United States

The pituitary gland harbors a population of stem cells. However, role and regulation of these cells remain poorly understood. We recently established organoids from mouse pituitary as a novel research tool to explore pituitary stem cell biology (Cox et al., J. Endocrinol. 2019; 240:287-308). In general, organoids represent 3D in vitro cell configurations that develop and self-organize from (single) tissue stem cells under well-defined culture conditions that typically mirror the stem cell niche and/or embryogenic processes. Organoids reliably recapitulate key aspects of the original organ, including of its stem cell compartment. Moreover, organoids are long-term expandable while retaining these properties. We demonstrated that pituitary organoids originate from the resident (SOX2+) stem cells, largely phenocopy these cells and retain the stemness phenotype during expansive culture. Interestingly, the organoids show confident in vivo translatability and, when developed from transgenically damaged gland, recapitulate the activation status of the stem cells as observed in situ following injury. Now, we found that the organoids also mirror the stem cells’ phenotype and biology in physiological conditions in which the stem cell compartment is either activated or compromised. Organoids from the neonatal maturing pituitary reproduce phenotypical and functional aspects of its activated stem cells, whereas organoids from aging gland mimic the declined functional state of the stem cells in old pituitary. Interestingly, this functional decay was found to be reverted during organoid culture, indicating that the old pituitary stem cells retain intrinsic functionality but are in vivo restrained by an obstructive microenvironment, not present in the organoid culture. Indeed, using single-cell transcriptomics and in vivo analysis, we found that the aging pituitary suffers from a prevailing inflammatory state (inflammaging) which appears to raise the threshold for stem cell activation. Interestingly, comparison of young and old pituitary led us to the discovery of pituitary stem cell activators. Finally, we found that activated parameters of organoid formation are also observed when tumorigenesis takes place in the gland, again mimicking the in situ stem cell activation that is occurring in this perturbed, pathological condition.Taken together, we identified, and applied, our new pituitary organoid model as advanced and powerful tool to gain profound insight into pituitary stem cell behavior across life and disease, which is expected to eventually translate into restorative and rejuvenative tactics when pituitary function is compromised by damage or age. In this context, our single-cell transcriptome database has strong potential to unveil appealing targets.?? 

ENDO 2021

Novel Pituitary Organoid Model as Powerful Tool to Unravel Pituitary Stem Cell Biology Across Ages and Disease

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ENDO 2020

ENDO is the place where leading experts, researchers, and the most respected clinicians in the field come together to share the latest findings in hormone science and discuss breakthroughs in clinical care.

technical paper

Exosomes, small extracellular vesicles carrying lipids, proteins, DNA and RNA, enable intercellular communication. Pituitary-derived exosomes have not been well validated, and as no human pituitary cell lines are available, we characterized exosomes derived from rat somatotroph tumor cells (GH1 and GH3). Rat FR and H9C2 cells were used as non-pituitary controls. Exosomes were isolated from serum-free culture supernatants by combining ultrafiltration and ultracentrifugation to eliminate hormone contamination. Derived exosomes were analyzed by NanoSight to visualize, size, and count particles. Exosomal proteins were extracted and exosome markers including TSG101, ALIX, CD63, HSP70, HSP90 detected by Western Blot. The exosome inhibitor GW4869 (10 µM, 30 h) reduced exosome release (up to 81%), whereas treating cells with hydrocortisone (0.1 µM, 72 h) increased exosome production (up to 42%) in GH1 and GH3 cells. Exosomal shuttle RNA characterized by RNA-Seq showed distinct pituitary vs non-pituitary exosome RNA profiles. Selected miRNAs assessed in exosomes and corresponding cells by qRT-PCR validated exosomal RNA-seq and suggested that miRNA signatures in exosomes and in respective cells of origin were concordant. Next, we explored downstream signaling of GH1-derived exosomes (GH1-exo) in vitro and in vivo and studied biological actions in normal hepatocytes and in malignant cells. As evidenced by mRNA-seq, GH1-exo distinctly altered signaling pathways in rat primary hepatocytes, vs pathways elicited by GH or PRL (0.5 µg/mL, 24 h). GH1-exo, FR-exo or vehicle were intravenously injected to 4-week-old female Wistar rats twice weekly for 4 weeks (5*109 exo/200 g, n=3), and livers dissected for mRNA-seq. Among GH1-exo specifically regulated genes, EIF2AK/ATF4, involved in cAMP responses and amino acid biosynthesis, were attenuated. In hepatocytes, GH1-exo suppressed up to 65% of nascent protein synthesis and reduced forskolin (10 µM)-stimulated cAMP activity by 19%, while GH (0.01-1 µg/mL) did not affect this pathway. Notably, GH1-exo also attenuated malignant cell motility. Both GH1-exo incubation or GH1 cell co-culture (48 h) suppressed migration, invasion and wound healing of HCT116 cancer cells by up to 70%. In contrast, treatment with rGH (0.5 µg/mL) increased HCT116 motility. Intravenous administration of GH1-exo (1010 exo/mouse, twice a week for 5 weeks) decreased metastatic tumor volume by 40% in nude mice harboring splenic HCT116 implants (5*105 cells/mouse, n=10), and especially abrogated hepatic metastases. mRNA-seq of GH1-exo treated HCT116 cells vs controls indicated dysregulated p53 and MAPK pathways, which may partially explain mechanisms underlying motility attenuation. The results elucidate novel biological actions of somatotroph adenoma cell-derived exosomes and suggest exosomes as non-hormonal messengers produced by pituitary tumors.?? 

Pituitary Somatotroph Adenoma Cell-Derived Exosomes: Characterization of Novel Non-Hormonal Functions

Background: In a prior Phase III, randomized-withdrawal study, osilodrostat, a potent oral 11β-hydroxylase inhibitor, provided rapid and sustained normalization of mean urinary free cortisol (mUFC) in most patients (pts) with CD. Now, we report efficacy and safety results from another Phase III study of osilodrostat in pts with CD that included an upfront, double-blind, randomized, placebo-controlled phase (LINC 4: NCT02697734). Methods: Adults with CD with mUFC >1.3 x ULN were randomized 2:1 to osilodrostat 2 mg bid or matching placebo for a 12-week (W) double-blind period, with dose adjustments at W2, 5 and 8 (range 1-20 mg bid) based on efficacy and tolerability; dose matching and adjustments were managed by independent endocrinologists. From W12 to W48, all pts received open-label osilodrostat, with dose adjustments permitted (range 1-30 mg bid). At W48, pts could enter an optional extension. Primary endpoint: proportion of randomized pts in each arm who received ≥1 treatment dose with mUFC ≤ULN at W12. Results: 73 pts were randomized and received osilodrostat (n=48) or matching placebo (n=25; baseline median [range] mUFC 2.5 x ULN [0.7-12.5] vs 2.2 x ULN [0.2-18.9]). 77% of osilodrostat recipients achieved mUFC ≤ULN at W12 vs 8% of placebo recipients (OR 43.4; 95% CI 7.1-343.2; P<0.0001). At W36, 81% (95% CI 69.9-89.1) of osilodrostat recipients had mUFC ≤ULN (key secondary endpoint). Median time to first controlled mUFC response was 35 days (95% CI 34‒52) for pts randomized to osilodrostat. Median duration of osilodrostat exposure at data cut-off (Feb 25, 2020) was 71.7 vs 62.3 weeks for pts randomized to osilodrostat and placebo (median [IQR] dose 4.7 [3.8-9.0] vs 6.0 mg/day [3.7-9.7]). Up to W12, 3 osilodrostat pts discontinued, 1 because of an AE (arthralgia), vs 0 with placebo. The most common (≥30%) AEs occurring by W12 were decreased appetite (38% osilodrostat vs 16% placebo), arthralgia (35% vs 8%) and nausea (31% vs 12%). AEs related to hypocortisolism and adrenal-hormone-precursor accumulation occurred in 15% vs 0% and 44% vs 36% of osilodrostat and placebo pts; most were grade 1/2 and resolved with dose reduction/interruption and/or concomitant medication. During the overall study period, the most common (≥30%) AEs occurring on osilodrostat treatment were arthralgia (45%), decreased appetite (45%), fatigue (38%), nausea (37%) and headache (33%). Improvements in cardiovascular- and metabolic-related parameters, including systolic and diastolic blood pressure and HbA1c, were observed with osilodrostat treatment at W12 and W48.?Conclusion: Osilodrostat was superior to placebo at normalizing mUFC levels at W12 (77% vs 8%). Improvements in mUFC levels were sustained at W36. Few pts discontinued because of AEs; hypocortisolism-related AEs were infrequent and manageable. We conclude that osilodrostat is a highly effective and well-tolerated treatment for pts with CD.?? 

Osilodrostat Is an Effective and Well-Tolerated Treatment for Cushing's Disease (CD): Results From a Phase III Study With an Upfront, Randomized, Double-Blind, Placebo-Controlled Phase (LINC 4)

Metopirone Treatment in Endogenous Cushing's Syndrome
Results from a Prospective Multicenter, Open-Label, Phase III/IV Study: PROMPT

Background: Based on the CHIASMA OPTIMAL study, oral octreotide capsules (OOC) were recently approved in the US as a long-term maintenance therapy for patients with acromegaly previously responding to injectable octreotide or lanreotide, somatostatin receptor ligands (SRLs). Results on longer-term efficacy and safety of OOC from the first 48 weeks of the open-label extension (OLE) of this study are presented here. Methods: Eligible patients had the option to enroll in the OLE of CHIASMA OPTIMAL following the double-blind placebo-controlled (DPC) period; 90% of patients who received OOC in the DPC period enrolled. All patients entering the OLE were initiated on a 60 mg/day dose of OOC and titrated up or down based on insulin-like growth factor I (IGF-I) level and/or acromegaly signs or symptoms. End points in the OLE were exploratory and included the proportion of patients who completed week 48 of the OLE, the proportion who completed as responders (defined as average IGF-I ≤1.0 × upper limit of normal [ULN] at weeks 46/48), and changes in IGF-I from baseline of DPC and OLE until week 48 of the OLE; multiple imputation (MI) was used for missing data. Results: Forty patients entered the OLE (n=20 each; OOC and placebo). Median exposure to OOC in the OLE was > 1 year for those who had been on placebo in the DPC and ≤ 21 months for those who had been on OOC. Dosing of OOC at the end of their participation in the OLE was 40 mg, n=3; 60 mg, n=10; and 80 mg, n=27. In those who received OOC during the DPC, 90% (n=18) completed 48 weeks of the OLE. Of responders at the end of the DPC period (n=14), 92.6% maintained response at OLE week 48. In patients from the OOC group who completed the DPC on study drug, average IGF-I using MI was 0.91 and 0.90 × ULN at OLE baseline and week 48, respectively. The mean change in IGF-I from the baseline of the DPC to OLE week 48 was 0.06 × ULN in patients who completed the DPC on OOC (n=19). In those who received placebo during the DPC, 70% (n=14) completed 48 weeks of the OLE. Of responders at the end of the DPC (n=5), 100% maintained response at OLE week 48. In patients from the placebo group who completed the DPC and did not revert to prior injectable therapy (n=9), the average IGF-I values were 1.09 and 0.87 × ULN at OLE baseline and week 48 respectively, using MI. The mean change in IGF-I from the baseline of the DPC to OLE week 48 was 0.08 × ULN in patients who completed the DPC on placebo (n=9). The most common treatment-emergent adverse events (TEAEs) were gastrointestinal; most were mild or moderate. The incidence of TEAEs was similar between patients who were on OOC or placebo during the DPC. The safety profile during the OLE did not show new concerns with increased duration of drug exposure. Conclusion: Long term maintenance of biochemical response to OOC is durable as assessed following ≤ 21 months of treatment. The OOC safety profile in the extension study is consistent with that of injectable SRLs but without injection-related AEs.?? 

One-Year Outcomes of the Open-Label Extension of CHIASMA OPTIMAL, a Phase 3 Study of Oral Octreotide Capsules in Acromegaly

Glucagon-Like Peptide-1 (GLP-1) Receptor Agonists Reduce Fluid Intake in Primary Polydipsia

Background: Studies comparing the timing and pace of puberty in overweight/obese girls (OW/OB) vs normal weight girls (NW) have produced conflicting results; some suggest earlier activation of the central components of the reproductive axis in OB while others are more consistent with a peripheral source of estrogen (e.g. adipose tissue) driving puberty in OB. Importantly, there have been no longitudinal assessments of both clinical and biochemical pubertal markers in OB vs. NW. Methods: 90 healthy pre-menarchal girls (26 OW/OB, 54 NW) from the community, aged 8.2-14.7 years, completed 2.8 ± 1.7 (mean, SD) study visits over the course of 4 years. Visits included dual-energy x-ray absorptiometry to calculate percent total body fat (TBF), Tanner staging, breast ultrasound for morphological staging (BMORPH; stages A-E), pelvic ultrasound, hand x-ray (bone age, BA), blood tests for reproductive hormones, and urine collection to determine a vaginal maturation index (VMI), an index of estrogen exposure in urogenital epithelial cells. Menarchal status was determined at each visit and via follow-up questionnaires. The effect of TBF on hormones and markers of estrogen action, the pace of breast maturation, and age at menarche were determined using a mixed, multi-state, or Cox proportional hazards model, respectively. Mixed and Cox models controlled for BMORPH at visit 1 (V1) and race. Results: NW girls were older than OW/OB (11.3 vs. 10.2 yrs, p&lt;0.01) at V1, more likely to be non-Hispanic White (66 vs. 40%, p=0.03), and had more advanced breast morphology BMORPH (p&lt;0.01). LH, E2, VMI, BA, and ovarian and uterine volume increased with time with no effect of TBF. There was an interaction between time and TBF for FSH, INHB, E1, Total T, Free T, and A’dione (p&lt;0.05): levels were initially similar in all TBF groups, but after 1 yr, levels increased in girls with higher TBF, plateaued in girls with mid-range TBF, and decreased in girls with lower TBF. Girls with higher TBF progressed through BMORPH stage D (corresponding to growth/arborization of the breast ductal system), more slowly than girls with lower TBF but achieved menarche at a younger age (risk 1.04x higher per 1 unit increase in TBF). Conclusions: Intensive reproductive phenotyping of girls during the pubertal transition reveals that both the neuroendocrine and ovarian components of the axis are generally preserved in girls with higher TBF but that the axis appears to be activated earlier than in girls with lower TBF. In late puberty, however, girls with higher TBF demonstrate subtle differences in standard hormonal (e.g. serum FSH, INHB, and androgen) and clinical (e.g. delayed growth of breast bud) markers of puberty. Investigation of the mechanistic basis for these differences and their potential clinical consequences for girls with higher TBF deserves further study.?? 

Longitudinal Investigation of Pubertal Milestones and Hormones as a Function of Body Fat in Girls

Backround:Collagens are the most abundant proteins in the human body. In a growth plate, collagen types II, IX, X and XI are present. Defects in collagen genes cause heterogeneous syndromic disorders frequently associated with asymmetric short stature (e.g. Kniest dysplasia, spondyloepiphyseal dysplasia). Less is known about nonsyndromic collagenopathies - data about their frequency and subtle phenotypic signs are sparse, the information about their response to growth hormone (GH) treatment is lacking completely.?Aim:To evaluate the frequency of collagenopathies in familial short stature (FSS) children and to describe their phenotype, including growth hormone (GH) treatment response.?Methods:Out of 522 individuals treated in our center with GH from the indication of primary GH deficiency (GHD) or small for gestational age short stature (SGA-SS), 87 children with FSS fulfilled the inclusion criteria (pre-treatment height ≤-2 SD in both patient/their shorter parent, signed written informed consent) and were enrolled to the study. Next-generation sequencing was performed to search for variants in COL2A1, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1 and COL11A2 genes. The results were evaluated using ACMG guidelines. The phenotype of children with (likely) pathogenic variants was described including the short-term GH treatment response (growth velocity and body-height SDS increase over three years of treatment). For statistical evaluation, parametric tests were used, p-values <0.05 were considered significant.?Results:A (likely) pathogenic variant in one of the collagen genes was found in 10/87 (11.5%) children. Their age was 12.5 years (median, range 6-17 years), their pre-treatment height was -3.1 SD (-2.4 to -4.3 SD). Their birth length (median -2.8 SD; range -0.7 to -4.1 SD) was more severely affected than birth weight (median -2.1 SD; range -1.0 to -2.7 SD). Eight children were treated with GH from SGA-SS indication, the remaining 2 were classified as mild GHD (maximal stimulated GH concentration 8.0 and 9.7 ug/l, normal brain MRI and examination of other pituitary hormones). Detailed anthropometric examination described mild asymmetry with shorter limbs and mild bone dysplasia signs (scoliosis, more pronounced lumbar lordosis, genua valga, limited elbow extension) in 2/10 and 4/10 affected children, respectively. Growth velocity improved from a median of 5.3 cm/year to 8.7 cm/year after one year of treatment (p<0.001, paired-sample T-test), height improved from a median of -3.1 SD to -2.2 SD after three years of therapy (p=0.001, ANOVA repeated measures analysis of variants).?Conclusion:Nonsyndromic collagenopathies are a frequent cause of FSS. The short-term response to GH treatment is promising.?Supported by the Ministry of Health, Czech Republic, grant number NV18- 07-00283 and by the research project of the Grant Agency of Charles University of Prague, GAUK 976718. ?? 

Familial Short Stature - a Novel Phenotype of Growth Plate Collagenopathies

Introduction: The genetic basis of human growth regulation has only been partly elucidated thus far. Therefore, finding causative genes in patients with short stature help in understanding precise pathophysiological mechanisms, establishing genotype-phenotype relationships and optimizing treatment. In order to extend our knowledge about the genes involved in short stature, we studied a unique cohort of consanguineous families with children with short stature from Sulaymani in Kurdistan, Iraq. Patients and Methods: Fifty-five consanguineous families, with children shorter than -2.3SDS at the time of examination (median height -3.3SDS, range -2.3 to -15SDS), who were examined at the endocrine outpatient clinic of the Department of Pediatrics, Sulaymani University College of Medicine between January 2018 and February 2020, were included in the study. In families with more than one child with short stature, the shorter sibling was selected as the proband (median age 8 years, range 1 - 15 years). Three probands were subsequently excluded due to the diagnosis of Turner’s syndrome and Edward’s syndrome. Consent was obtained from all families and probands’ DNA was analyzed by Whole Exome Sequencing (WES) methods. The data were processed by a bioinformatic pipeline and detected variants were filtered using variant analysis software. Selected potentially pathogenic variants were confirmed using Sanger sequencing methods and evaluated by the American College of Medical Genetics (ACMG) standards and guidelines. Results: A monogenic cause of short stature, which explained the patient phenotype, was elucidated in 13 of 26 families who were analyzed thus far. Seven families had multiple affected children making a total of 22 patients with a positive genetic diagnosis. Pathogenic or likely pathogenic variants (according to the ACMG standards) were found in genes involved in the GH-IGF-1 axis (GHR), in the extracellular matrix of the growth plate (COL1A2, MMP13, LTBP3, and ADAMTS17), in the regulation of chondrocytes (NPR2 and CTSK), transporter coding genes (SLC34A3), and other genes (PTCH1, GALNS, DNACJ21, ZSWIM6, GNPTG). Among them, there are 9 novel variants and 10 homozygous variants including variants in genes causing syndromic short stature. Unexpectedly, we successfully identified three cases of autosomal dominant short stature (variants in genes NPR2, COL1A2, PTCH1) as well. The remaining probands from 26 families are still being analyzed. Conclusion: With the help of NGS methods, we have successfully elucidated the genetic cause of short stature in nearly 50% of patients who were analyzed thus far. These results further strength the concept that genes affecting the growth plate (chondrocytes and the extracellular matrix) play a crucial role in growth regulation. Acknowledgements: The study was co-funded by grants AZV 18-07-00283 and GAUK 340420.?? 

Growth Plate Genes Are Key Regulators of Growth: Lessons Learned From Children of Consanguineous Families From Kurdistan, Iraq

Surgical approach of atypical genitalia according to adult patients with Differences of Sexual Development. Introduction: Differences of sexual development (DSD) define congenital diseases in which there is an atypical development of chromosomal, gonadal or anatomical sex, and may present varying degrees to genital atypia. There has been a discussion about the ideal time for surgical approach of atypical genitalia. S, because some non-governmental entities argue that the surgical approach should be delayed until adulthood after the patient's consent. Objective/methodology: To analyze the perspectives of adult DSD patients followed at a reference center in São Paulo on the surgical approach to correct atypical genitalia, through a semi-directed interview. Results: Thirty-seven adult patients with atypical genitalia were interviewed. Patients’ mean age was 36 years. 70% of them had atypical genitalia diagnosed at birth. The patients’ median age at the genitoplasty approach was 5 years (1 to 35 years). The median time interval between the beginning of the follow-up at the referral center and the surgical procedure was 1.9 years. When asked about the ideal period/age for genitoplasty, 72.2% considered the childhood, 16.7% cited when they’re teenagers, 8.3% in adulthood and 2.8% did n’t know. The discomfort reported by the patients related to atypical genitalia decreased after the surgical approach: from 3.8 to 2.9 p < 0.01 (on a scale of 1 “without discomfort’ to 4 “extreme discomfort”). Insecurity about the appearance of genitalia and functionality during sexual intercourse influences negatively affective relationships. Four (10.8%) patients presented gender dysphoria, all of them with 46 ,XY DSD, three with partial gonadal dysgenesis (all approached surgically before being admitted to our referral service) and one with 5-alpha-reductase 2 deficiency. Conclusion: Most 46 ,XY DSD patients considered childhood the ideal time to correct their atypical genitalia. An early follow-up in a reference center and an adequate evaluation by a multidisciplinary may influence the positive results associated to the surgical approach of the atypical genitalia in childhood and the low prevalence of gender dysphoria in adulthood.?? 

Self-Reported Feelings of Adult Patients With Differences of Sex Development (DSD) Regarding Genital Surgical Procedures

Gnas Heterozygous Inactiviation Affects Calcitonin Receptor Bioactivity in a Mouse Model of Albright Hereditary Osteodystrophy based upon Parental Inheritance

Novel Pituitary Organoid Model as Powerful Tool to Unravel Pituitary Stem Cell Biology Across Ages and Disease

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