Activin A and Wnt-dependent specification of human definitive endoderm cells

Activin/Nodal and Wnt signaling are known to play important roles in the regional specification of endoderm. Here we have investigated the effect of the length of stimulation with Activin A plus Wnt3a on the development of hepatic and pancreatic progenitors from the definitive endoderm (DE) cells derived from human pluripotent stem cells (hPSC). We show that DE-cells derived from hPSC with 3 days high Activin A and Wnt3a treatment were able to differentiate further into both tested endodermal lineages. When prolonging the DE-induction protocol from 3 to 5 or 7 days, almost pure DE-marker positive cell populations were obtained. However, these cells had an impaired pancreatic differentiation capacity, while they still developed into hepatocyte-like cells. Further propagation of the DE-cells in the presence of Wnt3a and Activin A led to the complete loss of differentiation capacity into hepatic or pancreatic lineages. When Wnt3a was removed after 24 h from the initiation of the differentiation, the cells were able to differentiate into PDX1+/NKX6.1+ pancreatic progenitors even with longer DE induction time while efficiency of hepatic differentiation was lower. Our results suggest that both the length and the timing of Wnt3a treatment during DE induction are crucial for the final differentiation outcome. Although it is possible to derive apparently pure DE cells with prolonged Activin A/Wnt-stimulation, their progenitor capacity is restricted to a limited time window.     

The applications of induced pluripotent stem (iPS) cells in drug development

The introduction of induced pluripotent stem (iPS) cells has been a milestone in the field of regenerative medicine and drug discovery. iPS cells can provide a continuous and individualized source of stem cells and are considered to hold great potential for economically feasible personalized stem cell therapy. Various diseases might potentially be cured by iPS cell-based therapy including Parkinson’s disease, Alzheimer’s disease, Huntington disease, ischemic heart disease, diabetes and so on. Moreover, iPS cells derived from patients suffering from unique incurable diseases can be developed into patient- and disease-specific cell lines. These cells can be used as an effective approach to study the mechanisms of diseases, providing useful tools for drug discovery, development and evaluation. The development of suitable methods for the culture and expansion of iPS cells and their differentiated progenies make feasible modern drug discovery techniques such as high-throughput screening. Furthermore, iPS cells can be applied in the field of toxicological and pharmacokinetics tests. This review focuses on the applications of iPS cells in the field of pharmaceutical industry.     

Three-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes

Background aims

Human induced pluripotent stem cells (hiPSCs) are becoming increasingly popular in research endeavors due to their potential for clinical application; however, such application is challenging due to limitations such as inferior function and low induction efficiency. In this study, we aimed to establish a three-dimensional (3D) culture condition to mimic the environment in which hepatogenesis occurs in vivo to enhance the differentiation of hiPSCs for large-scale culture and high throughput BAL application.

Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.     

人多能干细胞向红细胞的诱导分化

目前,体外生成人红细胞的实验技术较为复杂,为优化诱导步骤,采用两步法将人多能干细胞诱导分化为红细胞。首先,利用人多能干细胞(包括Rh阴性A型的脐带间充质干细胞(hUCMSC~(Rh-A))和人iPS(hiPS)细胞)在BVF培养液中进行诱导分化得到CD31~~+和CD34~+的阳性细胞群。经PCR和流式细胞仪检测CD31和CD34的表达发现,hUCMSC~(Rh-A)细胞诱导得到的CD31和CD34阳性细胞率分别是5.3%和22.7%;hiPS细胞诱导得到的CD31和CD34阳性细胞率分别是31.2%和8.2%。第二步,将获得的CD31~+和CD34~+的阳性细胞群在多种生长因子的作用下经过36 d诱导,分化为成熟红细胞。经吉姆萨染色检测得到的红细胞在形态和大小上与正常人红细胞相近,且存在血细胞去核的现象。荧光定量RT-PCR检测到了globin的表达,其中β-globin的表达量占20%以上。将得到的红细胞收集到离心管中,自然沉降后可见红色的红细胞沉淀。上述研究为大量制备人红细胞提供了新的有效的技术方法。     

Modelling familial dysautonomia in human induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have considerable promise as a novel tool for modelling human disease and for drug discovery. While the generation of disease-specific iPS cells has become routine, realizing the potential of iPS cells in disease modelling poses challenges at multiple fronts. Such challenges include selecting a suitable disease target, directing the fate of iPS cells into symptom-relevant cell populations, identifying disease-related phenotypes and showing reversibility of such phenotypes using genetic or pharmacological approaches. Finally, the system needs to be scalable for use in modern drug discovery. Here, we will discuss these points in the context of modelling familial dysautonomia (FD, Riley-Day syndrome, hereditary sensory and autonomic neuropathy III (HSAN-III)), a rare genetic disorder in the peripheral nervous system. We have demonstrated three disease-specific phenotypes in FD-iPS-derived cells that can be partially rescued by treating cells with the plant hormone kinetin. Here, we will discuss how to use FD-iPS cells further in high throughput drug discovery assays, in modelling disease severity and in performing mechanistic studies aimed at understanding disease pathogenesis. FD is a rare disease but represents an important testing ground for exploring the potential of iPS cell technology in modelling and treating human disease.     

The genomic stability of induced pluripotent stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of human patients with defined factors, hold promise for regenerative medicine because they can provide a renewable source of autologous cells for cell therapy without the concern for immune rejection. In addition, iPSCs provide a unique opportunity to model human diseases with complex genetic traits, and a panel of human diseases have been successfully modeled in vitro by patient-specific iPSCs. Despite these progresses, recent studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to the immunogenicity of some cells differentiated from iPSCs. The oncogenic potential of iPSCs is further underscored by the findings that the critical tumor suppressor p53, known as the guardian of the genome, suppresses induced pluripotency. Therefore, the clinic application of iPSCs will require the optimization of the reprogramming technology to minimize the genetic and epigenetic abnormalities associated with induced pluripotency.     

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.     

T lineage differentiation from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have potential to differentiate into T lymphocytes, however, the actual ability of iPS cells to develop into T lineages is not clear. In this study, we co-cultured iPS cells on OP9 cells expressing the Notch ligand Delta-like 1 (DL1), the iPS cells differentiated into T lymphocytes. In addition, in vitro stimulation of iPS cell-derived T lymphocytes resulted in secretion of IL-2 and IFN-γ. Moreover, adoptive transfer of iPS cell-derived T lymphocytes into Rag-deficient mice reconstituted their T cell pools. These results indicate that iPS cells are able to follow the normal program of T cell differentiation.     

Biomaterial aided differentiation and maturation of induced pluripotent stem cells

Engineering/reprogramming differentiated adult somatic cells to gain the ability to differentiate into any type of cell lineage are called as induced pluripotent stem cells (iPSCs). Offering unlimited self-renewal and differentiation potential, these iPSC are aspired to meet the growing demands in the field of regenerative medicine, tissue engineering, disease modeling, nanotechnology, and drug discovery. Biomaterial fabrication with the rapid evolution of technology increased their versatility and utility in regenerative medicine and tissue engineering, revolutionizing the stem cell biology research with the property to guide the process of proliferation, differentiation, and morphogenesis. Combining traditional culture platforms of iPSC with biomaterials aids to overcome the limitations associated with derivation, proliferation, and maturation, thereby could improve the clinical translation of iPSC. The present review discusses in brief about the reprogramming techniques for the derivation iPSC and details on several biomaterial guided differentiation of iPSC to different cell types with specific relevance to tissue engineering/regenerative medicine.     

猪诱导性多能干细胞技术的研究进展及应用前景

猪作为实验材料,具有由于来源方便、基因序列与人类的相近及其在畜牧业中的重要地位等优势,成为国内外研究的热点,但是猪的胚胎干细胞(Embryonic stem cells,ESC)建系方面的研究进展缓慢。诱导性多能干细胞(induced pluripotent stem cells,iPSC)技术的诞生,开创了体细胞重编程的全新方法。猪iPSC体系的建立将为家畜ESC体系的建立奠定基础,同时也对提高猪转基因克隆的效率,高效育种和保种,乃至生物医学领域均产生深远的影响。文章综述了iPSC技术的主要进展,重点阐述了猪iPSC技术的现状及其在生物医学和畜牧业中的应用前景,以期为从事该领域研究的科研人员提供参考。     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca(2+) transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) 'Ca(2+)-release units', which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.     

Generation of functional gut-like organ from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have the pluripotency to differentiate into broad spectrum derivatives of all three embryonic germ layers. However, the in vitro organ differentiation potential of iPS cells to organize a complex and functional “organ” has not yet been demonstrated. Here, we demonstrate that mouse iPS cells have the ability to organize a gut-like organ with motor function in vitro by a hanging drop culture system. This “induced gut (iGut)” exhibited spontaneous contraction and highly coordinated peristalsis accompanied by a transportation of contents. Ultrastructural analysis identified that the iGut had large lumens surrounded by three distinct layers (epithelium, connective tissue and musculature). Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICCs, enteric pacemaker cells), was observed in the wall of the lumen and formed a distinct and dense network. The neurofilament immunoreactivity was identified to form large ganglion-like structures and dense neuronal networks. The iGut was composed of all the enteric components of three germ layers: epithelial cells (endoderm), smooth muscle cells (mesoderm), ICCs (mesoderm), and enteric neurons (ectoderm). This is the first report to demonstrate the in vitro differentiation potential of iPS cells into particular types of functional “organs.” This work not only contributes to understanding the mechanisms of incurable gut disease through disease-specific iPS cells, but also facilitates the clinical application of patient-specific iPS cells for novel therapeutic strategies such as patient-specific “organ” regenerative medicine in the future.