Characterization of Non-hormone Expressing Endocrine Cells in Fetal and Infant Human Pancreas

Abstract

Context: Previously, we identified chromograninA positive hormone-negative (CPHN) cells in high frequency in human fetal and neonatal pancreas, likely representing nascent endocrine precursor cells. Here, we characterize the putative endocrine fate and replicative status of these newly formed cells.Objective: To establish the replicative frequency and transcriptional identity of CPHN cells, extending our observation on CPHN cell frequency to a larger cohort of fetal and infant pancreas.Design, Setting, and Participants: 8 fetal, 19 infant autopsy pancreata were evaluated for CPHN cell frequency; 12 fetal, 24 infant/child pancreata were evaluated for CPHN replication and identity.Results: CPHN cell frequency decreased 84% (islets) and 42% (clusters) from fetal to infant life. Unlike the beta-cells at this stage, CPHN cells were rarely observed to replicate (0.2 ± 0.1 vs. 4.7 ± 1.0%, CPHN vs. islet hormone positive cell replication, p < 0.001), indicated by the lack of Ki67 expression in CPHN cells whether located in the islets or in small clusters, and with no detectable difference between fetal and infant groups. While the majority of CPHN cells express (in overall compartments of pancreas) the pan-endocrine transcription factor NKX2.2 and beta-cell specific NKX6.1 in comparable frequency in fetal and infant/child cases (81.9 ± 6.3 vs. 82.8 ± 3.8% NKX6.1+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.9; 88.0 ± 4.7 vs. 82.1 ± 5.3% NKX2.2+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.4), the frequency of clustered CPHN cells expressing NKX6.1 or NKX2.2 is lower in infant/child vs. fetal cases (1.2 ± 0.3 vs. 16.7 ± 4.7 clustered NKX6.1+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01; 2.7 ± 1.0 vs. 16.0 ± 4.0 clustered NKX2.2+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01).Conclusions: The frequency of CPHN cells declines steeply from fetal to infant life, presumably as they differentiate to hormone-expressing cells. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells.Precis: CPHN cell frequency declines steeply from fetal to infant life, as they mature to hormone expression. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells.

Alterations in Beta Cell Identity in Type 1 and Type 2 Diabetes

“…While it is potentially plausible that these cells were formerly β cells that have undergone dedifferentiation or transdifferentiation, this is less likely given their distribution and increased frequency as scattered cells in exocrine pancreas in the setting of diabetes; it is therefore possible that they represent partially differentiated, newly formed endocrine cells. In support of this, we further reported abundant scattered CPHN cells in the exocrine compartment of human fetal and neonatal pancreas [8••], the highest frequency being in fetal pancreas compared to neonatal [90], potentially suggesting that the increased frequency of CPHN cells containing the β cell–specific transcription factors NKX6.1 or NKX2.2 in both T1D and T2D may be indicative of attempted β cell regeneration.…”

Validating expression of beta cell maturation-associated genes in human pancreas development

“…CHGA was selected for normalization because it is expressed at high levels in both alpha and beta cells ( Supplemental Figure S4A ), its expression precedes that of hormones in endocrine cell development, and it is not lost with de-differentiation in beta cell failure. For these reasons, we felt that CHGA rather than a gene like INS was better for normalization, because insulin expression itself is lower in immature cells and is lost in de-differentiated states ( Talchai et al, 2012 ; Moin et al, 2018 ; Ramond et al, 2018 ). Thus, the ratio of UCN3 / CHGA was used to normalize to the fraction of RNA derived from the endocrine population only.…”

“…Chromogranin A (CHGA) and chromogranin B (CHGB) are present in dense core secretory granules of many endocrine cell types. CHGA expression is present early in the development of pancreatic endocrine cells ( Supplemental Figure S4A ) ( Talchai et al, 2012 ; Moin et al, 2018 ; Ramond et al, 2018 ). CHGB has been shown to regulate beta cell secretory granule trafficking, and loss of CHGB impairs GSIS and proinsulin processing ( Bearrows et al, 2019 ).…”