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Research Containing: Cell Differentiation

Engineering Retina from Human Retinal Progenitors (Cell Lines)

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell-cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr(2)e(3), expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription-polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell line retains its capacity to differentiate into multiple cell types holds great promise for the use of tissue-specific adult stem cells for therapy.

Related URLs:
<Go to ISI>://WOS:000266344600020

Attachment to Cytodex Beads Enhances Differentiation of Human Retinal Progenitors in 3-D Bioreactor Culture

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Retinal degenerations are the leading cause of genetically inherited blindness. One of the strategies currently being tested for the treatment is cell/tissue transplantation. As such stem cells and tissue engineered constructs are of great importance. This report describes the growth of multipotential human retinal progenitors (cell line) in a 3-D bioreactor culture vessel with (adhesive substrate) laminin coated collagen 1/cytodex beads and without adhesive substrate (beadless culture). The study demonstrates that progenitors are capable of growth and differentiation in the bioreactor with or without beads. The presence of adhesive substrate accelerates and enhances photoreceptor differentiation in the bioreactor, reflected by significantly higher level expressions of several photoreceptor specific proteins; N acetyl transferase (AaNat), rhodopsin and cone transducin GNB3. Both monomeric and dimeric forms of rhodopsin are expressed in cells attached to beads, whereas, only the monomeric form is expressed in beadless culture. Similarly, a different isomeric form of tyrosine hydroxylase (a doublet) is expressed in cell bead attached cultures. Co-culturing retinal progenitors with retinal pigment epithelium (RPE) in cell bead cultures further stabilizes the photoreceptor phenotype and rhodopsin expression. Most of the retinal neuronal phenotypes are confirmed by an expression of specific proteins. The adhesive substrate in the form of collagen 1, laminin coated cytodex beads, could be just an effector for stabilization or a positive signal, modulating extracellular matrix (ECM) molecules and/or neurotrophins. In the future, the bioreactor culture system could be utilized to grow retina-like structures from ciliary epithelium by incorporating biodegradable substrates.

Related URLs:
<Go to ISI>://WOS:000301640300005

Dynamic Cultivation of Human Mesenchymal Stem Cells in a Rotating Bed Bioreactor System Based on the Z (R) RP Platform

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three-dimensional functional tissue-like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three-dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stein cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram (R). Furthermore, hydroxyapatite coated Sponceram (R) was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP-expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor-system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram (R) compared to hydroxyapatite coated Sponceram (R). With this study, we Present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1762-1771 2009

Related URLs:
<Go to ISI>://WOS:000273152000025

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity (μ g) may modulate the proliferation and differentiation. We investigated the application of μ g to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μ g for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in I G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers per-formed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μ g may be potentially beneficial in the fields of stem cell biology and somatic cell therapy. © 2005 Elsevier Ltd. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000229063800018

Three-dimensional culture environments enhance osteoblast differentiation

by cfynanon 9 June 2015in Biology & Biotechnology No comment

PURPOSE: In previous work from our laboratory, we demonstrated that the three-dimensional (3D) cell cultures developed in simulated microgravity environments enhanced osseous-like aggregate formation and accelerated preosteoblast cell differentiation. Thus, as described here, we hypothesize that aggregate formation and mineralization would occur with fewer than 10 x 10(6) cells as previously described. MATERIALS AND METHODS: Human preosteoblastic cells were cultured at different concentrations in a rotary wall vessel to simulate microgravity for 7 days. Aggregate size was assessed, and mineralization and collagen expression detected using Von Kossa and Masson Trichrome staining. Scanning electron microscopy was used for structural and elemental analysis. Immunohistochemistry was used to detect expression of the osteogenic markers BSPII and osteopontin (OP). RESULTS: Size and calcium expression were dependent upon cultured cell number (p < 0.01). Calcium and collagen expression were detected throughout the aggregate, but organization was independent of cell number. Aggregates had similar microscopic structural patterns demonstrating organized development. Presence of BSPII and OP showed that the aggregates share common differentiation proteins with in vivo bone formation. CONCLUSIONS: These results may lead to novel bone engineering techniques associated with dental treatment.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=med4&AN=18573152
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:medline&id=pmid:18573152&id=doi:10.1111%2Fj.1532-849X.2008.00330.x&issn=1059-941X&isbn=&volume=17&issue=7&spage=517&pages=517-21&date=2008&title=Journal+of+Prosthodontics&atitle=Three-dimensional+culture+environments+enhance+osteoblast+differentiation.&aulast=Boehrs&pid=%3Cauthor%3EBoehrs+J%3C%2Fauthor%3E&%3CAN%3E18573152%3C%2FAN%3E

Effect of simulated weightlessness on osteoprogenitor cell number and proliferation in young and adult rats

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Experiments with rats flown in space or hind limb unloaded (HU) indicate that bone loss in both conditions is associated with a decrease in bone volume and osteoblast surface in cancellous and cortical bone. We hypothesize that the decrease in osteoblastic bone formation and osteoblast surface is related to a decrease in the number of osteoprogenitors and/or decreased proliferation of their progeny. We tested this hypothesis by evaluating the effect of 14 days of HU on the number of osteoprogenitors (osteoblast colony forming units; CFU-O), fibroblastic colony forming units (CFU-F), and alkaline phosphatase-positive CFU (CFU-AP) in cell populations derived from the proximal femur (unloaded) and the proximal humerus (normally loaded) in 6-week-old and 6-month-old rats. To confirm the effect of unloading on bone volume and structure, static histomorphometric parameters were measured in the proximal tibial metaphysis. Effects of HU on proliferation of osteoprogenitors were evaluated by measuring the size of CFU-O. HU did not affect the total number of progenitors (CFU-F) in young or adult rats in any of the cell populations. In femoral populations of young rats, HU decreased CFU-O by 71.0% and mean colony size was reduced by 20%. HU decreased CFU-AP by 31.3%. As expected, no changes in CFU-O or CFU-AP were seen in cell populations from the humerus. In femoral cell populations of adult rats, HU decreased CFU-O and CFU-AP by 16.6% and 36.6%, respectively. Again, no effects were seen in cell populations from the humerus. In 6-week-old rats, there was a greater decrease in bone volume, osteoblast number, and osteoblast surface in the proximal tibial metaphysis than that observed in adult rats. Both trabecular thickness and trabecular number were decreased in young rats but remained unaffected in adults. Neither osteoclast number nor surface was affected by unloading. Our results show that the HU-induced decrease in the number of osteoprogenitors observed in vitro parallels the effects of HU on bone volume and osteoblast number in young and old rats in vivo, suggesting that the two may be interdependent. HU also reduced CFU-O colony size in femoral populations indicating a diminished proliferative capacity of osteoblastic colonies.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=med4&AN=15664015
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:medline&id=pmid:15664015&id=doi:&issn=8756-3282&isbn=&volume=36&issue=1&spage=173&pages=173-83&date=2005&title=Bone&atitle=Effect+of+simulated+weightlessness+on+osteoprogenitor+cell+number+and+proliferation+in+young+and+adult+rats.&aulast=Basso&pid=%3Cauthor%3EBasso+N%3C%2Fauthor%3E&%3CAN%3E15664015%3C%2FAN%3E

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