Derivation and comparative analysis of human pluripotent ESCs, iPSCs and SSCs: Convergence to an embryonic phenotype
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 specific aims of the funded proposal # RL1-00670 entitled “Derivation and comparative analysis of human pluripotent hESCs, hIPSCs and hSSCs: Convergence to an embryonic phenotype” are to: 1) Derive additional hSSC (human spermatogonial stem cell) and hIPSC (human induced pluripotent stem cell) lines. 2) Compare hSSCs, hESCs and hIPSCs in terms of critical molecular, genetic and developmental characteristics. 3) Incorporate well-characterized first-generation hSSCs and IPSCs into a Stanford human pluripotent stem cell bank for broad distribution to the scientific community. We have made substantial progress over the last year with the complete characterization of one hSSC line, generation of multiple iPSCs and comparisons to hESCs in terms of critical molecular, genetic and developmental properties. We have also established the Stanford RENEW BioBank where along with a collection of properly consented oocytes and embryos, we have established a reservoir of primary somatic cell lines and pluripotent stem cell lines for distribution. Several manuscripts have resulted from our progress in the last year (2 published, 2 submitted for publication and 1 in the final stages of preparation). In particular, we have generated data regarding the use of human SSCs for regenerative medicine and/or fertility preservation, the use of an external cell surface marker called SSEA3 to isolate a population of ordinary somatic cells (from skin biopsies) that are preferentially-able to be genetically reprogrammed and we have derived several unique iPSC lines that suggest they may be particularly useful for pharmacological and toxicological studies of Parkinson’s Disease (PD). We have found that although many characteristics of hESCs, hSSCs, and hiPSCs are similar, there are some fundamental differences that predict that different cell types will be more useful for particular applications than others.
During the last funding period, we have made substantial progress on our goals to derive multiple human pluripotent stem cell lines, to characterize their ability to differentiate to diverse cell types and to bank lines for distribution to other researchers. We have found that human spermatogonial stem cell lines differentiate to different cell types in the dish (in vitro) but have limited differentiation abilities in vivo in transplantation to murine models. In contrast, both human embryonic stem cell lines and induced pluripotent stem cell lines show ability to differentiate to diverse somatic lineages and the germ line both in vitro and in vivo upon transplantation.
We have exceeded our goals for derivation of lines and characterization of their properties in vitro. In the next year, we will finalize characterization in order to deposit all lines, in a very-well characterized form into a central bank for distribution broadly in California and to other researchers, as well.
The specific aims of the funded proposal # RL1-00670 entitled “Derivation and comparative analysis of human pluripotent hESCs, hIPSCs and hSSCs: Convergence to an embryonic phenotype” as proposed were to: 1) Derive additional hSSC (human spermatogonial stem cell) and hIPSC (human induced pluripotent stem cell) lines. 2) Compare hSSCs, hESCs and hIPSCs in terms of critical molecular, genetic and developmental characteristics. 3) Incorporate well-characterized first-generation hSSCs and IPSCs into a Stanford human pluripotent stem cell bank for broad distribution to the scientific community. Over the entire funding period, we have made substantial progress and have continued to meet or exceed our goals in many areas. However, we have also met a couple challenges that we are now addressing especially in of final characterization of lines and in banking for distribution of lines quite broadly. Our goal is to decisively address issues regarding the definition of hSSCs and pluripotency; we note that mouse SSCs clearly are pluripotent in every sense and we likely have to adjust conditions further to accomplish full pluripotency with hSSC lines. We expect that a comparison of our hSSC lines, with the hiPSCs derived from them will allow adjustments to be made, as necessary. However, we continue to observe as we also initially reported that hSSC lines do not readily form (they are "null" for) teratomas in vivo. We note that we have had several publications that indicate our progress as follows (asterisks indicate new submissions):
1. Byrne JA, Nguyen HN, Reijo Pera RA (2009) Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts. PLoS One 4, e7118. PMC2744017
2. *Panula S, Medrano JV, Kee K, Bergstrom R, Nguyen HN, Byers B, Wilson KD, Wu JC, Simon C, Hovatta O, Reijo Pera RA (2010) Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells. Hum Mol Genet 20:752-62.
3. *HN Nguyen, Byers B, Cord B, Shcheglovitov A, Byrne J, Gujar P, Kee K, Schuele B, Dolmetsch RE, Langston W, Palmer TD, Reijo Pera RA (2011) LRRK2 mutant iPSC-Derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell Stem Cell 8, 1–14.
4. *B Byers, B Cord, HN Nguyen, B Schule, J Byrne, JW Langston, RA Reijo Pera, T Palmer (2011) Emergence of an early disease phenotype from Parkinson’s disease induced pluripotent stem cells. PLoS One (in press).
5. JV Medrano, C Ramathal, HN Nguyen, C Simon, RA Reijo Pera (submitted) Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro from both hESCs and iPSCs. Stem Cells.
As also noted in the last report, we have generated intellectual property on: 1) use of human SSCs for regenerative medicine and/or fertility preservation, 2) use of SSEA3 to isolate a preferentially-reprogrammable somatic cell sub-population, and 3) derivation of unique iPSC lines from patients with genetically-defined Parkinson’s Disease (PD) for pharmacological and toxicological studies. Overall progress will continue with the final establishment of banked lines that are able to be broadly distributed.
This is the final report for the proposal with aims to: 1) Derive human spermatogonial stem cell (hSSC) and hIPSC (human induced pluripotent stem cell) lines from the same individual. 2) Compare hSSCs, hIPSCs and hESCs in terms of critical molecular, genetic and developmental characteristics. 3) Incorporate well-characterized cell lines into a Stanford human pluripotent stem cell bank for broad distribution to the scientific community. We have completed these aims with production of lines, publication of key findings in scientific journals and leveraging of research to obtain non-CIRM funds to perpetuate the research and maintain staff. We developed key assays that allow assessment of the total potential of different cell lines for disease modeling and understanding of human development. Efforts are underway to bring findings to bear on clinical problems in reproductive and general health. We anticipate that results will allow us to especially elucidate and address problems of male infertility in cases of germ cell (spermatogenic) loss in the face of cancer, autoimmune disorders and/or injury.
- PLoS One (2011) SNCA Triplication Parkinson's Patient's iPSC-derived DA Neurons Accumulate alpha-Synuclein and Are Susceptible to Oxidative Stress. (PubMed: 22110584)
- Stem Cells (2011) Divergent RNA-Binding Proteins, DAZL and VASA, Induce Meiotic Progression in Human Germ Cells Derived In vitro. (PubMed: 22162380)
- Hum Mol Genet (2011) Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells. (PubMed: 21131292)
- Cell Stem Cell (2011) LRRK2 Mutant iPSC-Derived DA Neurons Demonstrate Increased Susceptibility to Oxidative Stress. (PubMed: 21362567)
- Stem Cells (2009) Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. (PubMed: 18927477)
- PLoS One (2009) Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts. (PubMed: 19774082)