Biomedical subjects

Sergei A Kuznetsov

Publications and source records attributed to Sergei A Kuznetsov.

10 recordsLinked to original sources

PubMed2006

Sensitive and specific method for detecting G protein-coupled receptor mRNAs.

G protein-coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 mug of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines.

Cell Line↗

PubMed2006

In vivo bone formation by human bone marrow stromal cells: reconstruction of the mouse calvarium and mandible.

Bone marrow stromal cells (BMSCs) contain a subset of multipotent cells with the potential to repair hard-tissue defects. Mouse BMSCs, combined with a collagen carrier, can close critical-sized homologous mouse calvarial defects, but this new bone has a poor union with the adjacent calvarium. When human BMSCs are transplanted for the purpose of engineering new bone, best results can be achieved if the cells are combined with hydroxyapatite/tricalcium phosphate (HA/TCP) particles. Here, we demonstrate that transplantation of cultured human BMSCs in conjunction with HA/TCP particles can be used successfully to close mouse craniofacial bone defects and that removal of the periosteum from the calvarium significantly enhances union with the transplant. Transplants were followed for up to 96 weeks and were found to change in morphology but not bone content after 8 weeks; this constitutes the first description of human BMSCs placed long-term to heal bone defects. New bone formation continued to occur in the oldest transplants, confirmed by tetracycline labeling. Additionally, the elastic modulus of this engineered bone resembled that of the normal mouse calvarium, and our use of atomic force microscopy (AFM)-based nanoindentation offered us the first opportunity to compare these small transplants against equally minute mouse bones. Our results provide insights into the long-term behavior of newly engineered orthotopic bone from human cells and have powerful implications for therapeutic human BMSC transplantation.

Animals↗

PubMed2006

Postnatal skeletal stem cells.

Postnatal skeletal stem cells are a subpopulation of the bone marrow stromal cell network. To date, the most straightforward way of assessing the activity of skeletal stem cells within the bone marrow stromal cell (BMSC) population is via analysis of the rapidly adherent, colony-forming unit-fibroblast (CFU-F), and their progeny, BMSCs. Several in vitro methods are employed to determine the differentiation capacity of BMSCs, using osteogenic and adipogenic "cocktails" and staining protocols, and pellet cell culture for chondrogenic differentiation. However, true differentiation potential is best determined by in vivo transplantation in either closed or open systems. By in vivo transplantation, approximately 10% of the clonal strains are able to form bone, stroma, and marrow adipocytes, and are true skeletal stem cells. Furthermore, when derived from patients or animal models with abnormalities in gene expression, they recapitulate the disease phenotype on in vivo transplantation. Although ex vivo expansion of BMSCs inevitably dilutes the skeletal stem cells, when used en masse, they are attractive candidates for reconstruction of segmental bone defects, and as targets for gene therapy.

Adult Stem Cells↗

PubMed2006

Canine cranial reconstruction using autologous bone marrow stromal cells.

Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects.

Animals↗

PubMed2004

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow.

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.

Animals↗

PubMed2004

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.

Alleles↗

PubMed2004

Bone formation in transplants of human bone marrow stromal cells and hydroxyapatite-tricalcium phosphate: prediction with quantitative CT in mice.

PURPOSE: To determine whether quantitative computed tomography (CT) can be used to estimate the extent of new bone formation in hydroxyapatite-tricalcium phosphate (HA-TCP)-based transplants. MATERIALS AND METHODS: Bone-forming transplants were generated by attaching cultured human bone marrow stromal cells to aliquots of HA-TCP particles and were placed in subcutaneous pockets in immunocompromised mice. After 8 weeks, the transplants were individually imaged; each scan included a phantom. Overall bone mineral density (BMD) of each transplant was obtained. Hematoxylin-eosin-stained sections of the same transplants were then examined histologically, which is the reference standard for assessing bone formation. The extent of bone in each transplant was scored on a semiquantitative scale ranging from 0 to 4 by three independent blinded observers; the bone score for each transplant was calculated by averaging the three observer scores. BMD was compared with the histologically determined bone score for each transplant. Statistical evaluations included (a) calculation of empiric receiver operating characteristic curves to determine optimum BMD thresholds and (b) determination of the relationship between BMD and bone score, including derivation of Pearson correlation coefficients. RESULTS: One hundred twenty transplants were evaluated. Average BMD of 600 mg/cm3 K2HPO4 or more was noted in transplants with appreciable bone formation (bone score > or = 3), while average BMD of less than 600 mg/cm3 K2HPO4 was seen in transplants with poor bone formation (bone score < 3) (P <.001). Among transplants with appreciable bone formation, the BMD was proportional to the extent of mineralized matrix present in the new bone. CONCLUSION: Use of quantitative CT offers a practical approach for the noninvasive determination of new bone formation in mineralizing bone marrow stromal cells and HA-TCP transplants.

Animals↗

PubMed2003

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles.

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.

Actins↗

PubMed2003

Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells.

Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells.

Actins↗

PubMed2002

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis.

It is widely believed that microtubule- and F-actin-based transport of cytoplasmic organelles and membrane fusion is down-regulated during mitosis. Here we show that during the transition of Xenopus egg extracts from interphase to metaphase myosin V-driven movement of small globular vesicles along F-actin is strongly inhibited. In contrast, the movement of ER and ER network formation on F-actin is up-regulated in metaphase extracts. Our data demonstrate that myosin V-driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of F-actin in partitioning, positioning, and membrane fusion of the ER during cell division.

Actins↗