Immature Mesenchymal Cells Research Articles

Modulation of Osteonectin and Osteopontin Expression during Fracture Healing of Mouse Bone Tissue.

We investigated the expression of osteonectin and osteopontin during fracture healing of mouse tibiae by in situ hybridization (ISH) and the effects of fibrin(ogen) on the expression of bone matrix proteins by Northern blot analysis. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibiae. On days 2, 4, 7 and 14 after the operation, the fractured bones were excised, fixed with 4% paraformaldehyde (PFA) and decalcified with 20% EDTA to prepare 5-μm sections. Digoxigenin (DIG) labeled single-stranded DNA probes generated by uni-directional polymerase chain reaction (PCR) were used for ISH. Immunohistochemistry revealed fibrin(ogen) at the site of fracture hematoma 2-4 days after fracture and subsequent accumulation of mesenchymal cells. On days 4-14 after the fracture, osteonectin signals were predominantly expressed in proliferating chondrocytes at the endochondral ossification site and in osteoblasts of the marginal woven bone. Osteopontin was expressed on osteoblasts lining the surface of the marginal woven bone at the mid-late phase of fracture healing. In vitro, Northern blot analysis showed that treatment of the mesenchymal cells, C3H10T1/2, with fibrin(ogen) enhanced the steady-state level of osteonectin and osteopontin gene expression. These data suggested that fibrin(ogen) in the hematoma may be important for inducing bone matrix genes in immature mesenchymal cells at the early phase of fracture repair.

Cells with osteoblastic phenotypes can be explanted from human gingiva and periodontal ligament.

Considerable phenotypic heterogeneity has been reported in gingival fibroblasts. Similarly, cells from the periodontal ligament (PDL) can be isolated with different phenotypes. Although it has been suggested that cells from the gingiva do not contribute to the formation of hard tissue, it is theoretically possible that under appropriate stimuli, immature mesenchymal cells in gingiva could differentiate along an osteoblastic pathway. Differentiation of immature mesenchymal cells into osteoblasts following stimulation with osteoinductive factors has been demonstrated in muscle. We undertook experiments to establish whether cells with osteoblastic characteristics could be identified from human gingiva as well as from human periodontal ligament. Some cell populations from each of these tissues were found to have high basal alkaline phosphatase activity, to release osteocalcin in response to 1,25(OH)2 VitD3, and to form a mineralized matrix. Thus, cells can be isolated from the gingiva and PDL that exhibit phenotypic markers, which taken together are characteristic of osteoblastic cells. Other cell populations derived from the PDL and gingival connective tissue were isolated that had fibroblastic characteristics. These studies support the concept that gingival tissue can give rise to cells which may differentiate along either a fibroblastic or an osteoblastic pathway.

Morphological analogies of fetal prostate stroma and stromal nodules in BPH.

A "reawakening" of ontogenetic processes in the development of BPH is still in debate. Therefore, morphological analogies of fetal prostate stroma and nodular stromal proliferates in BPH were investigated. Fetal prostates (n = 30; weeks 12-40 of gestation) and stromal nodules of benign prostatic hyperplasia (n = 375 from autopsies, n = 100 from biopsies) were investigated by histo- and immunohistochemistry with special regard to cytoskeletal (alpha-actin, desmin, myosin, and vimentin), neuronal (S-100 protein), neuroendocrine (neuron-specific enolase), leukocytic (CD3, CD20, and CD68) and vascular (CD34, BMA-120, and factor VIII) antigens. The developing fetal prostate stroma consists of immature mesenchymal cells up to week 17 of gestation, followed by fibroblastic and fibromuscular stromal cells up to week 25 of gestation and predominantly smooth-muscular cells until the end of gestation. Stromal nodules occur as immature mesenchymal, fibroblastic, fibromuscular, and smooth-muscular, suggestive of a maturational process. The fetal prostate stroma and the stromal nodules present, with an increasing degree of maturation, a similar vascular pattern and a similar occurrence of CD3 (T-lymphocytes), CD20 (B-lymphocytes), CD68 (macrophages), S-100, and neuron-specific enolase-positive cells. The data suggest that ontogenetic processes are recapitulated in the development of stromal nodules in benign prostatic hyperplasia, supporting the idea of a "re-awakening" of fetal processes in BPH.

The role of type I collagen in the regulation of the osteoblast phenotype.

Evidence from a variety of sources indicates that the extracellular matrix forms an important part of a feedback loop governing the migration, proliferation, and differentiation of the cells that produce it. In keeping with this, we showed previously that the extracellular matrix of a multipotential mesenchymal clonal cell line (ROB-C26) induced to differentiate into a more osteoblastic cell type by the addition of exogenous retinoic acid produces an extracellular matrix capable of osteoinductive activity in vivo and of stimulating alkaline phosphatase activity in vitro. Since type I collagen is the major structural component of this extracellular matrix, we sought to determine whether and to what extent this protein is responsible for the previously observed inductive/stimulatory activity. To this end, C26 cells are cultured on plastic, in the presence of retinoic acid, on a type I collagen film, or on an extracellular matrix from retinoic acid-treated C26 cells, and cell differentiation is assessed by measuring changes in the abundance of a number of osteoblast-related mRNAs. These determinations are made by RNAse protection assay after 3 or 6 days of incubation and include measurements of the RNAs for type I collagen, alkaline phosphatase, osteopontin, transforming growth factor alpha 1 and beta 2, and Vgr-1/BMP-6. In addition, C26 cells are incubated in the presence of retinoic acid and several established inhibitors of the synthesis or assembly of extracellular matrix components and the effects on induced alkaline phosphatase activity determined. Our data show that while the collagen substrate mimics some of the effects of retinoic acid and the extracellular matrix, it cannot reproduce all of them. Specifically, while the latter two culture conditions increase the abundance of all six mRNAs, type I collagen film increases the levels of only three of the six (collagen I, alkaline phosphatase, and osteopontin). Moreover, while type I collagen film produces an increase in alkaline phosphatase message, it falls to produce a similar change in alkaline phosphatase activity, an effect seen with both retinoic acid and extracellular matrix. However, interruption of collagen I synthesis by cis-4-hydroxy-L-proline blocks the increase in alkaline phosphatase activity associated with retinoic acid treatment. Thus, it appears likely that type I collagen is a necessary but, by itself, insufficient factor to elicit the comprehensive expression of the osteoblastic phenotype in immature mesenchymal cells.

Spindle cell lipoma in a 14-month-old girl

This case report concerns a girl with spindle cell lipoma of the neck. Spindle cell lipoma is a variant of lipoma and was first described by Enzinger and Harvey in 1975. It occurs chiefly in males between 40 and 70 years of age. It is a benign lesion that can be cured by excision, and local recurrence is rare. Spindle cell lipoma is composed of adipocytes and non-fat-storing immature mesenchymal cells. The condition is uncommon in adults and had not been reported to occur in children.

Lipomatous Tumors of the Uterus: Clinico-pathological Features of 10 Cases with Immunocytochemical Study of Histogenesis

The clinico-pathological and immunocytochemical findings of 10 uterine fatty tumors (1 pure lipoma and 9 lipoleiomyomas) are referred. This kind of tumor is more frequent in older postmenopausal women, treated for a preoperative diagnosis of uterine leiomyoma. Macroscopically the tumor may show different consistence and colour as a consequence of the amount of lipomatous component. The microscopical detection of areas of perivascular immature mesenchymal cells with differentiation into adipocytes supports the hypothesis of "neometaplasia" of the lipomatous component derived from immature perivascular cells. On the contrary, the evidence of multivacuolation in smooth muscular cells and the presence of muscle markers in typical mature adipocytes in lipoleiomyomas, as revealed by immunocytochemistry, suggests the hypothesis of direct transformation of smooth muscle cells into adipocytes.

Comparative histological studies of bone and cartilage formations induced by various BMP-carrier composites.

Five different types of carriers combined with bone morphogenetic protein (BMP) were implanted into the dorsal subcutaneous tissue in the rat in order to study and compare the osteoinductive capacity and histological patterns. The carriers were: insoluble bone matrix (IBM), porous particles of hydroxyapatite (PPHAP), wet and dry collagen beads (CBw, CBd), fibrous collagen membrane (FCM), and fibrous glass membrane (FGM). The carriers alone were implanted for control studies. Each composite and its surrounding tissues were removed after 1, 2 and 3 weeks, and were studied by routine histology. Our results demonstrated that BMP-IBM, BMP-CBd, BMP-FCM and BMP-FGM induced bone tissue in a process that resembled an endochondral ossification mode; in BMP-IBM and BMP-CBd there was an early initiation of the process, in contrast with BMP-FCM and BMP-FGM where a delayed endochondral ossification was observed. These systems, with the exception of BMP-FGM, also induced bone formation through a direct ossification mode. On the other hand, BMP-PPHAP and BMP-CBw induced bone formation in a process that resembled an intramembranous ossification. BMP-carrier composites induce immature mesenchymal cells to become either osteogenetic or chondrogenetic cells, or both, depending on the physicochemical nature of the carrier that affected the microenvironment for cell differentiation. The presen study suggests that BMP-FCM, BNIP-PPHAP and BMP-CBd could be applicable carriers to explore the osteoinductive function of BMP, since no side-effects caused by the carrier were observed.

Pleomorphic Rhabdomyosarcoma in Adulthood

Currently, pleomorphic rhabdomyosarcoma (RMS) in adults is considered to be extremely rare or nonexistent. The authors have identified 11 cases of pleomorphic RMS using the following criteria: pleomorphic sarcoma occurring within voluntary muscle, large polygonal or strap-like cells with copious eosinophilic cytoplasm, desmin and myoglobin immunoreactivity, or ultrastructural evidence of sarcomeric differentiation. Ten patients were male, the median age at presentation was 56 years (range, 27-84), and the thigh (seven cases) was the most common site. Of eight cases with clinical follow-up, one patient is alive at 20 months, and seven died 2 to 28 months following diagnosis. The tumors were generally patternless, but several had storiform areas. Cross-striations were not identified. Immunostaining for muscle-related antigens was positive as follows: desmin (in 10 of 11 cases), myoglobin (in 10 of 11 cases), actin HHF-35 (in all 11 cases), smooth-muscle actin (in six of eight cases), sarcomeric actin (in six of nine cases), and fast myosin (in five of five cases). Staining for S-100 protein was negative in all cases. On electron microscopy (six cases), two tumors had well-differentiated rhabdomyoblasts with sarcomeres, Z-disks, and hexagonal arrays of myofilaments; three were poorly differentiated; and one contained immature mesenchymal cells only. Pleomorphic RMS can be distinguished from other pleomorphic sarcomas provided that well-fixed tumor tissue is available for immunohistochemical staining and electron microscopy. We consider that this distinction is important in view of the poor prognosis associated with pleomorphic RMS in this series.

Uterine sarcomas in CBA mice induced by combined treatment with 1,2-dimethylhydrazine and estradiol dipropionate. Light and electron microscopy.

Three uterine sarcomas induced by combined treatment (1,2-dimethylhydrazine and estradiol dipropionate) in CBA mice were examined by light and electron microscopy. Histologically the tumours consisted either of loose areas with stellate cells or of solid cellular areas. These were formed of elongated cells of the fibroblastic type and rounded cells with clear nuclei and abundant cytoplasm without clearly discernible bodies. Ultrastructurally, undifferentiated mesenchymal cells and cells with the features of fibroblasts and myofibroblasts were distinguished. The tumours described bear resemblance to human endometrial stromal sarcomas and are interpreted as sarcomas originating from immature mesenchymal cells differentiating mainly in the direction of cells of the fibroblastic type. Certain similarities with fibrous histiocytomas are noted.

The fate of calvarias implanted into the epidural spaces of rats. A preliminary study.

The histologic changes occurring in newborn rat calvarias implanted into the epidural space of adult rats were observed to determine whether this method was adequate as an experimental model of new bone formation in the spinal canal. Two weeks after implantation, all implanted calvarias of newborn rats grew with ossification in the epidural space. The grafted calvarias continued to grow gradually 6 weeks after this procedure. Six months later, 40% of the grafted calvarias showed progressive bone formation with osteoblasts, chondrocytes, and immature mesenchymal cells. In the other 60%, the implanted calvarias had developed into mature trabecular bone. Twelve months after the procedure, the calvarias had developed into mature bone in all cases, although the size of the grafted calvarias tended to decrease in size. It was concluded that this experiment had devised a suitable model for understanding the process of new bone formation in the spinal canal.

Immunohistochemical characterization of extracellular matrix components of yolk sac tumors

Proteoglycans (PGs) were isolated from yolk sac tumor and chondroitin sulfate large PG (core molecule with a molecular weight congruent to 200,000) and small PG (core molecule with a molecular weight congruent to 50,000) were detected. Immunohistochemical localization of PGs in three yolk sac tumors was investigated using monoclonal antibodies raised against both small and large PGs, which were purified from human ovarian fibroma capsule and a yolk sac tumor, respectively. The localization of large PG was observed to be distinct from that of small PG. A markedly positive reaction for antibody against large PG was observed in myxomatous areas, perivascular and perivesicular portions; hyaline globules were the most intensely reactive. In the areas showing a polyvesicular vitelline tumor pattern, the compact connective tissue stroma consisted of small PGs. It is conceivable that large PGs are synthesized by immature mesenchymal cells and also by epithelial-like cells as a basement membrane component, whereas small PGs are synthesized by mature fibroblastic cells synthesizing collagen. Immunohistochemical localization of other extracellular matrix components (laminin, fibronectin, type I-IV collagen) was also studied in relation to PG localization.

Spindle cell and pleomorphic lipoma: an immunohistochemical study and histogenetic analysis.

Twenty-two spindle cell lipomas and seven pleomorphic lipomas were investigated immunohistochemically in order to study the differentiation of the non-adipocytic elements. In all cases, neither spindle cells nor pleomorphic cells reacted with antibodies to a monocyte/macrophage antigen (MAC-387), fibronectin, laminin or type IV collagen. The absence of demonstrable basement membrane material argues against the possible prelipoblastic nature of these cells. With the antibody to S-100 protein, spindle cells were immunonegative, whereas pleomorphic cells sometimes revealed an intracytoplasmic weak to moderate staining reaction. In the light of what is known about the development of adipose tissue, our results would support the hypothesis of Bolen and Thorning (Am J Surg Pathol 1981; 5: 435-441) that spindle cell lipoma is composed of adipocytes and non-fat storing immature mesenchymal cells. It would appear that pleomorphic lipoma is similarly derived but that in some cases adipocytic differentiation is also abnormal. The characteristic clinical distribution of these two types of tumour may be of relevance in determining the cause of these unusual benign patterns of differentiation.

Embryonal rhabdomyosarcoma in nude mice and in vitro.

A transplantable tumor strain designated YNnu and a cultured cell line designated YN were established from the human paratesticular rhabdomyosarcoma of a 15-year-old boy. In vitro the cells showed ultrastructural features of immature mesenchymal cells, and a few cells were labeled by antibodies to myoglobin and/or desmin. In nude mice, the cells became to contain numerous myofibrils with Z-bands, and considerable number of cells reacted with anti-myoglobin and/or anti-desmin antibodies. We conclude that spindle-shaped or small round cells are the most immature rhabdomyoblast with multiplicity for cellular differentiation.

Congenital Granular-Cell Neoplasms

Congenital granular-cell tumors are uncommon benign neoplasms of unknown cause that develop primarily in gingival tissue and usually do not recur after surgical excision. Untreated, these neoplasms either cease to grow or "spontaneously" regress after birth. In this report, we describe an unusual case of a child with several congenital granular-cell neoplasms that arose on the lips and continued to increase in size as the child grew. Histologic examination of the neoplasms revealed them to be composed of granular and mesenchymal cells associated with abundant collagen fibers and prominent vascular structures. Ultrastructural study disclosed typical granular cells and a preponderance of immature mesenchymal cells, some of which appeared to be transitional or early granular cells. We interpreted these findings to mean that primitive mesenchymal cells are the proliferative elements and precursors of granular cells in congenital granular-cell neoplasms. The presence of numerous immature mesenchymal cells corroborates the clinical impression that the lesions were growing. Based upon the histological and ultrastructural findings reported here, and a review of the literature, we favor a mesenchymal or endothelial-cell origin for congenital granular cell neoplasms.

Combined ultrastructural and immunohistochemical studies on a mouse rhabdomyosarcoma.

Light microscopic and ultrastructural observations, using immunoperoxidase staining for myoglobin and actin were performed on pleomorphic rhabdomyosarcoma in an ICR-male mouse aged 98 weeks, which arose in the striated muscle of the lumbar region. The tumor consisted of three types of cells showing varying degrees of myofibrinogenesis. The small round or elongated mononucleated cells (type I), which predominated in number, were immature mesenchymal cells. Some of these cells contained small amount of perinuclear intermediate filaments (9-11 nm in diameter). The eosinophilic plump cells (type II) exhibited one or two irregular nuclei and broad cytoplasm containing both types of myofilaments, actin and myosin filaments, not arranged to form myofibrils. The multinucleated giant cells (type III) alone possessed a Well developed element of tumoral myofibrils, Z-band. C-type virus like particles were rarely found in cytoplasmic vesicles or intercellular spaces of all types of cells. Myoglobin-positive staining was detected in most of the type III cells, and a few of the type II cells, while actin-positive staining was seen in most of both types cells. However, the type I cells were consistently negative for these two markers.

A comparative ultrastructural study of chondrosarcoma, chordoid sarcoma, chordoma and chordoma periphericum

The present study is based on electron microscopical examinations of 15 conventional chondrosarcomas, 1 clear cell chondrosarcoma, 3 mesenchymal chondrosarcomas, 2 so-called chordoid sarcomas (extraskeletal myxoid chondrosarcoma), 4 sacrococcygeal chordomas, 2 ecchordoses and 1 neoplasm of tibia with features of a true peripheral chordoma (parachordoma). The neoplastic cells from various types of chondrosarcoma shared a number of features with nonneoplastic chondrocytes as e.g. a well-developed rough endoplasmic reticulum and microvillous cytoplasmic processes. In clear-cell chondrosarcoma, glycogen accumulation in the tumour cells was a prominent feature. The cells of mesenchymal chondrosarcoma usually showed the characteristics of immature mesenchymal cells. In contrast, chordomas commonly contained physaliferous cells with two types of vacuoles in their cytoplasm. The first type can be most adequately characterized as intracytoplasmic pseudoinclusions of intercellular substance, whereas the other type, glycogen-containing, single membrane-bound vacuoles most probably correspond to autophagosomes (cytolysosomes). Only vacuoles of the first type were recorded in the so-called chordoid sarcoma. They were also seen in chondrosarcomas. In contrast, both types of vacuoles were identified in the above-mentioned tibial tumour which, in addition, showed even other cytological characteristics of chordoma. The findings presented here have demonstrated distinct structural relationships between chordoid sarcoma and chondrogenic tumours. On the other hand, our observation of the uncommon tibial neoplasm indicates the possibility that tumours identical with chordoma may occur at sites other than the axial skeleton.

Ultrastructure and immunohistochemical localization of estradiol in three thecomas

Three ovarian thecomas were studied by ultrastructural and immunohistochemical techniques. In each tumor, a small number of tumor cells stained for estradiol. Vacuolated as well as plump non-vacuolated tumor cells were positive, but spindle-shaped cells were negative. Ultrastructural examination showed two principle cell types. Type I cells were immature mesenchymal cells that differed from typical steroid-secreting cells because only a minority had conspicuous smooth endoplasmic reticulum, although mitochondria often had tubular cristae. Type II cells were distinguished by abundant intermediate (10-nm) microfilaments and round mitochondria with incomplete cristae and empty centers. Focal smooth muscle differentiation was present in each tumor. Adherens-type intercellular junctions, including desmosomes in one case, and degenerate cells with markedly vesiculated cytoplasmic membrane systems were also present. Lipid was not abundant, but it was more conspicuous in degenerate cells and in type II cells. Thecoma cells thus closely resemble both ovarian stromal cells and theca interna cells, which are known to be capable of steroidogenesis. The localization of estradiol in a minority of tumor cells in each thecoma suggests that thecoma cells, too, are capable of steroid synthesis and supports the popular concept that hyperestrogenism in patients with thecomas may be the result of estradiol secretion by these tumors.

Ultrastructural observations on the histogenesis of localized fibrous tumours of the pleura (benign mesothelioma).

Five localized fibrous tumours of the pleura (benign mesothelioma) were studied ultrastructurally in order to elucidate their histogenesis. The histological subtypes of this benign fibrous lesion of the visceral pleura, i.e. the cellular, the collagenous, and the hyaline, were separately analysed. The tumours are composed of undifferentiated mesenchymal cells, intermediate and differentiated fibroblasts as well as collagenous interstitial tissue. The varying distribution of these cell elements account for the various histological subtypes. Morphological similarities between the mesenchymal tumour cells and the superficial mesothelial cells, which are always separated from the true tumour tissue by an intact basement membrane, were not observed. The different cellular elements can be regarded as parts of a continuous spectrum of cytodifferentiation, in which the mature fibroblasts are derived via intermediate forms from the undifferentiated cells. It is concluded that the localized fibrous tumours of the pleura arise from immature mesenchymal stem cells, which seems to be normally found in the submesothelial layer of the visceral pleura.

Monoclonal antibodies to E92, an endothelial cell surface antigen.

Two hybridoma-derived monoclonal antibodies have been developed that react with an antigen of molecular weight 92,000 daltons on the surface of human endothelial cells. Cultured human umbilical vein endothelial cells were used for immunization, but the antigen is present on arterial, venous and capillary endothelium, as determined by biotin-avidin immunoperoxidase staining of tissue sections. With this technique, other cell types in the tissues which were examined were not reactive, except for scattered fibroblasts and histiomonocytic cells, trophoblastic cells of the placenta, and benign immature mesenchymal cells in a renal cystadenocarcinoma. By cytofluorography, the antibodies were found to be unreactive with granulocytes, T lymphocytes, B lymphocytes, and the majority of monocytes. Fibroblasts were reactive with the antibodies, but the fluorescence tracings indicated a lower density of antigen on these cells than on endothelial cells. Immunoreactivity of fibroblasts could be decreased by treatment of the cells with thrombin, trypsin, or neuraminidase, whereas these enzymes did not affect the immunoreactivity of endothelial cells. The reactive antigen (E92) does not appear to be any of several previously described endothelial cell proteins, because of its molecular weight and its absence on other cell types. The presence of E92 on trophoblastic cells of the placenta and immature mesenchymal cells, as well as fibroblasts and endothelial cells, may indicate that it is a primitive antigen of mesodermal tissue that is lost by most cell types during differentiation.

サルあご関節関節円板における実験的微細損傷の治ゆ経過に関する組織学的観察

The purpose of the present paper is to elucidate the healing process of the experimental microtrauma at the articular disc of the temporomandibular joint in monkey and to determine the area where there is any repair. Twelve animals were used and two of them were used as control. In other monkeys, both temporomandibular joints were opened surgically and the discs were injured by a special type of knife for microtrauma. Two kinds of microtrauma were produced. One was within the confine of the articular surface of the disc and the other was beyond the articular surface over the lateral synovial lining. The animals were sacrificed at one, two, three, four, six and twelve weeks after operation. Block dissections of the articular apparatus were taken and examined histologically. Results showed that no repair was demonstrated in the injury which was confined in the articular surface but, when the wound communicated with the lateral synovial lining, immature mesenchymal cells began to invade along the bottom of incisive cleavage from the lining synovium adjacent to the wound. After two weeks these cells reach the incisive cleavage near the central portion of the articular disc. After six weeks, from the synovial membrane to the central portion of the articular disc, fibroblasts differentiated to form fine collagen fibers in the incision wound space. After twelve weeks, the component of the fibers were increased in the same area. But, the original fibrous structure of the articular disc still couldn't be observed.