Characterization of heterozygous ATTR Tyr114Cys amyloidosis-specific induced pluripotent stem cells.

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC-MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.

Gut-liver microphysiological systems revealed potential crosstalk mechanism modulating drug metabolism.

The small intestine and liver play important role in determining oral drug's fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study, we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid, enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

A specific plasma amino acid profile in the Insulin2Q104del Kuma mice at the diabetic state and reversal from hyperglycemia.

The metabolites in the plasma serve as potential biomarkers of disease. We previously established an early-onset diabetes mouse model, Ins2+/Q104del Kuma mice, under a severe immune-deficient (Rag-2/Jak3 double-deficient in BALB/c) background. Here, we revealed the differences in plasma amino acid profiles between Kuma and the wild-type mice. We observed an early reduction in glucogenic and ketogenic amino acids, a late increase in branched-chain amino acids (BCAAs) and succinyl CoA-related amino acids, and a trend of increasing ketogenic amino acids in Kuma mice than in the wild-type mice. Kuma mice exhibited hyperglucagonemia at high blood glucose, leading to perturbations in plasma amino acid profiles. The reversal of blood glucose by islet transplantation normalized the increases of the BCAAs and several aspects of the altered metabolic profiles in Kuma mice. Our results indicate that the Kuma mice are a unique animal model to study the link between plasma amino acid profile and the progression of diabetes for monitoring the therapeutic effects.

Selective proteasome degradation of C-terminally-truncated human WFS1 in pancreatic beta cells.

Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1 proteins by studying the stability of full-length wild-type (WT) WFS1, a missense mutant P724L, and two C-terminally truncated mutants, W837X and Y652X. We compared their stability by overexpressing them in MIN6 and HEK293T cells. The C-terminally truncated mutants W837X and Y652X are degraded more rapidly than the missense P724L mutant or wild-type WFS1 in MIN6 cells. In contrast, Y652X is more stable than WT or other mutant WFS1 proteins in HEK293T. In conclusion, we found that C-terminally truncated WFS1 mutants are selectively degraded in a cell type-specific manner.

Activation of cAMP (EPAC2) signaling pathway promotes hepatocyte attachment.

Primary Human Hepatocyte (PHH) remains undefeated as the gold standard in hepatic studies. Despite its valuable properties, partial attachment loss due to the extraction process and cryopreservation remained the main hurdle in its application. We hypothesized that we could overcome the loss of PHH cell attachment through thawing protocol adjustment and medium composition. We reported a novel use of a medium designed for iPSC-derived hepatocytes, increasing PHH attachment on the collagen matrix. Delving further into the medium composition, we discovered that removing BSA and exposure to cAMP activators such as IBMX and Forskolin benefit PHH attachment. We found that activating EPAC2, the cAMP downstream effector, by S-220 significantly increased PHH attachment. We also found that EPAC2 activation induced bile canaliculi formation in iPS-derived hepatocytes. Combining these factors in studies involving PHH or iPS-hepatocyte culture provides promising means to improve cell attachment and maintenance of hepatic function.

The Effect of Vitamin D3 and Valproic Acid on the Maturation of Human-Induced Pluripotent Stem Cell-Derived Enterocyte-Like Cells.

Cytochrome P450 3A4 (CYP3A4) is involved in first-pass metabolism in the small intestine and is heavily implicated in oral drug bioavailability and pharmacokinetics. We previously reported that vitamin D3 (VD3), a known CYP enzyme inducer, induces functional maturation of iPSC-derived enterocyte-like cells (iPSC-ent). Here, we identified a Notch activator and CYP modulator valproic acid (VPA), as a promotor for the maturation of iPSC-ent. We performed bulk RNA sequencing to investigate the changes in gene expression during the differentiation and maturation periods of these cells. VPA potentiated gene expression of key enterocyte markers ALPI, FABP2, and transporters such as SULT1B1. RNA-sequencing analysis further elucidated several function-related pathways involved in fatty acid metabolism, significantly upregulated by VPA when combined with VD3. Particularly, VPA treatment in tandem with VD3 significantly upregulated key regulators of enterohepatic circulation, such as FGF19, apical bile acid transporter SLCO1A2 and basolateral bile acid transporters SLC51A and SLC51B. To sum up, we could ascertain the genetic profile of our iPSC-ent cells to be specialized toward fatty acid absorption and metabolism instead of transporting other nutrients, such as amino acids, with the addition of VD3 and VPA in tandem. Together, these results suggest the possible application of VPA-treated iPSC-ent for modelling enterohepatic circulation.

Protocol to generate human pluripotent stem cell-derived pancreatic ß cells through methionine and zinc deprivation.

Efforts have been made to establish a differentiation protocol mimicking pancreatic development and to derive pancreatic ß cells for regenerative medicine. Here, we present an optimized pancreatic ß cell differentiation procedure using human pluripotent stem cells. We describe steps for a short 5-h methionine deprivation pretreatment followed by the application of zinc-deprived media at definitive endoderm differentiation stages to improve differentiation efficiency. The application of methionine and zinc deprivation facilitates the generation of functional pancreatic ß cells. For complete details on the use and execution of this protocol, please refer to Sim et al. (2022).1.

Generation of induced pluripotent stem cell-derived beta-cells in blood amino acids-like medium.

Traditional cell culture media do not accurately represent the availability of the nutrients in plasma. They usually contain a supraphysiological concentration of nutrients such as glucose, amino acids, etc. These high nutrients can alter the metabolism of cultured cells and induce metabolic phenotypes that do not reflect in vivo conditions. We demonstrate that the supraphysiological levels of nutrients interfere with endodermal differentiation. Refinement of media formulations has a potential application in maturity modulation of stem cell-derived ß-cells (SC-ß) generation in vitro. To address these issues, we established a defined culture system to derive SC-ß-cells using a blood amino acids-like medium (BALM). Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into the definitive endoderm, pancreatic progenitors, endocrine progenitors, and SC-ß in BALM-based med. The differentiated cells secreted C-peptide in vitro in response to high glucose levels and expressed several pancreatic ß-cell markers. In conclusion, amino acids at the physiological levels are sufficient for deriving functional SC-ß cells.

Promoting pancreatic ß cell proliferation: A powerful key for realizing regenerative therapy for diabetes.

This commentary is on a recent report on the regulation mechanism of beta cell proliferation.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor ß pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

Methionine metabolism regulates pluripotent stem cell pluripotency and differentiation through zinc mobilization.

Pluripotent stem cells (PSCs) exhibit a unique feature that requires S-adenosylmethionine (SAM) for the maintenance of their pluripotency. Methionine deprivation in the medium causes a reduction in intracellular SAM, thus rendering PSCs in a state potentiated for differentiation. In this study, we find that methionine deprivation triggers a reduction in intracellular protein-bound Zn content and upregulation of Zn exporter SLC30A1 in PSCs. Culturing PSCs in Zn-deprived medium results in decreased intracellular protein-bound Zn content, reduced cell growth, and potentiated differentiation, which partially mimics methionine deprivation. PSCs cultured under Zn deprivation exhibit an altered methionine metabolism-related metabolite profile. We conclude that methionine deprivation potentiates differentiation partly by lowering cellular Zn content. We establish a protocol to generate functional pancreatic ß cells by applying methionine and Zn deprivation. Our results reveal a link between Zn signaling and methionine metabolism in the regulation of cell fate in PSCs.

COVID-19-associated pulmonary aspergillosis in hemodialysis patients.

Background: Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) is a fatal complication in the general population. However, there are few reports on CAPA in patients undergoing hemodialysis (HD). Methods: This retrospective observational cohort study was conducted at a single center between December 2020 and June 2021. We enrolled 21 HD patients with COVID-19 undergoing treatment and divided them into two groups, CAPA and non-CAPA (COVID-19 with and without pulmonary aspergillosis), and evaluated their characteristics, clinical outcomes and comorbidities. Results: The log-rank test revealed that the 90-day survival rate after the initiation of treatment for COVID-19 was significantly lower in the CAPA (n = 6) than in the non-CAPA group (n = 15) (P = 0.0002), and the 90-day mortality rates were 66.6% and 0% in the CAPA and non-CAPA groups, respectively. In the CAPA group, four patients died due to respiratory failure (on Days 6 and 20), gastrointestinal bleeding (Day 8) and sepsis (Day 33); the reverse transcription-polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remained positive when they died. The remaining two patients survived and the negative conversion of RT-PCR for SARS-CoV-2 was confirmed on Days 10 and 15. The negative conversion of serum (1, 3)-ß-d-glucan (BDG) was confirmed on Day 15 in one patient; the BDG remained positive on Day 64 in the other. Conclusions: CAPA is a fatal complication in HD patients and the general population. Therefore, clinicians should consider the possibility of testing for CAPA in patients undergoing HD. Mycological workups may be helpful for the early detection of CAPA.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system.

Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

Generation of Human-Induced Pluripotent Stem Cell-Derived Functional Enterocyte-Like Cells for Pharmacokinetic Studies.

We aimed to establish an in vitro differentiation procedure to generate matured small intestinal cells mimicking human small intestine from human-induced pluripotent stem cells (iPSCs). We previously reported the efficient generation of CDX2-expressing intestinal progenitor cells from embryonic stem cells (ESCs) using 6-bromoindirubin-3'-oxime (BIO) and (3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT) to treat definitive endodermal cells. Here, we demonstrate the generation of enterocyte-like cells by culturing human iPSC-derived intestinal progenitor cells on a collagen vitrigel membrane (CVM) and treating cells with a simple maturation medium containing BIO, DMSO, dexamethasone, and activated vitamin D3. Functional tests further confirmed that these iPSC-derived enterocyte-like cells exhibit P-gp- and BCRP-mediated efflux and cytochrome P450 3A4 (CYP3A4)-mediated metabolism. We concluded that hiPS cell-derived enterocyte-like cells can be used as a model for the evaluation of drug transport and metabolism studies in the human small intestine.

Recent progress in pancreatic islet cell therapy.

Human pluripotent stem cells (PSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising cell sources in regenerating pancreatic islets through in vitro directed differentiation. Recent progress in this research field has made it possible to generate glucose-responsive pancreatic islet cells from PSCs. Single-cell RNA sequencing techniques have been applied to analyze PSC-derived endocrine beta-cells, which are then compared with human islets. This has led to the identification of novel signaling pathways and molecules involved in lineage commitment during pancreatic differentiation and maturation processes. Single-cell transcriptomics are also used to construct a detailed map of in vivo endocrine differentiation of developing mouse embryos to study pancreatic islet development. Mimicking those occurring in vivo, it was reported that differentiating PSCs can generate similar islet cell structures, while metabolomics analysis highlighted key components involved in PSC-derived pancreatic islet cell function, providing information for the improvement of in vitro pancreatic maturation procedures. In addition, cell transplantation into diabetic animal models, together with the cell delivery system, is studied to ensure the therapeutic potentials of PSC-derived pancreatic islet cells. Combined with gene-editing technology, the engineered mutation-corrected PSC lines originated from diabetes patients could be differentiated into functional pancreatic islet cells, suggesting possible autologous cell therapy in the future. These PSC-derived pancreatic islet cells are a potential tool for studies of disease modeling and drug testing. Herein, we outlined the directed differentiation procedures of PSC-derived pancreatic islet cells, novel findings through transcriptome and metabolome studies, and recent progress in disease modeling.

VMAT2 Safeguards ß-Cells Against Dopamine Cytotoxicity Under High-Fat Diet-Induced Stress.

Vesicular monoamine transporter 2 (VMAT2) uptakes cytoplasmic monoamines into vesicles for storage. VMAT2 plays a role in modulating insulin release by regulating dopamine levels in the pancreas, although the exact mechanism remains elusive. We found that VMAT2 expression in ß-cells specifically increases under high blood glucose conditions. The islets isolated from ß-cell-specific Vmat2 knockout (ßVmat2KO) mice show elevated insulin secretion levels in response to glucose stimulation. Under prolonged high-fat diet feedings, the ßVmat2KO mice exhibit impaired glucose and insulin tolerance and progressive ß-cell dysfunction. Here we demonstrate VMAT2 uptake of dopamine to protect dopamine from degradation by monoamine oxidase, thereby safeguarding ß-cells from excess reactive oxygen species (ROS) exposure. In the context of high demand for insulin secretion, the absence of VMAT2 leads to elevated ROS in ß-cells, which accelerates ß-cell dedifferentiation and ß-cell loss. Therefore, VMAT2 controls the amount of dopamine in ß-cells, thereby protecting pancreatic ß-cells from excessive oxidative stress.

Detailed analysis at a single-cell level of cells undergoing pancreatic differentiation.

Pancreatic cells derived from pluripotent stem cells using the stem cell-derived-ß (SC-ß) differentiation protocol are composed of four main cell types, namely ß-like cells (SC-ß-cells), α-like cells, enterochromaffin-like cells and non-endocrine cells. Single-cell dissociation and reaggregation depleted non-endocrine cells and improved ß-cell function. Veres et al. succeeded in efficiently purifying SC-ß by magnetic cell sorting using CD49a antibody and reaggregation, so that the final SC-ß-cell ratio increased to 80%.

Plasticity of histone modifications around Cidea and Cidec genes with secondary bile in the amelioration of developmentally-programmed hepatic steatosis.

We recently reported that a treatment with tauroursodeoxycholic acid (TUDCA), a secondary bile acid, improved developmentally-deteriorated hepatic steatosis in an undernourishment (UN, 40% caloric restriction) in utero mouse model after a postnatal high-fat diet (HFD). We performed a microarray analysis and focused on two genes (Cidea and Cidec) because they are enhancers of lipid droplet (LD) sizes in hepatocytes and showed the greatest up-regulation in expression by UN that were completely recovered by TUDCA, concomitant with parallel changes in LD sizes. TUDCA remodeled developmentally-induced histone modifications (dimethylation of H3K4, H3K27, or H3K36), but not DNA methylation, around the Cidea and Cidec genes in UN pups only. Changes in these histone modifications may contribute to the markedly down-regulated expression of Cidea and Cidec genes in UN pups, which was observed in the alleviation of hepatic fat deposition, even under HFD. These results provide an insight into the future of precision medicine for developmentally-programmed hepatic steatosis by targeting histone modifications.

Fetal androgen signaling defects affect pancreatic ß-cell mass and function, leading to glucose intolerance in high-fat diet-fed male rats.

We previously demonstrated that androgen signaling expands pancreatic ß-cell mass in the sexual maturation period (Am J Physiol Endocrinol Metab 314: E274-E286, 2018). The aim of this study was to elucidate whether fetal androgen signaling plays important roles in ß-cell mass development and ß-cell function in adulthood, defects of which are associated with type 2 diabetes mellitus. In the pancreas of male fetuses, androgen receptor (AR) was strongly expressed in the cytoplasm and at the cell membrane of Nkx6.1-positive ß-cell precursor cells but was markedly reduced in insulin-positive ß-cells. Administration of the anti-androgen flutamide to pregnant dams during late gestation reduced ß-cell mass and Ki67-positive proliferating ß-cells at birth in a male-specific manner without affecting body weight. The decrease of ß-cell mass in flutamide-exposed male rats was not recovered when rats were fed a standard diet, whereas it was fully recovered when rats were fed a high-fat diet (HFD), at 6 and 12 wk of age. Flutamide exposure in utero led to the development of glucose intolerance in male rats due to a decrease in insulin secretion when fed HFD but not standard diet. Insulin sensitivity did not differ between the two groups irrespective of diet. These results indicated that the action of fetal androgen contributed to ß-cell mass expansion in a sex-specific manner at birth and to the development of glucose intolerance by decreasing the secretion of insulin in HFD-fed male rats. Our data demonstrated the involvement of fetal androgen signaling in hypothesized sex differences in the developmental origins of health and disease by affecting pancreatic ß-cell function.

A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells.

Insulin-producing and -secreting cells derived from mouse pluripotent stem cells (PSCs) are useful for pancreatic development research and evaluating drugs that may induce insulin secretion. Previously, we have established a differentiation protocol to derive insulin-secreting cells from mouse embryonic stem cells (ESCs) using a combination of growth factors, recombinant proteins, and a culture substratum with net-like fibers. However, it has not been tested which materials and diameters of these fibers are more effective for the differentiation. Therefore, the present study aimed to produce net-like culture substratum formed from polyamide (PA) and polyacrylonitrile (PAN) fibers. Substrata were delineated into PA100, 300, 600, PAN100, 300, and 600 groups based on fiber diameters. The differentiation efficiencies of mouse ESCs cultured on the substrata were then examined by insulin 1 (Ins1) expression. Expression was found to be highest in PA300 differentiated cells, indicating the potential to produce high levels of insulin. To understand any differences in substratum properties, the adsorption capacities of laminin were measured, revealing that PA300 had the highest for it. We next examined the stage of differentiation affected by incubation with PA300. This showed that Sox17- and Pdx1-GFP-positive cells increased during the first step of differentiation. To show the production of insulin without absorption from the medium, we confirmed the expression of insulin C-peptide after differentiation. Finally, we tested the effects of PA300 on the differentiation of human-induced PSC, and found more Sox17-positive cells with the PA300 substratum at the definitive endoderm stage. Furthermore, these cells expressed insulin C-peptide and had glucose-responsive C-peptide secretion. In summary, our study identified and validated a novel substratum which is suitable for pancreatic differentiation of mouse and human PSCs.