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    • The pituitary gland is considered the master

    2018-10-20

    The pituitary gland is considered the master gland of hormone function. Hypopituitarism is a disease condition with insufficient or absent function of the pituitary gland. Pituitary tumors are the most common cause but many other triggers can induce pituitary dysfunction including inborn genetic defects, hmg-coa reductase trauma, immune and infectious diseases, or radiation therapy. The prevalence of hypopituitarism has been estimated at 46 per 100,000 (Regal et al., 2001), but this is likely an underestimation. The consequences of pituitary dysfunction are particularly serious in children where they can lead to severe learning disabilities, growth and skeletal problems, as well as effects on puberty and sexual function (Chemaitilly and Sklar, 2010). Chronic hypopituitarism requires lifelong complex hormone replacement therapies that are very costly and compromise quality of life. Furthermore, static delivery of hormones can only poorly mimic the dynamic secretion of the intact pituitary gland, which reacts to feedback mechanisms such as the hypothalamic-pituitary-adrenal (HPA) axis or the circadian clock. Therefore, there is a considerable clinical need to direct current treatment paradigms toward a more physiological and complete hormone replacement therapy (Smith, 2004). It is conceivable that replacing the damaged cells via cell transplantation can restore pituitary function and permanently cure chronic hypopituitarism. Previous work in mouse ESCs has shown that anterior pituitary cells, capable of hormone secretion, can be generated in 3D cultures by recapitulating some of the complex morphogenetic interaction between the developing hypothalamic and oral ectoderm tissues in vitro (Suga et al., 2011). Our laboratory has recently reported a first attempt at generating functional adenohypophyseal cells from human PSCs (Dincer et al., 2013), and very recently pituitary cells have been generated from hPSCs using a 3D organoid approach (Ozone et al., 2016). While these studies represent a promising proof of concept, current protocols remain inefficient, poorly defined, and unsuitable for developing current good manufacturing practice (cGMP)-compatible culture conditions that will be eventually required for human therapeutic use.
    Results
    Discussion A goal of our study was the development of a defined and highly efficient protocol to generate anterior pituitary lineages that obviates the need for co-culture or complex media formulations and that should be suitable for clinical-grade cell manufacturing. Using fluorescence-activated cell sorting (FACS)-purified SIX1::H2B-GFP+ placode cells, we demonstrate that pituitary fate can be induced at the expense of the default lens fate upon exposure to SHH, FGF8, and FGF10. Under these conditions, induction occurs in the absence of any hypothalamic cells previously thought to be critical for pituitary specification. While the signals essential for pituitary cell-fate induction from hPSCs do not require the presence of hypothalamic lineage cells, the interaction with the hypothalamic anlage is likely important for the complex tissue movements characteristic of pituitary gland development such as the formation of the Rathke\'s pouch. In future studies it may be interesting to combine directed differentiation and organoid culture techniques to study developmental tissue interactions and to retain 3D cytoarchitecture. However, our current strategy offers obvious advantages for cell manufacturing of highly defined, therapeutically relevant cell types at scale. The cGMP-ready PIP presented here has several major advantages over the previously published protocol (Dincer et al., 2013). It is well established that KSR can exhibit considerable lot-to-lot variability (Rao, 2008; Schwartz et al., 2011). The protocol presented here overcomes these challenges and yields improved overall differentiation efficiencies under conditions of minimal lot-to-lot variability based on testing >10 different lots of Essential8 for hiPSC maintenance and >5 different lots of Essential6 for differentiation.