Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates

We investigated the mechanism by which germline cells are recruited in every asexual reproductive cycle of the budding tunicate Polyandrocarpa misakiensis using a vasa homolog (PmVas) as the germline-specific probe. A presumptive gonad of Polyandrocarpa arose as a loose cell aggregate in the ventral hemocoel of a 1-week-old developing zooid. It developed into a compact clump of cells and then separated into two lobes, each differentiating into the ovary and the testis. The ovarian tube that was formed at the bottom of the ovary embedded the oogonia and juvenile oocytes, forming the germinal epithelium. PmVas was expressed strongly by loose cell aggregates, compact clumps, and peripheral germ cells in the testis and germinal epithelium. No signals were detected in growing buds and less than 1-week-old zooids, indicating that germ cells arise de novo in developing zooids of P. misakiensis. Cells of the loose cell aggregates were 5–6 μm in diameter. They looked like undifferentiated hemoblasts in the hemocoel. To examine the involvement of PmVas in the germline recruitment at postembryonic stages, both growing buds and 1-week-old developing zooids were soaked with double-stranded PmVas RNA. The growing buds developed into fertile zooids expressing PmVas, whereas the 1-week-old zooids developed into sterile zooids that did not express PmVas. In controls (1-week-old zooids) soaked with double-stranded lacZ RNA, the gonad developed normally. These results strongly suggest that in P. misakiensis, PmVas plays a decisive role in switching from coelomic stem cells to germ cells.     

Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates

The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

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.     

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.     

The differentiation of hepatocyte-like cells from monkey embryonic stem cells

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.     

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.     

Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro

H. Kataoka, T. K. Kobayashi, S. Amano, E. Yamada, M. Ishida, R. Kushima and H. Okabe Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro Objective: Primary culture of CD34 positive stem cells collected from human peripheral blood was performed with and without supplementation with concentrated ascitic fluid; morphological and immunocytochemical pictures of cultured cells were taken chronologically and compared. Methods: CD34‐positive stem cells collected from peripheral blood were cultured for 1, 24 and 48 hours. Concentrated ascitic fluid was added to the plates for the 24‐and 48‐hour cultures. For immunocytochemical studies, CD34, AE1/AE3, Ber‐Ep4 (EA), EMA, EGFR, CD31, CA125 and D2‐40 monoclonal antibodies were used. Results: After culture, small round cells with naked nuclei began to enlarge and to exhibit various changes in the cytoplasm and nucleus. Supplementation with concentrated body cavity fluid enhanced these changes. CD34‐positive cells with small round cell features were detected 1 hour after culture and these had no epithelial or mesothelial markers. After 24 hours, CD34‐positive cells had disappeared and cells weakly positive for EGFR, EMA, CA125 and D2‐40 were detected. Cells with strong and moderate positive reactions for EGFR, AE1/AE3, EA, EMA, D2‐40 and CA125 were detected after 48 hours. Supplementation with concentrated body cavity fluid increased the intensity and number of positive cells for these markers compared with the control group. The positive reaction, not only for the epithelial markers such as EGFR and AE1/AE3, but also for mesothelial markers such as CA125 and D2‐40, was found to be increased in small numbers of cells in direct proportion to the duration of the primary culture of the peripheral blood cells. CD31, characteristically expressed in endothelial cells, was negative in the cultured cells. Conclusion: Supplementation of peripheral blood CD34‐positive stem cells with body cavity fluid in vitro enhanced their differentiation toward cells of an epithelial or mesothelial phenotype, concomitant with loss of immunoreactivity for CD34. It is assumed that the routine cytological observation of cells obtained from body cavity fluid might cause possible cytomorphological and immunophenotypical changes due to the action of the growth factors contained in the body cavity fluid.     

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.     

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.     

Ginsenoside-Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells

Neural stem cells cultured as neurospheres were used to assess the effects of P. notoginseng on the production of neurons and glia. The crude saponins (PNS) and ginsenoside-Rd promote the differentiation of neurospheres into astrocytes. Ginsenoside-Rd increases the production of astrocytes in a dose-dependent manner. On the other hand, both PNS and ginsenoside-Rd induce a weak but significant effect by decreasing the number of neurons. The other ginsenosides do not induce any differentiation on both neurons and astrocytes.     

Role of Vasa, Piwi, and Myc-expressing coelomic cells in gonad regeneration of the colonial tunicate, Botryllus primigenus

In the colonial tunicate, Botryllus primigenus Oka, gonads consist of indifferent germline precursor cells, the primordial testis and ovary, and mature gonads, of which the immature gonad components can be reconstructed de novo in vascular buds that arise from the common vascular system, although the mechanism is uncertain. In this study, we investigated how and what kinds of cells regenerated the gonad components. We found that few Vasa-positive cells in the hemocoel entered the growing vascular bud, where their number increased, and finally developed exclusively into female germ cells. Simultaneously, small cell aggregates consisting of Vasa(-) and Vasa(±) cells appeared de novo in the lateral body cavity of developing vascular buds. Double fluorescent in situ hybridization showed that these cell aggregates were both Piwi- and Myc-positive. They could form germline precursor cells and a primordial testis and ovary that strongly expressed Vasa. Myc knockdown by RNA interference conspicuously lowered Piwi expression and resulted in the loss of germline precursor cells without affecting Vasa(+) oocyte formation. Myc may contribute to gonad tissue formation via Piwi maintenance. When human recombinant BMP 4 was injected in the test vessel, coelomic Piwi(+) cells were induced to express Vasa in the blood. We conclude, therefore, that in vascular buds of B. primigenus, female germ cells can develop from homing Vasa(+) cells in the blood, and that other gonad components can arise from coelomic Vasa(-)/Piwi(+)/Myc(+) cells.     

转录因子对干细胞高效定向分化为β细胞的作用

移植干细胞分化的胰岛β细胞能逆转糖尿病鼠的高血糖症,为细胞疗法临床应用于糖尿病治疗提供依据。但如何诱导干细胞高效、定向分化为具有分泌功能的胰岛β细胞,仍是当今世界难题。就影响干细胞分化为胰岛β细胞的关键转录因子PDX-1、Ngn3、NeuroD1、MafA的作用及机制进行综述,为干细胞高效定向分化提供新的思路。     

Multilineage differentiation of muscle-derived stem cells from GFP transgenic mice

It is unclear whether green fluorescent protein (GFP) expression is maintained during the course of multilineage differentiation of muscle-derived stem cells (MDSCs). We isolated MDSCs from GFP-transgenic mice and transferred them to chondrogenic, neurogenic or myogenic media. Multilineage differentiation was examined by morphological observation, histological staining, immunocytochemical staining, real-time RT-PCR and Western blot. Both differentiated cells and non-differentiated cells maintained stable GFP expression until the cells exhibited a senescent phenotype. Thus, MDSCs from GFP-transgenic mice have multilineage potential in vitro and that GFP expression does not influence the multilineage potential of MDSCs (or vice versa).     

Chemical and physical stimuli induce cardiomyocyte differentiation from stem cells

In this study we investigated cardiomyocyte differentiation of rat bone marrow-mesenchymal stem cells (BM-MSCs) by treating the stem cells with conditions mimicking that of myocardial infarction. The extract from infarcted rat myocardium contained the biochemical factors arising after infarction. The cardiac contraction and relaxation were simulated by applying 4% strain at 1 Hz to the stem cells. We found that the extract from infarcted myocardium or 4% strain each alone could induce cardiomyocyte differentiation of BM-MSCs, as shown by expression of cardiomyocyte-specific genes including α-actin, connexin 43, Nkx2.5, MEF2c, GATA4, α-MHC, and Troponin I. Furthermore, a combination of the extract and 4% strain had stronger effects on cardiomyocyte differentiation than what either treatment alone had. Our results suggest that this in vitro model system simulates the local cardiac environment cues after infarction and may be useful in identifying the biochemical and physical factors involved in cardiomyocyte differentiation.     

Carbon nanotubes promote neuron differentiation from human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine and transplantation therapy due to their self-renewal and pluripotent properties. We report that 2D thin film scaffolds composed of biocompatible polymer grafted carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells into neuron cells while maintaining excellent cell viability. According to fluorescence image analysis, neuron differentiation efficiency of poly(acrylic acid) grafted CNT thin films is significant greater than that on poly(acrylic acid) thin films. When compared with the conventional poly-l-ornithine surfaces, a standard substratum commonly used for neuron culture, this new type thin film scaffold shows enhanced neuron differentiation. No noticeable cytotoxic effect difference has been detected between these two surfaces. The surface analysis and cell adhesion study have suggested that CNT-based surfaces can enhance protein adsorption and cell attachment. This finding indicates that CNT-based materials are excellent candidates for hESCs’ neuron differentiation.