Thursday, May 5, 2011

5/6 TE-RegenMed-StemCell feed

     
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Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA.
May 5, 2011 at 7:30 AM
 

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA.

J Vis Exp. 2011;(50):

Authors: Dai B, Dahmani F, Cichocki JA, Swanson LC, Rasmussen TP

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H4(1). The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature (2,3). We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

PMID: 21540828 [PubMed - in process]

   
   
iPS cells forgive but do not forget.
May 5, 2011 at 7:30 AM
 

iPS cells forgive but do not forget.

Nat Cell Biol. 2011 May;13(5):523-5

Authors: Barrero MJ, Izpisua Belmonte JC

Induced pluripotent stem (iPS) cells offer the possibility to generate patient-specific cell types for use in regenerative medicine. However, a long-lasting question remains: are iPS and embryonic stem cells equivalent? iPS cells retain a transcriptional memory of their origin, which is now shown to endure with passages and to correlate with defects in the re-establishment of DNA methylation. Both selective pressure and genomic environment may account for these defects.

PMID: 21540851 [PubMed - in process]

   
   
A novel Janus-type AT nucleoside with benzoyl protecting groups forming a pleated-sheet structure.
May 5, 2011 at 7:30 AM
 

A novel Janus-type AT nucleoside with benzoyl protecting groups forming a pleated-sheet structure.

Acta Crystallogr C. 2011 May;67(Pt 5):o175-8

Authors: Pan MY, Wu XH, Luo DB, Huang W, He Y

The title compound, 5-amino-8-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-dione methanol monosolvate, C(32)H(25)N(5)O(9)·CH(4)O, which crystallized slowly from methanol, exhibits an anti conformation with a glycosyl-bond torsion angle of χ = -141.28 (17)°. The furanose moiety adopts an N-type sugar puckering ((3)T(4)). The corresponding pseudorotation phase angle and maximum amplitude are P = 24.5 (2)° and τ(m) = 38.3 (2)°, respectively. In the solid state, one methanol molecule acts as a bridge joining adjacent nucleoside molecules head-to-head, leading to a pleated-ribbon supramolecular structure, with the base moieties located in the centre of the ribbon and the sugar residues protruding to the outside of the layers, as in a DNA helix. The pleated-ribbon supramolecular structure is tethered together into a two-dimensional infinite pleated-sheet structure through aromatic stacking between the nucleobase planes and the benzene rings of the benzoyl protecting groups on the 5'-OH group of furanose.

PMID: 21540544 [PubMed - in process]

   
   
Isolation and culture of cells from the nephrogenic zone of the embryonic mouse kidney.
May 5, 2011 at 7:30 AM
 

Isolation and culture of cells from the nephrogenic zone of the embryonic mouse kidney.

J Vis Exp. 2011;(50):

Authors: Brown AC, Blank U, Adams DC, Karolak MJ, Fetting JL, Hill BL, Oxburgh L

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

PMID: 21540822 [PubMed - in process]

   
   
Harnessing the potential of induced pluripotent stem cells for regenerative medicine.
May 5, 2011 at 7:30 AM
 

Harnessing the potential of induced pluripotent stem cells for regenerative medicine.

Nat Cell Biol. 2011 May;13(5):497-505

Authors: Wu SM, Hochedlinger K

The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.

PMID: 21540845 [PubMed - in process]

   
   
The effects of dynamic loading on the intervertebral disc.
May 5, 2011 at 6:13 AM
 

The effects of dynamic loading on the intervertebral disc.

Eur Spine J. 2011 May 4;

Authors: Chan SC, Ferguson SJ, Gantenbein-Ritter B

Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of 'physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally 'discogenic' cells for tissue engineering purpose.

PMID: 21541667 [PubMed - as supplied by publisher]

   
   
The effect of pore size on tissue ingrowth and neovascularization in porous bioceramics of controlled architecture in vivo.
May 5, 2011 at 6:13 AM
 

The effect of pore size on tissue ingrowth and neovascularization in porous bioceramics of controlled architecture in vivo.

Biomed Mater. 2011 Feb;6(1):015007

Authors: Feng B, Jinkang Z, Zhen W, Jianxi L, Jiang C, Jian L, Guolin M, Xin D

The purpose of this study was to investigate the role of pore size on tissue ingrowth and neovascularization in porous bioceramics under the accurate control of the pore parameters. For that purpose, β-tricalcium phosphate (β-TCP) cylinders with four different macropore sizes (300-400, 400-500, 500-600 and 600-700 µm) but the same interconnection size (120 µm) and unchangeable porosity were implanted into fascia lumbodorsalis in rabbits. The fibrous tissues and blood vessels formed in scaffolds were observed histologically and histomorphometrically. The vascularization of the porous bioceramics was analyzed by single-photon emission computed tomography (SPECT). It is found that pore size as an important parameter of a porous structure plays an important role in tissue infiltration into porous biomaterial scaffolds. The amount of fibrous tissue ingrowth increases with the decrease of the pore size. In four kinds of scaffolds with different macropore sizes (300-400, 400-500, 500-600 and 600-700 µm) and a constant interconnection size of 120 µm, the areas of fibrous tissue (%) were 60.5%, 52.2%, 41.3% and 37.3%, respectively, representing a significant decrease at 4 weeks (P < 0.01). The pore size of a scaffold is closely related to neovascularization of macroporous biomaterials implanted in vivo. A large pore size is beneficial for the growth of blood vessels, and the diameter of a pore smaller than 400 µm limits the growth of blood vessels and results in a smaller blood vessel diameter.

PMID: 21206002 [PubMed - indexed for MEDLINE]

   
   
Neural tissue engineering using embryonic and induced pluripotent stem cells.
May 5, 2011 at 6:13 AM
 

Neural tissue engineering using embryonic and induced pluripotent stem cells.

Stem Cell Res Ther. 2011 Apr 15;2(2):17

Authors: Willerth SM

ABSTRACT: With the recent start of the first clinical trial evaluating a human embryonic stem cell-derived therapy for the treatment of acute spinal cord injury, it is important to review the current literature examining the use of embryonic stem cells for neural tissue engineering applications with a focus on diseases and disorders that affect the central nervous system. Embryonic stem cells exhibit pluripotency and thus can differentiate into any cell type found in the body, including those found in the nervous system. A range of studies have investigated how to direct the differentiation of embryonic cells into specific neural phenotypes using a variety of cues to achieve the goal of replacing diseased or damaged neural tissue. Additionally, the recent development of induced pluripotent stem cells provides an intriguing alternative to the use of human embryonic stem cell lines for these applications. This review will discuss relevant studies that have used embryonic stem cells to replicate the tissue found in the central nervous system as well as evaluate the potential of induced pluripotent stem cells for the aforementioned applications.

PMID: 21539726 [PubMed - as supplied by publisher]

   
   
Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro.
May 5, 2011 at 6:13 AM
 

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro.

Biosci Rep. 2011 Jun;31(3):199-210

Authors: Bryan N, Andrews KD, Loughran MJ, Rhodes NP, Hunt JA

One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.

PMID: 20840080 [PubMed - indexed for MEDLINE]

   
   
Rejuvenation of Nucleus Pulposus Cells Using Extracellular Matrix Deposited by Synovium-Derived Stem Cells.
May 5, 2011 at 6:13 AM
 

Rejuvenation of Nucleus Pulposus Cells Using Extracellular Matrix Deposited by Synovium-Derived Stem Cells.

Spine (Phila Pa 1976). 2011 May 2;

Authors: He F, Pei M

ABSTRACT: Study Design. After plating for six passages on either plastic flasks or extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs), expanded nucleus pulposus (NP) cells were evaluated for redifferentiation capacity.Objective. The aim was to assess the feasibility of using ECM deposited by a tissue-specific stem cell to provide a 3D microenvironment for NP cell rejuvenation.Summary of Background Data. Autologous disc cell-based therapy is a promising approach for intervertebral disc (IVD) regeneration. Unfortunately, the current in vitro expansion of NP cells in monolayer results in dedifferentiation of these cells.Methods. Primary NP cells were plated on either plastic flasks or ECM for six consecutive passages. At each passage, cell numbers were counted for proliferation rate, cell phenotype was evaluated using flow cytometry, and cell differentiation status was assessed using real-time PCR. The pellets from expanded NP cells at passages 1, 4, and 6 were incubated in a serum-free defined medium for 14 days. Redifferentiation capacity of the expanded NP cells was evaluated using histology, biochemistry, and real-time PCR.Results. NP cells expanded on ECM grew much faster with a smaller size and fibroblast-like shape compared to those on plastic flasks. ECM-treated NP cells acquired an enhanced CD90 expression and higher mRNA levels of types I, II, and X collagen and aggrecan, as well as a robust redifferentiation capacity, evidenced by dramatically increased type II collagen, aggrecan, and Sox9 and decreased type I collagen for up to six passages.Conclusion. SDSC-derived ECM can provide a tissue-specific microenvironment for the rejuvenation of NP cells with a higher proliferation rate and redifferentiation capacity. These characteristics may play a role in improving an autologous disc cell-based minimally invasive therapeutic approach toward physiological reconstruction of a biologically functional disc in the clinical setting.

PMID: 21540772 [PubMed - as supplied by publisher]

   
   
In vivo tracking of stem cell by nanotechnologies: future prospects for mouse to human translation.
May 5, 2011 at 6:13 AM
 

In vivo tracking of stem cell by nanotechnologies: future prospects for mouse to human translation.

Tissue Eng Part B Rev. 2011 Feb;17(1):1-11

Authors: Villa C, Erratico S, Razini P, Farini A, Meregalli M, Belicchi M, Torrente Y

Advances in stem cell research have provided important understanding of the cell biology and offered great promise for developing new strategies for tissue regeneration. Dynamic determination of stem cell migration and distribution in real time is essential for optimizing treatments in preclinical models and designing clinical protocols. Recent developments in the use of nanotechnologies have contributed to advance of the high-resolution in vivo imaging methods, including the positron emission tomography, the single-photon emission computed tomography, the magnetic resonance imaging, and microcomputed tomography. This review examines the use of nanotechnologies for stem cell tracking, the many contrast agents, and detectors that have been proposed and suggest future directions for mouse to human translation of these techniques, for both therapeutic and diagnostic purposes.

PMID: 20846051 [PubMed - indexed for MEDLINE]

   
   
Fabrication of porous scaffolds with a controllable microstructure and mechanical properties by porogen fusion technique.
May 5, 2011 at 6:13 AM
 

Fabrication of porous scaffolds with a controllable microstructure and mechanical properties by porogen fusion technique.

Int J Mol Sci. 2011;12(2):890-904

Authors: Tan Q, Li S, Ren J, Chen C

Macroporous scaffolds with controllable pore structure and mechanical properties were fabricated by a porogen fusion technique. Biodegradable material poly (d, l-lactide) (PDLLA) was used as the scaffold matrix. The effects of porogen size, PDLLA concentration and hydroxyapatite (HA) content on the scaffold morphology, porosity and mechanical properties were investigated. High porosity (90% and above) and highly interconnected structures were easily obtained and the pore size could be adjusted by varying the porogen size. With the increasing porogen size and PDLLA concentration, the porosity of scaffolds decreases, while its mechanical properties increase. The introduction of HA greatly increases the impact on pore structure, mechanical properties and water absorption ability of scaffolds, while it has comparatively little influence on its porosity under low HA contents. These results show that by adjusting processing parameters, scaffolds could afford a controllable pore size, exhibit suitable pore structure and high porosity, as well as good mechanical properties, and may serve as an excellent substrate for bone tissue engineering.

PMID: 21541032 [PubMed - in process]

   
   
Biotech in medicine--the topic of the Olten Meeting 2010.
May 5, 2011 at 6:13 AM
 

Biotech in medicine--the topic of the Olten Meeting 2010.

Chimia (Aarau). 2011;65(1-2):100-3

Authors: Heinzelmann E

Since 1998, the biotechnet--the national network of competence in biotech research--has been helping partners from industry to access excellence in R&D, giving them optimal support at low cost. Its annual 'Olten Meeting' is a dynamic hub for companies and research institutes as it highlights the latest trends in biotech. On November 24, 2010, the topical subject was biotech in medicine.

PMID: 21469455 [PubMed - indexed for MEDLINE]

   
   
Soluble and insoluble signals sculpt osteogenesis in angiogenesis.
May 5, 2011 at 6:13 AM
 

Soluble and insoluble signals sculpt osteogenesis in angiogenesis.

World J Biol Chem. 2010 May 26;1(5):109-32

Authors: Ripamonti U

The basic tissue engineering paradigm is tissue induction and morphogenesis by combinatorial molecular protocols whereby soluble molecular signals are combined with insoluble signals or substrata. The insoluble signal acts as a three-dimensional scaffold for the initiation of de novo tissue induction and morphogenesis. The osteogenic soluble molecular signals of the transforming growth factor-β (TGF-β) supergene family, the bone morphogenetic/osteogenic proteins (BMPs/OPs) and, uniquely in the non-human primate Papio ursinus (P. ursinus), the three mammalian TGF-β isoforms induce bone formation as a recapitulation of embryonic development. In this paper, I discuss the pleiotropic activity of the BMPs/OPs in the non-human primate P. ursinus, the induction of bone by transitional uroepithelium, and the apparent redundancy of molecular signals initiating bone formation by induction including the three mammalian TGF-β isoforms. Amongst all mammals tested so far, the three mammalian TGF-β isoforms induce endochondral bone formation in the non-human primate P. ursinus only. Bone tissue engineering starts by erecting scaffolds of biomimetic biomaterial matrices that mimic the supramolecular assembly of the extracellular matrix of bone. The molecular scaffolding lies at the hearth of all tissue engineering strategies including the induction of bone formation. The novel concept of tissue engineering is the generation of newly formed bone by the implantation of "smart" intelligent biomimetic matrices that per se initiate the ripple-like cascade of bone differentiation by induction without exogenously applied BMPs/OPs of the TGF-β supergene family. A comprehensive digital iconographic material presents the modified tissue engineering paradigm whereby the induction of bone formation is initiated by intelligent smart biomimetic matrices that per se initiate the induction of bone formation without the exogenous application of the soluble osteogenic molecular signals. The driving force of the intrinsic induction of bone formation by bioactive biomimetic matrices is the shape of the implanted substratum. The language of shape is the language of geometry; the language of geometry is the language of a sequence of repetitive concavities, which biomimetizes the remodelling cycle of the primate osteonic bone.

PMID: 21540997 [PubMed - in process]

   
   
Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration.
May 5, 2011 at 6:13 AM
 

Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration.

Biomed Mater. 2011 Feb;6(1):015013

Authors: Xiao C, Zhou H, Liu G, Zhang P, Fu Y, Gu P, Hou H, Tang T, Fan X

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

PMID: 21252414 [PubMed - indexed for MEDLINE]

   
   
Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering.
May 5, 2011 at 6:13 AM
 

Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering.

Acta Biomater. 2011 Apr 27;

Authors: Lee GS, Park JH, Shin US, Kim HW

This study reports the preparation of novel porous scaffolds of calcium phosphate cement (CPC) combined with alginate, and their potential usefulness as a three-dimensional (3-D) matrix for drug delivery and tissue engineering of bone. An α-tricalcium phosphate-based powder was mixed with sodium alginate solution and then directly injected into a fibrous structure in a Ca-containing bath. A rapid hardening reaction of the alginate with Ca(2+) helps to shape the composite into a fibrous form with diameters of hundreds of micrometers, and subsequent pressing in a mold allows the formation of 3-D porous scaffolds with different porosity levels. After transformation of the CPC into a calcium-deficient hydroxyapatite phase in simulated biological fluid the scaffold was shown to retain its mechanical stability. During the process biological proteins, such as bovine serum albumin and lysozyme, used as model proteins, were observed to be effectively loaded onto and released from the scaffolds for up to more than a month, proving the efficacy of the scaffolds as a drug delivering matrix. Mesenchymal stem cells (MSC) were isolated from rat bone marrow and then cultured on the CPC-alginate porous scaffolds to investigate the ability to be populated by cells and their subsequent differentiation along the osteogenic lineage. It was shown that MSC increasingly actively populated and also permeated into the porous network with time of culture. In particular, cells cultured within a scaffold with a relatively high porosity level showed favorable proliferation and osteogenic differentiation. An in vivo pilot study of the CPC-alginate porous scaffolds after implantation into the rat calvarium for 6weeks revealed the formation of new bone tissue within the scaffold, closing the defect almost completely. Based on these results, the newly developed CPC-alginate porous scaffolds could be potentially useful as a 3-D matrix for drug delivery and tissue engineering of bone.

PMID: 21539944 [PubMed - as supplied by publisher]

   
     
 
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