Biomedical subjects

Keith G Danielson

Publications and source records attributed to Keith G Danielson.

15 recordsLinked to original sources

PubMed2006

TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc.

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.

Amino Acid Motifs↗

PubMed2006

Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment.

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1alpha, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1alpha were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1alpha subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1alpha protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1alpha protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1alpha transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1alpha is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1alpha can be used as a phenotypic marker of nucleus pulposus cells.

Adaptation, Physiological↗

PubMed2006

Osteogenic potential of adult human stem cells of the lumbar vertebral body and the iliac crest.

STUDY DESIGN: Marrow was aspirated from the vertebral body (VB) and iliac crest (IC) of patients undergoing lumbar spinal surgery, following an approved protocol. Progenitor cells were isolated using standard culture conditions and their osteogenic potential evaluated. OBJECTIVE: To evaluate the osteogenic potential of mesenchymal stem cells (MSCs) isolated from the bone marrow of the human VB. SUMMARY OF BACKGROUND DATA: IC marrow grafting during cervical discectomy and fusion procedure is associated with donor site morbidity. Since the VB contains marrow cells, it may be possible to circumvent this problem by using this tissue for osseous graft supplementation. However, there is paucity of information concerning the osteogenic potential of non-IC-derived progenitor cells. Herein, we address this issue. METHODS: Marrow samples from VB of patients undergoing lumbar spinal surgery were collected; marrow was also harvested from the IC. Progenitor cells were isolated and the number of colony forming unit-fibroblastic (CFU-F) determined. The osteogenic potential of the cells was characterized using biochemical and molecular biology techniques. RESULTS: Both the VB and IC marrow generated small, medium, and large sized CFU-F. Higher numbers of CFU-F were obtained from the VB marrow than the IC (P < 0.05). Progenitor cells from both anatomic sites expressed comparable levels of CD166, CD105, CD49a, and CD63. Moreover, progenitor cells from the VB exhibited an increased level of alkaline phosphatase activity. MSCs of the VB and the IC displayed similar levels of expression of Runx-2, collagen Type I, CD44, ALCAM, and ostecalcin. The level of expression of bone sialoprotein was higher in MSC from the IC than the VB. VB and IC cells mineralized their extracellular matrix to a similar extent. CONCLUSIONS: Our studies show that CFU-F frequency is higher in the marrow of the VB than the IC. Progenitor cells isolated from both sites respond in a similar manner to an osteogenic stimulus and express common immunophenotypes. Based on these findings, we propose that progenitor cells from the lumbar vertebral marrow would be suitable candidate for osseous graft supplementation in spinal fusion procedures. Studies must now be conducted using animal models to ascertain if cells of the VB are as effective as those of the IC for the fusion applications.

Bone Marrow Cells↗

PubMed2005

N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin.

Animals↗

PubMed2004

Fluorescent detection of differentially expressed cDNA using SYBR gold nucleic acid gel stain.

We describe herein a modified differential gene display (DGD) technique that can be rapidly and simply performed and that eliminates the need for radioactivity by fluorescent visualization of complementary deoxyribonucleic acid (cDNA) bands with SYBR gold nucleic acid gel stain. To streamline the DGD procedure, a number of modifications were employed. Ribonucleic acid isolated from differentially treated populations of human trabecular bone-derived mesenchymal progenitor cells was reverse-transcribed into cDNA using oligo-dT primer, and subsequent amplification of differentially expressed cDNAs was done using arbitrary 25-mer primers and oligo-dT9 30-mer primers. Moderate-sized nondenaturing 6% polyacrylamide gels (30 x 20 cm) of 1.5-mm thickness were used for easier handling and increased sample loading capacity. Gels were subjected to electrophoresis overnight, stained with SYBR gold, and visualized and photographed using a commercially available gel imager. DNA bands ranging in size from 100 to 400 bp were visualized directly on an ultraviolet transilluminator, excised from the gel, and reamplified. The cDNA amplicons were subcloned, sequenced, and gene sequences were identified by a Basic Local Alignment Search Tool of genomic databases. Overall, this rapid and functional method proved quite effective for identification of novel genes that may be of interest in studies of cartilage and bone differentiation.

DNA, Complementary↗

PubMed2003

Biological response of chondrocytes cultured in three-dimensional nanofibrous poly(epsilon-caprolactone) scaffolds.

Nanofibrous materials, by virtue of their morphological similarities to natural extracellular matrix, have been considered as candidate scaffolds for cell delivery in tissue-engineering applications. In this study, we have evaluated a novel, three-dimensional, nanofibrous poly(epsilon-caprolactone) (PCL) scaffold composed of electrospun nanofibers for its ability to maintain chondrocytes in a mature functional state. Fetal bovine chondrocytes (FBCs), maintained in vitro between passages 2 to 6, were seeded onto three-dimensional biodegradable PCL nanofibrous scaffolds or as monolayers on standard tissue culture polystyrene (TCPS) as a control substrate. Gene expression analysis by reverse transcription-polymerase chain reaction showed that chondrocytes seeded on the nanofibrous scaffold and maintained in serum-free medium supplemented with ITS+, ascorbate, and dexamethasone continuously maintained their chondrocytic phenotype by expressing cartilage-specific extracellular matrix genes, including collagen types II and IX, aggrecan, and cartilage oligomeric matrix protein. Specifically, expression of the collagen type IIB splice variant transcript, which is indicative of the mature chondrocyte phenotype, was up-regulated. FBCs exhibited either a spindle or round shape on the nanofibrous scaffolds, in contrast to a flat, well-spread morphology seen in monolayer cultures on TCPS. Organized actin stress fibers were only observed in the cytoplasm of cells cultured on TCPS. Histologically, nanofibrous cultures maintained in the supplemented serum-free medium produced more sulfated proteoglycan-rich, cartilaginous matrix than monolayer cultures. In addition to promoting phenotypic differentiation, the nanofibrous scaffold also supported cellular proliferation as evidenced by a 21-fold increase in cell growth over 21 days when the cultures were maintained in serum-containing medium. These results indicate that the biological activities of FBCs are crucially dependent on the architecture of the extracellular scaffolds as well as the composition of the culture medium, and that nanofibrous PCL acts as a biologically preferred scaffold/substrate for proliferation and maintenance of the chondrocytic phenotype. We propose that the PCL nanofibrous structure may be a suitable candidate scaffold for cartilage tissue engineering.

Actins↗

PubMed2003

Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk.

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.

Animals↗

PubMed2003

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2.

Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen-activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non-chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein-1 (AP-1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP-1. SRF activity, on the other hand, was up-regulated in confluent monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP-1 and SRF activity whereas higher concentrations of this inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c-Fos and c-Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2 treated micromass cultures resulted in reduced activity of both genes. Our findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP-2 to positively regulate chondrogensis.

Animals↗

PubMed2003

A simple, high-yield method for obtaining multipotential mesenchymal progenitor cells from trabecular bone.

In vitro cultures of primary, human trabecular bone-derived cells represent a useful system for investigation of the biology of osteoblasts. Our recent discovery of the multilineage mesenchymal differentiation potential of trabecular bone-derived cells suggests the potential application of these cells as mesenchymal progenitors for tissue repair and regeneration. Such applications are crucially dependent on efficient cell-isolation protocols to yield cells that optimally proliferate and differentiate. In this study, we describe a simple, high-yield procedure, requiring minimal culture expansion, for the isolation of mesenchymal progenitor cells from human trabecular bone. Moreover, these cells retain their ability to differentiate along multiple mesenchymal lineages through successive subculturing. Cell populations isolated and cultured as described here allow the efficient acquisition of a clinically significant number of cells, which may be used as the cell source for tissue-engineering applications.

Aged↗

PubMed2003

Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone.

The in vitro culture of human trabecular bone-derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase-treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone-derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73(+), STRO-1(+), CD105(+), CD34(-), CD45(-), CD144(-) cell population resident within collagenase-treated, culture-processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. When cultured as described, the trabecular bone-derived cells display stem cell-like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long-term in vitro culture.

Adipocytes↗

PubMed2002

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells.

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-beta1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated proteoglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, beta-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor gamma2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.

Adipose Tissue↗

PubMed2002

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.

Alkaline Phosphatase↗

PubMed2002

In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells.

Cartilage constructs were fabricated by press-coating D,D-L,L-polylactic acid polymer blocks of 1 x 0.5 x 0.5 cm onto high-density cell pellets of 1.5 x 10(6) human mesenchymal stem cells (mhMSCs) isolated from the femoral head of patients undergoing total hip arthroplasty. Following attachment of the cell pellets to the polymer surfaces, chondrogenesis was induced by culturing the constructs for 3 weeks in a serum-free, chemically defined, chondrogenic differentiation medium supplemented with transforming growth factor beta-1 (TGF-beta1). Histochemical analysis showed that the press-coated pellets formed cell layers composed of morphologically distinct, chondrocyte-like cells, surrounded by a fibrous, sulfated proteoglycan-rich extracellular matrix. Immunohistochemical analysis detected collagen type II and cartilage proteoglycan link protein within the extracellular matrix. Expression of the cartilage-specific marker genes collagen types II, IX, X, and XI, and aggrecan was detected by RT-PCR. Scanning electron microscopy revealed organized and spatially distinct zones of cells within the cell-polymer constructs, with the superficial layer resembling compact hyaline cartilage. The fabrication method of press-coating biodegradable polymers with mhMSCs allows the in vitro production of cartilage constructs without harvesting chondrocytes from intact articular cartilage surfaces. These constructs may be applicable as prototypes for the reconstruction of articular cartilage defects in humans.

Adult↗

PubMed2002

Different osteochondral potential of clonal cell lines derived from adult human trabecular bone.

Cells derived from human trabecular bones have been shown to have multipotential differentiation ability along osteogenic, chondrogenic, and adipogenic lineages. In this study, we have derived two clonal sublines of human trabecular bone cells by means of stable transduction with human papilloma virus E6/E7 genes. Our results showed that these clonal sublines differ in their osteochondral potential, but are equally adipogenic, indicative of the heterogeneous nature of the parental cell population. The availability of these cell lines should be useful for the analysis of the mechanisms regulating the differentiation of adult mesenchymal progenitor cells.

Adult↗

PubMed2002

Human marrow-derived mesenchymal progenitor cells: isolation, culture expansion, and analysis of differentiation.

A number of adult mesenchymal tissues contain subpopulations of undifferentiated cells, which retain the capacity to differentiate along multiple lineages. These mesenchymal progenitor cells may be cultured in an undifferentiated state and, when given the appropriate signals, differentiate into an expanding list of several mesenchymal and a few ectodermal derived tissues. The maintenance and propagation of the multipotential nature of these progenitor cell populations are crucially dependent on the isolation protocol, the culture expansion conditions, particularly the properties of the fetal bovine serum supplement in the culture medium. This article describes a method for selection of the appropriate serum lot, and introduces a simplified isolation technique to optimize the yield of progenitor cells that maintain the capability of undergoing multilineage differentiation in response to appropriate cues. Cell populations isolated and culture expanded in this manner, by virtue of their multiple differentiation potential, should serve as ideal candidate cells for tissue engineering applications for the repair and regeneration of tissue damaged by disease and or trauma.

Adipocytes↗