Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells

Background: The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined. Methodology/Principal Findings: P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a \u27ground\u27 state after the inhibition had been removed. Conclusions/Significance: Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular \u27fate\u27 decision making processes. © 2011 Krawetz et al

Inhibition of Rho Kinase Regulates Specification of Early Differentiation Events in P19 Embryonal Carcinoma Stem Cells

The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed.Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular 'fate' decision making processes

Enhanced bone repair using embryonic stem cells in a mouse model of impaired fracture healing

Bibliography: p. 173-183Some pages are in colour.Includes copy of ethics approval and copyright permission. Original copies with original Partial Copyright Licence.Fractures that occur in osteoporotic bone typically demonstrate impaired healing or non-union of the bony ends and current treatment methods for these types of injuries are inadequate. The field of tissue engineering offers promising treatment alternatives for diseases and injuries that affect tissues with a limited capacity for repair. Embryonic stem (ES) cells offer advantages over other stem cells types in that these cells are pluripotent, possessing the ability to generate any cell type in the body, and have a high capacity for self-renewal. This thesis describes the development of a novel product for the augmentation of bone fracture repair. Using protocols previously developed by our group, we initially attempted to differentiate bioreactor-expanded ES cells into osteoblasts and chondrocytes in stirred suspension culture systems. Surprisingly, we found that the bioreactor culture environment promoted ES cell pluripotency, resulting in limited cell differentiation. Through further experimentation, we found that exposure to a collagen I extracellular matrix induced osteoblastic differentiation of ES cells with greater efficacy than medium supplementation. Furthern1ore, the cell-loaded collagen constructs formed mineralized tissue nodules following subcutaneous implantation into immune-compromised mice. Subsequently, we developed a fracture model, using both nonnal and osteoporotic mice, to test the efficacy of the constructs to contribute to bone repair at an orthotopic site in vivo. Bone formation was enhanced in the mice treated with cell-loaded collagen constructs and the implanted cells were identified at the fracture site for up to 8 weeks. The formation of a small ectopic soft tissue mass in some of the recipient mice suggests that the collagen I matrix reduced but did not eliminate the tumorigenic potential of the cells. To our knowledge, the studies presented herein are the first of their kind to demonstrate the efficacy of a stem cell therapy for fracture repair in osteoporotic bone in vivo. The findings presented in this thesis highlight the importance of using an orthotopic implantation system to test in vivo functionality of ES cell-derived cells and provide an important foundation for future studies looking to develop stem cell therapies for bone injuries in larger animal models and people

The decrease in endoderm differentiation observed in RA+Y-27632 treatment corresponds to maintenance of pluripotency.

<p>Total mRNA collected from samples was used in a qPCR with primers against the pluripotency marker Oct-4 (A). As expected, during directed differentiation into all three germ layers, the expression of Oct-4 is decreased. However, under RA+Y-27632 treatment (A) Oct-4 levels are more similar to that in undifferentiated cells. Immunofluorescent detection of Oct-4 in undifferentiated cells (B) and in cells treated with RA (C) RA plus Y-27632 (D) or RA with Y-27632 pre-treatment (E). qPCR with SSEA-1 probes showed similar levels and trends to that of Oct-4, with significant decreases observed during differentiation into any lineage (F). Furthermore, as with Oct-4 SSEA-1 mRNA expression remained at control levels during RA with Y-27632 treatment (F). Immunofluorescent detection of SSEA-1 in undifferentiated cells (G) and in those treated with RA (H) RA with Y-27632 (I) or RA with Y-27632 pre-treatment (J). FACS analysis demonstrates increased numbers of Oct-4 and SSEA-1 positive cells during RA induced differentiation and Y-27632 treatment (K). Scale bar equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.</p

Y-27632 enhances the differentiation of P19 cells into ectoderm.

<p>P19 cell aggregates were treated with RA+LIF to induced neural-ectoderm differentiation. Total mRNA was collected, converted to cDNA and used in a PCR to examine GFAP expression, a marker of ectodermal derived neural cell types (A). GFAP levels increased with RA+LIF treatment in the presence or absence of Y-27632. Increased GFAP expression was also detected in the DMSO treated groups supplemented with Y-27632. Immunofluorescent detection of GFAP in undifferentiated (B) RA+LIF (C) RA+LIF and Y-27632 (D) or RA+LIF with Y-27632 pre-treated cells (E). qPCR with NF-68 primers showed an increase in expression in cells exposed to Y-27632 during differentiation (F). Immunofluorescent detection of NF-68 in undifferentiated cells or those exposed to the following: (G) RA+LIF (H) RA+LIF and Y-27632 (I) or RA+LIF with Y-27632 pre-treatment (J). FACS analysis demonstrates increased numbers of GFAP- (trend) and NF-68-positive cells with Y-27632 treatment during RA+LIF induced differentiation (K). Scale bar equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.</p

Y-27632 treatment of P19 cells inhibits the differentiation of primitive endoderm.

<p>Monolayer grown P19 cells induced to differentiate with RA were collected for mRNA quantification and immunofluorescence. Total mRNA from all treatment groups was probed with a GATA-6 primer/probe, which is a marker of primitive endoderm. The RA treatment group displayed an increase of GATA-6, but decreased when RA was supplemented with Y-27632, with moderate recovery when cells where induced to differentiate after Y-27632 pre-treatment and removal of the inhibitor (A). Immunofluorescence of GATA-6 in undifferentiated cells (B) and in RA treated (C), RA+Y-27632 treated (D) or RA+Y-27632 pre-treated cells. Total mRNA converted to cDNA and probed with Troma-1, showed a dramatic decrease in expression when Y-27632 was present either during or before differentiation (F). Immunofluorescence of Troma-1 in undifferentiated (B) RA treated (C), RA+Y-27632 treated (D) and RA+Y-27632 pre-treated cells. FACS analysis demonstrates a significant decrease in GATA-6 and Troma-1 positive cells with Y-27632 present (K). Scale bars equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.</p

Differentiation of P19 cells into mesoderm is enhanced by Y-27632 treatment.

<p>P19 aggregates were treated with 1% DMSO to induce differentiation into mesoderm. Total mRNA was collected and probed with Brachyury, an early mesodermal marker. P19 cells treated with DMSO displayed an increase in Brachyury expression compared to cells treated with Y-27632 (A). Immunofluorescence analysis of cells either undifferentiated (B) or treated with DMSO treated (C) DMSO+Y-27632 treated (D) or DMSO treatment with Y-27632 pre-treatment (E) showing Brachyury localized to the nucleus. qPCR results for SMA show expression levels had increased following DMSO+Y-27632 treatment (F). Immunofluorescence of SMA in undifferentiated (G) DMSO treated (H) DMSO+Y-27632 treated (I) or DMSO treatment with Y-27632 pre-treatment (J). MyoD mRNA expression also increased with Y-27632 treatment in the presence of DMSO (K). Immunofluorescence of MyoD in undifferentiated cells (L) and in those treated with DMSO (M) DMSO+Y-27632 treated (N) or DMSO with Y-27632 pre-treatment (O). FACS analysis demonstrates a significant increase in Brachyury, SMA and MyoD positive cells with Y-27632 treatment (P). Scale bar equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.</p

Reduced Differentiation Efficiency of Murine Embryonic Stem Cells in Stirred Suspension Bioreactors

The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of leukemia inhibitory factor (LIF). Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs

Assessment of the Efficacy of MRI for Detectionof Changes in Bone Morphology in a MouseModel of Bone Injury

"This is the peer reviewed version of the following article: [Taha, M. A., Manske, S. L., Kristensen, E., Taiani, J. T., Krawetz, R., Wu, Y., Ponjevic, D., Matyas, J. R., Boyd, S. K., Rancourt, D. E. and Dunn, J. F. (2013), Assessment of the efficacy of MRI for detection of changes in bone morphology in a mouse model of bone injury. J. Magn. Reson. Imaging, 38: 231–237], which has been published in final form at [http://dx.doi.org/10.1002/jmri.23876]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."Purpose To determine whether magnetic resonance imaging (MRI) could be used to track changes in skeletal morphology during bone healing using high-resolution micro-computed tomography (μCT) as a standard. We used a mouse model of bone injury to compare μCT with MRI. Materials and Methods Surgery was performed to induce a burr hole fracture in the mouse tibia. A selection of biomaterials was immediately implanted into the fractures. First we optimized the imaging sequences by testing different MRI pulse sequences. Then changes in bone morphology over the course of fracture repair were assessed using in vivo MRI and μCT. Histology was performed to validate the imaging outcomes. Results The rapid acquisition with relaxation enhancement (RARE) sequence provided sufficient contrast between bone and the surrounding tissues to clearly reveal the fracture. It allowed detection of the fracture clearly 1 and 14 days postsurgery and revealed soft tissue changes that were not clear on μCT. In MRI and μCT the fracture was seen at day 1 and partial healing was detected at day 14. Conclusion The RARE sequence was the most suitable for MRI bone imaging. It enabled the detection of hard and even soft tissue changes. These findings suggest that MRI could be an effective imaging modality for assessing changes in bone morphology and pathobiology.Canadian Institutes of Health Research; Alberta Innovates Health Solutions Team in OsteoarthritisYe

Additional file 1: Figure S1. of p21−/− mice exhibit enhanced bone regeneration after injury

Mouse weights at each time point between age and sex matched strains. Figure S2. Mouse tibia dimensions in age and sex matched strains. Figure S3. Bone histomorphometry obtained via microCT scanning of radiation controls. Figure S4. Histological analysis of tibia of C57BL/6 (A) vs. p21−/− (D) mice 4 weeks after injury. Figure S5. Representative images of pits resorbed by osteoclasts. (DOCX 5619 kb