Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure.

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus. Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures.

While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N(5)-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.

Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions.

UNLABELLED: Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. INTRODUCTION: Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. METHODS: cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, ß-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. RESULTS: In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. CONCLUSIONS: The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

Attenuated reovirus displays oncolysis with reduced host toxicity.

BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. METHODS: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. RESULTS: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated σ1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. CONCLUSION: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.

Mesodermal fate decisions of a stem cell: the Wnt switch.

Stem cells are a powerful resource for cell-based transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and non-canonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis.

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.

Molecular multiple endpoint embryonic stem cell test--a possible approach to test for the teratogenic potential of compounds.

The embryonic stem cell test (EST) examines the cytotoxicity of chemical compounds on embryonic stem (ES) cells and 3T3.A31 fibroblasts. Additionally, the EST measures the ability of ES cells to differentiate into contracting cardiomyocytes following drug exposure. In this study, we introduce new endpoints to obtain a molecular multiple endpoint EST (mme-EST), enabling the identification of potential chemical effects on osteogenic, chondrogenic and neural differentiation in addition to the traditional endpoint of cardiomyocyte differentiation. Six compounds in three classes with known teratogenic in vivo potential were assayed with the mme-EST in a pilot study: penicillin G (non-teratogenic), 5-fluorouracil and retinoic acid (strongly teratogenic), diphenylhydantoin, valproic acid and thalidomide (moderately teratogenic). While the traditional EST measures a morphological endpoint, we included molecular markers of differentiation as endpoints. With the mme-EST, every compound could be classified correctly according to its known teratogenic potential in vivo. Penicillin G, 5-fluorouracil and diphenylhydantoin inhibited differentiation of all endpoints equally. Interestingly, valproic acid showed the strongest inhibition of neural differentiation, while thalidomide specifically inhibited osteogenic development. Retinoic acid, on the other hand, supported neural but inhibited chondrogenic and osteogenic differentiation concentration-dependently. Valproic acid and thalidomide, classified incorrectly with the established EST model, were classified correctly with the mme-EST according to their effects on specific endpoints. This pilot study indicates that the predictive value of the EST may be enhanced by including further differentiation endpoints.

Molecular markers in embryonic stem cells.

Embryonic stem cells are pluripotent cells derived from mouse blastocysts, which have the capacity to differentiate in vitro into a wide variety of cell types. Based on this potential the embryonic stem cell test (EST) has been developed, which represents an assay system for the classification of compounds for their teratogenic potential, based on the morphological evaluation of contracting myocard cells compared to the cytotoxic effects on undifferentiated stem cells and adult 3T3 fibroblasts. To expand the EST, the quantitative expression of the alpha- and beta-myosin heavy chain (MHC) genes under the influence of test compounds was studied employing real-time TaqMan PCR analysis. The molecular evaluation of the MHC genes allows a higher sensitivity for the classification of substances and the transfer of the EST to the molecular level allows to start experimental procedures at day 9 of culture. Thus, the modulated EST holds promise as a new easily quantifiable in vitro screening assay in teratology.