Organoids from pituitary as a novel research model toward pituitary stem cell exploration

Abstract

The pituitary is the master endocrine gland, harboring stem cells of which the phenotype and role remain poorly characterized. Here, we established organoids from mouse pituitary with the aim to generate a novel research model to study pituitary stem cell biology. The organoids originated from the pituitary cells expressing the stem cell marker SOX2 were long-term expandable, displayed a stemness phenotype during expansive culture and showed specific hormonal differentiation ability, although limited, after subrenal transplantation. Application of the protocol to transgenically injured pituitary harboring an activated stem cell population, resulted in more numerous organoids. Intriguingly, these organoids presented with a cystic morphology, whereas the organoids from undamaged gland were predominantly dense and appeared more limited in expandability. Transcriptomic analysis revealed distinct epithelial phenotypes and showed that cystic organoids more resembled the pituitary phenotype, at least to an immature state, and displayed in vitro differentiation, although yet moderate. Organoid characterization further exposed facets of regulatory pathways of the putative stem cells of the pituitary and advanced new injury-activated markers. Taken together, we established a novel organoid research model revealing new insights into the identity and regulation of the putative pituitary stem cells. This organoid model may eventually lead to an interesting tool to decipher pituitary stem cell biology in both healthy and diseased gland.

Experimental Evidence and Clinical Implications of Pituitary Adenoma Stem Cells

“…More recently, in vitro 3D multicellular structures (organoids), which better recapitulate phenotype and functions of the original tissue (62), have been proposed as a new experimental way to investigate PSC biology and differentiation pathways upon injury-activated stimuli (63). Organoid cells, derived from normal and GH-depleted adult mouse pituitary, mainly express Sox2 and E-cadherin, as previously described for PSCs (21,32) and retain Sox2 after expansion in culture, showing limited differentiation capacity.…”

“…Organoid cells, derived from normal and GH-depleted adult mouse pituitary, mainly express Sox2 and E-cadherin, as previously described for PSCs (21,32) and retain Sox2 after expansion in culture, showing limited differentiation capacity. Organoids from cell cultures of injured GHCre/iDTR pituitary show cystic structure, low proliferative activity, immature pituitary phenotype and alterations in specific pathways (i.e., Wnt/Lgr) as compared to dense undamaged pituitary organoid models, thus representing a valuable tool to study the regulation of putative PSCs in both normal and activated conditions (63).…”

“…As discussed in the previous paragraph, this topic is very puzzling using traditional mouse models, and novel approaches, such as zebrafish embryo, can address only few aspects of this matter (i.e., neoangiogenesis and cell migration). Recently, development of 3D organoid systems (63), derived from Sox2-expressing normal mouse pituitary cells allowed to maintain long-term growth and stem-like phenotype during the expansive culture but also to the ability to differentiate into hormone-producing subpopulations. The application of this methodology to PAs may contribute to definition of the actual tumor-initiating subpopulation, the cellular and molecular pathways underlying tumor growth and the possible acquisition of invasive features, overcoming limitations observed using animal models.…”

Molecular Mechanisms of Pituitary Cell Plasticity

“…Identification of the distinct subpopulations within a specific hormone cell type has been further facilitated through use of teleost transgenic models that demonstrate the role of heterotypic network communication in mediating gonadotrope function and plasticity ( 33 ). The ongoing development of pituitary tissue and cell culture techniques and the in vitro growth of pituitary stem/progenitor cells as “organoids” is directed toward development of an experimental model that is being utilized to address mechanisms of pituitary function that occur on an intermediate timescale, between the short term of cell culture and the lifetime of animal models ( 34 – 36 ). Organoid model developments include the use of human induced pluripotent stem cells (iPSCs) and the co-differentiation of hypothalamic and pituitary tissues in patient-specific organoids ( 37 , 38 ).…”

Stem Cells, Self-Renewal, and Lineage Commitment in the Endocrine System

“…Using an alternative approach, Dincer et al were able to induce a placodal fate in adherent hESCs cultures, ultimately producing functional corticotrophs, that secreted ACTH after subcutaneous implantation in mice (94). Preliminary attempts at generating pituitary organoids from adult mouse PSCs have been performed, though to date, these have lacked the degree of self-organization, and functional hormone release achieved by their ESCs-derived counterparts (95). Interestingly, in all strategies ACTH-secreting cells are the predominant differentiated endocrine cell type produced.…”