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Papers by Jorge Villanueva Araya
Matrix, 1989
Calvaria from 20-21 day old fetuses were obtained under sterile conditions and the endo- and exop... more Calvaria from 20-21 day old fetuses were obtained under sterile conditions and the endo- and exoperiosteum stripped off. Cells were dispersed by sequential collagenase-DNase treatment and suspended in 0.5% low Tm agarose in the presence of DMEM supplement with 10% FCS. After 4-5 days of incubation some 30% of these cells showed active synthesis of metachromatic extracellular matrix. Cells from skin, muscle and periosteum failed to show metachromatic matrix positive colonies to a comparable extent. The phenotypic expression of these cells was determined by analysis of collagen types. Eleven day old cultures were incubated in the presence of [3H]-proline plus beta-aminopropionitrile and ascorbic acid and the collagen extracted analyzed by polyacrylamide electrophoresis of their intact chains or CNBr-derived peptides. The results show that anchorage independence is a requirement for calvaria cells to express type II collagen. Type I collagen was preferentially expressed in monolayer culture or when pre-attached to a substrate before being cultured in agarose. Type II collagen was the predominant collagen when cells were cultured in agarose. Further characterization of cell populations was achieved by isopycnic centrifugation in a percoll gradient. Cell fractions were tested for their collagen phenotype when cultured in agarose. Cells recovered from densities 1.04 g/ml or higher synthesized type II collagen, while cells with densities lower than 1.04 g/ml synthesized mainly type I collagen. Isopycnic centrifugation appears to be a novel method for separation of phenotypically different cells from a heterogeneous population in fetal calvaria. The high density cell fractions may represent a mixture of pre-chondrocytes as well as pluripotential cells.
CÉLULAS MADRE Y MEDICINA DE REEMPLAZO CELULAR Stem Cells and Replacement Cellular Medicine, 2018
RESUMEN Las Células Madre (CM) son las responsables que aproximadamente 37,2 x 10 12 de células e... more RESUMEN
Las Células Madre (CM) son las responsables que aproximadamente 37,2 x 10 12 de células en un individuo promedio de 30 años en edad, con un peso de 70 Kg, 1.72m de altura y una superficie corporal de 1.85m2, provengan de una única célula: “el cigoto”1. Las CM se encuentran en todos los organismos pluricelulares y están encargadas de repoblar el tejido u órgano anfitrión durante el desarrollo, crecimiento, homeostasis, lesión y enfermedad. Debido al limitado potencial regenerativo de la naturaleza humana, se hace imperativo su estudio. En las últimas décadas ha habido un enorme avance en el conocimiento de la biología de las CM como también la habilidad de inducir la pluripotencia en células adultas, lo están permitiendo su acelerada traslación a la Medicina de reemplazo celular y terapia génica.
PALABRAS CLAVE: Células madre, Inducción de pluripotencia, Aislamiento de CM, Medicina de reemplazo celular, Terapia Génica.
ABSTRACT
Stem Cells (CM) are responsible for approximately 37.2 x 1012 cells in an average individual 30 years of age, with a weight of 70 kg, 1.72 m in height and a body surface area of 1.85m2, come from a single cell: "the zygote"1. The CM are found in all multicellular organisms and are responsible for repopulating the host tissue or organ during development, growth, homeostasis, injury and disease. Due to the limited regenerative potential of human nature, its study is imperative. In the last decades there has been an enormous advance in the knowledge of the biology of the CM as well as the ability to induce the pluripotency in adult cells, these are allowing its accelerated translation to the Medicine of cellular replacement and gene therapy.
KEYWORDS: Stem cells, Pluripotency induction, CM isolation, celular replacement Medicine, Gene Therapy.
Bacterial strains and plasmids. The strains, vectors, and recombinant plasmids used in this work ... more Bacterial strains and plasmids. The strains, vectors, and recombinant plasmids used in this work are described in . E. coli K38pGP1-2 and pT7-7 were kindly provided by S. Tabor (Harvard Medical School).
Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae ... more Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.
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Thesis Chapters by Jorge Villanueva Araya
Calvarial bone cells: The search for stem cells Jorge E. Villanueva PhD Dissertation University... more Calvarial bone cells: The search for stem cells
Jorge E. Villanueva
PhD Dissertation University of Southern California 0 (1993)
Dispersed calvarial bone cells from rat fetuses were cultured suspended in agarose and some 30% of them showed deposition of metachromic extracellular matrix. Anchorage independence was a requirement for calvarial cells to express type II collagen Cells recovered from densities 1.04 g percoll/ml or higher were able to mature like chondrocytes. This chondrogenic potential in agarose was affected by subcultivation in monolayers: fresh calvarial cells after 1-2 doubling population in monolayer produced type II collagen less than 15% of that synthesized by freshly suspended and after 3-4 doubling population in monolayer he type II collagen synthesis abolished. Also, monolayer sub-cultivation impaired the ability of calvarial cells to produce bone when implanted in rats while inside demineralized bone matrix (DBM) chambers. Moreover, both in vivo and in vitro chondrogenic potential of these cells were inversely depending upon the cell concentration. Bone development is tightly associated with vascularization. Endothelial cells enhanced bone formation by calvarial cells when implanted together in Millipore diffusion chambers. In chambers with calvarial cells alone cartilage was often seen. The calcium deposition was 70 times higher in chambers containing cell mixture than chambers containing either endothelial or calvarial cells alone. DBM-powder did not mimic similar effect on calvarial cell osteogenesis. It is not well known how angiogenesis may promote osteogenesis and a critical step is to define these events occurring between endothelial and bone cells under controlled genotype backgrounds. A cationic liposome was used for the introduction of E1A-gene to isolated rat endothelial and calvarial cell lines. Because of a low growth rate of the former cells, an improved media was designed to success in transforming these cells. Geneticin resistant and E1A-oncoprotein producing endothelial cell lines were established. In addition, a collection of drug-resistant calvaria cell lines were isolated of which morphological appearance, although similar to primary calvaria cell cultures, was very stable after extensive growth. Finally, the design of an alternative approach for the characterization of bone stem cells is proposed by using transient immortalization with a temperature sensitive oncogenic agent and scrutinizing among others the ability to mature like chondrocytes. (Copies available exclusively from Micrographics Department, Doheny Library, USC, Los Angeles, CA, 90089-0182.)
Keywords: Biological sciences, Molecular biology, bone cells
Mi mejor carta de recomendación by Jorge Villanueva Araya
Matrix, 1989
Calvaria from 20-21 day old fetuses were obtained under sterile conditions and the endo- and exop... more Calvaria from 20-21 day old fetuses were obtained under sterile conditions and the endo- and exoperiosteum stripped off. Cells were dispersed by sequential collagenase-DNase treatment and suspended in 0.5% low Tm agarose in the presence of DMEM supplement with 10% FCS. After 4-5 days of incubation some 30% of these cells showed active synthesis of metachromatic extracellular matrix. Cells from skin, muscle and periosteum failed to show metachromatic matrix positive colonies to a comparable extent. The phenotypic expression of these cells was determined by analysis of collagen types. Eleven day old cultures were incubated in the presence of [3H]-proline plus beta-aminopropionitrile and ascorbic acid and the collagen extracted analyzed by polyacrylamide electrophoresis of their intact chains or CNBr-derived peptides. The results show that anchorage independence is a requirement for calvaria cells to express type II collagen. Type I collagen was preferentially expressed in monolayer culture or when pre-attached to a substrate before being cultured in agarose. Type II collagen was the predominant collagen when cells were cultured in agarose. Further characterization of cell populations was achieved by isopycnic centrifugation in a percoll gradient. Cell fractions were tested for their collagen phenotype when cultured in agarose. Cells recovered from densities 1.04 g/ml or higher synthesized type II collagen, while cells with densities lower than 1.04 g/ml synthesized mainly type I collagen. Isopycnic centrifugation appears to be a novel method for separation of phenotypically different cells from a heterogeneous population in fetal calvaria. The high density cell fractions may represent a mixture of pre-chondrocytes as well as pluripotential cells.
CÉLULAS MADRE Y MEDICINA DE REEMPLAZO CELULAR Stem Cells and Replacement Cellular Medicine, 2018
RESUMEN Las Células Madre (CM) son las responsables que aproximadamente 37,2 x 10 12 de células e... more RESUMEN
Las Células Madre (CM) son las responsables que aproximadamente 37,2 x 10 12 de células en un individuo promedio de 30 años en edad, con un peso de 70 Kg, 1.72m de altura y una superficie corporal de 1.85m2, provengan de una única célula: “el cigoto”1. Las CM se encuentran en todos los organismos pluricelulares y están encargadas de repoblar el tejido u órgano anfitrión durante el desarrollo, crecimiento, homeostasis, lesión y enfermedad. Debido al limitado potencial regenerativo de la naturaleza humana, se hace imperativo su estudio. En las últimas décadas ha habido un enorme avance en el conocimiento de la biología de las CM como también la habilidad de inducir la pluripotencia en células adultas, lo están permitiendo su acelerada traslación a la Medicina de reemplazo celular y terapia génica.
PALABRAS CLAVE: Células madre, Inducción de pluripotencia, Aislamiento de CM, Medicina de reemplazo celular, Terapia Génica.
ABSTRACT
Stem Cells (CM) are responsible for approximately 37.2 x 1012 cells in an average individual 30 years of age, with a weight of 70 kg, 1.72 m in height and a body surface area of 1.85m2, come from a single cell: "the zygote"1. The CM are found in all multicellular organisms and are responsible for repopulating the host tissue or organ during development, growth, homeostasis, injury and disease. Due to the limited regenerative potential of human nature, its study is imperative. In the last decades there has been an enormous advance in the knowledge of the biology of the CM as well as the ability to induce the pluripotency in adult cells, these are allowing its accelerated translation to the Medicine of cellular replacement and gene therapy.
KEYWORDS: Stem cells, Pluripotency induction, CM isolation, celular replacement Medicine, Gene Therapy.
Bacterial strains and plasmids. The strains, vectors, and recombinant plasmids used in this work ... more Bacterial strains and plasmids. The strains, vectors, and recombinant plasmids used in this work are described in . E. coli K38pGP1-2 and pT7-7 were kindly provided by S. Tabor (Harvard Medical School).
Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae ... more Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.
and keywords not received
Calvarial bone cells: The search for stem cells Jorge E. Villanueva PhD Dissertation University... more Calvarial bone cells: The search for stem cells
Jorge E. Villanueva
PhD Dissertation University of Southern California 0 (1993)
Dispersed calvarial bone cells from rat fetuses were cultured suspended in agarose and some 30% of them showed deposition of metachromic extracellular matrix. Anchorage independence was a requirement for calvarial cells to express type II collagen Cells recovered from densities 1.04 g percoll/ml or higher were able to mature like chondrocytes. This chondrogenic potential in agarose was affected by subcultivation in monolayers: fresh calvarial cells after 1-2 doubling population in monolayer produced type II collagen less than 15% of that synthesized by freshly suspended and after 3-4 doubling population in monolayer he type II collagen synthesis abolished. Also, monolayer sub-cultivation impaired the ability of calvarial cells to produce bone when implanted in rats while inside demineralized bone matrix (DBM) chambers. Moreover, both in vivo and in vitro chondrogenic potential of these cells were inversely depending upon the cell concentration. Bone development is tightly associated with vascularization. Endothelial cells enhanced bone formation by calvarial cells when implanted together in Millipore diffusion chambers. In chambers with calvarial cells alone cartilage was often seen. The calcium deposition was 70 times higher in chambers containing cell mixture than chambers containing either endothelial or calvarial cells alone. DBM-powder did not mimic similar effect on calvarial cell osteogenesis. It is not well known how angiogenesis may promote osteogenesis and a critical step is to define these events occurring between endothelial and bone cells under controlled genotype backgrounds. A cationic liposome was used for the introduction of E1A-gene to isolated rat endothelial and calvarial cell lines. Because of a low growth rate of the former cells, an improved media was designed to success in transforming these cells. Geneticin resistant and E1A-oncoprotein producing endothelial cell lines were established. In addition, a collection of drug-resistant calvaria cell lines were isolated of which morphological appearance, although similar to primary calvaria cell cultures, was very stable after extensive growth. Finally, the design of an alternative approach for the characterization of bone stem cells is proposed by using transient immortalization with a temperature sensitive oncogenic agent and scrutinizing among others the ability to mature like chondrocytes. (Copies available exclusively from Micrographics Department, Doheny Library, USC, Los Angeles, CA, 90089-0182.)
Keywords: Biological sciences, Molecular biology, bone cells