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J J Minguell

Publications and source records attributed to J J Minguell.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a discrete population of human bone marrow-derived mesenchymal cells exhibiting properties of uncommitted progenitors.

Human bone marrow-derived mesenchymal progenitor cells (MPC) after ex vivo expansion give rise to a heterogeneous mixture of cells with distinct proliferative potential at various stages of differentiation. Here we show that when proliferative MPC were forced to metabolic death by exposure to 5-fluorouracil, the remaining subset (5-20%) contains a population of quiescent, uncommitted, and undifferentiated mesenchymal cells. The isolated cells self-renew and generate precursors committed at least to the adipogenic and osteogenic lineages. Taken together, these results demonstrate that within ex vivo-expanded bone marrow-derived MPC, there exist a discrete population of mesenchymal cells with properties of uncommitted progenitors. Because these cells are capable of engraftment into bone marrow, spleen, bone, and skeletal muscle after intravenous infusion and can be efficiently transduced with adenoviral vectors, they may represent an interesting option for cellular and gene therapies for a wide range of disorders of mesenchymal tissues.

Adipocytes↗
PubMed2001

Mesenchymal stem cells.

Within the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells. These cells can be ex vivo expanded and induced, either in vitro or in vivo, to terminally differentiate into osteoblasts, chondrocytes, adipocytes, tenocytes, myotubes, neural cells, and hematopoietic-supporting stroma. The multipotential of these cells, their easy isolation and culture, as well as their high ex vivo expansive potential make these cells an attractive therapeutic tool. In this work we will review the information dealing with the biology of mesenchymal progenitors as it has been revealed mainly by ex vivo studies performed with bone marrow-derived cells. The discussed topics include, among others, characteristics of mesenchymal progenitors, evidence for the existence of a vast repertoire of uncommitted and committed progenitors both in the bone marrow and in mesenchymal tissues, a diagram for their proliferative hierarchy, and comments on mobilization, microenvironment, and clinical use of mesenchymal progenitors. Despite the enormous data available at molecular and cellular levels, it is evident that a number of fundamental questions still need to be resolved before mesenchymal progenitors can be used for safe and effective clinical applications in the context of both cell and gene therapies.

Animals↗
PubMed2000

Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells.

OBJECTIVE: Based on their differentiation properties and facilely of ex vivo expansion, human bone marrow mesenchymal progenitor cells (MPC), are considered as attractive targets to deliver foreign genes to the bone marrow or other mesenchymal tissues. In this study we investigated the feasibility of transduce MPC with adenoviral vectors (Adv). METHODS: MPC were expanded ex vivo and transduced with replication-defective Adv-containing reporter genes (lacZ or GFP) under the control of CMV promoter. Transfection efficiency was assessed by microscopical scoring or by flow cytometry. Expression and involvement of Adv-attachment (CAR) and Adv-internalization (integrins alphav) receptors were evaluated by flow cytometric studies. RESULTS: Transgene expression analysis showed that only 19%+/-3% of cells expressed the transgenes at high levels. MPC express the attachment and internalization receptors required for Adv infection. While integrins alphavbeta3 and alphavbeta5 are expressed by all MPC, CAR is solely expressed by a fraction of low size cells. Antibodies against CAR and alphavbeta5, but not against alphavbeta3, blocked Adv-mediated gene transfer into MPC, showing that CAR and alphavbeta5 are required for infection. Because alphavbeta5, as compared with CAR, is overexpressed in MPC, the results suggest that the efficiency of Adv-mediated gene transfer into MPC depends on the level of CAR expression. CONCLUSION: These findings demonstrate that Adv may be useful to engineer a subpopulation of ex vivo expanded human mesenchymal progenitors, with a high level of transgene expression.

Adenoviridae↗
PubMed2000

Mesenchymal progenitor cells in human umbilical cord blood.

Haemopoiesis is sustained by two main cellular components, the haematopoietic cells (HSCs) and the mesenchymal progenitor cells (MPCs). MPCs are multipotent and are the precursors for marrow stroma, bone, cartilage, muscle and connective tissues. Although the presence of HSCs in umbilical cord blood (UCB) is well known, that of MPCs has been not fully evaluated. In this study, we examined the ability of UCB harvests to generate in culture cells with characteristics of MPCs. Results showed that UCB-derived mononuclear cells, when set in culture, gave rise to adherent cells, which exhibited either an osteoclast- or a mesenchymal-like phenotype. Cells with the osteoclast phenotype were multinucleated, expressed TRAP activity and antigens CD45 and CD51/CD61. In turn, cells with the mesenchymal phenotype displayed a fibroblast-like morphology and expressed several MPC-related antigens (SH2, SH3, SH4, ASMA, MAB 1470, CD13, CD29 and CD49e). Our results suggest that preterm, as compared with term, cord blood is richer in mesenchymal progenitors, similar to haematopoietic progenitors.

Adipocytes↗
PubMed2000

Production of soluble CD34 by human myeloid cells.

CD34, a glycophosphoprotein present in lymphohaematopoietic stem and progenitor cells, as well as in other cell types, exists in both transmembrane and intracytoplasmic forms. Transmembrane CD34 expression, which is high in the earliest haematopoietic precursors, decreases as cells mature. However, to our knowledge, there is no information on whether a decrease in transmembrane CD34 can also predict a release of the molecule from the cell membrane into the extracellular fluid. To investigate the above possibility, we studied conditions (incubation time, cell density and proliferative status) in human myeloid cells (lines KG-1a, KG-1 and cord blood-derived cells) that may cause a decrease in surface CD34 and the generation of a soluble form of the molecule. The latter, as demonstrated by Western blot analysis, adds more complexity to the proposed structural features and functional properties of CD34 in myeloid cells.

Antigens, CD34↗
PubMed2000

Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells.

This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high (95 and 85% respectively). Once attached, MCF-7 grow well on both monolayers. Morphology of MCF-7 cells, as analysed by light and epifluorescence microscopy, revealed that MCF-7 cells grow in clusters on 3T3, but disperse on MSC. Concomitant with the lost of their aggregation status, MCF-7 on MSC express low levels of the intercellular adhesion molecules, E-cadherin and epithelial-specific antigen (ESA). These results suggest that MSC represent an appropriate cell target to investigate the cellular and molecular events occurring at the interface of epithelial-marrow stromal interactions. Together, the model here described should permit to further evaluate the significance and prognostic impact of the shift of micrometastatic cells from a cluster-aggregated into a single-cell status.

Breast Neoplasms↗
PubMed2000

Biology and clinical utilization of mesenchymal progenitor cells.

Within the complex cellular arrangement found in the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal progenitor cells (MPC). These cells can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into at least seven types of cells: osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes and hematopoietic-supporting stroma. This broad multipotentiality, the feasibility to obtain MPC from bone marrow, cord and peripheral blood and their transplantability support the impact that the use of MPC will have in clinical settings. However, a number of fundamental questions about the cellular and molecular biology of MPC still need to be resolved before these cells can be used for safe and effective cell and gene therapies intended to replace, repair or enhance the physiological function of the mesenchymal and/or hematopoietic systems.

Animals↗
PubMed1999

Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells.

Bone marrow stroma provides the microenvironment for hematopoiesis and is also the source of mesenchymal progenitors (mesenchymal or marrow stromal cells [MSC]) that may serve as long-lasting precursors for bone, cartilage, lung, and muscle. While several studies have indicated the differentiation potential of MSC, few studies have been performed on the cells themselves. In an attempt to further expand our knowledge on these cells, we have performed studies on their cell cycle, immuno- and adhesive-phenotype, ex vivo expansion, and differentiation properties. MSC cultures have been initiated from human bone marrow low-density mononuclear cells and maintained in the absence of differentiation stimuli and hematopoietic cells. The homogenous layer of adherent cells thus formed exhibits a typical fibroblastlike morphology, a population doubling time of 33 h, a large expansive potential, and cell cycle characteristics including a subset (20%) of quiescent cells. The antigenic phenotype of MSC is not unique, borrowing features of mesenchymal, endothelial, and epithelial cells. Together, MSC express several adhesion-related antigens, like the integrin subunits alpha4, alpha5, beta1, integrins alphavbeta3 and alphavbeta5, ICAM-1, and CD44H. MSC produce and functionally adhere to extracellular matrix molecules. When incubated under proper stimuli, MSC differentiate into osteoblasts or adipocytes. Taken together, these results demonstrate that adherent marrow-derived cells cultured in the absence of hematopoietic cells and differentiation stimulus give rise to a population of cells with phenotypical and functional features of mesenchymal progenitors. The existence of a subset of quiescent cells in MSC cultures seems to be extremely significant, since their number and properties should be enough to sustain a steady supply of cells that upon proliferation and commitment may serve as precursors for a number of nonhematopoietic tissues.

Adipocytes↗
PubMed1997

Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients.

Transplantation of growth factor-mobilized peripheral blood progenitor cells (PBPC) is widely used in the treatment of several neoplastic diseases. While in PBPC harvests the presence of several accessory immune and tumor cells has been documented, that of stromal cells has not been reported. In the present study, we investigated for the presence of stromal cells in growth factor-mobilized PBPC harvests from breast cancer patients. Low-density cells from PBCP harvests in culture gave rise to an adherent layer containing fibroblast-like and large flat round cells. These cells express positive immunofluorescence staining for collagen I, collagen III, fibronectin, VCAM-1 (CD106), ICAM-1 (CD54) and mesenchymal antigens recognized by monoclonal antibodies, SH2 and SH3. PBPC-derived stromal cells do not express antigens CD34, CD45 and CD14. Stromal cells were detected in the PBPC harvests of 11/14 patients (median 0.63%; range 0.02-2.32) and their concentration correlates with the number of CD34+ cells in PBPC.

Antigens, CD34↗
PubMed1997

Hydrocortisone regulates types I and III collagen gene expression and collagen synthesis in human marrow stromal cells.

Hematopoiesis is the resultant of the orderly molecular and cellular interactions between progenitor cells and stroma. In vitro studies (Dexter-type cultures) have shown that initiation of hematopoiesis only occurs after establishment of a hydrocortisone-dependent layer of stromal cells. Although the molecular basis for the requirement of hydrocortisone are not well understood, data have shown that synthesis/assembly of extracellular matrix molecules (proteoglycans and fibronectin) is regulated by hydrocortisone. Since interstitial collagens are abundantly expressed in the marrow stroma, we investigated whether hydrocortisone may also modulate the expression of collagen types I and III. For these studies, human bone marrow fibroblast cultures were grown in standard culture medium either in the absence or presence of 10(-7) M hydrocortisone. Under both conditions, bone marrow fibroblasts synthesized collagen types I and III, and expressed the respective genes. However, hydrocortisone produced a decrease in the synthesis of interstitial collagens and also in the relative abundance of pro-alpha 1(I) and pro-alpha 1(III) mRNAs. The results of this study are consistent with the assumption that glucocorticoids regulate the expression of several extracellular matrix molecules in the marrow stroma and thus permit in vitro hematopoiesis to occur.

Bone Marrow↗
PubMed1996

The role of collagen in hematopoiesis.

Several types of collagen, including types I, III, IV, V and VI, are produced by bone marrow stromal cells. Current information indicates that changes in collagen production result in profound alterations in the capacity of hematopoietic precursors to proliferate and differentiate. Although not definitively established, collagen molecules may be involved in the establishment and conformation of the stroma-associated extracellular matrix and/or in adhesive interactions with progenitor cells. The dynamic role of collagen in hematopoiesis is indicated by the observation that collagen production and processing are regulated by several factors such as glucocorticoids, cytokines, collagenases and collagenase-inhibitory proteins.

Bone Marrow↗
PubMed1995

Modulation of the adhesion of hemopoietic progenitor cells to the RGD site of fibronectin by interleukin 3.

The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell-extracellular matrix (ECM) interactions amd also of cell-cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin-mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP-1 were used. Both of these cell lines have been shown to bind to FN-coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by alpha 5 beta 1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of alpha 5 and one antibody against beta 1 were used to study expression of the integrin. By flow cytometry the expression of alpha 5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP-1 cells the decrement was from 1.0 to 0.8. The loss of RGD-mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine-treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation.

Animals↗
PubMed1995

IL-3 increases surface proteoglycan synthesis in haemopoietic progenitors and their adhesiveness to the heparin-binding domain of fibronectin.

Haemopoietic progenitor cells (HPC) synthesize and accumulate a single type of membrane-associated chondroitin sulphate proteoglycan (MA-PG), which participates in HPC adhesiveness to fibronectin by interacting with its heparin-binding domain. Shortly after incubating cells with IL-3, we observed an increase in MA-PG synthesis in the multipotent (FDCP-mix) but not in the bipotent (FDCP-1) progenitor cell line. The charge density, hydrodynamic size, nature of the glycosaminoglycan (GAG) chains and stability of MA-PG from IL-3-treated and non-treated FDCP-mix cells were the same, suggesting that IL-3 affects the amount of MA-PG. The latter was evaluated by flow cytometry using monoclonal antibodies to the core protein and GAG residues. In all cases the mean fluorescence intensities were higher for IL-3-treated than for untreated cells. Cell adhesion studies to dishes coated with the fibronectin 40 kD fragment, containing the heparin-binding domain, demonstrated that adhesiveness of IL-3-treated cells was higher than that of untreated cells. These results suggest that in multipotent haemopoietic cells IL-3 regulates the amount of membrane-associated proteoglycans, which in turn modify the adhesive interactions of cells with the heparin-binding domain of fibronectin.

Animals↗
PubMed1995

The sulfation degree of membrane-associated proteoglycan from a hemopoietic cell line is determined by changes in the growth state of the cell.

Multipotential hemopoietic progenitor cells (FDCP-mix) proliferate in culture medium supplemented with horse serum. When transferred to a medium without serum, cells do not proliferate and enter a quiescent state. Both proliferative and quiescent cells synthesize only chondroitin sulfate proteoglycan (CS-PG) which is associated to the cell membrane. Incorporation of 35SO4 into CS-PG was 4-fold higher in quiescent than in proliferative cells. Flow cytometric studies using monoclonal antibodies which recognize the core protein or the CS chains, showed that the increased uptake of sulfate was not the consequence of an increase in the abundance of CS-PG. Further characterization demonstrated that CS-PG isolated from quiescent cells exhibited a slightly higher hydrodynamic size than CS-PG from proliferative cells. However, the glycosaminoglycan chains from PG derived from proliferative and quiescent cells have the same hydrodynamic size. Through ion-exchange chromatography we observed that the mean charge density of PG from quiescent cells was higher than in proliferative cells, suggesting a higher sulfation degree from PG synthesized by quiescent cells. This was confirmed by flow cytometric studies using monoclonal antibody 2B6, which recognizes the unsaturated terminal disaccharide of chondroitin-4-O-sulfate.

Animals↗
PubMed1994

Modifications in the synthesis of membrane-associated chondroitin sulfate proteoglycans in hemopoietic progenitor cells are accompanied by alterations in their adhesive properties.

In vitro studies in our laboratory have indicated that murine hemopoietic progenitor cell (HPC) lines, irrespective of their differentiation stage, synthesize and accumulate in the cell membrane a unique species of chondroitin sulfate proteoglycan (CS-PG). It has been postulated that CS-PG participates in HPC adhesion to pericellular stromal fibronectin by interacting with its heparin-promoting binding region. To further support this contention, we first attempted to modify CS-PG synthesis in HPC by the use of chlorate and p-nitrophenyl beta-D-xyloside, which inhibit sulfation and glycosaminoglycan (GAG) addition in proteoglycans, respectively. We then studied the effect that these modifications may have in the adhesive capacity of HPC to interact with fibronectin and its cell- and heparin-promoting binding chymotryptic fragments. Treatment with chlorate which resulted in a decreased sulfation of membrane-associated 35 S-labeled CS-PG, as judged by ion exchange chromatography, did not affect HPC adhesion to fibronectin or its fragments. However, beta-xyloside treatment which reduces the abundance of membrane-associated CS-PG, as evidenced by molecular sieve chromatography, produced a major and specific decrease in HPC adhesion to the heparin-promoting binding fragment of fibronectin. These results indicate that CS-PG are involved in HPC interaction with fibronectin, in a mode that seems to be dependent on the differentiation stage of HPC.

Animals↗
PubMed1994

[Ex vivo procedures associated with autologous hematopoietic stem cell transplantation].

In the last 15 years, autologous cryopreserved bone marrow has been extensively used to reestablish hematopoiesis after myeloablative therapies in patients with leukemia, lymphomas or solid tumors. Recently, the use of stem cells obtained from peripheral blood has become an additional source of autologous stem cells for patients, with bone marrow diseases as well as for patients with mammary or ovarian cancer, after high dose chemotherapy. The use of this therapy has created the need of implementing laboratory procedures (ex vivo) to concentrate, purify, cryopreserve and evaluate the hematopoietic stem cell and eliminate tumoral cells. This article reviews these procedures, available in Chile at the present time, and comments on the impact of its use in autologous bone marrow or peripheral blood stem cell transplantations in an estimated population of 300 patients per year.

Hematopoietic Stem Cell Transplantation↗