Gestational diabetes impacts fetal precursor cell responses with potential consequences for offspring

Abstract

Fetal programming has been proposed as a key mechanism underlying the association between intrauterine exposure to maternal diabetes and negative health outcomes in offspring. To determine whether gestational diabetes mellitus (GDM) might leave an imprint in fetal precursors of the amniotic membrane and whether it might be related to adverse outcomes in offspring, a prospective case-control study was conducted, in which amniotic mesenchymal stem cells (AMSCs) and resident macrophages were isolated from pregnant patients, with either GDM or normal glucose tolerance, scheduled for cesarean section. After characterization, functional characteristics of AMSCs were analyzed and correlated with anthropometrical and clinical variables from both mother and offspring. GDM-derived AMSCs displayed an impaired proliferation and osteogenic potential when compared with control cells, accompanied by superior invasive and chemotactic capacity. The expression of genes involved in the inflammatory response (TNFα, MCP-1, CD40, and CTSS) was upregulated in GDM-derived AMSCs, whereas anti-inflammatory IL-33 was downregulated. Macrophages isolated from the amniotic membrane of GDM mothers consistently showed higher expression of MCP-1 as well. In vitro studies in which AMSCs from healthy control women were exposed to hyperglycemia, hyperinsulinemia, and palmitic acid confirmed these results. Finally, genes involved in the inflammatory response were associated with maternal insulin sensitivity and prepregnancy body mass index, as well as with fetal metabolic parameters. These results suggest that the GDM environment could program stem cells and subsequently favor metabolic dysfunction later in life. Fetal adaptive programming in the setting of GDM might have a direct negative impact on insulin resistance of offspring.

Keywords: fetal precursors; gestational diabetes; offspring; placenta; programming; stem cells.

Figure 1

Histological features of the placental tissue from maternal and fetal sides obtained from gestational diabetes mellitus (GDM) and control women. Representative photomicrographs of terminal and intermediate villi in placental sections from pregnant control and GDM women, stained with Masson's trichrome to highlight connective tissue and collagen fibers (in green). Quantification of Masson's trichrome staining in villous stroma of placental sections is shown as percentage mean area ± SD (n = 6‐8 per group). Results are shown as mean ± SEM from independent donor experiments performed in duplicate. *P < .05 vs controls, **P < .01 vs controls

Figure 2

Gestational diabetes mellitus (GDM) affects the plasticity of fetal precursor cells from the amniotic membrane. A, MTT incorporation in proliferating amniotic mesenchymal stem cells (AMSCs) isolated from pregnant control and GDM women (n = 7‐9 per group). B, Representative photomicrographs of AMSCs isolated from pregnant control and GDM women (n = 4 per group), differentiated into adipocytes, osteocytes, and chondrocytes and stained with Oil Red, Alizarin Red, and Alcian Blue, respectively (magnification ×200). Quantification of the differentiation capacity was assessed by extracting the staining dyes and measuring the absorbance by spectrophotometry at 540 nm. C, Gene expression of adipogenic (ACACA, PPARy, FABP4, LPL), osteogenic (ALP, COL1α1), and chondrogenic (COMP, COL1α1, COL2α1) markers in AMSCs isolated from pregnant control and GDM women (n = 4 per group). In all cases, values of differentiated cells were normalized to their undifferentiated counterparts. Results are shown as mean ± SEM from independent donors experiments performed in duplicate. *P < .05 vs controls

Figure 3

Disturbances in amniotic mesenchymal stem cells (AMSCs) and amniotic membrane‐resident macrophages from placentas obtained from gestational diabetes mellitus (GDM) women. A, Gene expression analysis of the inflammatory markers TNFα, MCP‐1, CD40, CTSS, IL‐33, and PTGS2 in AMSCs isolated from pregnant control and GDM women (n = 8 per group). B, Prostaglandin E₂ (PGE2) levels in the conditioned medium of AMSCs from pregnant control and GDM women (n = 6‐7 per group). C, Migratory and invasive capacities of AMSCs isolated from pregnant control and GDM women assessed in Transwell assays (n = 7‐9 per group). D, Migration of THP‐1 and Jurkat cells to AMSC‐conditioned medium assessed in Transwell assays (n = 5‐7 per group). E, Migration of THP‐1 cells to GDM‐AMSC‐conditioned medium after incubation with anti‐MCP‐1 antibody. F, Gene expression analysis of pro‐inflammatory and chemotactic markers (TNFα, IL‐6, IL‐1β, MCP‐1, CCL3, and CD86) in amniotic membrane‐resident macrophages from pregnant control and GDM women (n = 6‐8 per group). Results are shown as mean ± SEM from independent donor experiments performed in duplicate. *P < .05 vs controls, **P < .01 vs control, ***P < .0001 vs controls

Figure 4

Phenotypic changes induced by a gestational diabetes mellitus (GDM)‐like environment rich in glucose, insulin, and palmitic acid on amniotic mesenchymal stem cells (AMSCs) obtained from control pregnant women. A, Gene expression analysis of the inflammatory markers TNFα, MCP‐1, CD40, IL‐1β, IL‐6, and IL‐33 in AMSCs isolated from control pregnant women stimulated with glucose (Gluc), insulin (Ins), and/or palmitic acid (PA; n = 4 per group). B, Migration capability of control AMSCs stimulated with glucose, insulin, and/or PA assessed by Transwell assays (n = 3 per group). C, Migration of THP‐1 cells to the conditioned medium of control AMSCs stimulated with glucose, insulin, and/or PA assessed in Transwell assays (n = 4 per group). D, MTT incorporation in proliferating control AMSCs stimulated with glucose, insulin, and/or PA (n = 4 per group). *P < .05 vs control, **P < .01 vs controls, ***P < .0001 vs controls