Reprogramming the Cell | Part 2: Road to a unipotent adult cell (Programming Cell)

Making of a unipotent adult cell is a complex journey beginning from fertilization of oocyte by a sperm and subsequent change in genetic information within the cell in a 'programmed' manner which allow totipotent zygote to differentiate into the whole human body. A 'programmed' manner involves series of changes involving initial transcriptional repression by means of RNA polymerase inhibition and Chromatin-based modification , in association with important epigenetic mechanisms which are regulated by various signaling pathways and other factors from surrounding cells (Niche). Such programmed mechanisms are critical for 1.) Generating the father of the stem cell known as primordial germ cell (PGC) from the zygote, 2.) Making ESC / ASC and Maintaining their fundamental properties - pluripotency and self-renewal, 3.) Transforming ESC into various adult cell types (somatic differentiation) and importantly 4.) Preserving and Propagating the master code of inheritance in future generations. With the advancement in molecular analytical methods, scientists now have ability to see behind the scene of this enigmatic cell program, trying to identify the key elements for possible therapeutic application(s) using reverse principle known as Reprogramming the Cell.

Formation of Germ Line Cells:
The most fundamental event in generation of a living being is formation of the germ line cells from the inner cell mass of an embryo which parallels conversion of totipotent cells of zygote into pluripotent germ line cells. The first generation of germ line cells are known as Primordial Germ Cells (PGC) which are not the actual stem cells as they do divide symmetrically (by mitosis) but do not have property of pluripotency. However, as PGC keeps on dividing, their subsequent daughter cells acquire ability of pluripotency under influence from transcriptional and epigenetic mechanisms. Such daughter cells are called Germline Stem Cells (GSC) which are actual embryonic stem cells (ESC) that undergo somatic differentiation to shape human body. Some PGCs are believed to migrate to developing gonads where they go under extensive genetic reprogramming (including reactivation of previously inactivated X-chromosome (Xi) in female PGCs) which is crucial for inheriting the original genome (and corresponding phenotype) into subsequent lineage.

Mechanisms underlying formation of PGCs:
Till date, there are very few experimental evidences of stem cell programming available from human embryo. Researchers have documented various signaling and genetic mechanisms underlying pluripotency using mouse models mainly. Key factors in entire germ line synthesis, subsequent pluripotency and inheritance are believed to inherit mainly from maternal genome and only some information from paternal side (DNA methylation, see below). Germ cells are thought to be formed well before somatic cell differentiation. Germ cells are formed from specialized pluripotent primitive ectoderm (PEct) of the inner cell mass (ICM) which usually separates from outer cell mass (OCM) between 8-cell and 16-cell stage of balstomeres. Two different theories exist for development of PGC: 1. ) Preformed theory which is seen in Drosophila, Caenorhabditis elegans, Xenopus, and zebrafish, suggests asymmetric division of maternal cytoplasm during oogenesis and/or fertilization to redirect few cells of zygote into germ line pool. 2.) Induction theory which is widely noted in mouse model, depicts synthesis of PGCs from their pluripotent precursors under influence of extra-embryonic ectodermic signals (i.e. Blimp1 expression and Hox gene downregulation, see below).

Somatic cell line repression and Pluripotency power:
Regardless of PGC development theory, earliest change is suppression of somatic cell line differentiation which is managed primarily by maternal genetic and epigenetic factors. Various mechanisms exist underlying repression of somatic cell line. Following are highlights from currently available evidences drawn from experimental animal studies:

• Transcriptional Repression: By Inhibition of RNA Polymerase II activity specific to somatic cell differentiation under direct effect of a maternally inherited germ plasm component PIE-1, germ cell less (gcl) and polar granule component (pgc) in Drosophila and C. elegans embryos
• Blimp1 (epigenetic modifier of ESC origin, function probably as a histone methyl transferase) expression is the starter event which precedes Hox gene downregulation. Together these two mechanisms are critical for development of pluripotent precursors from which PGCs arise. Cells that express Blimp1 do not express Hox gene but maintain expression of other transcripts, including Oct4, Nanog, and Sox2 - genes for pluripotency. Mutation of each of these genes cause defects in the establishment of pluripotency in vivo, and, conversely, overexpression of these genes can convert differentiated cells into pluripotent stem cells in vitro (Silva et al., 2006; Takahashi and Yamanaka, 2006)
• Chromatin-based epigenetic modification factors (i.e. Polycomb group (PcG) proteins such as Ezh2 and Eed and others) bring change in histone sturcture - losing H3-K9 dimethylation and DNA methylation as well as increase in H3-K27 trimethylation. These changes may be essential to erase parental imprints and to eventually activate the expression of differentiation genes for somatic cell line.

Following are unsolved puzzles in regard to Transcriptional and Epigenetic Modification of PGC:
1. ? Signals / Factors deciding loss of totipotency and switching certain cells to pluripotent germ cells
2. ? Factors helping GSC to regain totipotent state
3. ? Although PGC and GSC have Oct4 and other pluripotency genes, why are PGCs not pluripotent and their subsequent germ line generations have pluripotent ability?
4. ? Which factors make adult stem cell to loss its pluripotent ability?

Reference: (in-depth detail about Pathways of Pluripotency / Early Epigenetic Reprogramming / Loss of Totipotency and related topics)
1. Seydoux G,Braun RE | Pathway to Totipotency: Lessons from Germ Cells | Cell 2006(Dec 1);127:891-904
2. Surani NA,Hayashi K,Hajkova P | Genetic and Epigenetic Regulators of Pluripotency | Cell 2007(Feb 23);128:747-762
3. Hansis C | Totipotency, cell differentiation and reprogramming in humans | Reproductive BioMedicine Online 2006;13:551-557

Related posts: Reprogramming the Cell | Part 1: Basics of Stem Cell
To be continued.....