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| The mouse cornea as a transplantation site for live imaging of engineered tissue constructs.
April 3, 2010 at 6:19 AM
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| | The mouse cornea as a transplantation site for live imaging of engineered tissue constructs. Cold Spring Harb Protoc. 2010 Apr;2010(4):pdb.prot5416 Authors: Poché RA, Saik JE, West JL, Dickinson ME The field of tissue engineering aims to recapitulate healthy human organs and 3-D tissue structures in vitro and then transplant these constructs in vivo where they can be effectively integrated within the recipient patient and become perfused by the host circulation. To improve the design of materials for artificial tissue scaffolds, it would be ideal to have a high-throughput imaging system that allows one to directly monitor transplanted tissue constructs in live animals over an extended period of time. By combining such an assay with transgenic, cell-specific fluorescent reporters, one could monitor such parameters as tissue construct perfusion, donor cell survival, and donor-host cell interaction/integration. Here, we describe a protocol for a modified version of the classical corneal micropocket angiogenesis assay, employing it as a live imaging "window" to monitor angiogenic poly(ethylene glycol) (PEG)-based hydrogel tissue constructs. PMID: 20360370 [PubMed - in process] | | |
| Alginate-controlled formation of nano-scale calcium carbonate and hydroxyapatite mineral phase within hydrogel networks.
April 3, 2010 at 6:19 AM
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| | Alginate-controlled formation of nano-scale calcium carbonate and hydroxyapatite mineral phase within hydrogel networks. Acta Biomater. 2010 Mar 29; Authors: Xie M, Olderøy MO, Andreassen JP, Selbach SM, Strand BL, Sikorski P A one-step method was used to make nano-structured composites from alginate and calcium carbonate or calcium phosphate. Nanometer-scale mineral phase was successfully formed within the gel network of alginate gel beads, and the composites were characterized. It was found that calcite was the dominating polymorph in the calcium carbonate mineralized beads, while stoichiometric hydroxyapatite was formed in the calcium phosphate mineralized beads. A combination of electron microscopy, fourier-transform infrared spectroscopy, thermogravimetric analysis and powder X-ray diffraction showed that alginate played an active role in controlling mineral size, -morphology and -polymorph. For the calcium phosphate mineralized beads, alginate was shown to modulate stoichiometric hydroxyapatite with low crystallinity at room-temperature, which may have important applications in tissue engineering. The results presented in this work demonstrate important aspects of alginate-contro! lled crystallization, which contributes to the understanding of composite material design. PMID: 20359556 [PubMed - as supplied by publisher] | | |
| Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies.
April 3, 2010 at 6:19 AM
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| | Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies. Int J Mol Med. 2010 Feb;25(2):195-202 Authors: De Carlo E, Baiguera S, Conconi MT, Vigolo S, Grandi C, Lora S, Martini C, Maffei P, Tamagno G, Vettor R, Sicolo N, Parnigotto PP Increasing pancreatic islet survival and function is a starting point for obtaining a valuable bioartificial pancreas for the treatment of type 1 diabetes. In this context, decellularized matrices, obtained after the removal of tissue cellular part, are known to support in vitro adhesion, growth, and function of several cell types. We demonstrate that a homologous acellular pancreatic matrix is a suitable scaffold for rat islet cultures maintaining their long-term viability and function. Islets adhered to the pancreatic matrix showed a constant glucose-induced insulin release during long-term in vitro incubation, while islets cultured without a matrix or on the liver matrix showed a progressive reduction. In order to obtain implantable devices, acellular matrix/islet cultures were entrapped into poly(vinyl alcohol) (PVA)/ poly(ethylene glycol) (PEG) tubes obtained by the freezing/thawing procedure. Under this condition, an in vitro constant insulin release was det! ected. The devices were then implanted into diabetic rats where reduced insulin requirement was noted suggesting insulin secretory activity of islets contained in the device. Indeed, immunofluorescence confirmed the presence of insulin- and glucagon-producing cells into the explanted devices. These data show that PVA/PEG semi-permeable membrane can obtain devices that restore, at least in part, insulin secretion. PMID: 20043127 [PubMed - indexed for MEDLINE] | | |
| Engineering analysis and development of the spheroid reservoir bioartificial liver.
April 3, 2010 at 6:19 AM
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| | Engineering analysis and development of the spheroid reservoir bioartificial liver. Conf Proc IEEE Eng Med Biol Soc. 2009;2009:5985-8 Authors: McIntosh MB, Corner SM, Amiot BP, Nyberg SL A significant demand exists for a liver support device such as a Bioartifical Liver (BAL) to treat patients experiencing acute liver failure. This descriptive paper outlines the design and development of two of the key components of the Mayo Spheroid Reservoir Bioartificial Liver (SRBAL) system. One of the components is the multifunctional Spheroid Reservoir and the other is Multi-shelf Rocker. The Spheroid Reservoir provides an environment to support the viability and functionality of the hepatocyte spheroids at very high cell densities. The Spheroid Reservoir is the biologically active component of this extracorporeal liver support device. Since the Spheroid Reservoir is designed to support 200-400 grams of hepatocyte spheroids, a method to quickly produce large quantities of spheroids is required. The Multi-Shelf Rocker fulfills the production requirement by allowing the culturing of up to six liters of hepatocyte suspension in a conventional laboratory incubat! or. The SRBAL is designed to provide life sustaining liver-like function to patients in acute liver failure. PMID: 19965068 [PubMed - indexed for MEDLINE] | | |
| Whole organ decellularization - a tool for bioscaffold fabrication and organ bioengineering.
April 3, 2010 at 6:19 AM
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| | Whole organ decellularization - a tool for bioscaffold fabrication and organ bioengineering. Conf Proc IEEE Eng Med Biol Soc. 2009;2009:6526-9 Authors: Baptista PM, Orlando G, Mirmalek-Sani SH, Siddiqui M, Atala A, Soker S The use of synthetic and naturally-derived scaffolds for bioengineering of solid organs has been limited due to a lack of an integrated vascular network. Here, we describe fabrication of a bioscaffold system with intact vascular tree. Animal-donor organs and tissues, ranging in size up-to thirty centimeters, were perfused with decellularization solution to selectively remove the cellular component of the tissue and leave an intact extracellular matrix and vascular network. The vascular tree demonstrated sequential fluid flow through a central inlet vessel that branched into an extensive capillary bed and coalesced back into a single outlet vessel. In one example, the liver, we used central inlet vessels to perfuse human and animal liver cells through the bioscaffold to create a functional liver tissue construct in vitro. These results demonstrate a novel yet simple and scalable method to obtain whole organ vascularized bioscaffolds with potential for liver, kidney! , pancreas, intestine and other organs' bioengineering. These bioscaffolds can further provide a tool to study cells in their natural three-dimensional environment, which is superior for drug discovery platform compared with cells cultured in two-dimensional petri dishes. PMID: 19964173 [PubMed - indexed for MEDLINE] | | |
| Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z RP platform.
April 3, 2010 at 6:19 AM
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| | Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z RP platform. Biotechnol Prog. 2009 Nov-Dec;25(6):1762-71 Authors: Diederichs S, Röker S, Marten D, Peterbauer A, Scheper T, van Griensven M, Kasper C 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 stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram. Furthermore, hydroxyapatite coated Sponceram was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblast! s 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 compared to hydroxyapatite coated Sponceram. 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. PMID: 19795480 [PubMed - indexed for MEDLINE] | | |
| Intestinal epithelial cells in vitro.
April 3, 2010 at 6:19 AM
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| | Intestinal epithelial cells in vitro. Stem Cells Dev. 2010 Jan;19(1):131-42 Authors: Chopra DP, Dombkowski AA, Stemmer PM, Parker GC Recent advances in the biology of stem cells has resulted in significant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the difficulty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progenitor cells into the various intestinal lineages. Several short-term organ/organoid and epithelial cell culture models have been described. There is a dearth of long-term epithelial and/or stem cell cultures of intestine. With an expanding role of stem cells in the treatment of degenerative disorders, there is a critical need for additional efforts to develop in vitro models of stem/progenitor epithelial cells of intestine. The objective of this review is to recapitulate the cur! rent status of technologies and knowledge for in vitro propagation of intestinal epithelial cells, markers of the intestinal stem cells, and gene and protein expression profiles of the intestinal cellular differentiation. PMID: 19580443 [PubMed - indexed for MEDLINE] | | |
| Differentiation of dermal multipotent cells into odontogenic lineage induced by embryonic and neonatal tooth germ cell-conditioned medium.
April 3, 2010 at 6:19 AM
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| | Differentiation of dermal multipotent cells into odontogenic lineage induced by embryonic and neonatal tooth germ cell-conditioned medium. Stem Cells Dev. 2010 Jan;19(1):93-104 Authors: Huo N, Tang L, Yang Z, Qian H, Wang Y, Han C, Gu Z, Duan Y, Jin Y Stem cell-based therapy represents a novel and more advantageous modality of treatment for tooth defect or loss. However, this strategy is challenged in the circumstances where tooth-derived stem cells are not readily accessible. In present study we sought to explore the possibility of utilizing dermal multipotent cells (DMCs) easily available from skin tissue for odontogenic induction. Using the limiting dilution technique, colony-forming cell population was isolated and characterized by proliferative activity and multilineage differentiation potential. By exposure to conditioned medium of embryonic and neonatal tooth germ cells in culture, the proliferation and mineralization activity of DMCs was elevated, while the embryonic tooth germ cell-conditioned medium (ETGC-CM) produced more significant effects. Meanwhile, ETGC-CM-treated DMCs phenocopied the odontoblasts in vitro as indicated by specific lineage markers. Following in vivo transplantation as cell pellet! , ETGC-CM-treated DMCs were capable of producing blocks of mineralized tissues, which resembled those of dental pulp stem cell (DPSC) explants in the same subcutaneous pockets environment. These observations suggest that although more sufficient and continuous inductive microenvironment may be needed for undifferentiated DMCs to perform as odontoblasts, ETGC-CM-treated DMCs indeed acquire properties as those of DPSCs. Our work highlights the potential utility of DMCs as an alternative candidate cell source in hopes of developing more practical strategy of tooth regeneration research and offering promising opportunities for therapeutic approach. PMID: 19469666 [PubMed - indexed for MEDLINE] | | |
| Cytotherapy of osteonecrosis of the femoral head: a mini review.
April 3, 2010 at 6:03 AM
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| | Cytotherapy of osteonecrosis of the femoral head: a mini review. Int Orthop. 2010 Apr 2; Authors: Gao YS, Zhang CQ The pathogenesis and aetiology of nontraumatic osteonecrosis of the femoral head has not been revealed completely. However, with advances in stem cell research and regenerative medicine, it is believed that the onset of osteonecrosis of the femoral head probably has a cellular origin, and the possible therapy of osteonecrosis of the femoral head based on cytotherapy has great potential. In this review, the aetiology of osteonecrosis of the femoral head, animal experiments and clinical applications of cytotherapy are summarized and analysed. Current problems and future challenges are discussed. PMID: 20361186 [PubMed - as supplied by publisher] | | |
| Generation of induced pluripotent stem cells from human cord blood cells with only two factors: Oct4 and Sox2.
April 3, 2010 at 6:03 AM
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| | Generation of induced pluripotent stem cells from human cord blood cells with only two factors: Oct4 and Sox2. Nat Protoc. 2010 Apr;5(4):811-20 Authors: Giorgetti A, Montserrat N, Rodriguez-Piza I, Azqueta C, Veiga A, Belmonte JC Induced pluripotent stem cells (iPSC) provide an invaluable resource for regenerative medicine as they allow the generation of patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach is desirable, such as for cell therapy of acute conditions or when the patient's somatic cells are altered as a consequence of a chronic disease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carry minimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousand immunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for the derivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors (OCT4 and SOX2) in 2 weeks and without the need for additional chemical compounds. PMID: 20360773 [PubMed - in process] | | |
| Derivation of neural crest cells from human pluripotent stem cells.
April 3, 2010 at 6:03 AM
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| | Derivation of neural crest cells from human pluripotent stem cells. Nat Protoc. 2010 Apr;5(4):688-701 Authors: Lee G, Chambers SM, Tomishima MJ, Studer L Human pluripotent stem cell (hPSC)-derived neural crest (NC) cells present a valuable tool for modeling aspects of human NC development, including cell fate specification, multipotency and cell migration. hPSC-derived NC cells are also suitable for modeling human disease and as a renewable cell source for applications in regenerative medicine. Here we provide protocols for the step-wise differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into neuroectodermal and NC cells using either the MS5 coculture system or a novel defined culture method based on pharmacological inhibition of bone morphogenetic protein and transforming growth factor-beta signaling pathways. Furthermore, we present protocols for the purification and propagation of hPSC-NC cells using flow cytometry and defined in vitro culture conditions. Our protocol has been validated in multiple independent hESC and hiPSC lines. The average time required for! generating purified hPSC-NC precursors using this protocol is 2-5 weeks. PMID: 20360764 [PubMed - in process] | | |
| Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation.
April 3, 2010 at 6:03 AM
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| | Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation. Blood. 2010 Apr 1; Authors: Shao L, Sun Y, Zhang Z, Feng W, Gao Y, Cai Z, Wang ZZ, Look AT, Wu WS Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation, but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively indu! ced by irradiation in primitive hematopoietic cells and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy. PMID: 20360471 [PubMed - as supplied by publisher] | | |
| Wnt signaling has different temporal roles during retinal development.
April 3, 2010 at 6:03 AM
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| | Wnt signaling has different temporal roles during retinal development. Dev Dyn. 2010 Jan;239(1):297-310 Authors: Sánchez-Sánchez AV, Camp E, Leal-Tassias A, Mullor JL Differentiation of neural retinal precursor (NRP) cells in vertebrates follows an established order of cell-fate determination associated with exit from the cell cycle. Wnt signaling regulates cell cycle in colon carcinoma cells and has been implicated in different aspects of retinal development in various species. To better understand the biological roles of Wnt in the developing retina, we have used a transgenic and pharmacological approach to manipulate the Wnt signaling pathway during retinal development in medaka embryos. With the use of both approaches, we observed that during the early phase of retinal development Wnt signaling regulated cell cycle progression, proliferation, apoptosis, and differentiation of NRP cells. However, during later phases of retinal development, proliferation and apoptosis were not affected by manipulation of Wnt signaling. Instead, Wnt regulated Vsx1 expression, but not the expression of other retinal cell markers tested. Thus, t! he response of NRP cells to Wnt signaling is stage-dependent. PMID: 20014102 [PubMed - indexed for MEDLINE] | | |
| Human cord blood stem cell applications in cell therapy.
April 3, 2010 at 6:03 AM
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| | Human cord blood stem cell applications in cell therapy. J Stem Cells. 2009;4(2):95-103 Authors: Gu YC, Rao MS, Vemuri MC Human umbilical cord blood (UCB) is a valuable alternative source of ethically acceptable, clinically competent stem cells that is most likely closest to embryonic stem cells. Development of reliable methods for the expansion of cord blood stem cells is critical to ensure their clinical application. In the present article, advances in cord blood stem cell isolation, culture expansion methods through co-culture with human mesenchymal stem cells, culture optimization techniques with defined media and cord blood stem cell banking aspects have been reviewed. Refined methods of isolation as well as defined culture conditions of expansion that favor retention of stem cell phenotype and proper cryogenic storage can significantly increase the use of cord blood stem cells in human cell therapy applications. PMID: 20232595 [PubMed - indexed for MEDLINE] | | | | 
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