Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge. We have developed a multistage procedure by which hESCs can be directly differentiated to hepatocyte-like cells without embryoid body formation and the requirement of sodium butyrate. The hESC-derived hepatocyte-like cells (HLCs) exhibited characteristic hepatocyte morphology, expressed hepatocyte markers, including alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha, and possessed hepatocyte-specific activities, such as p450 metabolism, albumin production, glycogen storage, and uptake and excretion of indocyanine green. Hepatocyte growth factor was found to play a positive role in promoting hepatocyte differentiation. Our differentiation system has shown that hESCs can be differentiated to hepatocyte-like cells capable of executing a range of hepatocyte functions. Therefore, it presents a proof-of-principle of potential applications of using the hESC-derived hepatocytes. Additionally, the hESC-derived HLCs provide a unique model to study the mechanisms involved in human hepatocyte differentiation and liver function.     

Notch信号通路调控猕猴胚胎干细胞的胆管样细胞分化(英文)

猕猴胚胎干细胞(rhesus monkey embryonic stem(rES))与人胚胎干细胞有相似的生物学特性,因此是理想的临床前研究的替代模型。Notch信号通路在胆管及胆管上皮细胞的形成中有重要的作用,然而,有关Notch信号通路在ES细胞的胆向分化中的作用了解甚少。该实验以rES为模型,对Notch信号通路对ES细胞的胆向分化过程中的作用进行了较为系统的研究。rES在细胞因子ActivinA诱导作用下产生约80%的限定性内胚层细胞。以Matrigel作为细胞外基质,在含BMP4和FGF1的无血清培养体系中继续诱导5～7d,rES细胞来源的限定性内胚层细胞分化产生约胆管样细胞。分化的细胞表达胆管细胞的特异性蛋白((CK7、CK18、CK19、CK20和OV-6)及基因(GSTPi、IB4和HNF1β)。在胆管样细胞的分化过程中检测到了Notch1和Notch2基因及下游信号分子hes1和hes5的表达。用Notch抑制剂L-685458处理分化过程中的细胞可导致Notch1和Notch2基因及下游信号分子hes1和hes5的表达下降,同时CK19阳性的胆管样细胞分化比率也从90%下降至约20%。这一...     

Osteoblastic differentiation of monkey embryonic stem cells in vitro

Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification.     

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

Sodium butyrate-treated embryonic stem cells yield hepatocyte-like cells expressing a glycolytic phenotype

Embryonic stem cells serve as a promising technology to obtain specific cell types for a number of biomedical applications. Because traditional techniques, such as embryoid body formation result in a wide array of differentiated cells such as hepatic, neuronal, and cardiac lineages, strategies have been utilized which favor cell-specific differentiation to generate more uniformity. In the present study, we have investigated the use of sodium butyrate in a monolayer culture configuration to mediate hepatocyte differentiation of murine embryonic stem cells. Several functional assays used to characterize hepatocyte function (viz. urea secretion, intracellular albumin content, cytokeratin 18, and glycogen staining) were used to analyze the differentiating cell population, suggesting the presence of an enriched population of hepatocyte-like cells. Since mature hepatocytes mediate energy metabolism predominantly through oxidative means as opposed to hepatocyte precursors, which are primarily glycolytic, we have performed a kinetic analysis of glycolytic and functional capacity to characterize the differentiated cells. In conjunction with mitochondrial mass and activity measurements, we show that Na-butyrate-mediated differentiated cells mediate energy metabolism predominantly through glycolysis. This metabolic and mitochondrial characterization can assist in evaluating stem cell differentiation and may prove useful in identifying key regulatory molecules in mediating further differentiation.     

Cardiomyocyte differentiation from embryonic and adult stem cells

In recent years multiple reports indicating that embryonic as well as adult stem cells can differentiate to cardiomyocytes have ignited discussions on whether these stem cells could lead to new therapies for patients with heart disease. Recent developments have been made in the generation of cardiomyocytes from both embryonic and adult stem cells, and progress towards clinical applications in patients with heart failure has been made. Nevertheless, controversies surrounding safety and transdifferentiation issues will need to be overcome before these stem cell approaches can reach their full potential.     

Directed differentiation of dendritic cells from mouse embryonic stem cells

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.     

Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are mesoderm-derived cells that are considered a good source of somatic cells for treatment of many degenerative diseases. Previous studies have reported the differentiation of mesodermal MSCs into endodermal and ectodermal cell types beyond their embryonic lineages, including hepatocytes and neurons. However, the molecular pathways responsible for the direct or indirect cell type conversion and the functional ability of the differentiated cells remain unclear and need further research. In the present study, we demonstrated that valproic acid (VPA), which is a histone deacetylase inhibitor, induced an increase in the expression of endodermal genes including CXCR4, SOX17, FOXA1, FOXA2, GSC, c-MET, EOMES, and HNF-1β in human umbilical cord derived MSCs (hUCMSCs). In addition, we found that VPA is able to increase these endodermal genes in hUCMSCs by activating signal transduction of AKT and ERK. VPA pretreatment increased hepatic differentiation at the expense of adipogenic differentiation. The effects of VPA on modulating hUCMSCs fate were diminished by blocking AKT and ERK activation using specific signaling inhibitors. Together, our results suggest that VPA contributes to the lineage conversion of hUCMSCs to hepatic cell fate by upregulating the expression of endodermal genes through AKT and ERK activation.     

Hematopoietic differentiation of embryonic stem cells

This review is focused on methods that are used to derive hematopoietic cells from embryonic stem cells (ESCs). One of the strategies that have been recently used to achieve this goal is an approach of mimicking the hematopoietic niche in vitro by using hematopoiesis-supportive feeder cells, cocktails of soluble hematopoietic growth factors and a variety of matrices. While there is clear evidence that it is possible to derive hematopoietic stem cells (HSCs) and subsequently committed hematopoietic progenitors and mature cells from ESCs, there remains the need to address multiple issues including the efficiency of HSCs derivation in vitro and their proper functionality.     

Enhancement of cardiomyocyte differentiation from human embryonic stem cells

Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2'-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2'-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.     

胚胎干细胞向造血系统的分化

胚胎干细胞是指从囊胚期的内细胞团中分离出来的尚未分化的胚胎细胞,可分化形成各种组织类型。在合适的条件下,胚胎干细胞可发育成造血干细胞及各类成熟血细胞,为造血干细胞移植及血细胞输注开辟了新的来源,同时也为造血发生及造血调控研究提供了有效可靠的模型。本文将综述ES细胞向造血系统分化的诱导条件、调控机制及应用前景。     

Evaluation of hepatotoxicity of chemicals using hepatic progenitor and hepatocyte-like cells derived from mouse embryonic stem cells

Embryonic stem cell testing is an alternative model system to assess drug and chemical toxicities because of its similar developmental characteristics with in vivo embryogenesis and organogenesis. This study evaluated the toxicity of chemicals at specific developmental stages of mouse embryonic stem cell (ESC)-derived hepatic differentiation; hepatic progenitor cells (HPCs), and hepatocyte-like cells (HCs). The toxic effects of carbon tetrachloride (CCl₄), 5-fluorouracil (5-FU), and arsanilic acid (Ars) were evaluated by measuring the expressions of Cytokeratin (CK18) and GATA binding protein 4 (GATA-4) and the activities of aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) during the hepatic differentiation process. Non-toxic doses of three chemicals at a range of 25 to 500 μM for CCl₄, 12.5 to 800 nM for 5-FU and 6.25 to 400 mM for Ars were treated. In the CCl₄-treated group, significant decreases (P?<?0.05) of the marker expression were observed by more than 300 μM from day 10 in CK18 and by more than 400 μM of CCl₄ from day 22 in GATA-4, respectively. However, both markers were decreased (P?<?0.01) by treatments of all doses at day 40. In the 5-FU-treated group, the expressions of two proteins were not affected by any of the doses at day 10 and 22, whereas the GATA-4 expression was decreased (P?<?0.05) by more than 400 nM of 5-FU at days 28 and 40. In the Ars-treated group, the CK18 expression was inhibited (P?<?0.05) by more than 100 mM of Ars at day 22 but showed a tendency to recover. Although the GATA-4 was inhibited by all doses at day 22, the inhibition of GATA-4 recovered at days 28 and 40. ALP activities of three chemicals were significantly increased (P?<?0.05) by a dose-dependent manner. The activities of AST and LDH were prone to be increased by more than 300 μM of CCl_4, but not affected by all doses of 5-FU except for 800 nM of 5-FU in AST activities. In the Ars, the enzyme activities were significantly increased (P?<?0.05) by more than 50 μM of Ars in AST and more than 6.25 μM of Ars in LDH. The present results indicate that CCl₄ has a more toxic effect on HCs, whereas Ars is more toxic to HPCs. Additionally, in vitro alternative testing using ESC-derived HPCs and HCs could provide useful information on chemical toxicity during the hepatic differentiation process and could be a useful model system for assessing chemical hepatotoxicity.     

Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes

Recent success in the derivation of haploid embryonic stem cells (haESCs) from mouse via parthenogenesis and androgenesis has enabled genetic screening in mammalian cells and generation of gene-modified animals. However, whether haESCs can be derived from primates remains unknown. Here, we report the derivation of haESCs from parthenogenetic blastocysts of Macaca fascicularis monkeys. These cells, termed as PG-haESCs, are pluripotent and can differentiate to cells of three embryonic germ layers in vitro or in vivo. Interestingly, the haploidy of one monkey PG-haESC line (MPH1) is more stable compared with that of the other one (MPH2), as shown by the existence of haploid cells for more than 140 days without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Importantly, transgenic monkey PG-haESC lines can be generated by lentivirus- and piggyBac transposon-mediated gene transfer. Moreover, genetic screening is feasible in monkey PG-haESCs. Our results demonstrate that PG-haESCs can be generated from monkeys, providing an ideal tool for genetic analyses in primates.     

The differentiation of early embryonic stem cells into adipocytes-like cells

An early embryonic stem cell line, EES-6 cells, was established from 2-cell stage embryos of ddY mice. The cells were maintained in an undifferentiated state with D-MEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (GM) and 1 ng of leukemia inhibitory factor (LIF) without any feeder cells. In this study, EES cells were cultured with a medium containing embryotrophic factors (ETFs) which promoted the differentiation of EES cells into white and brown adipocytes-like cells for a period of 5 days. Lipid droplets in brown adipocyte-like cells were stained with Sudan III; however, large lipid-like droplets in white or brown adipocyte-like cells were unstained with either Sudan III or alcian blue. These findings have numerous possibilities for therapeutic use such as regeneration of skin and wound healing.