In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of ...human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells.
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•scRNA-seq of hESC differentiation into β cells in vitro•Progenitor and endocrine compartments express different WNT pathway components•APC deletion in mouse endocrine precursors prevents endocrine differentiation•Small molecule inhibition of the WNT pathway in vitro increases endocrine yield
In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric islet transplantation as a cure for type 1 diabetes. Sharon et al. use scRNA-seq to identify the cell populations that form during the process and uncover a role for WNT pathway inhibition during endocrine differentiation.
The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses ...human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.
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•Scalable suspension system is established to generate hepatic lineages from hEnSCs•scRNA-seq is used to delineate hEnSC-derived hepatic cells and their counterparts•E-heps and E-chos are transcriptomically and functionally comparable with adult cells•Encapsulated E-heps are able to rescue rats with acute liver failure
Feng et al. report a scalable serum-free suspension culture system that uses human endoderm stem cells as precursors for mass production of functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). Single-cell transcriptomics analysis and functional assessment reveal that E-heps and E-chos are comparable with adult counterparts.
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 β 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.
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•Methionine depletion of PSCs reduces protein-bound Zn through homocysteine•Zn depletion of PSCs mimics methionine depletion and shows impaired pluripotency•Zn depletion impairs proliferation and potentiates endoderm differentiation of PSCs•PSCs cultured in Zn depletion showed altered methionine metabolism
Pluripotent stem cells (PSCs) are highly dependent on methionine. Sim et al. show that Zn signaling lies downstream of methionine metabolism. Methionine depletion of PSCs triggers a decreased intracellular protein-bound Zn. Zn depletion of PSCs mimicked the effects of methionine depletion, leading to impaired pluripotency and proliferation.
Plasma cells provide high-affinity antibodies against invading pathogens. Although transcriptional and epigenetic mechanisms have been extensively studied for plasma cell differentiation, how these ...mechanisms respond to environmental cues remains largely unexplored. In this study, we show that ascorbic acid (vitamin C), an essential nutrient, is able to promote plasma cell differentiation and humoral immune response by enhancing TET2/3-mediated DNA demethylation. Ascorbic acid treatment during B cell activation has persistent effects on later plasma cell differentiation by predisposing germinal center B cells toward the plasma cell lineage. Conversely, ascorbic acid deficiency in vivo blocks plasma cell differentiation and attenuates the humoral immune response following antigen immunization. We further demonstrate that such effects of ascorbic acid on plasma cell differentiation require DNA methylcytosine oxidases TET2 and TET3. Our study thus reveals a previously uncharacterized link between essential nutrients and epigenetic regulation of plasma cell differentiation and humoral immune response.
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•Ascorbic acid promotes plasma cell differentiation•Ascorbic acid accelerates cytosine demethylation at Blimp1 enhancers•Ascorbic acid predisposes germinal center B cells toward later plasma cell lineage•Ascorbic acid’s effects on plasma cells are dependent on DNA demethylases Tet2 and Tet3
Ascorbic acid (vitamin C) has been proposed to enhance immunity, but its mechanism of action remains controversial. Qi et al. find that ascorbic acid functions as the co-factor of TET enzymes in germinal center B cells. By activating TET2 and TET3, ascorbic acid promotes cytosine demethylation at Blimp1 enhancers and potentiates the activated B cells toward plasma cell lineage, thus enhancing the humoral immune response.
A drastic transition at birth, from constant maternal nutrient supply in utero to intermittent postnatal feeding, requires changes in the metabolic system of the neonate. Despite their central role ...in metabolic homeostasis, little is known about how pancreatic β cells adjust to the new nutritional challenge. Here, we find that after birth β cell function shifts from amino acid- to glucose-stimulated insulin secretion in correlation with the change in the nutritional environment. This adaptation is mediated by a transition in nutrient sensitivity of the mTORC1 pathway, which leads to intermittent mTORC1 activity. Disrupting nutrient sensitivity of mTORC1 in mature β cells reverts insulin secretion to a functionally immature state. Finally, manipulating nutrient sensitivity of mTORC1 in stem cell-derived β cells in vitro strongly enhances their glucose-responsive insulin secretion. These results reveal a mechanism by which nutrients regulate β cell function, thereby enabling a metabolic adaptation for the newborn.
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•Nutritional changes after birth alter β cell function•β cells acquire glucose response via changes in mTORC1 nutrient sensitivity•Glucose response is independent of changes in canonical β cell markers expression•Nutrient-regulated mTORC1 activity controls stem cell-derived β cell function
At birth, metabolic adaptations prepare the newborn for its new nutritional environment. Helman et al. demonstrate a role of nutrient sensing by mTORC1 in adjusting insulin secretion in neonatal β cells. Exploiting this adaptative property of β cells results in improved glucose-responsive insulin secretion in human stem cell-derived β cells.
Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), ...induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.
Stem-cell-derived tissues could transform disease research and therapy, yet most methods generate functionally immature products. We investigate how human pluripotent stem cells (hPSCs) differentiate ...into pancreatic islets in vitro by profiling DNA methylation, chromatin accessibility, and histone modification changes. We find that enhancer potential is reset upon lineage commitment and show how pervasive epigenetic priming steers endocrine cell fates. Modeling islet differentiation and maturation regulatory circuits reveals genes critical for generating endocrine cells and identifies circadian control as limiting for in vitro islet function. Entrainment to circadian feeding/fasting cycles triggers islet metabolic maturation by inducing cyclic synthesis of energy metabolism and insulin secretion effectors, including antiphasic insulin and glucagon pulses. Following entrainment, hPSC-derived islets gain persistent chromatin changes and rhythmic insulin responses with a raised glucose threshold, a hallmark of functional maturity, and function within days of transplantation. Thus, hPSC-derived tissues are amenable to functional improvement by circadian modulation.
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•Epigenome dynamics of human-stem-cell-derived islet differentiation and maturation•Pioneer factors coordinate pervasive enhancer priming to steer endocrine cell fates•Core regulatory circuits identify LMX1B as critical for endocrine cell generation•Circadian rhythms trigger islet maturation via clock-controlled metabolic cycles
Epigenomic studies reveal mechanisms driving differentiation of human stem cells into pancreatic islets and uncover a role for circadian rhythms in their functional maturation. Circadian-clock-entrained islets gain metabolic oscillations and stable chromatin changes that trigger cyclic insulin responses with a raised glucose threshold and function within days of transplant.
Embryonic stem cells (ESCs) are derived from the inner cell mass of developing blastocysts, which have self-renewal ability and have the potential to develop or reconstitute into all embryonic ...lineages. Selenophosphate synthetase 1 (SEPHS1) is an essential protein in mouse early embryo development. However, the role of SEPHS1 in mouse ESCs remains to be elucidated. In this study, we generated Sephs1 KO ESCs and found that deficiency of SEPSH1 has little effect on pluripotency maintenance and proliferation. Notably, SEPHS1 deficiency impaired differentiation into three germ layers and gastruloid aggregation in vitro. RNA-seq analysis showed SEPHS1 is involved in cardiogenesis, verified by no beating signal in Sephs1 KO embryoid body at d10 and low expression of cardiac-related and contraction markers. Taken together, our results suggest that SPEHS1 is dispensable in ESC self-renewal, but indispensable in subsequent germ layer differentiation especially for functional cardiac lineage.
•Sephs1 KO mouse embryonic stem cells was successfully derived and cultured in 2i/LIF serum-free medium.•SEPHS1 deficiency has little effect in ESC self-renewal and pluripotency maintenance.•SEPHS1 deficiency impairs three germ layer differentiation.•Sephs1 plays an essential role in heart development.•SEPHS1 is important during gastrula formation.
The telencephalon is one of the most-elaborated tissues. A broad variety of cell types is produced by spatiotemporally regulated mechanisms and is involved, in different combinations, in subregional ...formation. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsal–ventral (D–V) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP, respectively). An in vitro differentiation system has been achieved using mouse embryonic stem cells, and major telencephalic neurons can be obtained in this way; however, in using the in vitro differentiation system, many telencephalic neuron subtypes remain undifferentiated, although some of them are related to neuronal diseases. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction. A continuous arrangement of Emx1+/Pax6−, Emx1+/Pax6+, and Emx1−/Pax6+ cells was achieved in Foxg1+ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1+ cells resided in the VP fraction. These findings suggested that the D–V axis of the pallium was recapitulated in the in vitro–derived pallium.
•Inhibition of the TGFbeta signal was efficient for neural induction from mouse embryonic stem cells.•A continuous arrangement of pallial subregions along the dorsal–ventral axis was achieved in Foxg1+ neuroepithelia in vitro.•Dbx1+ cells resided at the in vitro–derived pallial–subpallial boundary.
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.
•hiPSC-derived intestinal progenitors are differentiated into enterocytes•The collagen vitrigel membrane supports hiPSCs to mature into functional enterocytes•iPSC-derived enterocytes show efflux transporter and metabolizing enzyme activities•iPSC-derived enterocytes can be used as a human small intestine model
We established a culture procedure to generate hiPSC-derived enterocyte-like cells that can be used as a model for the evaluation of drug transport and metabolism studies in the human small intestine.
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