Biomedical subjects

A I Caplan

Publications and source records attributed to A I Caplan.

At least 55 records · Page 3Linked to original sources

PubMed1998

In vivo osteogenesis assay: a rapid method for quantitative analysis.

A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale scoring method gave an r2=0.767 while the Percentage Bone method gave a value of 0.902. These results indicate that scoring ceramic cubes by the percentage of pores containing bone gives a result that corresponds to image analysis measurements at nearly a 90% confidence level. Thus, the Percentage Bone method of scoring is an accurate and relatively quick scoring method for in vivo bone formation.

Animals↗

PubMed1998

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel.

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.

Animals↗

PubMed1998

The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells.

Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.

Adult↗

PubMed1998

Different response to osteo-inductive agents in bone marrow- and periosteum-derived cell preparations.

Rabbit bone marrow- and periosteum-derived cells were cultured in medium containing dexamethasone, bone morphogenetic protein-2 (BMP2) or both. The response of bone marrow-derived cells, measured as alkaline phosphatase expression, depended on the stage of growth. In subconfluent cultures, BMP2 had a greater effect than dexamethasone and treatment with both further increased enzyme activity. In confluent cultures, the effect of dexamethasone was greater than that of BMP2 and, when used together, they had an additive effect. The mineral deposition observed in these cultures did not have the typical structure of bone nodules. For periosteum-derived cells, dexamethasone did not increase the expression of alkaline phosphatase, while BMP2 did; treatment with both was less effective than treatment with BMP2 alone. Typical bone nodules were observed in cultures of periosteum-derived cells treated with dexamethasone and BMP2. These findings indicate that either osteoprogenitor cells from these two sources are intrinsically different or else non-progenitor cells in the preparations directly or indirectly affect the responsiveness to osteo-inductive agents.

Alkaline Phosphatase↗

PubMed1998

Articular cartilage repair. Rabbit experiments with a collagen gel-biomatrix and chondrocytes cultured in it.

To repair a full-thickness articular cartilage defect in rabbit knees, we developed a technique of using a collagen gel hardened by cultured allogeneic chondrocytes in it. The gel-chondrocyte composite accumulated an intense metachromatic matrix, and had elasticity and stiffness enough to be shaped easily after 2 weeks' culture in vitro. It was implanted into full-thickness articular cartilage defects. Histologic evaluation was performed up to 6 months after surgery, using a histologic grading scale composed of 5 categories. In the gel-chondrocyte composite implanted group, good repair was observed from as early as 1 day up to 6 months. On the other hand, in the empty control group, no repair was observed 1 day to 2 weeks after the defects were made. At 4 weeks, some repair occurred, but even at 6 months the repair was not good.

Animals↗

PubMed1997

LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors.

Human marrow-derived mesenchymal progenitor cells (hMPCs), which have the capacity for osteogenic and marrow stromal differentiation, were transduced with the myeloproliferative sarcoma virus (MPSV)-based retrovirus, vM5LacZ, that contains the LacZ and neo genes. Stable transduction and gene expression occurred in 18% of cells. After culture expansion and selection in G418, approximately 70% of neo(r) hMPCs co-expressed LacZ. G418-selected hMPC retain their osteogenic potential and form bone in vivo when seeded into porous calcium phosphate ceramic cubes implanted subcutaneously into SCID mice. LacZ expression was evident within osteoblasts and osteocytes in bone developing within the ceramics 6 and 9 weeks after implantation. Likewise, hMPCs transduced with human interleukin-3 (hIL-3) cDNA, adhered to ceramic cubes and implanted into SCID mice, formed bone and secreted detectable levels of hIL-3 into the systemic circulation for at least 12 weeks. These data indicate that genetically transduced, culture-expanded bone marrow-derived hMPCs retain a precursor phenotype and maintain similar levels of transgene expression during osteogenic lineage commitment and differentiation in vivo. Because MPCs have been shown to differentiate into bone, cartilage, and tendon, these cells may be a useful target for gene therapy.

Adult↗

PubMed1997

Human bone marrow-derived mesenchymal (stromal) progenitor cells (MPCs) cannot be recovered from peripheral blood progenitor cell collections.

The purpose of this study was to compare the ability to collect human bone marrow-derived mesenchymal (stromal) progenitor cells (MPC) from bone marrow versus peripheral blood hematopoietic progenitor cell (PBPC) collections using in vitro and in vivo assays. Ten milliliter samples of PBPC collections mobilized from 11 patients undergoing autotransplants using chemotherapy followed by G-CSF 5-10 micrograms/kg were evaluated using in vitro and in vivo assays for hematopoietic progenitors and MPCs. Additionally, 10 ml samples of unstimulated bone marrow aspirates as well as PBPC collected after mobilization using G-CSF 10 micrograms/kg obtained from 3 normal, histocompatible allogeneic donors were analyzed for hematopoietic progenitors and MPCs. The MPCs were isolated and culture-expanded as adherent cells in vitro and subsequently tested for the capacity to differentiate into mesenchymal phenotypes in vivo using calcium hydroxyapatite porous ceramic cubes implanted s.c. in athymic mice. Demineralized sections of these cubes were analyzed histologically for the appearance of bone and cartilage. Seven autotransplant subjects with cancer received G-CSF after chemotherapy administration, whereas 4 cancer patients and all 3 normal donors received G-CSF alone as the mobilizing regimen. For the autologous PBPC collections and the normal marrow aspirations, median hematopoietic progenitor content was in the normal range for our institution. MPCs were detected in in vitro cultures and as bone-positive ceramic cubes in samples of all 3 allogeneic donor bone marrows but in none of the 14 autologous and 6 allogeneic PBPC collections. In conclusion, MPCs could not be recovered in PBPC collections obtained from either normal donors or patients who underwent PBPC collections after mobilization therapy but could be obtained routinely from bone marrow samples. Although the role of transplanted MPCs is an area of clinical investigation, this study points out a fundamental differences in the population of cells transplanted after collection from bone marrow versus peripheral blood.

Adult↗

PubMed1997

Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

Bone marrow stroma contains multipotential mesenchymal progenitor cells which can differentiate into osteoblastic cells; we refer to these cells as mesenchymal stem cells (MSCs). Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) have been implicated in the osteogenic regulatory process by virtue of their mitogenic and differentiation activities, respectively. This study examines and compares the effects of bFGF and BMP-2 on dexamethasone (Dex)-dependent in vitro osteogenic differentiation of rat marrow-derived MSCs. A 6-day exposure to bFGF markedly stimulated cell growth and induced osteoblastic differentiation as shown by osteocalcin mRNA expression (day 14), bone nodule formation (day 18), and calcium deposition (day 18). These results indicate that bFGF enhances both mitogenic activity and osteogenic development of Dex-treated marrow MSCs. In contrast, BMP-2 did not induce osteogenesis as strongly as bFGF. Thus, exposure to BMP-2 slightly increased bone nodule number and calcium content compared with the control. Exposure of MSCs to both BMP-2 and bFGF induced expression of osteocalcin mRNA and mineralizing bone-like nodules as early as day 11 and resulted in enhancement of bone formation more markedly than either factor alone. Consistent with these results, porous calcium phosphate ceramic cubes implanted in vivo, which were loaded with MSCs pre-exposed to both bFGF and BMP-2, showed higher histologic score for bone formation than those with MSCs pre-exposed to either bFGF or BMP-2 alone. These data indicate that combined treatment with bFGF and BMP-2 synergistically enhances the osteogenic potency of bFGF in rat marrow MSC culture.

Animals↗

PubMed1997

Role of type X collagen on experimental mineralization of eggshell membranes.

Type X collagen is a transient and developmentally regulated collagen that has been postulated to be involved in controlling the later stages of endochondral bone formation. However, the role of this collagen in these events is not yet known. In order to understand the function of type X collagen, if any, in the process of biomineralization, the properties of type X collagen in eggshell membranes were further investigated. Specifically, calvaria-derived osteogenic cells were tested for their ability to mineralize eggshell membranes in vitro. Immunohistochemistry with specific monoclonal antibodies was used to correlate the presence or absence of type X collagen or its propeptide domains with the ability of shell membranes to be mineralized. The extent of mineralization was assessed by Von Kossa staining, scanning electron microscopy and energy-dispersive spectroscopy. The results indicate that the non-helical domains of type X collagen must be removed to facilitate the cell-mediated mineralization of eggshell membranes. In this tissue, intact type X collagen does not appear to stimulate or support cell-mediated mineralization. We postulate that the non-helical domains of type X collagen function in vivo to inhibit mineralization and thereby establish boundaries which are protected from mineral deposition.

Amino Acid Sequence↗

PubMed1997

Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell.

The eggshell of the chicken is a useful model to study matrix components which affect biomineralization. As an extension of our previous immunohistochemical work which suggested the presence of dermatan sulfate proteoglycans in the mineralized region of the eggshell, a study was undertaken to characterize these molecules biochemically. After demineralization with HCl and extraction with 4 M guanidinium chloride containing protease inhibitors, the extract was partitioned by anion exchange chromatography. Step elution with 0.25 M and 1.0 M sodium chloride resulted in the generation of two fractions, both of which contain chondroitinase-sensitive proteoglycans with molecular weights estimated at 200,000 by gel electrophoresis. The proteoglycans in each fraction have core proteins with molecular weights of approximately 120,000 and glycosaminoglycans with average molecular weights of 22,000. Based on differential sensitivity to chondroitinase ABC and AC II, these glycosaminoglycans contain a small proportion of dermatan sulfate. The disaccharide compositions of these glycosaminoglycans differ for the proteoglycans eluted with 0.25 M and 1.0 M sodium chloride. Those eluted with lower sodium chloride are enriched in unsulfated chondroitin and have much more 4-sulfated than 6-sulfated disaccharides; those eluted with 1.0 M sodium chloride contain primarily 4-sulfated disaccharides, a small amount of 6-sulfated disaccharides, and less unsulfated disaccharides than the proteoglycans eluted with 0.25 M sodium chloride. The large difference in the proportions of unsulfated chondroitin may be the reason for the elution at different sodium chloride concentrations. Both of the anion exchange column fractions contain other proteins in addition to the proteoglycans. These proteins are not separated from the proteoglycans by a second anion exchange column or by molecular sieve chromatography under dissociative conditions. Of particular interest is the observation that the eggshell proteoglycans and their core proteins are recognized by a monoclonal antibody which recognizes an epitope on the core protein of avian versican. This suggests that, in spite of the large differences in the sizes of the core proteins of versican and the eggshell proteoglycans, these core proteins share some homology. Because anionic molecules are thought to be important regulators of biomineralization, and because preparations like those analyzed in this study have been shown to influence in vitro calcium carbonate crystallization, the eggshell proteoglycans may play a role in eggshell mineralization.

Amino Acid Sequence↗

PubMed1997

Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

Adolescent↗

PubMed1997

Principles of cartilage repair and regeneration.

An experimental approach and logic are presented for the regeneration of skeletal tissues that focus on the recapitulation of embryonic events starting with an uncommitted progenitor cell population that the authors refer to as mesenchymal stem cells. The repair and regeneration of articular cartilage, which itself has no repair potential, is the subject of this presentation. Full thickness cartilage defects were created in the medial condyle of the distal femur. Self repair (empty defects), articular chondrocytes (allografts), and autologous mesenchymal stem cells were used and the results are reported in selected examples from more than 800 rabbit knees. The optimal number of the appropriate cells delivered in a supportive vehicle to a defect pretreated with a dilute trypsin solution to optimize the integration of repair with normal host cartilage provides a methodology in which regeneration of articular cartilage can be observed. The principles have relevance to the clinical repair and regeneration of cartilage and other skeletal defects.

Animals↗

PubMed1996

Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha.

We previously reported the purification, culture-expansion, and osteogenic differentiation potential of mesenchymal progenitor cells (MPCs) derived from human bone marrow. As a first step to establishing the phenotypic characteristics of MPCs, we reported on the identification of unique cell surface proteins which were detected with monoclonal antibodies. In this study, the phenotypic characterization of human marrow-derived MPCs is further established through the identification of a cytokine expression profile under standardized growth medium conditions and in the presence of regulators of the osteogenic and stromal cell lineages, dexamethasone and interleukin-1 alpha (IL-1 alpha), respectively. Constitutively expressed cytokines in this growth phase include G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11, while GM-CSF, IL-3, TGF-beta 2 and OSM were not detected in the growth medium. Exposure of cells in growth medium to dexamethasone resulted in a decrease in the expression of LIF, IL-6, and IL-11. These cytokines have been reported to exert influence on the differentiation of cells derived from the bone marrow stroma through target cell receptors that utilize gp130-associated signal transduction pathways. Dexamethasone had no effect on the other cytokines expressed under growth medium conditions and was not observed to increase the expression of any of the cytokines measured in this study. In contrast, IL-1 alpha increased the expression of G-CSF, M-CSF, LIF, IL-6 and IL-11 and induced the expression of GM-CSF. IL-1 alpha had no effect on SCF expression and was not observed to decrease the production of any of the cytokines assayed. These data indicate that MPCs exhibit a distinct cytokine expression profile. We interpret this cytokine profile to suggest that MPCs serve specific supportive functions in the microenvironment of bone marrow. MPCs provide inductive and regulatory information which are consistent with the ability to support hematopoiesis, and also supply autocrine, paracrine, and juxtacrine factors that influence the cells of the marrow microenvironment itself. In addition, the cytokine profiles expressed by MPCs, in response to dexamethasone and IL-1 alpha, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression.

Adult↗

PubMed1996

Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse.

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2Kb-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10(-7) M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells.

Adipocytes↗

PubMed1996

Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro.

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-beta 1 (TGF-beta 1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-beta 1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-beta 1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-beta 1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-beta 1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.

Alkaline Phosphatase↗

PubMed1996

Regulated expression of chondroitin sulfates at sites of epithelial-mesenchymal interaction: spatio-temporal patterning identified with anti-chondroitin sulfate monoclonal antibodies.

Chondroitin sulfate proteoglycans, cell surface and extracellular matrix molecules in both neural and non-neural tissues, are highly regulated during normal development. Entire proteoglycan molecules may be either up-regulated or down-regulated, or only the chondroitin sulfate glycosaminoglycan portions of these molecules may be modified. Subtle changes in the chemistries of chondroitin sulfate chains can now be identified through the use of a panel of anti-chondroitin sulfate monoclonal antibodies. Each of these antibodies recognizes specific chemical structures which are non-randomly dispersed along the lengths of chondroitin sulfate chains. The location of individual epitopes within defined domains in these chains is demonstrated through controlled treatments of aggrecan with chondroitinase ABC, whereby portions of these chains are removed from the non-reducing terminal ends and where the remainder of the chains remains covalently attached to the core protein. In these situations, some epitopes, such as those recognized by antibodies CS-56 and 6C3, can be removed without loss of other epitopes, such as that recognized by antibody 4C3. The independent expression of individual epitopes is demonstrated by immunocytochemical analyses of developing skin appendages in embryonic chicks and fetal humans. These are sites where highly patterned morphogenetic movements result from epithelial-mesenchymal interactions. In both chicks and humans, some epitopes are constitutively expressed while others are strictly regulated in the mesenchymal portions of the developing skin appendages. These data strongly suggest that chondroitin sulfate proteoglycans, including their chondroitin sulfate chains, have important roles in regulating these epithelial mesenchymal interactions. Furthermore, these data underscore the significance of the aforementioned observation that individual epitopes are located in specific domains within chondroitin sulfate chains. The highly organized expression of chondroitin sulfate proteoglycans in the development of the central nervous system strongly argues for a similar role for these molecules in the organs that comprise this system.

Animals↗

PubMed1996

Variable hormone responsiveness of osteoblast populations isolated at different stages of embryogenesis and its relationship to the osteogenic lineage.

A variable response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone treatment was observed for osteoblast cell populations isolated from 12- and 17-day-old embryonic chick calvariae. The younger embryonic cell population showed 2- and 5-fold inductions of osteocalcin and osteopontin gene expression, respectively, and a 25% inhibition of collagen gene expression when treated with 1,25-(OH)2D3. In contrast, these same genes all displayed approximately 80% inhibition of their expression when the older embryonic cell populations were treated with hormone. The hormone response was related to the appearance of the vitamin D3 receptor (VDR) and the developmental state of teh two cell populations by assessing the numbers of cells that were immunologically labeled for two osteoblast lineage, stage-specific surface makers (alkaline phosphatase and SB-5, an osteocyte marker) and the VDR. Using the sequence of marker presentation, with VDR appearing first, followed by alkaline phosphatase and then SB-5, models were tested using logistic regression analysis to validate this order of marker presentation and establish that the two embryonic ages of the cell populations represent discrete stages of their lineage. This analysis indicated that 1,25-(OH)2D3 treatment progressed the 12-day-old embryo cell populations along their lineage and that the hormone promoted the appearance of its own receptor (P < 0.001) However, the appearance of the VDR does not appear to be a determinant in the variable responses of the different embryonic aged cell populations to the hormone. These data quantitatively establish the unique nature of osteoblast cell populations within their lineage progression for cells isolated from embryos of different ages, such that cell populations isolated from younger embryos are comprised of primarily presumptive or immature osteoblasts, whereas cells isolated from older embryos are comprised of mature osteoblasts. These data also demonstrate that the genomic effects of 1,25-(OH)2D3 are dependent on the developmental stage of the osteoblast lineage, and the stimulatory actions of the hormone are targeted to immature osteoblasts, whereas the effect of the hormone on mature osteoblasts is inhibitory.

Animals↗

PubMed1996

Analysis of the developmental potential of conditionally immortal marrow-derived mesenchymal progenitor cells isolated from the H-2Kb-tsA58 transgenic mouse.

All nucleated cells from the adult transgenic mouse H-2Kb-tsA58 harbor the temperature-sensitive mutant gene for SV40 large T-antigen. Bone marrow cells from this transgenic mouse were isolated, expanded and cloned in vitro under conditions permissive to the expression of stable T-antigen. Clones of marrow-derived mesenchymal progenitor cells were tested in vitro and in vivo for their capacity to differentiate into mature mesenchymal phenotypes of bone, cartilage, muscle, adipose, and hematopoietic support cells (termed "stromacytes"). Mono-, bi-, and tri-potential clones were identified that were able to differentiate into bone, adipose and stromacyte phenotypes. A mixed population of cells showed some chondrocytic potential in vivo, however, no evidence of cartilage matrix production was detected in vitro for any of the immortomouse clones. These results support the hypothesis that marrow contains multiple progenitor cells that are part of a mesenchymal lineage.

Animals↗