RESUMO
Introduction: Measurement of pancreatic beta cell mass in animal models is a common assay in diabetes researches. Novel whole-organ clearance methods in conjunction with transgenic mouse models hold tremendous promise to improve beta cell mass measurement methods. Here, we proposed a refined method to estimate the beta cell mass using a new transgenic Tg(Pdx1-GFP) mouse model and a recently developed free-of-acrylamide clearing tissue (FACT) protocol. Methods: First, we generated and evaluated a Tg(Pdx1-GFP) transgenic mouse model. Using the FACT protocol in our model, we could quantify the beta cell mass and alloxan-induced beta cell destruction in whole pancreas specimens. Results: Compiled fluorescent images of pancreas resulted in enhanced beta cell mass characterization in FACT-cleared sections (2928869±120215 AU) compared to No-FACT cleared sections (1292372±325632 AU). Additionally, the total number of detected islets with this method was significantly higher than the other clearance methods (155.7 and 109, respectively). Using this method, we showed green fluorescent protein (GFP) expression confined to beta cells in Tg(Pdx1-GFP) transgenic. This enhanced GFP expression enabled us to accurately measure beta cell loss in a beta cell destruction model. The results suggest that our proposed method can be used as a simple, and rapid assay for beta cell mass measurement in islet biology and diabetes studies. Conclusion: The Tg(Pdx1-GFP) transgenic mouse in conjunction with the FACT protocol can enhance large-scale screening studies in the field of diabetes.
RESUMO
Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre-implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue-1 (LRH-1) and retinoic acid receptor gamma (RAR-γ), along with 2i/LIF (2a2iL) induces naïve-like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre-established hPSCs, and generation of new cell lines from blastocysts. 2a2iL-hPSCs match several defined criteria of naïve-like pluripotency and contribute to human-mouse interspecies chimeras. Activation of TGF-ß signaling is instrumental for acquisition of naïve-like pluripotency by the 2a2iL induction procedure, and transient activation of TGF-ß signaling substitutes for 2a to generate naïve-like hPSCs. We reason that 2a2iL-hPSCs are an easily attainable system to evaluate properties of naïve-like hPSCs and for various applications.
