected contributor to the ES research field, the collaborators include highly respected and productive researchers of the caliber of Drs Chang, Turek, Palmer and Weissman. It is hard to conceive of a more powerful combination of expertise and experience being applied to a research project of the nature and scope encompassed by the current application. There is little doubt, given the exceptional research facilities available to the applicant at Stanford coupled with the quality of the preliminary data that the applicant will deliver on the stated aims of this project.
• Under Aim 1, the proposal to generate pluripotent stem cell lines from spermatogonia is a well conceived strategy that puts the applicant in a competitive position. It is a strategy very much dependent upon the collaboration with Dr Turek. The applicant notes the current uncertainty surrounding the choice of the ideal feeder for the propagation of SSCs (mefs vs CD34+ TSFs). The research team is nevertheless well positioned to employ well considered alternative strategies if and when required.
• Similar uncertainties surround the choice of the optimal protocol to reprogram somatic cells to a pluripotent fate. The applicant has chosen to proceed with the combination of 4 transcription factors identified by the Thomson lab which avoids the use of c-myc. This again appears a sound choice. In terms of feasibility, there seems little doubt that the applicant will succeed, as have numerous other researchers using the Yamanaka or Thomson protocols, in generating iPSC cells. The current state of the art is rapidly changing but the collective experience of the collaborators involved in this project will allow them to make well informed choices as to modifications of the reprogramming protocol if necessary.
• The possibility of generating hESC from blastocysts 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 human SSC, IPSC and ESC on an isogenic background. Success in this endeavor would truly underscore the value of this project and it would be useful to know the likelihood of deriving such isogenic lines. Even if hESC lines cannot be made from this source this will not substantively detract from the quality or importance of the data generated through Aim 2.
• Under Aim 2, the comparison of gene expression patterns and epigenetic stability will likely yield novel data comparing the three categories of pluripotent cells. The applicant has a strong track record in this area and the capacity to conduct the proposed analyses is unquestioned. 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 and promise to generate novel comparative data.
• The assays which are to be employed to compare the mesendodermal differentiation of each stem cell type will make use of commercial antibodies and a panel of novel reagents developed in the Weissman lab in combination with RT-PCR analysis to examine the differentiation status of candidate progenitors cells for the mesodermal and endodermal lineages. Transplant assays are to be performed “as described for hematopoietic stem cells” to confirm the fate of the differentiated mesendodermal progenitors. These are critical since phenotype of cells in culture does not necessarily predict in vivo function. However the feasibility of these assays cannot be evaluated since details are not provided.
• Neuronal differentiation will be assessed in collaboration with the Palmer lab which has extensive experience in these assays.
Responsiveness to RFA:
This proposal will almost certainly yield pluripotent stem cell lines and is thus highly responsive to the RFA.
The intention of the applicant is to incorporate the newly derived stem cell lines, following extensive characterization, into the Stanford hESC bank for distribution to the broad scientific community. Dr Reijo Pera has considerable experience in distribution of NIH-approved hESCs through her role as PI on an NIH R24 national hESC distribution center and distribution of the newly developed lines will be managed through a web based resource developed at Stanford for this purpose.
Reviewer Three Comments
This proposal calls for the derivation of new pluripotent stem cell lines and comparison of self-renewal and differentiation properties between the new hESC, iPSC, and SSC (spermatogonial stem cells) derived lines. An interesting and innovative goal is to derive isogenic iPSC and SSC, and then compare with other hESC (no derivation of new hESC is included in this proposal, but will use early passage non-federal lines). They intend to assess whether hESC, iPSC, and SSC are different in pluripotency, differentiation, etc.
They will analyze extensive molecular, genetic, and developmental characteristics of the new lines, and then provide these well-characterized cell lines to the broader scientific community.
Design and Feasibility
The concept of having genetically matched iPSC and SSC is a novel approach, one that should facilitate a better understanding of the extent to which genetics may play a role in pluripotency and differentiation capacity, disease susceptibility, etc. They propose that hESC may be more like primordial germ cells, and hypothesize that iPS and SSC undergo a similar program (to hESC) of widespread destruction of mRNA, genome-wide demethylation, and nuclear activation during the reprogramming process. However, knowledge in this area is limited due to:
- Lack of SSC
- Diverse genetic background of hESC, iPSC, SSC
- Incomplete characterization of differentiation potential
This proposal will address at least some of these limitations. The plan is to derive a minimum of 3 “matched” SSC and iPS lines, and then perform extensive assays comparing the lines. This will include the following:
- Flow-based expression patterns (200 hybridoma panel screening)
- Transplantation studies
- PCR, also at single cell level
- Neuronal differentation
Of interest, they want to assess germ line potential, which is a relatively novel concept that has not currently been reported. They will evaluate epigenetic and imprinting status. All of the lines will be incorporated into the Stanford Pluripotent stem cell bank for distribution to other investigators.
The PI is well-funded, with specific topics of relevance to this proposal:
- Genetic analysis of germ cell differentiation
- Genetic epidemiology of ovarian aging.
- Differentiation of germ cells form hESC
- Human oocyte development (CIRM)
- Stanford training grant (CIRM)
- Stanford hESC Research (CIRM)
- Human oocyte development (Tobacco)
- Analysis of human imprinting (March of Dimes)
- Lots of relevant experience for PI and Co-investigators
- Good preliminary data
- Novel studies regarding germ cell potential
- Genetically matched iPSC and SSC
- Experience with banking and distribution of cells for research
This is an achievable amount of work in the proposed timeframe of this RFA, but there is still a considerable amount of work included in the proposal. As such, the PI is only listed for 10% effort, which appears to be insufficient. Other co-Investigators are only listed for 5%, 2%, 5%, 5%, 2%, giving the appearance that they were simply included on the application to impress the reviewers.
Responsiveness to RFA:
Responsiveness to RFA
This proposal is clearly responsive to some aspects of the RFA, scientifically for research studies. It is not likely that any of the new lines would be qualified for clinical use. SSC will obviously come only from male donors, while attempts will be made to also collect from embryo donors.
Reviewer Four Comments
Significance, feasibility and Strengths:
- PI and collaborators have extensive experience in the stem cell field as well as germ cell development
- Simple and straightforward aims while at the same time unique. No other group has hSSCs.
- Impressive preliminary data.
- Derivation of hSSCs and iPS cells from the same individual.
- PI will, among many other endpoints, compare the differentiation capabilities of the cells obtained. While endoderm, mesoderm and ectoderm derivatives can provide valuable information, PI is not proposing to make teratomas and compare the differentiation potential of the cells. This is likely due to a lack of teratoma formation when hSSC are used.
- Aim 3 is not really an aim but a requirement from CIRM.
Responsiveness to RFA: