Decoding the activated stem cell phenotype of the vividly maturing neonatal pituitary

The pituitary represents the endocrine master regulator. In mouse, the gland undergoes vivid maturation immediately after birth. Here, we in detail portrayed the stem cell compartment of neonatal pituitary. Single-cell RNA-sequencing pictured an active gland, revealing proliferative stem as well as hormonal (progenitor) cell populations. The stem cell pool displayed a hybrid epithelial/mesenchymal phenotype, characteristic of development-involved tissue stem cells. Organoid culturing recapitulated the stem cells’ phenotype, interestingly also reproducing their paracrine activity. The pituitary stem cell-activating interleukin-6 (IL-6) advanced organoid growth, although the neonatal stem cell compartment was not visibly affected in IL-6-/- mice, likely due to cytokine family redundancy. Further transcriptomic analysis exposed a pronounced WNT pathway in the neonatal gland, shown to be involved in stem cell activation and to overlap with the (fetal) human pituitary transcriptome. Following local damage, the neonatal gland efficiently regenerates, despite absence of additional stem cell proliferation, or upregulated IL-6 or WNT expression, all in line with the already high stem cell activation status, thereby exposing striking differences with adult pituitary. Together, our study decodes the stem cell compartment of neonatal pituitary, exposing an activated state in the vividly maturing gland. Understanding stem cell activation is key to potential pituitary regenerative prospects.


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As central hub of the endocrine system, the pituitary plays a quintessential role in governing the 44 endocrine glands throughout the body, thereby regulating key physiological processes including 45 growth, metabolism, fertility, stress and immunity. To execute this primordial function, the 46 pituitary has to properly develop into a compound gland of multiple endocrine cells, 47 encompassing somatotropes (producing growth hormone (GH)), corticotropes 48 (adrenocorticotropic hormone (ACTH)), lactotropes (prolactin (PRL)), gonadotropes (luteinizing 49 hormone (LH) and/or follicle-stimulating hormone (FSH)) and thyrotropes (thyroid-stimulating 50 hormone (TSH)) (Melmed, 2011;Willems et al., 2014). In the mouse, the pituitary undergoes an 51 intense growth and maturation process during the first postnatal weeks, with number and size of 52 hormone-producing cells substantively expanding following proliferation of committed 53 (progenitor) and endocrine cells (Carbajo-Pérez et al., 1990;Laporte et al., 2021;Sasaki, 1988; 54 Taniguchi et al., 2002;Zhu et al., 2015). Simultaneously, the local stem cell (SOX2 + ) compartment 55 shows signs of activation including elevated abundance and expression of stemness pathways 56 when compared to the adult pituitary stem cells (Gremeaux et al., 2012;Laporte et al., 2021). The 57 stem cells can give rise to new endocrine cells, a property which has been found most prominent 58 (although not extensive) during this neonatal period (Andoniadou et al., 2013;Rizzoti et al., 2013; 59 Zhu et al., 2015). In contrast, stem cells in the mature, adult pituitary are quiescent and do not 60 highly contribute to new endocrine cells during the (slow) homeostatic turnover of the gland 61 (Andoniadou et al., 2013;Laporte et al., 2021;Rizzoti et al., 2013;Vankelecom et al., 2014).

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However, the adult stem cells become activated following local injury in the gland, showing a 63 proliferative reaction and signs of differentiation toward the ablated cells, coinciding with 64 substantial regeneration (Fu et al., 2012). We recently identified interleukin-6 (IL-6) to be 6 enrichment of cell cycle processes in the Prolif SC versus the aggregate SC1 and SC2 clusters

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Interestingly, proliferative subclusters were also perceived for other cell types, moreover 114 exclusively in the neonatal gland. First, we discerned a proliferative Pit1/Pou1f1 + cell group. Pit1 115 represents the transcriptional regulator of hormone expression in somatotropes, lactotropes and 116 thyrotropes, and its expression also marks differentiation of progenitors within this so-called Pit1 + 117 lineage (Zhu et al., 2015). In addition, we distinguished a proliferative corticotrope subcluster.

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Both findings were corroborated by DEG/GO and hormone/Ki67 immunostaining analyses ( Figure   119 1 -figure supplement 1F; Supplementary file 1B,C and 2B,C). Strikingly, the neonatal AP also 120 contains gonadotrope progenitor and precursor subclusters as characterized by gradually 121 increasing gonadotrope markers (Nr5a1,Cga,Lhb,Fshb,Gnrhr) in parallel with declining stem cell 122 factors (Sox2, Krt8, Sox9) ( Figure 1A and B -figure supplement 1B). The gonadotrope lineage 123 arises last during embryonic development just before birth (Stallings et al., 2016), and some cells 124 in the neonatal AP indeed co-express SOX2 and the common gonadotropin subunit GSU ( Figure   125 1 -figure supplement 1G). In further support of an endocrine progenitor phenotype (as opposed 126 to a stem cell state), DEG analysis revealed upregulation of genes involved in hormone production 127 (e.g. Chga, Ascl1, Cga, Insm1) and enrichment of GO terms related to endocrine differentiation  140 Surprisingly, the stem cell compartment of the neonatal AP displays a highly mixed 141 epithelial/mesenchymal (E/M) phenotype, manifestly expressing both epithelial and 142 mesenchymal markers, which is clearly faded in the adult gland ( Figure 1C). In line, cells that co-7 express the epithelial markers cytokeratin 8 and 18 (CK8/18) and the mesenchymal marker 144 vimentin (VIM) were found in the marginal-zone (MZ) SOX2 + stem cell niche of the neonatal 145 pituitary, and were visibly less prominent in the adult gland ( Figure 1C). Similarly, co-expression 146 of SOX2 with VIM was more pronounced in the neonatal gland ( Figure 1C). In other developing 147 tissues, stem/progenitor cells also show a hybrid E/M character, indicative of their activation and 148 participation in the tissues' developmental process (Dong et al., 2018). Recently, a hybrid E/M 149 phenotype was also uncovered in the stem/progenitor cell cluster of the fetal human pituitary, 150 with the mesenchymal aspect lowering along further maturation (Zhang et al., 2020). Moreover, 151 epithelial-to-mesenchymal transition (EMT) has been reported to play a role in mouse pituitary 152 development through driving the exit of stem cells from the MZ toward the developing AP (Davis 153 et al., 2016;Yoshida et al., 2016

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The organoids, as before shown for adult AP (B. Cox et al., 2019), originated from the neonatal 180 tissue-resident SOX2 + stem cells. Seeding AP from neonatal SOX2 eGFP/+ reporter mice (expressing 181 enhanced green fluorescent protein (eGFP) in SOX2 + cells) generated only organoids that were

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Very recently, it has been reported that stem cells in early-postnatal (PD14) pituitary can function 199 as autocrine and paracrine signaling center, among others stimulating proliferation within the 200 own stem cell compartment (Russell et al., 2021). Interestingly, when co-cultured, neonatal (PD7) 201 AP organoids (as developed from WT mice) elevate the outgrowth of organoids from adult AP (as 202 established from ubiquitously tdTomato(tdT)-expressing R26 mT/mG mice) ( Figure 2D), coinciding 203 with increased proliferation of the adult AP organoid-constituting stem cells, showing a Ki67 + 204 index reaching the one of neonatal AP-derived organoids ( Figure 2D).

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Taken together, organoids from neonatal AP reflect the stem cell compartment of the gland at 206 this developmental stage regarding its activated and signaling-center phenotype.

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One factor that may be responsible for activating the stem cells in the neonatal pituitary is IL-6 208 which we recently uncovered as pituitary stem cell-activating factor in adult mouse (Vennekens 209 et al., 2021). Intriguingly, gene expression of Il6 is low in neonatal AP when compared to adult supplement 1F). Notwithstanding, IL-6 is still produced, and beneficial, for AP organoid 214 development. Indeed, a significantly smaller number of organoids grew from IL-6 knock-out (IL-6 -215 /-) versus WT neonatal AP, which was (partially) rescued by adding exogenous IL-6 ( Figure 2E).

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Moreover, although IL-6 addition did not significantly increase the number of organoids formed 217 from WT neonatal AP (most likely because the stem cell activation status is already high) ( Figure   218 2E), it strongly enhanced the passageability of the organoid cultures, from 4-5 passages without 219 IL-6 to at least 15 passages (6 months of expansive culture) with the cytokine ( Figure 2F), as also 220 observed before for adult AP (Vennekens et al., 2021). Addition of IL-6 is considered to 221 compensate for the decline in endogenous levels that is observed at passaging (

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To in vivo validate our organoid-based finding that the WNT pathway plays a role in stem cell 306 activation in the neonatal pituitary, we treated neonatal (WT) pups with the WNT pathway 307 (porcupine) inhibitor LGK-974 (LGK; Figure 3F). WNT target gene (Axin2, Lgr5) expression in the 308 AP diminished following LGK administration, thereby verifying its activity and efficacy at the level 309 of the pituitary ( Figure 3F). Interestingly, the number of proliferating SOX2 + cells decreased (

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The vivid neonatal pituitary shows swift and complete regeneration after local damage

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We have previously shown that somatotrope-ablation damage in the adult pituitary triggers acute 328 proliferative activation of the stem cell compartment and expression of GH in the SOX2 + cells, and 329 that the somatotrope population was eventually regenerated to 50-60% at 5-6 months after 330 damage infliction (Fu et al., 2012). Here, we investigated whether the neonatal gland, housing a 331 more activated ('primed') stem cell compartment, behaves differently regarding acute stem cell 332 reaction and regeneration.

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To start delving into the underlying mechanisms, we performed scRNA-seq analysis of damaged

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In conclusion, these data show that the vividly maturing neonatal gland more efficiently 372 regenerates after injury than the adult gland, but that the already highly activated stem cell

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We recently found that IL-6 is promptly upregulated in the adult pituitary, in particular in its stem 405 cells, following transgenically inflicted damage, associated with proliferative stem cell activation 406 (Vennekens et al., 2021). Intriguingly, IL-6 expression did not rise following damage in the 407 neonatal gland. Together, these findings suggest that the pituitary reacts to damage differently 408 according to developmental age. Along the same line, the dynamic neonatal pituitary more 409 16 efficiently (faster and more extensively) regenerates than the adult pituitary following the local 410 injury (Fu et al., 2012), likely due to the presence of activated ('primed') stem, committed 411 progenitor and just differentiated endocrine cells that all appear to contribute by increased 412 proliferation or differentiation (as supported by transcriptomic and in situ immunostaining 413 analyses). Of note, the somewhat lower somatotrope ablation grade in neonatal pituitary may 414 also partly contribute to the higher regeneration level. Interestingly, efficient regenerative 415 capacity is also present in other mouse organs at neonatal age (such as heart and cochlea), while 416 seriously declining or disappearing in adulthood (B. C. Cox et al., 2014;Lam et al., 2018). Detailed 417 unraveling of neonatal-pituitary regenerative mechanisms further needs profound studies, which 418 may find ground in our existing and to be extended single-cell transcriptomic analyses.

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IL-6 expression in the neonatal gland is low (much lower than in adult pituitary), and its absence 420 does not visibly affect the neonatally activated stem cell compartment. Either, IL-6 is not needed 421 in the neonatal gland to induce or sustain the activated stem cell phenotype, or its lack has been 422 taken over by cytokine family members such as LIF or IL-11. We provide support for the latter, 423 and also propose that the JAK/STAT pathway may be the common denominator that lies at the 424 basis of stem cell activation in the neonatal gland, hence also critical for organoid outgrowth (as    transgenes and abbreviated to GHCre/iDTR) as described in detail before (Fu et al., 2012).

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Offspring is genotyped for the presence of the Cre transgene by PCR using 5'-

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The AP of neonatal (PD7) mice was isolated and dispersed into single cells using trypsin (Thermo

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Fisher Scientific, Waltham, MA), all as previously described (Denef et al., 1978 483 (Butler et al., 2018). First, low-quality/dead cells and potential doublets (i.e. with less than 750 484 genes or more than 8,000 genes and more than 17.5% mitochondrial RNA; see cut offs in Figure   485 1

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To integrate our neonatal pituitary dataset with our previously published adult pituitary dataset 504 (Vennekens et al., 2021), we applied Seurat's reference-based integration approach, for which 505 the 'adult' condition was used as reference dataset when applying the FindIntegrationAnchors 506 function (as described above; (Stuart et al., 2019)). The top 30 PCs were selected and used for 507 UMAP dimensionality reduction (McInnes et al., 2018). Clusters were identified with the 508 FindClusters function with a clustering resolution set to 1.6. This analysis resulted in the 509 identification of 37 distinct clusters, which were annotated.

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Interactions between pairwise cell clusters were inferred by CellPhoneDB v.2.1.5, which includes 536 a public repository of curated ligands, receptors and their interactions (Efremova et al., 2020). We 537 ran the CellPhoneDB framework using a statistical method and detected ligand-receptor pairs that 538 were expressed in more than 20% of cells. Selected significant ligand-receptor pairs (P-value 539 ≤ 0.05 and mean value ≥ 0.5) are shown.

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Organoid cultures were passaged every 10-14 days; the organoids were incubated with TrypLE 549 Express (Thermo Fisher Scientific) and mechanically dispersed until organoid fragments were 550 obtained which were re-seeded in Matrigel drops as above.

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Images were recorded using a Leica DM5500 upright epifluorescence microscope (Leica

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The following figure supplement is available for figure 1: