1. Establishment of Pluripotency During Development
The transcription factor Cdx2 plays an essential role in the early stages of TE formation, as lack of zygotic Cdx2 transcripts causes hatching and implantation defects. We examine the impact of a defective TE, leading to Cdx2 depletion, on ICM pluripotency by evaluating the efficiency of the ICM to give rise to pluripotent embryonic stem cells (ESCs).
2. Pluripotency in Embryonic Stem Cells and Epiblast Stem Cells
Human ESCs (hESCs), like mouse ESCs (mESCs), can be derived from the ICM of preimplantation embryos. Both hESCs and mESCs share a number of cellular characteristics, such as pluripotent phenotype and self-renewal ability. Oct4, Nanog, and Sox2 are important in both human and mouse ESCs, as silencing of their gene expression results in cellular differentiation.
However, hESCs and mESCs exhibit markedly different responses to signaling pathways that support self-renewal. For example, mESCs require LIF/STAT3 signaling for self-renewal, whereas hESCs do not respond to LIF.
Epiblast stem cells (EpiSCs), which are derived from E5.5 or E6.5 mouse epiblasts, also express Oct4, Nanog, and Sox2 and exhibit features of pluripotency. EpiSCs are similar to hESCs in morphology and can be derived and maintained under conditions that support hESC self-renewal. hESCs share several molecular features with mESCs, but not with EpiSCs, including the expression of ICM/ESC marker genes (e.g., Rex1, Klf4) and lack of epiblast-specific gene expression (Fgf5). We are conducting comparative studies in hESCs, mEpiSCs, and mESCs to clarify the developmental status of human ESCs.
ESCs and EpiSCs differ in the regulation of Oct4. Although Oct4 is similarly expressed in both cells, the Oct4 promoter distal enhancer (DE) region exhibits higher activity in ESCs, whereas the proximal enhancer (PE) exhibits higher activity in EpiSCs. Oct4 promoter elements are differentially regulated during embryonic development, with DE active in ICM and primordial germ cells (PGCs) and the PE active in epiblasts. The basis for this "regulatory switch" is unknown.
ESCs and EpiSCs are considered to represent defined states of pluripotency. These cellular states can be interconverted by changing the medium conditions. During conversion, each cell line/state loses its distinct colony morphology and acquires the growth characteristics and signaling dependence as well as the Oct4 gene expression regulation of other cell state. We use Oct4 to define the regulatory networks in these two cell lines and to enhance our understanding of the biology of the switch during gastrulation.
3. Reprogramming to Pluripotency
Induction of pluripotency in multipotent neural stem cells
Induced pluripotent stem cells (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4. Our group subsequently demonstrated that Oct4 and Klf4, or c-Myc, or even Oct4 alone is sufficient to induce reprogramming of adult mouse neural stem cells to iPS cells—termed one-factor iPS cells (1F iPS cells). We further demonstrated the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor human NiPS cells, 1F hNiPS) by ectopic expression of Oct4 alone. These cells resemble hESCs in global gene expression profile, epigenetic status, as well as pluripotency both in-vitro and in-vivo. These findings demonstrate that Oct4 is sufficient to induce reprogramming of human neural stem cells to a cellular state of pluripotency. We are using the NSC system to obtain a better understanding of processes that occur during reprogramming.
Reprogramming of unipotent germline stem cells into pluripotent stem cells
We have recently developed a pluripotency induction culture system wherein unipotent male germline stem cells (GSCs) can give rise to pluripotent cells, termed germline-derived pluripotent stem (gPS) cells. The specific microenvironment in GSC culture, defined partly by the number of GSCs initially plated, determines successful conversion. This mouse model is expected to help elucidate the mechanisms underlying the reprogramming of unipotent cells into pluripotent cells as well as those underlying development of germline-related tumors, foreshadowing future derivation of human GSCs and their conversion into gPS cells. We are using the GSC system to obtain a better understanding of processes that occur during reprogramming.
Understanding the mechanisms of cellular reprogramming
The generation of iPS cells typically takes weeks and occurs with relatively low efficiency. The fusion of pluripotent ESCs with differentiated somatic cells also results in reprogramming. Although cell fusion results in hybrid cells with a tetraploid genome, which may hinder clinical applicability, this fast and relatively efficient reprogramming method presents a nice model for studying the reprogramming mechanisms.
Chromatin remodeling in cellular reprogramming
The varying efficiencies associated with different reprogramming methods suggest the presence of additional or alternate reprogramming factors that may reduce reprogramming duration and that may achieve efficiency equivalent to that of somatic cell nuclear transfer or fusion methods. Cell extract−based methods of reprogramming provide an opportunity to identify additional or alternate reprogramming factors with biochemical methods.
4. Derivation of Germ Cells from Pluripotent Stem Cells
In-vitro derivation and maturation of oocytes from pluripotent murine stem cells
An important achievement in developmental biology is the generation of a robust and reproducible in-vitro culture system that supports the induction and differentiation of a homogeneous population of functionally specific cells from totipotent or pluripotent stem cells. Our laboratory first demonstrated that mouse ESCs could differentiate along the female germline. We are currently optimizing our in-vitro system to study oocyte maturation and to derive fully functional oocytes. We expect to gain important insights into controlling germ cell development in a reproducible manner, thereby supporting the generation of a vast number of germ cells of a specific maturation state that are of acceptable quality for further use, such as the generation of oocytes for use in nuclear transfer techniques.
In-vitro spermatogenesis using established GSCs
Spermatogonial stem cells (SSCs) are male germline stem cells located at the basement membrane of seminiferous tubules in the testis. They can either self-renew to maintain the stem cell pool or differentiate into mature spermatogenic cells in a process called spermatogenesis. Recent studies have demonstrated the generation of haploid cells from testicular cells or ECSs. However, the generation of functional haploid cells from established undifferentiated GSC lines has not yet been demonstrated. Our laboratory has established GSC lines from adult mouse testis that can be maintained in long-term culture. Most importantly, these GSCs are fully functional, as they restore spermatogenesis upon transplantation into infertile mouse testis. Our in-vitro model is expected to broaden our understanding of the mechanisms underlying spermatogenesis and provide a valuable tool for potential clinical applications in the treatment of male infertility.
5. Disease modeling using pluripotent stem cells
To date, almost all preclinical research on human diseases has been carried out in animals. However, many diseases either cannot be modeled in animals or display significant differences when compared to human patients. Stem cells offer a unique opportunity to study diseases in-vitro. Pluripotent stem cells have the broadest potential and can be differentiated into all adult lineages, including those affected by disease. The recent discovery of human iPS cells has created the technology to derive pluripotent stem cell lines from patients with known pathophysiologies. Stem cells offer a virtually limitless source of specialized cells that all have known disease pathologies. This research enables the generation of detailed mechanistic studies as well as the development of new drugs that target specific phenotypes and novel pathways and that are more likely to be effective in the clinic.