Original ResearchHuman embryonic stem cell–derived pancreatic endoderm alleviates diabetic pathology and improves reproductive outcome in C57BL/KsJ-Lepdb/+ gestational diabetes mellitus mice
Introduction
Gestational diabetes mellitus (GDM) is a disease that occurs during pregnancy with pathologies including hyperglycemia, hyperinsulinemia, insulin resistance, and maldevelopment in fetuses [1]. Patients with GDM are typically diagnosed during the second trimester of pregnancy, without any prior signs of diabetes, and 3% to 5% of them remain diabetic even after pregnancy [2]. Besides maternal diabetes, GDM is also accompanied by maldevelopment of fetuses [3]. The mechanisms underlying GDM are not fully understood, but its origin is thought to be multifactorial involving both genetic and environmental factors [4]. Mutations in the pancreatic β-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) were reported to cause diabetes mellitus and hyperinsulinism [5]. High-saturated-fat diet was also shown to be able to induce glucose abnormalities among pregnant women [6].
Several animal models were used to study GDM [7]. The C57BL/KsJ-Lepdb/+ (abbreviated as db/+) mouse, created by a loss-of-function mutation in the leptin receptor gene Lepr [8], proved to be a promising model because the heterozygous mutant animals closely mimicked human GDM symptoms. Prior to pregnancy, they exhibited largely normal glucose tolerance until late gestation [9], [10]. Fetal development was also defective with fetus weight at term increased by 5% to 8% [11], [12].
Advances in stem cell technology have provided exciting development in diabetes treatment in animal models. Human embryonic stem cells (hESCs) were used to differentiate to pancreatic endoderm (PE) capable of treating diabetic mice [13], [14]. Pagliuca et al [15] have even demonstrated in a very recent study that a large-scale in vitro differentiation of human induced pluripotent stem cells generates glucose-responsive insulin-producing pancreatic β cells. These cells were able to secrete human insulin into the serum and ameliorate hyperglycemia after being transplanted in diabetic mice.
In this study, we started with differentiating hESCs to PE, followed by transplant into db/+ GDM mouse and assessed the effect on their diabetic symptoms and fetal development. We hypothesize that these hESC-derived PE transplant could alleviate diabetic symptoms and reproductive outcome in pregnant mice, as well as improve early development of fetuses and offspring.
Section snippets
Study design
We differentiated hESCs to PE following established methods [13]. The PE was then transplanted into db/+ female mice (db/+PE), whereas db/+ mice receiving no transplants (db/+) and wild-type (wt) mice were used as controls. Four weeks after transplant, breeding was conducted overnight in a 1:2 ratio; mating was confirmed by the presence of a vaginal mucous plug the following morning, which represented gestation day (GD) 0 (GD0). Blood glucose levels, plasma insulin levels, and body weight were
Characterization of hESC-derived PE
Following previously established 4-stage differentiation protocol [13], we differentiated H1 hESCs to PE (see Methods and materials). Using immunofluorescence, we were able to confirm the cells expressed appropriate markers throughout the differentiation. At the beginning of stage 1, H1 hESC expressed the pluripotency marker OCT4 [19] (Fig. 1A, top row). At stage 4, cells lost OCT4 and exhibited PDX1 expression (Fig. 1A, bottom row), a key pancreatic transcription factor [20]. We further
Discussion
Gestational diabetes mellitus affects nearly 10% of all pregnancies, depending on the population studied and the diagnostic tests used, and females suffering from GDM have elevated long-term risk of type 2 diabetes mellitus later in life [1]. Therefore, studies on the mechanism of GDM hold great clinical value. Stem cell therapy using hESCs and human-induced pluripotent stem cells has been carried out in animal models in various reports and was able to effectively treat diabetes [13], [15].
Acknowledgment
The authors thank Duo Chen and Guangya Wang for the discussion. This study was funded by Hebei Talented Graduate Student Funding. The authors declare that there are no conflicts of interest.
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